Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer

The Stat (signal transducer and activator of transcription) family of proteins are transcription factors known for their role as integrators of cytokine and growth factor receptor signaling and are required for cell growth, survival, differentiation, and motility [1, 2]. Stat activation is dependent upon tyrosine phosphorylation, which induces dimerization via reciprocal phosphotyrosine-SH2 (Src homology domain 2) interaction between two Stat molecules. Activated Stats translocate to the nucleus, where they bind to consensus promoter sequences of target genes and activate their transcription [3]. In normal non-transformed cells, Stat tyrosine phosphorylation is transient. However, in numerous cancer-derived cell lines and in an ever-growing number of primary tumors, Stat proteins (in particular, Stat3) are persistently tyrosine-phosphorylated [4].

Stat3 is constitutively activated in more than 50% of primary breast tumors and tumor-derived cell lines [5‐17]. Clinical studies demonstrated that elevated levels of tyrosine-phosphorylated Stat3 (pStat3) in patients with stage II breast cancer correlated with an incomplete response to neo-adjuvant chemotherapy (a poor prognostic feature) [7]. The evidence that activated Stat3 participates in a causal manner in breast tumorigenesis is based on studies from cell lines expressing high levels of pStat3. Inhibiting or removing Stat3 leads to increased apoptosis, chemosensitivity, and decreased angiogenesis both in cell culture and in xenograft models [5, 6, 10, 13, 14, 18‐22]. Furthermore, the expression of a constitutively active form of Stat3 (Stat3C) into immortalized human breast epithelial cells mediated de novo tumorigenesis, demonstrating the sufficiency of this transcription factor in promoting cellular transformation [16]. Stat3 activation can occur through the actions of many autocrine and paracrine growth factors (for example, interleukin-6 [IL-6], epidermal growth factor, platelet-derived growth factor, heregulin, vascular endothelial growth factor, and hepatocyte growth factor) and the Src non-receptor tyrosine kinase, leading to the prediction that its phosphorylation in breast cancer would be through these multiple, redundantly acting growth factors [6‐8, 12, 13, 18, 19, 23‐25]. Among these candidate cytokines, IL-6, which activates Stat3 through the glycoprotein 130/Janus kinase (gp130/Jak) pathway, is notable for its pleiotropic tumor-promoting activities, including anti-apoptotic, pro-invasive, and immune-stimulatory effects attributable to the activation of Stat3 target genes [26, 27]. Furthermore, high IL-6 levels have been linked to a poor prognosis in patients with advanced breast cancer, the pathogenesis of inflammatory breast cancer, and resistance to chemotherapy [26]. Our studies have revealed a prominent and non-redundant role for IL-6 in driving Stat3 activation in breast cancer.

By using animals deficient for Stat3 in certain tissues or by introducing dominant negative Stat3 constructs and anti-sense and short hairpin RNA Stat3 molecules into cell lines, many investigators have demonstrated a requirement for Stat3 in tumorigenesis [42]. Thus, inhibiting Stat3 activity or expression is likely to be an important therapeutic modality for a number of malignancies, including breast cancer. Targeting the Stat3 molecule directly is of significant interest, and indeed several small molecules that appear to target the Stat3 SH2 domain or the DNA-binding capacity of Stat3 have been developed [43‐45]. It has been assumed that the kinases responsible for Stat3 activation are many, and therefore targeting one kinase or receptor would not be of benefit for the treatment of a variety of tumors.

Here, we examined the mechanisms of Stat3 phosphorylation in breast cancer. We observed that high pStat3 levels in primary tumor specimens did not correlate with nuclear hormone receptor status (ER or PR) nor overexpression of the Her2neu receptor (Table 1). It has been suggested in murine models of breast cancer that aberrant Her2neu signaling may mediate Stat3 activation [22]. However, in primary human breast tumors, no such relationship was observed [7, 11, 17]. We examined pSrc and pEGFR levels by IHC in these tumor specimens, but less than 3% of the samples had elevated expression of these proteins, possibly for technical reasons as these values appear to be too low compared to other reports (data not shown) [7, 8]. To examine the potential mechanisms involved in mediating Stat3 phosphorylation, we examined breast cancer-derived cell lines expressing high levels of pStat3. Inhibition of EGFR and Src kinases did not have an effect on pStat3, suggesting that these kinases are not direct mediators of Stat3 phosphorylation in these breast cancer-derived cell lines. Our data regarding Src inhibition differ from those described by Garcia and colleagues [13] whereby treatment of MDA-MB-468 cells with an Src inhibitor (PD180970) resulted in inhibition of pStat3. Similarly, it was reported that treatment of MDA-MB-231 cells with an EGFR inhibitor (AG1478) inhibited Stat3 activation [19]. In our study, we used different inhibitors of both Src and EGFR at concentrations previously described to potently inhibit both of these kinases. The explanations for these discrepancies are not clear, but differences in inhibitor selectivity and the length of time for which cells are treated could explain our differing observations. Interestingly, dasatinib treatment of both lung and prostate cancer-derived cell lines did not affect pStat3 levels, suggesting that Src inhibition does not play a role in mediating Stat3 activation in these cancer-derived cell lines [38, 39]. In contrast to EGFR and Src inhibition, a pan-Jak inhibitor inhibited pJak2 and Stat3 phosphorylation (Figure 3). Furthermore, treatment of the cell lines with an anti-gp130 antibody significantly inhibited Stat3 phosphorylation (Figure 4). Given the previously described association between the EGFR family members and gp130 receptor, we attempted but were unable to show any physical association between EGFR and gp130 by immunoprecipitation experiments in MDA-MB-468 cells (which express the highest levels of EGFR) (data not shown) [19].

The cytokine receptor gp130 is the common signaling subunit for the IL-6 family of cytokines. The gp130 receptor is ubiquitously expressed in tissues and mediates numerous homeostatic functions, including immune response, inflammation, bone metabolism, and hematopoiesis [27]. IL-6, IL-11, OSM, LIF, and ciliary neurotropic factor are among the IL-6 family cytokines that use and require gp130 as a signaling subunit [27]. We determined that IL-6 was the essential cytokine mediating gp130 activation in MDA-MB-468 and MDA-MB-231 cells and the 4175 subline (Figure 4). We did not observe inhibition of pStat3 by the IL-6-blocking antibody in the MDA-MB-435 cell line nor was IL-6, LIF, or OSM detected in the CM from this cell line. We believe that IL-11 is responsible for mediating Stat3 activation because IL-11 mRNA levels were increased in this cell line compared to MDA-MB-468 cells (data not shown). However, we have yet to confirm by immunological means that IL-11 is mediating gp130 activation.

The data presented here suggest that the principal mediator of Stat3 activation in breast cancer is through the IL-6 family of cytokines activating the gp130/Jak signaling pathway. Stat3 activation through the gp130 receptor is regulated by both positive effectors such as IL-6 and negative regulators, including suppressor of cytokine signaling (SOCS) 1 and SOCS3, which inhibit Jak activity [46]. SOCS1 and SOCS3 promoters are frequently methylated in primary tumors and cancer-derived cell lines, resulting in decreased expression of these negative regulators of Jaks and leading to sustained Jak activation [47]. We hypothesize that even in the absence of IL-6 or IL-6 engagement with the gp130 receptor, Jaks may still be activated due to the lack of SOCS gene expression and can only be fully inhibited with a Jak inhibitor.

We propose that the IL-6/gp130/Jak signaling pathway plays a critical role in Stat3 activation in human breast cancer and that blockade of this pathway may be an important therapeutic modality in breast cancer.