Methods of treatment, prevention and prognosis of colorectal cancer

Abstract

The present invention relates to the treatment of cancer, including colorectal cancer (CRC), by inhibiting or blocking Annexin 1. The present invention also relates to the prevention of CRC in high risk patients by inhibiting or blocking Annexin A1. The present invention also relates to the reduction of chemotherapy resistance by inhibiting or blocking Annexin A1. The present invention also relates to the detection of poor prognosis in subjects with CRC by detecting or measuring the expression level of ANXA1 and / or the protein level of Annexin 1.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Patent Application Serial No. 62 / 672,989, filed May 17, 2018, and U.S. Patent Application Serial No. 62 / 796,349, filed January 24, 2019, both of which are incorporated herein by reference in their entirety.

[0003] Statement of Government Interest

[0004] This invention was made with government support under Grant Numbers CA192111, DE014924, DE023332, and CA197649-04 awarded by the National Institutes of Health. The United States government has certain rights in the invention. TECHNICAL FIELD

[0005] The present invention relates to the treatment and / or prevention of cancer, including colorectal cancer (CRC), by inhibiting or blocking Annexin 1. The present invention also relates to the reduction of chemotherapy resistance by inhibiting or blocking Annexin A1. The present invention also relates to the detection of poor prognosis in subjects with CRC by detecting or measuring the expression level of ANXA1 and / or the protein level of Annexin 1. BACKGROUND

[0006] In the United States, colorectal cancer (CRC) is the second leading cause of cancer deaths, affecting 1 in 20 people (ACS 2012). CRC has long been thought to be caused by the accumulation of host mutations over time, developing from premalignant adenomatous polyps to adenocarcinoma over approximately 10 years (Vogelstein and Kinzler 1993). Advances in microbiome detection technologies and human microbiome research have revolutionized our understanding of a variety of diseases, including CRC (Dulal and Keku 2014; Keku et al. 2015). However, there remains a need for more effective treatments for CRC, as well as other solid tumors.

[0007] Recent studies have identified the Gram-negative oral commensal bacterium Fusobacterium nucleatum (F. nucleatum or Fn) as an important factor in colorectal cancer (CRC) that is often associated with poor prognosis (Bullman et al., 2017; Yu et al., 2017). Fn stimulates colorectal tumorigenesis by binding of its FadA adhesion protein to E-cadherin, which then activates Wnt / β-catenin signaling (Rubinstein et al., 2013). E-cadherin is ubiquitously expressed, and it is not understood why the binding of FadA to E-cadherin on CRC cells is different from binding to other cells.

[0008] Fn has been detected in approximately 10-90% of CRC tissues (Tahara et al., 2014; Mima et al., 2016; Yu et al., 2016) and is more prevalent in the proximal colon compared to the distal colon (Mima et al., 2016a; Yu et al., 2016). Fn is often associated with advanced disease, chemotherapy resistance, metastasis, and poor prognosis (Bullman et al., 2017; Yu et al., 2017). Several studies support an etiological role of Fn in CRC (Yu et al., 2016; Rubinstein et al., 2013). Fn has been reported to promote CRC growth through its unique FadA adhesion protein, which binds to E-cadherin (CDH1) and activates Wnt / β-catenin signaling (Rubinstein et al., 2013). Binding of FadA to E-cadherin requires both the full pre-FadA consisting of 129 amino acid residues and the 111 amino acid residue signal peptide-free mFadA. Together they form the FadAc complex (Rubinstein et al., 2013; Xu et al., 2007). However, FadA does not promote growth of non-cancerous HEK293 cells even in the presence of E-cadherin (Rubinstein et al., 2013), raising the question of whether Fn-induced growth is CRC-specific and, if so, by what mechanism.

[0009] Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer syndrome and accounts for 1% of all colorectal cancer cases. The "F" stands for familial, meaning it is passed down in families; the "A" stands for adenomatous, referring to the type of polyps detected in the colon and small intestine that can turn into cancer; and the "P" stands for polyposis or a condition with many colon polyps. The gene for FAP is on the long arm of chromosome 5 and is called the APC gene.

[0010] Patients with FAP develop hundreds to thousands of colonic polyps, usually starting in the teenage years. All patients usually progress from colonic polyps to colorectal cancer by the age of 40. Patients with FAP must have their colon, and sometimes rectum, removed to prevent colon cancer. The only existing drug treatments, sulindac and celecoxib, have not been successful due to side effects and lack of efficacy. Thus, there is a need for better prevention and treatment options for FAP patients. SUMMARY

[0011] The present invention is based on the new finding set forth herein that the gram-negative oral anaerobic bacterium Fusobacterium nucleatum (F. nucleatum or Fn) is an important contributor to colorectal cancer. F. nucleatum stimulates the growth of colorectal cancer cells, but not pre-cancerous adenoma cells. Annexin A1, a previously unrecognized regulator of Wnt / β-catenin signaling, is a key component enabling F. nucleatum to exert its stimulatory effects. It is specifically expressed in proliferating colorectal cancer cells and involved in cyclin D1 activation. The FadA adhesion protein from F. nucleatum upregulates annexin A1 expression via E-cadherin. A positive feedback loop between FadA and annexin A1 is identified in cancer cells and is absent in non-cancer cells. A "two-hit" model of colorectal carcinogenesis is postulated, in which somatic mutations are the first hit and F. nucleatum is the second hit that exacerbates cancer progression after a benign cell becomes cancerous.

[0012] Embodiments of the present invention are based on annexin A1 (ANXA1) as a suitable therapeutic target, which is selectively expressed in proliferating CRC cells and specifically induced by Fn. Other aspects of the present invention include that Fn induces chemoresistance by activating annexin A1, and that chemoresistance can be overcome by blocking annexin A1. The present invention sets forth a new role of annexin A1 in regulating Wnt / β-catenin signaling. Given the widespread implication of Wnt / β-catenin in cancer and the increasing number of reports of Fn in different cancer types, including malignant solid tumors, including melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate cancer), pancreatic cancer, breast cancer, colon cancer (CRC), lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, and leukemia, annexin A1 is a new therapeutic target for reducing cancer cell proliferation and treating cancer.

[0013] Accordingly, one embodiment of the application is a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an agent that inhibits or blocks annexin Al.

[0014] Another embodiment of the application is a method of preventing cancer in a subject in need thereof (i.e., having a high risk of developing cancer), comprising administering a therapeutically effective amount of an agent that inhibits or blocks annexin Al. Subjects considered to have a high risk of developing colorectal cancer include subjects with familial adenomatous polyposis or FAP and subjects at risk of recurrence, e.g., subjects having high levels of annexin Al in colorectal cancer tissue.

[0015] Another embodiment of the application is a method of reducing chemoresistance of cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an agent that inhibits or blocks annexin Al.

[0016] Another embodiment of the application is a method of inhibiting proliferation of a cancer cell, comprising reducing expression of annexin Al on the cancer cell.

[0017] In certain embodiments, the cancer is colorectal cancer.

[0018] In certain embodiments, the cancer is selected from melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon or colorectal cancer (CRC), lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, and leukemia.

[0019] In certain embodiments, the agent includes, but is not limited to, an ANXA1-specific RNA interference (RNAi) molecule, including small interfering RNA (siRNA) and microRNA (miRNA).

[0020] In certain embodiments, the agent is an anti-annexin Al antibody, minibody, Fab or fragment, camelid antibody, nanobody, etc.

[0021] In certain embodiments, the agent is a small molecule capable of inhibiting the biosynthesis, intracellular translocation, upregulation, or release of annexin Al. In other embodiments, the small molecule is selected from indomethacin, thapsigargin, and sorafenib, and related structural analogs and derivatives.

[0022] The application also includes compositions and pharmaceutical compositions comprising an agent that inhibits or blocks annexin Al, and in certain embodiments, binding partners such as small molecules, peptides, and natural or synthetic polymer molecules.

[0023] In all of the foregoing embodiments, the administration of the agent that inhibits or blocks annexin A1 can comprise administration of the agent alone or in combination with other therapeutic agents, including but not limited to chemotherapeutic agents, targeted chemotherapeutic agents, and immunotherapies.

[0024] In addition, as shown herein, the level of annexin A1 expression in colorectal cancer is a poor prognosis predictor that is independent of cancer stage, grade, age, and gender.

[0025] Accordingly, another embodiment of the application is a method of detecting a poor prognosis and / or recurrence of colorectal cancer in a subject having colorectal cancer, the method comprising detecting the level of annexin A1 expression in a colorectal cancer tissue from the subject and comparing the level of expression to a reference level of annexin A1 expression in healthy colorectal tissue, wherein an increase in annexin A1 expression in the colorectal cancer tissue relative to the expression in healthy tissue indicates a poor prognosis and / or recurrence of colorectal cancer in the subject.

[0026] Accordingly, another embodiment of the application is a method of treating a subject having colorectal cancer, the method comprising detecting the level of annexin A1 expression in a colorectal cancer tissue from the subject and comparing the level of expression to a reference level of annexin A1 expression in healthy colorectal tissue, wherein an increase in annexin A1 expression in the colorectal cancer tissue relative to the expression in healthy tissue indicates a poor prognosis and / or recurrence of colorectal cancer in the subject, and actively treating the subject.

[0027] The active treatment for colorectal cancer can comprise administration of an agent that inhibits or blocks annexin A1 alone or in combination with a chemotherapeutic agent, a targeted chemotherapeutic agent, and / or an immunotherapy. Chemotherapeutic agents can include but are not limited to 5-fluorouracil, capecitabine, irinotecan, oxaliplatin, and the combination of trifluridine and tipyracil. Targeted chemotherapeutic agents include but are not limited to agents, compounds, or drugs that target VEGF, including bevacizumab, ramucirumab, and aflibercept; drugs that target EGFR, including cetuximab and panitumumab; and regorafenib. Immunotherapies include but are not limited to PD-1 inhibitors, including pembrolizumab and nivolumab; and CTLA-4 inhibitors, including ipilimumab. The addition of the agent that inhibits or blocks annexin A1 can reduce resistance to and / or increase the efficacy of these other therapeutic agents.

[0028] Determining expression of annexin A1 can be performed by any method known in the art, including but not limited to microarray, Southern blot, Northern blot, dot blot, primer extension, nuclease protection, subtractive hybridization, and separation of non-duplex molecules using, for example, hydroxyapatite, solution hybridization, filter hybridization, amplification techniques such as RT-PCR and other PCR-related techniques such as PCR with melt curve analysis and PCR with mass spectrometry, fingerprinting such as using restriction endonucleases and fingerprinting using structure-specific endonucleases. mRNA expression can also be analyzed using mass spectrometry techniques (e.g., MALDI or SELDI), liquid chromatography, and capillary gel electrophoresis. Any additional methods known in the art can be used to detect the presence or absence of a transcript.

[0029] Detection of annexin A1 expression can also be performed at the protein level by any method known in the art, including methods that produce qualitative results, such as methods in which the presence of the protein can be observed by the naked eye or other means, and / or methods that produce quantitative results. These methods include, but are not limited to, quantitative Western blot, immunoblot, quantitative mass spectrometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), and immunoenzymatic assay (IEMA), and sandwich assays using monoclonal and polyclonal antibodies.

[0030] The expression of annexin A1 in the colorectal cancer tissue can be compared to a reference value of the expression of annexin A1 in a healthy control tissue. The expression level can be measured as an absolute or relative level. BRIEF DESCRIPTION OF DRAWINGS

[0031] For the purpose of illustrating the present application, certain embodiments of the present application are depicted in the accompanying figures. However, the present application is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the figures.

[0032] Figure 1A-1 K shows that F. nucleatum preferentially binds to, invades and stimulates the growth of cancerous colorectal cells through annexin A1, which is selectively expressed in proliferating cancerous colorectal cells and is a novel CRC growth factor, and that ANXA1-specific siRNA inhibits tumor growth.

[0033] Figure 1A shows the results of cell proliferation assays following incubation of lung cancer cells PC-9, prostate cancer cells 22RV1, bladder cancer cells UMUC3, breast cancer cells MCF-7, non-cancerous cells AA / C1 ("C1") and AA / C1 / SB ("SB") of colonic adenoma origin or cancerous cells AA / C1 / SB / 10C ("10C") with wild-type Fusobacterium nucleatum 12230 (Fn), fadA deletion mutant US1 (US1) or E. coli DH5a (E. coli) at a multiplicity of infection (MOI) of 1000:1. Cells were counted at the times indicated. Cell numbers are mean ± SEM. Experiments were performed in triplicate and repeated three times. **p < 0.01, ***p < 0.001 compared to untreated controls; #p < 0.05, ###p < 0.001 compared to US1 -treated cells (two-way ANOVA).

[0034] Figure 1B Results are shown for attachment (left panel) and invasion (right panel) of wild-type Fusobacterium nucleatum 12230 (MOI = 50:1) on non-tumorigenic SB cells untreated or transfected with antisense or sense ANXA1 and on tumorigenic 10C cells untreated or transfected with control or ANXA1- or CDH1 -specific siRNA or both. Fn attachment and invasion on untreated SB cells were assigned as 100%; all other values are expressed as relative values to those obtained using untreated SB. Data are mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 (one-way ANOVA).

