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Breast cancer is now the most frequently diagnosed cancer and the leading global cause of cancer death in women. Despite advances in the diagnosis and treatment of human breast cancer, advanced stage, metastatic breast cancers are difficult to cure. Among the common target organs for breast cancer metastasis, post recurrence survival is worst when multiple metastasis was observed in the liver. Cancer stem cells (CSCs) are a sub-population of the cells that retain high tumorigenic capacity and, at the same time, are able to sustain the formation of tumors that recreate the cellular diversity of the parent lesions from which they have been originally isolated. We and others have shown that in epithelial malignancies such as breast and colorectal cancers, self-renewal ability of cancer cells with CSC properties is epigenetically regulated by their expression of microRNAs, such as with miR-200c, miR-142 and miR-221. Because of their highly tumorigenic properties, CSCs with are associated with metastatic progression, especially at the initial steps of metastases. We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. Comparison of gene expression profiles between the CD44+ human breast cancer cells metastasized to the liver (‘metastasized CSCs') and those in the primary site (‘primary CSCs') revealed that S100A10 was upregulated in metastasized CSCs than in primary CSCs. Knockdown of S100A10 in breast cancer cells reduced the matrix metalloproteinase activity and suppressed their invasion abilities in transwell cell invasion assays in which upper surface of transwell inserts were precoated with Matrigel. Knockdown of S100A10 suppressed, and overexpression of S100A10 enhanced the 3D organoid formation capacities of breast cancer PDX cells in vitro. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. Upregulation of S100 family protein expression is observed in many human cancers, including breast, lung, bladder and gastric cancers. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers. Citation Format: Yohei Shimono, Hisano Yanagi, Takashi Watanabe, Tatsunori Nishimura, Takanori Hayashi, Seiji Okada, Motoshi Suzuki, Kenji Kawada, Hironobu Minami, Noriko Gotoh. Role of S100A10 in the metastatic colonization of breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3083.

miRNAs are key players in the integrated regulation of cellular processes and shape many of the functional properties that define the “cancer stem cell” (CSC) phenotype. Little is known, however, about miRNAs that regulate such properties in human colorectal carcinoma. In this study, we compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (with CSC depletion) sorted in parallel from human primary colorectal carcinomas and identified miR-221 as the miRNA that displayed the highest level of preferential expression in EpCAM+/CD44+ cancer cells. High levels of miR-221 expression were associated with Lgr5+ cells in mouse colon crypts and reduced survival in patients with colorectal carcinoma. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. Finally, the most abundant splicing isoform of the human Quaking (QKI) gene, QKI-5, was identified as a functional target of miR-221; overexpression of miR-221–reduced QKI-5 protein levels in human colorectal carcinoma cells. As expected, overexpression of QKI-5 suppressed organoid-forming capacity in vitro and tumorigenic capacity of colorectal carcinoma PDX cells in vivo. Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer. Significance: These findings uncover molecular mechanisms underlying the maintenance of cancer stem cell properties in colon cancer.

