Methods and kits for diagnosing and treating cancer based on overexpression of the ADAMTSL5 gene

Abstract

The present invention relates to a method for diagnosing cancer in a mammal in need thereof. The method comprises the following steps: collecting a biological sample from the mammal; determining whether the ADAMTSL5 gene is overexpressed from the biological sample; and diagnosing cancer based on the determination of the overexpression of the gene. It also relates to a kit for determining the overexpression of the ADAMTSL5 gene in a biological sample obtained from a mammal, to a pharmaceutical composition for treating cancer comprising an agent targeting ADAMTSL5 or the ADAMTSL5 pathway and a pharmaceutically acceptable carrier, and to the use of ADAMTSL5 mRNA or protein as a cancer biomarker.

Description

[0001] Technical field of the invention

[0002] The present invention relates to a method for diagnosing cancer in a mammal in need thereof. The method of the present invention comprises the step of determining the expression level of a specific biomarker in a biological sample obtained from the mammal. The present invention also relates to a kit for determining such expression level and a pharmaceutical composition for inhibiting a specific biomarker pathway. Background of the Invention

[0004] Cancer is the second leading cause of death in the world. Nearly one sixth of all deaths worldwide are caused by cancer. For example, 8.8 million people died of cancer in 2015. Therefore, it is important to develop new methods for improving cancer diagnosis in patients who need it, and in particular for predicting their sensitivity to treatment in order to increase their treatment response, which will lead to increased survival expectancy.

[0005] Epigenetics is the study of changes in gene activity, which does not include modification of DNA sequences and can be propagated in cell division. Unlike mutations that affect DNA sequences, epigenetic modifications are reversible. It is well known that epigenetic abnormalities lead to the development and progression of human diseases, especially cancer. Epigenetic processes intervene in many events such as cell division, differentiation, survival and motility regulation. Changes in these mechanisms promote the transformation of healthy cells into cancer cells, and therefore any epigenetic abnormalities may be associated with tumorigenicity. Among the consequences related to cell epigenetic abnormalities, there are tumor suppressor gene silencing or overexpression of oncogenes (such as proto-oncogenes), which include abnormal DNA methylation or mutations of genes encoding enzymes responsible for chromatin modification. DNA methylation is an essential epigenetic mechanism that affects gene expression levels in cells. These changes can lead to rapid biological changes and the acquisition of malignant characteristics. Cancer scenarios are usually characterized by diffuse DNA hypomethylation and focal overmethylation in CpG-rich regions called CpG islands (CGIs). Typically, CGI overmethylation at promoters inhibits transcription of genes that act as tumor suppressors. Nevertheless, a large fraction of DNA methylation was also observed in gene body CGIs, with a positive correlation between methylation and upregulated gene expression. This implies that DNA hypermethylation in gene body CGIs is associated with upregulation of genes that would act as positive regulators of tumorigenicity, consistent with the reported functions of some of them in cancer cells. However, the implications of several other hypermethylated and upregulated genes in cellular tumorigenicity remain unknown.

[0006] These findings undermine the development of drugs that target epigenetic regulators (called epigenetic drugs or epi-medicine). So far, two main families of compounds have been developed: (i) compounds that inhibit DNA methylation, and (ii) compounds that target histone modifications. However, these types of compounds currently lack specific effects.

[0007] Based on the above, there is still a need to identify genes that can be used as new specific biomarkers and / or targets to be modulated with bioactive agents for cancer treatment. In particular, there is still a need to develop new methods for diagnosis, prognosis and treatment of cancer. SUMMARY OF THE INVENTION

[0009] According to a first aspect, the present invention relates to a method for diagnosing cancer in a mammal in need thereof. The method comprises the following steps:

[0010] - collecting a biological sample from said mammal;

[0011] - determining whether the ADAMTSL5 gene is overexpressed from the biological sample;

[0012] - Diagnosing cancer based on the determination of overexpression of said gene.

[0013] According to a second aspect, the present invention relates to a kit for determining overexpression of an ADAMTSL5 gene in a biological sample obtained from a mammal. The kit comprises at least one anti-ADAMTSL5 type antibody, a container for containing a biological sample, and a protocol for measuring overexpression of an ADAMTSL5 cancer marker gene, preferably a hepatocellular carcinoma marker gene, in a biological sample obtained from a mammal.

