among multiple signaling pathways, macro-molecular protein assemblies provide additional unique mechanisms for inducing events that would support the malignant phenotype. Such a non- traditional signaling mechanism has been identified for the EGFR, which has been detected in the cell nucleus and observed to function as a transcription factor [8,9]. Studies further revealed the nuclear EGFR complexes with Stat3 in breast cancer cells, and this complex induces specific genes, including the inducible nitric oxide synthase (iNOS) [10]. The additional EGFR function would compound its role as a mitogen and a promoter of cell survival, which all favor cancer. In that regard, the concurrent aberrant activation of EGFR and downstream signal mediators, including Src and Stat3, which occur with high frequencies in human cancers reflects an overall signaling complexity that supports the cancer phenotype. For example, with reference to pancreatic cancer, aberrant activation of EGFR occurs in 30–50% of cases [11], activated c-Src is noted in more than 70% of cases, and frequently accompanies EGFR overexpression [12], while aber- rant Stat3 activation is also highly prevalent [13,14,15]. Impor- tantly, our recent report that pancreatic cancer is more sensitive to the concurrent inhibition of aberrant Stat3 and EGFR or Src [16] shows the utilization of multiple aberrant signaling pathways for the maintenance of the cancer phenotype and how this influences the responsiveness to therapy. To extend our earlier studies [16], we sought to probe the molecular and functional interplay between Stat3, EGFR and Src and the underlying mechanisms of support of the pancreatic cancer phenotype. We herein provide evidence for a functional nuclear heteromeric EGFR, Src and Stat3 complex in pancreatic cancer cells, which promotes the induction of the c-Myc gene. Our report is the first on the identification of a nuclear EGFR, Src and Stat3 heteromeric complex that promotes the c-Myc gene induction. Understanding the dynamics of the EGFR, Src and Stat3 molecular interactions in pancreatic cancer would provide basis to design novel effective multiple-targeted therapy approaches for pancreatic cancer. Materials and Methods Cells and Reagents The human pancreatic cancer, Panc-1 and Colo-357 lines have all been previously described [16,17]. The immortalized human pancreatic duct epithelial cell (HPDEC) line was a kind gift of Dr. Tsao, OCI, UHN-PMH, Toronto) [18]. HPDEC were grown in Keratinocyte-SFM media supplemented with 0.2 ng EGF, 30 mg/ mL bovine pituitary extract and containing antimycol. All other cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% iron-supplemented bovine calf serum and 100 units/ml penicillin-streptomycin. Peptide synthesis The Stat3 SH2 domain peptide inhibitor (SPI), FISKERER- AILSTKPPGTFLLRFSESSK, the EGFR peptide motifs, pY1068 EGFR (pY1068), PEpYINQS and the pY1086EGFR (pY1086), PVpYHNQP were purchased from Peptide 2.0 (Fairfax, VA) at .95% purity. Nuclear Extract Preparation and Gel Shift Assays Nuclear extract preparation from cells was carried out as previously described [17]. Sub-cellular fractionation, and SDS-PAGE/Western Blot Analysis Western blotting analysis was performed as previously described [19,20] on whole-cell lysates, and on cytosolic and membrane A Functional Nuclear EGFR, Src and Stat3 Complex fractions, and on nuclear extracts. Sub-cellular fractions were prepared according standard protocol. Briefly, cells were washed using PBS, resuspended and lysed in low-salt buffer (20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM dithiothreitol, 1 mM TLA, 0.5 mM phenylmethylsulfonyl fluoride and 0.5% Nonidet P- 40), and centrifuged (13,000 x g, 4uC, 30 s) to obtain the cytosolic fraction. The pellet was washed three times with phosphate- buffered saline (PBS), resuspended in high-salt buffer (420 mM NaCl, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 20% Glycerol, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM dithiothreitol, 1 mM TLA and 0.5 mM phenylmethylsul- fonyl fluoride), incubated at 4uC for 30 min, with rocking, and centrifuged at (13,000 x g, 4uC, 30 min) to obtain the nuclear fraction (supernatant). The pellet obtained was washed three times with PBS, resuspended in RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1 mM TLA, and 0.5 mM phenylmethylsulfonyl fluoride), incu- bated for 30 min at 4uC with rocking, and centrifuged (13,000 x g, 4uC, 20 min). The supernatant was collected as the membrane fraction. Primary antibodies used are against pY845EGFR (Upstate Biotech, Millipore, Billerica, MA), pY705Stat3, Stat3, pY1068EGFR, pY1086EGFR, pY1173EGFR, EGFR, pY416Src, Src, and b-Actin (Cell Signaling Technology, Danvers, MA), and Tata-binding protein (TBP) (Santa Cruz Biotechnology, Santa Cruz, CA). The blocking peptides were purchased from the respective companies. Small-interfering RNA (siRNA) Transfection siRNA sequences for EGFR and Src were ordered from Dharmacon RNAi Technologies, Thermo Scientific (Lafayette, CO). Sequences used are: EGFR sense strand, 59-GAAGGAAACU- GAAUUCAAAUU-39; EGFR antisense strand, 59-pUUUGAAUU- CAGUUUCCUUCUU-39; control siRNA sense strand, 59-AGUAA- UACAACGGUAAAGAUU-39; and control siRNA antisense strand, 59-pUCUUUACCGUUGUAUUACUUU-39. The c-Src SMART- pool siRNA reagent (NM-005417, Catalog # M-003175-01-05) was used for Src. Transfection into cells was performed using 20 nM of EGFR siRNA or 25 nM of Src siRNA and 8 ml Lipofectamine RNAiMAX (Invitrogen Corporation, Carlsbad, CA) in OPTI-MEM culture medium (GIBCO, Invitrogen Corporation). Immunoprecipitation (IP), and Sequential Immunoprecipitation Studies These studies were performed as previously reported [21] using whole-cell lysates or nuclear extracts (250 mg total protein) and 5 mL of anti-EGFR or anti-Src polyclonal antibody or the monoclonal anti-Stat3 antibody (Cell Signaling Technology). For specificity, immunoblotting analysis using anti-EGFR, anti-Src and anti-Stat3 antibody was performed in the presence of the respective blocking peptide. Sequential IP studies were performed according to published procedures [10] with some modifications as follows: nuclear extracts, prepared as previously described [21], were subjected to a similar immunoprecipitation with respect to the first primary antibody, anti-EGFR antibody (Cell Signaling) or IgG (Santa Cruz) at 4uC overnight. The immunecomplex was then pelleted with 20 mL protein A/G agarose beads (Santa Cruz), washed three times using wash buffer A (0.1% SDS, 1% Triton X- 100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0), and then two times with wash buffer B (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0). Then the proteins were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3) and subjected to the second immunoprecip- itation by incubating with anti-Src antibody or IgG (Santa Cruz). PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19605