Identification of potential factors and regulatory pathways mediating STAT3 activation has important implications for the development and selection of molecularly targeted therapy in lung cancer. Our current findings demonstrate that the presence of pSTAT3 in lung adenocarcinoma tumor samples by immunohistochemical analysis correlates positively with expression of EGFR tyrosine kinase domain mutations. Owing to insufficient numbers of tumor specimens expressing somatic-activating mutations within the kinase domain, we were unable to determine whether pSTAT3 positivity correlated differentially with either ΔEGFR or L858R. Much of our data confirms prior observations regarding pSTAT3 and lung adenocarcinomas. Specifically, 50% of samples express variable levels of pSTAT3 by immunohistochemical analysis; a positive association exists between pSTAT3 and small tumor size, TTF1, pAKT, and pEGFR; and an inverse correlation exists between pSTAT3 and caspase-3 (14
). No association between pSTAT3 and survival was observed. A recent description of a murine model revealed inducible expression of EGFR kinase domain–activating mutations targeted to the lung epithelium gives rise to adenocarcinomas containing pSTAT3 and pAKT, demonstrating an association between this oncogene and activated STAT3 (26
). Of interest, expression of constitutively activated STAT3 targeted to the lung epithelium gives rise to de novo adenocarcinoma of the lung (C. Yan, unpublished observations).
The evidence demonstrating a direct association between EGFR and STAT3 is based primarily on work done in cell lines expressing high levels of EGFR, such as A431 and head and neck squamous cell carcinoma (HNSCC) cells (37
). The biochemical differences between cell lines expressing kinase domain–activating mutations within the EGFR versus wild-type EGFR include enhanced differential phosphorylation of specific tyrosine residues on the EGFR and alterations in the levels of pMAPK, pAKT, p-src, and pSTAT3 (8
). However, the mechanism of STAT3 phosphorylation in either mutant EGFR–transfected cell lines or cancer-derived cell lines is unclear, as inhibition of EGFR (with ZD), src (with dasatinib), or JAK2 (with AG490) activity does not lead to inhibition of STAT3 phosphorylation in cancer-derived cell lines (8
). Our current findings demonstrate that inhibition of JAKs with the use of a pan-JAK inhibitor (P6) completely abrogated STAT3 phosphorylation in all cancer-derived cell lines expressing mutant forms of EGFR (Figure ). Of importance, inhibition of neither EGFR activity (with ZD) nor src activity (with dasatinib-BMS) resulted in a change in pSTAT3 levels (Figure A and data not shown). The use of a pan-JAK inhibitor, rather than an inhibitor specific for only a single JAK, is likely critical, as multiple JAKs could be implicated in mediating STAT3 phosphorylation in an EGFR-dependent manner. The JAKs have been described as being associated with EGFR, but they are not required to mediate STAT phosphorylation in response to EGF (41
). P6 treatment of mutant EGFR cancer-derived cell lines did not markedly affect EGFR phosphorylation or pMAPK, which is a target of the EGFR (Figure A). Furthermore, the addition of EGF to these cell lines increases pEGFR, which was unaffected by P6 treatment (Figure B and data not shown). These data suggest an uncoupling between EGFR and JAKs.
Identification of the regulatory pathways leading to STAT3 activation has clear implications for the development of targeted therapy in lung cancer (13
). Of importance, we demonstrate that inhibition of JAK kinases with the use of the pan-JAK inhibitor P6 led to decreased proliferation and a G2
/M cell cycle block in 11-18, H1975, and H1650 cell lines (Figure C and data not shown). We demonstrate that STAT3 is likely the principal P6 target, as a partial knockdown of this protein in the H1650 cells led to a decrease in their proliferative capacity (Figure D). Inhibition of anchorage-independent growth by P6 was observed in all 4 cell lines, while ZD inhibited growth only in soft agar to comparable levels seen with P6 in H3255 cells (Figure , A and B). The H1975 cell line is resistant to the growth-inhibitory actions of ZD and dasatinib (8
). However, our data reveal that ZD, at a relatively higher concentration (5 μM), can inhibit the EGFR tyrosine phosphorylation and partially inhibit anchorage-independent growth of H1975 cells (Figure A and Figure B). This result is consistent with prior observations that ZD, at concentrations ranging from 5 to 10 μM, inhibits cell growth of H1975 (40
). As P6 is insoluble, we could not administer it in vivo. Thus, subsequently, we were only able to show that P6-treated cells in vitro are less capable of growing in vivo compared with DMSO-treated control cells (Figure , C and D). Nevertheless, these findings suggest that JAK inhibition may prove to be effective therapy in the treatment of cancers that depend on mutant EGFR activity, including those that may be less sensitive or resistant to TKIs.
