Vinicio Carloni

Androgen Receptor Expression in Prostate Carcinoma Cells Suppresses α6β4 Integrin-Mediated Invasive Phenotype1

Prostate cancer cells may lose androgen-sensitivity after androgen ablation therapy, becoming highly invasive and metastatic. The biological mechanisms responsible for higher tumurogenicity of androgen-independent prostate carcinomas are not entirely known. We demonstrate that androgen receptor regulation of adhesion and invasion of prostate cancer cells through modulation of α6β4 integrin expression may be one of the molecular mechanisms responsible of this phenomenon. We found that protein and gene expressions of α6 and β4 subunits were strongly reduced in the androgen-sensitive cell line LNCaP respect to the androgen-independent PC3 and that transfection of PC3 cells with a full-length androgen receptor expression vector resulted in a decreased expression of α6β4 integrin, reduced adhesion on laminin, and suppressed Matrigel invasion. Growth in soft agar was also suppressed in androgen receptor-positive PC3 clones. Treatment of androgen receptor positive clones with the synthetic androgen R1881 further...

Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A

Sperm motility is regulated by a complex balance between kinases and phosphatases. Among them, phosphatidylinositol 3-kinase (PI 3-kinase) has been recently suggested to negatively regulate sperm motility (Luconi, M., Marra, F., Gandini, L., Lenzi, A., Filimberti, E., Forti, G. and Baldi, E. (2001). Hum. Reprod. 16, 1931-1937). We demonstrate the presence and activity of PI 3-kinase in human spermatozoa and have investigated the molecular mechanism(s) by which the PI 3-kinase inhibitor, LY294002, triggers an increase in sperm motility. PI 3-kinase inhibition results in an increase in intracellular cAMP levels and in tyrosine phosphorylation of the protein kinase A-anchoring protein AKAP3. These effects finally result in a stimulation of protein kinase A (PKA) binding to AKAP3 in sperm tails through the regulatory subunit RIIβ. The increased binding of RIIβ to AKAP3 induced by LY294002 is mainly due to tyrosine phosphorylation of AKAP3, since it is completely blocked by the tyrosine kinase inhibitor erbstatin, which also reverses the effects of LY294002 on motility and suppresses PKA-AKAP3 interaction. The requirement of PKA binding to AKAP3 for sperm motility is confirmed by the reduction of motility induced by an inhibitor of RIIβ-AKAP3 binding, Ht31, whose effects on sperm motility and PKA binding to AKAP3 are reversed by LY294002. These results demonstrate that PI 3-kinase negatively regulates sperm motility by interfering with AKAP3-PKA binding, providing the first evidence of a molecular mechanism by which PKA can be targeted to sperm tails by interaction with tyrosine phosphorylated form of AKAP3.

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen‐sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR)

We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen‐independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3‐AR) through modulation of α6β4 integrin expression. The treatment with androgens further reduced invasion of the cells without modifying α6β4 expression, suggesting an interference with the invasion process by androgens. Here, we investigated EGF‐mediated signal transduction processes that lead to invasion in PC3‐AR cells. We show that EGF‐induced EGFR autotransphosphorylation is reduced in PC3‐AR cells compared to PC3 cells transfected only with the vector (PC3‐Neo). EGF‐stimulated PI3K activity, a key signaling pathway for invasion of these cells, and EGF‐PI3K interaction are also decreased in PC3‐AR cells and further reduced by treatment with androgen. Finally, we show that EGFR internalization process was reduced in PC3‐AR and LNCaP cells compared to PC3‐Neo. Investigations on the location of AR in PC3‐AR transfected cells were also conducted. Immunoconfocal microscopy and coimminoprecipitation studies demonstrated the presence of an interaction between EGFR and AR at membrane level in PC3‐AR and LNCaP cells. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less‐malignant phenotype by interfering with EGFR signaling leading to invasion through a mechanism involving an interaction between AR and EGFR.

