Composition for treating or preventing pancreatic cancer, method for treating or preventing pancreatic cancer, and method for detecting pancreatic cancer

Abstract

An abnormal expression of cytoplasmic Cep63 has been found to be enhanced in tumor cells of which the formation has been promoted due to p62 deficiency. Similarly, the expression of cytoplasmic Cep63 has been found to be enhanced in human pancreatic cancer. On the other hand, suppressing the expression of cytoplasmic Cep63 in the tumor cells and the human pancreatic cancer has been found to inhibit the proliferation thereof. Thus, a new target molecule contributing to p62-related tumor formation has been found, and cancer can be treated or prevented by suppressing the function of the molecule. Cancer can also be detected using the expression of the target molecule as an indicator.

Description

Composition for treating or preventing pancreatic cancer, method for treating or preventing pancreatic cancer, and method for detecting pancreatic cancer

[0001] The present invention relates to a composition for treating or preventing pancreatic cancer. The present invention also relates to a method for treating or preventing pancreatic cancer. The present invention also relates to a method for detecting pancreatic cancer.

[0002] Autophagy is a mechanism for the degradation of intracellular components, and is induced in response to various stresses to regulate physiological and pathological conditions. The relationship between autophagy dysfunction and tumorigenesis is being elucidated experimentally and clinically.

[0003] For example, Beclin 1 is a key regulator of autophagy, and its heterozygous knockout mice develop various types of cancer. Furthermore, mice lacking Atg5 or Atg7, which are important for the progression of autophagy, develop liver tumors.

[0004] Clinically, genetic mutations in various autophagy molecules have also been reported to be associated with human cancer. For example, Beclin 1 expression is reduced in some types of ovarian, breast, and prostate cancer due to monoallelic mutations. Atg5, LC3, and Fip200 have also been implicated in myeloma, glioblastoma, and breast cancer, respectively. These findings suggest that impaired autophagy leads to tumor progression, although the underlying mechanism remains unclear.

[0005] Like other autophagy-related molecules, p62 is also closely associated with tumorigenesis (Non-Patent Documents 1 and 2). p62 selectively transports autophagic cargo to the isolation membrane and promotes its degradation (Non-Patent Document 3). Clinically, p62 accumulation has been observed in various types of cancer, and experimental studies have also shown that p62 accumulation acts as an inducer of liver cancer and a driver of pancreatic cancer (Non-Patent Documents 4 and 5). Because autophagy modulation leads to p62 accumulation, tumorigenesis induced by autophagy modulation is thought to be mediated, at least in part, by p62. Naturally, in addition to p62 accumulation, factors such as oxidative stress from undigested abnormal organelles, the generation of necrotic cells, and failure of autophagic cell death are also involved in tumorigenesis induced by autophagy impairment (Non-Patent Documents 6, 7, and 8).

[0006] Furthermore, p62 plays an important role not only in autophagy but also in various stress responses (Non-Patent Documents 1 and 2). It is also believed that p62-induced tumorigenesis is primarily mediated by these non-autophagic mechanisms. For example, p62 directly binds to Keap1, dissociating Nrf2 from the Keap1-Nrf2 complex. The released transcription factor Nrf2 then translocates to the nucleus and transcribes antioxidant molecules, thereby contributing to tumorigenesis (Non-Patent Document 9). p62 also binds to MEKK3, Raptor, TRAF6, RIP1, and other proteins, and is involved in downstream signal transduction. These signals have also been reported to contribute to improved survival of cancer cells (Non-Patent Document 9).

[0007] Thus, it has been suggested that p62 plays an important role in tumorigenesis, although the details of the underlying mechanism remain unclear.

[0008] Taniguchi, K. et al., FEBS Lett., 2016, vol. 590, pp. 2375-2397; Moscat, J. and M. T. Diaz-Meco, Trends Biochem. Sci., 2012, vol. 37, pp. 230-236; Komatsu, M. and Y. Ichimura, FEBS Lett., 2010, vol. 584, pp. 1374-1378; Todoric, J. et al., Cancer Cell, 2017, vol. 32, pp. 824-839; Umemura, A. et al., Cancer Cell, vol. 29, pp. 935-948; Morselli, E. et al., Biochim. Biophys. Acta., 2009, vol. 1793, pp. 1524-1532; Degenhardt, K. et al., Cancer Cell, 2006, vol. 10, pp. 51-64; Shimizu, S. et al., Int. J. Mol. Sci., 2014, vol. 15, pp. 3145-3153; Sanchez-Martin, P. and M. Komatsu, J. Cell. Sci., 2018, 131: jcs222836; Tang, T. K., Nat. Cell Biol. , 2013, Vol. 15, pp. 1400-1402 Watanabe, Y. et al., Nat. Commun. , 2016, 7: 13508.

[0009] The present invention was made in light of these circumstances, and its purpose is to further elucidate the mechanism of tumorigenesis involving p62. A further object of the present invention is to identify new target molecules that contribute to tumorigenesis and to enable the treatment or prevention of cancer by inhibiting the function of these molecules. Another object of the present invention is to provide a method for detecting cancer using the expression of the target molecules as an indicator.

[0010] As mentioned above, p62 has been reported to function as a receptor for selective autophagy and enhance tumor formation. Therefore, the inventors first crossed p53-deficient (p53 knockout (KO)) mice, which are prone to tumor formation, with p62-deficient (p62KO) mice. Contrary to expectations, the results revealed that p62 deficiency promoted tumor formation in p53KO mice.

[0011] Although various cargoes have been reported for p62-bound autophagy, the present inventors focused on cytoplasmic Cep63 to elucidate the mechanism of this promotion. Cep63 is a centrosome-localized protein known to be important for centrosome duplication (Non-Patent Document 10). However, the present inventors previously found that Cep63 is also present in the cytoplasm and is degraded by p62-mediated autophagy in normal cells (Non-Patent Document 11). They also demonstrated that cytoplasmic Cep63 accumulates in cells in which autophagy does not occur or in cells lacking p62, leading to ectopically synthesized centrosomes.

[0012] Therefore, the present inventors focused on cytoplasmic Cep63 and conducted further research, and as a result, they found that tumor cells from the p62 / p53 double knockout (DKO) mice exhibited abnormal cytoplasmic expression of Cep63, which is necessary for centrosome formation, and furthermore, possessed multicentrosomes.

[0013] Furthermore, abnormal cytoplasmic Cep63 and multicentrosomes were observed in p62 / p53 double knockout cells in which tumor formation was induced by introducing mutant c-myc / Hras. Furthermore, when these cells were subjected to xenotransplantation experiments, large tumor formation was observed. Meanwhile, it was revealed that silencing cytoplasmic Cep63 significantly suppressed the growth of these tumors.

[0014] Interestingly, we found that cytoplasmic Cep63 levels were significantly increased in human pancreatic cancer, and furthermore, we demonstrated that silencing of cytoplasmic Cep63 suppressed the growth of xenografts derived from these pancreatic cancer cells.

[0015] The present invention is based on these findings, and more specifically provides the following.

[0016] <1> A composition for treating or preventing pancreatic cancer, comprising, as an active ingredient, a substance that suppresses the function of cytoplasmic Cep63.

[0017] <2> The composition according to <1>, wherein the substance that suppresses the function of cytoplasmic Cep63 is a nucleotide that binds to a transcription product of the Cep63 gene.

[0018] <3> The composition according to <2>, wherein the nucleotide comprises a nucleotide encoded by a DNA set forth in any one of SEQ ID NOs: 1 to 4, 26 to 31, and 35 to 37.

[0019] <4> A method for detecting pancreatic cancer, comprising the following steps (a) to (c): (a) detecting the amount of cytoplasmic Cep63 protein in a pancreas-derived sample isolated from a subject; (b) comparing the amount of protein detected in step (a) with a reference amount; and (c) determining that the subject has or is at risk of having pancreatic cancer, if the comparison in step (b) shows that the amount of protein in the subject is higher than the reference amount.

[0020] <5> The composition according to any one of <1> to <3>, characterized in that it is administered to a subject who has been determined to have or be at risk of having pancreatic cancer by the method according to <4>.

[0021] The present invention also relates to use of a substance that inhibits the function of cytoplasmic Cep63, a nucleotide that binds to a transcription product of the Cep63 gene, or a nucleotide containing a nucleotide encoded by DNA set forth in any of SEQ ID NOs: 1 to 4, 26 to 31, and 35 to 37, for the manufacture of a composition for treating or preventing pancreatic cancer.

[0022] The present invention also relates to a method for treating or preventing pancreatic cancer in a subject, which comprises administering to the subject a substance that suppresses the function of cytoplasmic Cep63, a nucleotide that binds to a transcription product of the Cep63 gene, or a nucleotide containing a nucleotide encoded by DNA set forth in any of SEQ ID NOs: 1 to 4, 26 to 31, and 35 to 37.

[0023] The present invention also relates to a method for treating or preventing pancreatic cancer in a subject determined to have or be at risk of having pancreatic cancer by the method described in <4>, which comprises administering to the subject a substance that suppresses the function of cytoplasmic Cep63, a nucleotide that binds to a transcription product of the Cep63 gene, or a nucleotide containing a nucleotide encoded by DNA set forth in any of SEQ ID NOs: 1 to 4, 26 to 31, and 35 to 37.

[0024] According to the present invention, by targeting cytoplasmic Cep63 and inhibiting its function, it is possible to treat or prevent pancreatic cancer. Furthermore, it is also possible to detect pancreatic cancer using the amount of cytoplasmic Cep63 protein as an indicator.

