Reduced expression of focal adhesion kinase in liver metastases compared with matched primary human colorectal adenocarcinomas.

The focal adhesion kinase (FAK) is implicated in integrin-mediated signal transduction pathways used in cell adhesion, cell motility, apoptosis, and anchorage-independent growth. Because cancer invasion and metastasis are thought to be associated with alterations in cellular adhesive and motile properties, we studied the expression of four focal adhesion proteins including FAK in matched samples of human normal colorectal mucosa (N), primary colorectal adenocarcinomas (T) and liver metastases (M) from 10 patients by Western blot analysis. This gave us the advantage of directly comparing levels of focal adhesion protein expression within the same genetic background. Average FAK expression level was significantly higher in T than in N and it was significantly lower in M than in T. Average paxillin expression level was also significantly higher in T than in N, but it was not significantly different between T and M. Similar results were obtained by immunohistochemical analyses of FAK and paxillin expression. Average vinculin and talin expression levels showed no significant differences among these three samples (N, T, and M). These data demonstrate that the FAK expression level increases in primary tumors compared with normal mucosa and decreases in liver metastases to the level of normal mucosa in the majority of human colorectal adenocarcinomas. Up- and down-regulation of FAK protein expression observed in this study may have a profound effect on the signal transduction.     

Extracellular pressure stimulates tumor cell adhesion in vitro by paxillin activation

Metastasizing colon cancer cells bind target tissues primarily via integrins. Extracellular pressure or shear stress stimulates integrin-mediated adhesion to matrix proteins or endothelial cells by activating the focal adhesion proteins FAK and Src. Because this effect is blocked by cytoskeletal perturbation and paxillin may link the cytoskeleton to the focal adhesion complex, we evaluated the role of paxillin in pressure-induced malignant colonocyte adhesion. We studied SW620 colon cancer cells and confirmed key results in Caco-2 colon cancer cells, primary human colon cancer cells, and a murine colonic adenocarcinoma. We evaluated adhesion to collagen at ambient and 15 mmHg increased pressure after 30 minutes, and paxillin, FAK, and Src phosphorylation in suspended cells prior to adhesion. Some cells were treated with siRNA to paxillin or FAK, or the Src inhibitor PP2. We also compared pressure-induced signals in suspended cells with adhesion-induced signals after adhesion to collagen. Pressure stimulated adhesion and paxillin phosphorylation in SW620 and Caco-2 cells and human primary colon cancer cells. Pressure also increased paxillin phosphorylation in murine carcinoma cells. SiRNA to paxillin decreased SW620 and Caco-2 paxillin without altering basal levels of phosphorylated paxillin. Paxillin reduction decreased basal adhesion to collagen, and inhibited pressure-stimulated adhesion, as well as paxillin, FAK397, FAK576, and Src476 phosphorylation. Neither PP2 nor siRNA to FAK inhibited induction of paxillin phosphorylation by pressure. In contrast, adhesion stimulated FAK, Src, and paxillin phosphorylation regardless of paxillin reduction. In summary, pressure induced paxillin phosphorylation in colon cancer cells. Paxillin reduction inhibited basal adhesion and blocked the pressure-mediated increase in adhesion, as well as pressure-induced FAK and Src signals, while adhesion-induced signals were preserved. Paxillin may be an upstream mediator of pressure-stimulated adhesion, important in metastasis.     

Focal adhesion kinase (pp125FAK) expression,activation and association with paxillin and p50CSK in human metastatic prostate carcinoma

