Overexpression of focal adhesion kinase (FAK) has been well correlated with tumor development and/or the maintenance of tumor phenotype. In addition, inappropriate activation of the extracellular regulated kinase (ERK) signaling pathway is common to many human cancers. In the present study, we investigated the interplay between FAK and ERK in androgen-independent prostate cancer cells (PC3 and DU145 cells). We observed that suppression of FAK expression using small interfering RNA–mediated knockdown decreased the clonogenic activity, whereas overexpression of FAK increased it. We also observed that detachment of PC3 and DU145 cells from their substrate induced tyrosine phosphorylation of FAK. ERK knockdown diminished FAK protein levels and tyrosine phosphorylation of FAK as well as FAK promoter-reporter activity. We also tested the effect of MEK inhibitors and small interfering RNA–mediated knockdown of ERK1 and/or ERK2 on cell proliferation, invasiveness, and growth in soft agar of PC3 and DU145 cells. Inhibition of ERK signaling grossly impaired clonogenicity as well as invasion through Matrigel. However, inhibition of ERK signaling resulted in only a modest inhibition of 3H-thymidine incorporation and no effect on overall viability of the cells or increased sensitivity to anoikis. Taken together, these data show, for the first time, a requirement for FAK in aggressive phenotype of prostate cancer cells; reveal interdependence of FAK and ERK1/2 for clonogenic and invasive activity of androgen-independent prostate cancer cells; suggest a role for ERK regulation of FAK in substrate-dependent survival; and show for the first time, in any cell type, the regulation of FAK expression by ERK signaling pathway. (Mol Cancer Res 2008;6(10):1639–48)

Prostate cancer is the second leading cause of cancer deaths among men in the United States. At present, no acceptable treatment exists for hormone-refractory tumors. Androgen withdrawal remains the only treatment option for men with advanced prostate cancer, however, most men who initially respond to hormone ablation therapy fail and the disease progresses (1).

Focal adhesion kinase (FAK) is an important intermediary of growth factor signaling, cell proliferation, migration, and invasion. Because the development of malignancy is often associated with perturbation in these processes, alteration in FAK activity in tumor cells is not surprising. Regulation of FAK activity is well understood and involves the phosphorylation of its residues, especially tyrosine phosphorylation (2), which controls a number of signaling cascade events involved in adhesion regulation, migration, and invasive phenotype as well as in growth and survival signaling. Although several studies with cell lines have shown the ability of FAK to regulate cell motility and invasion, these mechanisms cannot be wholly attributed to its kinase activity (3). Thus, it is important to determine the regulatory events controlling FAK activity and their role in tumor development or maintenance of the existing tumor phenotype.

Peptide growth factors and cytokines depend on mitogen-activated protein kinase (MAPK) signal transduction cascades for controlling cell growth and survival (4, 5). Several indirect lines of evidence suggest important roles for MAPK pathways in prostate biology (6, 7) and also show a critical role for MAPK signal transduction pathways in malignant transformation (8, 9). A marked increase in extracellular regulated kinase (ERK) activity was shown in human prostate adenocarcinomas compared with normal controls (10). MAPK signaling cascades could represent a final common pathway of many growth stimulators implicated in prostate cancer (11), and several studies indicate that FAK signaling through the ERK-MAPK pathway is required to maintain growth, at least in the case of cancer cells (12). Moreover, activated ERK localizes to focal adhesions but the functional significance of this localization has not been well understood. A recent study by Vomastek et al. (13) revealed that FAK regulates active ERK localization to focal adhesion, suggesting the existence of an important cross-talk between the FAK and ERK. In view of this cross-talk, we observed here for the first time that phosphorylation of FAK is regulated by ERK activity in prostate cancer cell lines. We found that FAK expression is increased and activated only in suspension culture. ERK also plays an important role in the regulation of FAK in prostate cancer cell line PC3, as inhibition of ERK resulted in a decrease in FAK promoter activity, expression, and tyrosine phosphorylation. Inhibition of ERKs and FAK with their respective inhibitors or small interfering RNAs (siRNA) resulted in a sharp decrease in anchorage-independent growth and invasion through Matrigel, but had little effect on cell proliferation.

Observations made in our study suggest that regulation of FAK through the ERK-MAPK pathway might control invasive and clonogenic phenotypes of the androgen-independent prostate cancer cell line, and may thus be a useful therapeutic target for cancer treatment.

FAK Is Required for Clonogenic Activity in PC3 Cells

FAK is an important intermediary of growth factor signaling serving in the maintenance of tumor phenotype. Hence, to determine the effects of FAK on colony formation, we tested the clonogenicity of PC3 cells treated with siRNA directed against FAK and with cells overexpressing FAK. Colony formation was significantly suppressed in FAK-depleted cells (Fig. 1A), whereas it was stimulated in cells transfected with HA-tagged wild-type FAK (Fig. 1C). Figure 1B shows reduced FAK expression in cells from representative experiments in which siRNA was used to inhibit FAK expression in PC3 cells. Cells transfected with an expression vector carrying HA-tagged FAK show increased the expression of full-length FAK protein as determined by Western blotting (Fig. 1D). These results suggest that increased FAK activity is an essential factor for the colony formation in PC3 cells.

