Inhibition of SRC tyrosine kinase as treatment for human pancreatic cancer growing orthotopically in nude mice.

PURPOSE: The Src family comprises a family of nonreceptor intracellular tyrosine kinases that mediate a variety of cellular pathways. Src kinases are overexpressed in a variety of human tumors, including cancer of the colon, breast, and pancreas, and they are an integral part of tumor cell signaling pathways associated with migration, proliferation, adhesion, and angiogenesis. EXPERIMENTAL DESIGN: We investigated whether the blockade of Src kinase by daily oral administration of the novel Src tyrosine kinase inhibitor AZM475271 [kindly provided by AstraZeneca (Macclesfield, United Kingdom)], alone or in combination with intraperitoneal gemcitabine, can inhibit growth and metastasis of orthotopically implanted human pancreatic carcinoma cells in nude mice. RESULTS: Treatment with AZM475271 alone reduced the primary pancreatic tumor volume by approximately 40%, whereas AZM475271 plus gemcitabine reduced tumor volume by 90%. Furthermore, treatment with AZM475271 and gemcitabine significantly reduced metastasis: none of eight animals who received the combination treatment had lymph node or liver metastases, compared with five of five and three of five animals, respectively, in the control group (P = 0.001). Src inhibition by AZM475271 (alone or with gemcitabine) was associated with significantly reduced tumor cell proliferation, decreased tumor microvessel density, and increased apoptosis in vivo. Moreover, these effects were all significantly increased when gemcitabine was combined with AZM475271 compared with gemcitabine alone. CONCLUSIONS: Src inhibition by AZM475271, either alone or in combination with gemcitabine, demonstrated significant antitumor and antimetastatic activity in an orthotopic nude mouse model for human pancreatic cancer. The combination of AZM475271 with gemcitabine sensitized tumor cells to the cytotoxic effect of gemcitabine.     

The FAK scaffold inhibitor C4 disrupts FAK-VEGFR-3 signaling and inhibits pancreatic cancer growth

Even with successful surgical resection and perioperative chemotherapy and radiation, pancreatic ductal adenocarcinoma (PDA) has a high incidence of recurrence. Tumor cell survival depends on activation of signaling pathways that suppress the apoptotic stimuli of invasion and metastasis. Focal adhesion kinase (FAK) is a critical signaling molecule that has been implicated in tumor cell survival, invasion and metastasis. We have previously shown that FAK and vascular endothelial growth factor receptor 3 (VEGFR-3) are overexpressed in cancer cells and physically interact to confer a significant survival advantage. We subsequently identified a novel small molecule inhibitor C4 that targeted the VEGFR-3-FAK site of interaction. In this study, we have shown that C4 disrupted the FAK-VEGFR-3 complexes in PDA cells. C4 treatment caused dose-dependent dephosphorylation and inactivation of the VEGFR-3 and FAK, reduction in cell viability and proliferation, cell cycle arrest and apoptosis in PDA cells. C4 increased the sensitivity of tumor cells to gemcitabine chemotherapy in vitro that lead to apoptosis at nanomolar concentrations of both drugs. C4 reduced tumor growth in vivoin subcutaneous and orthotopic murine models of PDA. The drug alone at low dose, decreased tumor growth; however, concomitant administration with low dose of gemcitabine had significant synergistic effect and led to 70% tumor reduction. Combination of C4 with gemcitabine had a prolonged cytostatic effect on tumor growth after treatment withdrawal. Finally, we report an anecdotal case of stage IV pancreatic cancer treated with gemcitabine in combination with C4 that showed a significant clinical response in primary tumor and complete clinical response in liver metastasis over an eight month period. Taken together, these results demonstrate that targeting the scaffolding function of FAK with a small-molecule FAK-VEGFR-3 inhibitor can be an effective therapeutic strategy against PDA.     