Tetraspanin CD81 is linked to ERK/MAPKinase signaling by Shc in liver tumor cells

Tetraspanins is a large family of membrane proteins that are implicated in cell proliferation, differentiation and tumor invasion. Specifically, the tetraspanin CD81 has been involved in cell proliferation but the mechanism is unknown. Here, we show that CD81 clustering stimulates ERK/MAPKinase activity and tyrosine phosphorylation of the adapter protein Shc in Huh7 cancer cells. In addition, overexpression of CD81 in HepG2 cells, NIH3T3 cells, and murine fibroblasts GD25 lacking the β1 family of integrins induces cell proliferation and ERK/MAPKinase activation. Linked with this event, we observed an increase in CD81-associated type II phosphatidylinositol 4-kinase activity. A mutant in the PTB domain of Shc failed to interact with phosphoinositides and localize to the plasma membrane thus blocking CD81-induced ERK/MAPKinase activation. Therefore, we conclude that CD81 stimulates synthesis of phosphoinositides with the recruitment of Shc to the plasma membrane via PTB domain, and this sequence of events induces activation of ERK/MAPKinase. These findings define a novel mechanism of ERK/MAPKinase activation and tumor cell proliferation.

Expression of transmembrane 4 superfamily (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration

BACKGROUND/AIMS Migration of activated hepatic stellate cells (HSC) is a key event in the progression of liver fibrosis. Little is known about transmembrane proteins involved in HSC motility. Tetraspanins (TM4SF) have been implicated in cell development, differentiation, motility and tumor cell invasion. We evaluated the expression and function of four TM4SF, namely CD9, CD81, CD63 and CD151, and their involvement in HSC migration, adhesion, and proliferation. METHODS/RESULTS All TM4SF investigated were highly expressed at the human HSC surface with different patterns of intracellular distribution. Monoclonal antibodies directed against the four TM4SF inhibited HSC migration induced by extracellular matrix proteins in both wound healing and haptotaxis assays. This inhibition was independent of the ECM substrates employed (collagen type I or IV, laminin), and was comparable to that obtained by incubating the cells with an anti-beta1 blocking mAb. Importantly, cell adhesion was unaffected by the incubation with the same antibodies. Co-immunoprecipitation studies revealed different patterns of association between the four TM4SF studied and beta1 integrin. Finally, anti-TM4SF antibodies did not affect HSC growth. CONCLUSIONS These findings provide the first characterization of tetraspanins expression and of their role in HSC migration, a key event in liver tissue wound healing and fibrogenesis.

Tetraspanin CD81-Regulated Cell Motility Plays a Critical Role in Intrahepatic Metastasis of Hepatocellular Carcinoma

BACKGROUND & AIMS Human hepatocellular carcinoma (HCC) can invade the portal vein and metastasize to other parts of the liver. Currently, the molecular and cellular mechanisms underlying intrahepatic metastasis of HCC are poorly understood. Tumor invasiveness could be considered an aspect of dysregulated motility, and the mechanisms that inhibit cell movement are considered to counteract the spreading of cancer cells through the liver. Accumulating observations suggest that the CD81 tetraspanin may have an inhibitory effect on cell movement. METHODS In the present study using both loss- and gain-of-gene function approaches, we verified that the functional interaction of tetraspanin CD81 with type II phosphoinositide 4-kinase (PI4KII) suppressed HCC cell motility by promoting the formation of CD81-enriched vesicles, non-endosomal intracellular structures, that sequestered actinin-4 with consequent remodeling of actin cytoskeleton. RESULTS We reported that HCC cells expressing CD81 showed an inability to metastasize compared with HCC cells with undetectable levels of CD81. CONCLUSIONS Taken together, these findings indicate that CD81 functions as a molecular organizer of membrane microdomains, whereby proteins such as PI4KII control actin remodeling and cell motility, establishing a role for these genes as negative modifiers of oncogenicity and HCC progression.

Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to transforming growth factor‐β1

1 Pentoxifylline (PTF) may act as a potential antifibrogenic agent by inhibiting cell proliferation and/or collagen deposition in cell type(s) responsible for the accumulation of extracellular matrix. The aim of the present study was to investigate at which level PTF may affect synthesis and degradation of type I collagen in human hepatic stellate cells (HSCs), a key source of connective tissue in fibrotic liver. 2 Procollagen type I synthesis and release were evaluated in cells maintained in serum free/insulin free medium for 48 h and then stimulated with transforming growth factor‐β1 (TGF‐β1) for different time periods in the presence or absence of PTF. TGF‐β1 caused an upregulation of procollagen I mRNA levels with a peak increase after 3–6 h of stimulation. This effect was followed by an increase in both the cell associated and the extracellular levels of the corresponding protein, with a peak effect at 9–12 h after the addition of TGF‐β1. Co‐incubation with PTF slightly but consistently reduced basal as well as stimulated procollagen I mRNA levels, with negligible effects on the cell‐associated expression of the corresponding protein. Conversely, PTF dose‐dependently reduced procollagen type I levels detected in supernatants from unstimulated and stimulated cells. 3 Pulse‐chase experiments employing L‐[3H]‐proline revealed that PTF was able to induce significantly the degradation of procollagen, mainly in the extracellular compartment. We next analysed the effect of PTF on the major pathway involved in type I collagen degradation. PTF did not affect the expression of metalloproteinase 1 (MMP‐1) mRNA both in basal and stimulated conditions, whereas it markedly reduced the expression of tissue inhibitor of metalloproteinase 1 (TIMP‐1) mRNA. Accordingly incubation with PTF increased the levels of ‘activated MMP‐1’ in cell supernatants in both basal and stimulated conditions. 4 These results suggest that the antifibrogenic action of PTF on human HSCs is mainly mediated by extracellular collagen degradation rather than by a reduction of collagen synthesis.

