Overexpression of alpha(v)beta6 integrin in serous epithelial ovarian cancer regulates extracellular matrix degradation via the plasminogen activation cascade

Recent evidence suggests that integrins are involved in the multi-step process of tumour metastasis. The biological relevance of alpha(v) integrins and associated beta-subunits in ovarian cancer metastasis was examined by analysing the expression of these cell surface receptors in nine ovarian cancer cell lines and also in the primary human ovarian surface epithelial cell line (HOSE). beta1, beta3 and beta5 subunits were present in all ten ovarian cell lines. beta6 subunit was present at varying levels in eight out of nine cancer cell lines but was absent in the HOSE cell line. Immunohistochemical staining showed that beta6 was present in both non-invasive (borderline) and high-grade ovarian cancer tissues but was absent in benign and normal ovarian tissue. High alpha(v)beta6 integrin expressing ovarian cancer cell lines had high cell surface expression of uPA and uPAR. Ovarian cancer cell lines expressing high to moderate level of alpha(v)beta6 integrin demonstrated ligand-independent enhanced levels of high molecular weight (HMW)-uPA and pro-matrix metalloproteinase 2 and 9 (pro-MMP-2 and pro-MMP-9) expression in the tumour-conditioned medium. High and moderate expression of alpha(v)beta6 integrin correlated with increased plasminogen-dependent degradation of extracellular matrix which could be inhibited by inhibitors of plasmin, uPA and MMPs or by monoclonal antibody against uPA, MMP-9 or alpha(v)beta6 integrin. These results suggest that endogenous de novo expression of alpha(v)beta6 integrin in ovarian cancer cells may contribute to their invasive potential, and that alpha(v)beta6 expression may play a role in ovarian cancer progression and metastasis.     

Phosphoinositide 3-OH kinase p85alpha and p110beta are essential for androgen receptor transactivation and tumor progression in prostate cancers

Phosphoinositide 3-OH kinases (PI3Ks) are a group of major intracellular signaling molecules. In our previous study, we found that inhibition of PI3K activity suppressed the androgen receptor (AR)-mediated gene expression in prostate cancer cells. The AR has been considered as a critical determinant for the development and progression of human prostate cancers. In this study, we sought to identify the PI3K isoforms involved in AR transactivation. Using a gene-specific small interference RNA (siRNA) approach, we determined that the regulatory isoform p85alpha and the catalytic isoform p110beta, but not p110alpha, were required for androgen-stimulated AR transactivation and cell proliferation in prostate cancer cells. Consistently, overexpression of wild-type p110beta but not p110alpha gene led to androgen-independent AR transactivation. Silencing p110beta gene in prostate cancer cells abolished tumor growth in nude mice. Of the dual (lipid and protein) kinase activities, p110beta's lipid kinase activity was required for AR transactivation. Further analysis by a chromatin immunoprecipitation assay showed that p110beta is indispensable for androgen-induced AR-DNA interaction. Finally, gene expression analysis of clinical specimens showed that both p85alpha and p110beta were highly expressed in malignant prostate tissues compared to the nonmalignant compartments, and their expression levels correlated significantly with disease progression. Taken together, our data demonstrated that p85alpha and p110beta are essential for androgen-stimulated AR transactivation, and their aberrant expression or activation might play an important role in prostate cancer progression.     

Integrins and their extracellular-matrix ligands in gastric-cancer

Extracellular matrix (ECM) proteins and their specific cellular receptors, play an important role in the regulation of epithelial morphogenesis and differentiation. Alterations in their expression and function have been found in a number of malignant tumours and these changes may help to explain their dedifferentiation and altered behaviour. In this study we have investigated expression and distribution of the epithelial beta 1 integrins (alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 1) and their ECM ligands (fibronectin, tenascin and laminin) in normal and neoplastic tissue. An up-regulation of two isoforms of fibronectin, and tenascin was seen in tumour associated matrix compared to normal stroma. Loss or down regulation of alpha integrin chains was seen more frequently in poorly differentiated carcinomas (alpha 2 p=0.002; alpha 3 p=0.013; alpha 6 p=0.0012) irrespective of tumour type (diffuse or intestinal) than in well/moderately differentiated tumours. Cell adhesion assays revealed that the ability of gastric carcinoma cell lines to bind matrix glycoproteins correlated to their degree of differentiation. Furthermore, poorly differentiated cell lines showed a down-regulation of alpha 2 and alpha 6 integrin expression. These data indicate that architectural and cytological differentiation in gastric carcinoma relates to altered patterns of expression of matrix glycoproteins and their receptors. The traditional Lauren classification seems to reflect these differences in cell-matrix interactions. Differing patterns of expression of those molecules involved in cell-matrix interactions may prove to be a more objective and biologically more relevant means of classifying gastric cancer.     

