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Biology of Human Tumors

Nicotine Reduces Survival via Augmentation of Paracrine HGF–MET Signaling in the Pancreatic Cancer Microenvironment

Daniel Delitto, Dongyu Zhang, Song Han, Brian S. Black, Andrea E. Knowlton, Adrian C. Vlada, George A. Sarosi, Kevin E. Behrns, Ryan M. Thomas, Xiaomin Lu, Chen Liu, Thomas J. George, Steven J. Hughes, Shannon M. Wallet and Jose G. Trevino
DOI: 10.1158/1078-0432.CCR-15-1256 Published April 2016

Abstract

Purpose: The relationship between smoking and pancreatic cancer biology, particularly in the context of the heterogeneous microenvironment, remains incompletely defined. We hypothesized that nicotine exposure would lead to the augmentation of paracrine growth factor signaling between tumor-associated stroma (TAS) and pancreatic cancer cells, ultimately resulting in accelerated tumor growth and metastasis.

Experimental Design: The effect of tobacco use on overall survival was analyzed using a prospectively maintained database of surgically resected patients with pancreatic cancer. Nicotine exposure was evaluated in vitro using primary patient–derived TAS and pancreatic cancer cells independently and in coculture. Nicotine administration was then assessed in vivo using a patient-derived pancreatic cancer xenograft model.

Results: Continued smoking was associated with reduced overall survival after surgical resection. In culture, nicotine-stimulated hepatocyte growth factor (HGF) secretion in primary patient-derived TAS and nicotine stimulation was required for persistent pancreatic cancer cell c-Met activation in a coculture model. c-Met activation in this manner led to the induction of inhibitor of differentiation-1 (Id1) in pancreatic cancer cells, previously established as a mediator of growth, invasion and chemoresistance. HGF-induced Id1 expression was abrogated by both epigenetic and pharmacologic c-Met inhibition. In patient-derived pancreatic cancer xenografts, nicotine treatment augmented tumor growth and metastasis; tumor lysates from nicotine-treated mice demonstrated elevated HGF expression by qRT-PCR and phospho-Met levels by ELISA. Similarly, elevated levels of phospho-Met in surgically resected pancreatic cancer specimens correlated with reduced overall survival.

Conclusions: Taken together, these data demonstrate a novel, microenvironment-dependent paracrine signaling mechanism by which nicotine exposure promotes the growth and metastasis of pancreatic cancer. Clin Cancer Res; 22(7); 1787–99. ©2015 AACR.

Translational Relevance

In addition to promoting carcinogenesis, sustained tobacco use is associated with accelerated tumor progression and reduced survival in both lung and head and neck cancers. This work first serves to validate this finding in pancreatic cancer. To address the mechanism underlying this phenomenon, we hypothesized that paracrine growth factor signaling within the stroma-rich pancreatic cancer microenvironment may be especially responsive to nicotine exposure. Through the use of human primary cell cultures and patient-derived xenografts, we investigate a novel, pathologic secretory response of the tumor-associated stromal cell to nicotine. Ultimately, this response leads to unrestrained HGF–MET signaling within the tumor microenvironment and the rapid accumulation of metastatic lesions. These results suggest strongly that nicotine administration, whether through tobacco use, replacement therapies or e-cigarettes, continues to promote the progression of pancreatic cancer even after a tumor has been established.

Introduction

An estimated 11% to 32% of pancreatic adenocarcinomas are attributed to tobacco use, representing an important cause of mortality in the United States (1, 2). Yet, the mechanisms linking smoking to the development and progression of pancreatic cancer remain poorly understood (3). Published reports demonstrate that continued smoking is associated with reduced survival in patients with lung and head and neck cancers (4, 5). These results suggest that sustained tobacco use promotes progression of these malignancies, but the effect of continued smoking on the progression of pancreatic cancer is unclear.

Tobacco use yields a multitude of toxins associated with carcinogenesis and tumor progression. Among these, nicotine has been characterized in both the initiation of cancer and progression of disease (6–10) but the effects on continue nicotine exposure on pancreatic cancer or its direct effect on the tumor microenvironment are poorly understood. Rationale for continued investigation of nicotine in cancer is supported by current trends in popular culture as e-cigarettes are promoted as a safer alternative to smoking despite the induction of comparable serum nicotine levels (11).