Activation of JAKs and STAT3 has been shown to occur through binding of the IL-6 family of cytokines to the gp130 receptor in many different cancer-derived cell lines, including myeloma; HNSCC; breast and prostate cancers; cholangiocarcinoma; and NSCLC (22
). Furthermore, high circulating levels of IL-6 have been found in patients who have metastatic breast and lung cancers (44
). Our current findings demonstrate that blockade of IL-6/IL-6R/gp130 signaling with the use of both sequestering antibodies and blocking antibodies inhibits STAT3 activation (Figure A). Furthermore, all cell lines produce very high levels of IL-6, which was rapidly secreted into the culture medium and could stimulate STAT3 phosphorylation in MCF-10A cells (Figure B). The degree of pSTAT3 inhibition with the blocking antibodies was not as profound as with P6 treatment. Furthermore, the blockade with both gp130 and IL-6 was only transient, lasting less than 24 hours (data not shown) (33
). To examine the consequences of IL-6 blockade on growth, we introduced an IL-6 shRNA construct into the lung cancer–derived cell lines. We demonstrated that reduced expression of IL-6 led to a decrease in pSTAT3, as well as a decrease in the in vitro and in vivo growth of H1975, H1650, and 11-18 cells (Figure ). Despite reduced production of IL-6, there was no effect on in vitro growth in the H3255 cell line, which is consistent with the lack of growth inhibition by P6 in this cell line (data not shown). Thus, blockade of the IL-6 pathway is sufficient to inhibit the growth of these cancer-derived cell lines and demonstrates the potential therapeutic benefit of IL-6 inhibition.
Given the previously described association between the EGFR family members and the gp130 receptor, we attempted but were unable to show any physical relation between EGFR and gp130 in H3255 (which expresses the highest levels of EGFR), 11-18, H1975, and H1650 cell lines (data not shown) (42
). To examine the relation between mutant EGFR and IL-6/gp130 receptor signaling, we introduced ΔEGFR into MCF-10A cells, and our current findings demonstrate an increased production of IL-6 mRNA and protein, with subsequent STAT3 activation and cellular transformation (Figure ). An immortalized breast epithelial cell line was chosen, as introduction of mutant EGFR into immortalized lung epithelial cells has resulted in an increase in pSTAT3 levels and anchorage-independent growth but no evidence of tumorigenesis (48
). As observed with the lung cancer–derived cell lines, STAT3 phosphorylation depends on IL-6/gp130/JAK signaling, but not on EGFR kinase activity in the ΔEGFR MCF-10A cells (Figure ). These novel findings suggest that ΔEGFR can drive expression of IL-6, which ultimately is responsible for mediating gp130/JAK/STAT3 signaling.
If mutant EGFR is capable of upregulating IL-6 synthesis, why is there no effect on pSTAT3 levels after inhibition of EGFR activity (Figures and )? The IL-6 protein is produced to high levels and is quite stable within the CM of these cancer-derived cell lines. Thus, decreasing its synthesis in the context of such excess would clearly have little to no consequence for STAT3 activation. To examine the contribution of mutant EGFR activity on IL-6 production and STAT3 activation, we first had to remove the IL-6–containing CM. After removal of CM, inhibition of the EGFR activity led to a partial decrease in IL-6 production and pSTAT3. This clearly demonstrates a requirement for mutant EGFR activity, but nonetheless supports the suggestion that other factors are mediating the regulation of IL-6 synthesis (Figure , A and B). Some of the described transcriptional mediators of the IL-6
gene include NF-κB, AP-1, C/EBPβ, and CREB, whose expressions and activity vary as a function of cell type and ligand stimulation (35
). The EGF has been shown to activate NF-κB and AP-1 transcription factors in HNSCC-derived cell lines (52
). It has been suggested that STAT3, in association with NF-κB, could bind to the IL-6 promoter through an NF-κB binding site and promote expression of the IL-6 gene (53
). Of interest, IL-6 production has been shown to depend on activated STAT3 in a mouse gastric model system (54
). We hypothesize that mutant EGFR leads to NF-κB activation and that STAT3 can potentiate IL-6 transcription through this critical interaction.
Our cell line data demonstrate a positive relation among mutant EGFR, IL-6, and pSTAT3. We examined lung adenocarcinoma TMAs for IL-6 expression and demonstrate a positive association between pSTAT3-positive samples (i.e., those with a staining score of +1, +2, or +3) and samples expressing moderate-to-high (+2 to +3) levels of IL-6. In summary, we demonstrate that the mechanism by which STAT3 is activated in lung adenocarcinomas is through mutant EGFR regulating expression of the IL-6 cytokine, which, in turn, activates the gp130/JAK pathway. Of importance, JAK kinase and IL-6 blockade leads to inhibition of growth and tumorigenesis, suggesting that the development of inhibitors of this pathway may be an important therapeutic target for these malignancies.