[0014] According to a third aspect, the present invention relates to a pharmaceutical composition comprising an agent targeting the ADAMTSL5 gene itself and / or a pathway in which ADAMTSL5 functions and a pharmaceutically acceptable carrier for treating cancer.

[0015] According to a fourth aspect, the present invention relates to the use of ADAMTSL5 protein as a cancer biomarker.

[0016] In a fifth aspect, the present invention relates to a method for treating cancer in a mammal in need thereof. The cancer is the same as those described above. The method includes a first step of diagnosing cancer in a mammal in need thereof, and a second step of treating cancer by administering an inhibitor of ADAMTLS5. The step of diagnosing cancer includes the same steps as those described above in the method for diagnosing cancer in a mammal in need thereof.

[0017] In a sixth aspect, the present invention relates to an in vitro method for monitoring the response of a mammal suffering from cancer to an anti-cancer treatment, comprising determining the expression level of ADAMTSL5 in a biological sample of the mammal at two or more time points during the anti-cancer treatment, wherein an equal or higher ADAMTSL5 expression level in the biological sample of the subject at a later time point compared to a reference value obtained in the biological sample of the subject at an earlier time point indicates that the subject is resistant to the anti-cancer treatment, whereas a lower ADAMTSL5 level indicates that the subject is responsive to the anti-cancer treatment.

[0018] Advantageously, the method for diagnosing cancer in a mammal in need thereof according to the present invention is characterized in that: - the cancer is selected from the group consisting of brain cancer, CNS cancer, colorectal cancer, breast cancer, lung cancer, skin cancer, kidney cancer, gastrointestinal cancer, myeloma, lymphoma, leukemia, cervical cancer, liver cancer and hepatocellular carcinoma, preferably the cancer is hepatocellular carcinoma; - the biological sample is selected from the group consisting of blood, biopsy tissue, serum, plasma, urine, feces, sputum, cerebrospinal fluid or supernatant from cell lysate, preferably the biological sample is tissue biopsy, blood, plasma, serum or urine; - overexpression of the ADAMTSL5 gene in the biological sample is determined by measuring the ADAMTSL5 protein level or mRNA level in the biological sample; - the ADAMTSL5 protein level in the biological sample is measured by adding at least one anti-ADAMTSL5 type antibody to the biological sample; - the anti-ADAMTSL5 type antibody is selected from the group consisting of unconjugated or conjugated to alkaline phosphatase horseradish peroxidase or to a fluorescent dye; - the ADAMTSL5 protein level in the biological sample is measured by using immunostaining, immunofluorescence, Western blotting or ELISA.

[0019] Advantageously, the kit for determining overexpression of the ADAMTSL5 gene of the invention is characterized in that: - the anti-ADAMTSL5 type of antibody is selected from uncoupled or coupled / conjugated to alkaline phosphatase, horseradish peroxidase or to a fluorescent dye.

[0020] Advantageously, the pharmaceutical composition of the invention is characterized in that: - the agent targeting ADAMTSL5 or the ADAMTSL5 pathway is selected from blocking antibodies, peptides, sh-RNA, si-RNA, microRNA, antisense RNA, chemical drugs, demethylating agents and agents regulating glycosylation and / or heparin binding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Other features and aspects of the present invention will be apparent from the following description and accompanying drawings, in which:

[0023] Figure 1 In clinically relevant cancer mouse models ( Alb-R26Met mice) and overexpression of ADAMTSL5;

[0024] Figure 2 In clinically relevant cancer mouse models ( Alb-R26 Met Overexpression of ADAMTSL5 mRNA and protein in WT mice;

[0025] Figure 3 Compared with cells with reduced ADAMTSL5 expression levels, cells from clinically relevant cancer mouse models ( Alb-R26 Met In vitro tumorigenic properties of hepatocellular carcinoma (HCC) cells established in vitro;

[0026] Figure 4 This indicates that HCC cells lose their in vivo tumorigenic properties after ADAMTSL5 downregulation;

