Insulin-like growth factor binding protein-1 (IGFBP-1) inhibits breast cancer cell motility

The breast cancer malignant phenotype is regulated by steroid hormones and peptide growth factors. We have shown previously that insulin-like growth factor-I (IGF-I) stimulates cell motility in a metastatic cell line, MDA-231BO. In this study, we show that neutralization of IGF action by a type I IGF receptor (IGFR1) blocking antibody or neutralization of IGF-I by IGFBP-1 reduced cell motility. However, in addition to inhibiting IGF effects, IGFBP-1 also diminished basal motility. Because IGFBP-1 contains a RGD motif important in binding of fibronectin to its alpha 5 beta 1 integrin receptor, we examined the effect of inhibiting integrin function on cell motility. As expected, disruption of fibronectin-integrin interactions interrupted basal motility in MDA-231BO cells. In addition, disruption of integrin function by an alpha 5 beta 1 blocking peptide also inhibited IGF stimulation of cell motility. To determine whether integrin function could interfere with IGF signaling, we used an alpha 5 beta 1 blocking peptide to show that in MDA-231BO cells integrin occupancy appeared necessary for phosphorylation of insulin receptor substrate-2 but not for IGFR1 activation. We conclude that IGFR1 and integrin action are linked in these breast cancer cells as disruption of integrin binding to its receptor influences IGF signaling pathways. Moreover, IGFBP-1 could have dual effects on cancer cell motility by disrupting both receptor systems.     

Insulin-like growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation that is modulated by alphavbeta3 integrin signaling

Insulin-like growth factor 1 (IGF-1) has been implicated in promoting mitogenic, metastatic and antiapoptotic phenotypes in several types of cancer. But little is known about the signal interaction of IGF-1 and integrin in the regulation of cervical cancer development and progression. This study is to investigate the regulatory mechanism of IGF-1 receptor (IGF-1R) signaling and its importance in cervical cancer formation. The growth and invasiveness of cervical cancer cells (SiHa and CaSki) were dose-dependently stimulated by IGF-1, whereas those of normal cervical epithelial cells were not. The immunoblot showed that IGF-1R proteins were abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells. IGF-1-stimulated invasion and proliferation were abolished by functional-blocking monoclonal antibody against IGF-1R, whereas these cellular functions were unaffected by either IgG or monoclonal antibody to insulin receptor. Functional-blocking monoclonal antibody against integrins alpha(v)beta3, but not alpha2 alpha3, alpha4 alpha6 beta1, beta4 or alpha2beta1, inhibited the IGF-1-stimulated invasion and proliferation in cervical cancer cells. alpha(v)beta3 integrin modulated IGF-1R phosphorylation by altering the rate of Src homology 2-containing phosphotyrosine phosphatase (SHP-2) recruitment to the activated IGF-1R. The modulation of alpha(v)beta3 occupancy also affected the activation of IGF-1R downstream-signaling elements, including activation of Akt and extracellular signal-regulated protein kinases 1/2 (Erk1/2). The treatment of blocking antibody of alpha(v)beta3 integrin or IGF-1R significantly inhibited tumor growth and caused tumor regression in SCID mice model. Immunoblots of tumor tissues confirmed that the phosphorylation of IGF-1R and downstream targets of Akt and Erk1/2 were remarkably decreased in SCID mice treated with blocking antibodies of alpha(v)beta3 or IGF-1R. Thus, these data suggest that the signal interaction between IGF-1R and alpha(v)beta3 integrin plays an important role in promoting the development and progression of cervical cancer.     

AlphaVbeta3 integrin regulates heregulin (HRG)-induced cell proliferation and survival in breast cancer

