CIP4 and Src in Promoting the Migration and Invasion of Breast Cancers

Cellular invasion represents a critical early step in the metastatic cascade, and many proteins have been identified as part of an “invasive signature.” The non-receptor tyrosine kinase Src is commonly upregulated in breast cancers, often in conjunction with overexpression of EGFR. Signaling from this pathway stimulates cell proliferation, migration, and invasion and frequently involves proteins that regulate the cytoskeleton. My data demonstrates that inhibition of Src, using the small-molecule inhibitor dasatinib, impairs cellular migration and invasion. Furthermore, Src inhibition sensitizes the cells to the effects of the chemotherapeutic doxorubicin resulting in dramatic, synergistic inhibition of proliferation with combination treatments. The Src-targeted protein CIP4 (Cdc42-interacting protein 4) associates with curved plasma membranes to scaffold complexes of Cdc42 and N-WASp. In these experiments, I show that CIP4 overexpression correlates with triple-negative biomarker status, cellular migration, and invasion of (breast cancer cells. Inhibition of CIP4 expression significantly decreases migration and invasion. Furthermore, I demonstrate the novel finding that CIP4 localizes to invadopodia, which are finger-like projections of the actin cytoskeleton that are associated with matrix degradation and cellular invasion. Depletion of CIP4 in invasive cells impairs the formation of invadopodia and the degradation of gelatin. Therefore, CIP4 is a critical component of the invasive phenotype acquired by human breast cancer cells. In this body of work, I propose a model in which CIP4 promotes actin polymerization by stabilizing the active conformation of N-WASp. CIP4 and N-WASp are both phosphorylated by Src, implicating this pathway in Src-dependent cytoskeletal rearragement. This represents a novel role for F-BAR proteins in migration and invasion.

p75 receptor-mediated neurotrophism: A new mechanism of melanoma cell survival and invasion

Trophism as a "clonal dominance" support mechanism for tumor cells is an unexplored area of tumor progression. This report presents evidence that the human melanoma low-affinity neurotrophin receptor (p75) can signal independently of its high-affinity tyrosine kinase counterparts, the TRK family of kinases. Signaling may be accomplished by a p75-associated purine-analog-sensitive kinase and results in enhanced invasion into a reconstituted basement membrane with a corresponding stimulation of matrix metalloproteinase-2 expression. Additionally, a "stress culture" survival assay was developed to mimic the growth limiting conditions encountered by melanoma cells in a rapidly growing primary tumor or metastatic deposit prior to neoangiogenesis. Under these conditions, p75, promotes the survival of high p75 expressing brain-colonizing melanoma cells. Extensive 70W melanoma cell-cell contact, which downregulates p75, immediately precedes the induction of cell death associated with diminished production of two key cell survival factors, bcl-2 and the p85 subunit of phosphoinositol-3-kinase, and an elevation in apoptosis promoting intracellular reactive oxygen species (ROSs). Since one function of bcl-2 may be to control the generation of ROSs via the antioxidant pathway, these cells may receive a apoptosis-prompting "double hit". 70W melanoma cell death occurred by an apoptotic mechanism displaying classical morphological changes including plasma membrane blebbing, loss of microvilli and redistribution of ribosomes. 70W apoptosis could be pharmacologically triggered following anti-p75 monoclonal antibody-mediated clustering of p75 receptors. 70W cells fluorescently sorted for high-p75 expression (p75$\sp{\rm H}$ cells) exhibited an augmented survival potential and a predilection to sort with the S + G2/M growth phase, relative to their low p75 expressing, p75$\sp{\rm L}$ counterparts. Apoptosis is significantly delayed by p75$\sp{\rm H}$ cells, whereas p75$\sp{\rm L}$ cells are exquisitely prone to initiate apoptosis. Importantly, the p75$\sp{\rm L}$ cells that survive apoptosis, highly re-expressed p75 and were remarkably responsive to exogenous NGF.^ These are the first data to implicate p75-mediated neurotrophism as an invasion and survival support mechanism employed by brain-metastatic cells. In particular, these results may have implications in little understood phenomena of tumor progression, such as the emergence of "clonal dominance" and tumor dormancy. ^

Cell surface $\beta$-1,4-galactosyltransferase function during invasion and metastasis

Metastasis is the complex process of tumor cell spread which is responsible for the majority of cancer-related deaths. Metastasis necessitates complex phenotypic changes, many of which are mediated by changes in the activities of cell surface molecules. One of these is cell surface $\beta$1,4-galactosyltransferase (GalTase), which is elevated on more highly metastatic cells. In this study, both molecular and biochemical methods were used to perturb and manipulate cell surface GalTase levels on K1735 murine melanoma cell lines in order to examine its function in metastasis.^ As expected, highly metastatic K1735 variants have higher cell surface GalTase than poorly metastatic variants. Stably transfected K1735 cell lines that overexpress surface GalTase were created. These cell lines were assayed for metastatic ability using an invasion chamber with Matrigel-coated filter inserts. Cells with increased surface GalTase were uniformly more invasive than neo transfected controls. With multiple cell lines, there was a direct correlation (r = 0.918) between surface GalTase activity and invasiveness. Homologous recombination was used to create K1735 cells with decreased levels of surface GalTase. These cells were uniformly less invasive than controls. Cell surface GalTase was inhibited using two different biochemical strategies. In both cases, inhibition of surface GalTase led to a decrease in in vivo metastatic ability of K1735 cells. This is the first direct experimental evidence addressing GalTase function in metastasis. These data provide several lines of independent evidence which show that cell surface GalTase facilitates invasion and metastasis. ^

S100A4 is a molecular mediator of endometrial carcinoma invasion

The molecular mechanisms of endometrail cancer invasion are poorly understood. S100A4, a member of the S100 Ca2+-binding protein family, was identified by oligonucleotide microarray qRT-PCR, and IHC, to be highly overexpressed in invasive endometrial carcinomas compared to non-invasive tumors. HEC-1A endometrial cancer cells transfected with S100A4 siRNA had undetectable S100A4 protein, decreased migration and invasion. The mechanism of S100A4 upregulation in endometrial cancer remains unclear. Methylation of the S100A4 gene was detected in benign endometrial glands and grade 1 tumors with no S100A4 expression. In contrast, grade 3 endometrioid tumors with high S100A4 expression showed no methylation of the gene. 5-Aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, induced the expression of S100A4 in the less invasive EC cell line, KLE, in which the S100A4 gene is hypermethylated and minimally expressed. S100A4 was induced during TGF-β1-triggered cell scattering in HEC-1A cells, in which S100A4 was demethylated. Transfection of HEC-1A cells with S100A4 siRNA significantly reduced the effect of TGF-β1 on basal migration and invasion. Our preliminary data suggested that this upregulation was mediated by the transcription factor Snail. One Snail binding consensus site was found in the region where DNA methylation was closely correlated with S100A4 gene expression. Chromatin immunoprecipitation assay confirmed the binding of Snail to this consensus site in HEC-1A cells. In SPEC2 endometrial cancer cells, loss of Snail leads to repressed S100A4 gene expression. Similar to S100A4, Snail was overexpressed in aggressive endometrial tumors. Our study suggested that the S100A4 gene was demethylated and further upregulated by the TGF-β1 and Snail pathway in invasive endometrial cancer. S100A4 could potentially serve as a good molecular marker for invasiveness and a target for therapeutic intervention for advanced endometrial cancer. ^

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.