Pp125^FAK, a protein tyrosine kinase (PTK) co-localized with integrins in focal adhesion plaques, is known to transduce signals involved in the regulation of cell adhesion and motility as well as the anchorage-independent growth of transformed cells. We investigated whether pp125^FAK could be part of a signalling pathway that contributes to the progression of human prostate carcinoma (PCa). Up-regulation of pp125^FAK expression, its activation by phosphorylation on tyrosine and its association with paxillin and p50^csk were preferentially observed in PCa tissues from patients with metastases, whereas normal and hyperplastic prostates and localized PCa tissues showed undetectable or low levels of both FAK mRNA and protein and an absence of pp125^FAK signalling complexes. The increase in expression and activation of pp125^FAK in metastatic PCa tissues was also corroborated by our findings in human PCa cell lines. Indeed, higher levels of pp125^FAK and FAK mRNA were observed in highly tumorigenic PC-3 cells as was the presence of activated pp125^FAK, as opposed to an inactive form in LNCaP cells, which have a lower tumorigenic ability. In addition, pp125^FAK formed signalling complexes with both paxillin and p50^csk in PC-3 cells as in metastatic PCa tissues. Together, our results show that an increase in FAK mRNA and protein, as well as pp125^FAK activation and association with signalling proteins, correlates with progression and invasion in human PCa tissues and cells. © 1996 Wiley-Liss, Inc.     

Rapamycin inhibits F-actin reorganization and phosphorylation of focal adhesion proteins

An early event of cell migration is characterized as the rapid reorganization of the actin cytoskeleton. Recently, we have demonstrated that rapamycin inhibits tumor cell motility. To understand the underlying mechanism, this study was set to determine whether rapamycin inhibition of cell motility is related to its prevention of F-actin reorganization. We found that rapamycin prevented type I insulin-like growth factor (IGF-I)-stimulated F-actin reorganization in human rhabdomyosarcoma (Rh30), Ewing sarcoma (Rh1), glioblastoma (U-373) and prostate carcinoma (PC-3) cells, and concurrently inhibited phosphorylation of focal adhesion proteins, including focal adhesion kinase (FAK), paxillin and p130(Cas) in the cells. The effect of rapamycin was blocked by expression of a rapamycin-resistant mutant of mTOR (mTORrr), but not a kinase-dead mTORrr. Downregulation of raptor mimicked the effect of rapamycin. Cells infected with a recombinant adenovirus expressing constitutively active and rapamycin-resistant mutant of p70 S6 kinase 1 (S6K1) conferred to resistance to rapamycin. Further, IGF-I failed to stimulate F-actin reorganization and phosphorylation of the focal adhesion proteins in the S6K1-downregulated cells. Expression of constitutively hypophosphorylated eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1-5A) inhibited IGF-I-stimulated F-actin reorganization, but did not alter the cellular protein or phosphorylation levels of the focal adhesion proteins. The results suggest that rapamycin inhibits IGF-I-induced F-actin reorganization and phosphorylation of the focal adhesion proteins by disruption of mTOR-raptor complex. Both S6K1 and 4E-BP1 pathways, mediated by the mTOR-raptor complex, are involved in the regulation of IGF-I-stimulated F-actin reorganization, but only the former controls IGF-I-stimulated phosphorylation of the focal adhesion proteins.     

Smad3 is overexpressed in advanced human prostate cancer and necessary for progressive growth of prostate cancer cells in nude mice.

PURPOSE: The purpose of this study was to investigate the potential role of Smad3, a key mediator of transforming growth factor-beta signaling, in progression of prostate cancer. EXPERIMENTAL DESIGN: Expression of Smad proteins was determined in human prostate cancer tissue array and cell lines. Growth and metastasis of cells overexpressing dominant-negative Smad3 (Smad3D) were studied to determine its role in tumor progression in mice. Cell growth, apoptosis, and expression of angiogenic molecules in tumor lesions were studied to determine potential pathways that Smad3 promotes tumor progression. RESULTS: Smad3 was overexpressed in human prostate cancer, which correlated with Gleason score and expression of proliferating cell nuclear antigen. Androgen-independent PC-3MM2 and DU145 cells expressed much higher levels of Smad3 than did androgen-dependent LNCaP, 22Rv1, and LAPC-4 cells. Overexpression of Smad3D in PC-3MM2 cells (PC-3MM2-Smad3D) had minimal direct effects on cell growth but attenuated effects of transforming growth factor-beta1 on gene expression and cell growth. Overexpression of Smad3D did not significantly alter tumor incidence but reduced tumor growth rate and metastasis incidence. Most cells in the control tumors, but not PC-3MM2-Smad3D tumors, were positively stained by an antibody to proliferating cell nuclear antigen. Microvessels and expression of angiogenic molecule interleukin-8 were significantly reduced in tumors from PC-3MM2-Smad3D cells. PC-3MM2-Smad3D tumors also expressed lower levels of vascular endothelial growth factor and platelet-derived growth factor. CONCLUSIONS: These data suggest that Smad3, through regulating angiogenic molecule expression in tumor cells, is critical for progression of human prostate cancer.     

