Gary E. Gallick

Src family kinases in tumor progression and metastasis

The Src family of non-receptor protein tyrosine kinases plays critical roles in a variety of cellular signal transduction pathways, regulating such diverse processes as cell division, motility, adhesion, angiogenesis, and survival. Constitutively activated variants of Src family kinases, including the viral oncoproteins v-Src and v-Yes, are capable of inducing malignant transformation of a variety of cell types. Src family kinases, most notably although not exclusively c-Src, are frequently overexpressed and/or aberrantly activated in a variety of epithelial and non-epithelial cancers. Activation is very common in colorectal and breast cancers, and somewhat less frequent in melanomas, ovarian cancer, gastric cancer, head and neck cancers, pancreatic cancer, lung cancer, brain cancers, and blood cancers. Further, the extent of increased Src family activity often correlates with malignant potential and patient survival. Activation of Src family kinases in human cancers may occur through a variety of mechanisms and is frequently a critical event in tumor progression. Exactly how Src family kinases contribute to individual tumors remains to be defined completely, however they appear to be important for multiple aspects of tumor progression, including proliferation, disruption of cell/cell contacts, migration, invasiveness, resistance to apoptosis, and angiogenesis. This review details the evidence for Src family activation in human tumors, and emphasizes possible consequences to tumor progression. Given the ability of Src and its family members to participate in so many aspects of tumor progression and metastasis, Src family kinases are attractive targets for future anti-cancer therapeutics.

Increase in activity and level of pp60c-src in progressive stages of human colorectal cancer.

Activation of the tyrosine kinase of the c-src gene product, pp60c-src, has been shown to occur in nearly every primary colorectal carcinoma, and is found as early as in polyps of high malignant potential. However, no studies have addressed potential pp60c-src changes which occur during progression. To examine this question, we have studied kinase activity and protein levels in 7 colonic polyps, 19 primary lesions, and 19 liver metastases relative to normal colonic mucosa. Significant increases in tyrosine kinase activity were seen as early as in colonic polyps of high malignant potential. Further increases were observed in activity and level in primary tumors. However, the greatest increases in activity and protein levels were observed in liver metastases. Additionally, six metastatic lesions were obtained in which synchronous primary tumor was resected. In each of these liver metastases, pp60c-src activity and level were significantly increased relative to the corresponding primary tumor, as well as to normal colonic mucosa. Our results demonstrate that progression of colon primary tumors to liver metastases correlates with increased pp60c-src kinase activity and protein level.

Development and Characterization of Gemcitabine-Resistant Pancreatic Tumor Cells

BackgroundPancreatic cancer is an exceptionally lethal disease with an annual mortality nearly equivalent to its annual incidence. This dismal rate of survival is due to several factors including late presentation with locally advanced, unresectable tumors, early metastatic disease, and rapidly arising chemoresistance. To study the mechanisms of chemoresistance in pancreatic cancer we developed two gemcitabine-resistant pancreatic cancer cell lines.MethodsResistant cells were obtained by culturing L3.6pl and AsPC-1 cells in serially increasing concentrations of gemcitabine. Stable cultures were obtained that were 40- to 50-fold increased in resistance relative to parental cells. Immunofluorescent staining was performed to examine changes in β-catenin and E-cadherin localization. Protein expression was determined by immunoblotting. Migration and invasion were determined by modified Boyden chamber assays. Fluorescence-activated cell sorting (FACS) analyses were performed to examine stem cell markers.ResultsGemcitabine-resistant cells underwent distinct morphological changes, including spindle-shaped morphology, appearance of pseudopodia, and reduced adhesion characteristic of transformed fibroblasts. Gemcitabine-resistant cells were more invasive and migratory. Gemcitabine-resistant cells were increased in vimentin and decreased in E-cadherin expression. Immunofluorescence and immunoblotting revealed increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells were activated in the receptor protein tyrosine kinase, c-Met and increased in expression of the stem cell markers CD (cluster of differentiation)24, CD44, and epithelial-specific antigen (ESA).ConclusionsGemcitabine-resistant pancreatic tumor cells are associated with EMT, a more-aggressive and invasive phenotype in numerous solid tumors. The increased phosphorylation of c-Met may also be related to chemoresistance and EMT and presents as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

HIF-1α, STAT3, CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas

Hypoxia stimulates a number of pathways critical to cancer cell survival, including the activation of vascular endothelial growth factor (VEGF) transcription. In normal fibroblasts, hypoxia-induced activation of the protein tyrosine kinase, Src, is required for VEGF expression. We show here in both pancreatic and prostate carcinoma cell lines cobalt chloride (used to mimic hypoxia) -induced VEGF expression requires Src activation and leads to increased steady-state levels of HIF-1α and increased phosphorylation of signal and transducer of transcription 3 (STAT3). STAT3 and hypoxia-inducible factor (HIF)-1α bind simultaneously to the VEGF promoter, where they form a molecular complex with the transcription coactivators CBP/p300 and Ref-1/APE. Expression of activated Src from an inducible promoter is sufficient to increase VEGF expression and form these STAT3/HIF-1α-containing promoter complexes. Inhibition of DNA binding by expression of either STAT3 or HIF-1α dominant negative mutants significantly reduces VEGF expression. These data suggest that the binding of both STAT3 and HIF-1α to the VEGF promoter is required for maximum transcription of VEGF mRNA following hypoxia.

