Taisaku Kuwahata

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.

M2-polarized tumor-associated macrophage infiltration of regional lymph nodes is associated with nodal lymphangiogenesis and occult nodal involvement in pN0 pancreatic cancer.

Objective Tumor-associated macrophages (TAMs) are reportedly involved in lymphangiogenesis in primary tumors, playing a crucial role in lymphatic metastasis. Furthermore, nodal lymphangiogenesis precedes and promotes regional lymph node (RLN) metastasis. We investigated the relationship of M2-polarized TAM infiltration of the RLNs, nodal lymphangiogenesis, and occult nodal involvement in pN0 pancreatic cancer. Methods Hematoxylin-eosin–stained primary tumor and regional LN specimens from 40 patients diagnosed with pN0 pancreatic cancer according to the pathological TNM classification were assessed. To evaluate lymphangiogenesis, lymphatic vessel density was measured by using D2-40 antibody. CD163 and cytokeratin AE1/AE3 antibodies were used to detect M2-polarized TAMs and isolated tumor cells in the RLNs, respectively. Results The nodal lymphatic vessel density had a strong association with the M2-polarized TAM density in the RLNs (P < 0.0001). Most of these TAMs expressed vascular endothelial growth factor C. Furthermore, in the RLNs, the M2-polarized TAM density was significantly associated with the incidence of isolated tumor cells (P = 0.0477). Conclusions M2-polarized TAM infiltration of RLNs is significantly associated with nodal lymphangiogenesis and occult nodal involvement in pN0 pancreatic cancer. Node-infiltrating M2-polarized TAMs may facilitate nodal lymphangiogenesis via the production of vascular endothelial growth factor C and thus promote RLN metastasis.

Epithelial–mesenchymal transition and mesenchymal–epithelial transition via regulation of ZEB-1 and ZEB-2 expression in pancreatic cancer†

Phenotypic plasticity of cancer cells via epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) is essential for tumor progression and metastasis.

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.

Significance of lymphangiogenesis in primary tumor and draining lymph nodes during lymphatic metastasis of pancreatic head cancer

The aim of this study was to investigate the significance of lymphangiogenesis in primary pancreatic tumors and in draining lymph nodes during lymphatic metastasis of pancreatic head cancers.

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)

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.

Advanced staging laparoscopy using single-incision approach for unresectable pancreatic cancer.

Purpose: As laparoscopy can detect imaging-occult metastatic lesions, it has been validated as a means of improving the assessment of tumor staging. Although controversy exists as to whether the procedure should be used routinely or selectively in pancreatic cancer patients, patients considered for treatment protocols for locally unresectable pancreatic cancer should be staged laparoscopically before initiation of therapy. We evaluate the feasibility and safety of advanced staging laparoscopy including peritoneal lavage cytology, laparoscopic ultrasound sonography (LUS), and LUS-guided biopsy through a single incision for locally advanced pancreatic cancer. Methods: Staging laparoscopy was performed in 44 patients with pancreatic cancer for deciding on treatment strategy. Our procedures included extensive peritoneal lavage of abdominal cavity for cytology, LUS for small metastasis detection, and tissue sample excision including LUS-guided biopsy. Eleven consecutive patients were treated with a single-incision staging laparoscopy approach (SI-SL group). The clinical parameters were compared between the SI-SL group and the multi-incision staging laparoscopy group (multi-incision group). Results: The mean operating time was longer and bleeding volume was less in the SI-SL group, although the differences were without statistical significance. The conversion rates to laparotomy were 9% in the SI-SL group and 30% in the multi-incision group. There were no severe postoperative complications. LUS-guided biopsy revealed malignancy for 3 patients in the SI-SL group. Conclusions: Advanced SI-SL is a feasible and safe alternative to the multi-incision approach for pancreatic cancer.

Abstract A52: Hypoxia inducible factor-1 alpha (HIF-1α) promotes migration and invasion of pancreatic cancer regulated by CD133 under hypoxia.