[0035] Figure 1C Figure is a graph of the results of qPCR analysis of villin 1 (VIL1) mRNA levels in 10C cells treated with control siRNA or VIL1 -specific siRNA, confirming knockdown of villin 1 (left panel) and attachment of Fusobacterium nucleatum 12230 to 10C cells treated with control siRNA or VIL1 -specific siRNA (right panel). Data are mean ± SD. Experiments were performed in triplicate and repeated twice. *p < 0.05 (student's t-test).

[0036] Figure 1DFigure 1A is a graph of the results of real-time qPCR analysis of ANXA1 expression using mRNA extracted from non-cancerous SB, cancerous 10C and human CRC cell lines HCT116, DLD1 and RKO grown to 50% or 100% confluence. All results were normalised to the ANXA1 mRNA level of 100% confluent SB cells, which was assigned a value of 1. Data are mean ± SEM. Experiments were performed in triplicate and repeated twice. **p<0.01 and ***p<0.001 (student’s t-test).

[0037] Figure 1E Figure 1C is a representative image of confocal microscopy analysis of annexin A1 immunofluorescence staining (green) and DAPI nuclear staining (blue) from SB and 10C cells grown to 20% (top panel) or 100% (bottom panel) confluence. A series of 20-50 consecutive images in the z-axis were stacked together to generate 3-D images at 400x magnification. Annexin A1 was most abundantly expressed on the outer layer of 20% confluent 10C (arrowed) compared to 100% confluent 10C or any confluent SB cells. Scale bar, x=l pm, y=l pm, z=l.6 pm. Experiments were repeated at least twice.

[0038] Figure 1F is a graph of the results of cell proliferation assays of non-tumorigenic SB and tumorigenic 10C adenoma-derived cells and human CRC cell lines HCT116, DLD1, SW480, HT29 and RKO untreated (black line with circles) or treated with control siRNA (grey line with triangles) or ANXA1 -specific siRNA (grey line with squares). Data are mean ± SEM. Experiments were performed in triplicate and repeated three times. *p<0.05, **p<0.01 and ***p<0.001 compared to untreated cells (two-way ANOVA).

[0039] Figure 1G Figure 1G is a graph of the results of cell proliferation assays of SB (left panel) and RKO (right panel) cells transfected with ANXA1 (grey line with squares) compared to control cells (black line with circles). Data are mean ± SEM. Experiments were performed in triplicate and repeated three times. *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA).

[0040] Figure 1HGraphs and images of xenograft tumor growth in nude mice after subcutaneous and bilateral inoculation of HCT116 cells transfected with control siRNA (left) or ANXA1 -specific siRNA (right). Tumor volumes were measured after 7 days post-injection (left graph, n=4). For each mouse, the tumor produced by ANXA1 siRNA-treated cells was normalized to the tumor produced by control siRNA-treated cells, which was assigned as 100%. Lines represent the mean values. *p<0.05 (unpaired t-test). Representative tumors are shown on the right graph: top, tumor produced by control siRNA-treated cells; bottom, tumor produced by ANXA1 -specific siRNA-treated cells.

[0041] Figure 1I Graphs and images of xenograft tumor growth in nude mice after subcutaneous and bilateral inoculation of DLD1 cells transfected with control siRNA (left) or ANXA1 -specific siRNA (right). Tumor volumes were measured after 7 days post-injection (left graph, n=3). For each mouse, the tumor produced by ANXA1 siRNA-treated cells was normalized to the tumor produced by control siRNA-treated cells, which was assigned as 100%. Lines represent the mean values. *p<0.05 (unpaired t-test). Representative tumors are shown on the right graph: top, tumor produced by control siRNA-treated cells; bottom, tumor produced by ANXA1 -specific siRNA-treated cells.

[0042] Figure 1J IL1 β, Nfkb2, Rantes, CCL20 and Cyclin D1 expression measured by real-time qPCR in MCF-7, AA / C1, AA / C1 / SB (SB) and AA / C1 / SB / 10C (10C) cells incubated with wild-type Fn 12230 for 3 hours and then extracted for RNA are shown. Data are mean ± SD. *p<0.05, **p<0.01 and ***p<0.001 compared to untreated cells.

[0043] Figure 1K Western blot analysis of E-cadherin and Annexin A1 expression in PC-9 lung cancer cells, 22RV1 prostate cancer cells, UMUC3 bladder cancer cells and MCF-7 breast cancer cells is shown. β-actin is included as an internal control.

[0044] Figure 2A-2 F shows that F. nucleatum selectively binds to cells expressing Annexin A1 and induces further Annexin A1 expression by FadA.

[0045] Figure 2A is representative images and graphs of flow cytometry analysis of SB and 10C cells after incubation with CFSE-labelled Fusobacterium nucleatum 12230 (Fn) or its fadA deletion mutant US1 (US1) for the indicated times followed by immunostaining with Annexin Al. Density plots are shown in the upper panels. x-axis, Annexin Al; y-axis, CFSE-labelled Fn or US1. Geometric mean of Annexin Al positive staining (line, scale on the right) and percentage of Annexin Al positive (solid bar) or negative (open bar) cells bound by Fn or US1 over the total number of cells analyzed (scale on the left) are shown in the lower panels. Data are mean ± SD. *p<0.05, ***p<0.001.

[0046] Figure 2B Figure 3 is a graph of the results of flow cytometry analysis of Annexin Al expression in 10C cells untreated or incubated with BSA (1000 pg / ml) or mFadA (1000 pg / ml) or FadAc (100, 300 or 1000 pg / ml). Data are mean ± SD. **p<0.01.

[0047] Figure 2C Figure 4 is a graph of the results of real-time qPCR analysis of ANXA1 mRNA levels in SB, 10C, HCT116 and DLD1 cells treated with wild-type Fusobacterium nucleatum 12230 (Fn) (dark lines with squares) or fadA deletion mutant US1 (light lines with circles) for the indicated lengths of time. Results were normalized to those obtained from untreated cells and are the mean of three independent experiments performed in triplicate, *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA).

[0048] Figure 2D Figure 5 is representative images of confocal microscopy of SB and 10C cells untreated or incubated with Fusobacterium nucleatum 12230 (Fn) for 1 hour at a MOI of 5:1. Annexin Al is stained in green (appears white in the images) while E-cadherin is stained in blue (appears grey in the images). Images are magnified 800x. Note the increased expression of Annexin Al in 10C compared to SB and its localization on the outer rim of the cell mass. Experiments were repeated three times. Scale bar, 250 nm.

[0049] Figure 2EStatistical analysis showing the association between exposure to F. nucleatum and upregulation of ANXA1 mRNA expression levels in colon cancer cells HT29. ANXA1 mRNA levels in HT29 cells in triplicate at baseline and after incubation with F. nucleatum 25586 were analyzed in RNA-seq datasets publicly available from the NCBI-GEO online repository (GSE90944) and containing global gene expression measurements from HT29 cells (Yu et al., 2017). The distribution of ANXA1 mRNA expression levels in the two sample groups (baseline vs. infected) was visualized using their log2 of TPM (transcripts per million) expression values as a measure using box-and-whisker plots. The difference in mean log2 TPM values between HT29 cells at baseline (n=3) and after incubation with F. nucleatum (n=3) was tested for statistical significance using a two-tailed t-test for continuous variables. The analysis revealed that HT29 cells exposed to F. nucleatum were characterized by an increase in ANXA1 mRNA expression levels compared to HT29 cells at baseline (p=0.01).

[0050] Figure 2F. Representative images and graphs of flow cytometry analysis of DLD1 and HCT116 cells after incubation with CFSE-labeled F. nucleatum 12230 (Fn) or its fadA deletion mutant US1 (US1) for the indicated times and subsequent immunostaining for annexin Al. Density plots are shown in the upper panels. x-axis, annexin Al; y-axis, CFSE-labeled Fn or US1. Geometric mean of annexin Al positive staining (blue line, scale on the right) and percentage of annexin Al positive (solid bars) or negative (open bars) cells bound by Fn or US1 over the total number of cells analyzed (scale on the left) are shown in the lower panels. Data are mean ± SD. *p<0.05, ***p<0.001.

[0051] Figures 3A-3L FadA, E-cadherin (CDH1), annexin Al and β-catenin are shown to form a complex in cancerous cells.

[0052] Figure 3A Flow cytometry analysis of annexin Al in 10C cells transfected with control siRNA (black dotted line) or CDH1-specific siRNA (red dotted line) and then either untreated or incubated with BSA (1000 pg / ml) or FadAc (1000 pg / ml) for 1 h. C, untreated control. Data are mean ± SD. Experiments were performed in triplicate and repeated twice. ***p<0.001 (two-way ANOVA).

[0053] Figure 3B Representative images are of 10C cells, either untreated (top) or after incubation with CFSE-labeled *Fusobacterium nucleatum* 12230 (red, bottom) and immunostained with annexin A1 (green, shown as white in the image) and E-cadherin (blue, shown as gray in the image). Images are magnified 1200x. A side view of the magnified image is shown on the far right. Note the enhanced annexin A1 expression in *Fusobacterium nucleatum*-bound cells and the co-localization of annexin A1, E-cadherin, and *Fusobacterium nucleatum* on the cell membrane (arrows). Experiments were repeated more than three times. Scale bar, 500 nm.

[0054] Figure 3C It is in conjunction with Alexa Fluor TM Representative images of 10C cells incubated with 488-conjugated BSA, mFadA, or FadAc (300 μg / ml; red) and subjected to confocal microscopy analysis after immunostaining with annexin A1 (green, shown as white in the image) and E-cadherin (blue, shown as gray in the image). Images are magnified 1200x. Note the enhanced expression of annexin A1 in response to FadAc and its colocalization with E-cadherin (arrows) compared to BSA and mFadA. Experiments were repeated twice. A side view is shown in the lower right of each image. Scale bar, 500 nm.

[0055] Figure 3D Western blot analysis of FadA, E-cadherin (CDH1), annexin A1 (ANXA1), and β-catenin in DLD1 cells is shown after incubation with FadAc (1000 μg / ml) for 15 or 120 min. C, untreated cells. Includes β-actin as an internal control. The experiment was repeated three times.

[0056] Figure 3E Western blotting using annexin A1 is shown. DLD1 cell lysates were incubated with FadAc (1000 μg / ml) for 15 or 120 min and mixed with agarose beads conjugated with rabbit anti-annexin A1 polyclonal antibody (α-annexin A1) or control rabbit IgG. FadA, E-cadherin (CDH1), annexin A1, and β-catenin were detected in the eluent by Western blotting. C, untreated control. The experiment was repeated three times.

[0057] Figure 3FBlots showing co-immunoprecipitation using FadA. DLD1 cell lysates were incubated with FadAc (1000 μg / ml) for 15 or 120 minutes and mixed with agarose beads coupled with mouse anti-FadA monoclonal antibody (a-FadA) or control mouse IgG. Eluates were tested by Western blot for FadA, E-cadherin (CDH1), Annexin Al and β-catenin. Experiments were repeated three times.

[0058] Figure 3G Graph showing results of flow cytometry analysis of β-catenin expression in 10C, HCT 116 and DLD1 cells after transfection with control siRNA (open bars) or ANXA1 -specific siRNA (solid bars) and subsequent incubation with Fusobacterium nucleatum 12230 at a MOI of approximately 20:1 for the indicated length of time. Geometric mean at 0 h for cells treated with control siRNA was designated as 1. Data are mean ± SD. ***p < 0.001. Experiments were performed in duplicate or triplicate and repeated 1-3 times. **p < 0.01, ***p < 0.001 (two-way ANOVA).

[0059] Figure 3H Representative images showing immunostaining of β-catenin in 10C cells after transfection with control siRNA or ANXA1 -specific siRNA and subsequent incubation with Fusobacterium nucleatum 12230 (Fn) for 2 h at a MOI of approximately 100:1. β-catenin was stained with Alexa fluor® 568 (red, shown as light grey in images), nuclei were stained with DAPI (blue, shown as dark grey in images). Images were taken with a confocal microscope at 800x magnification. -, no bacteria added. Note the increased expression of β-catenin in response to Fn in control siRNA-treated cells compared to ANXA1 siRNA-treated cells and its nuclear translocation.

[0060] Figure 3I Graph showing results of real-time qPCR analysis of E-cadherin (CDH1) mRNA levels in SB, 10C, HCT 116 and DLD1 cells after incubation with Fusobacterium nucleatum 12230 (Fn) (dark lines with squares) or fadA deletion mutant (US1) (light lines with circles) for the indicated length of time. All results were normalized to the results of untreated cells. Experiments were repeated twice. Scale bar, 200 nm.

[0061] Figure 3Jare representative images of confocal microscopy analysis of DLD1 cells untreated (upper panel) or after incubation with CFSE-labelled Fusobacterium nucleatum 12230 (red, lower panel) and immunostaining for Annexin A1 (green, shown as white on the image) and E-cadherin (blue, shown as grey on the image). Images were magnified 1200x. Arrows point to co-localization of Annexin A1, E-cadherin and Fn.

[0062] Figure 3K Results of real-time qPCR analysis of Cyclin D1 (CCND1) expression using mRNA extracted from cancerous 10C and human CRC cells HCT116 and RKO grown to 50% or 100% confluence are shown. Data are mean ± SEM. Experiments were performed in triplicate and repeated twice. *p<0.05, ***p<0.001 (student's t-test).