p190RhoGEF (Rgnef) promotes colon carcinoma tumor progression via interaction with focal adhesion kinase

Focal adhesion kinase (FAK) functions downstream of integrins and growth factor receptors to promote tumor cell motility and invasion. In colorectal cancer, FAK is activated by amidated gastrin, a protumorigenic hormone. However, it is unclear how FAK receives signals from the gastrin receptor or other G-protein-coupled receptors that can promote cell motility and invasion. The Rho guanine-nucleotide exchange factor p190RhoGEF (Rgnef) binds FAK and facilitates fibroblast focal adhesion formation on fibronectin. Here we report that Rgnef mRNA and protein expression are significantly increased during colorectal tumor progression. In human colon carcinoma cells, Rgnef forms a complex with FAK and upon gastrin stimulation, FAK translocates to newly-forming focal adhesions where it facilitates tyrosine phosphorylation of paxillin. short hairpin (shRNA)-mediated knockdown of Rgnef or FAK, or pharmacological inhibition of FAK activity, is sufficient to block gastrin-stimulated paxillin phosphorylation, cell motility, and invadopodia formation in a manner dependent upon upstream cholecystokinin-2 receptor expression. Overexpression of the C-terminal region of Rgnef (Rgnef-C, amino acid 1,279-1,582) but not Rgnef-CΔFAK (amino acid 1,302-1,582 lacking the FAK binding site) disrupted endogenous Rgnef-FAK interaction and prevented paxillin phosphorylation and cell motility stimulated by gastrin. Rgnef-C-expressing cells formed smaller, less invasive tumors with reduced tyrosine phosphorylation of paxillin upon orthotopic implantation, compared with Rgnef-CΔFAK-expressing cells. Our studies identify Rgnef as a novel regulator of colon carcinoma motility and invasion, and they show that a Rgnef-FAK linkage promotes colon carcinoma progression in vivo.     

ADAM15 supports prostate cancer metastasis by modulating tumor cell-endothelial cell interaction

Using human tumor and cDNA microarray technology, we have recently shown that the ADAM15 disintegrin is significantly overexpressed during the metastatic progression of human prostate cancer. In the current study, we used lentiviral-based short hairpin RNA (shRNA) technology to down-regulate ADAM15 in the metastatic prostate cancer cell line, PC-3. ADAM15 down-regulation dramatically attenuated many of the malignant characteristics of PC-3 cells in vitro and prevented the s.c. growth of PC-3 cells in severe combined immunodeficient (SCID) mice. By inhibiting the expression of ADAM15 in PC-3 cells, we showed decreased cell migration and adhesion to specific extracellular matrix proteins. This was accompanied by a reduction in the cleavage of N-cadherin by ADAM15 at the cell surface. Fluorescence-activated cell sorting analysis revealed reduced cell surface expression of the metastasis-associated proteins alpha(v) integrin and CD44. Furthermore, matrix metalloproteinase 9 secretion and activity were abrogated in response to ADAM15 reduction. In an in vitro model of vascular invasion, loss of ADAM15 reduced PC-3 adhesion to, and migration through, vascular endothelial cell monolayers. Using an SCID mouse model of human prostate cancer metastasis, we found that the loss of ADAM15 significantly attenuated the metastatic spread of PC-3 cells to bone. Taken together, these data strongly support a functional role for ADAM15 in prostate tumor cell interaction with vascular endothelium and the metastatic progression of human prostate cancer.     

