Cancer-associated fibroblasts contribute to cancer progression that is caused by epithelial-mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor-promoting cancer stroma. Here we report that a-smooth muscle actin-positive myofibroblast-like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC-derived myofibroblasts function to maintain tumorinitiating stem cell-like characteristics, including augmenting expression levels of various stemness-associated genes, enhancing sphere-forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both c-secretase inhibitor and siRNA directed against Jagged-1 attenuated MSC-associated E-cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC-derived myofibroblasts play important roles in regulating EMT and tumor-initiating stem cell-like properties of pancreatic cancer cells through an intermediating Notch signal. (Cancer Sci 2013; 104: 157-164) D uring tumor progression, epithelial-mesenchymal transition (EMT) contributes considerably to the malignant characteristics of tumors such as local invasion and distant metastasis.(1,2) Epithelial-mesenchymal transition has recently been reported as the key phenomenon that tightly regulates the stem cell-like characteristics of both normal and malignant cells. (3,4) Side population (SP) technology has been widely used to isolate the stem cell-enriched fraction in a variety of tissue. Side population cells are detected by their own ability to efflux Hoechst33342 dye through an ATP-binding cassette membrane transporter. We recently found that SP cells from pancreatic cancer cells are highly responsive to transforming growth factor-b (TGF-b)-mediated EMT, invasion, and metastasis.(5) Our results suggest that SP cells are enriched with cells that undergo mesenchymal-epithelial transition (MET) after TGF-b-associated EMT. Thus, our results indicated that an EMT ⁄ MET conversion is tightly linked to malignant potential in pancreatic cancer, such as invasion ⁄ metastasis. However, the mechanisms by which the EMT ⁄ MET status is regulated within a tumor in vivo remains undetermined.The tumor microenvironment consists of various stromal cells, including tumor-associated fibroblasts, endothelial cells, pericytes, adipocytes, and immune cells.(6) Among these cell types, cancer-associated fibroblasts (CAFs) and/or myofibroblasts have been recently implicated in regulating tumor progression, invasion, and metastasis. (7,8) Cancer-associated fibroblasts and myofibroblasts secrete a number of important inflammatory mediators, including MMP-2, -3, and -9, that can alter the stromal ECM and potentiate invasion, cell motility, and metastasis.

[Background] Side population (SP) cells are small fraction of cancer cells that have tumorigenic activity, chemotherapy resistance and metastatic potential. However, it remains to be clarified what characteristics SP cells possess. Here we report SP cells possess more invasive property than non-SP cells, occurring coincident with enhanced activation of a member of Rho-GTPase, Rac1. [Methods] Thirteen colon cancer cell lines were assessed for Rac1 activation by pull-down assay and the percentage of SP cell fraction within total number of cells by the flow cytometry-based side population (SP) technique to get invasive phenotype. Invasion assay was performed to evaluate SP cells’ behavior. Cell adhesion associated proteins such as E-cadherin and β-catenin and actin cytoskeleton were evaluated by immunocytochemistry [Results] We identified positive correlation between active Rac1 expression and SP cell frequency. In addition, Enhanced Rac1 activation and SP cell frequency were associated with poorly differentiated histology. Isolated SP cells expressed more active Rac1 as compared to non-SP cells, being associated with increased invasive activity in SP cells. Mesenchymal and amoeboid types of migration are based on reorganization of the actin cytoskeleton, but their requirements for Rho and Rac signaling differ. With respect to mesenchymal migration, membrane protrusions at the leading edge are formed in a Rac-dependent manner. Expression of a dominant-negative form of Rac in elongated cells inhibits the formation of such protrusive membrane structures, causing decreased cell migration. In contrast, Rho signaling is not essential for mesenchymal migration. With respect to amoeboid migration, the actin cytoskeleton is reorganized along the plasma membrane, causing dynamic membrane blebbing along the cell surface. Suppression of Rac1 activity by either dominant-negative Rac1 or dominant negative WAVE, a downstream mediator of Rac1 signaling reduced SP cell invasion activity. Over expression of constitutively active mutant of Rac1 enhanced SP cell motility. Furthermore, the E-cadherin attenuation and β-catenin enhancement were confirmed by immunohistocytochemistry after Rac1 inhibition. [Conclusions] We identified that Rac1 would appear to be a key molecule to mediate SP cells’ motility. We also implicate that suppression of Rac1 activity leads to reduction in number of invasive SP cells through restored E-cadherin function. Thus present study provides a concept for a new therapeutic strategy that targeting Rac1 molecule would be effective to suppress colon cancer invasive cell. Citation Format: Shinsuke Funakoshi, Toshifumi Azuma, Hajime Higuchi, Yasuo Hamamoto, Masayuki Adachi, Ayano Kabashima-Niibe, Akinori Hashiguchi, Hiromasa Takaishi, Toshifumi Hibi. Rac1 activation promotes invasive phenotype of side population in colon cancer cells by destabilizing the E cadherin-β-catenin ligation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4064. doi:10.1158/1538-7445.AM2013-4064