EGCG, a major component of green tea, inhibits tumour growth by inhibiting VEGF induction in human colon carcinoma cells.

Catechins are key components of teas that have antiproliferative properties. We investigated the effects of green tea catechins on intracellular signalling and VEGF induction in vitro in serum-deprived HT29 human colon cancer cells and in vivo on the growth of HT29 cells in nude mice. In the in vitro studies, (-)-epigallocatechin gallate (EGCG), the most abundant catechin in green tea extract, inhibited Erk-1 and Erk-2 activation in a dose-dependent manner. However, other tea catechins such as (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC) did not affect Erk-1 or 2 activation at a concentration of 30 μM. EGCG also inhibited the increase of VEGF expression and promoter activity induced by serum starvation. In the in vivo studies, athymic BALB/c nude mice were inoculated subcutaneously with HT29 cells and treated with daily intraperitoneal injections of EC (negative control) or EGCG at 1.5 mg day–1mouse−1starting 2 days after tumour cell inoculation. Treatment with EGCG inhibited tumour growth (58%), microvessel density (30%), and tumour cell proliferation (27%) and increased tumour cell apoptosis (1.9-fold) and endothelial cell apoptosis (3-fold) relative to the control condition (P< 0.05 for all comparisons). EGCG may exert at least part of its anticancer effect by inhibiting angiogenesis through blocking the induction of VEGF.

Down-regulation of Vascular Endothelial Growth Factor in a Human Colon Carcinoma Cell Line Transfected with an Antisense Expression Vector Specific for c-src

Vascular endothelial growth factor (VEGF) is implicated in the angiogenesis of human colon cancer. Recent evidence suggests that factors that regulate VEGF expression may partially depend on c-src-mediated signal transduction pathways. The tyrosine kinase activity of Src is activated in most colon tumors and cell lines. We established stable subclones of the human colon adenocarcinoma cell line HT29 in which Src expression and activity are decreased specifically as a result of a transfected antisense expression vector. This study determined whether VEGF expression is decreased in these cell lines and whether the smaller size and reduced growth rate of antisense vector-transfected cell lines in vivo might result, in part, from reduced vascularization of tumors. Northern blot analysis of these cell lines revealed that VEGF mRNA expression was decreased in proportion to the decrease in Src kinase activity. Under hypoxic conditions, cells with decreased Src activity had a <2-fold increase in VEGF expression, whereas parental cells had a >50-fold increase. VEGF protein in the supernatants of cells was also reduced in antisense transfectants compared with that from parental cells. In nude mice, subcutaneous tumors from antisense transfectants showed a significant reduction in vascularity. These results suggest that Src activity regulates the expression of VEGF in colon tumor cells.

Induction of VEGF in perivascular cells defines a potential paracrine mechanism for endothelial cell survival

Small tumor vessels are composed of endothelial cells (ECs) surrounded by pericytes. Pericytes are believed to be an EC survival factor, but their mechanism of action is unknown. One possible mediator, VEGF, promotes angiogenesis, EC proliferation, and EC permeability, and it protects ECs from apoptosis. We hypothesized that PDGF (platelet‐derived growth factor)‐BB, a cytokine released from tumor and ECs, mediates pericyte function by inducing VEGF, which in turn may affect EC survival. Using two pericyte‐like cell lines, 10T1/2 cells (murine pericyte cell line) and human vascular smooth muscle cells (hVSMCs), we showed that PDGF‐BB increased VEGF mRNA transcription. Although PDGF‐BB activated both the mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase (PI3‐K) pathways, activation of the PI3‐K pathway was the most important pathway for VEGF induction. Conditioned medium derived from colon cancer cells also induced VEGF in pericyte‐like cells via the PI3‐K pathway, which was blocked by SU6668, a tyrosine kinase inhibitor that blocks the receptors for PDGF, VEGF, and basic fibroblast growth factor. Conditioned medium from hVSMCs pretreated with PDGF‐BB prevented apoptosis of ECs, and this effect was partially abrogated by neutralizing antibodies to VEGF. These studies suggest that pericytes may protect ECs from apoptosis, in part, by cytokine signaling that increases VEGF.