[0025] This graph shows the tumor incidence rate in knockout (KO) mice at each age. In the figure, "3 mo." and "4 mo." represent the results of analyzing knockout mice at 3 and 4 months of age, respectively. The numbers at the top indicate the number of mice with tumors / total number of mice. These are micrographs showing the results of immunohistological analysis of Ki67 expression in thymocytes isolated from p62 / p53 double knockout (DKO) mice (3 and 4 months old). The top five panels show images stained with an anti-Ki67 antibody (green, under color display). The bottom five panels show the results of nuclear staining using DAPI (blue, under color display). Arrows indicate Ki67-positive cells. The scale bar indicates 5 μm. This graph shows the percentage of Ki67-positive cells in thymocytes (100 cells) from each knockout mouse. In the figure, the middle line (red, under color display) in each data point represents the mean value. The two lines above and below each data point (blue lines under the color display) represent the standard deviation (SD). The number of mice analyzed was four each for wild-type (WT), p53KO, and p62KO mice; seven for tumor-free p62 / p53DKO mice; and six for tumor-bearing p62 / p53DKO mice. This figure shows a representative histogram of the DNA content of thymocytes isolated from each knockout mouse, analyzed by FACS. This figure shows a histogram of the DNA content of thymocytes isolated from each mouse at 3 or 4 months of age, analyzed by FACS. Fixed thymocytes were stained with PI and analyzed by FACS. DNA abnormalities were determined based on the following criteria: (i) detection of an abnormal peak; (ii) detection of multiple peaks; and (iii) detection of a broad peak with less than 90% of cells in the G0 / G1 region. This is a graph showing the percentage of thymocytes in each knockout mouse in which DNA abnormalities were found. DNA abnormalities were determined based on the criteria shown in Figure 1F. The numbers at the top indicate the number of mice in which thymic DNA abnormalities were found / total number of mice. This is a photomicrograph showing representative results of immunofluorescent staining analysis of thymocytes isolated from 3- or 4-month-old mice.Nuclei were stained with DAPI (shown in blue under color coding). Scale bars represent 2 μm. Activation of Nrf2 and NFκB can be assessed by nuclear localization, but nuclear localization was not observed in thymocytes of any genotype. Photographs show the results of Western blotting analysis of the expression of each protein in thymocytes isolated from 3- or 4-month-old mice. Arrows indicate p-S6K (phosphorylated S6K). Activation of mTor and Nrf2 can be assessed by the expression levels of phosphorylated S6K and NQO1, respectively, but no differences were observed between genotypes. The same was true for the expression of A20, a regulator of NFκB. These micrographs show the results of immunofluorescent analysis of cytoplasmic Cep63 and centrosomes in thymocytes isolated from 3- and 4-month-old mice. In the color representation, areas stained with anti-Cep63 antibody are shown in red, and areas stained with anti-γ-tubulin antibody are shown in green. Nuclei stained with DAPI are shown in blue. The scale bar represents 1 μm. In thymocytes derived from p53KO mice, only mature centrosomes (co-localized Cep63 and γ-tubulin, indicated by arrows) are observed, whereas in thymocytes derived from p62 / p53DKO mice, cytoplasmic Cep63 (single-stained Cep63, indicated by arrowheads) is also present. This graph shows the results of measuring the number of cytoplasmic Cep63 per thymocyte (n>30). In the color representation, the red line indicates the average value. This is a micrograph showing the results of immunofluorescent analysis of centrosomes in thymocytes. Centrioles were stained using anti-γ-tubulin antibody (indicated in green in the color representation). Nuclei were stained using DAPI (shown in blue under color display). Arrows indicate multicentrosomal cells. The scale bar represents 5 μm. This graph shows the results of measuring multicentrosomal thymocytes (n>100). The middle line (red line under color display) for each data point represents the mean. The two lines above and below (blue lines under color display) for each data point represent SD. The number of mice analyzed was four each for wild-type (WT), p53KO, and p62KO mice, seven for tumor-free p62 / p53DKO mice, and six for tumor-bearing p62 / p53DKO mice.These are micrographs showing representative results of immunofluorescent staining of MEFs derived from each knockout mouse using anti-Cep63 antibody or anti-γ-tubulin antibody. In the color representation, areas stained with anti-Cep63 antibody are shown in red, and areas stained with anti-γ-tubulin antibody are shown in green. The scale bar represents 10 μm. A magnified image of the boxed area is shown below. The arrows and arrowheads indicate mature centrosomes and cytoplasmic Cep63, respectively. This is a graph showing the number of cytoplasmic Cep63 per cell in MEFs derived from each knockout mouse. n=30. In the color representation, the red line indicates the average value. The numbers at the top indicate the number of cells in which cytoplasmic Cep63 was detected / total number of cells. These are micrographs showing representative results of staining the centrosomes of MEFs derived from each knockout mouse with anti-γ-tubulin antibody. The arrows in the figure indicate centrosomes. The scale bar represents 10 μm. 3E-3G are graphs showing the percentage of multicentrosomal cells in MEFs derived from each knockout mouse. Each MEF was stimulated for 24 hours in the absence or presence of 100 nM Baf A1 and stained with γ-tubulin antibody. The percentage of multicentrosomal cells is shown as the mean + SD (n=3). These are micrographs showing the results of immunofluorescent staining of MEFs derived from p53KO mice. These are micrographs showing the results of immunofluorescent staining of MEFs derived from p62 / p53DKO mice. These are micrographs showing the results of immunofluorescent staining of MEFs derived from Atg5 / p53KO mice. In Figures 3E-3G, each MEF was stimulated with 10 μg / mL E64d and stained with anti-Cep63 antibody, anti-γ-tubulin antibody, or anti-Lamp2 antibody. Under color coding, areas stained with anti-Cep63 antibody are shown in red, areas stained with anti-γ-tubulin antibody are shown in green, and areas stained with anti-Lamp2 antibody are shown in gray. The scale bar represents 10 μm. A magnified image of the boxed area is shown on the right. The arrow indicates a mature centrosome. Cytoplasmic Cep63 is surrounded by Lamp2 in MEFs derived from p53KO mice (indicated by green arrowheads under color coding). In contrast, it is not surrounded in MEFs derived from p62 / p53DKO mice (indicated by white arrowheads).

[0023] Figure 1 shows a photograph depicting the results of Western blotting analysis of Ras G12V expression in tumorigenic p62 / p53DKO cells transfected with sh-Cep63, and a graph depicting the results of real-time PCR analysis of Cep63 expression. As a negative control for sh-Cep63, cells transfected with an empty vector (shRNA expression retroviral vector (pLMP vector)) were also analyzed (the results are shown as "Vector" in the figure). In Western blotting, α-tubulin was used as a loading control. In real-time PCR analysis, the amounts of Cep63 and gapdh mRNA were analyzed, and their ratios were measured. Figure 1 shows a micrograph depicting the results of immunofluorescence staining analysis of tumorigenic MEF cells (tumorigenic p62 / p53DKO cells) derived from p62 / p53DKO mice transfected with sh-Cep63. In the color representation, areas stained with anti-Cep63 antibody are shown in red, and areas stained with anti-γ-tubulin antibody are shown in green. Nuclei stained with DAPI are shown in blue. The dotted line indicates cell shape. The scale bar represents 2 μm. In tumorigenic p62 / p53 DKO cells, both mature centrosomes (co-localized Cep63 and γ-tubulin, indicated by arrows) and cytoplasmic Cep63 (single Cep63 staining, indicated by arrowheads) are observed, but the latter is reduced by introduction of sh-Cep63#3. On the other hand, no reduction in cytoplasmic Cep63 was observed even when an empty vector (pLMP vector) was introduced as a negative control for sh-Cep63. This graph shows the results of measuring the number of cytoplasmic Cep63 per cell in tumorigenic p62 / p53 DKO cells (n=30). As a negative control for sh-Cep63, cells into which an empty vector (pLMP vector) was introduced were also analyzed (the results are shown as "Vector" in the figure). The middle line (red line under color display) in each data point represents the average value. The two lines above and below each data point (blue lines under color display) represent the SD. The numbers at the top indicate the number of cells in which cytoplasmic Cep63 was observed / total number of cells. "#1", "#2", and "#3" indicate the analysis results for cells into which sh-Cep63#1, #2, and #3 were introduced, respectively.This is a graph showing the results of measuring the percentage of tumorigenic p62 / p53 DKO cells in which multicentrosomes were observed (n=100). As a negative control for sh-Cep63, cells into which an empty vector (pLMP vector) was introduced were also analyzed (the results are shown as "Vector" in the figure). Data are shown as the mean value + SD (n=3). This is a graph showing the proliferation rate of tumorigenic p62 / p53 DKO cells into which sh-Cep63 was introduced. The tumorigenic cells were plated at 1x10. ５The cells were seeded in 1000 xenografts, and 24 and 48 hours later, the cells were detached with trypsin and counted. The proliferation rate was calculated by dividing the cell number by the initial cell number. This graph shows the incidence of xenograft tumors and tumor weight. Tumor-forming p62 / p53DKO cells were injected subcutaneously into syngeneic mice, and tumors were removed 3 weeks later. Cells transfected with an empty vector (pLMP vector) were also analyzed as a negative control for sh-Cep63 (the results are indicated by "Vector" in the figure). The numbers at the top indicate the "number of tumors observed / total number of transplanted mice." The graph shows tumor weight. The middle line (red line under color display) in each data point represents the mean. The two lines above and below (blue lines under color display) in each data point represent SD. "#1," "#2," and "#3" indicate the analysis results for cells transfected with sh-Cep63#1, #2, and #3, respectively.