Inhibition of p53 function diminishes androgen receptor-mediated signaling in prostate cancer cell lines

Current therapy for advanced prostate cancer is mainly based on androgen deprivation, although most patients relapse to androgen-insensitive disease. Several mechanisms contributing to androgen-independent growth including alterations in the structure or expression of the androgen receptor (AR) and its cofactors have been identified. Recent evidence suggests that p53 is involved in androgen signaling. The analysis of the effect of p53 on androgen signaling was performed in 22Rv1 and LNCaP prostate cancer cells that express both p53 and AR. The overexpression of p53 diminished the androgenic response in both cell lines in a reporter gene assay. Conversely, the inhibition of p53 by three different p53 inhibitors, Pifithrin-1alpha (PFT-1alpha), an inhibitor of p53-dependent transactivation; MDM2, a regulator of p53 expression; and a dominant-negative N-terminally truncated p53 gene also reduced transactivation of androgen-dependent reporter genes. The inactivation of p53 by PFT-1alpha decreased AR-protein expression in both 22Rv1 and LNCaP cells. Our findings confirm that the overexpression of wild-type p53 decreases androgen function, whereas p53 expression at physiological levels stabilizes AR signaling. Thus, our findings suggest that there is a balance of AR and p53 expression during the androgen-dependent growth of prostate cancer, which is obliterated during further progression of the disease.     

Differential expression of integrin subunits in DU-145/AR prostate cancer cells

We have established a clonal DU-145 prostate cancer cell line (DU-145/AR) stably transfected with androgen receptor cDNA. We investigated the expression of integrin subunits, adhesion to extracellular matrices, the invasion of DU-145/AR prostate cancer cells. The expression of various integrin subunits and adhesion to various extracellular matrices in DU-145, DU-145/Neo and DU-145/AR cells were examined. The haptoinvasion and the haptotactic migration of these cells were investigated using a Transwell cell culture chamber assay. DU-145/AR cells exhibited lower expression of alpha6 and beta4 integrin subunits and higher expression of alpha2 and alpha5 than DU-145 cells. DU-145/AR cells showed significantly lower adhesion to fibronectin, laminin-1 and laminin-5 than DU-145/ Neo cells, whereas DU-145/AR cells showed higher adhesion to type I and type IV collagen. Haptoinvasion of DU-145/AR cells into Matrigel/fibronectin-coated filter was significantly reduced as compared with DU-145/Neo or DU-145 cells, but there was no significant difference between DU-145/AR and control cells in the haptotactic migration to fibronectin. Dihydrotestosterone (DHT) inhibited the invasive ability of DU-145/AR cells. These results indicate that androgen receptor may play a role in the regulation of adhesion to the extracellular matrices and invasion of prostate cancer cells through influencing the expression of specific integrin subunits.     

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.     

Increased gene expression levels of collagen receptor integrins are associated with decreased survival parameters in patients with advanced melanoma

Expression of collagen receptor integrins alpha1beta1 and alpha2beta1 has been associated with progression and metastatic potential of malignant melanoma. Integrin alpha2beta1 was originally characterized as a melanoma progression antigen. We have used real-time quantitative PCR to study the mRNA expression levels of three collagen receptor integrin chains, that is alpha1, alpha2 and alpha11 in metastases from 26 patients with melanoma. Interestingly, we find that survival after initiation of chemoimmunotherapy was significantly decreased in all patients whose tumours expressed high mRNA levels of alpha1 integrin, alpha2 integrin or alpha11 integrin when compared with lower tumour expression levels (P<0.05, log rank test). Moreover, those patients with high mRNA levels of all studied integrins had a significantly shorter survival from the appearance of the first metastasis than the patients with low levels of integrins (P<0.05). Furthermore, a high mRNA expression level of integrin alpha2 was found to be associated with poorer overall survival. High alpha2 mRNA levels (n=6) were associated with median survival of 35 months and low alpha2 mRNA levels (n=20), with median survival of 53 months (P=0.033). We conclude that collagen receptor integrins are important in the progression and prognosis of metastatic melanoma, and their measurements might be used as predictive markers when assessing disease progression.     

