Alterations in the focal adhesion kinase/Src signal transduction pathway correlate with increased migratory capacity of prostate carcinoma cells

Focal adhesion kinase (FAK) has been implicated in the regulation of cell migration. In addition, FAK expression is increased in a number of highly metastatic tumor cell lines. Therefore, we investigated the role of FAK in regulating migration of prostate carcinoma cell lines with increasing metastatic potential. We show that highly tumorigenic PC3 and DU145 cells exhibit intrinsic migratory capacity, while poorly tumorigenic LNCaP cells require a stimulus to migrate. Increased metastatic potential of PC3 and DU145 cells correlates with increased FAK expression, overall tyrosine phosphorylation and activity, as measured by autophosphorylation of tyrosine 397. However, in PC3 and DU145 cells, FAK autophosphorylation is adhesion dependent whereas a second site of tyrosine phosphorylation, tyrosine 861, a Src specific site, is uncoupled from adhesion-dependent signaling events. Finally, inhibiting the FAK/Src signal transduction pathway by over expressing FRNK (Focal adhesion kinase-Related Non-Kinase), an inhibitor of FAK activation, or treatment with PP2, a Src family kinase inhibitor, significantly inhibited migration of prostate carcinoma cell lines, demonstrating that tumor cell migration continues to be dependent on signals emanating from this pathway.     

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.     

Type I collagen receptor (alpha 2 beta 1) signaling promotes the growth of human prostate cancer cells within the bone

The most frequent site of prostate cancer metastasis is the bone. Adhesion to bone-specific factors may facilitate the selective metastasis of prostate cancer to the skeleton. Therefore, we tested whether prostate cancer bone metastasis is mediated by binding to type I collagen, the most abundant bone protein. We observed that only bone metastatic prostate cancer cells bound collagen I, whereas cells that form only visceral metastases failed to bind collagen. To confirm the relationship between collagen adhesion and bone metastatic potential, a collagen-binding variant of human LNCaP prostate cancer cells was derived through serial passage on type I collagen (LNCaP(col)). Fluorescence-activated cell sorting analysis showed that LNCaP(col) cells express increased levels of the integrin collagen I receptor alpha(2)beta(1) compared with LNCaP cells. Antibodies to the alpha(2)beta(1) complex inhibited LNCaP(col) binding to collagen, confirming that integrins mediated the attachment. Correspondingly, LNCaP(col) cells displayed enhanced chemotactic migration toward collagen I compared with LNCaP cells, an activity that could be blocked with alpha(2)beta(1) antibodies. To directly test the role of alpha(2)beta(1)-dependent collagen binding in bone metastasis, LNCaP and LNCaP(col) cells were injected into the tibia of nude mice. After 9 weeks, 7 of 13 (53%) mice injected with LNCaP(col) developed bone tumors, whereas 0 of 8 mice injected with LNCaP cells had evidence of boney lesions. LNCaP(col) cells were found to express increased levels of the metastasis-promoting RhoC GTPase compared with parental LNCaP. We conclude that collagen I attachment mediated by alpha(2)beta(1) initiates motility programs through RhoC and suggest a mechanism for prostate cancer metastasis to the bone.     

A new type of antimetastatic peptide derived from fibronectin.