[0063] Figure 3L Western blot analysis of Cyclin D1, Annexin A1 and β-actin in RKO cells transfected with control vector or ANXA1 is shown. Induction of Cyclin D1 in response to increased Annexin A1 was observed. Experiments were repeated twice.

[0064] Figures 4A-4G FadA and Annexin A1 are co-expressed in colorectal tumors in mice and humans is shown.

[0065] Figure 4A are graph and representative images of colorectal tumors developed in mice after treatment with PBS, E. coli DH5a (E. coli), fadA deletion mutant US1 (US1) or Fusobacterium nucleatum 12230 (Fn). Each symbol represents one mouse. Horizontal lines represent mean values. Representative tumors formed in the mouse colon are shown on the right, indicated by the blue arrow on the right. *p<0.05, **p<0.01 (one-way ANOVA).

[0066] Figure 4B APC min / + Graph of ANXA1 mRNA levels in mouse colon tumors (T) and normal colon tissue (N). Each symbol represents one mouse. Horizontal lines represent mean values. *p<0.05, **p<0.01 (two-way ANOVA).

[0067] Figure 4C are shown are graphs of APC min / +Plot of the positive correlation between fadA gene copy number (x-axis) and ANXA1 mRNA levels (y-axis) in mouse colon tumors (n=34; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicate.

[0068] Figure 4D Plot of the abundance of fadA in paired normal tissue and adenocarcinoma tissue from CRC patients (n=18) expressed as the ratio of fadA to total 16S rRNA gene determined by qPCR. Each symbol represents one patient. Horizontal lines indicate the mean. ** p<0.01 (paired t-test).

[0069] Figure 4E Plot of ANXA1 mRNA levels in paired normal tissue and adenocarcinoma tissue from CRC patients measured by qPCR. Each symbol represents one patient. Horizontal lines indicate the mean. ** p<0.01 (paired t-test).

[0070] Figure 4F Plot of the correlation between fadA gene copy number (x-axis) and ANXA1 mRNA levels (y-axis) in human colorectal adenocarcinoma tissue (n=18; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicate.

[0071] Figure 4G Representative images of confocal microscopy analysis of paired normal tissue and cancer tissue from two colon cancer patients. Cryosections were incubated with rabbit anti-Annexin A1 polyclonal antibody and 5G11 mouse anti-FadA monoclonal antibody. Slides were then stained with Alexa coupled donkey anti-rabbit antibody and Alexa coupled goat anti-mouse antibody, washed, and mounted in a mounting medium containing DAPI. Confocal microscopy images were scanned using a Nikon Ti Eclipse inverted microscope at 200x magnification for normal tissue and at 400x magnification for cancer tissue. Scale bar, 50 μm. Co-localization of FadA (red) and Annexin A1 (green) was observed in cancer but not in paired normal tissue.

[0072] Figures 5A-5C Anti-Annexin A1 antibody inhibits the growth of human colorectal cancer cells.

[0073] Figure 5AA graph showing the results of cell proliferation assays of human CRC cell line HCT116 untreated (black line with circles) or treated with control rabbit IgG (grey line with squares) or various different anti-annexin A1 antibodies including the anti-annexin A1 antibody (red line with triangles). Data are mean ± SEM. Experiments were performed in triplicate and repeated three times. *p<0.05, **p<0.01 and ***p<0.001 compared to untreated cells (two-way ANOVA).

[0074] Figure 5B is a graph of xenograft tumor growth in nude mice after subcutaneous bilateral inoculation of HCT 116 cells followed by daily (top panel) or every other day (bottom panel) treatment with anti-annexin A1 antibody or control. Tumor volume was measured every day until day 10 and also the percentage change from day 3 to day 4-10 is shown.

[0075] Figure 5C is an image of cells immunostained for annexin A1.

[0076] Figure 6 Various different annexin A1 knockout mice, including ANXA1 - / - APC min / + , ANXA1 + / - APC min / + and ANXA1 + / + APC min / + , are shown crossed with APC + / + APC min / + mice. Survival curves are shown for all three mice; ANXA1 + / - APC min / + vs ANXA1 + / + APC min / + ; ANXA1 - / - APC min / + vs ANXA1 + / - APC min / + ; ANXA1 - / - APC min / + .

[0077] Figures 7A-7C shows that annexin A1 is a new prognostic marker for colon cancer.

[0078] The relationship between ANXA1 mRNA expression levels and DFS was investigated in a database of 466 primary colon cancers assembled by merging four independent gene expression array datasets (GSE14333, GSE17538, GSE31595, GSE37892) from the NCBI-GEO online repository, as previously described (Dalerba et al., 2016). After stratifying patients into groups with high, intermediate and low ANXA1 expression using three different methods, the association between ANXA1 expression levels and DFS was tested using Kaplan-Meier survival curves.

[0079] Figure 7A Results based on the median of ANXA1 mRNA expression levels (high 50% vs. low 50%) are shown.

[0080] Figure 7B Results based on the quartile distribution of ANXA1 mRNA expression levels (high 25% vs. intermediate 50% vs. low 25%) are shown.

[0081] Figure 7C Results based on the ANXA1 mRNA expression threshold calculated using the StepMiner algorithm as previously described (Dalerba et al., 2011; Sahoo et al., 2007) (low vs. high) are shown. Overall, high ANXA1 mRNA expression levels were associated with a statistically significant decrease in DFS (p<0.001, log-rank test), regardless of the method used for stratification. The difference in ANXA1 mRNA expression levels did not appear to be associated with differences in the relative content of epithelial cells (i.e. tumor cell density) in each tumor of the biological samples analyzed, as revealed by the lack of correlation with the epithelial marker desmoplakin (DSP). DETAILED DESCRIPTION

[0083] Definitions

[0084] The terms used in this specification generally have their common ordinary meaning in the field of the application, and in the specific context where each term is used in the specification. Certain terms are discussed below or elsewhere in the specification in order to provide additional guidance to the practitioner in describing the methods of the application and how to use them. It also should be appreciated that the same thing can be said in more than one way. Thus, additional language besides what is below or what is otherwise presented in the specification can be used to express the same or similar concepts. Certain terminology can also be used in the description for the purpose of reference only and, thus, is not intended to limit the application. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the application or of any exemplified term. Likewise, the application is not limited to the preferred embodiments which are described herein.

[0085] The term "subject" as used in this application means an animal, such as a bird and a mammal, having an immune system. Mammals include canids, felines, rodents, bovids, equids, suids, ovines, and primates. Birds include, but are not limited to, poultry, songbirds, and birds of prey. Thus, the application can be used in veterinary medicine, for example, to treat companion animals, farm animals, laboratory animals in zoos, and wild animals. The application is particularly desirable for human medical applications.

[0086] The term "patient" as used in this application means a human subject. In certain embodiments of the application, the "patient" is a patient having cancer, more specifically colorectal cancer or FAP.

[0087] The terms "treat," "treatment," and the like, refer to alleviating, abrogating, ameliorating, or lessening at least one symptom of a disease or reversing the disease after its onset, arresting or slowing the growth of a tumor, arresting or slowing the spread of metastasis, reversing (at least partially) chemotherapy resistance, and any other means of subjective or objective improvement in the patient associated with the patient's cancer.

[0088] The terms "prevent," "prevention," and the like, refer to actions taken in advance of a clear disease or disorder onset in order to stop or minimize the disease or disorder from developing, or to slow the course of progression of the disease or disorder.

[0089] The term "in need thereof is a subject known or suspected to have cancer, in particular colorectal cancer, or at risk of developing cancer, in particular colorectal cancer.

[0090] A subject in need of treatment is one who already has the disease or disorder. A subject in need of prevention is one who has a risk factor for cancer, in particular colorectal cancer, including one who has FAP or a high level of annexin A1 in colorectal tissue indicative of a high risk of occurrence.

[0091] The term "agent" as used herein means a substance that produces or is capable of producing an effect, including but not limited to chemicals, drugs, biologicals, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

[0092] "Antibody," "fragment of an antibody," or "antibody fragment" are used interchangeably and mean one or more fragments or portions of an antibody that retain the ability to specifically bind to a particular antigen (Holliger et al., Nat. Biotech. (2005) 23(9): 1126). Antibodies of the present application can be antibodies and / or fragments thereof. Antibody fragments include Fab, F(ab')2, scFv, disulfide linked Fv, Fc, or variants and / or mixtures thereof. Antibodies can be chimeric, humanized, single chain, or bispecific. All antibody isotypes are encompassed by the present disclosure, including IgA, IgD, IgE, IgG, and IgM. Suitable IgG subtypes include IgG1, IgG2, IgG3, and IgG4. An antibody light or heavy chain variable region is composed of a framework region interrupted by three hypervariable regions known as complementarity determining regions (CDRs). The CDRs of an antibody or antigen binding portion of the present application can be of non-human or human origin. The framework of an antibody or antigen binding portion of the present application can be human, humanized, non-human (e.g., murine framework modified to reduce antigenicity in humans), or synthetic (e.g., consensus sequence).

[0093] The term "therapeutically effective amount" or "effective amount" encompasses an amount sufficient to ameliorate or prevent a symptom or sign of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular subject can vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0094] The terms "cancer," "tumor," "carcinoma," and "malignancy" refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, including adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias. More particular examples of such cancers include melanoma, lung cancer, head and neck cancer, renal cell carcinoma, colon cancer, colorectal cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatocellular carcinoma and hepatoma, bladder cancer, breast cancer, endometrial cancer, myeloma (e.g., multiple myeloma), salivary gland carcinoma, renal cancer such as renal cell carcinoma and Wilms' tumor, basal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, and esophageal cancer.

[0095] "Tumor" refers to a mass of tissue formed when cancer cells grow and reproduce, which can invade and destroy the normal, adjacent tissue. Cancer cells can break away from a malignant tumor and enter the bloodstream or lymphatic system, thereby allowing the cancer cells to spread from the primary tumor to form new tumors in other organs.

[0096] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, limitations of the measurement system, the exact purposes of the measurement, and / or the application for which the measurement is used. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean ranges within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value. Or, particularly for biological systems or processes, the terms can mean within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a value. In the application and in the claims, the

[0097] Abbreviations

[0098] MOI: multiplicity of infection

[0099] CRC: colon or colorectal cancer

[0100] FAP: familial adenomatous polyposis

[0101] Fn: Fusobacterium nucleatum or F. nucleatum

[0102] ANXA1: annexin A1

[0103] CDH1 : E-cadherin

[0104] Annexin Al as a target for the treatment and prevention of cancer and as a biomarker

[0105] The present invention is based in part on the finding that Fn selectively binds to proliferating CRC cells expressing annexin Al and that Fn binding in turn further induces annexin Al expression in an E-cadherin-dependent manner. The induced annexin Al forms a complex with FadA, E-cadherin and β-catenin, which is required for activation of β-catenin and stimulation of CRC growth.

[0106] In mice and humans, a positive correlation between FadA and annexin Al expression was observed in colorectal tumors. Thus, according to the present invention, annexin Al was identified as a key, previously unrecognized component in Fn-induced Wnt / β-catenin signaling. About 75% of CRCs are caused by mutations in the Wnt / β-catenin pathway. However, Wnt / β-catenin signaling is involved in a broad range of cellular functions. Due to this complexity, no Wnt inhibitor has been approved by the FDA for cancer treatment so far. Likewise, E-cadherin stimulation by F. nucleatum to stimulate CRC is ubiquitous, making it also an unsuitable therapeutic target. In contrast, annexin Al is a promising therapeutic target due to its selective expression in proliferating cancerous cells. Inhibition of annexin Al suppresses β-catenin signaling in cancerous cells without affecting non-cancerous cells, thus likely having lower adverse "off-target" effects.

[0107] Fn causes chemoresistance and promotes metastasis in CRC (Bullman et al., 2017; Yu et al., 2017), posing a major challenge for therapy. Antibiotic treatment is not ideal due to perturbing the normal intestinal microbiota. Thus, there is a need for preventing and / or treating Fn and Fn-related side effects in patients suffering from CRC. The present invention addresses these needs.

[0108] According to the present invention, annexin Al is a new therapeutic target for cancer therapy.

[0109] Annexin Al belongs to the annexin family of Ca2+-dependent phospholipid-binding proteins, has a molecular weight of 35-40 KD, and is present in both the cytoplasm and the cell membrane. Annexin Al has been suggested to play a role in resolution of inflammation (Peretti et al., 2009). Annexin Al has been postulated to be a tumor suppressor or promoter depending on the tumor type (Guo et al., 2013; Boudhraa et al., 2016). Although annexin Al has been associated with CRC (Onozawa et al., 2017; Su et al., 2010), its role in CRC is not clear.

[0110] The fundamental mechanistic differences between the interaction of F. nucleatum with cancerous and non-cancerous cells are shown herein. F. nucleatum preferentially binds to cancerous cells, aided by annexin Al, which is specifically expressed in proliferating CRC cells.

[0111] This is in line with previous reports by the inventors that while F. nucleatum can be detected in both colorectal adenoma and adenocarcinoma tissues, the fadA gene level is significantly higher in the latter than in the former (Rubinstein et al. 2013). While F. nucleatum cannot transform pre-cancerous cells into cancer, once benign cells become cancerous, they express high levels of annexin Al, which F. nucleatum activates Wnt / β-catenin signaling and stimulates growth.