Effect of the vascular endothelial growth factor receptor-2 antibody DC101 plus gemcitabine on growth,metastasis and angiogenesis of human pancreatic cancer growing orthotopically in nude mice

Vascular endothelial growth factor (VEGF) is the major pro-angiogenic factor for most tumors. VEGF expression has been shown to be associated with a poor prognosis in human pancreatic cancer. The purpose of our study was to determine the effect of blockade of VEGF receptor-2 activity with or without gemcitabine on tumor growth and metastasis in an orthotopic model of human pancreatic cancer in nude mice. Therapy with gemcitabine or DC101, a VEGF receptor-2 antibody, resulted in a significant reduction of primary pancreatic tumor growth compared to untreated controls. The combination of DC101 and gemcitabine inhibited primary pancreatic tumor growth and lymphatic metastasis to a greater degree than either agent alone. Treatment with DC101 decreased vessel counts and increased the area of hypoxic tumor tissue compared to controls. Immunofluorescent double staining for apoptotic endothelial cells demonstrated a significant increase in the number apoptotic endothelial cells 24 days after initiation of therapy with DC101 plus gemcitabine. DC101 plus gemcitabine also increased tumor cell death and decreased tumor cell proliferation in pancreatic tumors. These findings indicate that blockade of VEGF receptor activation interferes with the survival of tumor endothelial cells, resulting in a reduction of primary pancreatic tumor growth in nude mice. Furthermore, the data demonstrate that anti-VEGF receptor-2 therapy potentiates the tumoricidal effect of gemcitabine in this model. Anti-VEGF receptor-2 therapy in combination with gemcitabine may be a novel therapeutic approach for advanced pancreatic cancer.     

Rapamycin-induced endothelial cell death and tumor vessel thrombosis potentiate cytotoxic therapy against pancreatic cancer.

PURPOSE: Despite current chemotherapies, pancreatic cancer remains an uncontrollable, rapidly progressive disease. Here, we tested an approach combining a recently described antiangiogenic drug, rapamycin, with standard gemcitabine cytotoxic therapy on human pancreatic tumor growth. EXPERIMENTAL DESIGN: Tumor growth was assessed in rapamycin and gemcitabine-treated nude mice orthotopically injected with metastatic L3.6pl human pancreatic cancer cells. H&E staining was performed on tumors, along with Ki67 staining for cell proliferation and immunohistochemical terminal deoxynucleotidyl transferase-mediated nick end labeling and CD31 analysis. Rapamycin-treated tumor vessels were also directly examined in dorsal skin-fold chambers for blood flow after thrombosis induction. Cell death in human umbilical vein endothelial cells was assessed by flow cytometry after annexin-V staining. RESULTS: Rapamycin therapy alone inhibited tumor growth and metastasis more than gemcitabine, with remarkable long-term tumor control when the drugs were combined. Mechanistically, H&E analysis revealed tumor vessel endothelium damage and thrombosis with rapamycin treatment. Indeed, dorsal skin-fold chamber analysis of rapamycin-treated tumors showed an increased susceptibility of tumor-specific vessels to thrombosis. Furthermore, terminal deoxynucleotidyl transferase-mediated nick end labeling/CD31 double staining of orthotopic tumors demonstrated apoptotic endothelial cells with rapamycin treatment, which also occurred with human umbilical vein endothelial cells in vitro. In contrast, gemcitabine was not antiangiogenic and, despite its known cytotoxicity, did not reduce proliferation in orthotopic tumors; nevertheless, rapamycin did reduce tumor proliferation. CONCLUSIONS: Our data suggest a novel mechanism whereby rapamycin targets pancreatic tumor endothelium for destruction and thrombosis. We propose that rapamycin-based vascular targeting not only reduces tumor vascularization, it decreases the number of proliferating tumor cells to be destroyed by gemcitabine, thus introducing a new, clinically feasible strategy against pancreatic cancer.     

Pancreatic stellate cells: partners in crime with pancreatic cancer cells

Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression.     

The targeted immunocytokine L19-IL2 efficiently inhibits the growth of orthotopic pancreatic cancer