Mechanistically, our previous work has established an essential role for the transcriptional repressor known as inhibitor of differentiation-1 (Id1). We demonstrated that nicotine induces pancreatic cancer growth, metastasis, and chemoresistance through the induction of Id1 (12). However, this phenomenon was not characterized in the context of the tumor microenvironment which acts to promote tumor growth and chemoresistance through paracrine signaling events between pancreatic cancer cells and stromal elements (13, 14). Given the Src-dependent nature of Id1 induction described in our previous work, we chose to examine a known upstream mediator of Src activation with translational relevance in pancreatic cancer. Specifically, the mesenchymal–epithelial transition factor c-Met has emerged as a critical receptor tyrosine kinase (RTK) in cancer development and metastasis (15). For instance, recent investigations have isolated c-Met as a marker of pancreatic cancer stem cells (PCSC) and pharmacologic inhibition of c-Met reduced the expression of other PCSC markers, thereby restoring gemcitabine chemosensitivity (16, 17). In light of these findings, we asked if nicotine exposure could influence the tumor microenvironment through paracrine activation of c-Met signaling and determined whether activated c-Met may contribute to our previously characterized model of Id1 induced chemoresistance.

Our work demonstrates the first report of adverse prognostic effects associated with continued tobacco use in a prospectively maintained cohort of patients with potentially curative, surgically resected pancreatic cancer. Further, incorporation of signaling events between heterogeneous cell types within the pancreatic cancer microenvironment reveals novel, translational insights. We show that nicotine induces patient-derived tumor-associated stromal (TAS) cells to secrete HGF, resulting in the stimulation of c-Met and subsequent upregulation of Id1 expression in pancreatic cancer cells. Moreover, HGF–MET signaling induced by nicotine in this manner augments tumor growth and metastasis in a patient-derived pancreatic cancer xenograft model which integrates desmoplastic stromal elements, inherent in our proposed mechanism (18). Collectively, these results are corroborated by quantitative assessments of c-Met activation in resected pancreatic cancer specimens, demonstrating a significant reduction in survival for patients with phospho-Met–positive tumors. Taken together, these findings illustrate the translational relevance of c-Met activation in response to nicotine exposure in the tumor microenvironment. The data presented here demonstrate nicotine-induced tumor promoting effects through direct and paracrine signaling events, thus providing a global mechanism to the strong association between systemic administration of nicotine and pancreatic cancer tumor progression.

Materials and Methods

Cell culture, assays, and reagents

All human pancreatic cancer cell lines were authenticated within 6 months by short tandem repeat (STR) analysis. Human pancreatic cancer cell lines PANC-1, Mia-PaCa-2, and BxPC3 were obtained from the ATCC. The L3.6pl pancreatic cancer cell metastatic variant was derived as previously described (19, 20). The selection of L3.6plGemRes gemcitabine-resistant pancreatic cancer cells was conducted as previously described (12, 21). Cells were maintained in culture with Dulbecco's modified Eagle's medium/F12 (DMEM/F12) with 10% FBS (Atlanta Biologicals) and 0.6% penicillin/streptomycin and 5% CO2/95% air at 37°C. Patient-derived TAS cells were generated from direct culture of gross human pancreatic adenocarcinoma surgical specimens and maintained in DMEM/F12 plus 10% FBS as previously validated for pancreatic stromal cells (22–27). All primary TAS lines were confirmed to uniformly express high levels of α-smooth muscle actin (>99%) by immunocytochemistry and flow cytometry prior to experimentation (28). TAS lines demonstrated variable glial fibrillary acid protein (GFAP) expression (20%–60%) and did not express epithelial surface antigen or the immune cell marker CD45 (28). All experiments with TAS cells were performed on actively replicating cells between passages two and five. Gemcitabine (Eli Lilly) was suspended in Dulbecco's PBS (D-PBS) and used at concentrations for the half-maximal inhibitory concentration (IC50) previously published for pancreatic cancer cell lines (12, 29). Nicotine and recombinant human HGF stimulation were performed on cells that were rendered quiescent by serum starvation for 48 hours with a physiologic dose of 1 μmol/L nicotine (ref. 30; Sigma Aldrich) or 50 ng/mL recombinant human HGF (Millipore). Pancreatic cancer cell stimulation by TAS cells was performed by the addition of TAS-conditioned media and/or coculture assays using Transwell-Col plates (Costar) whereby pancreatic cancer cells were plated in a 24-well plate with TAS cells in the insert chambers as indicated. Phospho-Met immunofluorescence was performed at 48 hours on direct cocultures of TAS and pancreatic cancer cells plated simultaneously in 24-well plates. c-Met kinase inhibition was performed with crizotinib (Selleck Chemicals). Cellular viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Trevigen) according to the manufacturer's protocol.

Western blot

Lysates were prepared from pancreatic cancer cell lines, TAS or surgically resected tumor tissues and Western blots performed as previously described (12). Lysates were probed with either anti-Id1 antibody (BioCheck), anti-Met, anti-phospho-Met, and anti-GAPDH (Cell Signaling Technology), or anti-β-actin (Sigma-Aldrich).