[0112] On the basis of these results, a "two-hit" model of colorectal cancer was hypothesized, in which the accumulation of somatic cell mutations, such as increased expression of annexin Al, acts as the first "hit" and a microorganism, such as F. nucleatum, as the second "hit" exacerbates cancer progression. This model evolved from the well-established "adenoma-carcinoma" model (Fearon and Vogelstein 1990) and identifies a microorganism as a promoter of colorectal carcinogenesis. To support the "two-hit" model, ongoing research was conducted, which found that microorganisms promote cancer in susceptible hosts, i.e., after the first hit has occurred.

[0113] In the case of F. nucleatum, the stimulation of CRC is through an E-cadherin-mediated positive feedback loop of FadA and annexin Al, identified in cancerous cells and absent in non-cancerous cells. Increased expression of annexin Al in proliferating cancer cells enhances F. nucleatum binding, which in turn stimulates annexin Al expression and further enhances F. nucleatum binding, thereby activating β-catenin signaling.

[0114] It is also shown herein that annexin Al is a new biomarker for colon cancer recurrence, independent of cancer stage, grade, age, and gender. Annexin Al can be combined with cancer stage for improved prognostic stratification of colon cancer patients.

[0115] In addition, it is also shown herein that inhibition of annexin Al prolongs the life span of mice that have the same mutation as FAP patients (APC gene) in mice.

[0116] In summary, this is the first study to detail the molecular mechanisms of annexin A1 in cancer and elucidate its interaction with cancer-stimulating microbes for the first time. Given the widespread involvement of Wnt / β-catenin in cancer and the increasing reports of *Fusobacterium nucleatum* in various cancer types, annexin A1 represents a novel therapeutic target for different types of cancer associated with *Fusobacterium nucleatum*. Furthermore, although annexin A1 was identified through its interaction with *Fusobacterium nucleatum*, this study demonstrates that it is a CRC growth factor independent of the stated microbe. Its expression has also been detected in other cancer types. Figure 1K ). Methods and compositions of the invention for the treatment and prevention of cancer

[0117] As shown in this article, annexin A1 is a potential target for cancer therapy. This article also demonstrates that the use of annexin A1-specific short interfering RNA or siRNA inhibits the growth of cancer cells, including colorectal cancer cells.

[0118] siRNA is a double-stranded RNA molecule with a length of approximately 20-25 base pairs. It can interfere with the expression of complementary nucleotide sequences by degrading transcribed mRNA.

[0119] Furthermore, microRNAs or miRNAs are small non-coding RNAs, averaging 22 nucleotides, that regulate the expression of their target mRNA transcripts and can also be used in the methods of this invention. miRNAs function by binding to the 3'UTR of a gene, in this case, ANXA1.

[0120] Therefore, one embodiment of the present invention is a method for treating and / or preventing cancer, particularly colorectal cancer, in a desired subject, the method comprising administering annexin A1-specific short interfering RNA.

[0121] Another embodiment of the present invention is a method for treating and / or preventing cancer, particularly colorectal cancer, in a desired subject, the method comprising administering annexin A1-specific microRNA.

[0122] Another embodiment of the present invention is a method for treating and / or preventing cancer, particularly colorectal cancer, in a desired subject, the method comprising administering DNA encoding annexin A1-specific short interfering RNA.

[0123] Another embodiment of the present invention is a method for treating and / or preventing cancer, particularly colorectal cancer, in a desired subject, the method comprising administering DNA encoding annexin A1-specific microRNA.

[0124] Another embodiment of the present application is a method of preventing colorectal cancer in a subject having familial adenomatous polyposis (FAP), the method comprising administering a short interfering RNA specific for annexin A1.

[0125] Another embodiment of the present application is a method of preventing colorectal cancer in a subject having familial adenomatous polyposis (FAP), the method comprising administering a microRNA specific for annexin A1.

[0126] Another embodiment of the present application is a method of preventing colorectal cancer in a subject having familial adenomatous polyposis (FAP), the method comprising administering a DNA encoding a short interfering RNA specific for annexin A1.

[0127] Another embodiment of the present application is a method of preventing colorectal cancer in a subject having familial adenomatous polyposis (FAP), the method comprising administering a DNA encoding a microRNA specific for annexin A1.

[0128] Another embodiment of the present application is a method of reducing chemotherapeutic resistance of a cancer in a subject in need thereof, the method comprising administering a short interfering RNA specific for annexin A1.

[0129] Another embodiment of the present application is a method of reducing chemotherapeutic resistance of a cancer in a subject in need thereof, the method comprising administering a microRNA specific for annexin A1.

[0130] Another embodiment of the present application is a method of reducing chemotherapeutic resistance of a cancer in a subject in need thereof, the method comprising administering a DNA encoding a short interfering RNA specific for annexin A1.

[0131] Another embodiment of the present application is a method of reducing chemotherapeutic resistance of a cancer in a subject in need thereof, the method comprising administering a DNA encoding a short interfering RNA specific for annexin A1.

[0132] Another embodiment of the present application is a method of inhibiting proliferation of a cancer cell, the method comprising reducing expression of annexin A1 on the cancer cell by contacting the cell with a short interfering RNA specific for annexin A1.

[0133] Another embodiment of the present application is a method of inhibiting proliferation of a cancer cell, the method comprising reducing expression of annexin A1 on the cancer cell by contacting the cell with a microRNA specific for annexin A1.

[0134] Another embodiment of the application is a method of inhibiting proliferation of a cancer cell, comprising reducing expression of annexin Al on the cancer cell by contacting the cell with DNA encoding an annexin Al -specific short interfering RNA.

[0135] Another embodiment of the application is a method of inhibiting proliferation of a cancer cell, comprising reducing expression of annexin Al on the cancer cell by contacting the cell with DNA encoding an annexin Al -specific short interfering RNA.

[0136] An annexin Al -specific siRNA or miRNA or DNA encoding these RNAs that binds to annexin Al mRNA or the mRNA 3'UTR can be designed using sequence information.

[0137] The sequence of the ANXA1 gene can be found at the National Center for Biotechnology Database and can be used to make the interfering RNA molecules by methods known in the art. Annexin Al is encoded by the ANXA1 gene (Gene ID: 301) on chromosome 9.

[0138] The siRNA or microRNA or DNA can be made by recombinant methods known in the art. The siRNA or microRNA or DNA can also be modified to improve other desired properties, such as increased stability, decreased degradation in vivo, and increased cellular uptake. In addition, mi196a has been shown to down-regulate annexin Al (see Luthra et al., 2008).

[0139] For certain embodiments, the RNA or DNA will be targeted to a particular tissue or cell. In preferred embodiments, the tissue is a cancer and the cell is a cancer cell.

[0140] One method for delivering the nucleic acid is receptor-mediated endocytosis, in which the nucleic acid is coupled to a targeting molecule that can bind to a specific cell surface receptor, inducing endocytosis and transfer of the nucleic acid into the cell. Coupling is typically achieved by covalently linking polylysine to the receptor molecule, and then arranging for (reversible) binding of the negatively charged nucleic acid to the positively charged polylysine component.

[0141] Another method utilizes the transferrin receptor or folate receptor, which is expressed in many cell types. For example, in the production of microRNAs for use in the present administration method, the microRNAs can be manufactured to have a guide strand identical to the microRNA of interest and a passenger strand that is modified and linked to a molecule for improved cellular uptake.

[0142] Another method of administering the nucleic acid to the correct tissue is direct injection / particle bombardment, in which a syringe and needle are used to directly inject the nucleic acid into a specific tissue, such as cancerous tissue.

[0143] An alternative direct injection method utilizes particle bombardment ("gene gun") technology: nucleic acids are coated onto metal pellets and fired from a specially designed gun into cells. Successful gene transfer has been achieved in a number of different tissues using this method. These direct injection techniques are simple and relatively safe.

[0144] Another method of delivering microRNAs and siRNAs and DNA to the correct tissue or cell is the use of adeno-associated virus (AAV). Nucleic acids delivered in these viral vectors are expressed persistently, replacing the expression of microRNAs or siRNAs or DNA that are not expressed in the subject. AAV also has different serotypes, allowing tissue-specific delivery due to the natural tropism of each individual AAV serotype for different organs and the different cellular receptors that interact with each AAV serotype. In addition to the AAV serotype, the use of tissue-specific promoters for expression allows for even greater specificity.

[0145] Other mammalian viral vectors that can be used to deliver the RNA or DNA include onco-retroviral vectors, adenoviral vectors, herpes simplex viral vectors, and lentivirus.

[0146] Delivery vehicles such as liposomes, nanocapsules, nanoparticles, microparticles, microspheres, lipid particles, vesicles, and the like, can be used to introduce the compositions of the present application into suitable host cells. In particular, the transgene or protein delivered by the vector can be formulated for encapsulation in a lipid particle, liposome, vesicle, nanosphere or nanoparticle, and the like, for delivery.

[0147] The formation and use of liposomes is well known to those skilled in the art. Recently, liposomes with improved serum stability and circulating half-life have been developed (U.S. Patent No. 5,741,516). In addition, various methods of liposomes and liposome-like preparations have been described as potential drug carriers (U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0148] Liposomes have been successfully used for a large number of cell types that are normally resistant to transfection using other procedures. In addition, liposomes do not have the DNA size limitations typical of virus-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed examining the effectiveness of liposome-mediated drug delivery.

[0149] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also known as multilamellar vesicles (MLVs)). MLVs typically have diameters ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) containing an aqueous solution in the core with diameters ranging from 200 to 400 A.

[0150] Alternatively, nanocapsule formulations can be used. Nanocapsules can generally entrap substances in a stable and reproducible manner.

[0151] ​Nanoparticles are a colloidal carrier system that has been shown to improve the efficacy of encapsulated drugs by prolonging serum half-life. Polyalkylcyanoacrylate (PACA) nanoparticles are a polymeric colloidal drug delivery system that is being developed clinically (described in, e.g., Stella et al. (2000) J. Pharm. Sci., 89: 1452-1464; Brigger et al. (2001) Int. J. Pharm 214: 37-42; Calvo et al. (2001) Pharm. Res. 18: 1157-1166; and Li et al. (2001) Biol. Pharm. Bull. 24: 662-665). Biodegradable polyhydroxy acids such as copolymers of polylactic acid (PLA) and poly(lactide-co-glycolide) (PLGA) are being widely used in biomedical applications and have been approved by the FDA for certain clinical applications. In addition, nanoparticles have many desirable carrier characteristics, including: (i) a relatively high weight fraction of the drug to be encapsulated (loading) of the overall carrier system; (ii) a relatively high level of the drug used in the first step of the encapsulation process is incorporated into the final carrier (entrapment efficiency); (iii) the carrier has the ability to be lyophilized and reconstituted in solution without aggregation; (iv) the carrier is biodegradable; (v) the size of the carrier system is small; (vi) the carrier enhances the persistence of the particle. Nanoparticles can be synthesized using virtually any biodegradable shell known in the art. Such polymers are biocompatible and biodegradable and are modified desirably to increase the photochemical efficacy and circulation lifetime of the nanoparticle. In one embodiment, the polymer is modified with terminal carboxylic acid groups (COOH) that increase the negative charge of the particle and thus limit the interaction with negatively charged nucleic acids. The nanoparticles can also be modified with polyethylene glycol (PEG), which also increases the half-life and stability of the particle in circulation. Alternatively, the COOH groups can be converted to N-hydroxysuccinimide (NHS) esters for covalent coupling to amine-modified compounds.

[0152] Furthermore, it is shown herein that anti-annexin A1 antibodies inhibit the growth of colorectal cancer cells.

[0153] Accordingly, one embodiment of the present application is a method of treating and / or preventing cancer, in particular colorectal cancer, in a subject in need thereof, comprising administering an anti-annexin A1 antibody, minibody, Fab or fragment, camelid antibody or nanobody.

[0154] Another embodiment of the present application is a method of preventing colorectal cancer in a subject suffering from familial adenomatous polyposis (FAP), comprising administering an anti-annexin A1 antibody, minibody, Fab or fragment, camelid antibody or nanobody.

[0155] Another embodiment of the present application is a method of reducing chemotherapy resistance of a cancer in a subject in need thereof, the method comprising administering an anti- annexin A1 antibody, minibody, Fab or fragment, a camelid antibody or nanobody.

[0156] Another embodiment of the present application is a method of inhibiting proliferation of a cancer cell, the method comprising reducing expression of annexin A1 on the cancer cell by contacting the cell with an anti-annexin A1 antibody, minibody, Fab or fragment, a camelid antibody or nanobody.

[0157] Anti-annexin A1 antibodies that bind to annexin A1 can be designed using sequence information and conventional methods, such as hybridoma techniques or recombinant techniques. Antigen-binding fragments of whole antibodies (full-length antibodies) can be prepared by conventional methods. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of F(ab')2 fragments.

[0158] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced by, for example, conventional recombinant techniques.

[0159] The sequences of the ANXA1 gene and protein can be found at the National Center for Biotechnology Database, and can be used to make interfering RNA molecules and antibodies by methods known in the art. Annexin A1 is encoded by the ANXA1 gene on chromosome 9 (Gene ID: 301).

[0160] Other agents, including small molecules, can be used to block or inhibit annexin A1.

