The Mitogen-activated Protein (MAP) Kinases p38 and Extracellular Signal-regulated Kinase (ERK) Are Involved in Hepatocyte-mediated Phenotypic Switching in Prostate Cancer Cells*

Background: Epithelial mesenchymal phenotypic switching enables cancers to seed and survive in metastatic sites. Results: Both p38 and ERK1/2 MAP kinases need to be inhibited to allow for an epithelial reversion but activated to provide survival advantage in the face of chemotherapy. Conclusion: Distinct p38/ERK signaling outcomes are involved in hepatocytes-mediated MErT and tumor cells survival. Significance: Provides insights in understanding approaches to p38α or ERK1/2 activity regulation for cancer therapy. The greatest challenge for the seeding of cancer in metastatic sites is integration into the ectopic microenvironment despite the lack of an orthotopic supportive environment and presence of pro-death signals concomitant with a localized “foreign-body” inflammatory response. In this metastatic location, many carcinoma cells display a reversion of the epithelial-to-mesenchymal transition that marks dissemination in the primary tumor mass. This mesenchymal to epithelial reverting transition (MErT) is thought to help seeding and colonization by protecting against cell death. We have previously shown that hepatocyte coculture induces the re-expression of E-cadherin via abrogation of autocrine EGFR signaling pathway in prostate cancer (PCa) cells and that this confers a survival advantage. Herein, we show that hepatocytes educate PCa to undergo MErT by modulating the activity of p38 and ERK1/2. Hepatocytes inhibited p38 and ERK1/2 activity in prostate cancer cells, which allowed E-cadherin re-expression. Introduction of constitutively active MEK6 and MEK1 to DU145 cells cocultured with hepatocytes abrogated E-cadherin re-expression. At least a partial phenotypic reversion can be achieved by suppression of p38 and ERK1/2 activation in DU145 cells even in the absence of hepatocytes. Interestingly, these mitogen-activated protein kinase activities were also triggered by re-expressed E-cadherin leading to p38 and ERK1/2 activity in PCa cells; these signals provide protection to PCa cells upon challenge with chemotherapy and cell death-inducing cytokines. We propose that distinct p38/ERK pathways are related to E-cadherin levels and function downstream of E-cadherin allowing, respectively, for hepatocyte-mediated MErT and tumor cell survival in the face of death signals.

The greatest challenge for the seeding of cancer in metastatic sites is integration into the ectopic microenvironment despite the lack of an orthotopic supportive environment and presence of pro-death signals concomitant with a localized "foreignbody" inflammatory response. In this metastatic location, many carcinoma cells display a reversion of the epithelial-to-mesenchymal transition that marks dissemination in the primary tumor mass. This mesenchymal to epithelial reverting transition (MErT) is thought to help seeding and colonization by protecting against cell death. We have previously shown that hepatocyte coculture induces the re-expression of E-cadherin via abrogation of autocrine EGFR signaling pathway in prostate cancer (PCa) cells and that this confers a survival advantage. Herein, we show that hepatocytes educate PCa to undergo MErT by modulating the activity of p38 and ERK1/2. Hepatocytes inhibited p38 and ERK1/2 activity in prostate cancer cells, which allowed E-cadherin re-expression. Introduction of constitutively active MEK6 and MEK1 to DU145 cells cocultured with hepatocytes abrogated E-cadherin re-expression. At least a partial phenotypic reversion can be achieved by suppression of p38 and ERK1/2 activation in DU145 cells even in the absence of hepatocytes. Interestingly, these mitogen-activated protein kinase activities were also triggered by re-expressed E-cadherin leading to p38 and ERK1/2 activity in PCa cells; these signals provide protection to PCa cells upon challenge with chemotherapy and cell deathinducing cytokines. We propose that distinct p38/ERK pathways are related to E-cadherin levels and function downstream of E-cadherin allowing, respectively, for hepatocyte-mediated MErT and tumor cell survival in the face of death signals.
