Nicotine induces inhibitor of differentiation-1 in a Src-dependent pathway promoting metastasis and chemoresistance in pancreatic adenocarcinoma

Abstract

Smoking is a significant risk factor for pancreatic cancer, but the molecular mechanisms by which tobacco smoke components promote the growth and progression of these cancers are not fully understood. While nicotine, the addictive component of tobacco smoke, is not a carcinogen, it has been shown to promote the growth of non-small cell lung and pancreatic cancers in a receptor-dependent fashion. Here, we show that stimulation of pancreatic cancer cells with nicotine concentrations that are within the range of human exposure results in activation of Src kinase, which facilitated the induction of the inhibitor of differentiation-1 (Id1) transcription factor. Depletion of Id1 prevented nicotine-mediated induction of proliferation and invasion of pancreatic cancer cells, indicating that it is a major mediator of nicotine function. Nicotine could promote the growth and metastasis of pancreatic cancers orthotopically implanted into SCID mice; in addition, cells stably expressing a short hairpin RNA for Id1 did not grow or metastasize in response to nicotine. Nicotine could also confer resistance to apoptosis induced by gemcitabine in pancreatic cancer cells in vitro and depletion of Src or Id1 rendered the cells sensitive to gemcitabine. Further, nicotine could effectively inhibit the chemotherapeutic effects of gemcitabine on pancreatic tumors xenografted into mice. Clinical analyses of resected pancreatic cancer specimens demonstrated a statistically significant correlation between Id1 expression and phospho-Src, tumor grade/differentiation, and worsening overall patient survival. These results demonstrate that exposure to tobacco smoke components might promote pancreatic cancer progression, metastasis, and chemoresistance and highlight the role of Id1 in these processes.

Figure 1

Nicotine induces Src phosphorylation in pancreatic adenocarcinoma cells. (A) Expression of α7 subunit of nAChR as seen by reverse transcription-PCR in a variety of pancreatic cancer cell lines. (B) Quiescent L3.6pl metastatic pancreatic cancer cells stimulated with nicotine induce Src phosphorylation within 1 hour. (C and D) Nicotine induces Src phosphorylation in PANC-1 and acquired gemcitabine-resistant L3.6pl^GemRes pancreatic cancer cells with a maximal phosphorylation event at 1 hour with return to baseline within 4 hours.

Figure 2

Nicotine induces Id1 protein expression in pancreatic adenocarcinoma cells. (A and B) L3.6pl and acquired gemcitabine-resistant L3.6pl^GemRes demonstrate significant increases in Id1 expression when quiescent cells are exposed to 1 µM nicotine. (C) Similarly, nicotine induces Id1 expression in PANC-1 and Mia-PaCa-2 pancreatic cancer cells. (D) Nicotine stimulation leads to increased Id1 mRNA expression levels in a diverse sample of pancreatic cancer cells lines, suggesting that the induction is at the transcriptional level.

Figure 3

Nicotine induces Id1 expression in a Src-dependent manner and regulates proliferation and invasion of pancreatic adenocarcinoma cells. (A) Silencing of α7 nAChR inhibits nicotine-induced Src and Id1 expression L3.6pl pancreatic cancer cells. (B) Silencing of Src expression inhibits nicotine induction of Id1 expression in L3.6pl pancreatic cancer cells. (C) Pharmacologic inhibition of Src phosphorylation with dasatinib inhibits constitutive and nicotine-induced levels of Id1 expression in L3.6pl pancreatic cancer cells. (D) shRNA to Id1 expression demonstrates significantly less Id1 when compared to controls (inset). Nicotine promotes cell viability and silencing of Id1 significantly diminishes this effect. (E and F) Nicotine promotes invasion in L3.6pl and PANC-1 pancreatic cancer cells and silencing of Id1 significantly diminishes this effect as demonstrated by cells per high-power field (HPF) and hematoxylin staining of both pancreatic cancer cells on membrane (inset).

