PMC:2246177 / 30604-38741 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2246177","sourcedb":"PMC","sourceid":"2246177","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2246177","text":"Discussion\nAlthough traditionally known for its role in growth hormone signaling and mammary gland development, STAT5b has emerged as a therapeutic target due to its pivotal role in cancer. Inhibition of STAT5b, but not of STAT5a, in xenograft models of head and neck carcinomas via antisense oligonucleotides repressed tumor growth and hindered expression of the STAT5-regulated genes cyclin D1 and Bcl-XL [13,14]. STAT5b is more abundantly expressed than STAT5a in prostate cancer and breast cancer cell lines [12,31]. Inhibition of STAT5b via dominant-negative constructs and siRNA technology decreases DNA synthesis (Figure 6) [32], while exogenous expression of a basally active STAT5b mutant (Y740/743F) increases DNA synthesis of breast cancer cells [12]. Together, these data establish a fundamental role for STAT5b in the process of breast cancer tumorigenesis.\nSince activating mutations in STAT5b have not been found in breast cancer, it is important to look upstream to identify the kinases that regulate STAT5b, thus potentially leading to its increased activity in breast cancer cells. Both STAT5b and STAT3 are activated by several kinases overexpressed in breast cancer, including the EGFR, HER2, and c-Src. STAT3 was recently shown to also be phosphorylated by the nonreceptor tyrosine kinase Brk [1]. Since Brk is expressed in more than 60% of breast tumors but not in normal mammary tissue, it has been suggested to be a potential therapeutic target for breast cancer. Since evidence supports the involvement of both Brk and STAT5b in breast cancer proliferation, we investigated the ability of Brk to phosphorylate STAT5b and the biological significance of this activation.\nUsing synthetic peptides containing consensus motifs for the EGFR, insulin receptor, c-Src, or Abl tyrosine kinases, it was determined that the c-Src-preferred synthetic peptide is the best Brk substrate [17]. Given these data, it is not surprising that all of the identified Brk substrates (Sam68, SLM-1, SLM-2, STAP-2, paxillin, STAT3) are also c-Src substrates [1,30,33-35]. We have identified two more Brk substrates, STAT5a and STAT5b, which are also c-Src substrates (Figure 1). c-Src can mediate the phosphorylation of Y699 and Y725, however, Brk mediates Y699 phosphorylation but not Y725 phosphorylation (Figure 2b) [12,27]. A previous study demonstrated that Brk mediated phosphorylation of STAT3, but not of STAT1, STAT2, STAT5, or STAT6 [1]. These studies were performed in COS1 cells, however, not in breast cancer cells, and in vitro kinase assays were not performed for STATs other than STAT3. Using our previously characterized STAT5b-specific antibodies, we demonstrated here that Brk can also directly phosphorylate Y699 on STAT5b in breast cancer cell lines and in an in vitro kinase assay.\nExogenous expression of Brk in the Brk-negative breast cancer cell line BT-549 increased endogenous STAT5b transcriptional activity. Interestingly, the catalytically inactive K219M Brk mutant also significantly increased STAT5b transcriptional activity compared with vector alone, although not to the extent seen with wildtype Brk or the constitutively active (Y447F). In fact, Harvey and Crompton have previously reported that the kinase-inactive Brk mutant (K219M) could increase the proliferation of T47D cells compared with vector [22]. Since the K219M mutation disrupts the ATP-binding motif, but not the Src homology domain 2 or the Src homology domain 3, these data suggest that Brk has a role in intracellular signaling that does not require its kinase activity. In these cases, Brk may function as an adaptor protein. Finally, although the constitutively active Y447F Brk mutant was able to increase STAT5b transcriptional activity, it was not significantly higher than that seen with wildtype Brk. This mutation is at the presumptive autoinhibitory tyrosine phosphorylation site of Brk (Y447), equivalent to that identified in c-Src (Y527). Although the Y447F Brk increases phosphorylation of a synthetic peptide, the autophosphorylation of the activating tyrosine in Brk (Y342) is comparable with that seen with wildtype Brk [17,36]. Nevertheless, the Y447F Brk mutant has decreased transforming potential when compared with wildtype Brk in NIH3T3 cells [20]. Together, these data as well as the results we have presented here suggest that the regulation of Brk is more complex than originally thought, and probably involves its role as a kinase and an adaptor protein depending on the cell context.\nSince both c-Src and Brk tyrosine kinases are frequently overexpressed in breast cancer and since they both mediate Y699 phosphorylation of STAT5b, there is potential for these kinases to either substitute for one another or work together to activate STAT5b. As shown in Figure 5b, exogenous overexpression of c-Src, unlike Brk, did not enhance STAT5b transcriptional activity in the BT-549 cells, although we have reported that it does in other cell lines [12]. Exogenous expression of c-Src along with Brk, however, enhanced STAT5b transcriptional activity to a level greater than that with Brk alone. As the kinase-inactive c-Src did not enhance Brk-mediated STAT5b transcriptional activity, c-Src kinase activity may play a role in increasing the phosphorylation and functional activation of Brk. Together, these results demonstrate that c-Src and Brk do not merely substitute for one another in mediating STAT5b transcriptional activation. Rather, Brk can function independently of c-Src, or these two kinases can work together to enhance STAT5b activity.\nWhile Brk is not expressed in normal mammary epithelial cells, it is expressed in 60% of breast tumors – suggesting that Brk expression is regulated at the transcriptional level in breast cancer cells [16]. Furthermore, the DNA sequence of Brk isolated from gastrointestinal epithelial cells and that of Brk isolated from breast tumor cells are identical, suggesting that activating mutations in Brk are not accountable for Brk activity in breast cancer [18]. Located within the minimal functional promoter of Brk are NF-κB, Sp1, and STAT consensus binding sites, all known to play a role in tumorigenesis. Only NF-κB and Sp1, however, have thus far been shown to bind the Brk promoter [37]. Reports in human breast cancer samples have varied, showing that Brk correlates with HER2 and HER4 overexpression, as well as with estrogen receptor positivity [38-40]. There is also conflicting evidence showing a strong correlation between Brk staining by immunohistochemistry and tumor grade in one report [29], and another correlating Brk expression with long-term survival [39].\nOur mRNA and protein analysis across a panel of human breast cancer cell lines showed no correlation of Brk expression with estrogen receptor status or EGFR/HER2 overexpression (Figure 4). We have, however, shown that knockdown of Brk significantly decreased DNA synthesis in the EGFR-overexpressing and c-Src-overexpressing breast cancer cell lines, SKBr3 and BT-20 (Figure 6). The effect of Brk knockdown on inhibiting basal DNA synthesis was comparable with that seen with STAT5b knockdown in both cell lines. Furthermore, simultaneous knockdown of both Brk and STAT5b had the same effect on decreasing DNA synthesis as their individual knockdowns, suggesting that this kinase and substrate converge upon a pathway ultimately leading to proliferation. Ostander and colleagues have shown that Brk knockdown decreases cell growth as well as epidermal growth factor-induced (or heregulin-induced) migration of breast cancer cells [29]. Additionally, knockdown of Brk decreases epidermal growth factor-induced (or heregulin-induced) cyclin D1 expression in breast cancer cells [29]. As STAT5b is known to regulate cyclin D1 through consensus sites in the promoter, this pathway may be one mechanism by which Brk and STAT5b together regulate increases in DNA synthesis. Given the evidence that we have provided for the functionally relevant regulation of STAT5b by Brk, further pursuing the role of this Brk–STAT5b pathway in breast cancer may provide important novel therapeutic targets.","divisions":[{"label":"Title","span":{"begin":0,"end":10}}],"tracks":[]}