[0027] Figure 5 This indicates that ADAMTSL5 overexpression is associated with clinically relevant mouse models ( Alb-R26 Met In vivo acquisition of tumorigenic properties of immortalized hepatocytes from mice;

[0028] Figure 6 Demonstrating that clinically relevant cancer mouse models ( Alb-R26 Met ADAMTSL5 expression level in mice);

[0029] Figure 7 This indicated that ADAMTSL5 mRNA levels were high in 52% of HCC patients, with those associated with alcohol consumption accounting for the majority;

[0030] Figure 8 Demonstrated overexpression of ADAMTSL5 protein levels in 9 / 10 HCC patients compared with adjacent liver; and

[0031] Fig. 9 Figure 2 illustrates ADAMTSL5 mRNA levels in human cancer cell lines. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention relates to a method for diagnosing and prognosing cancer in a mammal in need thereof. First, the method comprises a step of collecting a biological sample from the mammal, then a step of determining whether the ADAMTSL5 gene / protein is overexpressed from the biological sample, and then according to a third step, diagnosing / prognosing cancer based on the determination of overexpression of the gene or protein.

[0034] According to the invention, mammals are in particular humans. However, all mammals are contemplated, even cats, dogs, horses or rodents, such as mice and rats.

[0035] According to a preferred embodiment of the present invention, the cancer is brain cancer, cancer of the central nervous system (CNS), colorectal cancer, breast cancer, lung cancer, skin cancer, gastrointestinal cancer, kidney cancer, myeloma, lymphoma, leukemia, cervical cancer, liver cancer, such as hepatocellular carcinoma (HCC). In particular, the cancer diagnosed according to the present invention is HCC.

[0036] According to the invention, the biological sample is selected from the group consisting of blood, tissue biopsy, serum, plasma, urine, feces, sputum, cerebrospinal fluid and supernatant from cell lysate. In particular, the biological sample used is tissue biopsy, blood, plasma, serum or urine.

[0037] According to a preferred embodiment of the present invention, overexpression of the ADAMTSL5 gene in a biological sample obtained from a mammal in need thereof is determined by measuring the ADAMTSL5 protein level or RNA level in the biological sample. The terms "upregulated", "upregulation", "overexpressed" or "overexpression" are used to mean that the expression, activity or level of a gene or an RNA transcript or protein product of a gene is higher relative to one or more controls (such as one or more positive and / or negative controls). In particular, levels are considered elevated when they are higher than those in control healthy tissues.

[0038] In fact, the mammalian genome contains 19 ADAMTS (A Disintegrin And Metalloproteinase with Thrombospondin) genes, numbered 1 to 20. Like its relatives, matrix metalloproteinases (MMPs) and ADAMs, ADAMTS belong to the metzincinprotease superfamily, named after the conserved methionine residue near the active site of zinc ion-dependent metalloproteinases. Representatives of the ADAMTS family are present in all metazoans, although to date, they have not been identified in unicellular organisms or plants. All ADAMTS are secreted extracellular enzymes with a compound domain organization, which contain from the amino terminus: a signal peptide, followed by a pre-region of variable length; a metalloproteinase domain; a disintegrin-like domain; a central type 1 thrombospondin sequence repeat (TSR) motif; and a cysteine-rich domain, followed by a spacer. Another family that has separated from ADAMTS is the seven ADAMTS-like genes (ADAMTSL), which encode proteins that resemble the ADAMTS auxiliary domain but lack its catalytic domain. These ADAMTSL proteins (including ADAMTSL 1 to 6 and papilin) ​​may play a role in regulating ADAMTS activity. ADAMTSL5 is a protein that has been discovered in the late 2000s and has been described as binding to fibrillin-1 and promoting fibril formation. As a key mechanism of growth factor regulation in the extracellular matrix, the role of ADAMTSL5 in microfibril formation has attracted much attention. ADAMTSL5 is more specifically a secreted protein with a unique domain composition, including N-terminal thrombospondin type 1 repeats, a cysteine-rich module, a spacer module, and a C-terminal netrin-like module, which is connected to the spacer by a proline-rich segment. ADAMTSL5 is thought to have been associated with certain diseases (such as psoriasis), but to date, ADAMTSL5 has not been associated with cancer as a potential biomarker or as a target for molecular therapy.