Background: Pancreatic cancer is a highly lethal disease and it is little sensitive to any current therapy. Over 17,000 patients died of pancreatic cancer, which is the fifth leading cause of cancer-related death in Japan. Multimodal treatments have completely failed to eradicate pancreatic cancer. A potential reason for the failure of the conventional therapeutic approach for pancreatic cancer might be explained by cancer stem cell (CSC) theory. CD133 (prominin-1) is a 5-transmembrane glycoprotein, which is well known as one of CSC markers in pancreatic cancer. We previously reported that CD133 expression in pancreatic cancer is correlated with poor prognosis and lymph node metastasis[1]. However, the function of CD133 is still obscure. On the other hand, pancreatic cancer is surrounded with desmoplastic morphology, which is under the low-oxygen condition because of its insufficient blood supply. In the present study, we hypothesized that CD133 plays a key role in invasion and metastasis for pancreatic cancer associated with HIF1-α expression. Materials and Methods: 1) We investigated the relationship between HIF-1α or CD133 expression in primary tumor tissues and survival for pancreatic cancer patients. 2) To clarify the mechanism of these relationships, we established a highly migratory cell line, Capan1M9, which is a subpopulation derived from Capan-1 cells by migration assay system. Over 90% of Capan1M9 cells express CD133[2]. Consequently, we established Capan1M9-GFP-shCD133 cells (CD133 knockdown) by lentiviral transduction method. Using these cell lines, other methods such as migration and invasion assays, wound healing assay, immunofluorescence staining, Western blot, and real-time quantitative RT-PCR were performed. Results: 1) Immunohistochemical study showed that HIF-1α expression positively correlated with poor prognosis and microvessel density in pancreatic cancer samples. 2) CD133+ cell population in Capan-1 showed more resistant to hypoxic condition (1% O2 and 0.1% O2) than CD133cell population by FACS analyses. This result indicated that CD133 contributed to the resistance of cancer cells in hypoxia. 3) We compared CD133+ cells (Capan-M9) and CD133-cells (shRNA-CD133-M9) under 1% O2 hypoxia. In hypoxia, CD133+ cells showed higher expression of HIF-1α than CD133cells. HIF-1α accumulation was observed in both of the nuclei of CD133+ and CD133cells under hypoxia. 4) HRE response was also showed high level in Luc-CD133+ cells than Luc-CD133-cells by luciferase assay in hypoxia. 5) Migration ability of CD133+ cells was higher than that of CD133-cells by wound healing assay and migration assay in hypoxic conditions. 6) Epithelial-mesenchymal transition (EMT)-related molecules such as Slug, N-cadherin and fibronectin were higher in Capan1-M9 (migratory subclone cells) than in Capan-1 (parental cells). Conclusion: These results indicated that HIF-1α stimulates migration and invasion of pancreatic cancer associating with EMT and CD133 expression under hypoxia. References: 1) Maeda S, et al. CD133 expression is correlated with lymph node metastasis and vascular endothelial growth factor-C expression in pancreatic cancer. Br J Cancer 98:1389-1397, 2008. 2) Ding Q, et al. Establishment of a highly migratory subclone reveals that CD133 contributes to migration and invasion through epithelial-mesenchymal transition in pancreatic cancer. Human Cell 25:1-8, 2012. Citation Format: Koki Maeda, Kosei Maemura, Shoji Natsugoe, Sonshin Takao, Qiang Ding, Makoto Yoshimitsu, Taisaku Kuwahata, Tomomi Hayashi, Shyuichiro Matsubara, Toru Obara, Yumi Miyazaki, Hiroyuki Shinchi. Hypoxia inducible factor-1 alpha (HIF-1α) promotes migration and invasion of pancreatic cancer regulated by CD133 under hypoxia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A52.

Abstract 2459: Interferon-α contributes to combined chemotherapy for CD133+ cancer stem cells in pancreatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objectives: Pancreatic cancer is a highly lethal disease because of high resistance to anti-cancer agents. Recent evidence suggests that a small population of cancer stem cells (CSCs) in solid tumors sustain tumor formation, self-renewal and drug resistance. CD133+ CSCs are reportedly essential for the development and perpetuation of pancreatic cancer and may account for resistance to standard chemotherapy drugs such as gemcitabine (GEM). Interferon-alpha (IFN-α) is a cytokine with pleiotropic effects, possessing direct cytotoxic and cytostatic effects on tumor cells. In the present study, we investigate the effect of IFN-α on combined chemotherapy in CD133+ pancreatic cancer cells. Methods: CD133+ and CD133− populations of Capan-1 cells, a human pancreatic cancer cell line, were isolated by FACS, and sphere formation assay and tumorigenic assay were performed. Capan-1 cells were treated by GEM with or without IFN-α and BrdU assay was used for cell-cycle analysis. Growth of xenograft tumors in nude mice was assessed after 3-weeks treatment with vehicle, GEM (1200 mg/kg/wk) alone, IFN-α (20000 U/mouse/every 2 days) alone or GEM combined with IFN-α. Results: 1) CD133+ population of Capan-1 cells showed CSC-like properties such as generation of spheres in the serum-free culture and tumorigenesis when implanted into NOD/SCID mice. 2) The growth inhibition by GEM treatment (IC50: 100 ng/mL) showed a significant difference between CD133+ and CD133− populations of Capan-1 cells, resulting in an increase of the CD133+ population. Similarly, CD133 protein levels increased in a time-dependent manner after GEM treatment by Western blot. 3) GEM treatment reduced S phase of both CD133+ and CD133− cells, and significantly induced apoptosis of CD133− cells more than that of CD133+ cells by BrdU assay. However, GEM treatment increased G0/G1 phase of CD133+ cells, indicating that CD133+ cells showed GEM resistance through the increase of G0/G1 phase. 4) All Capan-1 cells expressed IFN-α/β receptor II by FCM and immunohistochemical analyses and IFN-α (MED: 5000 U/mL) treatment reduced the CD133+ population ratio of Capan-1 cells. Importantly, IFN-α showed not only the decrease of G0/G1 phase but also the increase of S phase in CD133+ cells. Consequently, GEM combined with IFN-α treatment significantly increased the apoptotic phases in both CD133+ and CD133− cells. 5) In the in vivo experiment, GEM combined with IFN-α treatment similarly suppressed the tumor growth of Capan-1 cells. Conclusions: Our results indicate that IFN-α contributes to combined chemotherapy for CD133+ pancreatic cancer stem cells by modulating the cell cycle and/or cell proliferation, suggesting a clinical potential for treatment of pancreatic CSCs. Further investigation of the interactions between IFN-α and key regulatory pathways in CSC should be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2459. doi:10.1158/1538-7445.AM2011-2459