beta1A integrin expression is required for type 1 insulin-like growth factor receptor mitogenic and transforming activities and localization to focal contacts

The cells' ability to proliferate in response to growth factor stimulation is significantly altered during cancer progression. To investigate the mechanisms underlying these alterations in prostate cancer, the role and expression of beta1A integrin and type 1 insulin-like growth factor receptor (IGF-IR), known to contribute to cell proliferation and transformation, were analyzed. Using small interfering RNA oligonucleotides to down-regulate beta1A, we show that beta1A expression is required for IGF-IR-mediated prostate cancer cell proliferation and anchorage-independent growth. In vivo, using age-matched transgenic adenocarcinoma of mouse prostate (TRAMP) mice at different stages of prostate cancer [prostatic intraepithelial neoplasia, PIN; well-differentiated adenocarcinoma, WD; and poorly differentiated adenocarcinoma, PD], the expression of beta1A and of IGF-IR was studied. beta1A and IGF-IR expression levels were concurrently up-regulated in high PIN and WD, whereas their expression did not correlate in late-stage PD. In contrast to the up-regulated expression of beta1A, the levels of beta1C, a beta1 cytoplasmic variant that inhibits cell proliferation, were down-regulated in all stages of prostate cancer. A similar expression pattern was observed for a beta1C downstream effector, Grb2-associated binder-1 (Gab1) which is known to inhibit IGF-IR phosphorylation. To analyze in vitro the mechanistic implications of beta1A, beta1C, and Gab1 deregulation in prostate cancer, we investigated whether expression of either beta1 variant in beta1-null cells affected IGF-IR localization. We found that IGF-IR and beta1A were colocalized in highly specialized integrin signaling compartments, designated focal contacts. However, in the presence of beta1C, IGF-IR remained diffuse on the cell surface and did not localize to focal contacts. The findings that beta1 integrins and IGF-IR are concurrently deregulated and that expression of beta1 integrins is necessary to achieve appropriate IGF-IR intracellular distribution point to the important role that the cross-talk between these receptors may have during prostate cancer progression and will be helpful in formulating new therapeutic strategies.     

Role of the orphan nuclear receptor ROR alpha in the control of the metastatic behavior of androgen-independent prostate cancer cells

Orphan nuclear receptors constitute a subgroup of the superfamily of steroid/thyroid/retinoid receptors for which no endogenous ligand has been identified. The orphan nuclear receptor ROR alpha has been shown to be involved in the control of cell growth and differentiation. We have previously shown that, in DU 145 androgen-independent prostate cancer cells, ROR alpha activation brings about a significant decrease of cell proliferation and affects cell cycle progression through the modulation of cell cycle-related genes. The experiments here described have been performed to clarify whether ROR alpha might also be involved in the control of the metastatic behavior of DU 145 cells. We have shown that the thiazolidinedione derivative CGP 52608, the specific ROR alpha ligand and activator, reduces the ability of DU 145 cells to invade a reconstituted basement membrane (Matrigel). CGP 52608 also significantly decreased the capacity of prostate cancer cells to migrate towards a chemotactic stimulus (fibronectin), when plated in the upper compartment of a Boyden's chamber. Moreover, ROR alpha activation resulted in a decreased expression of alpha v beta 3 integrin and an increased level of expression of beta 4 integrin subunit. These findings indicate that the activation of the orphan nuclear receptor ROR alpha reduces the invasive and migratory capacities of androgen-independent prostate cancer cells, at least partially, by affecting integrin expression.     

Beta 1 integrin expression on human small cell lung cancer cells.