FIGURE 1.

Increased FAK activity is required for clonogenic activity. A and B. Cells were transfected with anti-FAK or control siRNA. Seventy-two hours later, cells were harvested and subjected to colony formation assay in 0.3% soft agar in complete growth medium (A) or processed for Western analysis with an antibody to FAK and GAPDH as a loading control (B). Cells were transfected with an HA-tagged FAK expression construct or vector and subjected to colony formation assay as above (C) or processed for Western analysis (D) with antibodies against total FAK, HA tag, and GAPDH to verify overexpression. All experiments were repeated at least twice. *, P < 0.05, compared with respective controls.

FIGURE 1.

Increased FAK activity is required for clonogenic activity. A and B. Cells were transfected with anti-FAK or control siRNA. Seventy-two hours later, cells were harvested and subjected to colony formation assay in 0.3% soft agar in complete growth medium (A) or processed for Western analysis with an antibody to FAK and GAPDH as a loading control (B). Cells were transfected with an HA-tagged FAK expression construct or vector and subjected to colony formation assay as above (C) or processed for Western analysis (D) with antibodies against total FAK, HA tag, and GAPDH to verify overexpression. All experiments were repeated at least twice. *, P < 0.05, compared with respective controls.

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FAK Is Activated and Its Expression Is Increased in Suspension Culture and Suppression of ERK1/2 Results in Decreased FAK Expression and Phosphorylation in Prostate Cancer Cells

FAK is known to activate the ERK pathway, which has been implicated in cell motility and invasiveness in other systems (14). Therefore, initial experiments were designed to examine the amount of stable protein and tyrosine phosphorylation status of FAK in PC-3 cells. Androgen-independent prostate cancer cells can be adapted to suspension cultures and are used as a model for metastatic growth of prostate cancer (15). In our experiments, we used a layer of 0.5% agarose made with complete medium as a substratum to prevent adherence and the cells were maintained in suspension in a liquid medium.

To determine the levels of FAK and FAK phosphorylation in PC3 and DU145 cells, we immunoprecipitated FAK protein from cells in adherent and suspension cultures and performed Western blot analysis against total FAK as well as phosphorylated FAK. In attached cells, the basal level of FAK tyrosine phosphorylation was too low to be detected in our assay. However, we observed increased amounts of FAK proteins in PC3 and DU145 cells grown in suspension for 24 and 48 hours, respectively (Fig. 2A and C). In addition to the increase in total protein, we also observed increased FAK phosphorylation in these cells grown in suspension (Fig. 2A-D). For studies on the effect of ERK on regulating FAK levels and phosphorylation, we immunoprecipitated FAK from control and ERK1/ERK2 siRNA knockdown cells, plated in adherent cultures as well as cells which had been replated under nonadherent conditions (on 0.5% agarose) for various times. The results are shown in Fig. 2A-E. In nonadherent cells in which ERK1/2 was knocked down, levels of FAK are decreased. Moreover, the band densitometry data show a highly significant, striking decrease in phosphorylated FAK in both PC3 and DU145 cells expressing ERK1/ERK2 siRNA (Fig. 2B and D). These results show the activation of FAK under nonadherent culture conditions, and a critical requirement of ERK1/2 in this process.

FIGURE 2.

FAK is increased and active in PC3 and DU145 cells in suspension culture and is regulated by ERK. A and C. Prostate cancer cells (A, PC3 cells; C, DU145 cells) were transfected with siRNA directed against ERK1, ERK2, or both simultaneously at 20 nmol/L final concentration, and FAK was immunoprecipitated after incubation in suspension for various times. After processing for Western analysis, immunoprecipitates were probed first with an antibody for total phosphotyrosine followed by an antibody for total FAK. B and D. Quantitative analysis of FAK activity in PC3 and DU145 cells presented in A and C, respectively. E. Prostate cancer cells (PC3, left; DU145, right) were transfected with siRNA directed against ERK1/ERK2 and replated as in A and C, and harvested at 72 h after replating. After processing for Western analysis, the blot was probed with antibodies against FAK, ERK1/2, and GAPDH. F. Prostate cancer cells (PC3, left; DU145, right) were transfected and replated as in A and C and harvested 72 h after replating. Immunoprecipitates and supernatants were probed in succession with antibodies for total phosphotyrosine, phosphorylated Y576, Y577, and total FAK. G. PC3 (left) and DU145 cells (right) were grown in suspension culture for 48 h and total cell lysates were analyzed for ERK1/2, phosphorylated ERK1/2, and GAPDH by Western blotting. Representative blots from at least three independent experiments involving multiple transfections in each.