[0033] Figure 1 is a photograph showing a representative example of the external appearance of a xenograft tumor obtained by transplanting tumorigenic p62 / p53DKO cells. Cells transfected with an empty vector (pLMP vector) were also analyzed as a negative control for sh-Cep63 (the results are indicated by "Vector" in the figure). The scale bar represents 10 mm. This is a micrograph showing the results of immunofluorescence staining analysis of tumorigenic MEF cells (tumorigenic Atg5 / p53DKO cells) derived from Atg5 / p53DKO mice transfected with sh-Cep63. Cells transfected with an empty vector (pLMP vector) were also analyzed as a negative control for sh-Cep63 (the results are indicated by "Vector" in the figure). In the color representation, areas stained with anti-Cep63 antibody are shown in red, and areas stained with anti-γ-tubulin antibody are shown in green. Nuclei stained with DAPI are shown in blue. The scale bar represents 2 μm. In tumorigenic Atg5 / p53DKO cells, both mature centrosomes (indicated by arrows) and cytoplasmic Cep63 (indicated by arrowheads) are observed, but the latter is reduced by introduction of sh-Cep63#3. On the other hand, no reduction in cytoplasmic Cep63 was observed when an empty vector was introduced. This graph shows the results of measuring the number of cytoplasmic Cep63 per cell in tumorigenic Atg5 / p53DKO cells (n=30).As a negative control for sh-Cep63, cells transfected with an empty vector (pLMP vector) were also analyzed (the results are shown under "Vector" in the figure). The middle line (red line under color display) in each data point represents the mean. The two lines (blue lines under color display) above and below each data point represent SD. The numbers at the top indicate "number of cells in which cytoplasmic Cep63 was observed / total number of cells." This graph shows the results of measuring the percentage of cells in which multicentrosomes were observed in tumorigenic Atg5 / p53 DKO cells (n=100). As a negative control for sh-Cep63, cells transfected with an empty vector (pLMP vector) were also analyzed (the results are shown under "Vector" in the figure). Data are shown as mean + SD (n=3). This graph shows the incidence of xenograft tumors and tumor weight. Tumorigenic Atg5 / p53DKO cells were injected subcutaneously into syngeneic mice, and tumors were removed 3 weeks later. Cells transfected with an empty vector (pLMP vector) were also analyzed as a negative control for sh-Cep63 (the results are indicated by "Vector" in the figure). The numbers at the top indicate the "number of tumors observed / total number of transplanted mice." The graph shows tumor weight. The middle line (red line under color display) in each data point represents the mean. The two lines above and below (blue lines under color display) in each data point represent SD. These photographs show representative examples of the external appearance of xenograft tumors obtained by transplanting tumorigenic Atg5 / p53DKO cells. Cells transfected with an empty vector (pLMP vector) were also analyzed as a negative control for sh-Cep63 (the results are indicated by "Vector" in the figure). The scale bar represents 10 mm. Figure 1 shows a photograph depicting the results of Western blotting analysis of Ras G12V expression in sh-Cep63-introduced tumorigenic Atg / p53DKO cells, and a graph depicting the results of real-time PCR analysis of Cep63 expression. Cells transfected with an empty vector (pLMP vector) were also analyzed as a negative control for sh-Cep63 (the results are indicated by "Vector" in the figure). In Western blotting, α-tubulin was used as a loading control.In real-time PCR analysis, the amounts of Cep63 and gapdh mRNA were analyzed and their ratios were measured. These are micrographs showing representative results of immunofluorescent staining of various human cancer cells using an anti-γ-tubulin antibody. Under color coding, areas stained with the anti-γ-tubulin antibody are shown in green. The scale bar represents 10 μm. Arrows indicate multicentrosomal cells. Enlarged images are shown below each image. These are graphs showing the percentage of multicentrosomal cells in various human cancer cells. n>100, evaluated from three or more independent experiments. Under color coding, the red line represents the average value. These are micrographs showing representative results of immunofluorescent staining of various human cancer cells using an anti-Cep63 antibody or an anti-γ-tubulin antibody. Under color coding, areas stained with the anti-Cep63 antibody are shown in red. Areas stained with the anti-γ-tubulin antibody are shown in green. The scale bar represents 5 μm. Both mature centrosomes (colocalized Cep63 and γ-tubulin, indicated by arrows) and cytoplasmic Cep63 (single Cep63 staining, arrowheads) are present. Figure 4 shows a graph showing the number of cytoplasmic Cep63 cells per cell in various human cancer cells. n = 30. Under color coding, the red line indicates the mean. High levels of cytoplasmic Cep63 were observed in pancreatic cancer cells. The percentage of multicentrosomal cells shown in Figure 4B correlates well with the number of cytoplasmic Cep63 cells per cell. Figure 4 shows representative micrographs of human clinical pancreatic cancer sections analyzed by immunofluorescence staining using anti-Cep63 or anti-γ-tubulin antibodies. Under color coding, areas stained with anti-Cep63 antibodies are shown in red. Areas stained with anti-γ-tubulin antibodies are shown in green. The scale bar represents 5 μm. Tumor cells contain both mature centrosomes (colocalization of Cep63 and γ-tubulin, indicated by arrows) and cytoplasmic Cep63 (single staining for Cep63, indicated by arrowheads), whereas the latter is rarely observed in non-cancerous areas. This graph shows the number of cytoplasmic Cep63 cells per cell in clinical cases of human pancreatic cancer. Non-cancerous areas (2 patients; 29 cells), grade 2 cancer (8 patients; 31 cells), and grade 3 cancer (6 patients; 25 cells) were analyzed. The middle line (red line under color display) in each data point represents the mean value.The two lines above and below each data point (blue lines under the color display) represent standard deviation. The number at the top indicates the number of cells with cytoplasmic Cep63 divided by the total number of cells. The characteristics of each grade are as follows: Grade 1 (low grade): Cancer cells closely resemble normal cells. They tend to grow slowly and are unlikely to spread. Grade 2 (intermediate grade): Cancer cells appear more abnormal and tend to grow slightly faster. Grade 3 (high grade): Cancer cells appear very abnormal and are likely to grow or spread rapidly. This photograph shows the results of Western blotting analysis of LC3 and p62 expression in human cancer cells. Human cancer cells (human pancreatic cancer-derived cell line: Panc1, human breast cancer-derived cell line: MCF7) were cultured and stimulated in medium containing 10 μM etoposide or medium containing 10 μM etoposide and 100 nM Baf A1. "M.W." indicates molecular weight. These are micrographs showing the results of immunofluorescence staining of Panc1 cells using an anti-LC3 antibody. Panc1 cells were cultured in a medium containing 10 μM etoposide or a medium containing 10 μM etoposide and 100 nM Baf A1, stimulated for 24 hours, and then stained with an anti-LC3 antibody. The area stained with the anti-LC3 antibody is shown in green under color coding. The scale bar represents 10 μm. These are micrographs showing representative results of analysis of the binding of endogenous p62 and Cep63 in human cancer cells using the Duolink® proximity ligation assay (PLA). Human cancer cells (Panc1 cells, MCF7 cells) were either untreated (NT), stimulated for 24 hours with E64d (10 μg / mL) and pepstatin (10 μg / mL), or stimulated with Baf A1 (100 nM). Next, the binding between p62 and Cep63 was visualized using the Duolink kit and anti-Cep63 and anti-p62 antibodies. Nuclei were stained with DAPI. The scale bar represents 10 μm. This graph shows the results of analyzing the binding between endogenous p62 and Cep63 in human cancer cells using the PLA method. n=500. The vertical axis of the graph represents the signal intensity per cell. In the color display, the red line for each data point represents the average value. The blue line represents the SD.These are micrographs showing the results of immunofluorescent staining of MCF7 cells or Panc1 cells stimulated with Baf A1 using anti-Cep63 and anti-p62 antibodies. Each of the three panels on the right shows a magnified image. The scale bar represents 2 μm. In MCF7 cells, dots where Cep63 and p62 co-localized (indicated by blue arrowheads in color display) were observed, whereas in Panc1 cells, only dots of Cep63 alone (indicated by white arrowheads) were observed. These are micrographs showing representative results of exogenously expressing GFP-Cep63 and Flag-p62 in human cancer cells, and analyzing the binding between these proteins using the PLA method. Human cancer cells (Panc1 cells, MCF7 cells) were stimulated for 24 hours under conditions of no treatment (NT), E64d (10 μg / mL), pepstatin (10 μg / mL), or Baf A1 (100 nM). The binding of GFP-Cep63 to Flag-p62 was detected using anti-GFP and anti-Flag antibodies. Nuclei were stained with DAPI. The scale bar represents 50 μm. This graph shows the results of PLA analysis of the binding of exogenous p62 to Cep63 in human cancer cells. n=500. The vertical axis of the graph represents the signal intensity per cell. In the color representation, the red line for each data point represents the mean value. The blue line represents the SD. These are micrographs showing the results of staining and analysis of GFP-p62-expressing MEFs with anti-Cep63 antibody, anti-GFP antibody, or anti-γ-tubulin antibody. Most cytoplasmic Cep63 (Cep63 single dot; indicated by blue arrowhead) colocalized with p62. These are micrographs showing the results of staining and analysis of GFP-Cep63 and Flag-p62-expressing Panc1 cells with anti-FLAG antibody, anti-GFP antibody, or anti-γ-tubulin antibody. Panc1 cells expressing GFP-Cep63 and Flag-p62 were stimulated with or without 100 nM Baf A1 for 4 hours. Next, the cells were stained with anti-FLAG antibody, anti-GFP antibody, or anti-γ-tubulin antibody. Cytoplasmic Cep63 (indicated by the white arrowhead) did not co-localize with p62, regardless of the presence or absence of Baf A1. Figure 1 shows a schematic diagram showing the structure of the p62 protein and proteins that interact with each domain.Figure 1 shows representative micrographs of the binding between p62 and RIP1 in human cancer cells (Panc1 cells, MCF7 cells) analyzed by PLA using the Duolink kit, anti-RIP1 antibody, and anti-p62 antibody. Nuclei were stained with DAPI. The scale bar represents 20 μm. Figure 2 shows graphs of the binding between p62 and RIP1 in human cancer cells (Panc1 cells, MCF7 cells) analyzed by PLA. n>100. The vertical axis of the graph represents signal intensity per cell. In the color representation, the red line for each data point represents the average value. The blue line represents SD. Figure 3 shows photographs of the knockdown of RIP1 in Panc1 cells confirmed by Western blotting. 1 is a graph showing the results of analyzing the binding of p62 to RIP1 in Panc1 cells transfected with siRNA targeting RIP1 (siRIP1) by PLA using a Duolink kit, anti-RIP1 antibody, and anti-p62 antibody. n>70. The vertical axis of the graph represents signal intensity per cell. Under color display, the red line for each data point represents the mean value. The blue line represents SD. 1 is a graph showing the results of analyzing the binding of p62 to Cep63 in Panc1 cells transfected with siRIP1 by PLA using a Duolink kit, anti-Cep63 antibody, and anti-p62 antibody. n>70. The vertical axis of the graph represents signal intensity per cell. Under color display, the red line for each data point represents the mean value. The blue line represents SD. Panc1 cells transfected with siRIP1 or control siRNA (siControl) were stained with anti-Cep63 or anti-γ-tubulin antibodies and observed under a fluorescence microscope. The scale bar represents 1 μm. Cytoplasmic Cep63 (single Cep63 staining, indicated by arrowheads) and mature centrosomes (colocalized Cep63 and γ-tubulin, indicated by arrows) were reduced by RIP1 knockdown. This graph shows the number of cytoplasmic Cep63 cells per cell for Panc1 cells transfected with siRIP1 or siControl. n = 90. The numbers at the top indicate the number of cells with cytoplasmic Cep63 detected divided by the total number of cells. Under color display, the red line for each data point represents the mean. The blue line represents SD.1 is a graph showing the percentage of multicentrosomal cells in Panc1 cells transfected with siRIP1 or siControl. n=100. This graph shows the results of analyzing the binding of p62 and Cep63 in MCF7 cells and Hs766T cells by PLA using the Duolink kit, anti-Cep63 antibody, and anti-p62 antibody. n>20. The vertical axis of the graph represents signal intensity per cell. In the color representation, the red line for each data point represents the mean value. The blue line represents SD. This graph shows the results of analyzing the binding of p62 and RIP1 in MCF7 cells and Hs766T cells by PLA using the Duolink kit, anti-RIP1 antibody, and anti-p62 antibody. n>20. The vertical axis of the graph represents signal intensity per cell. In the color representation, the red line for each data point represents the mean value. The blue line represents SD. 6A-6F are graphs showing the results of measuring the amount of Cep63 mRNA in Panc1 cells transfected with sh-Cep63 or an empty vector. The amounts of Cep63 and gapdh mRNA in sh-Cep63-transfected Panc1 cells were analyzed by real-time PCR, and their ratios were measured. Data are shown as mean + SD (n=3). Note that in Figures 6A-6F, the shRNA transfected into Panc1 cells was sh-Cep63#4. Furthermore, "Vector" in these figures indicates the results for cells transfected with an empty vector (negative control). These are photographs showing representative results of Panc1 cells transfected with sh-Cep63 or an empty vector stained with anti-Cep63 antibody or γ-tubulin antibody and observed under a fluorescence microscope. In the color representation, areas stained with anti-Cep63 antibody are shown in red. Areas stained with anti-γ-tubulin antibody are shown in green. The scale bar represents 2 μm. Panc1 cells contain both mature centrosomes (colocalized Cep63 and γ-tubulin, indicated by arrows) and cytoplasmic Cep63 (single-stained Cep63 dots, indicated by arrowheads), but the latter is reduced by sh-Cep63 transfection. This graph shows the number of cytoplasmic Cep63 per cell in Panc1 cells transfected with sh-Cep63 or an empty vector. n=30. Under the color representation, the red line indicates the average value. The numbers at the top indicate the number of cells in which cytoplasmic Cep63 was observed / total number of cells.1 is a graph showing the percentage of multicentric cells in Panc1 cells transfected with sh-Cep63 or an empty vector. n=100. Data are presented as the mean + SD (n=3). Photographs are showing representative examples of the external appearance of xenograft tumors obtained by transplanting Panc1 cells transfected with sh-Cep63 or an empty vector. sh-Cep63-introduced Panc1 cells were injected subcutaneously into nude mice, and tumors were removed 12 weeks later. The scale bar represents 10 mm. This is a graph showing the weight of xenograft tumors obtained by transplanting Panc1 cells transfected with sh-Cep63 or an empty vector. Data are presented as the mean + SD (n=5).

[0026] As shown in the Examples below, it has been revealed that abnormal cytoplasmic Cep63 expression is enhanced in tumor cells whose formation is promoted by p62 deficiency. Similarly, enhanced cytoplasmic Cep63 expression was also observed in human pancreatic cancer. On the other hand, it has also been revealed that suppressing the expression of cytoplasmic Cep63 in the tumor cells and human pancreatic cancer suppresses the growth of these tumors.

[0027] Therefore, the present invention relates to the use of a substance that inhibits the function of cytoplasmic Cep63 for the treatment or prevention of pancreatic cancer.