PURPOSE: Effective control of pancreatic cancer has been hampered primarily by the lack of tumor specificity of current treatment modalities. The highly specific antibody-mediated delivery of therapeutic agents to the tumor microenvironment might overcome this problem. We therefore investigated the therapeutic efficacy of the targeted immunocytokine L19-Interleukin-2 (L19-IL2), consisting of the human single-chain Fv antibody L19, which is highly specific for the extradomain B (ED-B) of fibronectin, and the human cytokine IL-2, in pancreatic cancer. EXPERIMENTAL DESIGN: Therapeutic effects of L19-IL-2, IL-2, and gemcitabine on tumor growth and metastasis were evaluated in orthotopic mouse models for pancreatic cancer. Immunohistochemistry was done to define ED-B expression, tumor necrosis, apoptosis, proliferation, and invasion of macrophages and natural killer (NK) cells. NK cells were depleted by i.v. injection of an anti-asialo-GM-1 antibody. RESULTS: ED-B is selectively expressed in human pancreatic cancer and in primary tumors and metastases of the mouse models. L19-IL-2 therapy was clearly superior to untargeted IL-2 or gemcitabine and inhibited tumor growth and metastasis with remarkable long-term tumor control. Therapeutic effects were associated with the induction of extensive tumor necrosis and inhibition of tumor cell proliferation. Immunohistochemistry revealed an increase of macrophages and NK cells in the tumor tissue, suggesting immune-mediated mechanisms. The functional relevance of NK cells for the therapeutic effect of the targeted immunocytokine L19-IL-2 was confirmed by NK cell depletion, which completely abolished its antitumor efficacy. CONCLUSIONS: These preclinical results strongly encourage the initiation of clinical studies using L19-IL-2 in pancreatic cancer.     

Targeting of urokinase plasminogen activator receptor in human pancreatic carcinoma cells inhibits c-Met- and insulin-like growth factor-I receptor-mediated migration and invasion and orthotopic tumor growth in mice

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)- and hepatocyte growth factor (HGF)-mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I- and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.     

Antiangiogenic treatment with three thrombospondin-1 type 1 repeats versus gemcitabine in an orthotopic human pancreatic cancer model.

PURPOSE: In this study, we investigated the antitumor efficacy of thrombospondin-1 three type 1 repeats (3TSR), the antiangiogenic domain of thrombospondin-1, in comparison and in combination with gemcitabine, in an orthotopic pancreatic cancer model. EXPERIMENTAL DESIGN: Human pancreatic cancer cells were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR, gemcitabine, 3TSR plus gemcitabine, or vehicle for 3 weeks. Subsequently, the effects of 3TSR and/or gemcitabine on tumor growth, tumor necrosis, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. RESULTS: After 3 weeks of treatment, 3TSR reduced tumor volume by 65%, and gemcitabine by 84%. Tumor volume was not statistically different between gemcitabine group and combinatorial treatment group. Extensive necrotic areas were observed in tumors from 3TSR-treated mice, whereas tumors from gemcitabine and combinatorially treated mice were less necrotic than control tumors. 3TSR reduced tumor microvessel density and increased tumor blood vessel endothelial cell apoptosis. In contrast, gemcitabine induced apoptosis and inhibited proliferation of cancer cells. CONCLUSION: 3TSR, the antiangiogenic domain of thrombospondin-1, showed comparable antitumor efficacy to gemcitabine in a human pancreatic cancer orthotopic mouse model. No synergistic effect was found when the two drugs were combined and possible reasons are discussed in detail. A delicate balance between normalization and excessive regression of tumor vasculature is important when initiating alternative combinatorial regimens for treatment of patients with pancreatic cancer.     

Transient gene silencing of galectin-3 suppresses pancreatic cancer cell migration and invasion through degradation of β-catenin

Pancreatic cancer is a leading cause of cancer-related mortality and often has a poor prognosis because of its late diagnosis, aggressive local invasion, early metastasis and poor response to chemotherapy. The chemotherapeutic agent gemcitabine is effective for treating advanced pancreatic cancer, but its efficacy remains less than satisfactory. It is expected that further investigation of pancreatic cancer cell invasion and development of strategies to block this process should improve the disease prognosis. In this study, we tested our hypothesis that galectin-3 (gal-3), a multifunctional member of the β-galactoside-binding protein family, may regulate pancreatic cancer cell motility and silencing of it inhibit cell motility. Previous studies demonstrated that this protein is associated with tumor cell adhesion, proliferation, differentiation, angiogenesis, apoptosis and metastasis. Here, we used gal-3 small interfering RNA (siRNA) to silence its expression in various pancreatic cancer cell lines to determine whether gal-3 regulates cell proliferation, migration and invasion in vitro. We found that silencing gal-3 reduced cellular migration and invasion, but failed to affect proliferation. In gal-3 siRNA-transfected cells, we detected a decrease in β-catenin expression, an important signal for cancer cell invasion, which was caused by downregulation of phosphorylated Akt and GSK-3β. We also found that matrix metalloproteinase (MMP)-2 expression was reduced by gal-3 silencing. These results indicate that gal-3-mediated invasion via MMP-2 regulated by β-catenin degradation is initiated by Akt phosphorylation in pancreatic cancer cells. Our results suggest that gal-3 can be a novel therapeutic target in pancreatic cancer.     