[0039] In the context of the present invention and in a preferred embodiment, the ADAMTSL5 protein level is measured by adding at least one antibody to the biological sample. Antibodies particularly useful for measuring the ADAMTSL5 protein level are of the anti-ADAMTSL5 type. Anti-ADAMTSL5 antibodies are particularly selected from uncoupled or coupled or conjugated to alkaline phosphatase, horseradish peroxidase (HRP) or to a fluorescent dye.

[0040] According to the present invention, the level of ADAMTSL5 protein present in a biological sample of a mammal in need thereof is measured, in particular, by using immunostaining, immunofluorescence, Western blotting or ELISA.

[0041] In biochemistry, immunostaining refers to the use of antibody-based methods to detect specific proteins in biological samples. Immunostaining covers a wide range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods. Immunofluorescence is a technique for light microscopy using a fluorescent microscope, and is primarily used for microbial samples. The technique uses the specificity of antibodies for their antigens to target fluorescent dyes to specific biomolecular targets within cells, and thus allows the distribution of target molecules to be visualized via the sample.

[0042] According to a second aspect, the present invention provides a kit for determining overexpression of the ADAMTSL5 gene in a biological sample obtained from a mammal. The kit comprises at least one anti-ADAMTSL5 type antibody and a container for containing the biological sample. The anti-ADAMTSL5 type antibodies used in the kit are the same as those described above.

[0043] According to the present invention, the level of ADAMTSL5 present in a biological sample of a mammal in need thereof can also be determined by measuring the level of ADAMTSL5 mRNA using, for example, microarray, RNA-seq, in situ hybridization, RNA-scope, and conventional semi-quantitative or quantitative RT-PCR.

[0044] According to a third aspect, the present invention relates to a pharmaceutical composition. The pharmaceutical composition comprises an agent targeting ADAMTSL5 itself or the ADAMTSL5 pathway and a pharmaceutically acceptable carrier for treating cancer. The term "ADAMTSL5 pathway" and its grammatical variations refer to a pathway in which the ADAMTSL5 gene is involved.

[0045] The reagents used for targeting the ADAMTSL5 pathway are particularly blocking antibodies, peptides, sh-RNA, si-RNA, microRNA, antisense RNA and chemical drugs. It also includes demethylating agents (such as decitabine) or reagents that regulate glycosylation and / or heparin binding. The demethylating agent of the present invention is a compound that causes genomic DNA hypomethylation by inhibiting DNA methyltransferase.

[0046] In a fourth aspect, the present invention relates to the use of ADAMTSL5 protein as a cancer biomarker. The cancer is the same as those described above.

[0047] In a fifth aspect, the present invention relates to a method for treating cancer in a mammal in need thereof. The cancer is the same as those described above. The method includes a first step of diagnosing cancer in a mammal in need thereof, and a second step of treating cancer by administering an inhibitor of ADAMTLS5. The step of diagnosing cancer includes the same steps as those described above in the method for diagnosing cancer in a mammal in need thereof.

[0048] In a sixth aspect, the present invention relates to an in vitro method for monitoring the response of a mammal suffering from cancer to an anti-cancer treatment, comprising determining the expression level of ADAMTSL5 in a biological sample of the mammal at two or more time points during the anti-cancer treatment, wherein an equal or higher ADAMTSL5 expression level in the biological sample of the subject at a later time point compared to a reference value obtained in the biological sample of the subject at an earlier time point indicates that the subject is resistant to the anti-cancer treatment, whereas a lower ADAMTSL5 level indicates that the subject is responsive to the anti-cancer treatment.