FIGURE 2.

FAK is increased and active in PC3 and DU145 cells in suspension culture and is regulated by ERK. A and C. Prostate cancer cells (A, PC3 cells; C, DU145 cells) were transfected with siRNA directed against ERK1, ERK2, or both simultaneously at 20 nmol/L final concentration, and FAK was immunoprecipitated after incubation in suspension for various times. After processing for Western analysis, immunoprecipitates were probed first with an antibody for total phosphotyrosine followed by an antibody for total FAK. B and D. Quantitative analysis of FAK activity in PC3 and DU145 cells presented in A and C, respectively. E. Prostate cancer cells (PC3, left; DU145, right) were transfected with siRNA directed against ERK1/ERK2 and replated as in A and C, and harvested at 72 h after replating. After processing for Western analysis, the blot was probed with antibodies against FAK, ERK1/2, and GAPDH. F. Prostate cancer cells (PC3, left; DU145, right) were transfected and replated as in A and C and harvested 72 h after replating. Immunoprecipitates and supernatants were probed in succession with antibodies for total phosphotyrosine, phosphorylated Y576, Y577, and total FAK. G. PC3 (left) and DU145 cells (right) were grown in suspension culture for 48 h and total cell lysates were analyzed for ERK1/2, phosphorylated ERK1/2, and GAPDH by Western blotting. Representative blots from at least three independent experiments involving multiple transfections in each.

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Because FAK can undergo tyrosine phosphorylation at multiple sites, we examined for changes in tyrosine phosphorylation at multiple sites in FAK protein. Results presented in Fig. 2F indicate that under nonadherent conditions, the tyrosine phosphorylation status of FAK is greatly reduced in ERK-suppressed cells, and specifically at tyrosine 576 and 577. The results (Fig. 2F) also show that FAK immunoprecipitation is quantitative, supporting the notion that FAK levels in immunoprecipitates accurately reflect cellular concentrations.

Because we observed that ERK plays an important role in regulating the activity of FAK under nonadherent culture conditions, it was important to determine the activation of ERK under these conditions. Results presented in Fig. 2G show increased phosphorylation of ERK1/2 when prostate cancer cells (PC3, left; DU145, right) were grown in suspension culture. Taken together, these results suggest that increased FAK activity in PC3 and DU145 cells under nonadherent conditions is a result of ERK activation.

ERK-MAPK Is Required for Expression of FAK in Prostate Cancer Cells

To address the mechanism of ERK-mediated regulation of FAK expression, we cotransfected PC3 cells with one of two FAK promoter-luciferase constructs and the inactive versions of ERK1/ERK2 or its vector control. In order to control for transfection efficiency and possible nonspecific effects of ERK inhibition on transcriptional or translational events, we included Renilla luciferase as a standard (results shown in Fig. 3A). The inclusion of inactivated ERK constructs reduced normalized FAK reporter activity by 30% to 40%, suggesting that the observed decrease in FAK protein levels in ERK-depleted cells could be due to decreased transcriptional activity. DU145 cells transfected with the inactivated forms of ERK in a similar manner showed reduced FAK promoter activity comparable to PC3 cells (Fig. 3A, bottom). Cotransfection of siRNA targeted against ERK1, ERK2, or in combination, with FAK promoter-luciferase constructs also significantly reduced the promoter activity both in PC3 (Fig. 3B, top) and DU145 (Fig. 3B, bottom). In this case, the degree of inhibition seems to correlate well with the relative amounts of ERK1 and ERK2 in PC3 cells. Taken together, these data show that the ERK1/2 MAPK pathway regulates expression as well as activity of FAK.

FIGURE 3.

Inhibition of ERK1/ERK2 function by siRNA resulted in decreased activity of FAK. A. Prostate cancer cells (PC3, top; DU145, bottom) were cotransfected with Renilla luciferase, one of two FAK promoter-firefly luciferase reporter constructs (p1173, p564), and either a combination of CMV promoter-driven ERK1 and ERK2 dominant-negative constructs (p1173 or p564 + ERK DN) or an equal amount of CMV-pcDNA3 vector. Firefly luciferase was normalized to Renilla luciferase and cellular protein. Columns, mean from two independent experiments involving multiple transfections in each; *, P < 0.05, from respective controls. B. Prostate cancer cells (PC3, top; DU145, bottom) were cotransfected with the indicated siRNA, p1173, and Renilla luciferase. Forty-eight hours later, cells were harvested as in A and Firefly luciferase was normalized to Renilla luciferase and cellular protein. Columns, mean from three independent experiments involving multiple transfections in each; *, P < 0.05, compared with respective controls.