[0161] Accordingly, one embodiment of the present application is a method of treating and / or preventing cancer, particularly colorectal cancer, in a subject in need thereof, the method comprising administering a small molecule that blocks or inhibits annexin A1.

[0162] Another embodiment of the present application is a method of preventing colorectal cancer in a subject having familial adenomatous polyposis (FAP), the method comprising administering a small molecule that blocks or inhibits annexin A1.

[0163] Another embodiment of the present application is a method of reducing chemotherapy resistance of a cancer in a subject in need thereof, the method comprising administering a small molecule that blocks or inhibits annexin A1.

[0164] Another embodiment of the application is a method of inhibiting proliferation of a cancer cell, the method comprising reducing expression of annexin A1 on the cancer cell by contacting the cell with a small molecule that blocks or inhibits annexin A1.

[0165] In certain embodiments, the small molecule includes, but is not limited to, indomethacin, celecoxib, and sorafenib, and related structural analogs and derivatives.

[0166] All of these agents can be used alone, in combination with each other, and / or in combination with other therapeutic agents.

[0167] All of the agents discussed herein can take the form of a pharmaceutical composition.

[0168] Another embodiment of the application is a pharmaceutical composition comprising an annexin A1 -specific siRNA and a pharmaceutically acceptable diluent, carrier, or adjuvant. Another embodiment of the application is a pharmaceutical composition comprising: an annexin A1 -specific siRNA; a vector, liposome, nanocapsule, nanoparticle, microparticle, microsphere, lipoplex, or vesicle; and a pharmaceutically acceptable diluent, carrier, or adjuvant.

[0169] Another embodiment of the application is a pharmaceutical composition comprising an annexin A1 -specific miRNA and a pharmaceutically acceptable diluent, carrier, or adjuvant. Another embodiment of the application is a pharmaceutical composition comprising: an annexin A1 -specific miRNA; a vector, liposome, nanocapsule, nanoparticle, microparticle, microsphere, lipoplex, or vesicle; and a pharmaceutically acceptable diluent, carrier, or adjuvant.

[0170] Another embodiment of the application is a pharmaceutical composition comprising DNA encoding an annexin A1 -specific siRNA and a pharmaceutically acceptable diluent, carrier, or adjuvant. Another embodiment of the application is a pharmaceutical composition comprising: DNA encoding an annexin A1 -specific siRNA; a vector, liposome, nanocapsule, nanoparticle, microparticle, microsphere, lipoplex, or vesicle; and a pharmaceutically acceptable diluent, carrier, or adjuvant.

[0171] Another embodiment of the application is a pharmaceutical composition comprising DNA encoding an annexin A1 -specific miRNA and a pharmaceutically acceptable diluent, carrier, or adjuvant. Another embodiment of the application is a pharmaceutical composition comprising: an annexin A1 -specific miRNA; a vector, liposome, nanocapsule, nanoparticle, microparticle, microsphere, lipoplex, or vesicle; and a pharmaceutically acceptable diluent, carrier, or adjuvant.

[0172] Another embodiment of the present application is a pharmaceutical composition comprising an anti-Ankyrin A1 antibody, minibody, Fab or fragment, a camelid antibody or nanobody and a pharmaceutically acceptable diluent, carrier or adjuvant.

[0173] Another embodiment of the present application is a pharmaceutical composition comprising a small molecule that blocks or inhibits Ankyrin A1 selected from the group consisting of indomethacin, lonafarnib and sorafenib and related structural analogs and derivatives and a pharmaceutically acceptable diluent, carrier or adjuvant.

[0174] The most preferred method of administration of the agents and compositions used in the methods of the present application is oral, intrathecal, nasal and parenteral, including intravenous. The agents must take a form appropriate for the chosen route of administration.

[0175] These pharmaceutical compositions for administration comprising one or more agents can comprise a therapeutically effective amount of the agent(s) and a pharmaceutically acceptable carrier.

[0176] The phrase "pharmaceutically acceptable" as used herein refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness, etc., when administered to a human, and are approved or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans, by a regulatory agency of the Federal or a state government.

[0177] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. These pharmaceutical carriers can be sterile liquids, such as saline solutions and oils, including those of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Adjuvants can be added to the RNA to protect it from degradation.

[0178] Pharmaceutical compositions suitable for oral administration can be in the form of capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquid) or emulsions. Tablets or hard gelatin capsules can contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules can contain vegetable oils, waxes, fats, semi-solid or liquid polyols. Solutions and syrups can contain water, polyols and sugars. Active agents intended for oral administration can be coated with or admixed with a material that delays the disintegration and / or absorption of the active agent in the gastrointestinal tract. Thus, sustained release over a period of several hours can be achieved and, if desired, the active agent can be protected from degradation in the stomach. Pharmaceutical compositions for oral administration can be formulated to release the active agent at a particular location in the gastrointestinal tract due to particular pH or enzymatic conditions.

[0179] Pharmaceutical compositions suitable for nasal and pulmonary administration can include solid carriers such as powders, which can be administered via rapid inhalation through the nose. Compositions for nasal administration can include liquid carriers such as sprays or drops. Alternatively, inhalation through the mouth can be achieved by a deep breath or a device through a mouthpiece. These compositions can include aqueous or oil solutions of the active ingredient. Compositions for inhalation can be supplied in specially adapted devices, including but not limited to pressurized aerosols, nebulizers or insufflators, which can be configured to provide a predetermined dose of active ingredient.

[0180] Another preferred form of administration is parenteral, including intravenous administration. Pharmaceutical compositions suitable for parenteral, including intravenous, administration include aqueous and non-aqueous sterile injection solutions or suspensions, which can contain anti-oxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of the subject. Other components that can be present in such compositions include water, alcohols, polyols, glycerin and vegetable oils. Compositions suitable for parenteral administration can be presented in unit-dose or multi-dose containers, such as sealed ampules and vials, and can be stored in freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. Suitable media for providing parenteral dosage forms of the present application are well known to those skilled in the art. Examples include: USP water for injection; aqueous media such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection and lactated Ringer's injection; water-miscible media such as ethanol, polyethylene glycol and polypropylene glycol; and non-aqueous media such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzocaine.

[0181] Other methods of administration include sublingual, vaginal, buccal or rectal administration, or transdermal administration to the subject.

[0182] The selection of a therapeutically effective dose is determined by a person of ordinary skill in the art taking into account several factors known to those of ordinary skill in the art. These factors include the specific form of the agent and its pharmacokinetic parameters such as bioavailability, metabolism, and half-life that have been established during the common development process that is used in obtaining regulatory approval of a pharmaceutical compound. Other factors to be considered in the dosage include the condition or disease to be treated or the benefit to be achieved in a normal individual, the body weight of the patient, the route of administration, whether the administration is short term or long term, drugs used concurrently, and other well- known factors that affect the efficacy of the agent being administered. Thus, the exact dose should be decided according to the judgment of the skilled artisan, the condition of each patient, and according to standard clinical techniques.

[0183] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" mean an amount of an RNA, antibody or small molecule or other agent and composition that, when administered to a cell, tissue, or subject, either alone or in combination with another therapeutic agent, is effective to elicit a measurable improvement in one or more symptoms of a disease or condition, such as a solid tumor cancer, or the progression of these diseases or conditions. A therapeutically effective dose also means an amount of the compound(s) adequate to achieve at least partial amelioration of symptoms, for example, an increase in the rate of treatment, cure, prevention, or amelioration of a relevant medical condition, or treatment, cure, prevention, or amelioration of these conditions. When applied to individual active ingredients, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, therapeutically effective dose refers to combined amounts of the active ingredients that produce the therapeutic effect. In situations where more than one active ingredient is administered concurrently, the dosage of each compound can be either the same or vary.

[0184] Combination methods and compositions of the invention for the treatment and prevention of cancer

[0185] Other embodiments of the present application include methods and compositions for treating and preventing cancer, wherein an agent that inhibits or blocks annexin A1 is used in combination with a therapeutically effective amount of other therapeutic agents including but not limited to chemotherapeutic agents, targeted chemotherapeutic agents, and immunotherapies.

[0186] Other embodiments of the present application include methods and compositions for reducing the chemoresistance of a cancer, wherein an agent that inhibits or blocks annexin A1 is used in combination with a therapeutically effective amount of other therapeutic agents including but not limited to chemotherapeutic agents.

[0187] In these embodiments, the agent that inhibits or blocks annexin A1 improves the efficacy of other therapies including but not limited to chemotherapeutic agents, targeted therapies, and immunotherapies and renders the cancer more susceptible to the other therapies.

[0188] As discussed herein, Fn contributes to chemotherapy resistance and promotes metastasis in CRC (Bullman et al., 2017; Yu et al., 2017), which poses a significant challenge to treatment. Antibiotic therapy is not ideal due to perturbation of normal intestinal flora. It is also shown herein that annexin A1 and Fn play a role together in the pathogenesis of cancer. Thus, targeting annexin A1 will improve the effectiveness of other cancer therapeutics.

[0189] Thus, another embodiment of the application is a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of an agent that inhibits or blocks annexin A1 and a therapeutically effective amount of an additional therapeutic agent, including but not limited to a chemotherapeutic agent, a targeted therapeutic agent, and an immunotherapy, and combinations thereof.

[0190] A "chemotherapeutic agent" or "chemotherapeutic drug" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include but are not limited to microtubule- targeting agents (MTAs), DNA-damaging agents, alkylating agents, antimetabolites, spindle poisons plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors.

[0191] The chemotherapeutic agent can be natural or synthetic.

[0192] In certain embodiments, the chemotherapeutic agent is a small molecule.

[0193] Chemotherapeutic agents for treating colorectal cancer can include but are not limited to 5-fluorouracil, capecitabine, irinotecan, oxaliplatin, and the combination of trifluridine and tipiracil.

[0194] Targeted chemotherapeutic agents block specific proteins or genes. Targeted therapeutic agents include but are not limited to denosumab, romidepsin, ofatumumab, pazopanib, everolimus, nilotinib, temisirolimus, lapatinib, sunitinib, dasatinib, vorinostat, erlotinib, bevacizumab, cetuximab, bortezomib, gefitinib, ibritumomab, alemtuzumab, imatinib, gemtuzumab, denileukin difitox, trastuzumab, rituximab, ramucirumab, ziv-aflibercept, panitumumab, and regorafenib.

[0195] Immunotherapies include but are not limited to PD-1 inhibitors including pembrolizumab and nivolumab and CTLA-4 inhibitors including ipilimumab.

[0196] The methods and compositions of the application can be used in combination with other anti-neoplastic agents or immunogenic agents in order to improve their efficacy, such as attenuated cancer cells, tumor antigens, antigen presenting cells pulsed with tumor-derived antigens or nucleic acids, such as dendritic cells, immunostimulatory cytokines (such as IL-2, IFNa2, GM-CSF) and cells transfected with genes encoding immunostimulatory cytokines such as, but not limited to, GM-CSF.

[0197] Cancers to be treated and / or prevented

[0198] The methods and compositions described herein can be used to treat cancer (i.e., to inhibit the growth or survival of tumor cells). As described herein, annexin Al has been identified as a Fn-independent tumor promoter. The promoting effect of Fn in CRC is due to its stimulation of annexin Al. Thus, the methods and compositions of the application can be used to treat cancers whose growth is enhanced by annexin Al, regardless of the presence of Fn.

[0199] Non-limiting examples of preferred cancers for treatment and / or prevention include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate cancer), pancreatic cancer, colon cancer or colorectal cancer (CRC), lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, and leukemia. In addition, the application includes refractory or recurrent malignancies whose growth can be inhibited using the compositions described herein.

[0200] The methods and compositions described herein can also be used to prevent cancer, in particular colorectal cancer, in patients with FAP, as well as in patients at risk of recurrence, such as subjects having high levels of annexin Al in colorectal cancer tissue.

[0201] Expression of annexin Al as an independent prognostic biomarker for CRC and methods for its detection

[0202] It is also described herein that an increased expression of ANXA-A1 mRNA in CRC tissue is associated with recurrence of colon cancer, independently of cancer stage, grade, age and gender (Example 8).

[0203] By using the differential expression of ANXA1, important predictions and determinations can be made regarding the severity of the disease and the treatment of the patient. Although the testing for this biomarker can be performed at any time after the diagnosis of CRC, preferably the testing will be performed as soon as possible after the clinician makes a positive diagnosis of CRC. In this way, the valuable insight into the disease can be used in the selection of the therapy.

[0204] Accordingly, in one embodiment of the application, the expression of ANXA1 can be tested. If the expression is increased compared to a reference value, the patient is identified as having a poor prognosis and should be considered for more aggressive treatment.

[0205] The aggressive treatment for colorectal cancer can include administration of an agent that inhibits or blocks annexin A1 alone or in combination with a chemotherapeutic agent, a targeted therapy agent, and / or an immunotherapy. Chemotherapeutic agents can include, but are not limited to, 5-fluorouracil, capecitabine, irinotecan, oxaliplatin, and the combination of trifluridine and tipiracil. Targeted therapy agents include, but are not limited to, drugs targeting VEGF including bevacizumab, ramucirumab, and aflibercept; drugs targeting EGFR including cetuximab and panitumumab; and regorafenib. Immunotherapies include, but are not limited to, PD-1 inhibitors including pembrolizumab and nivolumab and CTLA-4 inhibitors including ipilimumab. The addition of the agent that inhibits or blocks annexin A1 can reduce resistance to and / or increase the efficacy of these other therapeutic agents.