alpha(v)beta(3) integrin-overexpression in tumor associated vasculature is a marker of poor prognosis in breast cancer. A positive correlation between alpha(v)beta(3) integrin and overexpression of Heregulin (HRG), a growth factor associated with breast cancer aggressiveness was recently demonstrated. Here, we addressed the role of alpha(v)beta(3) in the proliferation and survival of HRG-overexpressing breast cancer models. Expression of the RGD-binding integrins alpha(v)beta(3), alpha(v)beta(5) and alpha(v)beta(6) was assessed in the HRG-overexpressing breast cancer cells MDA-MB-231, Hs578T (231/WT and Hs578T/WT, respectively) and derived cells transfected with the antisense orientation of the HRG-beta2 full-length cDNA (231/ASPOOL, 231/AS31 and Hs578T/AS15). Interestingly, only alpha(v)beta(3) expression was noticeably decreased by blockade of HRG expression in the 231/ASPOOL, 231/AS31 and Hs578T/AS15 cells. Small RGD-based peptidomimetic alpha(v)beta(3) antagonists significantly decreased cell viability and anchorage-dependent cell growth of HRG-overexpressing cells, while the low-HRG-expressing 231/AS31 cells did not show a significant response. Mechanistically, functional blockade of alpha(v)beta(3) impaired HRG-promoted hyperactivation of ERK1/ERK2 MAPK without altering the activation of AKT. Flow cytometric analysis of the cell cycle demonstrated that alpha(v)beta(3) antagonists significantly decreased S- and G2/M-phase subpopulations of 231/WT and control 231/VEC cells. Comparable, this effect was linked to an increase in the levels and nuclear translocation of the CDKs inhibitor p27(Kip1). Besides downregulating alpha(v)beta(3) and its driven signaling, HRG blockade led to decreased levels of CYR61 in 231/ASPOOL and 231/AS31 cells. Considering that CYR61 is sufficient to upregulate the expression of alpha(v)beta(3), we then assessed alpha(v)beta(3) levels in MDA-MB-231 cell derivatives expressing the antisense orientation of the CYR61 cDNA (231/CYR61AS-5 and 231/CYR61AS-8). Remarkably, downregulation of CYR61 drastically decreased the levels of alpha(v)beta(3) in the 231/CYR61-5 and 231/CYR61-8 cells, providing further evidence of a key role for CYR61 in HRG-dependent alpha(v)beta(3) overexpression. Moreover, blockade of CYR61 expression impaired the HRG-induced hyperactivation of ERK1/ERK2 MAPK without altering the activation status of AKT in MDA-MB-231 cells, thus resembling the effects exerted by the downregulation of HRG expression as well as by functional blockade of alpha(v)beta(3). These results indicate that HRG is regulating alpha(v)beta(3) levels as well as alpha(v)beta(3)-triggered signaling through its downstream effector, CYR61, in highly invasive breast cancer cells. Altogether, the data presented here provide evidence of a CYR61-regulatory role on alpha(v)beta(3) integrin expression in the modulation of uncontrolled growth of HRG-overexpressing breast carcinomas. This work supports additional studies concerning the use of integrin antagonists as dual therapeutic agents in breast cancer, targeting both, endothelial and tumor cells.     

Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumor cell survival.

Growth and dissemination of malignant melanoma has a profound impact on our population, and little is known concerning the mechanisms controlling this disease in humans. Evidence is provided that integrin alpha(v)beta3 plays a critical role in M21 melanoma tumor survival within human skin by a mechanism independent of its known role in angiogenesis. Antagonists of alpha(v)beta3 blocked melanoma growth by inducing tumor apoptosis. Moreover, M21 melanoma cell interactions with denatured collagen, a known ligand for alpha(v)beta3, caused a 5-fold increase in the relative Bcl-2:Bax ratio, an event thought to promote cell survival. Importantly, denatured collagen colocalized with alpha(v)beta3-expressing melanoma cells in human tumor biopsies, suggesting that alpha(v)beta3 interaction with denatured collagen may play a critical role in melanoma tumor survival in vivo.     

Studies on the expression of alpha(v)beta3 integrin receptors in non-malignant and malignant human cervical tumor tissues

Integrins are group of cell surface receptors, which mediate adhesion between cell-cell and cell-extracellular matrix proteins of the basement membrane. Integrins play an important role in cellular growth, development, morphology, signalling and also in tumor development. Among the integrin group of cell surface receptors one of the most important member is alpha(v)beta3 integrin receptor. Evidences say that the expression of this integrin receptor is regulated during tumor development. Large numbers of studies have been done to establish the role of alpha(v)beta3 integrin receptor in human melanoma systems. Expression of this receptor in metastatic but not benign melanomas suggests a role for this integrin in the regulation of tumor proliferation. Alpha(v)beta3 integrin receptor also plays a significant role in tumor angiogenesis, apoptosis and signal transduction process. Recent studies show that collagenase MMP-2 binds directly to integrin alpha(v)beta3 on the surface of invasive tumor cells and facilitates tumor cell invasion. In this present communication we studied the expression of alpha(v)beta3 integrin receptor in malignant and non-malignant cervical tumor tissues. Because receptor ligand interaction is a cell surface phenomenon the membrane fraction of tumor tissues was separated and the expression of alpha(v)beta3 integrin receptor was assayed by ELISA and immunoprecipitation from membrane extracted protein fraction. Comparative ELISA and immunoprecipitation clearly demonstrates much higher expression of alpha(v)beta3 vitronectin integrin receptor in the membrane fraction of malignant human cervical tumor tissues than nonmalignant tissue membrane fractions.     