Colorectal cancer stem cells (CSCs) have extensive abilities to initiate tumor formation, and are responsible for the progression and metastasis of colorectal cancers. MicroRNAs (miRNAs) are non-coding RNAs of 18-24 nucleotides that post-transcriptionally regulate expression of multiple genes by targeting their 3'untranslated regions (UTRs). miRNAs regulate various biological processes, and function as important regulators of CSC properties. miR-221 is one of the oncogenic miRNAs frequently upregulated in human cancers including colorectal cancer. Previous studies have shown that miR-221 enhances proliferation, migration, and invasion of cancer cells. However, the role of miR-221 in human colorectal CSCs is not fully elucidated. In this study, we analyzed the colorectal CSCs directly isolated from the surgical specimens of colorectal cancer patients to explore the role of miR-221 in CSCs. Expression profile of 754 miRNAs in the CD44+/EpCAM+ CSC and non-tumorigenic cancer cell (NTC) populations was analyzed by multiplex semi-quantitative PCR. Prognostic impact of miR-221 expression in CSCs was analyzed using the TCGA database. Target genes of miR-221 were validated by luciferase reporter assays and Western blot experiments. The roles of miR-221 and its target gene QKI-5 in colorectal CSCs were evaluated by organoid and xenotransplantation assays. Comparison of the expression levels of 754 miRNAs in the CSCs and NTCs resulted in the identification of 10 miRNAs upregulated or downregulated in CSCs compared to NTCs. Among them, miR-221 was most highly expressed in CSCs and its expression level was very low in NTCs and human normal colon epithelial cells of human colorectal cancer specimens (n=6, p Our results suggest that the miR-221/QKI5 axis plays important roles in the regulation of CSC properties in human colorectal cancers. Because miR-221 was preferentially and highly upregulated in human colorectal CSCs, but not in normal stem/progenitor cells, it is possible that miR-221 will be a biomarker that reflects amount and/or activity of CSCs in colorectal cancers and an attractive target to selectively attach colorectal CSCs by avoiding damages on their normal counterparts. Citation Format: Junko Mukohyama, Yohei Shimono, Piero Dalerba, Taichi Isobe, Qingjiang Hu, Debashis Sahoo, Naoki Shibuya, Hironobu Minami, Koshi Mimori, Yoshihiro Kakeji, Akira Suzuki. Epigenetic regulation of colorectal cancer stem cells by the miR-221/QKI5 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 488.

Introduction: Tumor microenvironment plays a key role in the development and progression of tumors. Although adipose tissue is a predominant component of tumor microenvironment in mammary tissue, and secretes various cytokines, chemokines and growth factors, roles of adipocytes in the breast cancers remain to be elucidated. Methods: In this study, we identified adipsin, an adipokine predominantly expressed in the adipose-derived stem cells (ADSCs) of breast cancer patients, enhanced the clonogenicity, proliferation, and cancer stem cell-like properties of human breast cancer patient-derived xenograft (PDX) cells. Human breast cancer PDXs were established by the engraftment of surgical specimens of the breast cancer patients into immunodeficient mice. Human mammary adipose-derived stem cells (ADSCs) were isolated from the enzymatically dissociated adipose tissues of the surgical specimens of breast cancer patients. Results: When ADSCs with diverse patient backgrounds were co-cultured with human breast cancer PDX cells, ADSCs significantly enhanced the clonogenicity of breast cancer PDX cells (n = 9; p < 0.05). We found that adipsin, an adipokine that is involved in the generation of C3a and C3b in the complement alternative pathway, was highly expressed in the cultured supernatants of ADSCs when compared to those of breast cancer PDX cells (n = 9; p < 0.05). Accordingly, the amount of C3a was significantly higher in the cultured supernatants of ADSCs when compared to those of breast cancer PDX cells (n = 9; p < 0.05). Suppression of adipsin by a specific inhibitor or knockdown in mammary ADSCs significantly suppressed the sphere-forming abilities of breast cancer PDX cells; reduced the mRNA expression levels of the cancer stem cell marker CD44 and the chemokine receptor CXCR4 (p < 0.01); and reduced the cell surface expression of CD44 in breast cancer PDX cells, suggesting that cancer stem cell-like properties were regulated by adipsin. Although the suppression of CXCR4 by a specific inhibitor showed a tendency to suppress the sphere-forming abilities of breast cancer PDX cells, the dual suppression of adipsin/C3a and CXCR4 by their specific inhibitors showed no additive effects on the suppression of the clonogenicity of breast cancer PDX cells. Conclusion: These observations suggest that ADSCs are important components of tumor microenvironment in human breast cancers, and secrete adipsin that functions as regulators of the clonogenicity, proliferation, and cancer stem cell-like properties of human breast cancer cells. Citation Format: Hideaki Goto, Yohei Shimono, Toru Mukohara, Yohei Funakoshi, Yoshinori Imamura, Seishi Kono, Shintaro Takao, Akira Suzuki, Hironobu Minami. Adipsin enhanced clonogenicity and proliferation of human breast cancer patient-derived xenograft cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1006.

