Tumors are stiffer than normal tissue, and tumors have altered integrins. Because integrins are mechanotransducers that regulate cell fate, we asked whether tissue stiffness could promote malignant behavior by modulating integrins. We found that tumors are rigid because they have a stiff stroma and elevated Rho-dependent cytoskeletal tension that drives focal adhesions, disrupts adherens junctions, perturbs tissue polarity, enhances growth, and hinders lumen formation. Matrix stiffness perturbs epithelial morphogenesis by clustering integrins to enhance ERK activation and increase ROCK-generated contractility and focal adhesions. Contractile, EGF-transformed epithelia with elevated ERK and Rho activity could be phenotypically reverted to tissues lacking focal adhesions if Rho-generated contractility or ERK activity was decreased. Thus, ERK and Rho constitute part of an integrated mechanoregulatory circuit linking matrix stiffness to cytoskeletal tension through integrins to regulate tissue phenotype.

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express α6β4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail–truncated α6β4 integrin. α6β4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of β4 integrin is necessary for basal and epidermal growth factor–induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional β1 and β4 integrin through activation of NFκB, and overexpression of NFκB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5–α6β4 integrin–RAC–NFκB signaling.

A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from preinvasive to invasive phenotype as it may occur ''spontaneously'' in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted basement membrane cultures. These cells remained noninvasive; however, unlike their nonmalignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher-grade tumors. To find functionally significant changes in transition from preinvasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between preinvasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP9, MMP13, MMP15, and MMP17 was up-regulated in the invasive cells. Using small interfering RNA-based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which preinvasive breast cells could acquire invasiveness in a metaplastic context. [Cancer Res 2008;68(5):1378-87]

Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genomewide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM. IntroductionMelanoma, the most lethal form of skin cancer, has increased in incidence and mortality over the last 3 decades. Metastatic disease that is not amenable to surgery is generally refractory to therapy and, therefore, ultimately lethal. Standard chemotherapy typically produces response rates on the order of 10%, and radiotherapy plays only a limited role in disease palliation. Despite these sobering facts, some optimism has been engendered by recent advances in our molecular understanding of the disease, particularly the finding that approximately 80% of metastatic melanomas (MMs) harbor mutually exclusive activating mutations of either N-RAS or B-RAF (reviewed in ref. 1). These lesions lead to activation of the RAF/MEK/ERK/MAPK pathway, which in turn controls the transcription of hundreds if not thousands of genes related to cellular proliferation, survival, and motility (2). Although work in murine models and pharmacological approaches have suggested that RAS-RAF activation is required not only for tumor formation, but also for tumor maintenance (3, 4), the cell-biological effects of ERK activation that are most relevant for tumor formation and progression have not been fully established.Arguably, the evidence for a clinically valuable anticancer immune response is stronger in MM than any other human malignancy (reviewed in refs. 5-8). Functional T cells restricted to melanoma antigens can be readily recovered from patients with MM, establishing the tumor's immunogenicity in humans (5-7, 9).

We show that mitogenic cells expressing T-type Ca 2ϩ channels (T-channels) are more sensitive to the antiproliferative effects of the drugs pimozide and mibefradil than cells without significant T-channel expression. The growth of Y79 and WERI-Rb1 retinoblastoma cells, as well as MCF7 breast cancer epithelial cells, all of which express T-channel current and mRNA for T-channel subunits, is inhibited by pimozide and mibefradil with IC 50 values between 0.6 and 1.5 M. Proliferation of glioma C6 cells, which show little T-channel expression, is less sensitive to these drugs (IC 50 ϭ 8 and 5 M for pimozide and mibefradil, respectively). Neither drug seems to alter cell cycle or the expression of cyclins. Although this strong correlation between T-channel expression and growth inhibition exists, the following results suggest that the drugs inhibit cell growth via different cytotoxic pathways: 1) pimozide and mibefradil have additive effects on T-channel current inhibition, whereas the antiproliferative activity of the drugs together is synergistic; 2) an increase in the number of apoptotic Y79 and MCF7 cells and a decrease in the mRNA for the antiapoptotic gene Bcl-2 is detected only in pimozide-treated cells, whereas in mibefradiltreated cells, the toxicity is primarily necrotic; and 3) growth inhibition by mibefradil is reduced in Y79 cells transfected with T-channel antisense and in differentiated Y79 cells (which have decreased T-channel expression), but growth inhibition by pimozide is affected to a lesser extent. These results suggest that pimozide and mibefradil inhibit cell proliferation via different cytotoxic pathways and that in the case of pimozide, it is unlikely that this effect is mediated solely by T-channel inhibition.