PURPOSE: We found previously that fibronectin (FN) has a cryptic functional site (YTIYVIAL sequence within the 14th type III repeat) opposing cell adhesion to extracellular matrix. A 22-mer FN peptide containing this site, termed FNIII14, inhibits beta1 integrin-mediated adhesion without binding to integrins. The present study shows that FNIII14 has the potential to prevent lymphoma cell metastasis. EXPERIMENTAL DESIGN: Antimetastatic effect of FNIII14 has been evaluated through in vitro or in vivo experiments. RESULTS: FNIII14 inhibited the integrin alpha4beta1-mediated B lymphoma Ramos cell adhesion to VCAM-1 on venule endothelial cells, as well as to FN. Murine T lymphoma L5178Y-ML25 cells, which are known to metastasize to liver and spleen, preferentially adhered to vitronectin (VN) and migrated toward VN concentration gradients. FNIII14 abrogated both the integrin alphavbeta3-mediated adhesion and migration of L5178Y-ML25 cells. Inhibition of the alphavbeta3mediated L5178Y-ML25 cell adhesion by FNIII14 was reversed by phenylarsine oxide, a protein tyrosine phosphatase inhibitor. In addition, FNIII14 abrogated the VN-stimulated tyrosine phosphorylation of intracellular signaling proteins, including focal adhesion kinase (p125(FAK)) and paxillin, suggesting that such a diversity of FNIII14 effects might be because of the negative regulation of p125(FAK) and paxillin tyrosine phosphorylation, which has been involved in adhesion signals transduced by different integrins. The in vivo experiment using a murine metastasis model showed that FNIII14 would inhibit liver and spleen metastases of L5178Y-ML25 cells at a dose much lower than that of RGDS. CONCLUSIONS: FNIII14 might be applicable as a new type of antimetastatic agent distinct from integrin-binding peptides.     

Inhibition of alpha(v)beta3 integrin survival signaling enhances antiangiogenic and antitumor effects of radiotherapy.

The involvement of alpha(v)beta3 and alpha(v)beta5 integrins in angiogenesis and the use of integrin antagonists as effective antiangiogenic agents are documented. Radiotherapy is an important therapy option for cancer. It has been shown that ionizing radiation exerts primarily antiangiogenic effects in tumors but has also proangiogenic effects as the reaction of the tumor to protect its own vasculature from radiation damage. Here, we show that combined treatment with S247, an Arg-Gly-Glu peptidomimetic antagonist of alpha(v)beta3 integrin, and external beam radiotherapy are beneficial in local tumor therapy. We found that radiation up-regulates alpha(v)beta3 expression in endothelial cells and consecutively phosphorylates Akt, which may provide a tumor escape mechanism from radiation injury mediated by integrin survival signaling. In the presence of S247, the radiation-induced Akt phosphorylation is strongly inhibited. Our studies on endothelial cell proliferation, migration, tube formation, apoptosis, and clonogenic survival show that the radiosensitivity of endothelial cells is enhanced by the concurrent administration of the integrin antagonist. The in vitro data are successfully translated into human glioma (U87), epidermoid (A431), and prostate cancer (PC3) xenograft models growing s.c. on BALB/c-nu/nu mice. In vivo, the combination of S247 treatment and fractionated radiotherapy (5 x 2.5 Gy) leads to enhanced antiangiogenic and antitumor effects compared with either monotherapies. These results underline the importance of alpha(v)beta3 integrin when tumors protect their microvasculature from radiation-induced damage. The data also indicate that the combination of integrin antagonists and radiotherapy represents a rational approach in local cancer therapy.     

The role of alpha(v)beta(3) in prostate cancer progression

Integrin alpha(v)beta(3) is involved in varied cell biological activities, including angiogenesis, cell adhesion, and migration on several extracellular matrix components. Although alpha(v)beta(3) is not typically expressed in epithelial cells, it is expressed in macrophages, activated leukocytes, cytokine-stimulated endothelial cells, osteoclasts, and certain invasive tumors. Interestingly, the adhesion and migration of breast cancer cells on bone matrix are mediated, in part, by alpha(v)beta(3). Similar to breast cancer cells, prostate cancer cells preferentially metastasize to the bone. The biological events that mediate this metastatic pattern of prostate cancer are not well defined. This review discusses the role alpha(v)beta(3) plays in prostate cancer progression, with specific emphasis on bone metastasis and on alpha(v)beta(3) signaling in prostate cancer cells. The data suggest that alpha(v)beta(3), in part, facilitates prostate cancer metastasis to bone by mediating prostate cancer cell adhesion to and migration on osteopontin and vitronectin, which are common proteins in the bone microenvironment. These biological events require the activation of focal adhesion kinase and the subsequent activation of PI-3 kinase/Akt signaling pathway.     