[0028] <Substances that inhibit the function of cytoplasmic Cep63> First, "substances that inhibit the function of cytoplasmic Cep63" will be described. In the present invention, "Cep63" is a protein also known as Centrosomal protein of 63 kDa (63 kDa centrosomal protein). If it is derived from a human, it is typically a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22 (the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 21 or 34). If it is derived from a mouse, it is typically a protein consisting of the amino acid sequence set forth in SEQ ID NO: 24 (the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 23). However, the sequence of a DNA (gene) encoding a protein can mutate in nature (i.e., non-artificially) due to mutations or the like. Therefore, in the present invention, such natural mutants can also be targets for functional inhibition.

[0029] As its name suggests, Cep63 is involved in centrosome duplication and is normally localized to the centrosome. However, the subject of the present invention is "cytoplasmic Cep63," which can induce abnormal centrosome synthesis by localizing and accumulating ectopically (in the cytoplasm). Furthermore, "inhibition of the function of cytoplasmic Cep63" includes not only inhibition of the function of the protein, but also inhibition of the expression of the Cep63 gene that contributes to the inhibition (such as transcriptional inhibition, translational inhibition, and degradation of the transcript).

[0030] In the present invention, the "substance that suppresses the function of cytoplasmic Cep63" is not particularly limited as long as it is a substance that is capable of suppressing the function of cytoplasmic Cep63. Examples of the substance include nucleotides that bind to transcription products of the Cep63 gene, genome editing systems that target the Cep63 gene, antibodies against the Cep63 protein, and peptides that have dominant-negative properties against the Cep63 protein.

[0031] Examples of "nucleotides that bind to the transcription product of the Cep63 gene" include nucleotides that have a sequence complementary or substantially complementary to the sequence of the transcription product (e.g., mRNA) of the Cep63 gene, or a part thereof. Here, "complementary" refers to nucleotide pairing selectivity due to hydrogen bonding between these nucleotides, where adenine (A) forms a pair with thymine (T) or uracil (U), and guanine (G) forms a pair with cytosine (C), when two polynucleotides anneal to each other. Furthermore, the term "substantially complementary sequence" refers to a sequence that has a degree of complementarity that allows it to bind to a transcription product of the Cep63 gene and suppress the function of cytoplasmic Cep63 under physiological conditions in pancreatic cancer cells of an individual to whom the compound is to be administered, and specifically refers to a sequence that has at least 80% complementarity over the entire length or a portion of the sequence of a transcription product of the Cep63 gene (for example, 85% or more complementarity, preferably 90% or more complementarity (91%, 92%, 93%, 94%), more preferably 95% or more complementarity (96%, 97%, 98%, 99%), and particularly preferably 100% complementarity).

[0032] More specifically, the "nucleotide binding to a transcription product of the Cep63 gene" of the present invention includes the following aspects: (1) siRNA for a transcription product of the Cep63 gene, (2) miRNA for a transcription product of the Cep63 gene, (3) antisense oligonucleotide for a transcription product of the Cep63 gene, and (4) a nucleotide having ribozyme activity against a transcription product of the Cep63 gene.

[0033] (1) siRNA Against Cep63 Gene Transcription Product In the present invention, "siRNA against Cep63 gene transcription product" is not particularly limited as long as it is a double-stranded RNA molecule (dsRNA) that specifically suppresses the expression of cytoplasmic Cep63. The double-stranded RNA portion in which RNAs in a dsRNA pair with each other is not limited to perfect pairing, and may contain unpaired portions due to mismatches (corresponding nucleotides are not complementary), bulges (there is no corresponding nucleotide in one strand), etc. Furthermore, the double-stranded RNA region in which RNAs pair with each other in a dsRNA may contain both bulges and mismatches.

[0034] The length of the siRNA of the present invention is not particularly limited as long as it can suppress the expression of the Cep63 gene and is non-toxic, but is, for example, 15 to 49 nucleotides, preferably 15 to 35 nucleotides, more preferably 17 to 30 nucleotides, more preferably 18 to 25 nucleotides, and even more preferably 19 to 23 nucleotides.

[0035] The siRNA of the present invention may be shRNA (small hairpin RNA, short hairpin RNA). The shRNA can be designed so that a portion thereof forms a stem-loop structure. For example, the shRNA can be designed to have a spacer sequence (e.g., 5 to 25 nucleotides) between the target sequence and the sequence complementary to the target sequence, with these sequences present in a single RNA strand, and to have a total length of, for example, 45 to 70 nucleotides.

[0036] The siRNA of the present invention may have additional nucleotides at the 5' or 3' end. The length of the additional nucleotides is usually 2 to 4 nucleotides. The additional nucleotides may be DNA or RNA, although using DNA may improve the stability of the nucleic acid. Examples of such additional nucleotide sequences include, but are not limited to, the sequences ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', and uuuuu-3'.

[0037] The siRNA of the present invention may have a protruding sequence (overhang) at the 3' end, specifically, one to which dTdT (dT: deoxythymidine) has been added. Alternatively, the siRNA may be blunt-ended with no additional end. The sense strand and the antisense strand of the siRNA may have different numbers of nucleotides, for example, "asymmetrical interfering RNA (aiRNA)" in which the antisense strand has overhangs at the 3' end and 5' end.

[0038] The target sequence of the siRNA of the present invention is not particularly limited as long as it can specifically suppress the expression of cytoplasmic Cep63, and candidates can be appropriately selected from sequences encoding Cep63 (e.g., the sequence set forth in SEQ ID NO: 21 or 34) in accordance with the rules described in, for example, Sayda M. Elbashir et al., Genes Dev., 2001, vol. 15, pp. 188-200. Furthermore, it is preferable to check the selected target sequence candidates for homology to a consecutive sequence of 16 to 17 nucleotides in mRNAs other than Cep63 using homology search software such as BLAST, thereby confirming the specificity of the selected target sequence.

[0039] As shown in the Examples below, from the viewpoint of being able to specifically suppress the expression of cytoplasmic Cep63, the target sequence of the siRNA of the present invention is preferably a sequence comprising any one of the sequences set forth in SEQ ID NOs: 1 to 4, 26 to 31, and 35 to 37. The target sequences of sh-Cep63#1 to #3 used in the Examples below are derived from mouse. The target sequences of #1 to #3 differ between mouse and human, and the human sequences corresponding to these mouse-derived sequences (SEQ ID NOs: 11 to 13) are the sequences set forth in SEQ ID NOs: 1 to 3, respectively. On the other hand, the target sequence of sh-Cep63#4 used in the Examples below is a human-derived sequence (SEQ ID NO: 4).

[0040] (2) miRNA Targeting a Transcription Product of the Cep63 Gene In the present invention, the "miRNA targeting a transcription product of the Cep63 gene" is not particularly limited as long as it inhibits translation of the gene encoding cytoplasmic Cep63. For example, it may inhibit its translation by pairing with the 3' untranslated region (UTR) of the target. Furthermore, the miRNA of the present invention may take the form of any of primary miRNA, pre-miRNA (precursor miRNA), and mature miRNA, but is preferably a pre-miRNA or mature miRNA, and more preferably a mature miRNA. Furthermore, the length of the miRNA is not particularly limited, but typically, pri-miRNA is several hundred to several thousand nucleotides in length, pre-miRNA is 50 to 80 nucleotides in length, and mature miRNA is 18 to 30 nucleotides in length.

[0041] Those skilled in the art can also select miRNA candidates using target prediction software on websites such as TargetScan (http: / / www.targetscan.org / vert_72 / ) and DIANA-micro-T-CDS (http: / / diana.imis.athena-innovation.gr / DianaTools / index.php?r=microT_CDS / index). Furthermore, miRNA candidates of the present invention can be obtained by searching a database related to miRNAs (TarBase (http: / / carolina.imis.athena-innovation.gr / diana_tools / web / index.php?r=tarbasev8 / index)).

[0042] (3) Antisense Oligonucleotide Against a Transcription Product of the Cep63 Gene In the present invention, an "antisense oligonucleotide against a transcription product of the Cep63 gene" refers to a nucleotide sequence that is complementary or substantially complementary to the sequence of the mRNA of the gene encoding Cep63, or a nucleotide containing a part thereof, and that has the function of inhibiting cytoplasmic Cep63 protein synthesis by binding to the mRNA to form a specific and stable duplex.

[0043] The antisense oligonucleotide of the present invention may be any of DNA, RNA, and DNA / RNA chimeras. When the antisense oligonucleotide is DNA, the RNA-DNA hybrid formed by the target RNA and the antisense oligonucleotide is recognized by endogenous ribonuclease H (RNase H) to cause selective degradation of the target RNA. Therefore, in the case of an antisense oligoDNA directed to degradation by RNase H, the target sequence may be not only a sequence in mRNA but also a sequence of an intron region in the initial translation product of the Cep63 gene. The intron sequence can be determined by comparing the genomic sequence with the cDNA sequence of the Cep63 gene using a homology search program such as BLAST or FASTA.

[0044] The length of the target region of the antisense oligonucleotide of the present invention is not limited as long as hybridization of the antisense oligonucleotide results in inhibition of translation into cytoplasmic Cep63 protein, and the target region may be the entire sequence of the mRNA encoding Cep63 or a portion thereof, but from the viewpoints of ease of synthesis, antigenicity, intracellular internalization, etc., the target region is preferably 10 to 40 nucleotides, and more preferably 15 to 30 nucleotides. More specifically, the 5'-terminal hairpin loop, 5'-terminal untranslated region, translation initiation codon, protein coding region, ORF translation termination codon, 3'-terminal untranslated region, 3'-terminal palindrome region, and 3'-terminal hairpin loop of the Cep63 gene can be selected as preferred target regions for the antisense oligonucleotide of the present invention, but the target region is not limited thereto.

[0045] Furthermore, the antisense oligonucleotide of the present invention may not only hybridize with the mRNA or initial transcription product of the Cep63 gene to suppress translation into protein, but may also bind to these genes, which are double-stranded DNA, to form a triplex and inhibit transcription into RNA (antigene).

[0046] (4) Nucleotides Having Ribozyme Activity Against Transcription Products of the Cep63 Gene In the present invention, "nucleotides having ribozyme activity against transcription products of the Cep63 gene" encompass not only RNAs having enzymatic activity for cleaving nucleotides, which are ribozymes in the narrow sense, but also DNAs as long as they have sequence-specific nucleic acid cleavage activity. The most versatile ribozymes are self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known. Hammerhead types exert their enzymatic activity with approximately 40 nucleotides, and by making several nucleotides (totaling approximately 10 nucleotides) on both ends adjacent to the hammerhead structure complementary to the desired cleavage site in the mRNA, they can specifically cleave only the target mRNA.

[0047] Furthermore, when the mRNA of the Cep63 gene itself has a double-stranded structure, the target sequence can be made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase (see, e.g., Warashina M. et al., PNAS, 2001, Vol. 98, No. 10, pp. 5572-5577). Furthermore, when a ribozyme is used in the form of an expression vector containing DNA encoding it, a hybrid ribozyme can also be formed by further linking a sequence of modified tRNA to promote translocation of the transcript into the cytoplasm (see, e.g., Kuwabara T. et al., Nucleic Acids Res., 2001, Vol. 29, No. 13, pp. 2780-2788).

[0048] Specific embodiments of the "nucleotides that bind to the transcription product of the Cep63 gene" of the present invention have been described above, but these nucleotides can be prepared appropriately by those skilled in the art using known techniques. For example, as described above, they can be prepared by determining the target sequence of the transcription product (e.g., mRNA) of the Cep63 gene and synthesizing a complementary sequence thereto using a commercially available automated DNA / RNA synthesizer.