Increased class 3 semaphorin expression modulates the invasive and adhesive properties of prostate cancer cells

The class 3 semaphorins, sema3A and sema3C, provide important guidance cues in cell development and in cancer; however, the role of these semaphorins in prostate cancer is not known. We report here that sema3A transfected cells exhibit decreased invasion and adhesion in Matrigel-based assays and that sema3C transfected cells exhibit increased invasive and adhesive characteristics. Important adhesion proteins were differentially modulated in sema3A and sema3C cells in a manner consistent with their subsequent invasive and adhesive characteristics. E-cadherin expression as determined by Western blot analysis was strongly upregulated in sema3A transfected cells, but strongly downregulated in sema3C transfected cells compared to untransfected and mock empty vector-transfected PC-3 cells. beta-catenin levels were not changed in sema3A transfected cells; however, sema3C transfected cells had lower expression of this protein. Sema3C transfected cells exhibited greater cellular membrane expression of certain alpha integrins as compared to untransfected and sema3A transfected cells, a characteristic associated with increased adhesion and invasion. These data indicate that the invasive ability of sema3A and sema3C transfected PC-3 cells is, in part, correlated with adhesion protein expression and adhesive ability to constituents of neighboring cells and the extracellular matrix.     

Reduced focal adhesion kinase and paxillin phosphorylation in BCR-ABL-transfected cells

BACKGROUND: BCR-ABL formation is critical to oncogenic transformation in chronic myelogenous leukemia and has been implicated as a key event leading to alterations in cytoskeletal structures and adhesion in the leukemic cells. The authors therefore investigated the effect of p210(BCR-ABL) on actin polymerization as well as on the expression and phosphorylation state of the adhesion proteins paxillin and focal adhesion kinase (FAK). METHODS: Transfection with BCR-ABL constructs abrogated the ability of NIH 3T3 fibroblasts to adhere and the cells underwent striking morphologic changes. RESULTS: Scanning electron microscopy revealed that the cells lost their elongated appearance and became rounded. This alteration was associated with significantly reduced actin polymerization. In addition, steady-state levels of paxillin and FAK protein were increased. However, while the overall level of phosphotyrosines was also increased, the amount of tyrosine phosphorylated paxillin and FAK was reduced in the BCR-ABL-transfected cells as compared to the parental cells. Culture on extracellular fibronectin matrix partially reversed the morphologic changes and resulted in a return, albeit incomplete, of filamentous actin in BCR-ABL-transfected 3T3 fibroblasts. In addition, phosphorylation of paxillin and FAK in the BCR-ABL-transfected NIH 3T3 cells was restored. CONCLUSIONS: The authors conclude that, in the current system, transfection of BCR-ABL attenuates FAK and paxillin phosphorylation and reduces actin polymerization, events accompanied by significant alterations in cellular morphology. The observation that exposure of the cells to fibronectin partially reverses all these changes suggests that the focal adhesion proteins and actin structures nevertheless remain responsive to signaling from the outside.     

Gene transfer of alpha1,3-fucosyltransferase increases tumor growth of the PC-3 human prostate cancer cell line through enhanced adhesion to prostatic stromal cells