Metastasis is the main cause of death in most cancers. It has been suggested that this complex metastatic cascade could be conceptually organized and simplified into two major phases: (i) physical translocation of a cancer cell from the primary tumor to the microenvironment of a distant tissue and then (ii) colonization (1). Therefore, understanding the many molecules and processes leading to successful colonization may lead to effective therapies for patients with already established metastases. It is established that successful dissemination results from a series of phenotypic switches that are regulated by the microenvironment (2,3). Cancer cells down-regulate E-cadherin to allow for translocation in a process known as epithelialto-mesenchymal transition (EMT) 2 (1). However, survival and colonization in the distant metastatic site, where there is a lack of orthotopic supportive environment and the presence of prodeath signals, requires a reversion of the phenotype, a mesenchymal-to-epithelial reverting transition (MErT) to reside among ectopic tissue epithelial cells. This is strongly supported by the accumulating evidence that show re-express E-cadherin in metastatic tumors (4 -7). Re-expression of E-cadherin is the crucial step in MErT.
Due to the key role of E-cadherin in human carcinoma progression, much effort has been devoted to understanding how E-cadherin is regulated during cancer progression, especially in EMTs. Various signaling molecules and transcription factors regulate the expression of E-cadherin (8). Accumulating evidence suggests that growth factor-induced EMT is the result of transcriptional reprogramming and chromatin remodeling (9,10). It has been shown that abrogated epidermal growth factor receptor (EGFR) activity recovers E-cadherin expression in prostate cancer cells (11,12). Moreover, by co-culturing the primary hepatocytes and carcinoma cells to recreate a key * This work was supported, in whole or in part, by the National Institutes of Health National Center for Advancing Translational Sciences Microphysiological Systems (MPS) program. This work was also supported by a Veterans Affairs Merit Award. 1  interaction extant in the liver microenvironment, it has been demonstrated that primary hepatocytes elicit E-cadherin cell surface expression in prostate carcinoma cells concomitant with down-regulation of EGFR signaling (12). These hepatocyte-induced E-cadherin re-expression in prostate and breast cancer cells increases the chemoresistant (13). EGFR activates several signaling cascades such as mitogenactivated protein kinases (MAPKs), PI3K-AKT, and JAK pathways. All of these kinase pathways are dysregulated in human tumors. The heterogeneity of the cellular models and the different experimental approaches result in the conflicting results of the roles of MAPK families in the genesis of EMTs. There is compelling evidence that ERKs and PI3K drive EMTs in many experimental systems (14 -16). Inhibition of ERK MAP kinase was able to completely restore E-cadherin cell-cell junctions in Ras-transformed breast epithelial cells (17). However, the role of SAPKs is less studied, although some reports indicate that JNK is an EMT inducer (18). p38 appears to promote EMT during development and in tumors (19 -21) but maintains E-cadherin expression in human primary mesothelial cells (22). However, the role of these kinases in disseminated cancer cells colonization in distant sites is unclear. Herein, we demonstrate that p38 and ERK1/2, but not JNK, JAK, or PI3K activation, are inhibited in prostate cancer cells in a hepatocyte microenvironment and result in the re-expression of E-cadherin. When challenged with chemotherapy and cell death-inducing cytokine, E-cadherin triggered p38 and ERK1/2 activity in PCa cells, which results in increased cells survival.