[0206] The presence or amount of gene expression can be compared to a reference value. In certain embodiments, the reference value is the level of gene expression of annexin A1 from a healthy control tissue. In certain embodiments, the healthy control tissue is a healthy colorectal tissue.

[0207] In certain embodiments, a tissue sample of colorectal cancer tissue from a subject having CRC is obtained.

[0208] Nucleic acids are extracted, isolated, and purified from cells of the tissue or body fluids by methods known in the art.

[0209] If desired, nucleic acid samples are prepared using known techniques. For example, samples can be treated to lyse cells using known lysis buffers, sonication, electroporation, and purified and amplified as desired, as will be appreciated by those skilled in the art. In addition, the reactions can be accomplished in a variety of different ways. The components of the reactions can be added simultaneously or sequentially in any order. In addition, the reactions can include a variety of different other reagents that can be useful in the methods and assays, including but not limited to salts, buffers, neutral proteins such as albumin, and detergents, which can be used to facilitate optimal hybridization and detection and / or reduce non-specific or background interactions. Depending on the sample preparation methods and purity, reagents to otherwise increase the efficiency of the assays such as protease inhibitors, nuclease inhibitors, and antimicrobials can also be used.

[0210] Following preparation, the mRNA or other nucleic acid is analyzed by methods known to those skilled in the art. In addition, where the nucleic acid is to be detected, the preferred method utilizes a cleavage or nicking technique to cleave the nucleic acid sample containing the target sequence to a size that is convenient to manipulate and hybridize to the target. This can be accomplished by shearing the nucleic acid with mechanical force, such as sonication, or by cleaving the nucleic acid with restriction endonucleases, or by any other method known in the art. However, in most cases, natural degradation that occurs during storage produces "short" oligonucleotides. Generally, the methods and assays of the present application can be performed on oligonucleotides as short as 20-100 base pairs, with 20 to 50 base pairs being preferred, and between 40 and 50, including 44, 45, 46, 47, 48, and 49 base pairs being most preferred.

[0211] Methods for examining gene expression are generally based on hybridization and include Southern blotting, Northern blotting, dot blotting, primer extension, nuclease protection, subtractive hybridization, and separation using non-double stranded molecules such as hydroxyapatite, solution hybridization, filter hybridization, amplification techniques such as RT-PCR and other PCR related techniques such as PCR with melting curve analysis and PCR with mass spectrometry, fingerprinting such as using restriction endonucleases, and using structure specific endonucleases. mRNA expression can also be analyzed using mass spectrometry techniques (e.g., MALDI or SELDI), liquid chromatography, and capillary gel electrophoresis. The presence or absence of transcripts can be detected using any other method known in the art.

[0212] For a general description of these techniques, see also Sambrook et al., 1989; Kriegler 1990; and Ausubel et al., 1990.

[0213] The screening and diagnostic methods of the present application can involve amplification of the target locus. The preferred method for target amplification of nucleic acid sequences is the use of a polymerase, particularly the polymerase chain reaction (PCR). PCR or other polymerase driven amplification methods obtain millions of copies of the relevant nucleic acid sequence, which can then be used as a substrate for probes or sequencing or in other assays.

[0214] Amplification using the polymerase chain reaction is particularly useful in embodiments of the present application. PCR is a rapid, general in vitro method for amplifying defined target DNA sequences present in a DNA source. Typically, the method is designed to allow selective amplification of a specific target DNA sequence from a heterogeneous collection of DNA sequences (e.g., total genomic DNA or a complex cDNA population). To allow this selective amplification, some prior DNA sequence information from the target sequence is required. This information is used to design two oligonucleotide primers (amplification primers) that are specific for the target sequence and are typically about 15-25 nucleotides long.

[0215] Alternatively, the Annexin A1 protein can be isolated and / or purified from a colorectal cancer tissue sample using any method known in the art, including but not limited to immunoaffinity chromatography.

[0216] Although any method known in the art can be used, preferred methods for detecting and measuring high levels of protein in a protein sample include flow cytometry, quantitative Western blotting, immunoblotting, quantitative mass spectrometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), and immunoenzymatic assay (IEMA), and sandwich assays using monoclonal and polyclonal antibodies.

[0217] Antibodies are the preferred method for detecting and measuring target or objective proteins in a sample. These antibodies can be commercially available or can be manufactured by routine methods known in the art. These antibodies can be monoclonal or polyclonal antibodies and fragments and immunologically equivalent equivalents thereof. The term "antibody" means both homomolecular entities as well as mixtures such as serum preparations composed of several homomolecular entities.

[0218] In preferred embodiments, these antibodies immunoprecipitate the objective protein from solution and react with the objective / target protein in Western blots, immunoblots, ELISAs, and other assays listed above.

[0219] Antibodies used in these assays can be labeled covalently or noncovalently with a reagent that provides a detectable signal. Any label and coupling procedure known in the art can be used. Labels include, but are not limited to, enzymes, fluorescent reagents, radiological labels, substrates, inhibitors, cofactors, magnetic particles, and chemiluminescent reagents. A large number of fluorescent materials are known and can be used as detectable labels. These include, for example, fluorescein, rhodamine, eosin, Texas red, AMCA blue, and Lucifer yellow. One particular detection material is an anti-rabbit antibody prepared in goats and coupled to fluorescein via isothiocyanate. Any desired target or binding partner can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any counting procedure currently available. Preferred isotopes can be selected from 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I and 186 Re. Enzymatic labels are also useful and can be detected by any of the colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gaseous measurement techniques currently in use. The enzyme is coupled to the selected particle by reaction with a bridging molecule such as carbodiimide, diisocyanate, glutaraldehyde, and the like. A number of enzymes useful in these procedures are known and can be utilized. In embodiments, the enzyme can be peroxidase, beta-glucuronidase, beta-D-glucosidase, beta-D-galactosidase, urease, glucose oxidase plus peroxidase, and alkaline phosphatase. Reference is made to U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 for their disclosure relating to alternative labeling materials and methods.

[0220] An alternative method for detecting protein markers is flow cytometric analysis of cells obtained from colorectal cancer tissue from the subject.

[0221] Kits

[0222] It is contemplated that all of the methods disclosed herein can take the form of a kit for use by a health care provider and / or diagnostic laboratory.

[0223] In certain embodiments, the present disclosure provides a kit comprising one or more probes and / or one or more antibodies described herein for detecting the expression level of ANXA1.

[0224] Assays for detecting and quantifying ANXA1 gene expression can be incorporated in kits. These kits can include probes for ANXA1, reagents for isolating and purifying nucleic acids from biological tissues or fluids, reagents for performing assays on the isolated and purified nucleic acids, instructions for use, and reference values for ANXA1 or means for obtaining reference values for ANXA1 in control samples.

[0225] Preferred embodiments of these kits have probes attached to a solid phase.

[0226] Assays for detecting and quantifying Annexin A1 protein can be incorporated in kits. These kits can include antibodies that recognize the peptide of interest, reagents for isolating and / or purifying proteins from biological tissues or fluids, reagents for performing assays on the isolated and purified proteins, instructions for use, and reference values for the amount or level of Annexin A1 or means for obtaining said reference values in control samples.

[0227] In another embodiment of the application, commercial test kits suitable for use by medical professionals can be prepared to determine the presence or amount of desired gene or protein activity, expression, or gene amplification in samples from patients with colorectal cancer.

[0228] In light of the above, an assay system for screening potential drugs that effectively modulate the activity or expression of ANXA1 is provided. The target can be introduced into a test system and promising drugs can also be introduced into the resulting cell culture, which is then examined for any changes in the target activity of the cells or proliferation or division of the cells resulting from the individual addition of the promising drug or from the increased effect of the amount of the known target. Examples

[0229] The application will be better understood from the Experimental Details which follow. However, one skilled in the art will readily recognize that the specific methods and results discussed are merely illustrative of the application and are not intended to limit the scope of the application as described in the following claims.

[0230] Example 1 - Materials and Methods for Examples 2-7

[0231] Bacterial strains and cell culture. E. coli DH5α was grown in LB medium at 37°C in air. Wild-type Fn 12230 and its fadA deletion mutant US1 were grown in Columbia medium supplemented with 5 μg / ml hemin, 1 μg / ml menadione at 37°C under anaerobic conditions (90% N2, 5% CO2 and 5% H2). To prepare CFSE-labeled Fn, the bacteria were grown to mid-log phase (A 600= 0.3) and washed twice with PBS, then incubated in PBS containing 50 μΜ 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Grand Island, NY) for 30 min at room temperature with gentle agitation. The labeled bacteria were washed 10 times with PBS and resuspended in PBS. The labeled bacteria were plated on trypticase soy agar supplemented with 5 μg / ml hematin, 1 μg / ml menadione, and 5% defibrinated sheep blood to enumerate viable bacterial cells. Cell cultures AA / C1, AA / C1 / SB (also referred to as SB), AA / C1 / SB / 10C (also referred to as 10C), HCT116, DLD1, SW480, HT29, and MCF-7 were maintained as previously described (Rubinstein et al., 2013; Williams 1990).

[0232] Plasmid construction and DNA / RNA transfection. Full-length ANXA1 was amplified by PCR using the primers listed in Table 1 and cloned into pcDNA TM 3.1(+) mammalian expression vector. Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer's instructions. Control siRNA, ANXA1 -specific siRNA, and CDH1 -specific siRNA were purchased from Invitrogen (CA). The siRNAs used in these experiments included: negative control: Invitrogen Stealth RNAi siRNA Negative Control, catalog number 12935300; Annexin siRNA: Invitrogen stealth siRNA (catalog number ANXA1 HSS100502) sequence UAA CCA UUA UGG CCU UAU GCA AGG C (SEQ ID NO: 1); and E-cadherin siRNA: Invitrogen stealth siRNA (catalog number CDH1 HSS101669) sequence UUA AUC UCC AGC CAGUUG GCA GUG U (SEQ ID NO: 2). Lipofectamine RNAi MAX (Invitrogen, CA) was used for siRNA transfection according to the manufacturer's instructions.

[0233] Antibodies. Annexin antibodies used in these experiments include: Annexin Al (Thermo Fisher Scientific, Cat# 71-3400); Annexin A2 (Thermo Fisher Scientific, Cat# PA5-27566); Annexin V (Thermo Fisher Scientific, Cat# PA5-27872); Annexin A6 (Thermo Fisher Scientific, Cat# 720161); and Annexin All (Thermo Fisher Scientific, Cat# PA5-68093).

[0234] Protein purification and conjugation. FadAc and mFadA were purified as previously described (Xu et al., 2007). To conjugate the proteins, 3 mg of FadAc, mFadA, and BSA (MP Biomedicals, Santa Ana, CA) were mixed with 10 mg / ml of Alexa Fluor 488 tetrafluorophenyl (TFP) ester (Invitrogen, CA) and vortexed at room temperature for 1 hour. After the reaction, unconjugated Alexa Fluor 488 TFP was removed using a PD-10 desalting column (GE Healthcare Life Sciences, Buckinghamshire, UK). TM 488 TFP. The amount of labeled protein was quantified using a spectrophotometer (NanoDrop Technologies, Wilmington, DE). TM

[0235] Cell proliferation assay. Cells were seeded in 24-well plates at a density of 5 x 10 4 cells per well. Cells were either left untreated or incubated with bacteria at a MOI of 1000: 1. Cell numbers were counted at the indicated time points using a hemocytometer as previously described (Rubinstein et al., 2013). Each experiment was performed in triplicate and repeated at least three times.

[0236] ​Cell culture attachment and invasion assays. The assays were performed as previously described (Han et al., 2000). Briefly, host cells were seeded in 24-well plates and grown to 80% confluency. Bacteria were added at a MOI of 50:1 and incubated for 1 h at 37°C and 5% CO2. After washing with PBS and lysis with water for 20 min, serial dilutions of the lysate were plated on blood agar plates to enumerate the number of viable bacteria. For invasion assays, bacteria were incubated with host cells for 3 h, followed by treatment with 300 pg / ml gentamicin and 200 pg / ml metronidazole for 1 h at 37°C. After washing with PBS, cells were lysed with water and intracellular bacterial counts were determined as described above. Levels of attachment and invasion were expressed as the percentage of bacteria recovered after cell lysis relative to the total number of bacteria initially added. Each experiment was performed in triplicate and repeated at least three times.

[0237] Flow cytometry. Approximately 2 x 10 5 cells were incubated with Fn or purified FadA in 35 mm dishes for the indicated length of time at 37°C. After washing with PBS, the cells were incubated with enzyme-free cell dissociation buffer (ThermoFisher Scientific) for 5 min at 37°C, followed by the addition of 1 ml DMEM. Cells were collected and centrifuged at 500 g for 3 min. The cell pellet was fixed with 75% cold ethanol and kept at -20°C. The pellet was again centrifuged at 2500 g for 10 min and washed with phosphate citrate buffer (200 mM Na2HP04, 100 mM citric acid, pH 7.4) and then blocked with PBS containing 2% skim milk for 1 h at room temperature. After washing with PBS, the cells were incubated with rabbit anti-annexin Al polyclonal IgG (1 :400 dilution, Invitrogen) or rabbit anti-β-catenin polyclonal IgG (1 :200 dilution, Invitrogen) or rabbit IgG isotype control (Invitrogen) for 1 h at room temperature. The cells were then washed with PBS and incubated with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1 : 1000 dilution, Invitrogen) for 1 h. After washing the cells with PBS, flow cytometry data were acquired by a BD LSR II flow cytometer and analyzed using Flow Jo software (Tree Star, San Carlos, CA).