FIGURE 3.

Inhibition of ERK1/ERK2 function by siRNA resulted in decreased activity of FAK. A. Prostate cancer cells (PC3, top; DU145, bottom) were cotransfected with Renilla luciferase, one of two FAK promoter-firefly luciferase reporter constructs (p1173, p564), and either a combination of CMV promoter-driven ERK1 and ERK2 dominant-negative constructs (p1173 or p564 + ERK DN) or an equal amount of CMV-pcDNA3 vector. Firefly luciferase was normalized to Renilla luciferase and cellular protein. Columns, mean from two independent experiments involving multiple transfections in each; *, P < 0.05, from respective controls. B. Prostate cancer cells (PC3, top; DU145, bottom) were cotransfected with the indicated siRNA, p1173, and Renilla luciferase. Forty-eight hours later, cells were harvested as in A and Firefly luciferase was normalized to Renilla luciferase and cellular protein. Columns, mean from three independent experiments involving multiple transfections in each; *, P < 0.05, compared with respective controls.

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ERK1/2 MAPK Plays an Essential Role in Regulating Clonogenic and Invasive Activity of Prostate Cancer Cell Lines

To assess the effects of MEK1/2 (which is an upstream activator of ERK1/2) inhibition on colony-forming ability, PC3 and DU145 cells were plated on a layer of 0.3% agarose in growth medium and exposed to MEK1/2 inhibitor, PD98059 (25 and 50 μmol/L) for 15 days. Incubation with 50 μmol/L of PD98059 suppressed colony formation by 85%, and by 50% at 25 μmol/L in PC3 cells (Fig. 4A), whereas 50 μmol/L of PD98059 suppressed colony formation by 75% in DU145 cells (Fig. 4C). Similar results were obtained with cells in which siRNA against ERK1/ERK2 was used and the results indicated that both ERK1 and ERK2 alone or in combination play a significant role in the colony formation of prostate cancer cell lines, PC3 (Fig. 4B) and DU145 (Fig. 4D).

FIGURE 4.

ERK1/ERK2 activity is essential for clonogenic activity in prostate cancer cell lines. PC3 (A and B) and DU145 (C and D) cells were treated with 25 and 50 μmol/L of PD98059 or transfected with siRNA directed against ERK1, ERK2, or both simultaneously at 20 nmol/L final concentration. Treated cells were then used for colony formation in soft agar as described in Materials and Methods. Each experiment was repeated at least thrice. *, P < 0.05, compared with respective controls.

FIGURE 4.

ERK1/ERK2 activity is essential for clonogenic activity in prostate cancer cell lines. PC3 (A and B) and DU145 (C and D) cells were treated with 25 and 50 μmol/L of PD98059 or transfected with siRNA directed against ERK1, ERK2, or both simultaneously at 20 nmol/L final concentration. Treated cells were then used for colony formation in soft agar as described in Materials and Methods. Each experiment was repeated at least thrice. *, P < 0.05, compared with respective controls.

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Another behavioral feature associated with tumor metastasis is an invasive phenotype of tumor cells. To test the role of the ERK1/2 MAPK signal transduction pathway in invasive behavior, PC3 and DU145 cells were plated in Matrigel-coated invasion chambers in the presence or absence of 50 μmol/L of PD98059 and exposed to a chemoattractant (1-10% gradient of fetal bovine serum). PD98059 dramatically decreased the ability of these cells to invade through a Matrigel matrix (Fig. 5A and C). Similar results were obtained with cells in which ERK was inhibited using siRNA against ERK1/ERK2, and taken together, indicate that both ERK1 and ERK2 alone or in combination contribute to the invasion of prostate cancer cell through Matrigel (Fig. 5B and D).

FIGURE 5.

ERK1/ERK2-dependent cell invasion and MMP-9 expression/activity in prostate cancer cell lines. PC3 (A and B) and DU145 (C and D) cells were treated with PD98059 or transfected with siRNA directed against ERK1, ERK2, or both simultaneously at 20 nmol/L final concentration. Treated cells were then allowed for the invasion through Matrigel as described in Materials and Methods. PC3 (E) and DU145 (F) invasion assay was done after anti–MMP-9/MMP-2 antibody addition as described in Materials and Methods. G. cDNA prepared from RNA isolated from prostate cancer cells (PC3 cells, left; DU145 cells, right) in the presence or absence of PD98059 for 24 or 48 h was subjected to reverse transcription-PCR with primers for both GAPDH and MMP-9. H. Gelatin zymogram for MMP-9 activity in PC3 cells after PD98059 treatment for 48 h. PC, positive control (human MMP-9 protein; Chemicon International). The image has been converted for clarity. I. Gelatin zymogram for MMP-9 activity after ERK1/ERK2 siRNA treatment for 72 h. PC, positive control (conditioning medium from bladder cancer cell, HTB9). The image has been converted for clarity. Both zymogram experiments were repeated twice and images from a representative experiment were used. *, P < 0.05, compared with respective controls.