Cell Lines and Cell Culture-American Type Culture Collection cell lines DU145, PC3 prostate cancer cells, MDA-MB-231 breast cancer cell lines, and NCI-H1299 and A549 lung cancer cell lines were cultured in media recommended by supplier. DU145-red fluorescence protein (RFP) and PC3-RFP were selected in growth medium with 1000 g/ml G418. Polyclones of DU145-shctrl-RFP and DU145-shEcad-RFP were selected in growth medium supplemented with 0.5 g/ml puromycin and 1000 g/ml G418. Human fibroblast HS-68 cells were cultured in DMEM with 1ϫ nonessential amino acids, 1ϫ sodium pyruvate, 2 mM l-glutamine, 1ϫ streptomycin/penicillin (Invitrogen).
caMEK1 and caMEK6 Mutagenesis-The constitutively active mutants of MEK1 (CA-MEK1, S218E/S222E) and MEK6 (CA-MEK6, S207E/S211E) were designed by substitution of the regulatory phosphorylation sites with glutamine as described previously (38). Mutations were generated by site-directed PCR and then cloned into expression vector pEYFP-N1 with a FLAG insertion (kindly gifted from Dr. Xu, University of Illinois at Chicago). The constructs were confirmed by DNA sequencing.
Co-culture-Primary human hepatocytes, obtained from excess pathologic specimens, were isolated and plated at 8 ϫ 10 5 cells per well in 6-well plates coated with 1% rat tail collagen in distilled H 2 O (BD Biosciences) and allowed to attach overnight. The next day 2 ϫ 10 4 RFP-labeled cancer cells were seeded onto hepatocyte monolayers. The co-cultured cells were maintained with hepatocyte maintenance media (Lonza). For fibroblast cocultures, the fibroblast monolayer was initially plated at 1 ϫ 10 5 cells per well in 6-well plates and seeded with 2 ϫ 10 4 the following day. Medium was replenished daily.
Cell Death Resistance Assay-On day 5 of co-culture cells were treated with camptothecin (CPT) or/and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) for 24 h, and the absolute living cell numbers were accessed by flow cytometry. Annexin V-488 (Invitrogen) was used to identify apoptotic cells, and absolute counting beads (Invitrogen) were used to count absolute viable cell numbers.
Immunoblot and Immunofluorescence-Hepatocytes and cancer cells were seeded on a coverslip precoated with 1% collagen and then fixed on day 5 with 4% formaldehyde followed by permeabilization with 0.1% Triton X-100. After blocking with 2% BSA, the cells were incubated with primary antibody at 4°C overnight and then with Alexa Fluor 488/647-conjugated secondary antibodies at room temperature for 1 h. DAPI was applied to stain the nucleus. For the Western blot, cells were lysed with radioimmune precipitation assay buffer with phosphatase inhibitor mixture II, III (Sigma) and protease inhibitor mixture (BD Biosciences).
Protein Chase Assays-DU145 cancer cells were seeded at 3 ϫ 10 5 cells/well in 6-well plates and treated with or without 10 M SB203580 or 50 M PD98059 for 24 h after the addition 100 ng/ml actinomycin D or 100 M cycloheximide for the indicated times. Cells were lysed with radioimmune precipitation assay buffer supplemented with a pan-kinase inhibitor mixture. 20 g of protein was used to assess protein levels by immunoblotting.

Hepatocyte-educated Prostate Cancer Cells Re-express E-cadherin and Other Epithelial Markers-
We have reported that hepatocyte co-culture can lead to up-regulation or re-expression of E-cadherin even in highly aggressive carcinoma cells (12,13). Herein, we show that this phenotypic switch extends to other epithelial markers of cell-cell cohesion including ZO-1 and connexin 43 (Fig. 1a). Most of single DU145 and PC3 cells developed a colony after 5 days of co-culture with hepatocytes. Microscopic observation of these prostate cancer cells in the presence of hepatocytes, but not fibroblasts or other cancer cells, revealed a change in cell morphology from elongated spindle-like shape to smaller cuboidal with reversion to an epithelial phenotype (Fig. 1b), indicative of a cell phenotypic change from mesenchymal to epithelial.
