Tomomi Hayashi

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

Abstract 3376: CD133 plays a critical role in epithelial-mesenchymal transition related to pancreatic cancer migration and invasion

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective: Pancreatic cancer is an exceptionally devastating and incurable disease due to early local invasion and distant metastasis. Accumulated evidence shows that certain cells within solid tumors may reactivate a latent embryonic progress, epithelial-mesenchymal transition (EMT), during cancer progression. Through EMT, pancreatic tumor epithelial cells can acquire mesenchymal traits which facilitate migration and invasion. Previously, we reported that CD133-expressing cancer stem-like cells play an important role in tumorigenesis and chemoresistance of pancreatic cancer. In the present study we investigate whether CD133 plays a role in EMT using highly migrated cells enriched by a migration assay system. Methods: Expression of EMT characteristic markers, E-cadherin and vimentin, was examined in pancreatic cancer cell line, Capan-1. Highly migratory cell line Capan1-M9 was established by migration and invasion assay confirmed. CD133 expression was detected by FACS, real-time RT-PCR and Western blot. EMT-related genes (slug, snail, E-cadherin, vimentin, occludin, desmoplakin) were examined by real-time RT-PCR and Western blot. shRNA-CD133-GFP was transfected into Capan1-M9 by lentivirus vector to knock down endogenous CD133. Results: Typical EMT, which showed suppressed E-cadherin and elevated vimentin levels, was observed in migrating Capan-1 cells. Using a migration transwell system, we specifically established that Capan1-M9 cells showed migratory and invasive abilities two- to three-fold, compared with parental cells. FACS analysis showed that CD133 was enriched by more than 90% in Capan1-M9 cells. Furthermore, the elevated mRNA and protein levels of CD133 were confirmed in Capan1-M9 by real-time RT-PCR and Western blot, respectively. Importantly, as demonstrated by real-time RT-PCR, slug and snail EMT-related transcription factors in Capan1-M9 were upregulated by seven- and two-fold, respectively, compared with parental cells. Simultaneously, levels of vimentin mRNA expression increased two-fold. By contrast, occludin and desmoplakin cell-cell junction molecules were suppressed to half their previous level. Finally, shRNA-CD133-GFP achieved 85% knockdown of endogenous CD133, which not only inhibited migration and invasion but also suppressed slug EMT-related transcription factor expression to less than 20%. Conclusions: Our results suggest that CD133 contributes to migration and invasion of pancreatic cancer cells through a process which might trigger EMT, especially via slug upregulation. By targeting CD133-induced EMT in pancreatic cancer, transition to an invasive phenotype leading to metastasis may be preventable. 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 3376. doi:10.1158/1538-7445.AM2011-3376

Abstract 4245: The role of cancer stem-like cells in epithelial-mesenchymal transition (EMT) for migration and invasion of pancreatic cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Objectives: In the last AACR meeting, we reported that CD133 as a marker of cancer stem cells (CSCs) plays an important role in tumorigenesis of a pancreatic cancer cell line, Capan-1. In the present study, epithelial-mesenchymal transition (EMT) is considered as morphological and physiological conditions in tumor progression such as invasion and metastasis. Our aim is to investigate whether CSCs play an important role in EMT for cell migration and invasion in pancreatic cancer. Methods: 1) Cell line: Capan-1 2) Antibodies; Slug, Snail, Vimentin, E-cadherin, HIF-1a, Desmoplakin and Occludin 3) Isolation of CD133-positive cells by a FACSAria 4) migration and invasion assays 5) Western blotting 6) RT-PCR 7) Immunohistochemical study 8) gemcitabine (GEM) treatment test Results: 1) We established a highly migratory subclone (Capan-1M9) was isolated from pancreatic cancer cell line, Capan-1, using the transwell migration assay system, showing that the distribution of CD133-positive cells reached more than 90%. 2) CD133+ population of Capan-1 cells in the parental cells showed higher tumorigenesis and invasiveness than CD133- population of cells. Similarly, Capan-1M9 cells showed more invasive and migratory than the parental cells. 3) Percentage of CD133+ population of cells increased more than that of CD133- population of cells under the hypoxia conditions (1% or 0.1% O2). 4) EMT-related transcription factors, Slug and Snail in Capan-1M9 were detected by Western blotting and real time RT-PCR. The expression of Snail mRNA in Capan-1M9 was 2 times, while Slug mRNA was 9 times more than that of parental cells. Also, Vimentin mRNA increased in Capan-1M9 cells compared with that in parental cells. Moreover, we found that desmoplakin and occludin mRNA decreased in Capan-1M9 cells. 5) In hypoxic conditions, most of Capan-1M9 cells showed EMT phenomenon and they induced significantly higher expressions of HIF-1a, Slug and Snail compared to the normoxic condition. 6) The cell proliferation of Capan-1 decreased by GEM treatment, but the ratio of CD133+ population of cells increased from 37.4 to 45.5% with the GEM treatment, showing that CD133+ cells have the ability of resistance against GEM treatment as the standard treatment for pancreatic cancer. Conclusions: Taken together, CD133-positive cells in Capan-1 could be the candidate for EMT-related population of cells, suggesting that cancer stem cells play an important role in invasion via EMT in hypoxia and have the chemo-resistant ability. In addition, Capan-1M9 may be a useful model for screening new targeted agents for CSCs of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4245.