Therapeutic significance of elevated tissue transglutaminase expression in pancreatic cancer.

PURPOSE: Tissue transglutaminase (TG2) is a multifunctional protein that is implicated in development of drug resistance and metastasis. Therefore, we examined therapeutic targeting of TG2 for inhibiting growth and metastasis of in vivo growing pancreatic ductal adenocarcinoma (PDAC) in nude mice. EXPERIMENTAL DESIGN: We implanted Panc-28 pancreatic cancer cells to induce orthotopic PDAC tumors in nude mice and determined the efficacy of liposomal TG2 small interfering RNA (siRNA) either alone or in combination with gemcitabine. RESULTS: We show that down-regulation of endogenous TG2 by siRNA could effectively block the growth of PDAC. Moreover, down-regulation of TG2 significantly enhanced the therapeutic efficacy of gemcitabine against PDAC and inhibited metastatic spread of the disease. The antitumor activity was related to inhibition of proliferation, angiogenesis, and Akt phosphorylation. CONCLUSION: siRNA-mediated down-regulation of TG2 represents a promising therapeutic approach for improved treatment of PDAC.     

Leptin signaling enhances cell invasion and promotes the metastasis of human pancreatic cancer via increasing MMP-13 production

Emerging evidence has suggested that leptin, an adipokine related to energy homeostasis, plays a role in cancer growth and metastasis. However, its impact on pancreatic cancer is rarely studied. In this study, we found that leptin''s functional receptor Ob-Rb was expressed in pancreatic cancer cell lines. Treatment with leptin enhanced the migration and invasion of pancreatic cancer cells but did not affect the proliferation of human pancreatic cancer cells. Leptin up-regulated the expression of matrix metalloproteinase-13 (MMP-13) via the JAK2/STAT3 signaling pathway. The overexpression of leptin was shown to significantly promote tumor growth and lymph node metastasis in a subcutaneous model and an orthotopic model of human pancreatic cancer, respectively. Furthermore, in human pancreatic cancer tissues, the expression of Ob-Rb was positively correlated with the MMP-13 level. The increased expression of either Ob-Rb or MMP-13 was significantly associated with lymph node metastasis and tended to be associated with the TNM stage in patients with pancreatic cancer. Our findings suggest that leptin enhances the invasion of pancreatic cancer through the increase in MMP-13 production, and targeting the leptin/MMP-13 axis could be an attractive therapeutic strategy for pancreatic cancer.     

Anti-Bv8 Antibody and Metronomic Gemcitabine Improve Pancreatic Adenocarcinoma Treatment Outcome Following Weekly Gemcitabine Therapy

Weekly gemcitabine therapy is the major treatment offered for patients with pancreatic adenocarcinoma cancer; however, relative resistance of tumor cells to chemotherapy, rapid regrowth, and metastasis are the main causes of death within a year. Recently, the daily continuous administration of chemotherapy in low doses – called metronomic chemotherapy (MC) – has been shown to inhibit primary tumor growth and delay metastases in several tumor types; however, its use as a single therapy is still in question due to its moderate therapeutic benefit. Here, we show that the combination of weekly gemcitabine with MC of the same drug delays tumor regrowth and inhibits metastasis in mice implanted orthotopically with pancreatic tumors. We further demonstrate that weekly gemcitabine, but not continuous MC gemcitabine or the combination of the two drug regimens, promotes rebound myeloid-derived suppressor cell (MDSC) mobilization and increases angiogenesis in this tumor model. Furthermore, Bv8 is highly expressed in MDSCs colonizing pancreatic tumors in mice treated with weekly gemcitabine compared to MC gemcitabine or the combination of the two regimens. Blocking Bv8 with antibodies in weekly gemcitabine-treated mice results in a significant reduction in tumor regrowth, angiogenesis, and metastasis. Overall, our results suggest that pro-tumorigenic effects induced by weekly gemcitabine are mediated in part by MDSCs expressing Bv8. Therefore, both Bv8 inhibition and MC can be used as legitimate ''add-on'' treatments for preventing post-chemotherapy pancreatic cancer recurrence, progression, and metastasis following weekly gemcitabine therapy.     