Figure 4

Nicotine induces a chemoresistant phenotype in pancreatic cancer cells and tumors. (A) Gemcitabine induces PARP cleavage in L3.6pl and Mia-PaCa-2 cells and not in acquired and innate resistant cells L3.6pl^GemRes and PANC-1, respectively (left panel). (B) Nicotine induces cell viability/proliferation in both sensitive L3.6pl and acquired chemoresistant L3.6pl^GemRes cells and nicotine induces a chemoresistant phenotype in the chemosensitive L3.6pl pancreatic cancer cell line. (C) Tumor growth monitored by luciferase activity (photons per second) in L3.6pl gemcitabine-chemosensitive cells demonstrates augmented growth in mice treated with nicotine, containment of tumor growth in mice treated with gemcitabine, and continued tumor growth in mice treated with nicotine and gemcitabine comparable to control with in vivo luciferase imaging of mice at termination of experiment demonstrating these effects (inlet). (D) Graph demonstrating the corresponding tumor volume (mm³) from each animal group. (E) Ex vivo imaging of livers, from each animal group, demonstrates significantly higher liver metastasis in mice treated with nicotine compared to control, no liver metastasis in mice treated with gemcitabine, and overall increased signal from liver in mice treated with nicotine and gemcitabine when compared to all groups as seen by quantification of luciferase activity (photons per second). (F) H&E staining of liver metastases (T) confirming metastatic deposits of pancreatic tumor adjacent to normal liver parenchyma (N).

Figure 5

Depletion of Id1 abrogates chemoresistant phenotype and tumorigenic properties of pancreatic cancer cells. (A) Depletion of Src and Id1 using siRNA induces PARP cleavage in L3.6pl^GemRes-acquired.resistant pancreatic cancer cells exposed to gemcitabine therapy. (B) Depletion of Id1 expression in innate chemoresistant PANC-1 cells induces PARP cleavage and resensitizes cells to the cytotoxic effects of gemcitabine. (C) Tumor growth monitored by luciferase activity (photons per second) over approximately 4 weeks demonstrates increased tumor burden in mice treated with nicotine, whereas Id1 depletion resulted in inhibition of tumor growth regardless of nicotine exposure. (D) Graph showing the corresponding tumor volume (mm³) from each animal group corresponding to in vivo luciferase imaging of mice at termination of experiment (inset) demonstrating the promoting effects of nicotine and the suppressive effects of Id1 silencing on primary tumor growth. (E) Nicotine-induced pSrc and Id1 expression levels in primary tumors in vivo while having no effect on Id1 expression in tumors originating from cells with stably transfected shRNA silencing of Id1. (F) Ex vivo quantification of luciferase activity (photons per second) from livers demonstrates significantly higher liver metastasis in mice treated with nicotine while demonstrating no liver metastasis in mice with tumors derived from Id1-depleted cells. (G) H&E staining of livers (N) with metastatic pancreatic tumor demonstrates increased expression of Id1 in metastatic deposits (M).

Figure 6

Id1 expression in resected human pancreatic adenocarcinoma. TMAs constructed from resected pancreatic ductal adenocarcinoma were stained for Id1, pSrc (activated form), and total Src using standard IHC techniques. A statistically significant correlation was observed between Id1 and tumor grade/differentiation and pSrc expression levels. No significant correlation was seen between total Src and Id1 expression.

Figure 7

Worsening tumor grade correlates with increased pSrc and Id1 expression levels and increased Id1 expression correlates with poor overall survival in pancreatic cancer patients. (A) Increased tumor grade level demonstrates a clear correlation with higher pSrc expression levels (score) with a Spearman correlation estimate of 0.31 (P < .001). (B) Increased tumor grade level is associated with increased Id1 expression (score) with a Spearman correlation estimate of 0.29 (P < .001). (C) Patients with high histologic grade have significantly worse survival than patients with low histologic grade (P = .0235). (D) Dichotomizing Id1 scores into low and high scores using the 75% score demonstrates significant correlation with overall patient survival, with high Id1 expression levels resulting in a worse overall survival (P = .0388). (E) Increased tumor grade level demonstrates a significant correlation with increased Id1 expression (algorithmic densitometric analyses) with Spearman correlation estimate of 0.24 (P = .0042). (F) Dichotomizing Id1 scores (determined by quantitative algorithm) into low and high scores with 75% score demonstrates a significant correlation with overall patient survival with high Id1 expression levels resulting in a worse overall survival (P = .0519).