Mara Fornaro

Fibronectin protects prostate cancer cells from tumor necrosis factor-alpha-induced apoptosis via the AKT/survivin pathway.

Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84→ Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via β1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-α (TNF-α). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-α-induced apoptosis. Evidence is provided that expression of β1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-α-induced apoptosis. In contrast, the β1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-α in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis.

Integrins and prostate cancer metastases

Integrins have emerged as modulators of a variety of cellular functions. They have been implicated in cell migration, survival, normal and aberrant cellular growth, differentiation, gene expression, and modulation of intracellular signal transduction pathways.In this review article, the structural and functional characteristics of integrins, their expression and their potential role in prostate cancer metastases will be discussed.

SHP-2 positively regulates myogenesis by coupling to the Rho GTPase signaling pathway

ABSTRACT Myogenesis is an intricate process that coordinately engages multiple intracellular signaling cascades. The Rho family GTPase RhoA is known to promote myogenesis, however, the mechanisms controlling its regulation in myoblasts have yet to be fully elucidated. We show here that the SH2-containing protein tyrosine phosphatase, SHP-2, functions as an early modulator of myogenesis by regulating RhoA. When MyoD was expressed in fibroblasts lacking functional SHP-2, muscle-specific gene activity was impaired and abolition of SHP-2 expression by RNA interference inhibited muscle differentiation. By using SHP-2 substrate-trapping mutants, we identified p190-B RhoGAP as a SHP-2 substrate. When dephosphorylated, p190-B RhoGAP has been shown to stimulate the activation of RhoA. During myogenesis, p190-B RhoGAP was tyrosyl dephosphorylated concomitant with the stimulation of SHP-2s phosphatase activity. Moreover, overexpression of a catalytically inactive mutant of SHP-2 inhibited p190-B RhoGAP tyrosyl dephosphorylation, RhoA activity, and myogenesis. These observations strongly suggest that SHP-2 dephosphorylates p190-B RhoGAP, leading to the activation of RhoA. Collectively, these data provide a mechanistic basis for RhoA activation in myoblasts and demonstrate that myogenesis is critically regulated by the actions of SHP-2 on the p190-B Rho GAP/RhoA pathway.

Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by β1 integrins

We show here that β1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The β1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, β1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, β1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the β1 cytodomain plays an important role in mediating β1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, β1A associates with IRS-1 and β1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.

Alternatively spliced variants: A new view of the integrin cytoplasmic domain

A large number of studies have underscored a major role for the integrin alpha beta cytoplasmic domains in the modulation of cell functions. Cytoplasmic domain variants of the beta 1, beta 3, beta 4, alpha 3, alpha 6 and alpha 7 subunits have been described. These molecules are generated by alternative splicing events and are expressed in a cell- or tissue-type specific manner. Some of these variants (beta 1C, beta 1D, alpha 6A and alpha 7A) are predominantly expressed upon differentiation and have been shown to be regulated during development. The studies on the structure-function relationship of the integrin variant subunits, published between 1989 and now, will be reviewed here for the first time. The results demonstrate that differences in the cytoplasmic domain do not affect either the alpha beta heterodimer formation or the ligand specificity. Instead, alternatively spliced integrin cytoplasmic domains appear to be essential modulators of receptor localization, cell proliferation and migration, as well as phosphorylation of signaling molecules. These observations lead to the current hypothesis that cell-type specific regulation of alternatively spliced integrin cytoplasmic domains may provide a highly specialized mechanism to control cell growth and intracellular signaling pathways.

SHP-2 regulates the phosphatidylinositide 3'-kinase/Akt pathway and suppresses caspase 3-mediated apoptosis.