Background: It is becoming clear that presence of PIK3CA mutations is associated with lower pathological complete response rate in patients with HER2-overexpressing breast cancer when treated with trastuzumab-based chemotherapy in neo-adjuvant settings. On the other hand, in in vitro studies using traditional 2-dimentional (2D) cell culture, differential cellular or biochemical response to trastuzumab between PIK3CA-mutant (mt) and -wild-type (wt) cells has not been clearly demonstrated. Further, while tumor shrinkage is occasionally observed in breast cancer patients who are treated with trastuzumab as a single agent, cyto-toxic effect of trastuzumab is not simulated in 2D culture models. Recently, many studies reported 3-dimensional (3D) cell culture mimics in vivo environment better than 2D culture. Therefore, we hypothesized that 3D culture better represents clinically-observed trastuzumab resistance associated with PIK3CA mutation than 2D culture, and decided to comparatively investigate cellular and biochemical response to trastuzumab in HER2-amlified PIK3CA-mt and -wt cell lines cultured in 2D and 3D environments. Method: HER2-amplified breast cancer cell lines, BT474 (PIK3CA-wt), and UACC893 and MDA-MB361 (PIK3CA-mt) were seeded (day 0) and allowed to grow in 2D and 3D (NanoCluture Plate®, ORGANOGENIX, Kanagawa, Japan) cell culture plates. On day 3, trastuzamab (10 µg/ml) and/or BKM120 (1 and 5 µM), a PI3K inhibitor, were added. The effect of the drugs on cell growth was evaluated with WST-8 assay on days 3 through 7. Apoptosis and cell signaling were evaluated using Western blot on day 6 and days 3 through 5, respectively. Result: In PIK3CA-wt BT474, treatment with trastuzumab led to decrease in cell number, indicating cyto-toxic effect, only in 3D culture but not in 2D culture. In PIK3CA-mt UACC893 and MDA-MB-361 cell lines, treatment with trastuzumab resulted in no cellular reduction either in 2D or 3D cultures. Consistently, increase in cleaved PARP, indicative for apoptosis, was observed only in 3D-cultured BT474 but not in 2D-cultured BT474 or two PIK3CA-mt cell lines. Furthermore, in BT474, greater decrease in phosphorylation of AKT (p-AKT) was observed in 3D culture than in 2D culture. In PIK3CA-mutant cell lines, trastuzumab did not change level of p-AKT regardless of cell culture conditions. In PIK3CA-mutant UACC893, combined treatment with trastuzumab and BKM120 resulted in greater increase in expression of cleaved PARP than either drug alone. Conclusion: Trastuzumab-induced inhibition of PI3K/AKT pathway and resultant apoptosis in HER2-overxpressing PIK3CA-wt cells may be observed in 3D culture, which may be simulating cyto-toxic effect of trastuzumab by itself observed in clinic. Furthermore, 3D cell culture represents the resistance to trastuzumab associated with PIK3CA mutation better than 2D cell culture. Citation Format: Takashi Tatara, Toru Mukohara, Rina Tanaka, Yohei Shimono, Masanori Toyoda, Naomi Kiyota, Midori Hirai, Yoshihiro Kakeji, Hironobu Minami. 3D culture may better represent trastuzumab resistance associated with PIK3CA mutation than 2D culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3165. doi:10.1158/1538-7445.AM2017-3165

The canonical WNT signaling plays a critical role in many adult stem cells, including those of the breast and intestine. The fact that the canonical WNT signaling is implicated in both stem cell self-renewal and cancer suggests that normal physiological regulator of stem cell functions might be “hijacked” in cancer. Adenomatous polyposis coli (APC) is a component of the destruction complex that destabilizes β-catenin and suppresses the activity of the canonical WNT signaling. MicroRNAs (miRNAs) are important regulators of stem cell functions. We have previously reported a set of 37 miRNAs that are upregulated or downregulated in human breast cancer stem cells (BCSCs, a CD44+CD24-/lowlineage- population of human breast cancer cells) as compared to non-tumorigenic breast cancer cells (NTCs). Among them, miR-200c targets BMI1 that is a critical regulator of the stem cell maintenance, and strongly impairs the functions of human BCSCs in vivo. In this study, we compared the expression profiles of miRNAs, mRNAs and proteins between BCSCs and NTCs isolated from the patient specimens of human breast cancers and patient-derived tumor xenografts (PDXs) established by their transplantation. Luciferase assays were performed using the plasmid in which the 3’UTR region of candidate mRNA was cloned downstream of a luciferase minigene. The effect of miRNAs on the activity of WNT signaling was evaluated using a TCF reporter plasmid. Finally, the abilities to form organoids and to form tumors in immunodeficient mice were evaluated using the human BCSCs infected with the miRNA inhibitor expressing lentivirus. We found that miR-142 was highly upregulated in BCSCs, but was hardly expressed in NTCs in the patient breast cancer specimens. We confirmed that miR-142 targeted the sequence within the 3’UTR of APC mRNA and suppressed APC protein expression. Accordingly, miR-142 activated the canonical WNT signaling pathway in an APC-suppression dependent manner. The results of mRNA and protein expression profiling of the BCSCs isolated from human breast cancer PDXs suggested that the canonical WNT signaling was activated in BCSCs. Finally, inhibition of miR-142 in the BCSCs suppressed the tumor growth in vivo. These results suggest that the miR-142, a miRNA frequently upregulated in human BCSCs, could provide at least a part of the molecular mechanism for aberrant activation of the canonical WNT signaling in breast cancer in which APC mutations are much less frequent than colon cancer. Citation Format: Yohei Shimono, Taichi Isobe, Andrei Turtoi, Junko Mukohyama, Toru Mukohara, Akira Suzuki, Vincent Castronovo, Hironobu Minami. MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2017-2890

Patient-derived tumor xenografts (PDXs) established by xenotransplantation of surgically-resected human cancer specimens are an attractive model to analyze the character of cancer cells within the patient cancer tissues. However, establishment of PDX is occasionally hampered by lymphomagenesis. Lymphomagenesis in the PDX has been observed in the xenotransplantation of several types of cancers, such as lung, liver, gastric, bladder, breast, prostate, and colorectal cancers. Lymphomagenesis in the PDX is caused by the proliferation of the EBV-infected lymphocytes under insufficient immunosurveillance in the immunodeficient mice. However, it is still difficult to predict lymphomagenesis before xenotransplantation. In this study, we analyzed the expression of EBV-related genes in the 15 surgical specimens of the consented colorectal cancer (CRC) patients and the PDXs established by their xenotransplantation. The CRC specimens and the established PDX tumors were histologically examined. Then, the specimens were immunohistochemically stained with anti-CD3 and CD20 antibodies. The presence of EBV was histologically evaluated by EBER in situ. The expression levels of EBV-related genes, such as EBNAs, LMP1, EBER, BamHI W region and EBV-associated microRNAs, were evaluated using the genomic DNA and mRNAs prepared from the CRC specimens and the established PDX tumors. Nine PDXs were established by the xenotransplantation. Histological examination showed that 7 of 9 (78%) PDX recapitulated histopathological characteristics of the patient CRC tissues. However, 2 of 9 (22%) PDXs exhibited the morphological characteristics of EBV-associated human diffuse large B cell lymphoma (DLBCL). Then, we confirmed that lymphoma was formed by clonal proliferation of human B-cell lymphocytes, and strongly positive for EBER. We investigated whether the expression of EBV-related genes in the patient specimen is associated with lymphomagenesis in the PDXs. Expression of EBV genes and RNAs in patient CRC tissues were not clearly associated with lymphomagenesis in the PDXs. BamHI W region is a major internal repeat in EBV genome and the PCR amplification of this region is useful to evaluate the presence and amount of EBV. When the EBV BamHI W region was detectable in the patient specimens, the transplantation of the samples resulted in lymphomagenesis in 2 out of 3 patient cancer specimens. In contrast, when this region was undetectable, no patient cancer specimens resulted in lymphomagenesis (0 out of 7 patient cancer specimens). These results suggest that the amount and/or presence of EBV itself in the patient cancer specimens is one of the factors that are associated with lymphomagenesis in the PDXs. Therefore, the PCR amplification of EBV BamHI W region is an attractive method to evaluate the risk of lymphomagenesis before xenotransplantation. Citation Format: Junko Mukohyama, Dai Iwakiri, Yoh Zen, Toru Mukohara, Hironobu Minami, Yoshihiro Kakeji, Yohei Shimono. Detection of EBV BamHI W region in surgical cancer specimen is a useful method to evaluate the risk of lymphomagenesis in patient-derived tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4822. doi:10.1158/1538-7445.AM2017-4822