[0049] siRNA can be prepared by synthesizing the sense and antisense strands of the target sequence on mRNA using an automated DNA / RNA synthesizer, denaturing them, and then annealing them. Alternatively, shRNA can be synthesized and then cleaved using the RNA-cleaving protein dicer.

[0050] Furthermore, the "nucleotides binding to the transcription product of the Cep63 gene" of the present invention may be not only naturally occurring RNA or DNA, but may also contain various chemical modifications to improve stability and specific activity (affinity with RNA). For example, in order to prevent degradation by hydrolases such as nucleases, the phosphate residues of each constituent nucleotide can be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, phosphorodithioate, etc. Furthermore, the hydroxyl group at the 2'-position of the sugar (ribose) of each nucleotide can be substituted with -OR (R=CH ３(2'-O-Me), CH ２ CH ２ OCH ３ (2'-O-MOE), CH ２ CH ２ NHC (NH) NH ２ , C.H. ２ CONHCH ３ , C.H. ２ CH ２ Furthermore, the nucleotide portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5'-position of the pyrimidine, or by substituting a carbonyl group at the 2'-position with a thiocarbonyl group.

[0051] The sugar moiety of RNA predominantly has two conformations, C2'-endo (S-type) and C3'-endo (N-type), and in single-stranded RNA, these two conformations exist in equilibrium, but when double-stranded RNA is formed, it is fixed to the N-type. Therefore, in order to impart strong binding ability to target RNA, BNA (LNA), an RNA derivative in which the sugar moiety conformation is fixed to the N-type by bridging the 2' oxygen and 4' carbon, is also preferably used. Furthermore, in addition to BNA, other non-natural nucleotides (artificial nucleotides, nucleotide analogs) such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), morpholino nucleic acid, tricyclo-DNA (tcDNA), 2'-O-methylated nucleic acid, 2'-MOE (2'-O-methoxyethyl)-modified nucleic acid, 2'-AP (2'-O-aminopropyl)-modified nucleic acid, 2'-fluorinated nucleic acid, and 2'F-arabinonucleic acid (2'-F-ANA) can also be suitably used.

[0052] Nucleotides containing such various modifications can also be chemically synthesized by known techniques.

[0053] Furthermore, the "nucleotide that binds to the transcription product of the Cep63 gene" of the present invention may also take the form of a nucleotide construct (expression cassette) that can express the nucleotide in vivo. The expression cassette of the present invention is not particularly limited as long as it is a polynucleotide into which the above-mentioned nucleotide is incorporated in an expressible state, and examples thereof include a polynucleotide containing a promoter, a sequence encoding the nucleotide, and, if necessary, a transcription termination signal.

[0054] The promoter is not particularly limited as long as it can induce the expression of the nucleotide, but from the viewpoint of accurately transcribing short RNAs such as siRNA, a Pol III promoter is preferred, and more specific examples include a mouse or human U6-snRNA promoter, a human H1-RNase P RNA promoter, and a human valine-tRNA promoter. Furthermore, a sequence of four or more consecutive Ts is used as a transcription termination signal.

[0055] The expression cassette constructed in this manner may be inserted into a plasmid vector or a viral vector, such as a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, or Sendai virus, or an animal cell expression plasmid.

[0056] <Composition for treating or preventing pancreatic cancer> Next, a composition for treating or preventing pancreatic cancer of the present invention, which contains the above-mentioned substance that inhibits the function of cytoplasmic Cep63 as an active ingredient, will be described.

[0057] In the present invention, "pancreatic cancer," which is the target of treatment, etc., refers to a malignant tumor that occurs in pancreatic tissue. Pancreatic cancer is pathologically classified into, but is not limited to, invasive pancreatic ductal carcinoma, pancreatic endocrine tumor, pancreatic acinar cell carcinoma, etc. depending on the tissue from which it occurs.

[0058] In the present invention, "treatment" includes not only complete recovery from pancreatic cancer, but also alleviation of symptoms related to pancreatic cancer and inhibition of its progression (so-called improvement). Furthermore, "prevention" includes inhibition of the occurrence of pancreatic cancer, delay in the onset of the disease, or inhibition of its recurrence.

[0059] The composition of the present invention may be in the form of a pharmaceutical composition (such as a drug, quasi-drug, or veterinary drug) or a reagent used for research purposes (for example, in vitro or in vivo experiments) and applied to model animals, cultured cells, etc.

[0060] The composition of the present invention can be formulated by known pharmaceutical methods depending on the above-mentioned compound as an active ingredient.For example, it can be used orally or parenterally as capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual tablets, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc.

[0061] In preparing these formulations, the pharmaceutical composition may be appropriately combined with a carrier that is pharmacologically or food- or drink-acceptable, specifically, physiological saline, sterilized water, vegetable oil, solvent, excipient, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, aromatic agent, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, solubilizing agent, or other additives.

[0062] Furthermore, when the substance of the present invention that inhibits the function of cytoplasmic Cep63 is in the form of a nucleotide, it can be incorporated into a delivery system such as a liposome or microsphere, and other molecules can be attached. Examples of such adducts include polycations such as polylysine, which act to neutralize the charge of the phosphate backbone, and hydrophobic substances such as lipids (phospholipids, cholesterol, etc.) that enhance interaction with cell membranes or increase nucleotide uptake. Examples of lipids that can be attached include cholesterol or its derivatives (cholesteryl chloroformate, cholic acid, etc.). Such substances can be attached to the 3' or 5' end of the nucleotide, or via a nucleotide, sugar, or intramolecular nucleoside bond. Other groups include capping groups specifically placed at the 3' or 5' end of the nucleotide to prevent degradation by nucleases such as exonucleases and RNases. Examples of such capping groups include hydroxyl-protecting groups known in the art (e.g., glycols such as polyethylene glycol and tetraethylene glycol).

[0063] The product (drug, reagent, etc.) of the composition of the present invention or its instruction manual may bear a label indicating that the product is used for the treatment or prevention of pancreatic cancer. Here, "bearing a label on the product or instruction manual" means that the label is affixed to the product itself, container, packaging, etc., or to an instruction manual, package insert, promotional material, other printed matter, etc. disclosing product information.

[0064] The composition of the present invention may also be in the form of a kit. For example, such a kit may include a kit in which the substance that suppresses the function of cytoplasmic Cep63 and the pharmacologically or food / beverage acceptable carrier, etc., typically exist as two or more substances, but can be prepared into a single composition by mixing or the like before being ingested by a subject.

[0065] <Method for treating or preventing pancreatic cancer> The present invention also provides a method for treating or preventing pancreatic cancer in a subject, comprising having the subject ingest a substance that suppresses the function of cytoplasmic Cep63, or a composition containing the substance as an active ingredient.

[0066] The "subject" of treatment, etc., in the present invention is not particularly limited and may be not only humans but also non-human animals. Non-human animals are also not particularly limited, and may include various livestock, poultry, pets, laboratory animals, etc. More specifically, vertebrates, preferably mammals, more preferably primates (humans, monkeys, chimpanzees, orangutans, gorillas, etc.), ungulates (cattle, horses, sheep, goats, etc.), and rodents (mice, rats, guinea pigs, etc.). Furthermore, the "subject" may not only be an individual suffering from pancreatic cancer, but also an individual undergoing or having undergone treatment for pancreatic cancer, an individual at risk of recurrence of pancreatic cancer, or an individual suspected of having pancreatic cancer. Furthermore, subjects determined to have or be at risk of having pancreatic cancer by the pancreatic cancer detection method of the present invention described below may also be suitable subjects.

[0067] The method of administration of the composition of the present invention is not particularly limited, and may be oral or parenteral. Parenteral administration includes, for example, local administration, intravenous administration, intra-arterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, and administration by infusion. Furthermore, when the substance that suppresses the function of cytoplasmic Cep63 of the present invention is in the form of a nucleotide, it can be administered locally using a gene gun, ultrasonic gene transfer, electroporation, a catheter, etc.

[0068] When ingesting the composition of the present invention, the amount of intake can be appropriately selected by a person skilled in the art depending on the subject's age, body weight, disease symptoms, health condition, dosage form of the composition, method of intake, etc. Furthermore, the composition may be administered or ingested once or multiple times (e.g., twice) per day. The administration or ingestion period can be discontinued depending on the degree of recovery from pancreatic cancer, but from the perspective of preventing recurrence, the administration or ingestion may be continued without discontinuation. Note that "continuous" may mean continuous daily administration or continuous administration at intervals.

[0069] Furthermore, the treatment methods of the present invention may be used in combination with other known treatment methods for pancreatic cancer. Examples of such treatment methods include surgical treatment, radiation therapy, and administration of a pancreatic cancer therapeutic agent. "Surgical treatment" is not particularly limited and involves the resection of tissue containing pancreatic cancer tissue. "Radiation therapy" is not particularly limited and includes stereotactic body radiation therapy (SBRT), intensity-modulated radiation therapy (IMRT), and particle beam therapy. Examples of known "pancreatic cancer therapeutic agents" include 5-FU, irinotecan, oxaliplatin, levofolinate, gemcitabine, TS-1, nab-paclitaxel, liposomal irinotecan, cisplatin, olaparib, erlotinib, leucovorin, or combinations thereof. Further examples include immunotherapeutic agents (immune checkpoint inhibitors, effector T cells, NK cell therapy, dendritic cells, DC vaccines, etc.).

[0070] <Method for detecting pancreatic cancer> As shown in the Examples below, increased expression of cytoplasmic Cep63 was observed in pancreatic cancer. Therefore, the present invention provides the following method for detecting pancreatic cancer.

[0071] A method for detecting pancreatic cancer, comprising the following steps (a) to (c): (a) detecting the amount of cytoplasmic Cep63 protein in a pancreas-derived sample isolated from a subject; (b) comparing the amount of protein detected in step (a) with a reference amount; and (c) determining that the subject has or is at risk of having pancreatic cancer, if the comparison in step (b) shows that the amount of protein in the subject is higher than the reference amount.

[0072] In the present invention, the term "subject" refers to an individual to be tested for pancreatic cancer using the method of the present invention, and is not particularly limited and may include not only humans but also non-human animals. Examples of such subjects include vertebrates, preferably mammals, more preferably primates (humans, monkeys, chimpanzees, orangutans, gorillas, etc.), ungulates (cows, horses, sheep, goats, etc.), and rodents (mice, rats, guinea pigs, etc.), but are typically humans. Furthermore, the term "subject" may refer not only to individuals suspected of having pancreatic cancer, but also to individuals currently suffering from pancreatic cancer, individuals undergoing or having undergone treatment for pancreatic cancer, and individuals at risk of recurrence of pancreatic cancer.

[0073] A "pancreas-derived sample isolated from a subject" may be a pancreas-derived sample (cells constituting the pancreas, tissues constituting the pancreas, or those contained in body fluids (blood, lymph, tissue fluid, etc.)) that has been extracted from a subject (e.g., a human body) and is completely isolated from the body of origin, and preferably includes cells constituting the pancreas and tissues constituting the pancreas.

[0074] When the sample according to the present invention is subjected to a method for detecting the amount of a protein described below, it may be further prepared appropriately in a form suitable for the method (e.g., tissue that has been subjected to formalin fixation, alcohol fixation, freezing, or paraffin embedding, or a cytoplasmic fraction isolated from the sample, or a protein solution thereof, etc.) Furthermore, those skilled in the art can select a known method for these preparations, taking into consideration the type and condition of the sample, etc.