FIGURE 5.

ERK1/ERK2-dependent cell invasion and MMP-9 expression/activity in prostate cancer cell lines. PC3 (A and B) and DU145 (C and D) cells were treated with PD98059 or transfected with siRNA directed against ERK1, ERK2, or both simultaneously at 20 nmol/L final concentration. Treated cells were then allowed for the invasion through Matrigel as described in Materials and Methods. PC3 (E) and DU145 (F) invasion assay was done after anti–MMP-9/MMP-2 antibody addition as described in Materials and Methods. G. cDNA prepared from RNA isolated from prostate cancer cells (PC3 cells, left; DU145 cells, right) in the presence or absence of PD98059 for 24 or 48 h was subjected to reverse transcription-PCR with primers for both GAPDH and MMP-9. H. Gelatin zymogram for MMP-9 activity in PC3 cells after PD98059 treatment for 48 h. PC, positive control (human MMP-9 protein; Chemicon International). The image has been converted for clarity. I. Gelatin zymogram for MMP-9 activity after ERK1/ERK2 siRNA treatment for 72 h. PC, positive control (conditioning medium from bladder cancer cell, HTB9). The image has been converted for clarity. Both zymogram experiments were repeated twice and images from a representative experiment were used. *, P < 0.05, compared with respective controls.

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Matrix metalloproteinases (MMP) are a family of enzymes whose function primarily relates to the degradation of extracellular matrix proteins, and which are necessary for cell invasion. Recent data have shown that MAPK plays a major role in inducing proteolytic enzymes that degrade the basement membrane (16). To determine the role of MMPs in cell invasion in our study, we included MMP-2 and MMP-9 antibodies along with the cells during the invasion assay. Anti–MMP-9 antibody inhibited PC3 and DU145 cell invasion by ∼60% to 70% compared with control IgG (Fig. 5E and F). We did not find a significant effect of MMP-2 antibody on cell invasion. Thus, increased production of MMP-9 by prostate cancer cells might be associated with increased invasion. Moreover, as can be seen in Fig. 5G, 50 μmol/L of PD98059 down-regulates the MMP-9 transcripts over a period of 24 hours. Zymography done with conditioned medium from PC3 cells grown in the presence of 50 μmol/L of PD98059 or cells transfected with ERK1/2 siRNA also indicates reduced MMP-9 activity in prostate cancer cell lines (Fig. 5H and I). Taken together, these results directly implicate the role of ERK1/2 MAPK pathway in clonogenic activity and maintenance of the invasive phenotype in prostate cancer cell lines.

ERK1/2 MAPK Signaling Pathway Participates in But Is Not Essential for the Growth and Survival of PC3 Cells, and Suppression of ERK1/2 Does Not Result in Anoikis of PC3 Cells

Earlier in our study, we observed that MEK1/2 inhibitor PD98059 or ERK1/ERK2 siRNA affected colony formation and invasion of prostate cancer cell lines PC3 and DU145. In an effort to rule out that these observed differences were not a result of changes in cell growth or survival, we determined thymidine incorporation and the status of apoptosis markers in these cells.

The MAPK module is composed of three protein kinases, p42/p44MAPK (ERK1/2), p38MAPK, and JNK cascades. Both p42 and p44MAPK are activated by dual phosphorylation on a threonine and a tyrosine residue, achieved by the dual-specificity kinase MKK1/2. Whereas MKK1/2 remains permanently in the cytoplasm, p42/p44MAPK are re-localized from the cytoplasm to the nucleus upon stimulation (17). Many of the targets of the ERKs are transcription factors and proteins responsible for entry into the cell cycle. In this context, Fig. 6A illustrates that treatment of PC3 cells with 25 μmol/L of PD98059 results in a dramatic reduction in the activity of both ERK1 and ERK2, whereas the levels of total p42 and p44 remain unchanged. The effect of PD98059 is maximal within 15 minutes of treatment and remains for as long as 30 minutes. Although cell numbers (data not shown) and 3H-thymidine incorporation (Fig. 6B) were consistently lower in cells treated with MEK inhibitors, the reduction in 3H-thymidine incorporation is minor (∼20%). To assess treatment specificity, cells were preincubated with a mixture of antisense oligonucleotides targeted to p42 and p44 or with a scrambled sequence, followed by the addition of 3H-thymidine. Even though the antisense oligonucleotides reduced the amount of total ERK1/2 available in the cells (Fig. 6C), the observed reduction in 3H-thymidine incorporation parallel those obtained with MEK1/2 inhibitors (Fig. 6D).

FIGURE 6.