The integrins are a supergene family of cell surface glycoproteins that promote cellular adhesion. Each member of the family is an alpha/beta heterodimer composed of a distinct alpha subunit noncovalently linked to one of at least six common beta subunits. These include the six beta 1 integrins (alpha 1-6/beta 1) which represent receptors for extracellular matrix proteins and the three beta 2 integrins (alpha L, alpha M, alpha X/beta 2) that are expressed by leukocytes and which bind to C3bi and/or endothelial ligands. Recently, it was reported that certain human tumor cells express the beta 1 integrins and that small cell lung cancer (SCLC) cell lines express the beta 2 integrin Mo1 (alpha M/beta 2). To extend these initial observations, we examined SCLC cell lines for integrin expression at the glycoprotein and mRNA levels and assessed the potential function of these integrins in promoting SCLC adhesion. An indirect immunofluorescence analysis of five SCLC cell lines (NCI-H187, H345, H146, H209, and N417) using alpha and beta subunit-specific monoclonal antibodies demonstrated the uniform expression of beta 1 (beta 1 much greater than beta 2 greater than or equal to beta 3 congruent to beta 4). Among the beta 1-associated alpha subunits, alpha 3 was uniformly expressed at high surface density by all five cell lines (as confirmed in H345 cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of anti-beta 1 and anti-alpha 3 immunoprecipitates), while alpha 5 was not detected. The leukocyte (beta 2-associated) alpha M and alpha L subunits were also variably expressed by the five lines. Consistent with the surface expression of beta 1 integrin gene products, beta 1 (but not beta 2) mRNA was detected in SCLC cells by Northern blot analysis. That beta 1 integrin expression was involved in SCLC adhesion was suggested by the adherence of H345 cells to laminin, a known ligand for the alpha 3 beta 1 integrin. Moreover, an antibody specific for the beta 1 subunit inhibited this adhesion, indicating that the beta 1 subunit promotes adhesion to laminin. We conclude that beta 1 integrin molecules are expressed by human SCLC cells (with uniform expression of alpha 3/beta 1) and promote their adhesion to laminin.     

Unique expression of integrin fibronectin receptors in human neuroblastoma cell lines.

Cultured human neuroblastoma cells can be classified morphologically into 3 types: neuroblastic (N), intermediate (I) and substrate adherent (S). Neuroblastoma cells of all types were found to attach and display distinct morphological characteristics on fibronectin, with S-type cells attaching better than N-type cells. Studies of the expression of integrin fibronectin receptors (alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1 and alpha V beta 1) were carried out using a total of 26 morphologically distinct cell lines and their subpopulations. Fluorescence-activated cell sorting (FACS) analysis and immunoprecipitation revealed that all S-type cells expressed abundant alpha 5 beta 1, while N-type cells barely expressed this molecule. Although alpha 3 beta 1 expression of S-type cells was also higher than that of N-type cells, some N-type cells had significantly increased levels of this molecule. alpha 4 beta 1 was found to be randomly expressed. All cell lines tested expressed alpha V beta 1. Human neuroblastoma cells, the majority of which are N-type cells with very low alpha 5 beta 1 expression, are also contrasted with other childhood cancer cells (rhabdomyosarcoma, Ewing's sarcoma, and glioma), all of which expressed high levels of alpha 5 beta 1. The characteristic expression of integrin fibronectin receptors may account for the clinically unique tumor behavior, and the immunohistochemical staining for integrins may become a useful alternative to conventional histology in differential diagnosis and a marker for prognosis in neuroblastoma.     

Osteopontin: possible role in prostate cancer progression.

Human prostate cancer has the propensity to metastasize to the bone where reciprocal cellular interactions between prostate cancer and bone cells are known to occur. Osteopontin (OPN), a noncollagenous bone extracellular matrix, is a secreted adhesive glycoprotein with a functional RGD cell-binding domain that interacts with the alpha(v)beta3 cell surface integrin heterodimer. OPN has been associated with malignant transformation as well as being ligand to the CD44 receptor. Polyclonal antibodies to human OPN (hOPN) were prepared, and specificity was shown by preabsorption with recombinant hOPN. The stimulatory effect of hOPN protein and the inhibitory effect of hOPN antibody on human prostate cancer cell lines LNCaP and C4-2 were assessed by induction or inhibition of anchorage-independent growth, respectively. Expression of hOPN mRNA in prostate cancer cell lines and human prostate cancer tissue specimens were measured by mRNA blot analysis. Protein expression was assessed by immunohistochemistry in human prostate cancer specimens and by Western blot analysis in prostate cancer cell lines. hOPN stimulated anchorage-independent growth of the human prostate cancer cell lines LNCaP and C4-2 in vitro. Antibodies to hOPN inhibited the growth-stimulatory effect by endogenous OPN, which can be overcome by the addition of exogenous hOPN. hOPN mRNA and protein are expressed in human prostate cancer cell lines in vitro and in clinical human prostate cancer specimens. These findings taken together suggest that OPN may act as a paracrine and autocrine mediator of prostate cancer growth and progression.     