[0238] Immunofluorescence staining. 1 x 10 3 - 1 x 10 5Aliquots of cells were seeded at 400 μl in a Nunc Lab-Tek II compartment slide system (Thermo Fisher Scientific) and allowed to grow for 2–5 days until the desired synergy was achieved. After washing with DMEM, the cells were then inoculated with CFSE-labeled Fn or Alexa Fluor. TM Cells were incubated for the specified time length with 488-conjugated FadAc, mFadA, and BSA (300 μg / ml). After washing 10 times with DMEM, the cells were fixed in PBS containing 4% paraformaldehyde at room temperature for 15 min, and then neutralized in PBS containing 1% glycine at room temperature for 15 min. After blocking for 1 hour at 4°C with PBS containing 2% skim milk and 0.3% Triton X-100, the cells were incubated overnight at 4°C with goat anti-human E-cadherin polyclonal antibody (1:400 dilution, R&D Systems, MN) and rabbit anti-annexin A1 polyclonal antibody (1:400 dilution, Invitrogen) or rabbit anti-β-catenin polyclonal IgG (1:400 dilution, Invitrogen) in blocking solution. After washing three times with PBS containing 0.3% Triton X-100, the cells were conjugated with Cy3-conjugated donkey anti-goat IgG (1:1000 dilution, Jackson Immuno Research, West Grove, PA) and Alexa. The conjugated donkey anti-rabbit IgG (1:1000 dilution, Invitrogen) was incubated, washed, and covered in a DAPI-containing fixation medium (Vector Laboratories, CA, USA). The sample was then observed using a Nikon Ti Eclipse inverted microscope via scanning confocal microscopy.

[0239] For immunofluorescence staining of human colon samples, paired frozen sections of tumor and normal tissue were obtained. The slides were fixed in 4% polyformaldehyde for 15 minutes and permeabilized with PBS containing 0.1% Triton X-100, followed by blocking nonspecific binding. The slides were then incubated with rabbit anti-annexin A1 polyclonal antibody (Thermo Fisher Scientific) and mouse anti-FadA monoclonal antibody 5G11 (Xu et al., 2007) or with isotype controls, namely rabbit IgG (Invitrogen) and mouse IgG (R&D Systems, Minneapolis, MN). After washing, the slides were incubated with Alexa... Conjugated donkey anti-rabbit antibody (Invitrogen) and Alexa The conjugated goat anti-mouse antibody (Invitrogen) was incubated, washed, and mounted on a DAPI-containing fixation medium (Vector Laboratories, CA, USA). The slide was then observed using a Nikon Ti Eclipse inverted microscope via scanning confocal microscopy.

[0240] Western blot analysis. RKO cells transfected with the ANXA1 expression vector or the expression vector alone were seeded in 6-well plates and grown for 2 days. Cells were washed with ice-cold PBS and then blotted with Haltib. TM Lysis was performed using a single-use mixture of protease and phosphatase inhibitors (Thermo Fisher Scientific, MA) in RIPA lysis buffer (EMD Millipore, Burlington, MA). Cell lysates were centrifuged at 13,000 x g for 10 min at 4 °C, and protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. 1 μg of total protein was separated using a NuPAGE™ 4-12% Bis-Tris gel (Thermo Fisher Scientific) and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). The membrane was blocked for 1 hour at room temperature with 5% skim milk in TBS (TBST) containing 0.1% Tween 20, and then incubated overnight at 4°C with antibodies against annexin A1 (Thermo Fisher Scientific, 71-3400, 1:4000 dilution), cyclin D1 (Thermo Fisher, catalog number 701421, 1:250 dilution), β-catenin (Thermo Fisher Scientific, 71-2700, 1:2000 dilution), or β-actin (Abcam, ab6276, 1:4000 dilution) in TBST containing 0.5% skim milk. After washing three times with TBST, the membrane was incubated with a second antibody conjugated to HRP in TBST for 1 hour at room temperature. After washing, immunoreactive bands were detected using ECL Western blot analysis substrates (Thermo Fisher Scientific).

[0241] Co-immunoprecipitation. DLD1 cells were incubated with 1 mg / ml FadAc for 0, 15 and 120 minutes, respectively. Rabbit anti-annexin Al antibody (Thermo Fisher Scientific, MA), mouse anti-FadA antibody 5G11 (Xu et al., 2007), rabbit IgG or mouse IgG were covalently bound to agarose resin using the Pierce Co-Immunoprecipitation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The cells were lysed, mixed with the antibody-coupled resin and incubated for 2 hours at room temperature. After washing, the complexes bound to the antibodies were eluted and checked by Western blot analysis as described above. After electrophoresis and transfer, the PVDF membrane was incubated with rabbit anti-annexin Al polyclonal antibody (Thermo Fisher Scientific), mouse anti-FadA monoclonal antibody 7H7, rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology), mouse anti-β-actin monoclonal antibody (Cell Signaling Technology) or rabbit anti-β-catenin polyclonal antibody (Thermo Fisher Scientific). After washing, the membrane was incubated with HRP-coupled goat anti-rabbit IgG antibody (Bio-Rad) or PolyHRP-coupled goat anti-mouse IgG antibody (Thermo Fisher Scientific). The membrane was washed and incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and bands were detected using a Chemidoc MP imaging system (Bio-Rad).

[0242] APC min / + Mouse model. APC min / + Mice were obtained from Jackson Laboratories. All mice were kept in top-tier filter cages in a room with a 12-hour light cycle, fed with autoclaved food and water ad libitum and manipulated in a laminar flow hood. Before bacterial inoculation, mice were provided with drinking water supplemented with antibiotics (1 g / L ampicillin and 1 g / L metronidazole) for 2 weeks. Bacterial cultures were grown and sedimented by centrifugation at 5000 g for 5 minutes, then resuspended in PBS at an estimated density of 2 x 10 10 CFU / ml. Aliquots of 50 μl of bacterial suspension were gavaged three times a week for 8 weeks. At the end of the treatment, animals were sacrificed and the colon was collected, washed with PBS and opened longitudinally. Tumors were counted. Tumors and normal intestinal tissue were collected for histological analysis and extraction of DNA and RNA.

[0243] Tumor xenografts. Four-week-old nude mice were purchased from Taconic Biosciences (NY, USA). A subcutaneous bilateral injection of 1 x 10 7 HCT 116 cells treated with control or ANXA1 -specific siRNA into the nude mice, with control cells injected on the left side and ANXA1 -knocked down cells injected on the right side. On days 7-9 post-injection, tumor length and width were measured using calipers, and tumor volume was calculated using the following formula: volume = (width) 2 x length / 2.

[0244] Clinical samples. A total of 18 CRC cases were retrieved from the archives at the Department of Pathology at Columbia University Medical Center. Hematoxylin and eosin (H&E) slides were evaluated to confirm the presence of colorectal adenocarcinoma. Frozen sections of tumor and paired normal tissue were used for DNA / RNA extraction and immunofluorescence staining.

[0245] DNA and RNA extraction and real-time quantitative PCR. RNA was extracted from cultured cells using the QIAGEN RNeasy Mini Kit (Germany) according to the manufacturer's instructions. DNA and RNA were extracted from normal and tumor tissue from mice and clinical samples using the All Prep DNA / RNA Mini Kit (Germany). The samples were added with lysis buffer and 0.1 mm glass beads (Mo Bio Laboratories, Carlsbed, CA) and then homogenized in a FastPrep-24 (MP Biomedicals) for 60 seconds. DNA and RNA concentrations were measured using a NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies, DE). For DNA, samples were diluted to 30 ng / μl and 1 μl was used for real-time qPCR. For RNA, reverse transcription was performed using the Superscript IV First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions. Real-time qPCR was performed in duplicate in a StepOnePlus (Applied Biosystems, CA) using primers listed in Table 1. To quantify gene copies, standard curves were generated using plasmids with the 16S rRNA gene or the fadA gene. For RNA, data were analyzed using the 2(-ΔΔC(T)) method (Livak et al., 2001) and normalized to the β-actin control.

[0246] Statistical analysis. Differences between groups were examined by two-tailed t-test, one-way or two-way ANOVA followed by Student-Newman-Keuls (SNK) test. For clinical samples, Kruskal-Wallis non-parametric test followed by Conover test was performed. p < 0.05 was considered statistically significant.

[0247] Table 1 - Primers used in this study

[0248]

[0249]

[0250] Example 2 - Fusobacterium nucleatum preferentially binds, invades and stimulates the growth of cancerous colorectal cells through annexin Al, and annexin Al is selectively expressed in proliferating cancerous colorectal cells, is a novel CRC growth factor and can be inhibited by siRNA

[0251] It has been previously shown that FadA cannot promote the growth of non-cancerous HEK293 cells, even in the presence of E-cadherin (Rubinstein et al., 2013).

[0252] To determine the specificity of the growth stimulation mediated by F. nucleatum, the effects of F. nucleatum strain 12230 (Fn 12230) on PC-9 lung cancer cells, 22RV1 prostate cancer cells and MCF7 breast cancer cells, which all express E-cadherin, and UMUC3 bladder cancer cells, which do not express E-cadherin, were tested. No growth stimulation was detected; on the contrary, F. nucleatum inhibited the proliferation of PC-9, 22RV1 and UMUC3 cells, presumably due to a toxic effect (Figure 1A). Thus, it is possible that FadA promotion of growth requires additional components specific to CRC.

[0253] To identify possible CRC components, a CRC progression model was used, which consists of a series of cell lines sequentially derived from a non-malignant human colonic adenoma (Williams 1990). AA / C1 is a slowly growing, non-tumorigenic adenoma cell line with low colony formation efficiency. Upon treatment with 1 mM sodium butyrate, it gives rise to the AA / C1 / SB cell line, which grows faster, has an increased colony formation efficiency, but is still non-tumorigenic in mice. Further mutagenesis of the AA / C1 / SB cells with N-methyl-N'-nitro-N-nitrosoguanidine resulted in the tumorigenic cell line AA / C1 / SB / 10C (Williams 1990). Fn 12230 accelerates the growth of the tumorigenic AA / C1 / SB / 10C (referred to as "10C"), but not the non-tumorigenic AA / C1 or AA / C1 / SB (referred to as "SB") (Fig. 1A). The growth stimulation requires FadA, since the fadA deletion mutant US1 is defective. Consistent with these findings, while all cell lines treated with Fn 12230 express high levels of pro-inflammatory markers, only the 10C cells exhibit high expression of the oncogene cyclin D1 Figure 1J ). Fn 12230 binds 10C with 75% higher efficiency and invades 150% more efficiently than the non-tumorigenic parent SB Figure 1B . These results are consistent with the inventors' previous finding that FadA gene levels (and Fn) are significantly higher in human colorectal cancer tissue than in adenoma tissue (Rubinstein et al. 2013). The results also suggest that 10C and SB can differ in their membrane components, which can explain the differential binding by Fn.

[0254] Comparative proteomic analysis revealed that two membrane proteins are increased in 10C compared to SB, annexin Al (ANAX1) and villin (Roth et al. 2010). Downregulation of annexin Al in 10C by siRNA effectively reduces Fn binding and invasion in a similar manner to inhibition of CDH1 Figure 1B ), while knockdown of villin had no effect Figure 1C . Transfection of ANAX1 into SB cells significantly increased Fn binding and invasion Figure 1B . These results suggest that annexin Al plays an important role in the interaction of Fn with the tumorigenic cells 10C.

[0255] Annexin Al was found to be selectively expressed in proliferating tumorigenic and CRC cells. In 10C and human CRC cells HCT116, DLD1 and RKO, ANAX1 gene expression was significantly higher in the non-confluent state compared to the confluent state, while no expression difference was observed in the non-tumorigenic SB Figure 1D). In the CRC cell lines, RKO expressed significantly less ANXA1 than the others. Immunofluorescence staining revealed that annexin A1 expression was on the outer layer of the mass in growing tumorigenic 10C cells ( Figure 1E ). In contrast, neither cell density-dependent expression nor spatial expression was observed in non-tumorigenic SB cells.

[0256] Downregulation of annexin A1 by siRNA inhibited the growth of 10C, HCT116, DLD1, SW480 and HT29, but not the non-tumorigenic SB or the human CRC cell line RKO, which expresses very little annexin A1 ( Figure 1D and 1F). Transfection of ANXA1 into SB and RKO significantly stimulated their proliferation ( Figure 1G ). When equal numbers of HCT116 cells treated with control or ANXA1 -specific siRNA were injected subcutaneously bilaterally into nude mice, inhibition of ANXA1 significantly attenuated tumor growth compared to controls ( Figure 1H ). Similar results were obtained with DLD1 ( Figure 1I ).

[0257] Since Fn was not included in these experiments ( Figures 1D-1H ), the results demonstrate that annexin A1 is a key growth factor for CRC independent of Fn and can be inhibited by siRNA.