Osteopontin increases lung cancer cells migration via activation of the alphavbeta3 integrin/FAK/Akt and NF-kappaB-dependent pathway

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which is abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and cell proliferation via interaction with its receptor, alphavbeta3 integrin. However, the effect of OPN on migration activity in human lung cancer cells is mostly unknown. Here we found that OPN increased the migration via activation of alphavbeta3 integrin in human lung cancer cells (A549 cells). Phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002), Akt inhibitor or ERK inhibitor (PD98059) inhibited the OPN-induced increase in the migration of lung cancer cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), p85 subunit of PI3K, serine 473 of Akt and ERK. In addition, treatment of A549 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) inhibited OPN-induced migration of lung cancer cells. Stimulation of A549 cells with OPN also induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OPN-mediated increases in IKK alpha/beta, IkappaBalpha and p65 Ser(536) phosphorylation were inhibited by Ly294002, Akt inhibitor and PD98059. Co-transfection with FAK, p85, Akt and ERK mutants also reduced the OPN-induced kappaB-luciferase activity. Taken together, these results suggest that OPN acts through alphavbeta3 integrin, which in turn activates the FAK, PI3K, Akt, ERK and NF-kappaB pathways, contributing to the migration of lung cancer cells.     

Aspirin inhibits highly invasive prostate cancer cells

Cell adhesion, proteolytic degradation and cell migration are interrelated processes responsible for the invasion and metastasis of cancer. One of the crucial molecules involved in cancer metastasis is urokinase-type plasminogen activator (uPA). An elevated concentration of uPA is a strong indicator of poor prognosis. In addition to the proteolytic activity of uPA, which degrades the extracellular matrix, uPA also binds to its receptor (uPAR) and controls cell adhesion and migration through the reorganization of actin cytoskeleton. We have recently demonstrated that constitutively active nuclear factor-kappa B (NF-kappaB) is responsible for the increased secretion of uPA and that inhibition of NF-kappaB suppresses secretion of uPA and cell migration of highly invasive cancer cells. Aspirin and other nonsteroidal anti-inflammatory drugs have been recently shown to have a chemopreventive effect in colon and pancreatic cancers. Here we show that aspirin inhibits NF-kappaB, resulting in the suppression of uPA secretion from the highly invasive human prostate cancer cells PC-3. Furthermore, aspirin inhibited migration of PC-3 cells, suggesting an effect on the uPA-uPAR signaling complex. Finally, aspirin suppressed adhesion of PC-3 cells to fibronectin (FN), which binds to an alpha3beta1 integrin receptor, and to vitronectin (VN), which binds to alphavbeta3 integrin receptor. Altogether, our data suggests that aspirin inhibits the formation of uPA-uPAR-FN-alpha3beta1 and uPA-uPAR-VN-alphavbeta3 complexes, resulting in the suppression of cell adhesion and cell motility of the highly invasive prostate cancer cells PC-3. These results indicate that aspirin may contribute directly to reducing invasion and metastasis of prostate cancers by inhibiting cell migration and invasion.     

Laminin-5 beta3A expression in LNCaP human prostate carcinoma cells increases cell migration and tumorigenicity