Alpha6beta1 integrin induces proteasome-mediated cleavage of erbB2 in breast cancer cells

ErbB2 and alpha6 integrin have been implicated in malignancy of breast cancer cells. Here we have determined the influence of alpha6beta1 integrin on erbB2 signaling in anchorage-independent growth, using MDA-MB435 breast cancer cells. Firstly, we transfected the cells with erbB2 cDNA, and isolated cells with high or low levels of alpha6beta1 integrin by cell sorting (alpha6H-ErbB and alpha6L-ErbB). We found that an erbB ligand, heregulin beta1, enhanced growth activity of alpha6L-ErbB cells, but not alpha6H-ErbB cells. Secondly, we established cells expressing a beta4 integrin deletion mutant (beta4-deltacyt), which selectively inhibited alpha6beta1 integrin expression and adhesion to laminin-1. Again, heregulin beta1 enhanced the growth of erbB2 cDNA-transfected beta4-deltacyt cells, but not mock cells. Western blot analysis revealed that heregulin beta1 stimulated phosphorylation of Akt and its downstream molecules, GSK3beta and p70S6kinase, and that the extent of phosphorylation was greater in ErbB2/beta4-deltacyt cells than ErbB2/mock cells. Furthermore, we found that the erbB2 cytoplasmic domain was truncated in ErbB2/mock cells, which was independent of ligand stimulation and adhesion, and was suppressed by proteasome inhibitors. These results suggest that alpha6beta1 integrin inhibits erbB2 signals by inducing proteasome-dependent proteolytic cleavage of the erbB2 cytoplasmic domain, and may thereby contribute to the regulation of tumor growth.     

Altered cell-matrix contact: a prerequisite for breast cancer metastasis?

The integrins are receptors that regulate interaction between epithelial cells and the extracellular matrix. Previous studies have shown that a reduction in the expression of the alpha2beta1, alpha3beta1, alpha6beta1, alpha(v)beta1 and alpha(v)beta5 integrins in primary breast cancer is associated with positive nodal status. In order to assess the functional significance of altered integrin expression, primary breast cancer cells were derived from individual patients with known tumour characteristics using immunomagnetic separation. Purified human fibronectin, vitronectin, laminin and type IV collagen were used to represent the principal extracellular matrix proteins in an in vitro adhesion assay. Primary breast cancer cells from lymph node-positive patients were significantly less adhesive to each of the matrix proteins studied (P<0.001, Mann-Whitney U-test). Matrix adhesion of primary breast cancer cells from node-negative patients was inhibited by appropriate integrin monoclonal antibodies (P<0.001, paired Wilcoxon test). Adhesion to fibronectin, vitronectin and laminin, but not type IV collagen, was influenced by the inhibitor arginine-glycine-aspartate, suggesting that breast cancer cell recognition of collagen IV is mediated through alternative epitopes. Weak matrix adhesion correlated with loss of integrin expression in tissue sections from corresponding patients assessed using immunohistochemistry. This study demonstrates a link between altered integrin expression and function in primary breast cancers predisposed to metastasize.     