Interestingly, different stages of converted PCa cells were found in hepatocytes microenvironment. The first stage was spindle-like with low E-cadherin expression levels, which was similar with parental PCa cells; the second stage was spindlelike with high E-cadherin in cytoplasm, and no E-cadherin was found on the membrane for the spindle-like PCa cells; the third stage was cuboidal-like with E-cadherin in the perinuclear, and the final stage was cuboidal-like with E-cadherin on the rim of cells. DU145 cells were found in the last two stages (Fig. 1c), and PC3 cells were founds in the all four stages (Fig. 1d) due to their different morphology. On the 3rd day, 24.1% PC3 colonies developed to a cuboidal-like phenotype, and only 1% of colonies found E-cadherin expressing on the cell membrane, but on the 5th day, 37.5% PC3 colonies were cuboidal cells and 17.5% were in colonies with E-cadherin on the cell membrane (Fig. 1e). The percentage of DU145 colonies with E-cadherin on the cell membrane was increased from 3.7% (day 3) to 57.5% (day 5) (Fig. 1f), which indicated hepatocytes educate PCa cells to develop an E-cadherin-dependent MErT in addition to simply re-expressing E-cadherin.
We have previously shown that E-cadherin expression in prostate cancer metastases is inversely correlated with the size of metastasis (13), suggesting a metastable nature to the reversion toward an epithelial phenotype. To explore the relationship between the colony size and E-cadherin expression patterns in vitro, the cell number per colony with different E-cadherin expression levels was determined. For DU145 cells, the colonies with E-cadherin on the cell membrane had a smaller size than in the cytoplasm (Fig. 1g); for spindle-like PC3 cells, most E-cadherin low-expressed cells were single or twocell colonies, but E-cadherin high expression colonies had the bigger size; for cuboidal PC3, E-cadherin expression levels was inversely correlated with the size of colonies, which was similar with DU145 (Fig. 1h). This suggests both a metastable nature of the phenotypic reversion and cell distance limit to the hepatocyte-induced changes.
Hepatocytes Suppress p38 and ERK1/2 Activation in DU145 Cells Result in E-cadherin Re-expression-Based on the above, we looked at what intermediary signaling in the cell could account for the changes in phenotype. Abrogation of EGFR signaling in prostate cancer cells can lead to up-regulation of E-cadherin (11), and co-culture with hepatocytes leads to sup-pression of the EGFR autocrine signaling in these cells indicative of the role of EGFR signaling cascades in E-cadherin regulation. As the EGFR pathway activates many intracellular signaling cascades including PI3K-AKT, ERK, JAK, or PKC, and PKC may activate p38 and JNK (23), we next explored which EGFR signaling cascade(s) might be involved in hepatocyteregulated E-cadherin re-expression in PCa cells. Negligible p-JNK, moderate p-p38, p-JAK2, and high p-ERK1/2 and p-AKT levels were found in parental DU145 cells. The presence of hepatocytes with the DU145 cells resulted in decreased levels of all mentioned above (Fig. 2, a-d). To look at which pathway(s) plays a dominant role in E-cadherin regulation in DU145 cancer cells, selective kinase inhibitors were applied, and relevant E-cadherin expression was detected. All inhibitors efficiently abrogated their targets (Fig. 2i). Inhibition of p38 or ERK MAPK activity increased E-cadherin expression at both the transcriptional and protein level, and although inhibition of PI3K or AKT decreased E-cadherin, inhibition of JAK did not affect E-cadherin (Fig. 2, e and f). Moreover, the lung cancer cell A549 responded similarly to DU145 to the kinase inhibitors (Fig. 2f). Knockdown of p38␣ or EKR1/2 expression by siRNA produced a similar up-regulation of E-cadherin (Fig. 2g).
To probe how p38 and ERK regulate E-cadherin expression, actinomycin was applied to block de novo RNA synthesis. SB203580 and PD98059 could not enhance E-cadherin expression in DU145 anymore. However, cycloheximide was applied to block new proteins synthesis; neither SB203580 nor PD98059 prevented E-cadherin degradation compared with control (Fig. 2h). These data suggested p38 and ERK regulate E-cadherin through RNA synthesis control and not at the protein level.