Elevated expression of sialyl Lewis X has been postulated to be a prognostic indicator of prostate cancer. However, direct evidence for the relationship between increased expression of sialyl Lewis X and malignancy of prostate cancer is still lacking. To determine whether increased levels of sialyl Lewis X leads to malignancy in prostate tumor, we transfected the human prostate cancer cell line PC-3 with alpha1,3-fucosyltransferase III (FTIII) to obtain stable transfectants, PC-3-FTIII lines, that highly express sialyl Lewis X. When inoculated in the prostate of nude mice, PC-3-FTIII cells produced large prostate tumors, while mock-transfected PC-3 cells, which are negative for sialyl Lewis X antigen, produced small prostate tumors. The aggressive tumor formation by PC-3-FTIII cells was inhibited by preincubation of the tumor cells with anti-sialyl Lewis X antibody, by the presence of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide but not by control peptide. PC-3-FTIII cells and mock-transfected PC-3 cells exhibited no significant difference in cell numbers when cultured in vitro. Remarkably, PC-3-FTIII adhered to prostatic stromal cells in vitro with higher affinity than mock-transfected PC-3. Such adhesion was inhibited by preincubation of PC-3-FTIII cells with antisialyl Lewis X antibody, by the addition of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide. However, anti-E-selectin, anti-P-selectin or anti-L-selectin antibodies did not inhibit the adhesion of PC-3-FTIII cells to the stromal cells. These results suggest that prostate cancer cells gain aggressiveness through adhesive interaction with prostatic stromal cells by a novel mechanism involving sialyl Lewis X.     

Repulsive guidance molecules, novel bone morphogenetic protein co-receptors, are key regulators of the growth and aggressiveness of prostate cancer cells

Repulsive guidance molecule (RGM) family members RGMA, RGMB and RGMC are GPI-linked membrane proteins recently identified as co-receptor of bone morphogenetic proteins (BMPs). BMPs are a group of proteins enriched in bone and play important roles in prostate cancer. The current study aimed to investigate roles played by RGMs in prostate cancer. Expression of RGMs was examined in prostate cancer cell lines and prostate cancer tissues using RT-PCR and immunohistochemical staining. Knockdown of each RGM in prostate cancer cells was performed using the respective anti-RGMA, RGMB and RGMC transgenes. A variety of in vitro function tests were employed to analyze the influence on cancer cell functions by RGM knockdown. The implications of RGM knockdown in BMP signalling were also examined using both Western blot and real-time quantitative PCR. Knockdown of RGMA had no effect on cell growth, migration and invasion, but promoted cell-matrix adhesion. Knockdown of RGMB and RGMC increased growth and adhesion, but only RGMB knockdown increased capacities of migration and invasion in PC-3 cells. Further investigations showed an increase in Smad-3 activation and reduced levels of Smad-1 in PC-3 cells by RGMB and RGMC knockdown, and also an up-regulation of ID1, a BMP target gene particularly in exposure to BMP7. RGMs play inhibitory roles in prostate cancer by suppressing cell growth, adhesion, migration and invasion. RGMs can coordinate Smad-dependent signalling of BMPs in prostate cancer cells.     

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

Background

Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines.

Methods

Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays.

Results

In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity.

Conclusions

We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

     

Inhibition of focal contact formation in cells transformed by p185neu.

Signaling pathways mediated by adhesive molecules are tightly associated with cytoskeletal organization and cell growth regulation. Focal adhesion kinase (FAK) plays a prominent role in the adhesion signaling pathway through its tyrosine kinase activity and protein-protein interaction with other signaling molecules, including src, paxillin, and p130CAS, and other proteins. We explored the roles of these signaling molecules in the transformation of B104-1-1 cells, an NIH/3T3-derived cell line transformed by activated rat p185neu. The cytoskeletal organization of the p185neu-transformed cells was disrupted, and their morphology was dramatically altered. FAK, paxillin, and p130CAS appeared to be tyrosine phosphorylated in both NIH/3T3 and B104-1-1. However, the phosphorylation levels of paxillin and p130CAS were lower in B104-1-1 cells than in NIH/3T3 cells. Surprisingly, the association between FAK and paxillin was enhanced in B104-1-1 cells, suggesting reorganization of protein-protein interaction modulated by protein phosphorylation. Our results showed that even though cellular transformation by src and neu has similar consequences, such as focal adhesion disassembly and increased metastasis potential, the molecular events underlying the signaling pathways can be dramatically different.     

