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Genetic alterations in the TGFβ signaling pathway in combination with oncogenic alterations lead to cancer development in the intestines. However, the mechanisms of TGFβ signaling suppression in malignant progression of intestinal tumors have not yet been fully understood. We have examined ApcΔ716 Tgfbr2ΔIEC compound mutant mice that carry mutations in Apc and Tgfbr2 genes in the intestinal epithelial cells. We found inflammatory microenvironment only in the invasive intestinal adenocarcinomas but not in noninvasive benign polyps of the same mice. We thus treated simple Tgfbr2ΔIEC mice with dextran sodium sulfate (DSS) that causes ulcerative colitis. Importantly, these Tgfbr2ΔIEC mice developed invasive colon cancer associated with chronic inflammation. We also found that TGFβ signaling is suppressed in human colitis–associated colon cancer cells. In the mouse invasive tumors, macrophages infiltrated and expressed MT1-MMP, causing MMP2 activation. These results suggest that inflammatory microenvironment contributes to submucosal invasion of TGFβ signaling–repressed epithelial cells through activation of MMP2. We further found that regeneration was impaired in Tgfbr2ΔIEC mice for intestinal mucosa damaged by DSS treatment or X-ray irradiation, resulting in the expansion of undifferentiated epithelial cell population. Moreover, organoids of intestinal epithelial cells cultured from irradiated Tgfbr2ΔIEC mice formed “long crypts” in Matrigel, suggesting acquisition of an invasive phenotype into the extracellular matrix. These results, taken together, indicate that a simple genetic alteration in the TGFβ signaling pathway in the inflamed and regenerating intestinal mucosa can cause invasive intestinal tumors. Such a mechanism may play a role in the colon carcinogenesis associated with inflammatory bowel disease in humans. Cancer Res; 75(4); 766–76. ©2015 AACR.

The intestinal epithelium maintains homeostasis by a self-renewal process involving resident stem cells, including Lgr5+ crypt-base columnar cells, but core mechanisms and their contributions to intestinal cancer are not fully defined. In this study, we examined a hypothesized role for KLF5, a zinc-finger transcription factor that is critical to maintain the integrity of embryonic and induced pluripotent stem cells, in intestinal stem-cell integrity and cancer in the mouse. Klf5 was indispensable for the integrity and oncogenic transformation of intestinal stem cells. In mice, inducible deletion of Klf5 in Lgr5+ stem cells suppressed their proliferation and survival in a manner associated with nuclear localization of β-catenin (Catnb), generating abnormal apoptotic cells in intestinal crypts. Moreover, production of lethal adenomas and carcinomas by specific expression of an oncogenic mutant of β-catenin in Lgr5+ stem cells was suppressed completely by Klf5 deletion in the same cells. Given that activation of the Wnt/β-catenin pathway is the most frequently altered pathway in human colorectal cancer, our results argue that KLF5 acts as a fundamental core regulator of intestinal oncogenesis at the stem-cell level, and they suggest KLF5 targeting as a rational strategy to eradicate stem-like cells in colorectal cancer. Cancer Res; 74(10); 2882–91. ©2014 AACR.

Background: The development of trastuzumab has had a major impact in treatment of breast cancer patients with HER2 overexpression. Despite clinical usefulness, intrinsic or acquired resistance to trastuzumab is a common clinical phenomenon. However, the mechanisms of resistance to trastuzumab have not been fully elucidated. Potential mechanisms include aberrant downstream signaling caused by loss of PTEN and/or PIK3CA mutation, alternative signaling from IGF-1R and masking with MUC4. The objective of this study was to determine the possible mechanisms of resistance to trastuzumab as neoadjuvant therapy in women with HER2-overexpressing operable breast cancer. Patients & methods: Patients with operable breast cancer received 12 cycles of weekly paclitaxel (80 mg/m2 IV) plus weekly trastuzumab (4mg/kg loading dose followed by 2 mg/kg IV) for 12 weeks before surgery. All tumors were HER2-positive by immunohistochemistry (IHC) or fluorescence in situ hybridization. Expressions of ER, PgR, Ki67, PTEN, phosphorylated IGF-1R (pIGF-1R) and MUC4 were performed by IHC in core needle biopsy samples at baseline. ER and PgR status was assessed using Allred score. For Ki67 labeling index, a total of 400 cells were counted from three consecutive high-power magnifications. PTEN, pIGF-1R and MUC4 expression level was scored semiquantitatively based on staining intensity. PIK3CA mutation status was evaluated by sequencing of PIK3CA exons 9 and 20 using PCR amplification and direct sequencing. pCR was defined as no residual invasive carcinoma in the breast. Results: Thirty-seven patients were enrolled and assessable for clinical and pathologic responses. ER and PgR were positive in 18 (48.6%) and 16 (43.2%) patients, respectively. The overall response rate was 86.5%, including a complete response in 21 patients and a partial response in 11 patients. The pCR rate was 48.6% (18/37). Negative, moderate and strong membranous expression of MUC4 was observed in 3 (8.1%), 18 (48.6%) and 12 (32.4%) patients, respectively. Membranous staining for pIGF1-R was negative in 24 (64.9%) patients. PTEN loss was observed in 33.3% (8/24) of the tumor examined. PIK3CA sequence analysis of the 13 tumors identified 2 mutations in exon 20 and 2 mutations in exon 9, corresponding to a PIK3CA mutation frequency of 30.8%. MUC4 and pIGF1-R status did not affect the pCR rate. PTEN loss and/or PIK3CA mutation were not significantly associated with pCR rate. pCR rate was significantly correlated with PgR negativity (p = 0.004) and higher Ki67 (p = 0.01). Conclusions: These data indicate that aberrant downstream signaling caused by loss of PTEN and/or PIK3CA mutation, alternative signaling from IGF-1R and masking with MUC4 were not important mechanisms of resistance to trastuzumab. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-22.