Cancer associate fibroblasts (CAFs), one of the major components of the cancer stroma, are prominently involved in tumor progression including epithelial to mesenchymal transition (EMT). Recently, bone marrow derived mesenchymal stem cells (MSCs) have been suggested as candidate for the source of tumor-promoting CAFs. We previously demonstrated that side population (SP) cells, which are enriched with so-called tumor initiating stem cell like cells (TISCs), play a pivotal role of local invasion and liver metastasis in pancreatic cancer (Int-J-Cancer 124:2771, 2009). Therefore, we formulated the hypothesis that MSCs regulate SP cells EMT and TISC-like properties such as tumor formation/progression. The AIM of the present study was to examine whether bone marrow-derived MSCs have ability to mediate EMT and/or tumor progression in pancreatic cancer SP cells. METHODS: MSCs were pre-treated with TGF-beta for 3 days. SP or non-SP cells were co-cultured in the presence or absence of TGF-beta-pretreated MSCs (Tb-MSCs) for 3 days. Real-time PCR experiments were performed to quantitate mRNA levels of EMT associated genes as well as so-called stemness related genes. PANC-1 derived SP or non-SP cells were co-injected with Tb-MSCs into NOD/SCID mice, and their tumorigenesity was evaluated. RESULTS: Co-culturing with Tb-MSCs dramatically induced EMT-associated mRNA alterations including suppression of E-cadherin and induction of slug in PANC-1 SP cells. In SP cells but not in non-SP cells, levels of stemness genes including CD133, Oct4, LGR5, were enhanced by co-culturing with Tb-MSCs. Tb-MSCs enhanced in vitro sphere forming activity as well as in vivo tumor formation in SP cells. These alterations including EMT-associated gene modifications and enhanced sphere formation were suppressed by Notch signal inhibition, suggested MSCs-associated regulation of EMT and TISC-like properties are mediated by Notch-signaling. In CONCLUSION, MSCs regulate EMT and they maintain TISC-like properties in pancreatic cancer SP cells via Notch signal-dependent mechanisms. Targeting Notch signaling would appear to be attractive to prevent MSCs and/or stromal cell-associated cancer progression. Citation Format: Ayano Kabashima-Niibe, Hajime Higuchi, Yumi Matsuzaki, Yo Mabuchi, Shinsuke Funakoshi, Masayuki Adachi, Yasuo Hamamoto, Hiromasa Takaishi, Toshifumi Hibi. Mesenchymal stem cells mediate epithelial-to-mesenchymal transition (EMT) and stemness regulation in pancreatic cancer via Notch signal. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 264. doi:10.1158/1538-7445.AM2013-264

BACKGROUND: Epithelial to Mesenchymal Transition (EMT) and Mesenchymal to Epithelial Transition (MET) may participate in pancreatic ductal adenocarcinoma (PDAC) metastasis. CD44 proteins are cell membrane proteins which function as co-receptors regulating many cellular functions. Some reports have shown that isoform switching from CD44 variant form (CD44v) to CD44 standard form (CD44s) takes place during the EMT. (Brown et al., J. Clin. Invest, 2011, Yae et al., Nat Commun., 2012). The AIM of this study was to explore the regulation of CD44 isoform switching in PDAC cells. Methods: The expression levels of CD44v and CD44s in PDAC cell lines were assessed by immunohistochemistry and immunoblot analysis. PDAC cell lines were implanted into immunodeficiency mice and treated with sulfasarazine for 14 days. Sulfasarazine, which inhibits glutamate-cysteine transport, reduces CD44v-dependent cell growth. CD44v or CD44s positive cells were isolated using flow cytometry and induced to undergo the EMT by TGF-β treatment (7.5ng/mL) for 72 hours. A doxycycline inducible system was employed to overexpress Slug, which promotes the EMT phenotype. To induce MET, the cells were released from TGF-β treatment and cultured for an additional 72 hours. E74-like ETS Transcription Factor 3 (ELF3) expression was knocked-down by siRNA transfection. RESULTS: Immunohistochemistry analysis revealed that CD44v protein is expressed specifically by PDAC with ductal structures within the complex tumor environment of these cancers. However, in contrast, CD44v expression was not observed in solitary infiltrating cancer cells. PANC-1, AsPC-1 and SUIT-2 PDAC cell lines expressed both CD44v and CD44s isoforms. Tumor mass in implanted PANC-1, AsPC-1 and SUIT-2 cells were reduced 69%, 50% and 36% respectively by sulfasarazine treatment following 2 weeks of therapy. However, CD44v null HPAC cells were resistant to sulfasarazine therapy following implantation. E-cadherin was expressed in CD44v positive cells but not in CD44s positive cells, and both CD44v and E-cadherin protein were reduced by TGF-β treatment. Slug over expressed cells were resistant to sulfasarazine treatment compared to non-Slug-induced cells. Removal of TGF-β restored both CD44v and E-cadherin expression levels which consistently occurs with MET. However, restoration of CD44v expression was diminished when ELF3 expression was reduced by siRNA transfection. CONCLUSION: The isoform switching between CD44v and CD44s was reversible. The expression of CD44v isoform appears to be controlled by ELF3 during MET. Citation Format: Ayano Kabashima-Niibe, Hiromasa Takaishi, Takanori Kanai, Hideyuki Saya, Gregory J. Gores, Hajime Higuchi. ELF3 modulates mesenchymal to epithelial transition through regulation of CD44 variant isoform expression in pancreatic cancer 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 857. doi:10.1158/1538-7445.AM2017-857