Knockout of α6β1-integrin expression reverses the transformed phenotype of hepatocarcinoma cells

Abstract Background & Aims: Hepatocellular carcinoma is a common complication in liver cirrhosis. The integrin α6β1, a receptor for the laminin family of extracellular matrix proteins, has been found to be overexpressed in hepatocarcinoma. In an effort to further characterize the involvement of α6β1-integrin in hepatocarcinoma progression and to study α6β1-mediated functions, a human hepatocarcinoma cell line, HepG2, that express high surface levels of α6β1 and uses only this integrin to mediate adhesion on laminin was identified. Methods: To assess the role of α6β1 in these cells, a cytoplasmic domain deletion mutant of the β4-integrin subunit by complementary DNA transfection was expressed. The expression of the mutant β4 subunit in association with endogenous α6 showed a dominant-negative effect on α6β1 expression. Results: Stable transfectants of HepG2 that expressed the mutant β4 subunit showed a reduced ability to adhere and migrate on laminin matrices and to invade Matrigel. Furthermore, transfected cells showed significantly lower growth rates and reduced anchorage-independent growth compared with mock-transfected cells. Conclusions: These findings on the expression and function of α6β1 in hepatocarcinoma cells emphasize the potential contribution of this laminin receptor in the neoplastic transformation of hepatocytes. GASTROENTEROLOGY 1998;115:433-442

Hepatic stellate cells and extracellular matrix in hepatocellular carcinoma: more complicated than ever.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer death. Recent epidemiological data indicate that the mortality rate of HCC will double over the next decades in the USA and Europe. Liver cancer progresses in a large percentage of cases during the clinical course of chronic fibro‐inflammatory liver diseases leading to cirrhosis. Therefore, HCC development is regarded as the result of different environmental risk factors each involving different genetic, epigenetic‐ and chromosomal alterations and gene mutations. During tumour progression, the malignant hepatocytes and the activated hepatic stellate cells are accompanied by cancer‐associated fibroblasts, myofibroblasts and immune cells generally called tumour stromal cells. This new and dynamic milieu further enhances the responsiveness of tumour cells towards soluble mediators secreted by tumour stromal cells, thus directly affecting the malignant hepatocytes. This results in altered molecular pathways with cell proliferation as the most important mechanism of liver cancer progression. Given this contextual complexity, it is of utmost importance to characterize the molecular pathogenesis of HCC, and to identify the dominant pathways/drivers and aberrant signalling pathways. This will allow an effective therapy for HCC that should combine strategies affecting both cancer and the tumour stromal cells. This review provides an overview of the recent challenges and issues regarding hepatic stellate cells, extracellular matrix dynamics, liver fibrosis/cirrhosis and therapy, tumour microenvironment and HCC.

The androgen receptor associates with the epidermal growth factor receptor in androgen-sensitive prostate cancer cells.

Many recent evidences indicate that androgen-sensitive prostate cancer cells have a lower malignant phenotype that is in particular characterized by a reduced migration and invasion. We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with the synthetic androgen R1881 further reduced invasion of the cells without, however, modifying alpha6beta4 expression on the cell surface, suggesting an interference with the invasion process in response to EGF. We investigated whether the presence of the AR could affect EGF receptor (EGFR)-mediated signaling in response to EGF by evaluating autotransphosphorylation of the receptor as well as activation of downstream signalling pathways. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. An interaction between EGFR and AR has been demonstrated by immunoconfocal and co-immunoprecipitation analysis in PC3-AR cells, suggesting a possible interference of AR on EGFR signalling by interaction of the two proteins. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signalling in response to EGF leading to invasion through a mechanism involving an interaction between AR and EGFR.