Real-time PCR

Total RNA was harvested from TAS, pancreatic cancer cells or patient tumors and purified using RNeasy Minikit plus DNase I treatment (Qiagen). After which RT-PCR was performed using the iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad, Hercules, CA). RNA was probed for HGF or c-Met (31), AchR subunits or GAPDH (Sigma Aldrich) using human-specific primers. Each reaction was performed in a total volume of 50 μL containing 2× SYBR Green, reverse transcriptase (Bio-Rad Laboratories), RNA template and 50 nmol/L of each specific primer utilizing the following thermocycler reaction: 50°C for 10 minutes, 95°C for 5 minutes, and 40 cycles of amplification (95°C for 10 seconds, 55°C for 30 seconds). All data were normalized to GAPDH expression. β1 nAChR subunit: forward primer, 5′-TCA GAA ATG GGT CCG CCC TG-3′; reverse primer, 5′-TCC TGT TTG AGC CAC ACA TTG GT-3′. α5 nAChR subunit: forward primer, 5′-CTA GGC TGA GGC TGC TGT CCC-3′; reverse primer, 5′-ATG GAG CAC TGA GTG TGA GTC GT-3′. α7 nAChR subunit: forward primer, 5′-TCC CCG GCA AGA GGA GTG AA-3′; reverse primer, 5′-GAG GGC GGA GAT GAG CAC AC -3′. α9 nAChR subunit: forward primer, 5′-ATG CAC CGG CCA TCA CCA AA-3′; reverse primer, 5′-GAT CTC CGC TGT CCA AGG CG -3′.

HGF and phospho-Met ELISA

As many as 106 TAS cells or 106 pancreatic cancer cells were plated in a 10-cm dish. After 24 hours of nicotine stimulation, culture media were collected and probed for HGF by ELISA (R&D Systems). For detection of phospho-Met in patient-derived tumor lysates, tumors were placed in cell-extraction buffer (Invitrogen) with complete Mini protease inhibitor cocktail (Roche) and lysed using the FastPrep-24 system according to the manufacturer's protocol (MP Biomedicals). Phospho-Met was determined using the PathScan Phospho-Met (panTyr) Sandwich ELISA (Cell Signaling Technology) according to the manufacturer's instructions.

siRNA knockdown/pharmacologic inhibition of c-Met expression/activity

Pancreatic cancer cells were transfected with control (100 pmol/L), c-Met (100 pmol/L), or c-Src (100 pmol/L) siRNA (Santa Cruz Biotechnology) using Oligofectamine reagent (Invitrogen). Pharmacologic inhibition of c-Met phosphorylation was performed using crizotinib (200 nmol/L). Western blot analysis was used to monitor the expression and phosphorylation levels of c-Met.

IHC and immunofluorescence

Sample processing and IHC staining was performed by the University of Florida's Molecular Pathology Core Facility. Briefly, patient tumors, xenografts and murine lung specimens were formalin-fixed and paraffin embedded. Sections measuring 5 μm were stained with hematoxylin and eosin (H&E). In some tissues, serial 5-μm sections were probed using anti-α5 AchR and anti-α7 AchR (Santa Cruz Biotechnology) or phospho-Met (Cell Signaling Technology) following antigen retrieval with citrate buffer pH 6.0 for tissue IHC analysis.

Immunofluorescence was performed on cells fixed for 10 minutes using paraformaldehyde followed by a 1 hour wash with 0.1% Triton X-100 in PBS with 3% BSA. Cells were then incubated overnight using a primary antibody to c-Met (Cell Signaling Technology). A goat anti-rabbit secondary antibody conjugated to AF647 was then applied with 4′,6-diamidino-2-phenylindole (DAPI) and Phalloidin stains (Life Technologies).

Surgical patient cohort

A review of an institutional review board–approved, prospectively maintained pancreatic cancer database at the University of Florida was performed. Informed consent was obtained from all patients. Data analyzed included all consecutive patients who underwent surgery for pancreatic adenocarcinoma with continued follow-up from 2000 to 2011 (n = 90). Histologic slides of pancreatic specimens were reviewed by a pathologist specializing in pancreatic cancer (C. Liu).

Statistical analysis

All statistical analyses were performed using SPSS version 22.0 (IBM SPSS Statistics for Windows; IBM Corp.). All in vitro and animal experimental groups were compared using the independent samples t test. Univariate and multivariate Cox proportional hazards models examined the effect of stage, grade, and smoking status on overall survival. All variables demonstrating an association with overall survival on univariate analysis (P < 0.20) were incorporated into multivariate analysis. A separate prospectively maintained database based on pancreatic cancer specimens obtained immediately upon resection (n = 26) was used to analyze phospho-Met levels in fresh tissue lysates. Patients were dichotomized to phospho-Met positive or negative based on a significant elevation of the intratumoral phospho-Met signal from background on ELISA. Kaplan–Meier survival curves were used to analyze overall survival, and the log-rank test was used to evaluate significance (P < 0.05).