Effects of ERK inhibitors on PC3 cell proliferation. A. PC3 cells were incubated overnight in serum-free medium with the indicated concentrations of PD98059. FCS was then added to a final concentration of 10% and cells incubated for the indicated times. Western blots of cell lysates were probed with an antibody specific to phosphorylated ERK1/2, stripped, and reprobed with an antibody recognizing total ERK1/2 protein. B. PC3 cells were plated at a low density and allowed to proliferate in the presence or absence of the indicated concentrations of PD98059 for 7 days. 3H-Thymidine was introduced into the cultures at the indicated times and cultures incubated for an additional 6 h. Cells were trypsinized, counted, and trichloroacetic acid–precipitable radioactivity was determined from an equal number of cells. C. PC3 cells were transfected with the indicated concentrations of scrambled (Scr) or antisense oligonucleotides (AS) directed against ERK1/2. After 24 h of incubation in serum-free medium, FCS was added to 10% and cells processed as in A above. D. PC3 cells were transfected with the indicated concentrations of scrambled (Scr) or antisense oligonucleotides (AS) directed against ERK1/2; 48 h after transfection, cells were incubated with 3H-thymidine for 6 h and processed as in A above. E. PC3 cells were transfected with the indicated siRNA and incubated for 48 to 72 h. At the end of this period, cells were trypsinized and replated on a substrate of 0.5% agarose for an additional 48 h. Cells were then processed for Western blotting and probed for poly(ADP-ribose) polymerase (PARP).

FIGURE 6.

Effects of ERK inhibitors on PC3 cell proliferation. A. PC3 cells were incubated overnight in serum-free medium with the indicated concentrations of PD98059. FCS was then added to a final concentration of 10% and cells incubated for the indicated times. Western blots of cell lysates were probed with an antibody specific to phosphorylated ERK1/2, stripped, and reprobed with an antibody recognizing total ERK1/2 protein. B. PC3 cells were plated at a low density and allowed to proliferate in the presence or absence of the indicated concentrations of PD98059 for 7 days. 3H-Thymidine was introduced into the cultures at the indicated times and cultures incubated for an additional 6 h. Cells were trypsinized, counted, and trichloroacetic acid–precipitable radioactivity was determined from an equal number of cells. C. PC3 cells were transfected with the indicated concentrations of scrambled (Scr) or antisense oligonucleotides (AS) directed against ERK1/2. After 24 h of incubation in serum-free medium, FCS was added to 10% and cells processed as in A above. D. PC3 cells were transfected with the indicated concentrations of scrambled (Scr) or antisense oligonucleotides (AS) directed against ERK1/2; 48 h after transfection, cells were incubated with 3H-thymidine for 6 h and processed as in A above. E. PC3 cells were transfected with the indicated siRNA and incubated for 48 to 72 h. At the end of this period, cells were trypsinized and replated on a substrate of 0.5% agarose for an additional 48 h. Cells were then processed for Western blotting and probed for poly(ADP-ribose) polymerase (PARP).

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Nonmalignant cells tend to undergo anoikis (suspension-induced cell death) when detached from their substratum, although malignant cells develop resistance (18). Because anoikis can be associated with ERK inhibition (19), we looked for markers of anoikis in PC3 cells treated with siRNA to ERK1/2 for 48 to 72 hours. Figure 6E suggests that ERK knockdown results in specific albeit modest cleavage of poly(ADP-ribose) polymerase after 48 hours; however, the amount of cleavage does not increase with further incubation times up to 96 hours and most cells remain viable using trypan blue assay (data not shown). Such a low level of apoptosis seems unlikely to account for cell death as a reason for the observed ERK-dependent decrease in colony-forming ability.

Taken together, these results suggest that ERK1/2 inhibition in PC3 cells either by siRNA or by MEK inhibitors does not result in a significant loss of cell viability. Therefore, the observed changes in clonogenicity and invasion may not be a reflection of suspension-induced prostate cancer cell death.

There is considerable interest and effort in the development of tyrosine kinase inhibitors as cancer therapeutics. FAK is a nonreceptor tyrosine kinase that provides signaling function at sites of integrin adhesion, which are involved in the regulation of cell migration/invasion (20). ERK activity is also known to be intimately involved in actin polymerization, and hence, cell motility (21, 22). Generally, increased FAK expression is associated with tumor development and poor clinical outcome, which highlights FAK as a useful therapeutic target. We found that decreased FAK activity in androgen-independent prostate cancer cells leads to impaired colony formation in soft agar (Fig. 1). These findings are in agreement with studies in other cancer cell types (14). Our data indicate that FAK protein expression, as well as its phosphorylation, is sensitive to ERK levels (Fig. 2A-G). Although many studies (23) have concentrated on the contributions of FAK towards ERK phosphorylation and activity, the regulation of FAK expression by ERK1/2 proteins is a novel observation (Fig. 2A-G) in our study. However, because knockdown of ERK resulted in decreased levels of FAK, both molecules may work together to promote the survival of cells in suspension and thus are required for clonogenic activity. The promoter region of FAK contains activator protein-1, activator protein-2, and Sp1 binding sites among others (24), suggesting that transcriptional regulation of FAK by ERKs might occur, and indeed, our results indicate that ERK knockdown or dominant-negative ERKs suppress FAK promoter activity in a reporter assay (Fig. 3). Increased FAK phosphorylation as a result of cell detachment is also novel and suggests a mechanism other than integrin engagement, perhaps through sustained Ras-independent ERK activity, as has been suggested for other anoikis-resistant cells (ref. 25; cf. Fig. 6). Recently, a role for a FAK-activating protein was suggested on the basis of structural evidence (26). Clearly, additional studies are needed to investigate these mechanisms.