Angiopoietin-2 stimulates breast cancer metastasis through the alpha(5)beta(1) integrin-mediated pathway

Acquisition of a metastatic phenotype by breast cancer cells includes alternations of multigenic programs that permit tumor cells to metastasize to distant organs. Here, we report that angiopoietin-2 (Ang2), a known growth factor, is capable of promoting breast cancer cell invasion leading to metastasis. Analysis of 185 primary human breast cancer specimens that include 97 tumors showing lymph node and/or distant metastasis reveals a significant correlation between the expression of Ang2 and E-cadherin, Snail, metastatic potential, tumor grade, and lymph-vascular invasion during breast cancer progression. Using a xenograft model, we show that overexpression of Ang2 in poorly metastatic MCF-7 breast cancer cells suppresses expression of E-cadherin and induces Snail expression and phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) promoting metastasis to the lymph nodes and lung. In cell culture, Ang2 promotes cell migration and invasion in Tie2-deficient breast cancer cells through the alpha(5)beta(1) integrin/integrin-linked kinase (ILK)/Akt, GSK-3beta/Snail/E-cadherin signaling pathway. Inhibition of ILK and the alpha(5)beta(1) integrin abrogates Ang2 modulation of Akt, GSK-3beta, Snail, and E-cadherin and Ang2-stimulated breast cancer cell migration and invasion. Together, these results underscore the significant contribution of Ang2 in cancer progression, not only by stimulating angiogenesis but also by promoting metastasis, and provide a mechanism by which breast cancer cells acquire an enhanced invasive phenotype contributing to metastasis.     

Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins

alpha(v) integrins have been shown to play an important role in epithelial-derived cell migration, cell growth and tumor invasion/metastasis, however their role on cells of hematopoietic origin is less clear. Epstein-Barr virus (EBV), a human herpesvirus associated with several lymphoproliferative disorders in man, induces expression of alpha(v) integrins on transformed B lymphocytes. In the studies reported here, we show that EBV infection increases alpha(v), beta3 and beta5 integrin subunit mRNAs as well as upregulates the expression of the alphavbeta3 integrin protein on human B cells. Among the nine different EBV proteins expressed in latently infected B cells (nuclear and plasma membrane-associated), only LMP1, LMP2A and EBNA2 were shown to selectively transactivate the alpha(v) integrin promoter. Treatment of EBV-transformed B cells with alpha(v) antisense oligonucleotides specifically reduced cell surface expression of alpha(v) integrins, inhibited cell growth in low serum, reduced cell invasion in matrigels and decreased expression of metalloprotease, MMP9. These studies indicate that alpha(v) integrins play a significant role in EBV-induced B-lymphocyte proliferation and invasion. Strategies to interfere with alphav integrin expression and/or function may therefore be of potential value in the treatment of EBV-associated lymphoproliferative disorders.     

Integrin alpha v beta 3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models

PURPOSE: Integrins alpha v beta 3 and alpha v beta 5 are important in tumor growth and angiogenesis and have been recently explored as targets for cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We explored the combination of integrin antagonist Cilengitide (EMD 121974) and ionizing radiation. METHODS AND MATERIALS: Levels of alpha v beta 3 were determined for human umbilical vein endothelial cells (HUVEC), as well as H157 and H460 human non-small-cell lung cancer cells, using FACS analysis and immunofluorescence imaging. Clonogenic assays, Western immunoblots probed for cleaved caspase 3, and Annexin-V probing were used to evaluate cell survival and apoptosis. A cell detachment assay and matrigel assay were used to further examine the effects of treatment. RESULTS: Human umbilical vein endothelial cells had the highest alpha v beta 3 level, followed by H157, and H460. Interestingly, we found that 5 Gy irradiation induced expression of alpha v beta 3 in all cell lines. Clonogenic assays showed a radiosensitizing effect with Cilengitide, and calculation of the dose enhancement ratio showed that the effect was highest in HUVECs (1.38), followed by H157 (1.19), and H460 (1.10), corresponding to the levels of target expression. There was an increase in apoptotic cells after combination treatment with Cilengitide and radiation, and there was an increase in detached cells after treatment with Cilengitide. Additionally, there was decreased endothelial tubule formation after combination treatment. CONCLUSIONS: We conclude that radiation induces expression of alpha v beta 3 integrin in endothelial and non-small-cell lung cancer models, and that integrin antagonist Cilengitide is a radiosensitizer in proportion to the levels of target integrin expression.     