Patient-derived murine xenografts

All animal studies were performed with approval from the University of Florida Institutional Animal Care and Use Committee. A 2×2 mm section of a surgically resected primary pancreatic adenocarcinoma or 2-mm core biopsy was implanted subcutaneously into 8-week-old female NOD-SCID IL2 receptor gamma chain knockout (NSG) mice (Jackson Laboratory; 20 mice). Mice were anesthetized using inhaled isofluorane during the procedure and administered two doses of buprenorphine immediately and 12 hours postoperatively. On postoperative day 5, mice with visible tumors were equally randomized by size to receive 1 mg/kg nicotine (n = 10) or an equal volume of PBS (n = 10) three times per week via intraperitoneal injections, as previously described (12). Tumor dimensions were measured three times per week using calipers. Tumor volumes were calculated using the equation: v = xy2/2, where v is volume, x is tumor length, and y is tumor width. Tumors were allowed to reach an endpoint of 2 cm in maximum diameter prior to euthanasia. Primary tumors and lungs were harvested for IHC analysis.

Results

Continued tobacco use following potentially curative pancreatic cancer surgery is associated with reduced overall survival

Continued tobacco use during cancer treatment is associated with reduced survival in both head and neck squamous cell cancers as well as small-cell lung cancers (4, 5). However, a comparable effect has not previously been demonstrated for patients battling pancreatic cancer. In order to determine the relationship between continued tobacco exposure after pancreatic cancer resection and survival, we analyzed postoperative survival in 90 consecutive patients with pancreatic cancer who underwent pancreaticoduodenectomy (PD) or distal pancreatectomy (DP) from 2000 to 2011 with curative intent. All patients had continued clinical follow-up at the University of Florida. Patients were categorized into three groups: those who continued to use tobacco (current smokers), had a remote tobacco history (former smokers), and had no history of tobacco use. Univariate analysis was performed using known prognostic clinicopathologic parameters. Positive lymph node ratio, previously established as a prognostic parameter in pancreatic cancer, proved to be a more significant predictor than N stage and was therefore incorporated into multivariate analysis of overall survival (32). All other variables demonstrating established trends with survival were then incorporated into a multivariate Cox regression. Our data demonstrated that poor tumor differentiation (HR, 2.03; P = 0.007) and continued tobacco abuse (HR 1.93; P = 0.040) significantly correlated with reduced overall survival (Table 1). Accordingly, quantitative assessment of smoking history in our pancreatic cancer population demonstrated a trend toward significance between estimated pack-years smoked and reduced survival on multivariate analysis (HR, 1.01 per pack-year; P = 0.082, data not shown). Thus, after controlling for tumor stage and grade, it was determined that continued tobacco use is independently associated with reduced overall survival in patients with surgically resected pancreatic cancer (Fig. 1A). Together, these data suggest that sustained tobacco use may directly influence the malignant progression of pancreatic cancer.

Figure 1.
Figure 1.

A, continued smoking reduces overall survival in patients with resected pancreatic cancer. Patients with pancreatic adenocarcinoma were followed postoperatively after pancreatic resection and a multivariate Cox proportional hazards model was used to evaluate the effects of continued tobacco abuse on overall survival. Continued smoking correlated with reduced survival in pancreatic cancer (HR 1.93; P = 0.040). P < 0.05 was considered statistically significant. B, TAS induces gemcitabine chemoresistance and Id1 expression in pancreatic cancer cells. TAS cells were cultured from human pancreatic tumors using the outgrowth method. Pancreatic cancer cells were cocultured with or without TAS in the presence or absence of gemcitabine at concentrations corresponding to LD50 values for each pancreatic cancer cell line. C, TAS coculture induces Id1 expression in pancreatic cancer cells. β-Actin was used as a loading control. D–F, TAS cells secrete HGF while pancreatic cancer cells express c-Met and Id1. D, qRT- pancreatic cancer R (left) and ELISA (right) were performed for HGF expression and secretion in TAS and pancreatic cancer cells. Data, mean ± SD of three independent experiments (*, P < 0.01 TAS vs. pancreatic cancer cell lines). E, protein lysates from TAS and pancreatic cancer cell lines indicated were harvested and probed for Id1 (right) and c-Met (left). β-Actin was used as a loading control. F, immunofluorescent stains viewed at 20× magnification were performed on cells in culture of the indicated cell type. c-Met is depicted here in red, while phalloidin staining appears in green. Phalloidin stains are displayed to indicate the characteristic stress fiber organization of TAS cells. Goat anti-mouse AF647-stained controls are displayed in the far right column. Scale bar, 10 μm.

View this table:
Table 1.

