Ischemia-induced K-ras mutations in human colorectal cancer cells: role of microenvironmental regulation of MSH2 expression.

Mutation of the K-ras gene is one of the most common genetic alterations in solid tumors, including colorectal cancer. The relatively late emergence of K-ras mutations in colorectal cancer is particularly striking in the class of mismatch repair-deficient tumors associated with early-onset microsatellite instability. We, therefore, tested the hypothesis that the microsatellite instability phenotype itself does not efficiently trigger K-ras mutations in colorectal cancer cells, but rather that tumor-associated microenvironmental conditions (e.g., hypoxia and hypoglycemia) contribute to this event by modulating genetic instability. We examined K-ras(G13D) mutation using PCR-RFLP analysis in two different microsatellite instability colorectal cancer cell lines (HCT116 and DLD-1) and their variants in which the mutant (but not the wild-type) K-ras allele has been genetically disrupted (Hkh-2 and Dks-8). We found K-ras(G13D) mutation to occur at far greater incidence in cells derived from xenografted tumors or exposed to conditions of combined hypoxia and hypoglycemia in vitro. Interestingly, this mutagenesis was neither enhanced by induced oxidative damage nor prevented by the antioxidant vitamin E. Moreover, the accumulation of K-ras mutations was paralleled by down-regulation of the key mismatch repair protein MSH2 in xenografted tumors, particularly in hypoperfused areas and under hypoglycemic conditions (in vitro). In contrast, the microsatellite stable colorectal cancer cell line Caco-2 neither accumulated K-ras mutations nor showed down-regulation of MSH2 under these conditions. Thus, our study suggests that ischemia may not simply select for, but can actually trigger, increased mutation rate in crucial colorectal cancer oncoproteins. This finding establishes a novel linkage between genetic instability, tumor ischemia, and genetic tumor progression and carries important implications for applying anticancer therapies involving tumor hypoxia (e.g., antiangiogenesis) in microsatellite instability cancers.

Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts.

Vascular endothelial growth factor (VEGF) occurs in at least five different isoforms because of alternative splicing of the gene. To investigate the roles of different VEGF isoforms in tumor blood vessel formation and tumorigenicity, the three major isoforms (VEGF(121), VEGF(165), and VEGF(189)) were overexpressed in an early-stage human melanoma cell line (WM1341B), which is VEGF-negative and nontumorigenic in immunodeficient mice. Although overexpression of VEGF(121) and VEGF(165) resulted in aggressive tumor growth, WM1341B cells transfected with VEGF(189) remained nontumorigenic and dormant on injection. Although tumor growth rate depended on the level and not the isoform of VEGF expressed, striking isoform-specific differences in vascular patterning were associated with VEGF(121)- versus VEGF(165)-dependent tumorigenic conversion of human melanoma. Thus, tumors overexpressing VEGF(165) generated dense, highly heterogeneous vessel networks that were distinctly different from those of tumors expressing VEGF(121) (poorly vascularized and necrotic). Paradoxically, although VEGF(165) expression appears to result in the most effective tumor perfusion, it is the expression of VEGF(121) that is observed during human malignant melanoma progression. Indeed, unbiased selection of spontaneously tumorigenic variants of WM1341B (by coinjection with Matrigel) led to predominant expression of the VEGF(121) isoform. The vascular patterning in these tumors (1341-P3N1, 1341-P3N2) resembled that of the VEGF(121)-transfected WM1341B tumors. These results suggest that, for reasons that remain to be elucidated, a "minimal" program of tumor vascularization may be favored during melanoma progression.

Antibody-based targeting of alternatively spliced tissue factor: a new approach to impede the primary growth and spread of pancreatic ductal adenocarcinoma.

Alternatively spliced Tissue Factor (asTF) is a secreted form of Tissue Factor (TF), the trigger of blood coagulation whose expression levels are heightened in several forms of solid cancer, including pancreatic ductal adenocarcinoma (PDAC). asTF binds to ß1 integrins on PDAC cells, whereby it promotes tumor growth, metastatic spread, and monocyte recruitment to the stroma. In this study, we determined if targeting asTF in PDAC would significantly impact tumor progression. We here report that a novel inhibitory anti-asTF monoclonal antibody curtails experimental PDAC progression. Moreover, we show that tumor-derived asTF is able to promote PDAC primary growth and spread during early as well as later stages of the disease. This raises the likelihood that asTF may comprise a viable target in early- and late-stage PDAC. In addition, we show that TF expressed by host cells plays a significant role in PDAC spread. Together, our data demonstrate that targeting asTF in PDAC is a novel strategy to stem PDAC progression and spread.

Host microenvironment in breast cancer development: inflammatory and immune cells in tumour angiogenesis and arteriogenesis.

Breast cancer progression is associated with and dependent upon robust neovascularization. It is becoming clear that tumour-associated 'normal' cells, such as immune/inflammatory cells, endothelial cells and stromal cells, conspire with cancer cells in promoting this process. In particular, infiltrating immune/inflammatory cells secrete a diverse repertoire of growth factors and proteases that enable them to enhance tumour growth by stimulating angiogenesis and, as we suggest here, by promoting 'tumour arteriogenesis' - enlargement of feeding vessels supplying the expanding tumour capillary bed. Macrophages and their chemoattractants (e.g. macrophage chemoattractant protein-1) are critical for the arteriogenic process in ischaemia, and probably also in breast neoplasia. A better understanding of these various cellular and molecular constituents of breast cancer neovascularization may be useful in designing more effective therapies.

Angiogenesis and the role of epigenetics in metastasis.