Interactions between extracellular matrix proteins and prostate carcinoma cells change dramatically during prostate tumor progression. We have concentrated on two key modifications that occur in the hemidesmosome in prostate carcinoma: loss of laminin-5 protein expression and altered basal cell polarity of the alpha6beta4 integrin. We previously demonstrated two cell line-specific isoforms (beta3A and beta3B) of the LAMB3 message. Cells expressing only the beta3B isoform did not translate the beta3 protein and were unable to assemble the laminin-5 trimer. One such cell line, LNCaP, was selected to determine whether restoration of the laminin-5 beta3A isoform would cause expression of a functional laminin-5 beta3 chain, assembly and secretion of the laminin-5 trimer, and reversion to a non-neoplastic phenotype. Laminin-5 beta3A cDNA was cloned and stably transfected into LNCaP cells. We observed the restoration of the beta3 protein, but a laminin-5 trimer was not secreted. Moreover, increased cell migration was demonstrated, and tumorigenicity was increased in SCID mice. A microarray analysis, performed between transfected and nontransfected LNCaP cells, showed most changing genes to be associated with signal transduction. The beta3 chain of laminin-5 may thus play an important role in signal transduction, which may enhance cell motility and tumorigenesis.     

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.     

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 alpha6beta4 integrin expression. The treatment with androgens further reduced invasion of the cells without modifying alpha6beta4 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.     

Biological activity of substrate-bound basic fibroblast growth factor (FGF2): recruitment of FGF receptor-1 in endothelial cell adhesion contacts

Substrate-bound FGF2 promotes endothelial cell adhesion by interacting with alpha(v)beta(3) integrin. Here, endothelial GM7373 cells spread and organize focal adhesion plaques on immobilized FGF2, fibronectin (FN), and vitronectin (VN). alpha(v)beta(3) integrin, paxillin, focal adhesion kinase, vinculin and pp60(src) localize in cell-substratum contact sites on FGF2, FN or VN. However, only immobilized FGF2 induces a long-lasting activation of extracellular signal-regulated kinases(1/2) (ERK(1/2)) and cell proliferation that was inhibited by the ERK(1/2) inhibitor PD 098059 and the tyrosine kinase (TK) inhibitor tyrphostin 23, pointing to the engagement of FGF receptor (FGFR) at the basal side of the cell. To assess this hypothesis, GM7373 cells were transfected with a dominant negative TK(-)-DeltaFGFR1 mutant (GM7373-DeltaFGFR1 cells) or with the full-length receptor (GM7373-FGFR1 cells). Both transfectants adhere and spread on FGF2 but GM7373-DeltaFGFR1 cells do not proliferate. Also, parental and GM7373-FGFR1 cells, but not GM7373-DeltaFGFR1 cells, undergo morphological changes and increased motility on FGF2-coated plastic. Finally, FGFR1, but not TK(-)-DeltaFGFR1, localizes in cell adhesion contacts on immobilized FGF2. In conclusion, substrate-bound FGF2 induces endothelial cell proliferation, motility, and the recruitment of FGFR1 in cell-substratum contacts. This may contribute to the cross talk among intracellular signaling pathways activated by FGFR1 and alpha(v)beta(3) integrin in endothelial cells.     

Integrin expression and ability to adhere to extracellular matrix proteins and endothelial cells in human lung cancer lines.

We examined the integrin expression in 19 human lung cancer cell lines with monoclonal antibodies to the integrin subunits alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, beta 1, beta 2, and beta 4. We measured their ability to adhere to the extracellular matrix (ECM) and human umbilical vein endothelial cells (HUVECs). Almost all lines expressed the beta 1 subunit and approximately half of the lines expressed the beta 4 subunit; by contrast, none expressed the beta 2 subunit. Subunits alpha 2, alpha 3, alpha 5 and alpha 6 were frequently expressed, whereas very few lines expressed alpha 1 and alpha 4. Most lines adhered strongly to ECM (type I collagen, laminin and fibronectin) in correspondence to their expression of integrins. Binding by most lines to fibronectin was completely inhibited by arginine-glycine-aspartic acid (RGD) peptide. Three lines that expressed few or no integrins had very weak ability to adhere to ECM. Strong binding to HUVECs was found in most lines, but the three lines had very little ability to adhere to HUVECs. Binding to HUVECs was strongly inhibited at 4 degrees C, under divalent cation-free conditions and by antibodies to the beta 1 subunit. These results suggest that lung cancer cells adhere to ECM and endothelial cells through integrins, especially the beta 1 subfamily.     