Survival analysis

TAS induces gemcitabine chemoresistance in pancreatic cancer cells through paracrine signaling

TAS cells have been implicated in the promotion of pancreatic cancer cell growth, invasion, metastasis, and chemoresistance via paracrine signaling through multiple growth factor pathways (33, 34). To investigate the role of TAS in chemoresistance, we harvested primary TAS cells from surgically resected pancreatic cancer specimens with an established outgrowth method (28). To determine the effects of cultured TAS on gemcitabine resistance in pancreatic cancer cells, coculture of TAS outgrowths with the pancreatic cancer cell lines was performed using Transwell inserts. PANC-1 and L3.6pl were selected for analysis as they represent both moderately resistant and highly sensitive cell lines to gemcitabine, respectively. As demonstrated in Fig. 1B, 48 hours of gemcitabine treatment reduced the viability of pancreatic cancer cells but coculture of pancreatic cancer cells with TAS resulted in gemcitabine chemoresistance. We have previously identified the Id1 transcriptional repressor as an essential mediator of nicotine-mediated pancreatic cancer cell chemoresistance (12). TAS-mediated induction of Id1 in pancreatic cancer cells was therefore evaluated. Indeed, TAS-conditioned media induced Id1 expression in pancreatic cancer cells in a time-dependent manner in a variety of pancreatic cancer cell lines (Fig. 1C). Thus, paracrine interactions between TAS and pancreatic cancer cells induce a similar chemoresistant phenotype as previously demonstrated with nicotine.

Nicotine treatment augments HGF–MET-mediated paracrine signaling between TAS and pancreatic cancer cells

Although c-Met has been established as a pancreatic cancer stem cell marker that contributes to chemoresistance, the responsible signaling events remain poorly defined (35). In order to determine whether paracrine HGF–MET signaling could result in downstream induction of Id1, we first determined the expression of HGF, c-Met, and Id1 by both TAS and pancreatic cancer cells. qRT-PCR and ELISA confirmed both expression and secretion of the HGF ligand in patient-derived TAS cultures, while representative pancreatic cancer cell lines displayed low to undetectable levels (Fig. 1D). Conversely, pancreatic cancer cell lines, but not TAS, expressed Id1 and c-Met (Fig. 1E). c-Met expression in pancreatic cancer cells alone was further supported by immunofluorescent microscopy, confirming the expression of c-Met in pancreatic cancer cells and the absence of c-Met in TAS even upon coculture with pancreatic cancer cells (Fig. 1F). These data support a potential paracrine signaling mechanism whereby HGF secreted by TAS is able to interact with its binding partner c-Met in pancreatic cancer cells to induce Id1-mediated chemoresistance.

We next asked if nicotine could influence the tumor microenvironment by increasing secretion of the HGF ligand in TAS cells and c-Met expression in pancreatic cancer cells. As in Fig. 2A, nicotine treatment stimulated HGF expression and secretion in TAS cells. Additionally, nicotine exposure led to increased c-Met levels in pancreatic cancer cells (Fig. 2B) and this c-Met induction appeared to be occurring at the posttranscriptional level, as qRT-PCR demonstrated no change in c-Met transcription with nicotine treatment (Supplementary Fig. S1). Previous work has identified Src as an early mediator of nicotine stimulation in cancer cells (12, 36). To assess whether the induction of c-Met was similarly dependent on Src expression, Src siRNA treatment was incorporated into nicotine stimulations, demonstrating that nicotine-mediated c-Met induction was indeed Src dependent in pancreatic cancer cells (Supplementary Fig. S2). We then questioned whether nicotine stimulation alone could lead to persistent c-Met activation in direct TAS/pancreatic cancer cell cocultures. Indeed, phospho-Met immunofluorescence at 48 hours in coculture demonstrated c-Met activation only in cocultures exposed to nicotine, which was abrogated by pharmacologic c-Met inhibition using crizotinib (Fig. 2C). Importantly, nicotine stimulation did not lead to c-Met activation in pancreatic cancer cells alone (Supplementary Fig. S3).

Figure 2.
Figure 2.

Nicotine augments expression of HGF and c-Met in TAS and pancreatic cancer cells, respectively. A, TAS were stimulated with nicotine and RNA, supernatants or protein lysates were harvested for ELISA (left) and qRT-PCR (right) for analysis of HGF expression. B, pancreatic cancer cells were stimulated with nicotine and Western blot analysis of c-Met induction was performed. β-Actin was used as a loading control. C, c-Met activity was evaluated in pancreatic cancer cells alone and direct TAS/pancreatic cancer cell coculture at 48 hours with and without nicotine stimulation (1 μmol/L) and crizotinib treatment (200 nmol/L). Phospho-Met staining (top, green) was superimposed upon phase-contrast images with DAPI stains shown separately (bottom, blue). D, qRT-PCR was also utilized to evaluate the expression of nicotinic AchR subunits within the TAS in the absence of nicotine exposure. Data, mean ± SD of three independent experiments.

