Enhanced sensitivity of pancreatic cancer cells to concurrent inhibition of aberrant signal transducer and activator of transcription 3 and epidermal growth factor receptor or Src

Abstract

Many molecular aberrations occur in pancreatic cancer. Although aberrant epidermal growth factor receptor (EGFR), Src, and signal transducer and activator of transcription 3 (Stat3) are implicated in pancreatic cancer, therapies that target only one of these entities are undermined by signaling cross-talk. In the human pancreatic cancer lines, Panc-1 and Colo-357, pY845EGFR, pY1068EGFR, pY1086EGFR, and pY1173EGFR levels and pY416c-Src are concurrently elevated with aberrantly active Stat3 in a complex signaling cross-talk. Thus, understanding the signaling integration would facilitate the design of effective multiple-targeted therapeutic modalities. In Panc-1 and Colo-357 lines, pY845EGFR, pY1068EGFR, and pY1086EGFR levels are responsive to c-Src inhibition in contrast to pY1173EGFR, which is EGFR kinase-dependent. Constitutively active Stat3 is sensitive to both EGFR and Src inhibition, but the early suppression of aberrantly active Stat3 in response to the inhibition of EGFR and Src is countered by a Janus kinase (Jaks)-dependent reactivation, suggesting that Jaks activity is a compensatory mechanism for Stat3 induction. The inhibition of EGFR, Src, or Stat3 alone induced weak biological responses. By contrast, the concurrent inhibition of Stat3 and EGFR or Src induced greater viability loss and apoptosis and decreased the migration/invasion of pancreatic cancer cells in vitro. Significantly, the concurrent inhibition, compared with monotargeting modality, induced stronger human pancreatic tumor growth inhibition in xenografts. We infer that the tumor growth inhibition in vivo is caused by the simultaneous suppression of the abnormal functions of Stat3 and EGFR or Src. These studies strongly suggest that the concurrent targeting of Stat3 and EGFR or Src could be a beneficial therapeutic approach for pancreatic cancer.

Fig. 1.

Immunoblotting analyses of Stat3, Src, and EGFR activities for effects of inhibitors. A and B, immunoblotting analysis of whole-cell lysates from cells untreated or treated with ZD or Das (Ai) or transfected with or without c-Src siRNA (Aii), EGFR siRNA (Aiii), or scrambled siRNA control (con) and probing for pY416c-Src (pY416Src), Src, pY845EGFR, and EGFR; and untreated or treated with ZD or Das and probing for pY1068EGFR (Bi), pY1086EGFR (Bii), and pY1173EGFR (Biii), and EGFR. Data are consistent with those obtained from four independent experiments.

Fig. 2.

EMSA and immunoblotting analyses for effects of inhibitors on Stat3 activation. EMSA analysis of Stat3 DNA-binding activity in Panc-1 (A) or Colo-357 (B) cells treated or untreated with the pan ErbB inhibitor PD169, ZD, Das, the Jak inhibitor AG490, the ErbB2-selective inhibitor AG879, or inhibitor combinations for the indicated times, or Panc-1 and Colo-357 cells treated for 24 h with S3I-201 (C), or immunoblotting analysis of whole-cell lysates from Panc-1 cells transfected with EGFR siRNA, Src siRNA, or scrambled siRNA (control) and probing for pStat3 or Stat3 (D). *, supershift analysis. Data are consistent with those obtained from three independent experiments.

Fig. 3.

Cell proliferation and viability studies for the effects of inhibitors. A, trypan blue exclusion/phase-contrast microscopy for viable Panc-1 or Colo-357 cells after treatment for 0 to 96 h with 1 μM ZD, 100 nM Das, or 50 μM S3I-201, or combinations. B, CyQuant cell proliferation assay for the viability of Panc-1 (left) or Colo-357 (right) cells in response to 48-h treatments with the designated concentrations of ZD, Das, and S3I-201, alone and in combinations. Values, mean and S.D. n = 4 experiments, each in triplicate.

Fig. 4.

Cell cycle distribution, cell growth, and colony survival and apoptosis studies for the effects of inhibitors. A, cells were untreated (control) or treated with ZD (1 μM), Das (100 nM), S3I-201 (50 μM), ZD + S3I-201, or Das + S3I-201 for 24 h, stained with propidium iodide, and analyzed by flow cytometry for cell cycle distribution. B, CyQuant cell proliferation assay for the viability of Panc-1 (i) and Colo-357 (ii) cells after treatments for 48 h with the designated concentrations of ZD, Das, S3I-201, and gemcitabine (Gem), alone and in combinations. C, number of colonies emerging from cells in culture (500/6-cm dish) untreated or treated once with ZD, Das, S3I-201 (S3I), or combinations and allowed to culture. D, annexin V binding/flow cytometry analysis of normal HPDEC, Panc-1, or Colo-357 cells treated or untreated with inhibitors or combinations. Values, mean and S.D. n = 4 experiments each in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 5.

Concurrent inhibition of Stat3 and EGFR or Src inhibits migration and invasion and suppresses c-Myc expression. A, effects of 24-h treatment with ZD, Das, and/or S3I-201 (S3I) on migration and invasion. B, immunoblotting analysis of whole-cell lysates for c-Myc and β-actin expression in Panc-1 cells after treatment with inhibitors for 24 h. Values, mean and S.D. n = 3 or 4 experiments each in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 6.

In vivo study of growth inhibition of human pancreatic cancer xenografts. Human pancreatic Colo-357 tumor-bearing mice were given ZD (75 mg/kg i.v.), Das (15 mg/kg i.v.), and S3I-201 (5 mg/kg i.v.) alone or in combination every 2 or 3 days for the first 2 weeks and then daily for 5 days each week for the next 3 weeks (A), or Panc-1 tumor-bearing mice were given ZD (75 mg/kg i.v.), Das (15 mg/kg i.v.), and S3I-201 (5 mg/kg i.v.) alone or in combination every 2 or 3 days, or given erlotinib (5 mg/kg i.v., every day) in combination with gemcitabine (100 mg/kg i.v., every 3 days) for 48 days (B). Tumor sizes, measured at 2- to 5-day intervals, were converted to tumor volumes and plotted against days. Values, mean and S.E.M. n = 7 or 8 mice per group. *, p < 0.05; **, p < 0.001.

Fig. 7.

A model of collaborative function of hyperactive EGFR, Src, and Stat3 in support of human pancreatic cancer. Hyperactive c-Src contributes to promoting the aberrant EGFR activation. Both hyperactive EGFR and Src induce constitutive Stat3 activation, whereas Jak activity contributes to promoting Stat3 signaling by serving as a compensatory mechanism. Aberrantly active Stat3 dysregulates the induction of target genes and, together with other potential mechanisms induced by the hyperactive EGFR and Src, induces cell growth, survival, and migration/invasion, thereby promoting pancreatic tumorigenesis and progression.