Addition of matrix metalloproteinase inhibition to conventional cytotoxic therapy reduces tumor implantation and prolongs survival in a murine model of human pancreatic cancer

Matrix metalloproteinases (MMPs) participate in basement membrane degradation, a critical step in invasion of cancer cells. We have previously shown that MMP inhibition of pancreatic cancers improves survival and decreases MMP production in vivo. The purpose of this study was to determine whether BB-94 was better than cytotoxic therapy and would increase the efficacy of cytotoxic therapy (gemcitabine) in a murine model of human pancreatic cancer. A human pancreatic adenocarcinoma cell line (HPAC) was injected into the pancreata of BALB/c nu/nu mice. The mice were randomized 7 days after cancer cell injection to receive vehicle control, BB-94, gemcitabine, or gemcitabine and BB-94 until death or sacrifice at 84 days. At necropsy, tumors were harvested, and the relative enzyme activities of MMP-2 and MMP-9 were determined by gelatin zymography. Active MMP-2 levels in serum were determined using an ELISA technique. Combination treatment with gemcitabine and BB-94 significantly reduced implantation rates and improved survival in mice with documented orthotopic tumors compared with either therapy alone or control. Tumor levels of active and latent MMP-2 were higher than those of MMP-9 in both treated and control mice. There was a significant reduction of tumor MMP-2 activity in mice treated with BB-94, gemcitabine, or gemcitabine and BB-94. Serum levels of active MMP-2 were reduced in all treated groups, with the greatest reduction occurring in mice treated with gemcitabine and BB-94. Combination therapy with gemcitabine and BB-94 reduces cancer implantation and improves survival compared to treatment with BB-94, gemcitabine, or vehicle control alone. MMP production was reduced in all treated groups, reflecting reduced tumor progression, which was particularly seen with combination therapy with gemcitabine and BB-94. This study supports combining MMP inhibition with cytotoxic therapy (gemcitabine) for patients with pancreatic cancer.     

CXCL12-CXCR4在胰腺癌侵袭转移中的作用

目前研究发现趋化因子及受体CXCL12-CXCR4生物轴在胰腺癌的侵袭转移中起重要作用.CXCL12-CXCR4可通过促进胰腺癌细胞增殖、增强癌细胞的迁移、促进细胞外基质蛋白降解和血管新生等机制而促进胰腺癌的侵袭转移.     

CXCL12-CXCR4在胰腺癌侵袭转移中的作用

目前研究发现趋化因子及受体CXCL12-CXCR4生物轴在胰腺癌的侵袭转移中起重要作用.CXCL12-CXCR4可通过促进胰腺癌细胞增殖、增强癌细胞的迁移、促进细胞外基质蛋白降解和血管新生等机制而促进胰腺癌的侵袭转移.     

CXCL12-CXCR4在胰腺癌侵袭转移中的作用

目前研究发现趋化因子及受体CXCL12-CXCR4生物轴在胰腺癌的侵袭转移中起重要作用.CXCL12-CXCR4可通过促进胰腺癌细胞增殖、增强癌细胞的迁移、促进细胞外基质蛋白降解和血管新生等机制而促进胰腺癌的侵袭转移.     

Silencing of STRN4 suppresses the malignant characteristics of cancer cells

The striatin family of proteins, comprising STRN, STRN3 and STRN4, are multidomain‐containing proteins that associate with additional proteins to form a large protein complex. We previously reported that STRN4 directly associated with protein kinases, such as MINK1, TNIK and MAP4K4, which are associated with tumor suppression or tumor progression. However, it remains unclear whether STRN4 is associated with tumor progression. In this report, we examined the role that STRN4 plays in cancer malignancy. We show that depletion of STRN4 suppresses proliferation, migration, invasion and the anchorage‐independent growth of cancer cells. In addition, STRN4 knockdown increases the sensitivity of pancreatic cancer cells to gemcitabine. Finally, we show that STRN4 knockdown suppresses the proliferation and metastasis of cancer cells in mice. Our results demonstrate a possible role of STRN4 in tumor progression.