[0049] Example: Materials and Methods:

[0050] In the following examples, genetically modified mice were used. In these mice, the expression level of the receptor tyrosine kinase (RTK) Met, which is activated in about 50% of human HCCs, was slightly increased above endogenous levels based on a genetic approach that allows regulation of gene expression in a tissue / time-specific manner - (R26 stopMet mice; Fan et al. PLoS Genetics 2015; Fan et al. Hepatology 2017). Recently, it was demonstrated that increased expression of methylated (Met) RTKs in the liver perturbs tissue homeostasis, leading to tumor initiation and progression to HCC (Alb-R26 Met ). Based on Alb-R26 Met Comparison of Met expression levels in liver tumors of human (n=32) and human (n=249) showed that in Alb-R26 Met Met levels in the genetic context (3.16 ± 0.06 relative to control livers) correspond to those found in approximately 20% of HCC patients (48 / 249). Analysis of 96 different genes in a panel of tumor samples (n = 32) showed that Alb-R26 Met Mouse models of liver tumorigenesis correspond to HCC patient subgroups, thus establishing Alb-R26 Met Clinical relevance of HCC mouse models. MetMouse models were used to study the effects of DNA methylation on transcriptional switches involved in tumorigenesis. A striking enrichment of genes that were both hypermethylated and overexpressed in CpG islands (CGIs) was identified. Among them was ADAMTSL5.

[0051] Example 1: Alb-R26 compared to control liver Met Hypermethylation in CGI of ADAMTSL5 gene and overexpression of mRNA ADAMTSL5 in tumor models.

[0052] Figure 1 (A) Schematic representation showing mouse ADAMTSL5 transcripts and CGIs from the UCSC genome browser and Refseq gene annotation based on the NCBI37 / mm9 mouse reference. The diagram allows visualization of exons, introns, and CGIs of ADAMTSL5, with gene body CGIs found to be hypermethylated in tumors highlighted as squares.

[0053] Figure 1 (B) is a schematic representation of the human ADAMTSL5 protein from Badel et al., Matric Biology, 2012. Different domains present in the ADAMTSL5 protein were reported, which may be involved in regulating the interaction of ADAMTSL5 with other proteins and the biological function of ADAMTSL5.

[0054] Figure 1 (C) and (D) illustrate control liver (n=3) and Alb-R26 Met Promoter CGI in tumors (n=10) Figure 1 (C)) and genome CGI ( Figure 1 (D) Methylation levels (β values) of ADAMTSL5 in the liver. Note that the methylation levels of ADAMTSL5 in the promoter CGI were similar in control and tumor samples, while in Alb-R26 compared with control livers. Met Methylation levels in somatic CGIs were significantly higher in tumors. In conclusion, tumors are characterized by hypermethylation of ADAMTSL5 in somatic CGIs.

[0055] Figure 1 (E) Illustration of the expression of RNA-seq in Alb-R26 compared with control livers. Met ADAMTSL5 mRNA expression levels in tumors. The graph shows read counts (on the right) and Log2FC (fold change) (on the left). Significance: *: P < 0.05; FDR (adjusted p value) = 4.31 E-15 In conclusion, tumors are characterized by overexpression of ADAMTSL5 mRNA.

[0056] Figure 1 (F) shows the expression of Alb-R26 in liver compared with control liver (n=5). Met ADAMTSL5 expression levels in tumors (n=16) by RT-qPCR. Note that in Alb-R26 compared to control livers Met High expression levels of ADAMTSL5 in tumors In conclusion, tumors are characterized by consistent overexpression of ADAMTSL5 mRNA.

[0057] Example 2: Alb-R26 Met Overexpression of ADAMTSL5 mRNA and protein in tumor models.

[0058] Figure 2 (A) shows tumor (from Alb-R26 Met Representative images of ADAMTSL5 mRNA expression levels (by RNA-scope) in livers of Alb-R26 mice and control livers (dissected from control mice). Met Strong staining was observed in tumors but not in control livers. In conclusion, similar to the data from RNA-seq and RT-qPCR analyses, tumors are characterized by consistent overexpression of ADAMTSL5 mRNA.

[0059] Figure 2 (B) shows that in tumor (from Alb-R26 Met Representative images of ADAMTSL5 protein levels by immunofluorescence (left) and immunostaining (right) in livers (dissected from control mice) and control livers (dissected from control mice). Note that ADAMTSL5 is expressed in Alb-R26 compared to control livers. Met In conclusion, tumors are characterized by consistent overexpression of ADAMTSL5 protein, coherent with high mRNA levels.

[0060] Example 3: Alb-R26 in which ADAMTSL5 is overexpressed Met In vitro tumorigenic properties of HCC cells. *: P<0.05; **: P<0.01; ***: P<0.001.