The Src homology domain 2 (SH2)‐containing tyrosine phosphatase SHP‐2 has been implicated in the regulation of the phosphatidylinositol 3′‐kinase (PI3K)/Akt pathway. The ability of SHP‐2 to regulate the PI3K/Akt pathway is suggested to result in the positive effect of SHP‐2 on cell survival. Whether SHP‐2 regulates insulin‐like growth factor‐1 (IGF‐1)‐dependent activation of Akt at the level of PI3K has yet to be established. Furthermore, the identification of the down‐stream apoptotic target engaged by SHP‐2 in cell survival also has yet to be determined. Here, we show that overexpression of a catalytically inactive mutant of SHP‐2 inhibited insulin‐like growth factor‐1 (IGF‐1)‐dependent PI3K and Akt activation. Consistent with the observation that SHP‐2 participates in pro‐survival signaling fibroblasts expressing a deletion within exon 3 of SHP‐2, which results in a truncation of the amino‐terminus SH2 domain (SHP‐2Ex3−/−), were hypersensitive to etoposide‐induced cell death. SHP‐2Ex3−/− fibroblasts exhibited enhanced levels of etoposide‐induced caspase 3 activity as compared to wild‐type fibroblasts and the enhanced level of caspase 3 activity was suppressed by a caspase 3‐specific inhibitor. Re‐introduction of wild‐type SHP‐2 into the SHP‐2Ex3−/− fibroblasts rescued the hypersensitivity to etoposide‐induced caspase 3 activation. The effects of abrogating SHP‐2 function on cell survival were not specific to the loss of the amino‐terminus SH2 domain of SHP‐2 since RNAi‐mediated knock‐down of SHP‐2 also reduced cell survival. Taken together, these data indicate that the catalytic activity of SHP‐2 is required to regulate the PI3K/Akt pathway and thus likely participates in anti‐apoptotic signaling by suppressing caspase 3‐mediated apoptosis. J. Cell. Physiol. 199: 227–236, 2004© 2003 Wiley‐Liss, Inc.

p27 kip1 acts as a downstream effector of and is coexpressed with the β 1C integrin in prostatic adenocarcinoma

Integrins are a large family of transmembrane receptors that, in addition to mediating cell adhesion, modulate cell proliferation. The beta1C integrin is an alternatively spliced variant of the beta1 subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain. We have shown previously that in vitro beta1C inhibits cell proliferation and that in vivo beta1C is expressed in nonproliferative, differentiated epithelium and is selectively downregulated in prostatic adenocarcinoma. Here we show, by immunohistochemistry and immunoblotting analysis, that beta1C is coexpressed in human prostate epithelial cells with the cell-cycle inhibitor p27(kip1), the loss of which correlates with poor prognosis in prostate cancer. In the 37 specimens analyzed, beta1C and p27(kip1) are concurrently expressed in 93% of benign and 84%-91% of tumor prostate cells. Forced expression of beta1C in vitro is accompanied by an increase in p27(kip1) levels, by inhibition of cyclin A-dependent kinase activity, and by increased association of p27(kip1) with cyclin A. beta1C inhibitory effect on cell proliferation is completely prevented by p27(kip1) antisense, but not mismatch oligonucleotides. beta1C expression does not affect either cyclin A or E levels, or cyclin E-associated kinase activity, nor the mitogen-activated protein (MAP) kinase pathway. These findings show a unique mechanism of cell growth inhibition by integrins and point to beta1C as an upstream regulator of p27(kip1) expression and, therefore, a potential target for tumor suppression in prostate cancer.

β1C Integrin in Epithelial Cells Correlates with a Nonproliferative Phenotype: Forced Expression of β1C Inhibits Prostate Epithelial Cell Proliferation