While it is becoming recognized that screening of oncology drugs on a platform using two-dimensionally (2D)-cultured cell lines is unable to precisely select clinically active drugs, three-dimensional (3D)-culture systems are emerging and show potential for better simulating the in vivo tumor microenvironment. The aim of this study was to reveal differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We first evaluated differences between 2D- and 3D-cultured breast cancer cell lines by assessing drug sensitivity, oxygen status, and expression of Ki-67 and caspases. Three cell lines (BT-549, BT-474, and T-47D) developed dense multi-cellular spheroids (MCSs) in 3D-culture, and tended to show greater resistance to paclitaxel and doxorubicin than in 2D-culture. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB-231) developed only loose MCSs in 3D-culture, and showed drug sensitivities similar to those in 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved PARP expression in 2D- than in 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected cells from paclitaxel-induced apoptosis. Hypoxia, as measured with phosphorescent LOX-1, was observed only in dense 3D-MCSs. BT-549 had considerably fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0 dormant sub-population may be responsible for its drug resistance in 3D-culture. In addition, BT-474 had a lower level of caspase-3 in 3D- than in 2D-culture, suggesting that the 3D-environment was anti-apoptotic. We next examined whether these findings were reproduced in primary cells utilizing a breast cancer patient-derived xenograft (PDX). The fresh disaggregated PDX tissue developed dense MCSs in 3D-culture. We compared staining for Ki-67, and caspase-3 and -8 in 2D- and 3D-cultured primary cells obtained from the PDX, and the original patient tumor (Ki-67 only): 2D-cultured cells showed much greater proportions of Ki-67-positive and caspase-3-positive cells than the others. 3D-cultured cells tended to show greater resistance to paclitaxel and doxorubicin compared to the others. Residual spheroids after exposure to paclitaxel did not grow after removal of the drug but majority of cells in the spheroids appeared alive based on their excluding trypan blue dye. In the residual spheroids, greater population of cells showed phosphorylated histone H2AX expression than those in the spheroids unexposed to paclitaxel, suggesting that chemotherapy-induced senescence associated with drug resistance. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important characteristics of tumor grown in vivo, namely hypoxia, dormancy, anti-apoptotic features, and their resulting drug resistance. 3D primary culture will be a model for the study of chemotherapy-induced senescence. Citation Format: Yoshinori Imamura, Toru Mukohara, Yohei Shimono, Yohei Funakoshi, Naoko Chayahara, Masanori Toyoda, Naomi Kiyota, Shintaro Takao, Seishi Kono, Tetsuya Nakatsura, Hironobu Minami. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 319. doi:10.1158/1538-7445.AM2015-319

In pathway-targeted cancer drug therapies, the relatively rapid emergence of drug-tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. The aim of this study is (1) to clarify the subpopulations of DTPs in EGFR mutated non-small lung cancer (NSCLC) cells that have different roles in the resistance to epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI) and (2) to develop effective treatments. In this study, we used two EGFR mutated lung adenocarcinoma cell lines, PC9 and II-18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 month. We analyzed the mRNA expression of the stem cell related markers by qRT-PCR and the expression of the cellular senescence associated proteins by Western blotting. Then, we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by the glucose metabolism targeting therapies and a stem-like cell targeting drug, withaferin A. The time-dependent upregulation of the stem cell-related markers was observed until day 24. Western blot analysis revealed that the cellular senescence associated proteins were upregulated in a similar time course. Senescent cells secrete various inflammatory cytokines and chemokines, a phenomenon known as SASP. Recently, SASP has been reported to contribute to the emergence and maintenance of cancer stem-like cells. Using FACS analysis, DTPs were divided into the two populations