Constitutively Active MEK1 or MEK6 Abrogates Hepatocyteinduced E-cadherin Re-expression in DU145 Cells-Because hepatocytes enhanced E-cadherin expression in DU145 cells and this was phenocopied by suppressing p38 and ERK1/2 activity, demonstrating sufficiency, we queried whether abrogation of these was required for re-expression of E-cadherin, or whether activation of these intermediary signalers could overcome the hepatocyte influence. We introduced constitutively active MEK6 (caMEK6, activates p38 constitutively) and MEK1 (caMEK1, activates ERK1/2 constitutively) into DU145 cells. CaMEK6-expressing DU145 cells showed a notable increase in p38 phosphorylation levels in comparison to empty vector (EV)-expressing cells (Fig. 3a) as well as caMEK1-activated ERK1/2 (Fig. 3b). We next applied these cells co-cultured with hepatocytes. As expected, constitutively active MEK1 or MEK6 abrogated, at least partially, hepatocyte-induced E-cadherin reexpression in DU145 cells (Fig. 3, c and d). Thus each intermediary kinase was both sufficient and required.
Hepatocytes Render DU145 Resistant to Death Induced in an E-cadherin-dependent Manner-E-cadherin re-expression in the liver microenvironment increases the chemoresistance of breast and prostate cancer cells after treatment with chemotherapeutic agents such as staurosporine and camptothecin (13). TRAIL is a member of the tumor necrosis factor family that preferentially kills tumor cells. Chemotherapeutic agents enhance TRAIL-induced apoptosis in PCa cells (24,25). On the 5th day of co-culture, cells were treated with drugs for 24 h, and APRIL 18, 2014 • VOLUME 289 • NUMBER 16

JOURNAL OF BIOLOGICAL CHEMISTRY 11155
then surviving DU145 cells were assessed by flow cytometry; annexin-V was applied to label apoptotic cells, and the absolute living cell number was calculated. Reverted DU145 cells became more resistant to killing after the treatment of TRAIL (Fig. 4a) or co-treatment of TRAIL and CPT (Fig. 4b). Downregulation of E-cadherin by stably introducing shRNA abrogated this resistance to death, but no difference was noted in cell survival between parental control and E-cadherin K D DU145 cells in the face of the same cell death challenge (Fig. 4c). This is not due to simply being in G 0 or mitotic arrest, as the E-cadherin knockdown cells presented a similar proliferation rate compared with control cells when co-cultured with hepatocytes (Fig. 4d). This indicates this cell death resistance was proliferation-independent.
Select MAPK Effectors Are Required for Chemoresistance in DU145 Cells-To investigate the molecular mechanism of E-cadherin-related cell survival, the various MAP kinases were selectively inhibited in the co-culture cells, and then the cells were treated with CPT. Inhibition of p38 and ERK1/2 activities (Fig. 5c) but not JNK and PI3K (data not shown) abrogated DU145 cells survival advantage. This observation was also confirmed with PD153035-induced DU145 cell survival. After 48 h of PD153035 treatment to induce E-cadherin re-expression, DU145 cells displayed chemoresistance to CPT in a manner dependent on intact p38 and ERK (Fig. 5b). To eliminate that the kinase inhibitor per se altered cell survival, these inhibitors were applied to the parental DU145 cells followed by challenge with CPT. P38, JNK, and PI3K inhibitors did not alter cell survival, but ERK inhibitor improved cell survival from cell death (Fig. 5a). This effect was shown to be due to limiting cell proliferation, as a counting of viable cell numbers before and after inhibitors treatment demonstrated that the ERK inhibitor limited the increase in cell number (data not shown). These data suggested p38 and ERK1/2 played roles in the PCa cell survival via E-cadherin. Additionally, high levels of ERK1/2 and p38 phosphorylation were detected in DU145 cells treated with CPT and TRAIL. All challenges stimulated ERK1/2 and p38 phosphorylated in control cells but not E-cadherin shRNA expression cells (Fig. 5d).