Runx2–smad signaling impacts the progression of tumor‐induced bone disease

Runx2, a master regulator of osteogenesis, is abnormally expressed in advanced prostate cancer. Here, we addressed Runx2 contribution to formation of prostate cancer‐related osteolytic and osteoblastic bone lesions by mediating TGFβ/BMP signaling through direct interaction with Smads. Further, we examined involvement of the Runx2–Smad complex in mediating tumor growth and distal metastasis. To identify Runx2–Smad‐specific mechanisms of prostate tumor activity in bone, we generated PC3 prostate cancer cell lines expressing Runx2‐WT or one of two mutant proteins (Runx2‐HTY and Runx2‐ΔC) that each disrupt the Runx2–Smad interaction, either directly through a point mutation or by deletion of the functional C‐terminus, respectively. Intratibial tumors generated from these cells revealed that Runx2‐WT‐expressing cells resulted in predominantly osteolytic disease, whereas cells expressing mutant proteins exhibited tumors with mixed osteolytic/osteoblastic lesions. Extent of bone loss and woven bone formation was assessed by radiography and micro‐computed tomography. Bioluminescent imaging showed the presence of labeled prostate cancer cells in the lung at the latest time point examined, with Runx2‐WT group exhibiting increased incidence of tumor cells in lung. Notably, disruption of the Runx2–Smad interaction significantly reduced incidence and size of lung tumors. Altered expression of Runx2 target genes involved in invasion, growth, adhesion and metastasis supported our findings. Thus, our studies demonstrate that Runx2 in prostate cancer cells plays a significant role in intratibial prostate cancer‐related tumor growth and bone loss through mechanisms mediated by the Runx2–Smad signaling pathway. This work expands upon the potential importance of Runx2 as a therapeutic target in cancer.     

Gene expression of angiogenic factors correlates with metastatic potential of prostate cancer cells

We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-beta2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3-5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.     

Constitutive activation of MAPK/ERK inhibits prostate cancer cell proliferation through upregulation of BRCA2

BRCA2 is central to an utterly diverse biological behavior elicited after integrin-mediated normal and prostate cancer cell adhesion to basement membrane (BM) and extracellular matrix (ECM) proteins. Unlike normal cells, adhesive stimuli in cancer cells activate PI 3-kinase/AKT signaling resulting in BRCA2 degradation and unchecked cancer cell proliferation and metastasis. However, the precise mechanisms involved in normal BRCA2 homeostasis are unknown. We investigated ERK and AKT phosphorylation in normal (PNT1A) and cancer (PC-3) prostate cells after adhesion to ECM and the effects upon BRCA2 and cell proliferation. PNT1A cell adhesion to ECM triggered MAPK/ERK signaling resulting in upregulation of BRCA2 mRNA and protein, with negligible effects upon cell proliferation. Disruption of MAPK/ERK with PD98059 prevented any BRCA2 upregulation inhibiting DNA synthesis below basal levels. PC-3 cells exhibited a defective MAPK/ERK pathway that was unresponsive to adhesion to the ECM, which instead triggered PI 3-kinase/AKT signaling leading to BRCA2 protein depletion and cell proliferation. Reconstitution of MAPK/ERK by recombinant expression of a constitutively active form of MAPK kinase 1 (MEK1) effectively reversed the neoplastic phenotype by increasing BRCA2 expression and preventing any aberrant cell proliferation at rest and upon interaction with ECM proteins. Our results suggest that aberrant loss of MAPK/ERK activity in prostate cancer may play a pivotal role in the malignant phenotype, and provide evidence that interventions aimed at bypassing the signaling block are able to effectively reverse neoplastic unchecked cell proliferation.     

