The work presented here establishes an important, previously undiscovered role for STAT5b in the migration of highly aggressive breast cancer cells. Knockdown of STAT5b inhibits migration of BT-549 and MDA-MB-231 human breast cancer cells to serum and fibronectin (Figures and ). This inhibition can be rescued by expression of wt-, Y699F-, or dn-STAT5b, but not with the SH2 domain mutant R618K-STAT5b (Figure ). Upon attachment to fibronectin, STAT5b-knockdown cells form multiple, contractile protrusions resulting in loss of directionality and inefficient migration (Figures and ).
Knockdown of STAT5b inhibits migration of both BT-549 and MDA-MB-231 breast cancer cell lines to serum, whereas knockdown of STAT5a has no effect (Figure ). These results may seem contradictory to those published by Sultan and colleagues [10
], which reported a suppressive effect of STAT5a on breast cancer cell migration. However, in those studies, STAT5a was overexpressed in the moderately migratory BT-20 and T-47D breast cancer cell lines, which contain little to no endogenous STAT5a. The STAT5a and STAT5b expression pattern in those cell lines is very different from that seen in MDA-MB-231 cells, and consequently, the signaling may differ. Moreover, the role of endogenous STAT5b was not investigated in the studies of Sultan and associates. We and others found that STAT5b is the predominant STAT5a/b protein expressed in breast cancer cell lines and tissues, and that STAT5b, not STAT5a, mediates proliferation of breast cancer cells [10
]. Differential expression or activity (or both) of STAT5a and STAT5b has been reported in other cancer model systems [9
]. STAT5b levels and phosphorylation are elevated in SCCHN tumors compared with control mucosa, whereas STAT5a levels to do not change [24
]. Consistent with these findings, antisense inhibition of STAT5b, but not STAT5a, inhibits in vivo
growth of SCCHN xenografts [24
]. In prostate cancer, differential STAT5a and STAT5b protein expression can be correlated with metastatic potential. STAT5a is expressed in nonmetastatic C1D mouse prostate cancer cells, but not in their metastatic C2H counterparts, whereas STAT5b is expressed in both [9
]. Additionally, STAT5a is expressed in LNCaP human prostate cancer cells but not the more highly migratory PC-3 prostate cancer cell line, but STAT5b levels are comparable [9
]. For these reasons, it is imperative to determine the individual contributions of STAT5a and STAT5b, as they may have distinct functions in tumorigenesis.
After establishing the necessity of STAT5b for maximal migration of breast cancer cells to serum, we investigated the mechanism by which this occurs. Interestingly, the migratory function of STAT5b does not require phosphorylation at tyrosine 699 (Y699) or the C-terminal transactivation domain (TAD), evidenced by equivalent rescue of migration with either wt-, Y699F-, or dn-STAT5b (Figure ). Phosphorylation of Y699 is a hallmark of STAT5b transcriptional activation. If this residue is mutated to a phenylalanine, such that it cannot be phosphorylated (Y699F), the resulting STAT5b mutant is transcriptionally inactive [17
]. Dn-STAT5b is also transcriptionally inactive because of the loss of the TAD and the ability to bind transcriptional cofactors [14
]. Although STAT proteins are predominantly thought of as transcription factors that function in the nucleus, recent work identified a non-transcriptional, cytoplasmic role for unphosphorylated STAT3 in regulating tubulin dynamics [26
]. Unphosphorylated STAT dimers take on an antiparallel configuration through interactions between the DNA-binding domains [27
]. In this conformation, the SH2 domains are on opposite sides of the structure and are free to interact with phosphorylated tyrosines of other proteins. The necessity of the SH2 domain of STAT5b in migration is consistent with a cytoplasmic function of unphosphorylated STAT5b. We propose that STAT5b uses its SH2 domain to act as a scaffolding protein, bringing together signaling molecules necessary for efficient, directional migration. Although migration is inhibited to similar levels after STAT5b knockdown or blocking of β1
-integrin (Figure ), it remains to be determined whether STAT5b interacts directly with β1
-integrin or if this effect is indirect. There are many tyrosine-phosphorylated proteins in the cytoplasm involved in migratory signaling with which STAT5b may interact, and future studies are aimed at uncovering these associations.
Loss of STAT5b leads to a polarity defect that impedes directional movement (Figure ). During spreading on fibronectin, STAT5b-knockdown cells take on a remarkable phenotype distinguished by the formation of multiple, unstable protrusions (Figure ). No defect is found in initial attachment, and once attached, protrusions extend rapidly. However, these protrusions are highly dynamic, and over time, they contract back into the cell. This phenotype is indicative of disrupted equilibrium between Rho family GTPases. Rac is localized predominantly at the leading edge, with Rho in the tail. Normal ratios of Rho and Rac lead to Rac-mediated formation and spreading of broad lamellipodia in the front of the cell, followed by Rho-mediated tail retraction, overall resulting in directional migration toward a stimulus [29
]. Increased Rho activity in the front of the cell would disrupt this equilibrium and could account for the multiple protrusions and increased contraction at the front of the cell seen with STAT5b knockdown. Based on the data presented here, we postulate that unphosphorylated STAT5b mediates migration of breast cancer cells through regulation of cytoplasmic Rho GTPase family signaling.
In summary, these studies are the first to report a role for STAT5b in the migration of breast cancer cells. It is well established that STAT5b positively regulates breast cancer cell proliferation and survival, two processes important for initial tumor formation and growth. These data implicate STAT5b in the later stages of tumorigenesis also, such as migration. Future studies will further elucidate the mechanism by which STAT5b exerts its effect on migration, thereby broadening our understanding of how STAT5b promotes tumorigenesis and possibly metastasis. This will facilitate the long-term goal of defining conditions whereby STAT5b would be an effective therapeutic target for the treatment of breast cancer.