Given the physiologic response of TAS and pancreatic cancer cells to nicotine treatment, the repertoire of nicotine receptors and their subunits on these cell types was briefly evaluated. Our previous work has already established that pancreatic cancer cells express high levels of the α7 nAchR subunit, which was required for subsequent Id1 induction (12). Interestingly, qRT-PCR assay of the nAchR subunits β3, α5, α7, and α9 on a representative sample of patient-derived TAS cells revealed high expression of the α5 nAchR subunit (Fig. 2D). While the mechanistic role of these subunits in nicotinic signaling within TAS cells remains speculative, these original observations support the presence of nicotinic receptors on TAS cells and therefore support a potential role for nicotine in the pancreatic tumor microenvironment.

TAS-mediated paracrine signaling induces Id1 expression through an HGF–MET-dependent mechanism

We have previously demonstrated the importance of Id1 expression in pancreatic cancer progression and chemoresistance (12). To assess whether Id1 expression could be influenced by TAS cells in a c-Met–dependent manner, pancreatic cancer cells were silenced of c-Met followed by stimulation with TAS-conditioned media. Inhibition of c-Met expression abrogated the observed induction of Id1 by TAS-conditioned media (Fig. 3A), suggesting a c-Met–dependent-Id1 signaling axis. To determine whether the Id1 induction observed was specifically due to the HGF ligand and no other potential soluble mediators within the TAS-conditioned media, we performed the following experiments. Pancreatic cancer cells were stimulated with physiologic levels of HGF (50 ng/mL) alone and Id1 expression evaluated (Fig. 3B). Id1 expression was induced in a similar time-dependent manner as with the addition of TAS-conditioned media. This HGF induction of Id1 was abrogated by siRNA knockdown of c-Met (Fig. 3C) in a manner similar to the results noted in Fig. 3A. Further, Id1 induction in response to HGF (Fig. 3D) or TAS-conditioned media (Fig. 3E) was abrogated by the FDA-approved targeted c-Met tyrosine kinase inhibitor crizotinib. These results demonstrate that pancreatic cancer cell Id1 induction from adjacent TAS signaling is dependent on activation of the HGF–MET cascade. Taken together, we suggest that nicotine may enlist the stromal compartment to induce chemoresistance by augmenting the c-Met signaling pathway in pancreatic cancer cells.

Figure 3.
Figure 3.

TAS-mediated induction of Id1 expression in pancreatic cancer cells is c-Met dependent. A (left to right), pancreatic cancer cell lines were transfected with c-Met or control siRNA and either exposed to TAS-conditioned media or media control. Protein lysates were harvested, and the expression of c-Met was evaluated by Western blot. B, HGF induces Id1 expression in pancreatic cancer cells. C (left to right), pancreatic cancer cell lines were transfected with c-Met or control siRNA and were exposed to HGF, and the expression of Id1 was evaluated by Western blot. D (left and right), crizotinib inhibits HGF induction of c-Met phosphorylation in pancreatic cancer cells. E (left and right), pancreatic cancer cell lines were exposed to c-Met inhibitor crizotinib, TAS-conditioned media, or combination. Protein lysates were harvested and probed for Id1 expression by Western blot. β-Actin was used as a loading control. Data, immunoblots of three independent experiments.

Nicotine administration induces tumor growth and metastasis and promotes HGF expression and c-Met activation

Our previous data demonstrated the effects of nicotine on primary cancer cell growth in an orthotopic pancreatic cancer xenograft model (12), but this model is unable to suggest an effect of nicotine on the more representative stromal-rich tumor microenvironment. Therefore, to support our in vitro data, we examined the effects of nicotine on pancreatic cancer and its closely related stromal environment by utilizing a patient-derived pancreatic cancer xenograft model, previously validated to preserve both genetic and epigenetic elements of the original tumor (37). Prior to our in vivo experiments with nicotine, confirmation of the presence of nicotinic receptors in surgically resected pancreatic cancer tissue specimens utilized in our xenograft model was performed. Supporting our in vitro data, α7 subunits localized to pancreatic cancer cells, while α5 displayed a more diffuse pattern, staining the surrounding, desmoplastic stromal elements (Fig. 4A).

Figure 4.
Figure 4.