[0258] Example 3 - Selective binding of F. nucleatum to annexin A1 -expressing cells and induction of further annexin A1 expression by FadA in a positive feedback loop

[0259] When Fn was incubated with 10C, it immediately showed preferential binding to annexin A1 -expressing cells from as early as 5 min after incubation, while no binding preference was detected in SB (Fig. 2A, compare solid and open bars). Annexin A1 expression increased with continued Fn incubation, with a significant induction at 60 min, which persisted until 120 min (Fig. 2A). This induction required FadA, as the fadA deletion mutant US1 was defective, and purified FadAc induced annexin A1 in a dose-dependent manner ( Figure 2B ).

[0260] Notably, similar results were obtained after reanalyzing a recently published publicly available RNA-seq dataset containing gene expression data from human HT29 CRC cells incubated with different strains of F. nucleatum ATCC 25586 (Yu et al., 2017) ( Figure 2ESimilar observations were made using HCT116 and DLD1 cells (Figure 2F). As shown by the real-time qPCR results, the induction appears to be at the transcriptional level. Figure 2C The kinetics of transcriptional activation are enhanced by annexin A1-positive cells and the percentage of cells binding to Fn (which will increase the rate of activation). Figure 2C This was confirmed by comparison with Figures 2A and 2F. These results suggest a positive feedback loop in which Fn converts annexin A1-negative cells to annexin A1-positive cells, which further enhances its own binding. Figure 1D Consistent with observations in the study, Fn induces annexin A1 (Fn) at the outer layer of the cell mass. Figure 2D No annexin A1 induction was detected in non-tumorigenic SB cells (Fig. 2A, 2C, 2D), indicating that the induction is specific to tumorigenic and CRC cells. The fadA deletion mutant US1 does not induce annexin A1 expression. However, it also exhibits preferential binding to annexin A1-positive cells (Fig. 2A, 2F), suggesting that annexin A1 may mediate the binding of additional Fusobacterium nucleatum components to cancer cells.

[0261] Example 4 - Formation of a complex of FadA, E-cadherin, annexin A1, and β-catenin in cancer cells

[0262] It has been previously shown that FadA binds to E-cadherin on CRC cells (Rubinstein et al., 2013). Therefore, the interaction between FadA, E-cadherin, and annexin A1 was investigated. FadA-induced annexin A1 expression is mediated by E-cadherin (…). Figure 3A Although Fn does not affect E-cadherin expression at the transcriptional level, as determined by real-time qPCR ( Figure 3I Analysis using confocal microscopy revealed that in 10C and DLD1 cells, Fn and FadAc colocalized with E-cadherin and annexin A1 on the cell membrane and intracellularly. Figure 3B and 3C This is consistent with previous reports that FadAc binding leads to the internalization of cadherins (vascular endothelial cadherin and E-cadherin) (Rubinstein et al., 2013). Compared with cells treated with mFadA or BSA, FadAc-treated 10C cells not only showed increased annexin A1 expression, but also increased co-localization of E-cadherin and annexin A1. Figure 3C Western blot analysis confirmed that E-cadherin, annexin A1, and β-catenin increased synchronously after incubation with FadAc. Figure 3D), indicating that activation of β-catenin can involve a multi-component complex. In fact, all four components can be co-immunoprecipitated ( Figure 3E , 3F ). In 10C and DLD1 cells, knockdown of annexin Al by siRNA abrogated β-catenin activation and its nuclear translocation ( Figures 3G-3J ). Thus, annexin Al is an essential component in the annexin Al-E-cadherin-Fn complex and FadA-mediated oncogenic response that activates Wnt / β-catenin signaling.

[0263] The present inventors have also previously reported that binding of F. nucleatum to E-cadherin on CRC cells activated β-catenin signaling, leading to overexpression of oncogenes such as cyclin Dl (CCND1) (Roth et al., 2010). In cancerous 10C, HCT116 and DLD1 cells, activation of β-catenin expression by F. nucleatum was abrogated when ANXA1 was knocked down by siRNA ( Figure 3G ). Nuclear translocation of β-catenin was also inhibited in 10C ( Figure 3H ). These data indicate that annexin Al is required for activation of Wnt / β-catenin signaling.

[0264] Cyclin Dl gene expression also exhibited cell density dependence in 10C and HCT116 cells, being significantly higher in non-confluent than in confluent state, consistent with ANXA1 expression (compare Figure 1D and 3K ). In contrast, no expression difference was detected in RKO cells in which ANXA1 was not detected (compare Figure 1D and 3K ). However, when ANXA1 was transfected into RKO cells, an increase in cyclin Dl expression was observed, indicating a driving role of annexin Al in oncogene expression ( Figure 3L ). These observations support the role of annexin Al in modulating β-catenin signaling.

[0265] Example 5 - Co-expression of FadA and annexin Al in colorectal tumors in mice and humans

[0266] Using APC min / + The correlation between FadA and annexin Al was investigated in vivo in mice. C57BL / 6 APC min / +Mice developed significantly more tumors in the colon Figure 4A ), confirming the driver role of FadA in tumorigenesis. In all treatment groups, significantly higher levels of ANXA1 mRNA were detected in tumors compared to normal colon tissue from the same mouse, with the highest levels observed in the group treated with wild-type Fn Figure 4B ). A positive correlation between fadA and ANXA1 was detected in Fn-induced tumors, with a correlation coefficient of 0.43 (p=0.01; Figure 4C ). In CRC patients, higher levels of fadA and ANXA1 were detected in adenocarcinoma tissue compared to adjacent normal tissue Figure 4D , 4E ), with a correlation coefficient of 0.62 (p<0.001; Figure 4F ). Immunofluorescence staining of paired tumor and normal tissue confirmed this finding, with significantly higher levels of FadA and Annexin A1 protein detected in tumors. Co-localization of FadA and Annexin A1 was observed in tumors but not in normal tissue Figure 4G . These results further support the role of FadA and Annexin A1 in tumorigenesis.

[0267] Example 6 - Annexin A1 can be inhibited by anti-Annexin A1 antibodies, thereby inhibiting the growth of cancer cells in vitro and in vivo

[0268] Similar to the results shown for siRNA in Example 2 and Figures IF, 1H and 1I, downregulation of Annexin A1 by Annexin A1 antibodies inhibited tumor growth both in vitro and in vivo.

[0269] Downregulation of Annexin A1 by anti-Annexin A1 antibodies inhibited the growth of HCT116. However, antibodies against Annexin A2, Annexin V, Annexin A6 and Annexin A11 had no effect on the growth of the cells Figure 5A .

[0270] Aliquots of 2-8 x 10 6 Aliquots of 2-8 x 10 2 x length / 2. In both treatment regimens, inhibition of Annexin A1 significantly attenuated tumor growth compared to control Figure 5B ). Tumors treated with anti-Annexin A1 antibodies did not show Annexin A1 in the tumor tissue Figure 5C .

[0271] Example 7 - Elimination of annexin A1 significantly prolonged lifespan in a mouse model of familial adenomatous polyposis (FAP).

[0272] Anxa1 - / - Mice were obtained from Dr. Mauro Perretti at Queen Mary University (London, UK). These mice were then compared with Apc mice that carried a mutation in one copy of the tumor suppressor gene APC and developed spontaneous tumors in the small and colon. min / + Mice bred in Jackson Laboratory to produce Anxa1 + / - Apc min / + and Anxa1 - / - Apc min / + Group.

[0273] Elimination of annexin A1 in APC mutant mice extended their lifespan in a dose-dependent manner, including ANXA1. - / - APC mutant mice have a significantly longer lifespan than ANXA1 mice. + / + APC mutant mice ( Figure 6 ).

[0274] Example 8 - Annexin A1 is a novel prognostic biomarker for colorectal cancer

[0275] As previously described (Dalerba et al., 2016), the relationship between ANXA1 mRNA expression levels and recurrence risk in CRC was investigated in a database of 466 primary colorectal cancers obtained by assembling four independent gene expression array datasets (GSE14333, GSE17538, GSE31595, GSE37892) from the NCBI-GEO online resource. The association was first tested using Kaplan-Meier survival curves, employing three different patient stratification methods: (1) based on the median ANXA1 mRNA expression level (… Figure 7A (2) Iquartile distribution based on ANXA1 mRNA expression levels Figure 7B (3) Based on the expression threshold calculated using the StepMiner algorithm as previously described (Dalerba et al., 2011; Sahoo et al., 2007). Figure 7C The association between ANXA1 mRNA expression levels and relapse risk was examined using both univariate and multivariate analyses based on the Cox proportional hazards approach, with ANXA1 mRNA expression levels modeled as a continuous variable.

[0276] Regardless of the method used for stratification, high levels of ANXA1 mRNA expression were associated with a statistically significant decrease in disease-free survival (DFS) rate and a significant increase in risk of relapse (p<0.001, log-rank test). Differences in ANXA1 mRNA expression levels did not appear to be associated with differences in the relative content of epithelial cells (i.e. tumor cell density) in each tumor, as revealed by the lack of visual correlation with the epithelial marker desmoplakin (DSP). Importantly, the association between high levels of ANXA1 mRNA expression and increased risk of relapse remained statistically significant even after a series of multivariate analyses (Cox proportional hazards method) excluding as possible confounding variables the clinical stage, the pathological grade, the age and the sex (Table 2) and when ANXA1 expression was modeled as a continuous variable (Table 3) and when tested in the entire dataset (n=466; p<0.001) and in the subset of patients for which the pathological grading of the corresponding tumor was annotated (n=216; p<0.001).

[0277] The hazard ratio (HR) of disease relapse associated with increased levels of ANXA1 expression was 1.44 (95% CI 1.24-1.68; p<0.001) when tested together with the clinical stage, the age and the sex in the entire group of patients (n=466) and 1.56 (95% CI 1.21-2.02; p<0.001) when tested together with the clinical stage, the pathological grade, the age and the sex in the subset of patients for which the pathological grading was annotated (n=216).

[0278] Table 2 - Association between ANXA1 mRNA expression levels and risk of relapse in all colon cancer patients (n=466)

[0279]

[0280] 1 HR: hazard ratio

[0281] 2 CI: confidence interval

[0282] 3 ANXA1 mRNA levels and age were modeled as continuous variables

[0283] 4 M / F: male vs. female

[0284] *** p<0.001

[0285] Table 3 - Association between ANXA1 mRNA expression levels and risk of relapse in colon cancer patients for which information on tumor grade was available (n=216)

[0286]

[0287]

[0288] 1 HR: Hazard Ratio

[0289] 2 CI: Confidence Interval

[0290] 3 ANXA1 mRNA levels and age were modelled as continuous variables

[0291] 4 M / F: Male vs Female

[0292] *** p<0.001

[0293] References

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[0323] incorporated by reference

[0324] All references cited herein are incorporated by reference to the extent that a specific and individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent is so identified by reference. This incorporation by reference is made pursuant to 37 C.F.R. § 1.57(b)(1) by the Applicant, and is intended to apply to each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is expressly acknowledged to comply with 37 C.F.R. § 1.57(b)(2), even though no specific statement of incorporation by reference is immediately adjacent to the citation of each of these references. The general incorporation by reference statement made herein is not in any way diminished by the inclusion of specific incorporation by reference statements, if any, in this specification. Citation of references herein does not constitute an admission that the referenced reference is prior art to the present application, nor does it constitute any admission as to the correctness or date of availability of the contents of any of these publications or documents. The entire contents of each patent file mentioned herein, including correction certificates, patent application files, scientific articles, government reports, websites, and other references, are incorporated herein by reference in their entirety for all purposes. In the event of a conflict in terminology, the present specification controls.

[0325] The scope of the application is not intended to be limited to the particular embodiments described in this document. Indeed, various modifications of the application, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0326] The written description above is considered sufficient to enable one of ordinary skill in the art to practice the application. Various modifications of the application, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0327] equivalents

[0328] The application can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are to be considered in all respects only as illustrative and not restrictive. In various different embodiments of the methods and systems of the application, where the term "comprising" is used in the description of the steps or components recited, it is also contemplated that the methods and systems can consist essentially of, or consist of, the recited steps or components. Furthermore, it should be understood that the order of steps or order for performing certain actions are immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0329] In this specification, the singular encompasses the plural unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification will control.

[0330] All percentages and ratios used herein are by weight, unless otherwise specified.

Claims

1. Use of a therapeutically effective amount of an agent that inhibits or blocks annexin A1 as the sole active ingredient in the preparation of a medicament for preventing selectively annexin A1 (ANXA1) cancers in subjects with familial adenomatous polyposis by inhibiting the growth of tumor cells, wherein the agent that inhibits or blocks annexin A1 is selected from sorafenib and anti-annexin A1 antibodies, and wherein the cancer is selected from colorectal cancer (CRC).

2. The use according to claim 1, wherein the pharmaceutical agent further comprises nanoparticles, lipid particles, or vesicles.

3. The use according to claim 1, wherein the agent further comprises microparticles.

4. The use according to claim 1, wherein the pharmaceutical agent further comprises liposomes, nanocapsules, or microspheres.

5. The use according to claim 1, wherein the pharmaceutical agent further comprises a pharmaceutically acceptable carrier.

6. The use according to claim 1, wherein the pharmaceutical agent further comprises a pharmaceutically acceptable diluent or adjuvant.

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