Bombesin and gastrin releasing peptide increase tyrosine phosphorylation of focal adhesion kinase and paxillin in non-small cell lung cancer cells

The effects of some oncogenes, growth factors and neuropeptides are mediated by tyrosine phosphorylation of focal adhesion kinase (p125(FAK)) and paxillin cytoskeletal proteins. In this study the ability of bombesin/gastrin releasing peptide (BB/GRP) to stimulate tyrosine phosphorylation of p125(FAK) and paxillin in non-small cell lung cancer (NSCLC) H1299 cells was investigated. BB, 100 nM caused increased p125(FAK) and paxillin tyrosine phosphorylation maximally after 1 min. The effect of BB on p125(FAK) and paxillin tyrosine phosphorylation was concentration-dependent, being half maximal at 4-8 nM. Also, 100 nM GRP, GRP(14-27) but not GRP(1-16) increased p125(FAK) and paxillin tyrosine phosphorylation indicating that the C-terminal of GRP is essential. BW2258U89, a GRP receptor antagonist, caused a dose-dependent inhibition of BB-stimulated p125(FAK) and paxillin tyrosine phosphorylation with an IC50 value of 3 microM. Cytochalasin D (0.3 microM), which inhibits actin polymerization, reduced the ability of BB to stimulate tyrosine phosphorylation of p125(FAK) and paxillin. Genistein (50 microM) and H-7 (50 microM), which are kinase inhibitors, reduced the tyrosine phosphorylation of p125(FAK) and paxillin stimulated by BB. Also, treatment of NCI-H1299 cells with FAK antisense resulted in decreased FAK tyrosine kinase activity and proliferation. These results suggest that p125(FAK) is an important enzyme for NSCLC proliferation.     

Diverse biological effect and Smad signaling of bone morphogenetic protein 7 in prostate tumor cells

We found that bone morphogenetic protein (BMP) 7, a member of the BMP family, was strikingly up-regulated during the development of primary prostatic adenocarcinoma in the conditional Pten deletion mouse model. To determine the relevance of this finding to human prostate cancer, we examined the expression of BMPs and BMP receptors (BMPR) as well as the responsiveness to recombinant human BMP7 in a series of human prostate tumor cell lines. All prostatic cell lines tested expressed variable levels of BMP2, BMP4, and BMP7 and at least two of each type I and II BMPRs. In all cases, BMP7 induced Smad phosphorylation in a dose-dependent manner, with Smad5 activation clearly demonstrable. However, the biological responses to BMP7 were cell type specific. BPH-1, a cell line representing benign prostatic epithelial hyperplasia, was growth arrested at G1. In the bone metastasis-derived PC-3 prostate cancer cells, BMP7 induced epithelial-mesenchymal transdifferentiation with classic changes in morphology, motility, invasiveness, and molecular markers. Finally, BMP7 inhibited serum starvation-induced apoptosis in the LNCaP prostate cancer cell line and more remarkably in its bone metastatic variant C4-2B line. Each of the cell lines influenced by BMP7 was also responsive to BMP2 in a corresponding manner. The antiapoptotic activity of BMP7 in the LNCaP and C4-2B cell lines was not associated with a significant alteration in the levels of the proapoptotic protein Bax or the antiapoptotic proteins Bcl-2, Bcl-xl, and X-linked inhibitor of apoptosis. However, in C4-2B cells but not in LNCaP cells, a starvation-induced decrease in the level of survivin was counteracted by BMP7. Taken together, these findings suggest that BMPs are able to modulate the biological behavior of prostate tumor cells in diverse and cell type-specific manner and point to certain mechanisms by which these secreted signaling molecules may contribute to prostate cancer growth and metastasis.     

Human prostate cancer cells adhere specifically to hemoglobin: a possible role in bone-specific metastasis

From the supernatant of rabbit bone marrow, we isolated an organ-specific factor, which was related with the metastasis of prostate cancer to the bone and examined its adhesion to prostate cancer cells (PC-3). Molecular weight and amino acid sequence analyses of the active component obtained by high performance liquid chromatography revealed that a component identical to the alpha chain of hemoglobin accounted for 80% of the biological activity. Hemoglobin showed over 50% adhesion to PC-3 cells but only 10% adhesion to human colon cancer cell lines, representative of organ non-specific metastasis, and leukemia cells line, representative of a non-solid tumor. Some substance in the bone marrow may promote the first step of adhesion of cancer cells to bone marrow in the metastasis of prostate cancer to the bone, possibly an amino acid sequence or some tertiary structure similar to hemoglobin.