[0075] As described above, the "cytoplasmic Cep63" to be detected in the present invention is Cep63 localized in the cytoplasm, and includes natural variants thereof. Furthermore, the cytoplasmic Cep63 to be detected in the present invention includes not only the full-length amino acid sequence but also partial peptides thereof.

[0076] The "amount of protein" detected in the present invention may be not only an absolute amount but also a relative amount. Examples of relative amounts include the ratio to the total protein amount (e.g., the total protein amount of a cell, the total protein amount of a cytoplasmic fraction). Examples of relative amounts include the protein amount ratio (a numerical value expressed in so-called arbitrary units (AU)) based on the measurement method or measurement device used for detection. The relative amount may also be, for example, a value calculated based on the amount of a reference protein. The "reference protein" according to the present invention may be any protein that is stably present in a sample and whose amount varies only slightly between different biological samples, such as an endogenous control (internal standard) protein.

[0077] Those skilled in the art can appropriately employ known techniques to "detect the amount of protein." Examples of such known techniques include immunohistochemical staining (such as immunofluorescence staining), imaging cytometry, enzyme-linked immunosorbent assay (ELISA), CLEIA (chemiluminescent enzyme immunoassay), latex agglutination, antibody array, immunoblotting, immunochromatography, flow cytometry, radioimmunoassay, immunoprecipitation, and other antibody-based detection techniques (immunological methods), as well as mass spectrometry.

[0078] In the "immunological method", an antibody that binds to Cep63 is used, the antibody is brought into contact with cytoplasmic Cep63, and the protein amount of cytoplasmic Cep63 is detected using the binding ability of the antibody to cytoplasmic Cep63 as an indicator.

[0079] "Mass spectrometry" refers to a measurement method using a mass spectrometer in which a peptide sample (the aforementioned biological sample) is ionized using an ion source, and the ionized peptide sample is moved in a vacuum in an analytical section using electromagnetic force or time-of-flight differentials to separate and detect the peptides according to their mass-to-charge ratio. The ionization method using an ion source can be appropriately selected from EI, CI, FD, FAB, MALDI, ESI, and other methods. Furthermore, the separation method for the ionized peptide sample in the analytical section can be appropriately selected from magnetic deflection, quadrupole, ion trap, time-of-flight (TOF), Fourier transform ion cyclotron resonance, and other separation methods. Tandem mass spectrometry (MS / MS), which combines two or more mass spectrometry methods, and triple quadrupole mass spectrometry can also be used. In particular, selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer allows multiple pancreatic cancer markers to be measured simultaneously in a single measurement. Furthermore, a mass spectrometer may be used alone, or in combination with liquid chromatography (LC) or high-performance liquid chromatography (HPLC), to separate and purify peptides constituting a target protein to obtain a sample.

[0080] In the detection method of the present invention, the amount of protein detected in this manner is compared with a reference amount of the same protein. Those skilled in the art can set such a reference amount by appropriately selecting a statistical analysis method suitable for the detection method. Examples of statistical analysis methods include the Mann-Whitney U test, t-test, analysis of variance (ANOVA), Kruskal-Wallis test, Wilcoxon test, odds ratio, hazard ratio, Fisher's exact test, receiver operating characteristic analysis (ROC analysis), and classification and decision tree analysis (CART analysis). Furthermore, normalized or standardized and normalized data can also be used for comparison.

[0081] There are no particular limitations on the "reference amount" to be used for comparison, and a person skilled in the art can use it as a standard in accordance with the above-mentioned detection method and statistical analysis method to distinguish between pancreatic cancer and a non-cancerous pancreas (e.g., a healthy pancreas), or to determine the stage of pancreatic cancer (Grades 1 to 3 (see the brief explanation for [Figure 4F] above; early, intermediate, late, etc.)). This can be set as a so-called cutoff value.

[0082] In clinical practice, classification by cancer stage is widely used to indicate the degree of progression of pancreatic cancer. There are two classification methods for staging pancreatic cancer: the classification based on the Japan Pancreas Society's Pancreatic Cancer Guidelines and the international UICC (Union for International Cancer Control) classification. In both classifications, pancreatic cancer is divided into four stages, I to IV, and the stage is determined based on factors such as tumor size, the presence or absence of infiltration into adjacent organs (e.g., the duodenum, bile duct, portal vein system, celiac artery, superior mesenteric artery, etc.), the presence or absence of lymph node metastasis, and the presence or absence of distant metastasis. The higher the stage number, the more advanced the pancreatic cancer. In the present invention, "early stage" refers to pancreatic cancer up to stage I, "intermediate stage" refers to pancreatic cancer at stages II and III, and "late stage" refers to pancreatic cancer at stage IV.

[0083] Examples of the reference amount for distinguishing between pancreatic cancer and non-cancerous pancreas include the median or mean protein amount of cytoplasmic Cep63 detected in individuals not affected by pancreatic cancer (e.g., healthy individuals). Alternatively, the reference amount may be a value determined by comparing the protein amounts of cytoplasmic Cep63 in individuals not affected by pancreatic cancer and individuals affected by pancreatic cancer (e.g., a value between the protein amount in individuals not affected by pancreatic cancer and the protein amount in individuals affected by pancreatic cancer).

[0084] The reference amount for determining the stage of pancreatic cancer may be, for example, a value determined by comparing the protein amounts of cytoplasmic Cep63 in a group of individuals suffering from early-stage pancreatic cancer, a group of individuals suffering from intermediate-stage pancreatic cancer, and a group of individuals suffering from late-stage pancreatic cancer. Furthermore, the reference amount for determining the stage (intermediate to late stage) of pancreatic cancer may be, for example, the median or average protein amounts of cytoplasmic Cep63 detected in a group of individuals suffering from stage I pancreatic cancer.

[0085] The term "higher than the reference amount" can be appropriately determined by those skilled in the art based on the above-mentioned statistical analysis method. For example, the detected amount of protein is higher than the corresponding reference amount, preferably the difference is recognized as statistically significant (e.g., P<0.05). Another example is that the detected amount of protein is at least twice (preferably at least five times, at least ten times) the corresponding reference amount.

[0086] Furthermore, the accuracy of the pancreatic cancer detection method of the present invention can be improved by combining it with a known pancreatic cancer detection method. Such "known pancreatic cancer detection methods" are not particularly limited, and include diagnostic imaging methods, histological examination methods, blood hormone examination methods, and methods using known pancreatic cancer markers as indicators. Examples of "diagnostic imaging methods" include abdominal ultrasound, abdominal computed tomography (CT), endoscopic retrograde cholangiopancreatography (ERCP), magnetic resonance cholangiopancreatography (MRCP), and endoscopic ultrasound (EUS). Examples of histological examination methods include FNA biopsy and EUS-FNA. Examples of "blood hormone examination methods" include methods of measuring the levels of hormones such as insulin, gastrin, glucagon, and VIP in the blood. Examples of "methods using known pancreatic cancer markers as indicators" include methods using, as indicators, the expression of carbohydrate antigens such as CA19-9, Span-1, CA50, CA242, Dupan-2, TAG-72, and urinary fucose, as well as CEA, POA, and TPS.

[0087] Furthermore, pancreatic cancer detection is usually performed by a physician (including someone under the physician's instructions), and the data on the protein amount and the like are useful for diagnosis, including determining the timing of treatment by the physician. Therefore, the method of the present invention can also be described as a method for collecting data on the protein amount for diagnosis by a physician, a method for presenting the data to a physician, a method for comparing and analyzing the protein amount with a reference amount, and a method for assisting a physician in diagnosis.

[0088] <Agent for detecting pancreatic cancer> As described above, in the detection method of the present invention, pancreatic cancer can be detected by detecting the amount of cytoplasmic Cep63 protein using an antibody that binds to Cep63. Therefore, the present invention provides an agent for detecting pancreatic cancer by the above-mentioned method, which agent comprises an antibody that binds to Cep63.

[0089] The "antibody" contained in the agent of the present invention may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody. "Antibody" includes all classes and subclasses of immunoglobulins. "Polyclonal antibody" is an antibody preparation containing different antibodies against different epitopes. "Monoclonal antibody" refers to an antibody (including antibody fragments) obtained from a substantially homogeneous population of antibodies. In contrast to polyclonal antibodies, monoclonal antibodies recognize a single determinant on an antigen. In the present invention, a "functional fragment" of an antibody refers to a portion (partial fragment) of an antibody that specifically recognizes a target protein. Specific examples include Fab, Fab', F(ab')2, variable region fragments (Fv), disulfide-linked Fv, single-chain Fv (scFv), sc(Fv)2, diabodies, multispecific antibodies, and polymers thereof.

[0090] The antibody of the present invention, if it is a polyclonal antibody, can be obtained by immunizing an animal with an antigen (Cep63, a partial peptide thereof, or cells expressing these, etc.) and purifying the antiserum by conventional means (e.g., salting out, centrifugation, dialysis, column chromatography, etc.).

[0091] Monoclonal antibodies can also be produced by the hybridoma method or recombinant DNA method, a representative example of which is the method of Kohler and Milstein (Nature, 256:495 (1975)). The recombinant DNA method involves cloning DNA encoding the antibody according to the present invention from hybridomas, B cells, or the like, incorporating it into an appropriate vector, and then introducing it into host cells (e.g., mammalian cell lines, Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody according to the present invention as a recombinant antibody (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997 Wiley, P. Shepherd and C. Dean, Monoclonal Antibodies, 2000 Oxford University Press; Vandamme A. M. et al., Eur. J. Biochem. 192:767-775 (1990)).

[0092] The antibody may be provided in a form immobilized on a carrier for use in various immunological techniques. Examples of such solid-phase carriers include insoluble carriers in the form of beads, microplates, test tubes, sticks, test strips, or test pieces made of materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, cellulose, Sepharose, glass, metal, ceramics, or magnetic materials. The immobilization can be carried out by appropriately binding the antibody or the like to the solid-phase carrier using known methods such as physical adsorption, chemical binding, or a combination of these.

[0093] Furthermore, the antibody may be labeled with a labeling substance according to the detection technique in various immunological methods, such as enzymes (β-D-glucosidase, luciferase, HRP, etc.), luminescent substances (luminol, luciferin, lucigenin, etc.), fluorescent substances (FITC, FAM, DEAC, R6G, TexRed, Cy5, etc.), ３ H. １４ C. ３２P. ３５ S. １２３ Examples of suitable affinity substances include radioisotopes such as I, and affinity substances such as biotin and streptavidin.

[0094] In addition to the antibody, the pharmaceutical agent of the present invention may contain other components acceptable for use as a composition. Examples of such other components include pharmacologically acceptable carriers or diluents (sterilized water, physiological saline, vegetable oil, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, antiseptics, etc.). Examples of excipients that can be used include lactose, starch, sorbitol, D-mannitol, and sucrose. Examples of disintegrating agents that can be used include starch, carboxymethylcellulose, and calcium carbonate. Examples of buffering agents that can be used include phosphates, citrates, and acetates. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, and tragacanth. Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of stabilizers that can be used include propylene glycol, diethylin sulfite, and ascorbic acid. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. Examples of antiseptics that can be used include sodium azide, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc.

[0095] In addition to the antibody or drug of the present invention, a substrate necessary for detecting the label, a solution for dissolving sample proteins (protein dissolution reagent), a buffer solution (diluent, washing solution) used for diluting and washing the sample, a reagent for stopping the label detection reaction (reaction quencher), a positive control (e.g., each pancreatic cancer marker, preparation), a negative control, an isotype control antibody for the antibody of the present invention, etc. can be combined to form a kit for detecting pancreatic cancer. Examples of such kits include a kit for detecting pancreatic cancer comprising an antibody of the present invention and at least one item selected from an isotype control antibody for the antibody, a positive control, and a negative control. Furthermore, when an unlabeled antibody is used as the antibody preparation, a labeled substance that binds to the antibody (e.g., secondary antibody, protein G, protein A, etc.) can be combined. Furthermore, such a kit can include instructions for use of the kit.