Expression of the alpha6beta4 integrin provides prognostic information in bladder cancer

Integrins are cell surface receptors for extracellular matrix components that may participate in metastatic processes. Normal urothelial tissues show a polarized expression of alpha6beta4 integrin on basal cells at their junction with the lamina propria. We have previously shown that bladder cancers frequently overexpress one member of the integrin family, the alpha6beta4 integrin. In this study, we evaluated the level of alpha6beta4 integrin expression in bladder cancer specimens from 57 patients and correlated the expression level with patient survival. Expression was evaluated by immunoperoxidase staining. Three patterns of alpha6beta4 expression were observed: negative (13 patients); strong overexpression throughout the tumor cells (21 patients); and weak expression that most closely resembled expression in normal urothelium (23 patients). Individuals with weak staining tumors had a statistically significantly better survival (p=0.041) than patients whose tumors exhibited either no expression or strong overexpression. These data indicate that evaluation of the expression of alpha6beta4 integrin may provide valuable prognostic information on clinical outcome in patients with bladder cancer.     

Migration-promoting role of VEGF-C and VEGF-C binding receptors in human breast cancer cells

Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic factor over-expressed in highly metastatic, cyclooxygenase (COX)-2 expressing breast cancer cells. We tested the hypothesis that tumour-derived VEGF-C may play an autocrine role in metastasis by promoting cellular motility through one or more VEGF-C-binding receptors VEGFR-2, VEGFR-3, neuropilin (NRP)-1, NRP-2, and integrin alpha9beta1. We investigated the expression of these receptors in several breast cancer cell lines (MDA-MB-231, Hs578T, SK-BR-3, T-47D, and MCF7) and their possible requirement in migration of two VEGF-C-secreting, highly metastatic lines MDA-MB-231 and Hs578T. While cell lines varied significantly in their expression of above VEGF-C receptors, migratory activity of MDA-MB-231 and Hs578T cells was linked to one or more of these receptors. Depletion of endogenous VEGF-C by treatments with a neutralising antibody, VEGF-C siRNA or inhibitors of Src, EGFR/Her2/neu and p38 MAP kinases which inhibited VEGF-C production, inhibited cellular migration, indicating the requirement of VEGF-C for migratory function. Migration was differentially attenuated by blocking or downregulation of different VEGF-C receptors, for example treatment with a VEGFR-2 tyrosine kinase inhibitor, NRP-1 and NRP-2 siRNA or alpha9beta1 integrin antibody, indicating the participation of one or more of the receptors in cell motility. This novel role of tumour-derived VEGF-C indicates that breast cancer metastasis can be promoted by coordinated stimulation of lymphangiogenesis and enhanced migratory activity of breast cancer cells.     

Beta1 integrins modulate cell adhesion by regulating insulin-like growth factor-II levels in the microenvironment

The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The beta1C and beta1A integrins, two cytoplasmic variants of the beta1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the beta1C variant, an inhibitor of cell proliferation, which is down-regulated in prostate cancer, up-regulates insulin-like growth factor-II (IGF-II) mRNA and protein levels. In contrast, beta1A does not affect IGF-II levels. We provide evidence that beta1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the beta1C cytoplasmic domain contains the structural motif sufficient to increase cell adhesion to Laminin-1. This autocrine mechanism that locally supports cell adhesion to Laminin-1 via IGF-II is selectively regulated by the beta1 cytoplasmic domain via activation of the growth factor receptor binding protein 2-associated binder-1/SH2-containing protein-tyrosine phosphatase 2/phosphatidylinositol 3-kinase pathway. Thus, the concurrent local loss of beta1C integrin, of its ligand Laminin-1, and of IGF-II in the tumor microenvironment may promote prostate cancer cell invasion and metastasis by reducing cancer cell adhesive properties. It is, therefore, conceivable that reexpression of beta1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype.