Yumi Miyazaki

mTOR plays critical roles in pancreatic cancer stem cells through specific and stemness-related functions

Pancreatic cancer is characterized by near-universal mutations in KRAS. The mammalian target of rapamycin (mTOR), which functions downstream of RAS, has divergent effects on stem cells. In the present study, we investigated the significance of the mTOR pathway in maintaining the properties of pancreatic cancer stem cells. The mTOR inhibitor, rapamycin, reduced the viability of CD133+ pancreatic cancer cells and sphere formation which is an index of self-renewal of stem-like cells, indicating that the mTOR pathway functions to maintain cancer stem-like cells. Further, rapamycin had different effects on CD133+ cells compared to cyclopamine which is an inhibitor of the Hedgehog pathway. Thus, the mTOR pathway has a distinct role although both pathways maintain pancreatic cancer stem cells. Therefore, mTOR might be a promising target to eliminate pancreatic cancer stem cells.

Establishment of a highly migratory subclone reveals that CD133 contributes to migration and invasion through epithelial–mesenchymal transition in pancreatic cancer

Pancreatic cancer is a lethal disease because of invasion and early metastasis. Although CD133, a marker of cancer stem cells (CSCs) in a variety of solid tumors, has been studied in recent decades, its function remains obscure. Recent reports suggest that epithelial–mesenchymal transition (EMT) may be related to the properties of CSCs. In this study, we investigated whether CSC markers are associated with EMT. For Capan1M9, a highly migratory cell subclone established from human pancreatic cancer cell line Capan-1, CD133 expression, migration, and invasion were greater than for the parent cells. In Capan1M9 cells, the EMT-related transcription factors Slug and Snail were up-regulated, and N-cadherin and fibronectin were also substantially increased. In contrast, occludin and desmoplakin were suppressed. Knockdown of endogenous CD133 in the Capan1M9 cells led to Slug suppression and reduction of migration and invasion. Taken together, CD133 has an important role in migration and invasion by facilitating EMT in pancreatic cancer cells.

CD133 facilitates epithelial-mesenchymal transition through interaction with the ERK pathway in pancreatic cancer metastasis

BackgroundPancreatic cancer is a lethal disease due to the high incidence of metastasis at the time of detection. CD133 expression in clinical pancreatic cancer correlates with poor prognosis and metastasis. However, the molecular mechanism of CD133-regulated metastasis remains unclear. In recent years, epithelial-mesenchymal transition (EMT) has been linked to cancer invasion and metastasis. In the present study we investigated the role of CD133 in pancreatic cancer metastasis and its potential regulatory network.MethodsA highly migratory pancreatic cancer cell line, Capan1M9, was established previously. After shRNA was stable transducted to knock down CD133 in Capan1M9 cells, gene expression was profiled by DNA microarray. Orthotopic, splenic and intravenous transplantation mouse models were set up to examine the tumorigenesis and metastatic capabilities of these cells. In further experiments, real-time RT-PCR, Western blot and co-immunoprecipitate were conducted to evaluate the interactions of CD133, Slug, N-cadherin, ERK1/2 and SRC.ResultsWe found that CD133+ human pancreatic cancer cells were prone to generating metastatic nodules in in vivo models using immunodeficient mice. In contrast, CD133 knockdown suppressed cancer invasion and metastasis in vivo. Gene profiling analysis suggested that CD133 modulated mesenchymal characteristics including the expression of EMT-related genes, such as Slug and N-cadherin. These genes were down-regulated following CD133 knockdown. Moreover, CD133 expression could be modulated by the extracellular signal-regulated kinase (ERK)1/2 and SRC signaling pathways. The binding of CD133 to ERK1/2 and SRC acts as an indispensable mediator of N-cadherin expression.ConclusionsThese results demonstrate that CD133 plays a critical role in facilitating the EMT regulatory loop, specifically by upregulating N-cadherin expression, leading to the invasion and metastasis of pancreatic cancer cells. Our study provides a novel insight into the function of CD133 in the EMT program and a better understanding of the mechanism underlying the involvement of CD133 in pancreatic cancer metastasis.

miR-30 family promotes migratory and invasive abilities in CD133(+) pancreatic cancer stem-like cells.

Pancreatic cancer is a deadly disease with a poor prognosis. Recently, miRNAs have been reported to be abnormally expressed in several cancers and play a role in cancer development and progression. However, the role of miRNA in cancer stem cells remains unclear. Therefore, our aim was to investigate the role of miRNA in the CD133+ pancreatic cancer cell line Capan-1M9 because CD133 is a putative marker of pancreatic cancer stem cells. Using miRNA microarray, we found that the expression level of the miR-30 family decreased in CD133 genetic knockdown shCD133 Capan-1M9 cells. We focused on miR-30a, -30b, and -30c in the miR-30 family and created pancreatic cancer cell sublines, each transfected with these miRNAs. High expression of miR-30a, -30b, or -30c had no effect on cell proliferation and sphere forming. In contrast, these sublines were resistant to gemcitabine, which is a standard anticancer drug for pancreatic cancer, and in addition, promoted migration and invasion. Moreover, mesenchymal markers were up-regulated by these miRNAs, suggesting that mesenchymal phenotype is associated with an increase in migration and invasion. Thus, our study demonstrated that high expression of the miR-30 family modulated by CD133 promotes migratory and invasive abilities in CD133+ pancreatic cancer cells. These findings suggest that targeted therapies to the miR-30 family contribute to the development of novel therapies for CD133+ pancreatic cancer stem cells.

Interferon-alpha modulates the chemosensitivity of CD133-expressing pancreatic cancer cells to gemcitabine

Pancreatic cancer is a lethal disease as current chemotherapies with gemcitabine (GEM) are still insufficient. Accumulating evidence suggests that cancer stem cells (CSC) are responsible for chemoresistance and that CD133 is one of the CSC markers in pancreatic cancer. Interferon‐alpha (IFN‐α), a cytokine with pleiotropic effects, has direct cytotoxic and cytostatic effects on tumor cells. The aim of the present study was to investigate whether IFN‐α can modulate the chemosensitivity of a human pancreatic cancer cell line, Capan‐1, to GEM. Cell cycles were evaluated for response to GEM with and without IFN‐α by BrdU assay. GEM inhibited Capan‐1 cell growth in a dose‐dependent manner. GEM (IC50; 100 ng/mL) treatment reduced the number of both CD133+ and CD133− cells in the S phase, induced apoptosis of CD133− cells more than that of CD133+ cells and increased accumulation of CD133+ cells into the G0/G1 phase. These results infer that CD133+ cells take shelter into the G0/G1 phase from GEM treatment. IFN‐α modulated CD133+ cells from the G0/G1 phase to the S phase. Consequently, apoptosis was accelerated in both CD133+ and CD133− cells after IFN‐α combined with GEM treatment. Furthermore, GEM combined with IFN‐α treatment showed a significant tumor suppressive effect in the in vivo study. Importantly, CD133+ cells showed CSC‐like properties, such as generation of spheres, highly invasive ability and high tumorigenesis. These results suggest that IFN‐α, as a modulator, could contribute to the treatment of CD133+ cancer cells and be effective in combined chemotherapies with GEM for pancreatic cancer stem‐like cells. (Cancer Sci 2012; 103: 889–896)

Slug contributes to gemcitabine resistance through epithelial-mesenchymal transition in CD133+ pancreatic cancer cells

CD133-positive pancreatic cancer is correlated with unfavorable survival despite current development of therapy. Slug acts as a master regulator of epithelial-mesenchymal transition (EMT) which is the essential process in cancer progression. The aim of this study was to investigate the role of Slug in gemcitabine treatment for CD133-positive pancreatic cancer cells. We used a previously established pancreatic cancer cell line expressing high level of CD133 (Capan-1M9), which also expresses high level of Slug. We generated Slug knock-down subclone (shSlug M9) from this cell line, and compared expression of EMT-related genes, migration, invasion and gemcitabine resistance between two cell lines. Slug knock-down in CD133-positive pancreatic cancer cell line led to the reduction of migration and invasion ability. Furthermore, Slug knock-down sensitized CD133-positive pancreatic cancer cell line to gemcitabine. These results suggest that Slug plays an important role in not only invasion ability through EMT but also gemcitabine resistance of CD133-positive pancreatic cancer cells.

CD133 Modulate HIF-1α Expression under Hypoxia in EMT Phenotype Pancreatic Cancer Stem-Like Cells.

Although CD133 is a known representative cancer stem cell marker, its function in tumor aggressiveness under hypoxia is not fully known. The aim of this study is to demonstrate that CD133 regulates hypoxia inducible factor (HIF)-1α expression with tumor migration. The CD133+ pancreatic cancer cell line, Capan1M9, was compared with the CD133− cell line, shCD133M9, under hypoxia. HIF-1α expression levels were compared by Western blot, HIF-1α nucleus translocation assay and real-time (RT)-PCR. The hypoxia responsive element (HRE) was observed by luciferase assay. The migration ability was analyzed by migration and wound healing assays. Epithelial mesenchymal transition (EMT) related genes were analyzed by real-time RT-PCR. HIF-1α was highly expressed in Capan1M9 compared to shCD133M9 under hypoxia because of the high activation of HRE. Furthermore, the migration ability of Capan1M9 was higher than that of shCD133M9 under hypoxia, suggesting higher expression of EMT related genes in Capan1M9 compared to shCD133M9. Conclusion: HIF-1α expression under hypoxia in CD133+ pancreatic cancer cells correlated with tumor cell migration through EMT gene expression. Understanding the function of CD133 in cancer aggressiveness provides a novel therapeutic approach to eradicate pancreatic cancer stem cells.

