Elevated Expression of Stromal Palladin Predicts Poor Clinical Outcome in Renal Cell Carcinoma

The role that stromal renal cell carcinoma (RCC) plays in support of tumor progression is unclear. Here we sought to determine the predictive value on patient survival of several markers of stromal activation and the feasibility of a fibroblast-derived extracellular matrix (ECM) based three-dimensional (3D) culture stemming from clinical specimens to recapitulate stromal behavior in vitro. The clinical relevance of selected stromal markers was assessed using a well annotated tumor microarray where stromal-marker levels of expression were evaluated and compared to patient outcomes. Also, an in vitro 3D system derived from fibroblasts harvested from patient matched normal kidney, primary RCC and metastatic tumors was employed to evaluate levels and localizations of known stromal markers such as the actin binding proteins palladin, alpha-smooth muscle actin (α-SMA), fibronectin and its spliced form EDA. Results suggested that RCCs exhibiting high levels of stromal palladin correlate with a poor prognosis, as demonstrated by overall survival time. Conversely, cases of RCCs where stroma presents low levels of palladin expression indicate increased survival times and, hence, better outcomes. Fibroblast-derived 3D cultures, which facilitate the categorization of stromal RCCs into discrete progressive stromal stages, also show increased levels of expression and stress fiber localization of α-SMA and palladin, as well as topographical organization of fibronectin and its splice variant EDA. These observations are concordant with expression levels of these markers in vivo. The study proposes that palladin constitutes a useful marker of poor prognosis in non-metastatic RCCs, while in vitro 3D cultures accurately represent the specific patient's tumor-associated stromal compartment. Our observations support the belief that stromal palladin assessments have clinical relevance thus validating the use of these 3D cultures to study both progressive RCC-associated stroma and stroma-dependent mechanisms affecting tumorigenesis. The clinical value of assessing RCC stromal activation merits further study

Carcinoma Matrix Controls Resistance to Cisplatin through Talin Regulation of NF-kB

Extracellular matrix factors within the tumor microenvironment that control resistance to chemotherapeutics are poorly understood. This study focused on understanding matrix adhesion pathways that control the oral carcinoma response to cisplatin. Our studies revealed that adhesion of HN12 and JHU012 oral carcinomas to carcinoma matrix supported tumor cell proliferation in response to treatment with cisplatin. Proliferation in response to 30 µM cisplatin was not observed in HN12 cells adherent to other purified extracellular matrices such as Matrigel, collagen I, fibronectin or laminin I. Integrin β1 was important for adhesion to carcinoma matrix to trigger proliferation after treatment with cisplatin. Disruption of talin expression in HN12 cells adherent to carcinoma matrix increased cisplatin induced proliferation. Pharmacological inhibitors were used to determine signaling events required for talin deficiency to regulate cisplatin induced proliferation. Pharmacological inhibition of NF-kB reduced proliferation of talin-deficient HN12 cells treated with 30 µM cisplatin. Nuclear NF-kB activity was assayed in HN12 cells using a luciferase reporter of NF-kB transcriptional activity. Nuclear NF-kB activity was similar in HN12 cells adherent to carcinoma matrix and collagen I when treated with vehicle DMSO. Following treatment with 30 µM cisplatin, NF-kB activity is maintained in cells adherent to carcinoma matrix whereas NF-kB activity is reduced in collagen I adherent cells. Expression of talin was sufficient to trigger proliferation of HN12 cells adherent to collagen I following treatment with 1 and 30 µM cisplatin. Talin overexpression was sufficient to trigger NF-kB activity following treatment with cisplatin in carcinoma matrix adherent HN12 cells in a process disrupted by FAK siRNA. Thus, adhesions within the carcinoma matrix create a matrix environment in which exposure to cisplatin induces proliferation through the function of integrin β1, talin and FAK pathways that regulate NF-kB nuclear activity

Differential Expressions of Adhesive Molecules and Proteases Define Mechanisms of Ovarian Tumor Cell Matrix Penetration/Invasion

Epithelial ovarian cancer is an aggressive and deadly disease and understanding its invasion mechanisms is critical for its treatment. We sought to study the penetration/invasion of ovarian tumor cells into extracellular matrices (ECMs) using a fibroblast-derived three-dimensional (3D) culture model and time-lapse and confocal imaging. Twelve ovarian tumor cells were evaluated and classified into distinct groups based on their ECM remodeling phenotypes; those that degraded the ECM (represented by OVCAR5 cells) and those that did not (represented by OVCAR10 cells). Cells exhibiting a distinct ECM modifying behavior were also segregated by epithelial- or mesenchymal-like phenotypes and uPA or MMP-2/MMP-9 expression. The cells, which presented epithelial-like phenotypes, penetrated the ECM using proteases and maintained intact cell-cell interactions, while cells exhibiting mesenchymal phenotypes modified the matrices via Rho-associated serine/threonine kinase (ROCK) in the absence of apparent cell-cell interactions. Overall, this study demonstrates that different mechanisms of modifying matrices by ovarian tumor cells may reflect heterogeneity among tumors and emphasize the need to systematically assess these mechanisms to better design effective therapies

Genetic ablation of Cav1 differentially affects melanoma tumor growth and metastasis in mice. Role of Cav1 in Shh heterotypic signaling and transendothelial migration.

Both cell-autonomous and non-cell-autonomous factors contribute to tumor growth and metastasis of melanoma. The function of caveolin-1 (Cav1), a multifunctional scaffold protein known to modulate several biologic processes in both normal tissue and cancer, has been recently investigated in melanoma cancer cells, but its role in the melanoma microenvironment remains largely unexplored. Here, we show that orthotopic implantation of B16F10 melanoma cells in the skin of Cav1KO mice increases tumor growth, and co-injection of Cav1-deficient dermal fibroblasts with melanoma cells is sufficient to recapitulate the tumor phenotype observed in Cav1KO mice. Using indirect coculture experiments with fibroblasts and melanoma cells combined with cytokine analysis, we found that Cav1-deficient fibroblasts promoted the growth of melanoma cells via enhanced paracrine cytokine signaling. Specifically, Cav1-deficient fibroblasts displayed increased ShhN expression, which heterotypically enhanced the Shh signaling pathway in melanoma cells. In contrast to primary tumor growth, the ability of B16F10 melanoma cells to form lung metastases was significantly reduced in Cav1KO mice. This phenotype was associated mechanistically with the inability of melanoma cells to adhere to and to transmigrate through a monolayer of endothelial cells lacking Cav1. Together, our findings show that Cav1 may regulate different mechanisms during primary melanoma tumor growth and metastatic dissemination

Integration of SNP and mRNA arrays with microRNA profiling reveals that MiR-370 is upregulated and targets NF1 in acute myeloid leukemia

Abstract Background: Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied. Results: Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1. Conclusions: NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR- 370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors