Here, we examined the consequences of persistently activated Stat3 in prostate tumorigenesis and determined that the expression of Stat3-C is sufficient to mediate tumorigenesis and the migration of immortalized but nontransformed prostate epithelial cells. A comparison of the morphologies of the control and Stat3-C expressing cells revealed a shift in the distribution of filipodia toward a greater abundance of lamellipodia and, correspondingly, an increase in the migratory capacity of the cells (Fig. ). Stat3 has been shown to play a critical role in cell migration through both transcriptional and nontranscriptional mechanisms (12
Tumorigenesis involves the acquisition of genetic and epigenetic changes that cause the aberrant loss or gain of functions by cellular proteins. The consequences include the ability of tumor cells to proliferate, resist apoptosis, demonstrate angiogenic potential, migrate, and invade, as well as the ability of these cells to evade immune surveillance. Activated Stat3 has been linked to all of these processes in part through the transcriptional regulation of critical target genes, including those for c-myc, cyclin D1, matrix metalloproteinase 9 (MMP-9), MMP-2, vascular endothelial growth factor, Bcl-xL
, Mcl-1, survivin, and LIV-1, as well as the repression of p53 and Fas (6
). We and others previously demonstrated that Stat3-C can transform immortalized fibroblasts and breast epithelial cells, in part as a consequence of increased cyclin D1 and MMP-9 expression (6
). However, the levels of neither of these targets nor those of many of the above-mentioned targets (MMP-2, survivin, c-myc, and LIV-1) were increased in our Stat3-C-expressing RWPE-1 cells, suggesting that additional Stat3 targets which are tissue or tumor specific exist (data not shown).
In order to identify potential Stat3-C-regulated transcripts which may contribute to the Stat3-C-mediated transformation and increased migration of prostate epithelial cells, we found many potentially interesting up- or down-regulated transcripts in these cells by Affymetrix analysis (see Table S1 in the supplemental material). In addition to the levels of ITGβ6, FN, and TNC, we confirmed by RT-PCR that those of MMP-3, thrombospondin 1, S100 A9, nicotinamide N
-methyltransferase, and brain-heart-protocadherin were also increased, while the levels of cyclin D1, Stat1, p73, and myeloid cell leukemia sequence 1 were unchanged in Stat3-C-containing cells compared to those in pBabe vector control cells (data not shown). Many of these proteins have known roles in EMT, migration, and tumorigenesis (3
We focused our attention on ITGβ6 and its ligand FN as possible targets, given their known roles in cell migration and tumorigenesis (1
). TNC is also a ligand for ITGαvβ6, may modulate the affinity of FN for ITGαvβ6, and is implicated in tumorigenesis and the metastatic spread of cancer cells (28
). An increase in the expression of TNC in the Stat3-C-containing cells relative to that in the pBabe vector control cells was observed by Western blotting (data not shown). However, we did not examine the role of TNC in these processes given the lack of commercially available purified reagents and functional blocking antibodies. We attempted to decrease the levels of TNC by using small interfering RNA but did not see a significant decrease in mRNA levels. The expression of fibronectin has been associated with malignant transformation and resistance to the apoptosis of tumor cells (36
). The accumulation of FN in a number of malignancies, including breast, lung, and pancreatic cancers, in particular by the peritumoral stromal cells, has been noted previously (21
). The treatment of breast cancer-derived MCF-7 cells with OSM leads to a Stat3-dependent increase in cell migration, with a concomitant increase in FN production, after 72 h of OSM treatment (61
). We observed a relatively rapid increase in FN mRNA levels after 4 h of OSM stimulation of RWPE-1 cells, suggesting that Stat3 may directly regulate the transcription of the FN gene in this cell type (Fig. ). The addition of FN to our cultured prostate epithelial cells resulted in an increase in migration, with a change in the phenotype of the pBabe vector control cells to one approximating the phenotype of the Stat3-C-expressing cells, including a decrease in E-cadherin levels, which is a hallmark of an EMT (Fig. ) (31
). These data suggest that activated Stat3 is likely to be a regulator of FN production, which is partly responsible for both an EMT and enhanced cell migration.
Integrins are essential mediators of tumorigenesis, adhesion, and migration. ITGαvβ6 levels in particular have been found to be increased in many tumors, including colorectal tumors, squamous-cell carcinomas, and pancreatic and breast tumors (2
). Recently, it was determined that an EMT of colorectal cells also leads to an increase in the expression of ITGαvβ6 through the Ets-1 transcription factor. Furthermore, the migration of these cells on FN requires ITGαvβ6, and a correlation between ITGαvβ6 and aggressive colorectal tumors was determined (4
). Here, we demonstrate that Stat3-C-containing prostate epithelial cells express high levels of ITGβ6 mRNA and protein. The activation of Stat3 by OSM led to an increase in the levels of ITGβ6 mRNA within 4 h of stimulation. Furthermore, both the inhibition of Stat3 activity and a decrease in the abundance of Stat3 by short hairpin RNA in DU145 cells (a cancer-derived human prostate epithelial cell line) resulted in a decrease in the ITGβ6 mRNA levels. Increased ITGβ6 expression in MCF10A cells (a human breast epithelial cell line) expressing Stat3-C was observed, suggesting that activated Stat3 may participate in regulating ITGβ6 expression in more than one tumor type (data not shown) (13
). An examination of the ITGβ6 promoter revealed many optimal (TTN5
AA, where N represents any nucleotide) Stat3 binding sites (Fig. ). Activated Stat3 may bind to the ITGβ6 promoter and stimulate the expression of an ITGβ6 promoter-luciferase gene construct, suggesting that ITGβ6 is a direct transcriptional target of Stat3 (Fig. ).
The engagement of integrins with extracellular matrix proteins results in the regulation of the small GTPases (19
). The activity of the small GTPases (Rac, Rho, and Cdc42) regulates the dynamic organization of the actin cytoskeleton, which determines cell morphology and regulates cell migration (50
). Here, we demonstrate that the addition of exogenous FN can enhance cell migration (Fig. ). Huang et al. have shown that the ITGαvβ6-mediated migration of murine keratinocytes on FN is blocked by an anti-ITGαvβ6 antibody (29
). Here, we show that the Stat3-C expressing cells migrated better than pBabe control cells and that this phenotype was enhanced in the presence of FN. Significantly, the enhanced migratory phenotype was inhibited by a function-blocking antibody, 6.3G9, to ITGαvβ6 (Fig. ). Furthermore, culturing Stat3-C-expressing cells in the presence of 6.3G9 led to an increase in E-cadherin expression approximating the expression seen in the pBabe vector control cells (Fig. ). We hypothesize that activated Stat3 may promote cell migration and an EMT of RWPE-1 cells as a function of the coordinated regulation of ITGαvβ6 and FN.
We examined primary prostate tissue samples for the expression of activated Stat3 and ITGαvβ6. We observed that a relatively small fraction of cancer samples expressed high levels of activated Stat3, and no significant correlation between activated Stat3 and clinical parameters was noted (Fig. ). This finding is in contrast to observations which show that a high percentage of samples express activated Stat3 and that it correlates with a high Gleason score and an advanced pathological stage (15
). A possible explanation is provided by the labile nature of the pYStat3 signal in primary cancer tissues (16
). Specifically, we observed that the pYStat3 signal in cancer samples decreased within 30 min of procurement (data not shown). Thus, we may be underestimating the number of pYStat3-positive samples in our tissue microarrays. The incidence of strong expression (staining score, +2 to +3) of ITGαvβ6 in primary prostate cancer cells was low (10%), while the overall level of expression was 23%, and no significant correlation between high levels of pYStat3 and levels of ITGαvβ6 was observed. However, high levels of ITGαvβ6 expression as well as pYStat3 were found within basal cells of both nonneoplastic ducts and those with intraepithelial neoplasia, although the degree of positive correlation did not achieve statistical significance. The role these basal cells play in the pathogenesis of prostate cancer is unknown. Furthermore, it remains unclear whether the histologically benign-appearing ducts adjacent to cancer are not already in the process of transformation due to field effects. In summary, we have identified and characterized novel targets of activated Stat3 that contribute to Stat3-mediated migration and may play a role in prostate tumorigenesis.