Abstract 1533: The roles of novel tumor-associated macrophages (TAMs) in invasion and metastasis of pancreatic cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Pancreatic cancer (PC) is a lethal disease due to its systemic metastasis. In the recent years, compelling evidence has emerged that the complex crosstalk between stromal cells and cancer cells in the tumor microenvironment may regulate tumor growth, invasion and metastasis. The tumor microenvironment of PC consists mainly of a dense, desmoplastic stroma rich in collagen fibers, extracellular matrix proteins, fibroblasts, and infiltrating inflammatory cells. M2 macrophages, so-called tumor-associated macrophages (TAMs), infiltrate and interact with cancer cells. However, the interaction between PC and TAMs are not fully understood underlying mechanism. Our evidence suggests that the expression of FRβ is limited to activated macrophages in human malignancy, such as PC. Recently, we are trying to investigate the role of FRβ+ TAMs in PC. The aim of this study is to investigate how PC cells cooperate with FRβ+ TAM infiltration in the invasive front. Methods: Tumor samples were obtained from 76 patients with PC. None of these patients had received any preoperative chemotherapy or radiotherapy. Both FRβ+ and tumor-infiltrating (CD68+) macrophages were examined in each tumor specimen by immunohistochemical and immunofluorescence staining using a newly developed anti-human FRβ monoclonal antibody and CD68 antibody. Results: 1) FRβ+ macrophages showed a heterogeneous distribution with foci of high density in PC. In contrast, they were rarely observed in noncancerous pancreatic tissues. Compared with CD68+ macrophages, a larger population of FRβ+ macrophages existed in the invasive front. Although the numbers of CD68+ and FRβ+ macrophages were both higher in the invasive front compared to the central portion of pancreatic tumors, the proportion of FRβ+ macrophages to CD68+ macrophages in the invasive front (71.8%) was significantly higher than that in the central portion (44.6%). 2) We evaluated the expression of VEGF in tumor-infiltrating macrophages by double immunofluorescence staining. The majority of FRβ+ macrophages showed distinct VEGF expression. By contrast, lower numbers of CD68+ macrophages showed VEGF expression. The mean proportion of FRβ+ macrophages with VEGF expression (70.1%) was significantly higher than that of CD68+ macrophages with VEGF expression (40.5%). 3) A high number of FRβ+ macrophages showed a positive association with high MVD, high incidence of hematogenous metastasis and a poor prognosis in PC patients. Conclusions: Our findings revealed for the first time that FRβ+ macrophages are a novel subset of TAMs in PC and may play an important role in the tumor microenvironment in association with systemic metastasis through the interaction with tumor cells and vessels. Therefore, FRβ+ macrophages may be promising a targeted therapy for PC. Citation Format: Takao Sonshin, Qiang Ding, Taku Nagai, Hiroshi Kurahara, Makoto Yoshimitsu, Yumi Miyazaki, Ryoko Imakiire, Shoko Ueno, Hiromi Tokushige, Shyuichiro Matsubara, Toru Obara, Takami Matsuyama. The roles of novel tumor-associated macrophages (TAMs) in invasion and metastasis of pancreatic cancer. [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 1533. doi:10.1158/1538-7445.AM2013-1533