The expression of the β 1C integrin, an alternatively spliced variant of the β 1 subunit, was investigated in human adult and fetal tissues. In the adult, β 1C immunoreactivity was found in nonproliferative, differentiated simple, and/or pseudostratified epithelia in prostate glands and liver bile ducts. In contrast, β 1C was undetectable in stratified squamous epithelium of the epidermis and/or in hepatocytes. Luminal prostate epithelial cells expressed β 1C in vivo and in vitro , but no β 1C was seen in basal cells, which are proliferating cells. Fetal prostate expressed β 1C in differentiated glands that had a defined lumen, but not in budding glands, indicating that β 1C is a marker of prostate epithelium differentiation. The β 1C and the common β 1A variants are differentially distributed: β 1A was found in luminal and basal epithelial as well as in stromal cells in the prostate. In the liver, β 1C and β 1A were coexpressed in biliary epithelium, whereas vascular cells expressed only β 1A . Because we found β 1C in nonproliferative and differentiated epithelium, we investigated whether β 1C could have a causal role in inhibiting epithelial cell proliferation. The results showed that exogenous expression of a β 1C , but not of a β 1A , cytoplasmic domain chimeric construct, completely inhibited thymidine incorporation in response to serum by prostate cancer epithelial cells. Consistent with these in vitro results, β 1C appeared to be downregulated in prostate glands that exhibit regenerative features in benign hyperplastic epithelium. These data show that the presence of β 1C integrins in epithelial cells correlates with a nonproliferative, differentiated phenotype and is growth inhibitory to prostate epithelial cells in vitro . These findings indicate a novel pathophysiological role for this integrin variant in epithelial cell proliferation.

Regulation of mRNA and Protein Levels of β1 Integrin Variants in Human Prostate Carcinoma

Alterations of integrin expression levels in cancer cells correlate with changes in invasiveness, tumor progression, and metastatic potential. The beta1C integrin, an alternatively spliced form of the human beta1 integrin, has been shown to inhibit prostate cell proliferation. Furthermore, beta1C protein levels were found to be abundant in normal prostate glandular epithelium and down-regulated in prostatic adenocarcinoma. To gain further insights into the molecular mechanisms underlying abnormal cancer cell proliferation, we have studied beta1C and beta1 integrin expression at both mRNA and protein levels by Northern and immunoblotting analysis using freshly isolated neoplastic and normal human prostate tissue specimens. Steady-state mRNA levels were evaluated in 38 specimens: 33 prostatic adenocarcinomas exhibiting different Gleasons grade and five normal tissue specimens that did not show any histological manifestation of benign prostatic hypertrophy. Our results demonstrate that beta1C mRNA is expressed in normal prostate and is significantly down-regulated in neoplastic prostate specimens. In addition, using a probe that hybridizes with all beta1 variants, mRNA levels of beta1 are found reduced in neoplastic versus normal prostate tissues. We demonstrate that beta1C mRNA down-regulation does not correlate with either tumor grade or differentiation according to Gleasons grade and TNM system evaluation, and that beta1C mRNA levels are not affected by hormonal therapy. In parallel, beta1C protein levels were analyzed. As expected, beta1C is found to be expressed in normal prostate and dramatically reduced in neoplastic prostate tissues; in contrast, using an antibody to beta1 that recognizes all beta1 variants, the levels of beta1 are comparable in normal and neoplastic prostate, thus indicating a selective down-regulation of the beta1C protein in prostate carcinoma. These results demonstrate for the first time that beta1C and beta1 mRNA expression is down-regulated in prostate carcinoma, whereas only beta1C protein levels are reduced. Our data highlight a selective pressure to reduce the expression levels of beta1C, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.

SHP-2 activates signaling of the nuclear factor of activated T cells to promote skeletal muscle growth

The formation of multinucleated myofibers is essential for the growth of skeletal muscle. The nuclear factor of activated T cells (NFAT) promotes skeletal muscle growth. How NFAT responds to changes in extracellular cues to regulate skeletal muscle growth remains to be fully defined. In this study, we demonstrate that mice containing a skeletal muscle–specific deletion of the tyrosine phosphatase SHP-2 (muscle creatine kinase [MCK]–SHP-2 null) exhibited a reduction in both myofiber size and type I slow myofiber number. We found that interleukin-4, an NFAT-regulated cytokine known to stimulate myofiber growth, was reduced in its expression in skeletal muscles of MCK–SHP-2–null mice. When SHP-2 was deleted during the differentiation of primary myoblasts, NFAT transcriptional activity and myotube multinucleation were impaired. Finally, SHP-2 coupled myotube multinucleation to an integrin-dependent pathway and activated NFAT by stimulating c-Src. Thus, SHP-2 transduces extracellular matrix stimuli to intracellular signaling pathways to promote skeletal muscle growth.