The major obstacle to devising effective ways to treat cancer is its heterogeneity and genetic instability. It was originally postulated that targeting the process of tumor angiogenesis could circumvent this problem, as it involves genetically stable epigenetically controlled host stroma. Thus, anti-angiogenic approaches should be applicable across various tumor types and organ sites, including metastases. However, early clinical experience with this therapy revealed unexpectedly distinct responses between different tumors and organ sites. Here we propose that the heterogeneity of pre-clinical and clinical results obtained with anti-angiogenic agents stems from the deep functional linkage that may exist between genetic and epigenetic tumor progression. Thus, epigenetic processes regulating tumor associated host blood vessels (such as tumor microenvironment) display unstable, heterogeneous and progressive characteristics to an extent comparable with (and causally linked to) the instability of the cancer cell genome. As well, many known epigenetic factors (such as hypoxia, inflammation, expression of growth factors, etc.) may have genetic causes and consequences (e.g., oncogene expression, loss of tumor suppressor genes). This reciprocal interrelationship and heterogeneity may translate into site and stage specific changes in angiogenesis regulation, and angiogenesis dependence, ultimately to changes in the metastatic ability/efficiency of cancer cells, even in the same patient. A better understanding of the linkage between genetic and epigenetic events in growth and metastasis of various cancers may result in more effective use of anti-angiogenic therapy in future.

Tissue factor regulation by epidermal growth factor receptor and epithelial-to-mesenchymal transitions: effect on tumor initiation and angiogenesis.

ErbB oncogenes drive the progression of several human cancers. Our study shows that in human carcinoma (A431) and glioma (U373) cells, the oncogenic forms of epidermal growth factor receptor (EGFR; including EGFRvIII) trigger the up-regulation of tissue factor (TF), the transmembrane protein responsible for initiating blood coagulation and signaling through interaction with coagulation factor VIIa. We show that A431 cancer cells in culture exhibit a uniform TF expression profile; however, these same cells in vivo exhibit a heterogeneous TF expression and show signs of E-cadherin inactivation, which is coupled with multilineage (epithelial and mesenchymal) differentiation. Blockade of E-cadherin in vitro, leads to the acquisition of spindle morphology and de novo expression of vimentin, features consistent with epithelial-to-mesenchymal transition. These changes were associated with an increase in EGFR-dependent TF expression, and with enhanced stimulation of vascular endothelial growth factor production, particularly following cancer cell treatment with coagulation factor VIIa. In vivo, cells undergoing epithelial-to-mesenchymal transition exhibited an increased metastatic potential. Furthermore, injections of the TF-blocking antibody (CNTO 859) delayed the initiation of A431 tumors in immunodeficient mice, and reduced tumor growth, vascularization, and vascular endothelial growth factor expression. Collectively, our data suggest that TF is regulated by both oncogenic and differentiation pathways, and that it functions in tumor initiation, tumor growth, angiogenesis, and metastasis. Thus, TF could serve as a therapeutic target in EGFR-dependent malignancies.

Dynamic stromal-epithelial interactions during progression of MCF10DCIS.com xenografts.

MCF10DCIS.com cells form comedo type ductal carcinoma in situ in immune-deficient mice before forming invasive ductal carcinoma. As the lesions mature, both stromal and epithelial cells undergo phenotypic changes detected by immunohistochemistry. Myofibroblasts are present before the formation of carcinoma in situ and after development of invasive carcinoma. MCF10DCIS. com lesions develop a myoepithelial layer prior to exhibiting a basement membrane surrounding the ductal mass. TGFbeta1 is initially expressed by the epithelial cells but is expressed by stroma in invasive carcinoma. Stromal derived factor-1 is detected in epithelial cells in early carcinoma in situ but is produced in stromal cells in invasive carcinoma. The receptor CXCR4 is expressed by epithelial cells in the xenografts at all times, as is the hepatocyte growth factor receptor c-met. MCF10DCIS.com xenografts illustrate the dynamic interplay of epithelium and stroma in the development of carcinoma in situ and subsequent invasive carcinoma. Although the phenotype of the epithelial cells may be dependent upon the stroma, the malignant epithelium induces the development of the stroma necessary for progression to the invasive stage. (c) 2007 Wiley-Liss, Inc.

Oncogenic events regulate tissue factor expression in colorectal cancer cells: implications for tumor progression and angiogenesis.

Tissue factor (TF) is the primary cellular initiator of blood coagulation and a modulator of angiogenesis and metastasis in cancer. Indeed, systemic hypercoagulability in patients with cancer and TF overexpression by cancer cells are both closely associated with tumor progression, but their causes have been elusive. We now report that in human colorectal cancer cells, TF expression is under control of 2 major transforming events driving disease progression (activation of K-ras oncogene and inactivation of the p53 tumor suppressor), in a manner dependent on MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI3K). Furthermore, the levels of cell-associated as well as circulating (microvesicle-associated) TF activity are linked to the genetic status of cancer cells. Finally, RNA interference experiments suggest that TF expression is an important effector of the K-ras-dependent tumorigenic and angiogenic phenotype in vivo. Thus, this study establishes a causal link between cancer coagulopathy, angiogenesis, and genetic tumor progression.

Effect of p53 status on tumor response to antiangiogenic therapy.

The p53 tumor suppressor gene is inactivated in the majority of human cancers. Tumor cells deficient in p53 display a diminished rate of apoptosis under hypoxic conditions, a circumstance that might reduce their reliance on vascular supply, and hence their responsiveness to antiangiogenic therapy. Here, we report that mice bearing tumors derived from p53(-/-) HCT116 human colorectal cancer cells were less responsive to antiangiogenic combination therapy than mice bearing isogenic p53(+/+) tumors. Thus, although antiangiogenic therapy targets genetically stable endothelial cells in the tumor vasculature, genetic alterations that decrease the vascular dependence of tumor cells can influence the therapeutic response of tumors to this therapy.