Expression and function of CYR61, an angiogenic factor, in breast cancer cell lines and tumor biopsies

We have previously shown that expression of heregulin (HRG) is closely correlated with breast cancer progression. We have subsequently isolated Cyr61, a ligand for the alpha(v)beta3 integrin that is differentially expressed in HRG-positive cells, and have shown that it is expressed in all of the invasive and metastatic breast cancer cell lines tested. Preliminary evaluation of Cyr61 expression in breast tumor biopsies revealed expression of Cyr61 in about 30% of invasive breast carcinomas. Significantly, we demonstrated that Cyr61 is a downstream effector of HRG action, because a Cyr61-neutralizing antibody abolished the ability of HRG-expressing cells to migrate in vitro. Furthermore, we have shown that HRG-expressing cells denote higher levels of alpha(v)beta3 expression, and we have established that Cyr61 action is mediated, at least in part, through its receptor alpha(v)beta3, because a functional blocking antibody of the alpha(v)beta3 blocked the Matrigel outgrowth of HRG-expressing cells. These results strongly suggest that Cyr61 is necessary for HRG-mediated chemomigration and that Cyr61 plays a functional role in breast cancer progression, possibly through its interactions with the alpha(v)beta3 receptor.     

The COOH-terminal globular domain of fibrinogen gamma chain suppresses angiogenesis and tumor growth

Fibrinogen is a major plasma protein (350 kDa) that induces proliferative signals by serving as a scaffold to support the binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during wound healing, angiogenesis, and tumor growth. Fibrin(ogen) degradation products generated during fibrinolysis are implicated in tissue injury. The fibrinogen gamma chain has a COOH-terminal globular domain (gamma C, residues 151-411 of the gamma chain, 30 kDa) to which several integrin cell adhesion receptors (e.g., platelet alpha(IIb)beta(3), endothelial alpha(v)beta(3), and leukocyte alpha(M)beta(2)) bind. Integrins play a critical role in signal transduction from fibrin(ogen). We found that gamma C and its truncation mutant (designated gamma C399tr), with a deletion of the COOH-terminal 12 residues, induced apoptosis of endothelial cells and blocked tube formation of endothelial cells. DLD-1 human colon cancer cells that secrete gamma C or gamma C399tr grew at similar levels in vitro but grew much slower in vivo than mock-transfected cells. The recombinant purified gamma C399tr fragment markedly suppressed tumor growth, development of intratumoral vasculature, and tumor metastasis in vivo in the highly metastatic Met-1 breast cancer model. The determinant responsible for binding to endothelial cells is cryptic in native fibrinogen but is exposed in gamma C and gamma C399tr. These results suggest that fibrinogen has a novel cryptic determinant, which can exert apoptosis-inducing activity on endothelial cells when exposed, and polypeptides containing this determinant have therapeutic potential.     

Aberrant expression of integrin and erbB subunits in breast cancer cell lines

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.     

The pattern of metastasis of human melanoma to the central nervous system is not influenced by integrin alpha(v)beta(3) expression

We investigated the effect of integrin alpha(v)beta(3) expression on the metastatic pattern of human melanoma cells in the central nervous system (CNS). For this purpose, we developed a hematogenous CNS melanoma metastasis model in nude mice using a modified internal carotid artery infusion technique. This protocol revealed 2 different patterns of CNS metastasis. The integrin alpha(v)beta(3)-expressing melanoma lines Mel57 and Zkr nearly exclusively produced metastases in the brain parenchyma, whereas cells of the BLM and MV3 lines, devoid of integrin alpha(v)beta(3) expression, preferentially metastasized to dura mater and leptomeninges. Treatment with hyaluronidase to obtain single BLM cell suspensions did not influence the metastatic pattern, indicating that this was not simply the result of entrapment of tumor cell aggregates in large-sized leptomeningeal vessels. The role of integrin alpha(v)beta(3) expression in the process of metastasis was tested by transfection of BLM, but did not lead to an altered pattern of metastasis. We did observe, however, slower growth of the transfected tumors, although the in vitro growth rate was unaltered, indicating a reduction in tumorigenicity. We conclude from our findings that CNS metastasis of melanoma cells in the mouse xenograft model occurs in at least 2 different but very reproducible patterns. Although it is predicted that adhesion of tumor cells to endothelial cells plays a role in this phenomenon, tumor cell integrin alpha(v)beta(3) expression per se does not explain the difference in metastatic behavior in the CNS. We assume that other, as yet unknown factors, must be involved.     