[0096] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. The examples were carried out using the materials and methods shown below.

[0097] (Antibodies and Compounds) For the antibodies used in this example, see Table 1 below. In Table 1, "IF" and "WB" indicate the dilution concentrations of each antibody in immunofluorescence staining and Western blotting.

[0098]

[0099] Baf A1, E64d, and pepstatin A were purchased from Sigma. PI and 7AAD were purchased from BD. All other compounds were purchased from Nacalai.

[0100] (Animals) For details on p62 knockout (KO) mice, Atg5KO mice, and p53KO mice, see Torii, S. et al., EMBO Rep., 2016, Vol. 17, pp. 1552-1564; Komatsu, M. et al., Cell, 2007, Vol. 131, pp. 1149-1163; and Kuma, A. et al., Nature, 2004, Vol. 432, pp. 1032-1036. p62 / p53 double knockout (DKO) mice were prepared by mating. All animals were kept in a SPF animal facility. This experiment was approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University and was conducted in accordance with the committee's guidelines.

[0101] (Cell culture and gene transfer) Wild-type (WT), p53KO, p62KO, and p62 / p53DKO primary thymocytes were collected from each mouse. Mouse embryonic fibroblasts (MEFs) and human cancer cell lines were cultured in a medium containing L-glutamine (2 mM), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), HEPES / Na ＋ The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with erythrocyte colony-stimulating factor (10 mM; pH 7.4), 2-mercaptoethanol (0.05 mM), penicillin (100 U / mL), streptomycin (100 μg / mL), and fetal bovine serum (10%).

[0102] The retroviral plasmid (pbabe-c-mycT58A+HrasG12V; Addgene, catalog number: #11130) was introduced into MEFs using Plat-E cells. Specifically, the plasmid was introduced into Plat-E retroviral packaging cells, and the medium was collected 48, 60, and 72 hours later. The culture supernatant containing the retrovirus was then collected and added to MEFs.

[0103] Transfection into Panc1 cells (a cell line derived from human pancreatic cancer) and MCF7 cells (a cell line derived from human breast adenocarcinoma) was carried out using transfection reagents (Lipofectamine 3000 and Viafect), respectively.

[0104] Other plasmids were introduced into MEFs using electroporation transfection (Neon® Electroporation System).

[0105] In the siRNA experiment, a transfection reagent (Lipofectamine RNAiMAX) was used, and cells (1 × 10 ６ ) was transfected with 300 pmol of siRNA.

[0106] The target sequences of the introduced siRNAs targeting human Cep63 are as shown in Table 2 below. Note that the "position" in Table 2 indicates the position in the Cep63 cDNA of the sequence targeted by each siRNA (the position in the sequence set forth in SEQ ID NO: 21). Furthermore, the numbers in parentheses indicate the position in the Cep63 mRNA of the sequence targeted by each siRNA (the position in the sequence set forth in SEQ ID NO: 34). The sequence set forth in SEQ ID NO: 21 corresponds to the sequence consisting of nucleotides 86 to 2112 set forth in SEQ ID NO: 34.

[0107]

[0108] The sequence of the introduced siRNA targeting human RIP1 was 5'-UACCACUAGUCUGACGGAUAAA-3' (SEQ ID NO: 25). Stealth RNAi Negative Control Low GC Duplexes was used as a control siRNA.

[0109] (Suppression of Cep63 gene expression by shRNA-expressing retroviral vector) A synthetic oligonucleotide targeting Cep63 was cloned into an shRNA-expressing retroviral vector (pLMP vector; see Dickins, R.A. et al., Nat. Genet., 2005, vol. 37, pp. 1289-1295). The target sequence of the shRNA is as follows:

[0110]

[0111] Table 3 shows shRNAs targeting mouse-derived Cep63 and their corresponding shRNAs targeting human-derived Cep63. In the table, "position" indicates the position in the Cep63 cDNA of the sequence targeted by each shRNA (for human-derived shRNAs, the position in the sequence set forth in SEQ ID NO: 21 is indicated, and for mouse-derived shRNAs, the position in the sequence set forth in SEQ ID NO: 23 is indicated). In this example, sh-Cep63#1 to 3 targeted mouse-derived Cep63 (SEQ ID NO: 11 to 13) and sh-Cep63#4 targeted human-derived Cep63 (SEQ ID NO: 4) were used.

[0112] Xenograft and tumorigenesis experiments: Tumorigenesis of p62 / p53DKO mouse-derived MEFs and Atg5 / p53DKO mouse-derived MEFs was achieved by introducing the mutant c-myc (T58A) gene and the mutant Hras (G12V) gene. Furthermore, sublines expressing sh-Cep63 were generated from these tumorigenic MEFs.

[0113] For xenografting, tumor-transforming MEFs were cultured at 3 x 10 in 150 μL of HBSS. ５ Panc-1 cells were suspended in 150 μL of Matrigel at a density of 1 × 10 ７ The cells were then suspended to a density of 1000. Each of the suspended cells was then subcutaneously injected into syngeneic mice, and tumors were removed and analyzed after 3 or 12 weeks.

[0114] (Immunostaining) Cells were washed twice with PBS, fixed with 4% PFA, and incubated for 30 minutes at room temperature in PBS containing 1% BSA and 0.05% Triton X-100. Primary antibodies diluted with 1% BSA were then added, and the cells were incubated for approximately 1.5 hours at room temperature. The cells were washed twice, and the corresponding secondary antibodies (labeling compounds: Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 633) were added. Immunostaining was performed using a confocal microscope (Carl Zeiss, model number: LSM710).

[0115] To stain for centrosomal proteins, cells were cultured on cover slips, fixed in cold methanol (-20°C) for 15 minutes, and rehydrated with PBS. Observations were performed using a confocal microscope (Carl Zeiss, model number: LSM710). In some experiments, analysis was performed using an imaging cytometer (GE Healthcare, product name: IN Cell Analyzer 2200). For centrosome number counts, cells with three or more centrosomes were considered to have an excess of centrosomes.

[0116] Paraffin-embedded human cancer tissue sections were purchased from MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. and BioMax Life Sciences, Inc. After deparaffinization using standard methods, the sections were autoclaved in 10 mM citrate buffer, subjected to blocking treatment, and immunostained using anti-Cep63 antibody and anti-γ-tubulin antibody.

[0117] (DNA aneuploidy analysis) Fixed thymocytes were stained with PI or 7AAD and analyzed by flow cytometry. DNA abnormalities were determined based on the following criteria (Figure 1F): (i) detection of an abnormal peak; (ii) detection of multiple peaks; and (iii) detection of a broad peak with less than 90% of cells in the G0 / G1 region.

[0118] (Protein Binding Analysis) Duolink® Proximity Ligation Assay (PLA) was performed according to the manufacturer's instructions. Specifically, cells were cultured on glass coverslips and fixed with cold methanol (-20°C) for 15 minutes. Next, the cells were blocked with PBS containing 0.02% Triton X-100 and 1% BSA, and primary antibodies were added. The cells were washed, and the PLA probe was added, followed by hybridization, ligation, and amplification at 37°C. Fluorescent images were captured and analyzed using an IN Cell Analyzer 2200 or BZX-800.

[0119] (Statistical Processing) Data are shown as mean + standard deviation. Statistical analysis was performed using Prism software (GraphPad Software). Comparison of two sets of data was performed using an unpaired two-tailed Student's t-test. Comparison of multiple sets of data was performed using one-way analysis of variance (one-way ANOVA) and Tukey's post-hoc comparison test. A P value of less than 0.05 was considered to indicate a statistically significant difference between two groups.

[0120] The results of experiments conducted using the above materials and methods are shown below.

[0121] <Tumor formation in p62 / p53 DKO mice> p62 is known to have the ability to induce carcinogenesis. Therefore, the present inventors crossed p53KO mice, which are carcinogenesis model mice, with p62KO mice and analyzed whether deletion of the p62 gene could suppress carcinogenesis.

[0122] Although tumor formation sites vary in p53KO mice depending on their genetic background, the C57BL / 6J strain used in this example is known to be prone to thymic tumor formation (see Harvey, M. et al., FASEB J., 1993, Vol. 7, pp. 938-943).

[0123] Analysis of 3-month-old mice revealed tumor formation in 8.3% of p53KO mice, but no tumor formation in p62KO mice. Surprisingly, tumor formation was observed in 34% of mice lacking both genes (Figure 1). As shown in Table 4 below, tumors were observed in various organs, including the spleen and skeletal muscle, but the most common was the thymus. This is consistent with the characteristics reported in previous studies in C57BL / 6J mice (Harvey, M. et al., 1993). Similar results were observed in 4-month-old mice (Figure 1A).

[0124]

[0125] Table 4 shows the sites of tumor formation in p62 / p53 DKO mice (3 or 4 months old) and the tumor incidence at those sites (number of mice with tumors at each site / total number of mice).

[0126] Furthermore, staining with Ki67, a marker for proliferating cells, revealed that Ki67 expression was detected in both deficient mice, and was further enhanced in the tumor-bearing thymus (Figs. 1B and 1C).

[0127] Furthermore, DNA histogram analysis revealed that almost all thymocytes were in the G0 / G1 phase in wild-type (WT), p53KO, and p62KO mice, whereas abnormal distributions were observed in thymocytes from both p53 and p62-deficient mice (Fig. 1D and 1E). The detection rate of such abnormal distributions was 39% in cells derived from the thymus of both p53 and p62-deficient mice without tumor formation, and 100% in cells derived from the thymus of both p53 and p62-deficient mice (Fig. 1G).

[0128] These results revealed that, contrary to the above prediction, tumor formation was promoted by deleting p62 under p53-deficient conditions.

[0129] <Increased Number of Cytoplasmic Cep63 and Centrosomes in p62 / p53 DKO Mice> Next, we analyzed how tumor formation due to p53 deficiency is promoted by p62 deficiency.

[0130] p62 has been shown to be an important molecule in selective autophagy and various stress responses. Regarding the latter, p62 activates Nrf2 by dissociating it from the Keap1-Nrf2 complex, resulting in the translocation of activated Nrf2 into the nucleus (Sanchez-Martin, P. and M. Komatsu, J. Cell. Sci., 2018, 131:jcs222836). However, nuclear translocation of Nrf2 was not observed in the thymus of any of the mice used in this study. Although p62 is also involved in the mTor and NF-kB pathways, activation of these pathways was not observed (Figures 2A and 2B). These findings demonstrate that no function of p62 other than autophagy is involved in thymic tumorigenesis.

[0131] Although various cargoes have been reported for p62-bound autophagy, the present inventors focused on cytoplasmic Cep63. Cep63 is a centrosomally localized protein known to be important for centrosome duplication (Tang, T.K., Nat. Cell Biol., 2013, Vol. 15, pp. 1400-1402). However, the present inventors previously found that Cep63 is also present in the cytoplasm and is degraded by p62-mediated autophagy in normal cells (Watanabe, Y. et al., Nat. Commun., 2016, 7:13508). It has also been shown that cytoplasmic Cep63 accumulates in cells in which autophagy does not occur or in cells lacking p62, resulting in ectopically synthesized centrosomes. Since abnormalities in centrosome number can cause chromosomal abnormalities and carcinogenesis, we hypothesized that cytoplasmic Cep63 may be involved in promoting tumorigenesis due to p62 deficiency.