depending on the surface expression of CD133. CD133high DTPs were characterized by the higher expression of stem cell related markers. On the other hand, CD133low DTPs were characterized by higher expression of the cellular senescence associated proteins. Rapamycin is reported to suppress SASP. Gefitnib and rapamycin effectively suppressed the emergence of CD133high DTPs. We found CD133low DTPs showed higher glucose metabolic state, consistent with the previous reports that show senescence-associated metabolic reprogramming is induced by anticancer therapies. We targeted this metabolic change using a glucose transporter inhibitor (phloretin) and a glycolytic blocker (2DG). These agents successfully suppressed the emergence of CD133high DTPs. Finally, withaferin A could inhibit the emergence of CD133high DTPs. In conclusion, we found that the EGFR-TKI-induced DTPs in EGFR mutated NSCLC cells were composed of at least two distinct cell populations: CD133high DTPs with stem-like characters and CD133low DTPs with cellular senescent characters. CD133low DTPs supported CD133high DTPs via SASP and blocking SASP effectively suppressed the emergence of CD133high DTPs. Furthermore, our results suggest that the glucose metabolism targeting therapies and withaferin A can be new therapeutic candidates to ablate DTPs. Citation Format: Kei Kunimasa, Tatsuya Nagano, Yohei Shimono, Shuntaro Tokunaga, Daisuke Tamura, Motoko Tachihara, Kazuyuki Kobayashi, Yoshihiro Nishimura. The glucose metabolism targeting therapies and withaferin A eliminate epidermal growth factor tyrosine kinase inhibitor-induced drug-tolerant persisters in non-small lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 748. doi:10.1158/1538-7445.AM2015-748

The canonical Wnt/β-catenin pathway plays a crucial role in driving proliferation of normal colon epithelial cells and colon cancer cells. Tcf4, which is encoded by the Tcf7l2 gene, is a critical downstream molecule of the Wnt/β-catenin pathway. Although several downstream target genes of the Wnt/β-catenin pathway have been identified (e.g. CD44, Lgr5 and EphB2 receptor which mark both normal colon stem cells (NCSCs) and colon cancer stem cells (CoCSCs)), upstream regulators of this pathway remain poorly characterized. MicroRNAs (miRNAs) are endogenous small noncoding RNAs which suppress translation by binding to specific seed sequences in 3′untranslated region (3′UTR) of target mRNAs. In this study, we aimed to find miRNAs that are highly expressed during normal colonic epithelial differentiation and can also regulate the canonical Wnt/β-catenin pathway; in addition, their roles in the regulation of proliferation and differentiation in both normal colon and colon cancer are investigated. We isolated similar numbers of immature progenitor cells (EpCAMhigh CD44+; crypt base) and mature cells (EpCAMhigh CD44− CD66a+; top of the crypt) from both normal and colon cancer primary samples by FACS and performed real-time PCR. In normal human colon, we found that Tcf4 is highly expressed at the crypt base and is absent or low at the top of the crypt. In primary human colon cancer, Tcf4 expression is higher in tumorigenic cells (the fraction containing the CoCSCs) than the non-tumorigenic cells. Furthermore, we found several miRNAs that are differentially expressed in the top and bottom of the crypt of human and murine normal colon epithelium. One of the miRNAs, miR-203, is downregulated both in human NCSCs and CoCSCs. MiR-203 suppresses Tcf7l2 expression by specifically targeting its 3′UTR, as shown with a luciferase reporter. Western blotting showed that Tcf4 protein expression was decreased in colon cancer cell lines transfected with miR-203. Moreover, miR-203 suppresses Wnt/β-catenin signaling as assayed by TOP-flash. Overexpression of miR-203 inhibited the organoid-formation of murine normal colon and human xenograft CoCSCs in vitro. Furthermore, miR-203 suppressed in vivo tumorigenic capacity of human CoCSCs when injected subcutaneously in NOD/SCID interleukin-2 receptor γ chain null (Il2rg(-/-)) immunodeficient mice. In conclusion, miR-203 is downregulated in both in NCSCs and CoCSCs as compared to their more mature progeny, and regulates the canonical Wnt/β-catenin pathway by suppressing the expression of Tcf4. These findings suggest that normal stem/progenitor cells and CoCSCs share a similar molecular machinery to regulate their proliferation capacity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1012. doi:1538-7445.AM2012-1012