Bimolecular interaction of insulin-like growth factor (IGF) binding protein-2 with alphavbeta3 negatively modulates IGF-I-mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the alpha v beta 3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and alpha v beta 3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7 beta 3, which overexpressed the alpha v beta 3 integrin. Collectively, these results indicate that alpha v beta 3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.     

Changes in the levels of integrin and focal adhesion kinase (FAK) in human melanoma cells following 532 nm laser treatment.

Despite the increase in laser therapy, concern remains that sublethal treatment of pre-malignant lesions may adversely affect the biological behaviour of surviving cells. Integrin receptors mediate interaction of cells with the extracellular matrix and their occupation leads to focal adhesion kinase (FAK) activation. Using our previously established model we have now investigated subcellular changes and compared integrin and FAK concentrations, the degree of FAK phosphorylation and its association with the beta1 integrin in laser vs. non-laser treated cells. We treated cells with laser generated from a frequency doubled Q-switched (Nd:YAG) laser system (532 nm) at 0.4 J/cm2 twice per week for 4 weeks. Using cell lysates we performed Western immunoblotting 24 hr later to detect integrin subunits and FAK proteins and immunoprecipitation to investigate FAK phosphorylation and its association with beta1. Cell morphology was examined using electron microscopy. SK23 and G361 cells exhibited an 3.4- and 11.2-fold increase, respectively, in FAK protein following laser treatment. FAK phosphorylation in SK23 cells was increased by 82%, whereas FAK phosphorylation in G361 cells was reduced slightly (2%). Furthermore, both alpha3 and 4 integrins were up-regulated, by approximately 4-fold and 7- to 9-fold, respectively. In addition, the beta1 integrin was proteolysed in both cell lines and the levels of FAK associated with beta1 was increased (2.1- and 2.7-fold, respectively). Finally, laser treatment of SK23 cells caused an increased number of cell processes. Sublethal 532 nm laser light thus induces changes in integrin and FAK concentrations and subsequently influences cellular attachment and morphology.     

Expression and functional role of CCR9 in prostate cancer cell migration and invasion.

PURPOSE: Metastasis is responsible for most cancer-related deaths; hence, therapies designed to minimize metastasis are greatly needed. The precise cellular and molecular mechanisms used by cancer cells for metastasis are not fully understood; however, the metastatic spread of neoplastic cells is probably related to the ability of these cells to migrate, invade, home, and survive locally. The migration of tumor cells shares many similarities with leukocyte trafficking, which is regulated by chemokine receptor-ligand interactions. The current study evaluates the molecular mechanisms of CCL25 and CCR9 in prostate cancer cell migration and invasion. EXPERIMENTAL DESIGN: In the current study, real-time quantitative polymerase chain reaction, flow cytometry analysis, and in vitro migration as well as invasion chamber analysis (with and without antibody-mediated inhibition) were used to ascertain the biological and functional significance of CCR9 expression by normal prostatic epithelial cells (PrEC) or prostate cancer cell lines (LNCaP-10995 and PC3). RESULTS: We report that functional CCR9 is highly expressed by LNCaP cells and modestly, yet significantly, expressed by PC3 cells when compared with PrEC cells. Neutralization of CCL25-CCR9 interactions impaired the migration and invasion potential of the LNCaP and PC3 cell lines. CCL25 differentially modulated the expression of collagenase-1 or matrix metalloproteinase (MMP)-1, collagenase-3 (MMP-13), stromalysin-2 (MMP-10), stromalysin-3 (MMP-11), and gelatinase-A (MMP-2), but not MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, or MMP-14 in prostate cancer cells. CONCLUSIONS: These studies suggest that the expression and activation of CCR9 affect cancer cell migration, invasion, and MMP expression, which together may affect prostate cancer metastasis.     