[0132] Indeed, staining of thymocytes for Cep63 and a centrosomal marker (γ-tubulin) revealed that Cep63 was localized only in the centrosomes of thymocytes from p53KO mice. However, in thymocytes from p62 / p53DKO mice, Cep63 expression was observed not only in the centrosomes but also in the cytoplasm (Figures 2C and 2D). Furthermore, this cytoplasmic Cep63 was also observed in thymocytes from non-tumorigenic p62 / p53DKO mice (Figures 2E and 2F). Thus, the increase in cytoplasmic Cep63 and centrosome number was observed regardless of the presence or absence of tumor formation, suggesting that these increases are not the result of tumor formation.

[0133] The role of Cep63 in tumorigenesis: Increased cytoplasmic Cep63 and centrosomes were observed in the thymus of p53 / p62 DKO mice, and tumor formation was also observed. Therefore, we analyzed whether increased cytoplasmic Cep63 and centrosomes actually affect tumorigenesis.

[0134] To analyze this, we first performed xenograft experiments using p62 / p53DKO MEFs, which were tumorigenic due to the expression of mutant c-myc and Hras. As a result, similar to the thymus, these double-deficient MEFs also showed higher cytoplasmic Cep63 and centrosome numbers than single-deficient MEFs (Figures 3A-3D).

[0135] Furthermore, when the lysosomal protease inhibitor E64d was added, Cep63, which is degraded by autophagy, was observed to colocalize with lysosomes in p53-deficient MEFs, but not in both deficient MEFs (Figures 3E and 3F). Similar findings were observed in Atg5 / p53-deficient MEFs (Figure 3G), demonstrating that autophagy deficiency and p62 deficiency also lead to increased cytoplasmic Cep63 and centrosome numbers in MEFs.

[0136] To analyze the relationship between cytoplasmic Cep63 and tumorigenesis, we attempted to knockdown Cep63 in tumorigenic p53 / p62-deficient MEFs using shRNA (three sequences #1 to #3 shown in Table 3) (Figure 3H). As a result, we observed cases in which Cep63 knockdown reduced the number of cytoplasmic Cep63 and centrosomes (Figures 3I to 3K). However, it was revealed that such knockdown did not affect centrosomal Cep63 or cell proliferation (Figure 3L).

[0137] Specifically, in tumorigenic MEFs, effective sh-Cep63 targeting only cytoplasmic Cep63 suppressed tumor formation after xenografting, but did not affect growth on plates. This demonstrated the involvement of cytoplasmic Cep63 in three-dimensional tumor formation and malignant progression. Furthermore, although it is thought that growth would be reduced if effective sh-Cep63 acted on centrosomal Cep63, the lack of effect on growth on plates also demonstrated that effective sh-Cep63 does not affect centrosomes.

[0138] Next, tumorigenic p53 / p62-deficient MEFs with cytoplasmic Cep63 knockdown were subcutaneously transplanted into mice, and tumor weights were analyzed 3 weeks later. While tumor formation was observed in all transplanted vector control cells, tumor formation was suppressed in the knockdown cells, and the tumors formed were smaller (Figures 3M and 3N). Similar results were obtained in experiments using Atg5 / p53-deficient MEFs (Figures 3O-3T).

[0139] These results demonstrate that cytoplasmic Cep63 is involved in tumorigenesis and tumor growth. Furthermore, it was revealed that Cep63 degradation by autophagy mediated by p62 and Atg5 is important for suppressing tumorigenesis.

[0140] Increased centrosome number in human pancreatic cancer cell lines. When the number of multicentrosomal cells was counted in various human cancer cell lines, multicentrosomal cells were observed in many cells, and the proportion of multicentrosomal cells correlated with the number of cytoplasmic Cep63 (Figures 4A to 4D). These results suggest that centrosome formation from cytoplasmic Cep63 is the cause of multicentrosome formation in cancer cells.

[0141] Among various cancer cell lines, pancreatic cancer cell lines such as Panc1, Hs766T, and Capan2 were found to have high numbers of cytoplasmic Cep63 and multicentrosomes (Figures 4A-4D). Similar increases in cytoplasmic Cep63 were also confirmed in stained sections from human pancreatic cancer patients (Figures 4E and 4F). Therefore, dysregulation of cytoplasmic Cep63 and increased centrosome numbers are considered to be characteristic features of pancreatic cancer cells.

[0142] Two possible causes of increased cytoplasmic Cep63 in pancreatic cancer cells are autophagy failure and inadequate recognition by p62.

[0143] Therefore, we first stimulated cells with the DNA-damaging agent etoposide, and measured autophagy activity using LC3 and p62 as indicators. We then compared these with the breast cancer cell line MCF7, which had a normal number of centrosomes. The results showed that the pancreatic cancer cell line Panc1 also showed an increase in LC3-II and a decrease in p62, similar to MCF7 (Figures 4G and 4H). We also analyzed whether the lysosomal inhibitor Baf A1 affected the progression of autophagy, but found no abnormalities (Figures 4G and 4H), confirming that the autophagy mechanism was intact in Panc1 cells.

[0144] Next, the binding between p62 and Cep63 was analyzed. Specifically, the binding between the two molecules was analyzed using the PLA method, which can detect fluorescence when two molecules are in close proximity.

[0145] As a result, fluorescent signals indicating the binding of both molecules were detected in untreated MCF7 cells, but when autophagic degradation was inhibited with the protease inhibitor E64d / pepstatin or the lysosomal inhibitor Baf A1, the signal was enhanced (Figures 4I and 4J). Furthermore, colocalization of Cep63 and p62 was detected in MCF7 cells treated with Baf A1 (Figure 4K). These results indicate that in MCF7 cells with a normal number of centrosomes, cytoplasmic Cep63 forms a p62-Cep63 complex and is degraded by autophagy.

[0146] In contrast, no PLA fluorescent signal was detected in Panc1 cells, even after autophagy inhibition (Figures 4I and 4J). Immunostaining also showed no colocalization of Cep63 and p62 (Figure 4K), revealing that Cep63 and p62 do not bind in Panc1 cells. This may be due to mutations in the Cep63 or p62 genes in Panc1 cells. Therefore, exogenous Cep63 and p62 were introduced and analyzed by PLA. As a result, signals were detected in MCF7 cells using exogenous Cep63 and p62, but not in Panc1 cells (Figures 4L and 4M). Furthermore, colocalization was not observed by immunostaining (Figures 4N and 4O). These results suggest that an as-yet-unidentified factor in Panc1 cells interferes with the interaction between p62 and Cep63 and contributes to the increase in cytoplasmic Cep63.

[0147] <Inhibition of p62-Cep63 binding by RIP1 in pancreatic cancer cells> p62 is known to bind to various molecules via various domains it possesses (see FIG. 5A; Taniguchi, K. et al., FEBS Lett., 2016, Vol. 590, pp. 2375-2397; Moscat, J. and M.T. Diaz-Meco., Trends Biochem. Sci., 2012, Vol. 37, pp. 230-236). In previous studies, the present inventors have demonstrated that Cep63 binds to p62 via the ZZ domain (see Watanabe, Y. et al., Nat. Commun., 2016, 7:13508). The ZZ domain has also been reported to bind to RIP1. Therefore, we speculated that RIP1 might inhibit the binding of Cep63 to p62 in Panc1 cells.

[0148] Therefore, we analyzed the binding of RIP1 and p62 using PLA and confirmed strong binding in Panc1 cells (Figures 5B and 5C). Furthermore, suppression of RIP1 expression with siRNA attenuated the PLA signal indicating binding between p62 and RIP1 (Figures 5D and 5E), while importantly, enhanced binding between p62 and Cep63 (Figure 5F). Furthermore, we observed a decrease in the number of cytoplasmic Cep63 and a decrease in the proportion of multicentrosomal cells (Figures 5G-5I). Furthermore, strong binding between p62 and RIP1 and weak binding between p62 and Cep63 were also observed in another pancreatic cancer cell line, Hs766T cells (Figures 5J and 5K).

[0149] These findings demonstrate that RIP1 inhibits the binding of p62 to Cep63 in pancreatic cancer cells, thereby causing an increase in cytoplasmic Cep63 and an increase in the number of centrosomes.

[0150] The role of Cep63 in human pancreatic cancer. We analyzed the effect of cytoplasmic Cep63 on tumorigenesis in Panc1 cells by introducing sh-Cep63#4 into Panc1 cells and knocking down Cep63. As a result, knocking down Cep63 in Panc1 cells reduced the number of cytoplasmic Cep63 and the proportion of multicentrosomal cells (Figures 6A-6D). Furthermore, we xenografted these cells into nude mice and analyzed their tumorigenicity, revealing that knocking down Cep63 suppressed tumor growth (Figures 6E and 6F).

[0151] <Knockdown of Cep63 by siRNA in pancreatic cancer cells> We attempted to develop additional siRNAs for knocking down Cep63. First, 11 candidate sequences that target Cep63 and have minimal effect on other genes were selected (see Table 2). Furthermore, siRNAs consisting of these candidate sequences were prepared and transfected into Panc1 cells. Proteins were then extracted from each cell and subjected to Western blotting analysis using an anti-Cep63 antibody to evaluate the effect of the candidate siRNAs on suppressing Cep63 expression.

[0152] As a result, si-8, si-2, si-4, si-9, and si-5 were found to have a strong inhibitory effect on Cep63 expression, to the extent that the Cep63-derived band disappeared in Western blotting. Furthermore, si-7 was found to have a partial inhibitory effect, although it was weaker than the siRNA. On the other hand, si-1 and si-3 showed no significant difference in the expression level of Cep63 compared to the negative control, and no inhibitory effect on Cep63 was found.

[0153] As described above, p62 exerts its anti-carcinogenic function by promoting the degradation of cytoplasmic Cep63 by autophagy. On the other hand, it has been revealed that p62 deficiency causes an increase in centrosomes and promotes carcinogenesis, particularly in pancreatic cancer cells.

[0154] As described above, according to the present invention, by targeting cytoplasmic Cep63, antitumor activity against pancreatic cancer can be exerted. Furthermore, the amount of cytoplasmic Cep63 can be used as an indicator to determine the presence or absence of pancreatic cancer. Therefore, the present invention is useful for treating or preventing pancreatic cancer, as well as for diagnosing it.

Claims

1. A composition for treating or preventing pancreatic cancer, comprising as an active ingredient a substance that inhibits the function of cytoplasmic Cep63.

2. The composition according to claim 1, wherein the substance that inhibits the function of cytoplasmic Cep63 is a nucleotide that binds to the transcription product of the Cep63 gene.

3. The composition according to claim 2, wherein the nucleotide comprises a nucleotide encoded by a DNA set forth in any one of SEQ ID NOs: 1 to 4, 26 to 31 and 35 to 37.

4. A method for detecting pancreatic cancer, comprising the following steps (a) to (c): (a) detecting the amount of cytoplasmic Cep63 protein in a pancreas-derived sample isolated from a subject; (b) comparing the amount of protein detected in step (a) with a reference amount; and (c) determining that the subject has or is at risk of having pancreatic cancer, if the comparison in step (b) shows that the amount of protein in the subject is higher than the reference amount.

5. A composition according to any one of claims 1 to 3, characterized in that it is administered to a subject who has been determined to have or be at risk of having pancreatic cancer by the method according to claim 4.

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