[0061] Figure 3 (A) Alb-R26 used for molecular and functional studies MetSchematic diagram of the establishment of HCC cell lines (Fan et al. Hepatology, 2017). This figure summarizes how to generate HCC cell lines for their molecular characterization and for their bioassays. According to the protocol, HCC cell lines were established and reported in Fan et al. Hepatology, 2017 from different Alb-R26 Met Liver tumors were generated from mouse dissections. Briefly, the dissected tumors were minced, incubated in a solution containing collagenase and DNase, and tissue debris was removed using a 100 μm sterile filter. The cells were then cultured directly under adherent conditions or under non-adherent conditions for 1 week in William E medium containing supplements to enrich for cells with tumorigenic properties. After the cultured cells were expanded, a portion was frozen and stored as a reserve, while another portion was characterized by molecular (biochemistry, RT-qPCR) and functional (its ability to form tumors in nude mice).

[0062] Figure 3 (B) Reports the expression of three Alb-R26 proteins in liver compared with control liver. Met Graph of ADAMTSL5 expression levels (by RT-qPCR) in HCC cell lines (HCC3, HCC13, and HCC14). In conclusion, HCC cells are characterized by consistent overexpression of ADAMTSL5 mRNA as shown in tumors.

[0063] Figure 3 (C) shows the expression of Alb-R26 stably transfected with a plasmid carrying the shRNA targeting sequence (also carrying the puromycin gene for selection of stable clones) relative to the control. Met mRNA expression level of ADAMTSL5 in HCC cells. Cells were transfected with the target plasmid and then exposed to puromycin for 7 days after 48 hours to select cells in which the plasmid had been stably integrated. Clones in which the expression level of ADAMTSL5 was downregulated by the shRNA targeting sequence were identified by RT-qPCR. In conclusion, the shRNA targeting sequence for ADAMTSL5 resulted in an effective downregulation of ADAMTSL5 mRNA levels in HCC cells, which interfered with ADAMTSL5 protein expression levels.

[0064] Figure 3 (D) illustrates the results of an anchorage-independent growth assay (soft agar assay) performed using HCC control cells or HCC cells carrying shRNA sequences targeting ADAMTSL5. This assay demonstrates the ability of cells to form colonies under non-adherent conditions, thus revealing their in vitro tumorigenic properties. The results show that compared with control cells, Alb-R26 cells carrying shRNA sequences targeting ADAMTSL5 showed a significant increase in the growth rate of HCC cells. MetThe number of colonies formed by HCC cells was reduced. In conclusion, high levels of ADAMTSL5 in HCC cells are required for their tumorigenic properties in vitro.

[0065] Figure 3 (E) is a depiction of an anchorage-independent growth assay (soft agar assay) showing partial rescue of ADAMTSL5-targeted Alb-R26 with conditioned medium from control cells. Met In vitro tumorigenic properties of HCC cells. The results showed that extracellular ADAMTSL5 confers tumorigenicity to cells expressing low levels of ADAMTSL5. In conclusion, ADAMTSL5 is a secreted protein that exerts its functions in the extracellular environment and can also act in a cell-nonautonomous manner.

[0066] Figure 3 (F) Alb-R26 derived from control and ADAMTSL5-targeted Met Representative images (left) and quantification (right) of tumor spheres of HCC cells (right). Note that, compared with Alb-R26 Met HCC shAdamts15 Compared with cells (carrying shRNA sequences targeting ADAMTSL5), the number and size of tumor spheres generated from control cells were higher. In conclusion, high levels of ADAMTSL5 in HCC cells confer self-renewal ability.

[0067] Example 4: In Alb-R26 Met Downregulation of ADAMTSL5 in HCC cells resulted in loss of tumorigenic properties of HCC cells in vivo. *: P<0.05; **: P<0.01; ***: P<0.001.

[0068] Figure 4 (A) contains Alb-R26 Met HCC cells (upper) or Alb-R26 Met Images of tumors dissected from nude mice xenografts of -shADAMTSL5 HCC cells (bottom).