DISCUSSION
In patients with advanced cancer, widespread manifestation of distant metastases is a major cause of cancer-related deaths. Despite this important clinical problem, little is known about the mediators that promote tumor outgrowth in the metastatic organ. The role of the MErT in cancer metastasis is controversial (2,8). Most likely this is due to cellular heterogeneity and the complex multistep process of cancer development and progression and its likely reversion back to a mesenchymal phenotype when metastatic nodules grow out (7). Thus, it is hard to capture MErT in vivo and in vitro.
We successfully induced MErT in prostate cancer cells via co-culturing with human hepatocytes, which elicited E-cadherin and other epithelial cell markers such as ZO-1 and connexin 43 re-expressing on the cell membrane. We found that this PCa cell phenotypic conversion appeared to be a process of education by hepatocytes (Fig. 1, c-f) in that there were intermediary forms of morphological shapes with E-cadherin being mainly expressed in intracellular vesicles. It could be that the low E-cadherin expressing spindle-like PCa cells with high motility, prevented clustering. It appears that over time these E-cadherin low, spindle-like, high motility PCa cells converted to E-cadherin high, cuboidal, low motility. E-cadherin expression level in PCa cells was inversely correlated with size of colony ( Fig. 1, g and h), which was consistent with a previous study (7). Our study highlights the role of the cancer cell extrinsic microenvironment prevailing in the metastatic organ as a major promoter of survival and outgrowth of disseminated tumor cells by induction of MErT.
To intervene in this phenotypic plasticity, the molecular triggers need to be discerned. Although we had reported earlier that for prostate cancers, disruption of autocrine EGFR signaling was sufficient for E-cadherin re-expression (11,12), the intracellular pathways have not been defined. EGFR activates several signaling cascades such as MAPKs, PI3K-AKT, and JAK pathways in cancers. ERK and AKT are the essential downstream effectors of EGFR pathway. Moreover, EGFR also activates the JAK and PKC pathway, with PKC further activating p38 and JNK (23). Herein, we queried whether the various MAP kinase pathways may be involved. Pharmacologic inhibitors suggested roles for the ERK and p38 pathways, with p38 being the most prominent, suppression of which allowed for E-cadherin up-regulation and MErT. The counter to this was achieved by constitutive activation of p38 using a MEK6 construct that prevented E-cadherin up-regulation. Although MEK6 activates all p38 isoforms (26), and both p38␣ and p38␤ are sensitive to SB203580 (27), we used siRNA to define p38␣ as the key isoform. There are four genes that encode p38 MAPKs: MAPK14 (that encodes p38␣), MAPK11 (that encodes p38␤), MAPK12 (that encodes p38␥), and MAPK13 (that encodes p38␦) (28). p38␣ and p38␤ are closely related proteins that could have overlapping functions. Whereas p38␣ is highly abundant in most cell types, p38␤ seems to be expressed at very low levels, and its contribution to p38 MAPK signaling is not clear. p38␥ and p38␦ are only expressed in specific tissues (29,30). Most of the published literature, including our study on p38 MAPKs, refers to p38␣. Consistent with our implications of p38 being involved in tumor progression, several negative regulators of p38 MAPK signaling have been found to be overexpressed in human tumors and cancer cell lines (31,32), supporting a tumor suppressor function of p38␣. However, increased levels of phosphorylated p38␣ have been correlated with malignancy in various cancers (33)(34)(35)(36); this could lead to a mesenchymal phenotype. Unlike the unsettled literature on the role of p38 signaling, ERK1/2 MAPKs are widely accepted as the tumor promotors.