Targeting Src Family Kinases Inhibits Growth and Lymph Node Metastases of Prostate Cancer in an Orthotopic Nude Mouse Model

Aberrant expression and/or activity of members of the Src family of nonreceptor protein tyrosine kinases (SFK) are commonly observed in progressive stages of human tumors. In prostate cancer, two SFKs (Src and Lyn) have been specifically implicated in tumor growth and progression. However, there are no data in preclinical models demonstrating potential efficacy of Src inhibitors against prostate cancer growth and/or metastasis. In this study, we used the small molecule SFK/Abl kinase inhibitor dasatinib, currently in clinical trials for solid tumors, to examine in vitro and in vivo effects of inhibiting SFKs in prostate tumor cells. In vitro, dasatinib inhibits both Src and Lyn activity, resulting in decreased cellular proliferation, migration, and invasion. In orthotopic nude mouse models, dasatinib treatment effectively inhibits expression of activated SFKs, resulting in inhibition of both tumor growth and development of lymph node metastases in both androgen-sensitive and androgen-resistant tumors. In primary tumors, SFK inhibition leads to decreased cellular proliferation (determined by immunohistochemistry for proliferating cell nuclear antigen). In vitro, small interfering RNA (siRNA)-mediated inhibition of Lyn affects cellular proliferation; siRNA inhibition of Src affects primarily cellular migration. Therefore, we conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer and that Src and Lyn activities affect different cellular functions required for prostate tumor growth and progression.

ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma

Background Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends. Methodology/Principal Findings We have isolated populations of cells with high ALDH activity (ALDHhigh) and/or CD133 cell surface expression from human xenograft tumors established from multiple patient tumors with pancreatic adenocarcinoma (direct xenograft tumors) and from the pancreatic cancer cell line L3.6pl. Through fluorescent activated cell sorting (FACs)-mediated enrichment and depletion of selected pancreatic cancer cell populations, we sought to discriminate the relative tumorigenicity of cell populations that express the pancreatic CSC markers CD133 and aldehyde dehydrogenase (ALDH). ALDHhigh and ALDHlow cell populations were further examined for co-expression of CD44 and/or CD24. We demonstrate that unlike cell populations demonstrating low ALDH activity, as few as 100 cells enriched for high ALDH activity were capable of tumor formation, irrespective of CD133 expression. In direct xenograft tumors, the proportions of total tumor cells expressing ALDH and/or CD133 in xenograft tumors were unchanged through a minimum of two passages. We further demonstrate that ALDH expression among patients with pancreatic adenocarcinoma is heterogeneous, but the expression is constant in serial generations of individual direct xenograft tumors established from bulk human pancreatic tumors in NOD/SCID mice. Conclusions/Significance We conclude that, in contrast to some previous studies, cell populations enriched for high ALDH activity alone are sufficient for efficient tumor-initiation with enhanced tumorigenic potential relative to CD133+ and ALDHlow cell populations in some direct xenograft tumors. Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDHhigh cell populations. ALDHhigh/CD44+/CD24+ or ALDHlow/CD44+/CD24+ phenotypes do not appear to significantly contribute to tumor formation at low numbers of inoculated tumor cells. ALDH expression broadly varies among patients with pancreatic adenocarcinoma and the apparent expression is recapitulated in serial generations of direct xenograft tumors in NOD/SCID. We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.

Src activation regulates anoikis in human colon tumor cell lines

Src is a non-receptor protein tyrosine kinase, the expression and activity of which is increased in >80% of human colon cancers with respect to normal colonic epithelium. Previous studies from this and other laboratories have demonstrated that Src activity contributes to tumorigenicity of established colon adenocarcinoma cell lines. Src participates in the regulation of many signal transduction pathways, among which are those leading to cellular survival. In this study, we addressed the potential role of Src activation to a specific aspect of tumor cell survival, resistance to detachment-induced apoptosis (anoikis). Using five colon tumor cell lines with different biologic properties and genetic alterations, we demonstrate that expression and activity of Src corresponds with resistance to anoikis. Enforced expression of activated Src in subclones of SW480 cells (of low intrinsic Src expression and activity) increases resistance to anoikis; whereas decreased Src expression in HT29 cells (of high Src expression and activity) by transfection with anti-sense Src expression vectors increases susceptibility to anoikis. In contrast, increasing or decreasing Src expression had no effect on susceptibility to staurosporine-induced apoptosis in attached cells. PD173955, a Src family-specific tyrosine kinase inhibitor, increases the susceptibility of HT29 cells to anoikis in a dose- and time-dependent manner. Increasing Src expression and activity led to increased phosphorylation of Akt, a mediator of cellular survival pathways, whereas decreasing Src activity led to decreased Akt phosphorylation. In colon tumor cells with high Src activity, the PI3 kinase inhibitor LY 294002 sensitized cells to anoikis. These results suggest that Src activation may contribute to colon tumor progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis.