Prevention of Akt phosphorylation is a key to targeting cancer stem-like cells by mTOR inhibition.

CD133 expression in pancreatic cancer correlates with poor prognosis and increased metastasis. CD133+ pancreatic cancer cells exhibit cancer stem cell (CSC)-like properties. We established a CD133+ cell-rich subline from Capan-1 pancreatic cancer cells as a pancreatic CSC model and compared the effects of KU-0063794, a dual mTORC1/mTORC2 inhibitor, against those of mTORC1-specific rapamycin. We found that KU-0063794 prevents sphere formation, a self-renewal index, at high concentrations. Rapamycin inhibited sphere formation but to a lesser degree. In the present study, we aimed to determine the mechanistic roles of mTOR complex 2 (mTORC2) in maintaining CSC-like properties. By examining the PI3K/Akt/mTOR signaling pathway, we observed lower Akt phosphorylation in KU-0063794-treated cells. Phosphorylation of mTORC1 downstream effectors was inhibited by both inhibitors. Thus, mTORC2 activates Akt and modulate stem-like properties, whereas mTORC1 downstream signaling correlates directly with stem-like properties.

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

UNLABELLED: 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.

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.

[A case of rectal cancer which showed complete response to chemotherapy for para-aortic lymph node metastases remaining after surgery, for which resection of Virchow's lymph node recurrence led to long-term survival].

A 55-year-old man with a positive fecal occult blood test visited our department, and after a thorough medical evaluation, was diagnosed with Stage IV Rs rectal cancer with marked para-aortic lymph node metastasis. In December 2007, the patient underwent low anterior rectal resection with D3 lymph node dissection, but the para-aortic lymph nodes were left. The metastatic lymph nodes showed a complete response(CR)to post-operative chemotherapy with FOLFOX, FOLFIRI, IRIS, and irinotecan+cetuximab, and the complete response was sustained for 18 months after surgery. Later, he developed Virchow's lymph node metastasis, which was also resected. At present, 5 years after the first surgery, the patient, whose chemotherapy has been discontinued, is alive without recurrence. It appears that using key drugs, such as 5-fluorouracil, leukovorin, oxaliplatin, irinotecan, and cetuximab, and performing aggressive salvage surgery for Virchow's lymph node recurrence, led to long-term recurrence-free survival.

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.

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 (IC(50); 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.

Clinical significance of folate receptor ß-expressing tumor-associated macrophages in pancreatic cancer.

PURPOSE: To examine the appearance and distribution of folate receptor ß-expressing (FRß+) macrophages in the pancreatic tumor microenvironment and their relationship to metastasis and prognosis in pancreatic cancer patients. METHODS: Tumor samples were obtained from 76 patients with pancreatic cancer who underwent curative resection. None of these patients had received any preoperative chemotherapy or radiotherapy. Both FRß+ and tumor-infiltrating (CD68+) macrophages were examined in each tumor specimen by immunohistochemical and immunofluorescence staining using a newly developed anti-human FRß monoclonal antibody and CD68 antibody. The appearance, distribution, expression of vascular endothelial growth factor (VEGF) on FRß-expressing or CD68+ macrophages, and tumor microvessel density (MVD) were assessed. Log rank test and Cox proportional hazard regression were used to investigate the associations among CD68+ or FRß+ macrophages, clinicopathologic factors, and overall survival. RESULTS: FRß+ macrophages were prominent in the perivascular regions of the tumor-invasive front and a specific subset with VEGF expression in the CD68+ macrophages. A high number of FRß+ macrophages showed a positive association with high MVD, a high incidence of hematogenous metastasis, and a poor prognosis in pancreatic cancer patients. CONCLUSIONS: FRß+ macrophages are a novel subset of tumor-associated macrophages in pancreatic cancer and may play an important role in the tumor microenvironment in association with systemic metastasis through the interaction with tumor cells and vessels. FRß+ macrophages may be promising a targeting therapy for pancreatic cancer.

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

UNLABELLED: BACKGROUND AND OBJECTIES: Phenotypic plasticity of cancer cells via epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) is essential for tumor progression and metastasis. METHODS: Tissue samples were obtained from 76 pancreatic head cancers. We assessed the expression of E-cadherin, vimentin, ZEB-1, and ZEB-2 by immunohistochemical and immunofluorescence staining. Next, 147 metastatic lymph nodes from 45 pancreatic cancers with low expression of E-cadherin were obtained and divided into two categories according to the maximum diameter of the metastases: 2 mm or more and less than 2 mm. RESULTS: High expressions of ZEB-1 and ZEB-2 in the primary tumors were significantly associated with repression of E-cadherin (P = 0.0007), and poorer prognosis (P = 0.0322). Forty-three (29.3%) of the 147 metastatic tumors from pancreatic cancers with low expression of E-cadherin showed high E-cadherin expression. Cancer cells in the larger metastases showed high expression of E-cadherin (P = 0.0061) and low expression of ZEB-1 (P = 0.0170) and ZEB-2 (P = 0.0036) compared with those in the smaller metastases. CONCLUSIONS: In primary pancreatic tumors and metastatic lymph nodes, high and low expression of ZEB-1 and ZEB-2 was associated with mesenchymal and epithelial phenotype of cancer cells, respectively.

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.

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.

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

BACKGROUND AND OBJECTIVES: 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. METHODS: Specimens were obtained from 70 patients. To evaluate lymphangiogenesis, we measured lymphatic vessel density (LVD) using D2-40 antibody in the primary tumors and in the draining lymph nodes. AE1/AE3 antibody was used to detect tiny, histologically negative metastases in lymph nodes. RESULTS: Patients with high LVD of primary tumors had significantly higher incidence of node metastasis (P = 0.0006) and lower postoperative survival rate (P = 0.0066) than those with low LVD. Intranodal LVDs increased with increasing size of the intranodal metastases. The LVDs of non-metastatic nodes in patients with node metastasis were also significantly higher than those of non-metastatic nodes in patients without node metastasis (P < 0.0001). The LVDs of peripancreatic nodes in patients with paraaortic node metastases were significantly higher than those in patients without paraaortic metastasis (P < 0.0001). CONCLUSIONS: Lymphangiogenesis in primary tumors and draining lymph nodes is essential for efficient spread of tumor cells through the lymphatic system. Thus, inhibition of lymphangiogenesis could limit lymphatic dissemination of tumor cells.