Identification of heat shock protein 60 as a molecular mediator of alpha 3 beta 1 integrin activation

The alpha 3 beta 1 integrin is involved in the adhesion of metastatic breast cancer cells to the lymph nodes and to osteoblasts in the bone. Regulation of the affinity or avidity of integrins for their ligands may result from conformational changes induced by changes in the microenvironment of the integrin. Two surface proteins, 55 and 32 kDa, coimmunoprecipitated with the alpha 3 beta 1 integrin from breast carcinoma cells. The 55-kDa protein preferentially associated with the active form of the alpha 3 beta 1 integrin. The protein was identified as HSP60 using two-dimensional electrophoresis and mass spectrometry and confirmed by reimmunoprecipitation of the integrin immune complex with an anti-HSP60 antibody. In cell spreading assays on a thrombospondin-1 substrate, addition of exogenous-recombinant HSP60 was sufficient to specifically activate alpha 3 beta 1 integrin but not to activate function of alpha 2 beta 1, alpha v beta 3, alpha 4 beta 1, or alpha 5 beta 1 integrins. Furthermore, mizoribine, an HSP60-binding drug, blocked activation of the alpha 3 beta 1 integrin induced by insulin-like growth factor 1 (IGF1) or exogenous recombinant HSP60 and inhibited the association of HSP60 with the integrin. Additionally, inhibiting the surface expression of endogenous HSP60 by nonactin inhibited activation of the alpha 3 beta 1 integrin by IGF1. These data demonstrate that HSP60 binding is sufficient to activate alpha 3 beta 1 integrin function and suggest that association of endogenous HSP60 with alpha 3 beta 1 integrin is necessary for IGF1-induced activation.     

Src phosphorylates Tyr284 in TGF-beta type II receptor and regulates TGF-beta stimulation of p38 MAPK during breast cancer cell proliferation and invasion

Genetic and epigenetic events often negate the cytostatic function of transforming growth factor-beta (TGF-beta) in mammary epithelial cells (MEC), which ultimately enables malignant MECs to proliferate, invade, and metastasize when stimulated by TGF-beta. The molecular mechanisms underlying this phenotypic conversion of TGF-beta function during mammary tumorigenesis remain poorly defined. We previously established alpha(v)beta(3) integrin and Src as essential mediators of mitogen-activated protein kinase (MAPK) activation, invasion, and epithelial-to-mesenchymal transition stimulated by TGF-beta in normal and malignant MECs. Mechanistically, beta(3) integrin interacted physically with the TGF-beta type II receptor (TbetaR-II), leading to its tyrosine phosphorylation by Src and the initiation of oncogenic signaling by TGF-beta. We now show herein that Src phosphorylated TbetaR-II on Y284 both in vitro and in vivo. Interestingly, although the expression of Y284F-TbetaR-II mutants in breast cancer cells had no effect on TGF-beta stimulation of Smad2/3, this TbetaR-II mutant completely abrogated p38 MAPK activation by TGF-beta. Accordingly, Src-mediated phosphorylation of Y284 coordinated the docking of the SH2 domains of growth factor receptor binding protein 2 (Grb2) and Src homology domain 2 containing (Shc) TbetaR-II, thereby associating these adapter proteins to MAPK activation by TGF-beta. Importantly, Y284F-TbetaR-II mutants also abrogated breast cancer cell invasion induced by alpha(v)beta(3) integrin and TGF-beta as well as partially restored their cytostatic response to TGF-beta. Our findings have identified a novel alpha(v)beta(3) integrin/Src/Y284/TbetaR-II signaling axis that promotes oncogenic signaling by TGF-beta in malignant MECs and suggest that antagonizing this signaling axis may one day prove beneficial in treating patients with metastatic breast cancers.     

The desmoplastic reaction surrounding hepatic colorectal adenocarcinoma metastases aids tumor growth and survival via alphav integrin ligation