Nicotine augments tumor growth and metastasis in a patient-derived pancreatic adenocarcinoma xenograft model. A, primary human pancreatic cancer specimens and normal pancreas controls were subjected to IHC analysis of α5 and α7 nicotinic AchR subunit staining. Images are viewed at 10× magnification. B, animals with palpable patient-derived tumors were administered physiologic doses of nicotine or control. Tumor dimensions were measured with calipers 3 times per week, and volumes were calculated over 45 days. Data, mean ± SEM (*, P < 0.05 vs. control). C–E, xenograft lysates from nicotine-treated and control animals were subjected to qRT-PCR for HGF expression (C), ELISA (D), and Western blot analysis (E) for c-Met activation. F, two representative lungs from nicotine-treated and control animals were subjected to H&E analysis, viewed at 20× magnification.

Tumor-bearing mice that received physiologic doses of nicotine (1 mg/kg) experienced significantly augmented tumor growth as compared with nicotine-free controls (Fig. 4B). Supporting our in vitro results, analysis of xenograft lysates revealed significantly increased HGF expression in tumors from nicotine-treated mice compared with those from controls (Fig. 4C). Next, we determined if nicotine induced activation of c-Met within the tumor microenvironment. Tumor-bearing mice treated with nicotine demonstrated consistently elevated levels of phosphorylated c-Met (Fig. 4D and E). Additionally, nicotine administration induced pulmonary metastasis by H&E examination in 5 of 8 (62%) treated animals, as opposed to none of the (0 of 8) controls (Fig. 4F). Hepatic metastasis was not observed in either group. Together, the in vivo data support the mechanisms delineated in vitro whereby nicotine augments the HGF–MET signaling cascade by induction of HGF secretion in TAS and c-Met expression in pancreatic cancer cells. These data support a role for nicotine on a more representative intact pancreatic tumor and its supporting microenvironment, strongly correlating with accelerated tumor growth and metastasis in vivo.

Phosphorylated c-Met directly correlates with reduced overall survival in pancreatic cancer

Given the relationship between activated c-Met and tumor growth and metastasis in our xenograft model, the prognostic value of activated c-Met levels in resected human pancreatic cancer specimens was evaluated. Specifically, activated c-Met content in fresh tumor lysates from a cohort of 26 surgically resected patients with pancreatic cancer was quantitatively evaluated using an ELISA (Fig. 5A). In addition, phospho-Met expression was confirmed by IHC staining on representative samples (Fig. 5A, inset). Here, elevated intratumoral phospho-Met levels correlated with reduced survival when evaluated in a continuous fashion using a Cox proportional hazards model (HR 11.1, P = 0.049). Kaplan–Meier survival curves were generated upon dichotomization of tumors to phospho-Met–positive and –negative groups based on significantly elevated phospho-Met signals from background on ELISA. Notably, not one single patient with a positive phospho-Met cancer survived 1 year postoperatively (median OS 6.1 vs. 15.2 months; P = 0.028; Fig. 5A). Taken together, these results indicate that c-Met activation, implicated by our model of nicotine-mediated pancreatic cancer progression, portends a poor prognosis in pancreatic cancer.

Figure 5.
Figure 5.

Activated c-Met in pancreatic cancer specimens correlates with reduced postoperative survival. A, intratumoral c-Met activation was measured using a phospho-Met ELISA on tumor lysates, and representative samples were verified using IHC (insets). Kaplan–Meier survival curves were generated through stratification of expression or not of phospho-Met within the primary tumor of 26 patients with pancreatic adenocarcinoma for whom clinical follow-up was obtained. Significance was determined using log-rank analysis. B, a summary model is depicted by which nicotine stimulates HGF secretion in TAS while directly inducing c-Met expression in pancreatic cancer cells, thereby augmenting c-Met activation in a paracrine manner.

Discussion

Data presented here provide strong evidence of an association between nicotine exposure and reduced survival in patients with resected pancreatic cancer. Additionally, we demonstrate an equally important role for nicotine on the desmoplastic pancreatic cancer tumor microenvironment. Specifically, in addition to promoting pancreatic cancer growth and chemoresistance directly through the induction of Id1, nicotine concurrently amplifies paracrine HGF–MET signaling in the tumor microenvironment. This mechanism functions through the induction of HGF in TAS cells and subsequent c-Met activation in pancreatic cancer cells, which is both necessary and sufficient for TAS-induced Id1 expression in pancreatic cancer cells (Fig. 5B). Accordingly, induction and upregulation of the HGF–MET axis in patient-derived pancreatic cancer xenografts following nicotine treatment was associated with rapid tumor growth and metastasis. Supporting our preclinical work, when we evaluated our surgically resected patient population, c-Met activation in human pancreatic cancer specimens was a predictor of poor survival.