[0069] Alb-R26 was injected subcutaneously into the flank of nude mice. Met or Alb-R26 Met -shAdamtsL5 HCC cells (5x10 6 xenograft studies. Figure 4 (A) contains images of tumors dissected 8 weeks after xenograft establishment. Note that Alb-R26 was injected subcutaneously in Met HCC cells formed tumors in nude mice, and Alb-R26 was injected subcutaneously into the nude mice. MetThe tumors in mice expressing -shADAMTSL5 HCC cells were greatly reduced, indicating that the tumorigenic properties of the cells were impaired in vivo.

[0070] Figure 4 (B) contains xenograft growth curves reporting the mean tumor volume per group measured weekly. In conclusion, downregulation of ADAMTSL5 levels in HCC cells (achieved by stable transfection of shRNA targeting sequences) interferes with the in vivo tumorigenic properties of HCC cells. In summary, these results suggest that downregulation of ADAMTSL5 expression levels interferes with tumor establishment / evolution.

[0071] exist Figure 4 In (C), quantitative analysis of tumor volumes dissected 8 weeks after cell injection is shown. Each point corresponds to the volume of each tumor. In conclusion, downregulation of AdamtsL5 levels in HCC cells (achieved by stable transfection of shRNA targeting sequences) interferes with tumorigenic properties in vivo.

[0072] Example 5: Immortalization-R26 Met Sensitized hepatocytes acquire tumorigenic properties in vivo after overexpression of ADAMTSL5.

[0073] Figure 5 (A) Immortalization-A26 Met Schematic representation of the establishment of primed hepatocytes (embryonic hepatocytes carrying elevated levels of Met RTK and immortalized with SV40 large T antigen). Briefly, R26 Met Cultured hepatocytes from embryonic livers were immortalized by infection with a retrovirus carrying the SV40 large T antigen (plus a neomycin gene for selection of stable clones) and then subsequently treated for 7 days with a medium that allows hepatocytes to be depleted of other cell types. Met Hepatocytes were sensitized because wild-type Met levels were elevated 3-fold, although they were not tumorigenic due to the inability to form tumors in xenografts.

[0074] Figure 5 (B) Image of a mouse containing xenografts 11 weeks after establishment. Note that immortalized-R26 Met hepa overAdamts15 Nude mice that were overexpressed with ADAMTSL5 developed tumors on both flanks.

[0075] Immortalized-R26 by subcutaneous injection Met Control (immortalized-R26 Met hepa WT ) or overexpression of ADAMTSL5 (immortalized-R26 Met hepa overAdamts15 )Hepatocytes (5x106 Xenograft studies were performed on both flanks of nude mice using 400 cells.

[0076] Figure 5 (C) contains xenograft growth curves reporting the mean tumor volume for each group measured weekly.

[0077] exist Figure 5 (D) shows the quantitative analysis of tumor volumes dissected 11 weeks after cell injection. Each dot corresponds to the volume of each tumor. Note that Met hepa WT In contrast, all mice injected with immortalized-R26 Met hepa overAdamts15 In conclusion, overexpression of ADAMTSL5 levels in immortalized hepatocytes that are not tumorigenic due to their inability to form tumors in xenografts is not sufficient to confer tumorigenic properties to the cells in vivo.

[0078] Example 6: Alb-R26 Met ADAMTSL5 expression levels in tumors at early and late stages.

[0079] Figure 6 (A) is a graphic representation of tissue samples used for RT-qPCR analysis of ADAMTSL5 levels. The samples used were: control wild-type liver (WT), Alb-R26 Met Healthy liver, early and late Alb-R26 Met Tumor.

[0080] exist Figure 6 In (B), the graph reports the expression levels of ADAMTSL5, AFP (alpha-fetoprotein), GPC3 (Glypican-3) (two HCC markers), and Ki67 (proliferation marker) (by RT-qPCR). Note that in wild-type and Alb-R26 Met In healthy livers, ADAMTSL5 and other markers analyzed were expressed at significantly lower levels. In contrast, at early stages, Alb-R26 Met Tumors already have elevated transcript levels. In conclusion, ADAMTSL5 transcript levels can be used to distinguish healthy samples from tumor samples that are already in an early tumorigenic state.

[0081] Example 7: High ADAMTSL5 mRNA levels were present in 52% of HCC patients, with those associated with alcohol consumption accounting for the majority.

