(Fig. 6A(ii), EGFR/Src). By contrast, PCR analysis showed no appreciable detection of TWIST, a gene previously reported to be EGFR target [22] in the EGFR chromatin immunecomplex, while the gene was detected in the Stat3 and Src chromatin-immunopre- cipitates (Fig. S3). These differences may be tumor cell-type dependent. To verify the specificity of the immunoreagents, the non-specific IgG was similarly used in the ChIP assay, and subsequent PCR analysis showed no detectable levels of the c-Myc gene (Fig. 6A, IgG). Taken together with the sequential immuno- precipitation data in Figure 2B), these studies demonstrate that EGFR, Src and Stat3 form a detectable heteromeric complex that is associated with the expression of c-myc in pancreatic cancer cells. To further study the potential involvement of the heteromeric EGFR, Src and Stat3 complex in the regulation of genes, we performed immunoblotting analysis of known regulated genes, including c-Myc. Results showed moderate or no significant change in the expression of c-Myc, Cyclin D1, iNOS, and VEGF upon the siRNA knockdown of EGFR or Src alone (Fig. 6B). By contrast, the concurrent knockdown of EGFR with Stat3 inhibition (by S3I-201) [25], or the concurrent knockdown of Src with Stat3 inhibition, or the concurrent EGFR knockdown with Src inhibition (by Das) resulted in a strong suppression of c- Myc expression (Fig. 6C(ii)). The bands corresponding to the expression levels of c-Myc were quantified, analyzed by Im- ageQuant, and represented as percent of control (Fig. 6C(ii), % numbers in parenthesis). Results show over 76% suppression of c- Myc expression (Fig. 6C(ii)) following the concurrent modulation of any two of EGFR, Src and Stat3, except for the siRNA knockdown of EGFR and Src together, which only showed 20% decrease. The moderate change in the c-Myc expression in response to the co-transfection with EGFR siRNA and c-Src siRNA may be due to the fact that we could only achieve partial knockdown of Src (Fig. 6C(i)), although EGFR is significantly suppressed by the EGFR siRNA (Fig. 6C(i)). In contrast, the use of the pharmacological inhibitor, Das, which strongly inhibits Src activity, in combination with siRNA knockdown of EGFR strongly suppressed c-Myc expression. These studies together suggest the possibility that the c-Myc gene is regulated by EGFR, Src and Stat3 complex in a manner that is susceptible to concurrent modulation of any two of the EGFR, Src and Stat3 proteins, but not to the inhibition of EGFR, Src or Stat3 alone. Discussion Aberrations in the EGFR, c-Src and Stat3 signaling pathways occur with a high frequency in many human cancers [14,15,38,39,40,41] and are associated with poor prognosis. Notably, constitutively-active Stat3 induces dysregulation of gene expression, contributing to the altered gene expression profile that is a hallmark of cancer. The details of aberrantly-active Stat3- mediated dysregulation of gene expression continue to be elucidated and the initial studies indicate the mechanisms are more complex. The present studies strongly suggest that in forming a nuclear, transcriptionally-active EGFR, Src, Stat3 heteromeric complex, the EGFR and Src proteins cooperate with Stat3 to promote the altered gene expression. Such cooperation between Stat3 and other proteins for the transcriptional induction of genes has similarly been observed in other systems. Specifically, Stat3 cooperates with NF-kB to induce certain genes [42,43]. Furthermore, there have been reports of a nuclear EGFR pathway [8,9], in which a nuclear EGFR-Stat3 complex promotes the induction of iNOS in breast cancer cells [10]. These reports together with our present findings indicate the complicated nature of the mechanisms by which aberrantly-active Stat3 might A Functional Nuclear EGFR, Src and Stat3 Complex dysregulate gene expression in cancer cells. In this context, the present studies extend our earlier report [16] in suggesting that nuclear heteromeric EGFR, Src and Stat3 complex regulates the c-Myc gene in pancreatic cancer cells. It is likely that other genes may by induced by the EGFR, Src and Stat3 nuclear complex and we note that iNOS, VEGF, and Cyclin D1 are reported to be induced by the EGFR/Stat3 complex or Stat3 [10,44,45] and could well be candidates for the regulation by the EGFR, Src and Stat3 complex. Taken together, these studies provide a novel mechanism for the de-regulation of gene expression in cancer cells. The observation that the EGFR, Src and Stat3 complex is also detected in the cytoplasm raises the possibility that it is formed extra-nuclear and transported into the nuclear space. Previous reports have described inherent EGFR nuclear localization mechanisms, including facilitation via the endosomal sorting machinery and the interaction with importins a1/b1 [31], and several nuclear translocation mechanisms have also been proposed for Stat3, including EGFR-mediated endocytosis [6,31]. Although any of these processes could facilitate the nuclear translocation of the heteromeric EGFR, Src, Stat3 complex should it be formed outside of the nucleus, the present data excludes the possibility that EGFR-mediated endocytosis is involved. Whether the other nuclear translocation pathways proposed for Stat3, including the utilization of the Ran-GTPase [4], Sec61 translocon [46], and chaperoning by MgcRacGAP [5] are involved in promoting the nuclear transport of the complex remains to be studied. Our findings also do not preclude the assembly of the complex de novo in the nucleus. Present data also suggest that only a portion of the intracellular EGFR, Src and Stat3 protein pools are utilized in the formation of the nuclear complex, raising the possibility that there may be diverse cellular pools of EGFR, Src, or Stat3 with different accessibility limitations. There also could be different pools of pre- associated complexes of the three proteins, a possibility that will be consistent with the report that cytoplasmic Stat3 exists as complexes with accessory scaffolding proteins [47]. Such pre- formed complexes would not only facilitate the signal induction [48], but may also serve to stabilize the proteins. The incidence of signaling cross-talk has long been known, and the associations of EGFR with Src [49], Src with Stat3 [20,50], and EGFR with Stat3 [26,27] at the plasma membrane and the perinuclear space [6] have been reported. Specifically, Stat3 binds to pY1068 and pY1086 motifs of EGFR [26,27], while Src binds to Y845EGFR. In cancer cells, aberrant Stat3 activation is promoted by hyperactive protein tyrosine kinases, including EGFR and Src [15,40,41], and evidence has indicated that c-Src phosphorylates Y845EGFR, Y1068EGFR, and Y845EGFR motifs in pancreatic cancer cells [16,27,51]. Present data reveals that the pY1068EGFR and pY1086EGFR motifs and the Stat3 SH2 domain amino acid residues 588–615 are essential for EGFR-Stat3 interaction within the context of the heteromeric EGFR, Src and Stat3 complex. It remains to be determined what the exact configuration is for the heteromeric complex. Also, the present data does not exclude the possibility that other accessory proteins could be present in the complex together with EGFR, Src and Stat3. We had recently reported about the functional cooperation between EGFR, Src and Stat3 in promoting and supporting pancreatic cancer, wherein the cancer phenotype and the expression of c-Myc in the cancer cells were both insensitive to the inhibition of EGFR, Src or Stat3 alone [16]. While it is possible that the c-Myc gene may be regulated cooperatively by EGFR, Src and Stat3 through mechanisms independent of each other, our study also raises the possibility of a complex transcriptional regulation by mechanisms that involve the nuclear EGFR, Src and Stat3 heteromeric complex. PLoS ONE | www.plosone.org 11 May 2011 | Volume 6 | Issue 5 | e19605