It has been shown that inhibition of AKT activity restores E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells (39), which is in contrast with our findings in DU145 prostate cancer cells. We speculated that this may be due to different carcinoma cell lines controlling E-cadherin levels via diverse mechanisms. In OSCC cells the E-cadherin promoter is hypermethylated, but in the prostate lines E-cadherin levels are reduced at the protein and transcriptional levels. To confirm our speculation, other cancer cells, breast carcinoma MAD-MB-231 cells and lung adenocarcinoma H1299 cells, were treated with AKT inhibitor for 48 h, which restored E-cadherin mRNA expression levels by 2.8-and 5.2-fold, respectively (data not shown).
In our study both p38 and ERK1/2 displayed biphasic functions. First, PCa cells with high basal MAPKs levels underwent MErT either by inhibiting ERK1/2 and p38 activities or by hepatocyte co-culture that suppressed such activities (Fig. 2). Confounding this simple linkage, upon re-expression of E-cadherin .01. f, immunoblot for E-cadherin expression in DU145 and A549 cells with the same treatment above. The columns are presented as the mean Ϯ S.E. DU145, n ϭ 6; A549, n ϭ 3. g, immunoblot for E-cadherin expression in DU145 cells exposed to 80 pM control, p38␣ siRNA pool, ERK1, or/and ERK2 siRNA pool after 48 h. The columns are presented as the mean Ϯ S.E. n ϭ 3 each in triplicate. h, DU145 cells were pretreated with 100 ng/ml actinomycin for 4 h followed by 10 M SB203580 or PD98059 for 48 h. Cells were sampled with radioimmune precipitation assay buffer with a pan-protease inhibitor mixture. 20 g of total protein/well was used to assess E-cadherin expression. i, cycloheximide (CHX) chase assay. DU145 cells were exposed to SB203580 or PD98059 for 24 h followed by 100 M cycloheximide for indicated times. E-cadherin expression was detected by Western blot. j, kinase inhibitor efficiency was assessed by Western blot. DU145 cells were pretreated with the indicated kinase inhibitor for 1 h and then treated with or without 5 nM human EGF for 5 min. Active kinases were blotted with specific phospho antibodies. p-STAT3 was detected as a downstream effector of JAK2. Results shown are representative of at least three separate experiments.  the epithelial PCa cells activate ERK1/2 and p38 secondary to E-cadherin binding. This provides resistance to chemotherapeutics and death factors (Fig. 5e). Of further interest, exposure of the PCa cells to either CPT or TRAIL increased the levels of phosphorylated ERK1/2 and p38 (Fig. 5e), suggesting a compensatory change to protect the cells. This can be modeled by invoking specific temporospatial signaling cascades for each aspect. However, the main implication for designing approaches to limit MErT is that it is critically important to carefully consider the tumor stage before attempting to modulate p38␣ or ERK1/2 activity for cancer therapy.
One caveat is noted to the cell survival aspect of this signaling network. Suppression of cellular proliferation and/or metabolism suppression also could provide survival advantage (8,37). Our finding showed the similar relative cell numbers of control and E-cadherin shRNA introduced PCa cells co-culturing with hepatocytes (Fig. 4d). Although these studies do not conclusively demonstrate that the epithelial PCa cells have the same proliferation rate as the mesenchy-mal cells, it implies that the survival advantage arises from the intrinsic survival signals but not proliferation suppression or cell quiescence.
In summary, the data herein paint a picture of varied and stage-specific activation of select MAP kinases during tumor progression. First, high levels of ERK1/2 and p38 activity assist in establishing a mesenchymal phenotype required for dissemination from the prostate. Upon metastatic seeding, this is suppressed to allow for survival in a hostile environment, but separate signaling cascades that use these same intermediaries provide at least some of the survival signals. Last, it is possible, if not likely, that during the later reversion back to an aggressive mesenchymal phenotype, the PCa again up-regulates ERK1/2 and p38 to drive this outgrowth.
Acknowledgments-We thank members of the Wells and Roy laboratories for stimulating discussions and suggestions.