Epidermal growth factor receptor activation in androgen-independent but not androgen-stimulated growth of human prostatic carcinoma cells.

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.     

T-cell receptor gamma chain alternate reading frame protein (TARP) expression in prostate cancer cells leads to an increased growth rate and induction of caveolins and amphiregulin.

Previously, we showed that prostate and prostate cancer cells express a truncated T-cell receptor gamma chain mRNA that uses an alternative reading frame to produce a novel nuclear T-cell receptor gamma chain alternate reading frame protein (TARP). TARP is expressed in the androgen-sensitive LNCaP prostate cancer cell line but not in the androgen-independent PC3 prostate cancer cell line, indicating that TARP may play a role in prostate cancer progression. To elucidate the function of TARP, we generated a stable PC3 cell line that expresses TARP in a constitutive manner. Expression of TARP in PC3 cells resulted in a more rapid growth rate with a 5-h decrease in doubling time. cDNA microarray analysis of 6538 genes revealed that caveolin 1, caveolin 2, amphiregulin, and melanoma growth stimulatory activity alpha were significantly up-regulated, whereas IL-1beta was significantly down-regulated in PC3 cells expressing TARP. We also demonstrated that TARP expression is up-regulated by testosterone in LNCaP cells that express a functional androgen receptor. These results suggest that TARP has a role in regulating growth and gene expression in prostate cancer cells.     

Akt1 mediates prostate cancer cell microinvasion and chemotaxis to metastatic stimuli via integrin β(3) affinity modulation

Background:Activation of Akt and increased expression of integrin β(3) are the two most important changes that have been linked to the attainment of metastatic potential by prostate cancer cells. However, a direct link between Akt activity and inside-out activation of integrin β(3) in mediating prostate cancer cell metastatic properties is not established.Methods:Using functional and biochemical approaches, we examined the role of Akt1 in the affinity modulation of integrin β(3) in prostate cancer cells.Results:Although expression of murine TRAMP and human PC3 cells with constitutively active Akt1 (CA-Akt1) enhanced their affinity for integrin α(v)β(3) specific ligands and motility on various extracellular matrix proteins, the reverse was observed with the expression of dominant-negative Akt1 (DN-Akt1). Although enhanced motility and transendothelial migration of CA-Akt1-expressing cells were blunted by co-expression with DN-integrin β(3) or upon pre-treatment with integrin β(3)-blocking antibodies (LM 609), impaired motility and transendothelial migration of DN-Akt1-expressing cells were rescued by pre-treatment of prostate cancer cells with integrin β(3)-activating antibodies, AP7.4.Conclusion:Our data is the first to demonstrate a link between Akt1 activity and affinity modulation of integrin β(3) in the regulation of prostate cancer cell motility, transendothelial migration and chemotaxis to metastatic stimuli.     

Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases

KAI1/CD82, a tetraspanin protein, was first identified as a metastasis suppressor in prostate cancer. How loss of CD82 expression promotes cancer metastasis is unknown. Restoration of CD82 expression to physiological levels in the metastatic prostate cell line PC3 inhibits integrin-mediated cell migration and invasion, but does not affect integrin expression. Integrin-dependent activation of the receptor kinase c-Met is dramatically reduced in CD82-expressing cells, as is c-Met activation by its ligand HGF/SF. CD82 expression also reduced integrin-induced activation and phosphorylation of the cytoplasmic tyrosine kinase Src, and its downstream substrates p130Cas and FAK Y861. Inhibition of c-Met expression or Src kinase function reduced matrigel invasion of PC3 cells to the same extent as CD82 expression. These data indicate that CD82 functions to suppress integrin-induced invasion by regulating signaling to c-Met and Src kinases, and suggests that CD82 loss may promote metastasis by removing a negative regulator of c-Met and Src signaling.