PURPOSE: The treatment of metastatic colorectal carcinoma represents a major clinical challenge. We investigated the hypothesis that the desmoplastic reaction within the liver elicited by metastatic adenocarcinoma, characterized by collagen I deposition and altered collagen IV distribution, promotes the growth and survival of hepatic colorectal carcinoma metastases. EXPERIMENTAL DESIGN: Partial hepatectomy specimens for metastatic colorectal adenocarcinoma were examined immunohistochemically for differential integrin expression. Human colorectal adenocarcinoma cell lines HT-29, KM12SM, and KM12c were grown on wild-type collagen I or IV, or cleavage-resistant r/r collagen I, and assessed for their growth, survival, and resistance to 5-fluorouracil. The effect of alpha(v)beta(3) and alpha(v)beta(5) integrin blockade by neutralizing antibodies was examined. RESULTS: Collagen I, in contrast to collagen IV, significantly enhanced the growth, survival, and chemoresistance of colorectal carcinoma cells. Blockade of the alpha(v)beta(3) and alpha(v)beta(5) integrins significantly reduced colorectal carcinoma cell proliferation on collagen, especially in the cell line with the most metastatic potential. These in vitro findings correlated with the pattern of integrin expression identified within resected hepatic colorectal carcinoma metastases. Using matrix metalloproteinase-resistant r/r collagen I as a dominant negative ligand for alpha(v) integrins, we showed a key role for this integrin-ligand interaction in mediating the survival and proliferation of colorectal carcinoma cells. CONCLUSIONS: Desmoplasia has an important role in the development of hepatic colorectal carcinoma metastasis. The interaction between integrin and collagen I is identified as a potential therapeutic target.     

PTHrP increases xenograft growth and promotes integrin alpha6beta4 expression and Akt activation in colon cancer

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines. Expression of PTHrP and phosphatidylinositol 3-kinase (PI3-K) pathway components correlates with the severity of colon carcinoma. Here we observed a positive effect of endogenous PTHrP on LoVo (human colon cancer) cell proliferation, migration, invasion, integrin alpha6 and beta4 expression, and p-Akt levels. There was a direct correlation between PTHrP expression and anchorage-independent cell growth. PTHrP significantly increased xenograft growth; tumors from PTHrP-overexpressing cells showed increased expression of integrins alpha6 and beta4, and PI3-K pathway components. The higher expression of PTHrP in human colon cancer adenocarcinoma vs. normal colonic mucosa was accompanied by increased integrin alpha6 and beta4 levels. Elevated PTHrP expression in colon cancer may thus upregulate integrin alpha6beta4 expression, with consequent PI3-K activation. Targeting PTHrP might result in effective inhibition of tumor growth, migration, and invasion.     

Coordinated expression of integrin subunits,matrix metalloproteinases (MMP), angiogenic genes and Ets transcription factors in advanced-stage ovarian carcinoma: a possible activation pathway?

The events that mediate tumor progression in ovarian carcinoma are poorly understood to date. This review summarizes our results studying metastasis-associated molecules in advanced-stage ovarian carcinomas, details the co-expression of mRNA of these genes, and discusses their prognostic role. Fifty-five primary and metastatic FIGO stage III-IV ovarian carcinomas were analyzed for the expression of alpha v and beta1 integrin subunits, the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP, the MMP inhibitor TIMP-2, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), PEA3 and Ets-1 using mRNA in situ hybridization. Tumor and adjacent stromal cell expression was scored. The association between integrin subunit expression and the expression of MMP, TIMP-2, angiogenic genes, PEA3 and Ets-1 was statistically analyzed. Alpha v integrin subunit mRNA expression in carcinoma cells showed significant association with that of MMP-2 and IL-8 in this cellular compartment, while the presence of beta1 integrin subunit mRNA showed similar association with that of PEA3, Ets-1, IL-8, bFGF and MMP-2. Expression of beta1 integrin subunit mRNA in stromal cells was associated with that of TIMP-2 and Ets-1 in this compartment. In addition, significant intercellular associations were found between alpha v integrin subunit mRNA expression in carcinoma cells and stromal cell expression of Ets-1, as well as between stromal cell expression of alpha v integrin subunit and labeling for IL-8 in carcinoma cells. The presence of beta1 integrin subunit mRNA in carcinoma cells showed a significant association with that of Ets-1, IL-8 and bFGF in stromal cells, while the presence of beta1 integrin subunit mRNA in stromal cells was associated with tumor PEA3 mRNA expression. To the best of our knowledge, this is the first evidence for coordinated autocrine and paracrine expression of members of these four families of metastasis-associated genes in human cancer. The results of this analysis support experimental data regarding cross-talk between carcinoma cells and peritumoral fibroblasts. They also suggest the existence of a putative activation sequence of metastatic genes, involving the beta1 (and possibly alpha v) integrin subunits, IL-8, PEA3, Ets-1 and MMP in ovarian carcinoma.     

Overexpression of platelet-type 12-lipoxygenase promotes tumor cell survival by enhancing alpha(v)beta(3) and alpha(v)beta(5) integrin expression

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.