[0082] exist Figure 7In (A), the upper figure reports a cohort of HCC patients (371 patients) with the following ADAMTSL5 mRNA levels: 21% of HCC patients were characterized by downregulation of ADAMTSL5 (white, left), 27% were characterized by no change (white with black lines, middle), and 52% had upregulated ADAMTSL5 mRNA levels (black, right). Lower figure: A chart reporting 3 subgroups of HCC patients according to low, unchanged, and high ADAMTSL5 expression levels (indicating numbers and percentages). The data correspond to an RNA-seq study of a cohort of 371 HCC patients (from TCGA). It was noted that ADAMTSL5 was overexpressed in 193 of the 371 HCC patients (52%; Log2FC>1; FDR<0.05). In conclusion, high ADAMTSL5 transcript levels can be detected in more than 50% of HCC patients.

[0083] exist Figure 7 (B), the table reports the presence (black lines) or absence of major HCC risk factors in all 371 analyzed patients. Note that HCC patients with elevated ADAMTSL5 levels are significantly associated with drinking, although not exclusively. In conclusion, high mRNA levels of ADAMTSL5 are present in a large proportion of patients characterized by multiple risk factors, with those associated with drinking accounting for the majority.

[0084] Example 8: The ADAMTSL5 protein level was overexpressed in the vast majority of HCC analyzed patients.

[0085] The figure reports immunostaining with anti-ADAMTSL5 antibody in tumor samples compared to adjacent liver. Note that there are strong ADAMTSL5 protein levels (dark staining; performed with Fast Red) in all analyzed HCC patients (except patient #8). In conclusion, high ADAMTSL5 protein levels characterize the vast majority of HCC patients.

[0086] Example 9: Overexpression of ADAMTSL5 in a panel of human cancer cell lines.

[0087] The graph shows the mRNA expression levels of ADAMTSL5 in a panel of human HCC cell lines, MKN (gastric cancer) cell lines, human breast cell lines, HELA (human cervical) cancer cell lines, and HEK (human embryonic kidney) cell lines. The average values ​​are from 3 independent experiments. In conclusion, high mRNA levels of ADAMTSL5 are present in cancer cells of different origins.

Claims

1. Use of an agent for determining overexpression of the ADAMTSL5 gene in the preparation of a kit for diagnosing hepatocellular carcinoma in a mammal in need thereof, the diagnosis comprising the following steps: collecting a biological sample from the mammal; determining whether the ADAMTSL5 gene is overexpressed from the biological sample; Hepatocellular carcinoma is diagnosed based on the determination of overexpression of the gene.

2. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, biopsy tissue, urine, feces, sputum or cerebrospinal fluid.

3. The use of claim 1, wherein the biological sample is a biopsy tissue, plasma, serum or urine.

4. The use according to claim 1, 2 or 3, wherein the reagent for determining overexpression of the ADAMTSL5 gene is a reagent for determining the ADAMTSL5 protein level or mRNA level.

5. The use according to claim 4, wherein the reagent for determining the level of ADAMTSL5 protein is at least one anti-ADAMTSL5 type antibody.

6. The use according to claim 5, wherein the anti-ADAMTSL5 type antibody is selected from unconjugated or conjugated with alkaline phosphatase horseradish peroxidase or with a fluorescent dye.

7. The use of claim 4, wherein the ADAMTSL5 protein level in the biological sample is measured by using immunostaining, immunofluorescence, Western blotting or ELISA.

8. The use of claim 4, wherein the kit comprises at least one anti-ADAMTSL5 type antibody, a container for containing the biological sample, and instructions for measuring overexpression of the ADAMTSL5 hepatocellular carcinoma marker gene in the biological sample obtained from the mammal.

9. The use according to claim 8, wherein the anti-ADAMTSL5 type antibody is selected from unconjugated or conjugated with alkaline phosphatase, horseradish peroxidase or with a fluorescent dye.

10. The use of claim 4, wherein the ADAMTSL5 mRNA level in the biological sample is measured by a methodology that can be used to detect mRNA in the biological sample, including microarray, RNA-seq, in situ hybridization, RNA-scope, and conventional semi-quantitative or quantitative RT-PCR.

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