Abstract 294: CD133/ERK axis mediates invasion and metastasis through epithelial to mesenchymal transition in pancreatic cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Over 90% of pancreatic cancer-associated mortality is due to metastasis, which involves several serial steps from disseminating of primary cancer cells to colonizing in the distant organs. CD133, a marker of cancer stem cells (CSCs) in various solid tumors including pancreatic cancer, has been studied for recent decade. However, the role of CD133 is still obscure. It has been supposed that the epithelial-mesenchymal transition (EMT) is one of CSC properties in the process of metastasis. The present study is to investigate the regulatory function of CD133 in migration and invasion of pancreatic cancer (PC). Methods: We previously reported the establishment of endogenous CD133high PC cells derived from a human PC cell line, Capan-1, at AACR 2011 Meeting. CD133high fraction was sorted by FACS, and CD133 expression was detected by real-time RT-PCR and Western blot. shRNA-CD133-GFP or shRNA-Slug-GFP was transfected into CD133high PC cells to establish CD133knock-down or Slug knock-down PC cells by lentivirus vector to knock down CD133 or Slug, resulting in 80-85% knock-down of endogenous CD133 or Slug. To examine EMT, we used migration and invasion assay, wound healing assay, DNA microarray, real-time RT-PCR and Western blot. Results: FACS analysis showed that CD133 was enriched by more than 90% in CD133high PC cells. CD133high PC cells showed migratory and invasive abilities two- to three-fold compared with parental cells or CD133knock-down PC cells. Expressions of Slug, N-cadherin and fibronectin were upregulated in the CD133high PC cells, which are characteristic components of EMT by DNA microarray and real-time RT-PCR, but these were downregulated in CD133knock-down PC cells. In addition, N-cadherin could be downregulated in Slug knock-down PC cells but no change of CD133 expression. We found that not only N-cadherin also Slug and CD133 were down regulated significantly by ERK inhibitor (U0126) administration. Further we treated the CD133high PC cells with N-cadherin neutral antibody, the wound healing speed was significantly slower than that without antibody neutralization. Conclusion: Taken together, N-cadherin plays an important role in migration and invasion. CD133/ERK axis and Slug interacted as a modulation loop on N-cadherin expression. Further study should shed new light to understand the orchestrated network associated with CD133 of pancreatic cancer metastasis. These insights on CD133 regulation could be a promising novel targeted therapy for pancreatic cancer. Citation Format: Qiang Ding, Makoto Yoshimitsu, Koichiro Tsukasa, Yumi Miyazaki, Shyuichiro Matsubara, Toru Obara, Sonshin Takao. CD133/ERK axis mediates invasion and metastasis through epithelial to mesenchymal transition in pancreatic cancer. [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 294. doi:10.1158/1538-7445.AM2013-294

Abstract 1486: Role of Slug in chemoresistance, invasion and metastasis of CD133-expressing pancreatic cancer.

Background: Pancreatic cancer is a devastating disease despite current development of therapy. Accumulating evidence suggests that cancer stem cells (CSCs) are responsible for recurrence and chemoresistance. CD133, a marker of CSCs in various solid tumors including pancreatic cancer, has been studied for recent decade. However, the role of CD133 is still obscure. Epithelial-mesenchymal transition (EMT), which is the essential process in the wound healing, development and cancer progression, is one of the CSC characteristics. However, the role of EMT in the pancreatic cancer progression is not sufficiently elucidated. Slug is a member of the Snail family of zinc finger transcription factors and plays a role in the EMT. Slug is demonstrated to be overexpressed in numerous cancers, and involved in migration and tumorigenesis. Recent study demonstrated that EMT was directly suppressed by downregulation of the transcription of Slug (Chakrabarti et al, Nat Cell Biol, 2012). In this study, we investigated the relationship between Slug and biological properties in pancreatic cancer cells. Materials and Methods: We established a highly migratory cell population with endogenous CD133 high derived from Capan-1 cells by migration assay system. Over 90% of CD133 high cells expressed CD133 by flow cytometer. Consequently, we established CD133 knock-down and Slug knock-down by lentiviral transduction method. We used gemcitabine (GEM) for chemosensitivity assay. To examine the role of Slug, we used migration and invasion assay, wound healing assay, DNA microarray, real-time RT-PCR and Western blot. Results: 1) DNA microarray and real-time RT-PCR revealed higher expression of EMT-related genes, such as Slug and N-cadherin, in CD133 high cells compared to CD133 knock-down cells, suggesting that CD133 knock-down related to downregulation of Slug. 2) Slug knock-down cells showed more sensitive to GEM by MTT assay compared to parental cells (CD133 high cells), but no change of CD133 expression. 3) Slug knock-down cells showed less migratory and invasive abilities than CD133 high cells. 4) CD133 high cells showed more in vivo metastasis compared to CD133 knock-down cells using immunodeficient mice. In vivo experiment using Slug knock-down cells is now ongoing. Conclusion: Our results suggest that Slug plays an important role in chemoresistance, invasion and metastasis. Citation Format: Koichirou Tsukasa, Qiang Ding, Makoto Yoshimitsu, Yumi Miyazaki, Toru Obara, Shyuichiro Matsubara, Koki Maeda, Sonshin Takao. Role of Slug in chemoresistance, invasion and metastasis of CD133-expressing pancreatic cancer. [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 1486. doi:10.1158/1538-7445.AM2013-1486