ERK activity is also known to be intimately involved in actin polymerization, and hence, cell motility (21, 22). Inappropriate signaling of the Ras-Raf-MEK-ERK pathway is associated with many human tumors, and pathway mutations conferring constitutive activity are extremely common in certain tumor types (27). Although such Ras or Raf mutants are rare in human prostate tumors (7, 28), other data indicate altered regulation of this pathway in cancerous cells (29). Increased ERK activation has been correlated with human prostate adenocarcinoma (10), but it may be more characteristic of early lesions rather than of late stage cancer (9). Ras signaling is implicated in tumor cell invasion and metastasis (30). NIH3T3 cells expressing constitutively active Ras are tumorigenic in nude mice, but are metastatic only if Raf and MEK are activated downstream (31). Several studies have associated MMP expression with MEK activation or ERK-activated transcription factors (32, 33). In agreement, our results suggest that MEK inhibition suppresses anchorage-independent growth (Fig. 4A-D) and invasion (Fig. 5A-D) of prostate cancer cells. The resultant suppression of invasion by MEK inhibitor was due to decreased MMP-9 transcription and activity (Fig. 5G-I) as the addition of MMP-9 antibody decreased invasion (Fig. 5E and F). Thus, invasion and MMP-9 are interdependent phenomena in case of prostate cancer cell lines. In support, changes in the clonogenicity and invasion of PC3 cells are not related to the loss of growth and survival of the cells as a result of treatment with MEK inhibitor (Fig. 6A-D). This emphasizes our observations that the changes are a direct result of interactions between FAK and ERK and not as an incidental loss of cell number due to ERK inhibition.

Phosphorylation of the proapoptotic BH3 protein BimEL by ERK results in its degradation and inability to interact with Bax (34), a mechanism known to be partly responsible for anoikis resistance in breast cancer cell lines (25). Although increased sensitivity to anoikis is an attractive explanation for decreased colony formation in ERK-depleted cells, little poly(ADP-ribose) polymerase cleavage was evident in these cells for at least 96 hours after placing cells in suspension, suggesting that apoptosis is, at least, not an early event (Fig. 6E). In our experiments, caveolin-1, a scaffolding protein reported to be a tumor promoter in prostate cancer (LNCaP) cells and negatively regulated by ERK1/2 (35), showed no changes in expression following ERK depletion (data not shown).

In summary, our results show a critical role for FAK in clonogenic activity, an important role for ERK1/2 signaling cascade in clonogenic and invasive phenotypes, and regulation of FAK expression and activation by ERK in androgen-independent prostate cancer cells. Taken together, these results suggest that these cascades may offer novel molecular targets to clinical outcome, and particularly, in the response to therapeutic regimens.

Cell Line and Reagents

PC3 and DU145 (36) cells, tumorigenic, androgen-independent prostate cancer cell lines isolated from bone and brain metastases, respectively, were purchased from the American Type Culture Collection and maintained in a 1:1 mixture of DMEM and Ham's F12, supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2. PD98059 (EMD Biosciences) was dissolved in DMSO as 50 mmol/L stock solutions. Antibodies directed against ERK1/2, phosphorylated ERK1/2, and poly(ADP-ribose) polymerase were from Cell Signaling Technology. Antibody against glyceraldehyde-3′-phosphate dehydrogenase (GAPDH) was from Chemicon. Anti-FLAG M2 antibody was from Sigma-Aldrich. Anti-human MMP-2 and MMP-9 antibodies were purchased from Santa Cruz Biotechnology, and control IgG mouse antibody was from eBioscience.

Plasmids and Oligonucleotides

Antisense oligonucleotides directed against ERK1/2 (5′-GCCGCCGCCGCCGCCAT-3′), or scrambled sequence (5′-CGCGCGCTCGCGCACCC-3′) were transfected with Lipo TAXI transfection reagent (Stratagene) according to the instructions of the manufacturer. siRNAs directed against ERK1 and ERK2 (37) were obtained from Integrated DNA Technologies and were introduced into cells with HiPerFect (Qiagen) at 20 nm final concentration according to the recommendations of the manufacturer. siRNA directed against FAK was obtained from Ambion, Inc. Control siRNAs used were either directed against lamin A/C (Dharmacon) or un-BLASTable sequence (Ambion). Boyden chamber or soft agar assays were initiated 48 h after transfection.