Background Concurrent trastuzumab with paclitaxel/fluorouracil, epirubicin, and cyclophosphamide (P/FEC) chemotherapy as neoadjuvant treatment revealed the high pathologic complete response (pCR) rate of 54.5% (Buzdar, 2007). Deregulation of the phosphatidylinositol 3-kinase (PI3K) pathway either through loss of PTEN or mutation of the catalytic subunit alpha of PI3K (PIK3CA) might confer resistance to trastuzumab. The objective of this study was to determine the efficacy of concurrent administration of trastuzumab and paclitaxel wtihout anthracycline as preoperative chemotherapy. The correlation between deregulation of PI3K and resistance to trastuzumab was also investigated. Patients & methods Patients with HER2-positive, operable breast cancer received 12 cycles of weekly paclitaxel (80 mg/m2) and trastuzumab (4mg/kg loading dose then 2 mg/kg) for 12 weeks before surgery. PTEN status was evaluated by immunohistochemistry. PTEN staining intensity scores was recorded on an integer scale from 0 to 2+ (0; no staining, 1+; reduced staining, and 2+; equal staining as compared to the internal control). Sequencing of PIK3CA exons 9 and 20 was done by PCR amplification and direct sequencing. pCR was defined as no residual invasive carcinoma in the breast. Results Twenty-eight patients were enrolled and assessable for clinical and pathologic responses. The overall response rate was 92.9%, including a complete response in 13 patients and a partial response in 13 patients. The pCR rate was 53.6% (15/28). Twenty-three patients (82.1%) underwent breast concerving surgery. Progesteron receptor (PgR) status was significantly correlated with pCR (p=0.025). Eight of 24 patients (33.3%) were scored PTEN negative. PIK3CA mutations were identified in 4 of 13 patients (30.8%). There was no significant difference in pCR rate and PTEN loss/PIK3CA mutation. Correlation of PgR status and pCR Conclusions These data indicate that the combination of trastuzumab and paclitaxel without anthracycline is effective preoperative chemotherapy with high pCR rate. PTEN loss and/or PIK3CA mutation were not useful predictors of resistance to trastuzumab. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-11-04.

Background and Aim: Intrahepatic cholangiocarcinoma (IHCC) is the second most prevalent malignancy in the liver, and is one of the most aggressive malignancies characterized by a poor prognosis. It is believed that IHCC is an epithelial cell malignancy arising from cholangiocytes composing the intrahepatic bile ducts. However, patients with viral hepatitis develop IHCC, suggesting that IHCC is derived from transformed hepatocytes. Indeed, recent studies have revealed that IHCC can arise from Notch-mediated conversion of hepatocytes in mice. The new 3D culture system called organoid culture has been developed, which allows long-term expansion of Lgr5-positive stem cells. To clarify whether IHCC cells harbor the potency of conversion into functional hepatocytes, we established organoids derived from human IHCC and cultured them in different culture conditions. Methods: We established organoids derived from human IHCC and cultured these organoids with expansion medium (EM) containing R-spondin 1. Conversion of IHCC cells into hepatocytes was conducted by organoid culture with differentiation medium (DM) containing BMP7, DAPT, FGF19 and dexamethasone. Results: Gene set enrichment analysis (GSEA) showed that genes associated with hepatocyte differentiation signature were significantly enriched in IHCC organoids cultured with DM compared with those cultured with EM. IHCC organoids cultured with DM induced up-regulation of hepatocyte markers albumin, HNF4a and CYP3A4, and acquired mature hepatocyte functions including albumin secretion, bile acid production and increased CYP3A4 activity. Removal of R-spondin 1, a ligand of Wnt signaling pathway, from EM enhanced hepatocyte differentiation. IHCC organoids cultured with DM significantly reduced the malignant potential in vitro and in vivo compared with those cultured with EM. Further studies using thioacetamide (TAA)-induced IHCC model mice showed that TAA-induced inflammation in the liver promoted recruited macrophages to secrete Wnt3a. Conclusions: These findings indicated that IHCC cells can be converted into functional hepatocytes by inhibition of Wnt signaling pathway. Activation of Wnt signaling pathway by recruited macrophages may play a critical role in the malignant transformation of hepatocytes into IHCC, which could be an important therapeutic target for IHCC. Citation Format: Toshiaki Nakaoka, Yoshimasa Saito, Toshihide Muramatsu, Hidenori Ojima, Yae Kanai, Yuko Sugiyama, Masaki Kimura, Takanori Kanai, Toshiro Sato, Hidetsugu Saito. Intrahepatic cholangiocarcinoma cells can be converted into functional hepatocytes by inhibition of Wnt signaling pathway [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 5768. doi:10.1158/1538-7445.AM2017-5768