Electrophoresis, Western Blotting, and 3H-Thymidine Incorporation Assays

Assay were done as described (38). Quantitation of crystal violet–stained cells was done by drying stained plates, solubilizing the stain in 2-ethoxyethanol, followed by absorbance determination at 570 nm.

Immunoprecipitation

FAK was immunoprecipitated from cell lysates by overnight incubation at 4°C with 2 μg of antibody (polyclonal C-20, Santa Cruz Biotechnology) prebound to protein A/G beads (Oncogene Research Products, EMD). Following Western blotting, tyrosine-phosphorylated FAK was detected with 4G10 monoclonal antibody (Upstate, Millipore). Blots were then stripped and reprobed with polyclonal antibody to FAK and/or Y576 and Y577 phosphorylated FAK (Cell Signaling).

Measurement of Clonogenic Potential

Assays for growth in soft agar were done as described by Rizzino (39). Colony-forming and invasiveness assays were quantified using the Gel Doc system and Quantity One software (Bio-Rad). Results shown are means of at least two independent assays done in quadruplicate. ANOVA was used for statistical analysis.

Plasmid and Reporter Assay

The p1173 and p564 FAK promoter-luciferase reporter constructs was a kind gift from Drs. V. Golubovskaya and W. Cance (University of Florida). PC3 cells were transiently transfected with 20 nm siRNA against ERK1/ERK2 and 0.5 μg of luciferase reporter vector along with 10 ng of Renilla luciferase expression plasmid using Effectene transfection reagent (Qiagen) according to the instructions of the manufacturer. Luciferase activity was measured using the dual luciferase kit (Promega Corporation) with Monolight 2010 Luminometer (Analytical Luminescence Laboratory).

Matrigel Invasion Assay

The in vitro invasion assays were carried out in BD BioCoat Matrigel chambers (Transwell, Corning) according to Repesh et al. (40). PD98059 was added as indicated (or transfected cells were used) and the chambers incubated for 24 h. In another set of experiments, 100 μg/mL of either anti-human MMP-2 or anti-human MMP-9 IgG or mouse IgG were added in the upper compartment of the cells (41). For analysis, the inside of the chamber was swabbed to remove nonmigratory cells. The membrane was then rinsed with PBS, cells fixed and stained with 0.4% crystal violet in 0.2 mol/L of citric acid for 20 min, and rinsed extensively with water. Membranes were excised, dried, mounted on slides, photographed, and counted. Results shown are means of at least two independent assays done in triplicate. Statistics were determined by ANOVA.

Reverse Transcription-PCR

Total RNA was extracted using RNEasy mini kit (Qiagen). RNA (1 μg) was used to prepare cDNA using iScript second-strand cDNA synthesis kit (Bio-Rad Laboratories). Synthesized cDNA (50 ng) was used to amplify MMP-9 or GAPDH mRNA using human gene–specific primers.

MMP-Zymography

Cells in semiconfluent cultures (∼80% confluent) were placed into serum-free medium, treated with PD98059 or ERK1/ERK2 siRNA or both (after 24 h of transfection, old medium was replaced with serum-free medium) as indicated, cultured for an additional 48 h, the conditioned medium was concentrated (Microcon YM-10 centrifugal filter; Millipore), and separated on 7% SDS polyacrylamide gel containing 0.1% (w/v) gelatin under nonreducing conditions. Zymogram for MMP-9 was done according to Bernhard and Muschel (42). Pure human MMP-9 (50 ng) protein or conditioned medium from bladder cancer cells (HTB9) were used as positive controls.

No potential conflicts of interest were disclosed.

Grant support: NIH/NCI-P20 CA103680 (H. Koul, Pilot Project PI), NIH RO1-54084 (H. Koul), and University of Colorado Denver, School of Medicine and Department of Surgery Academic Enrichment Funds (P. Maroni and H. Koul).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

T.R. Johnson, L. Khandrika, and B. Kumar contributed equally to this work.

The construct expressing HA-tagged FAK under the control of a CMV promoter was a kind gift from Dr. J.L. Guan (Departments of Internal Medicine-MMG and Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI). We are very grateful to Drs. V. Golubovskaya (Department of Surgery, School of Medicine, University of Florida, Gainesville, FL) and W. Cance (University of Florida, Miami, FL) for their gift of the p1173 and p564 FAK promoter-luciferase reporter constructs, and to Dr. M. Cobb (Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX) for the gift of dominant-negative ERK1/2 constructs.

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