Our mechanistic findings support the work of others investigating the effects of nicotine in cancer. Chellappan and colleagues have demonstrated nicotine-induced proliferation and invasion in a variety of cancer types (7, 8, 38). Our previous work built on this foundation, by demonstrating that nicotine-mediated pancreatic cancer growth, metastasis and chemoresistance is Id1 dependent (12). Similarly, our findings regarding the significance of activated c-Met in pancreatic cancer corroborate the work of Simeone and colleagues identifying c-Met as a putative marker of pancreatic cancer stem cells (17, 35). In addition, c-Met inhibition with such tyrosine kinase inhibitors as crizotinib has demonstrated success in preclinical work and is currently used by multiple clinical trials (16), lending further translational relevance to the investigation of the role of HGF–MET signaling in pancreatic cancer. Given the mixed results of c-Met inhibitors in clinical trials thus far, patient selection will be critical to delineate proper application of these therapies (39). Here, we present preliminary data supporting a simple clinical tool, a recent history of nicotine exposure, as a part of that selection process.

Here, we demonstrate that the mechanism by which nicotine increases c-Met levels in pancreatic cancer cells is Src dependent. Previous work has identified β-arrestin as a key scaffolding protein allowing nAChR-mediated activation of Src (36). The direct binding of β-arrestin and Src demonstrated in these investigations would suggest a role for Src as an early mediator of the nAChR response. We find that c-Met induction is downstream of Src and occurs at the posttranscriptional level. Control of c-Met levels in this manner has been demonstrated previously through sequestration of the E3-ubiquitin ligase responsible for the ubiquitination of c-Met in the Golgi apparatus (40). However, the precise interplay between Src and c-Met upon nicotine stimulation remains speculative at this point.

Although we report strong correlations between smoking and survival as well as intra-tumoral phospho-Met levels and survival, the link between smoking and c-Met activity in patient tumors has not yet been characterized but is critical to our future work. Importantly, nicotine-mediated upregulation of c-Met occurs regardless of basal c-Met expression levels amongst pancreatic cancer cell lines. However, because there is high variability in observed c-Met expression in human pancreatic cancer specimens, a large number of patients would be required to confirm the relationship between nicotine exposure and c-Met activation within human pancreatic cancer specimens, which we are currently accumulating.

An additional limitation of this work is the lack of pharmacologic inhibition of c-Met in vivo. However, the correlation between nicotine administration, tumor growth, intratumoral HGF expression, and phospho-Met levels suggests that nicotine induces HGF–MET signaling within the tumor microenvironment, which is a known promoter of tumor growth and metastasis (34). While the addition of nicotine with c-Met tyrosine kinase inhibition in a patient-derived xenograft model with an intact tumor microenvironment is a future direction, there are published reports demonstrating the importance of the HGF/c-Met cascade in pancreatic cancer in vivo (35, 41–43).

Crizotinib is known to inhibit multiple tyrosine kinase receptors. Thus, despite abrogation with c-Met knockdown, TAS-mediated Id1 induction in pancreatic cancer cells may not be solely dependent on HGF–MET signaling. A recent analysis of crizotinib activity in pancreatic cancer cell lines implicated ALK inhibition as the primary mechanism of action rather than direct effects on c-Met (44). In this study, ALK inhibition contributed to reduced pancreatic cancer cell viability in isolated crizotinib-treated pancreatic cancer cells. Conversely, others have demonstrated that c-Met represents the therapeutic target in pancreatic cancer and that gemcitabine acts synergistically with crizotinib in an orthotopic xenograft model (41). Both investigations utilized a xenograft model using established pancreatic cancer cell lines, which lack the human fibrous stromal component responsible for HGF production. Therefore, HGF–MET paracrine signaling is not clinically applicable in the context of isolated pancreatic cancer cells in vitro or injected pancreatic cancer cell xenografts. Our data would therefore suggest that c-Met inhibition may still be a more prominent mechanism mediating the antitumor effects of crizotinib in an intact tumor microenvironment containing up to 80% HGF-secreting stromal cells by mass, which is supported by multiple recent investigations (41, 45–47).

Accumulating evidence suggests a prominent role for the induction of Id1 as a central event in angiogenesis and tissue regeneration (48, 49). However, the relationship between HGF and Id1 appears to be specific to the environment in question. Recent experiments in hepatoma cell lines suggest that HGF induces cell-cycle arrest via the inhibition of Id1 expression in this population (50). On the other hand, our findings are consistent with other literature that demonstrated that HGF–MET–Id1 signaling supports a prosurvival, regenerative mechanism in epithelial cells (15). In addition, investigations into fracture repair have also demonstrated active paracrine HGF stimulation, which leads to Id1 expression and subsequent wound healing (51). Rafii and colleagues examined Id1 in the context of hepatic regeneration, that paradoxically suggested that Id1 expression by endothelial cells leads to HGF secretion which then fuels c-Met activation and proliferation in nearby hepatocytes (52). Alternatively, Apte and colleagues recently demonstrated that HGF secreted from TAS fuels endothelial cell proliferation and tube formation, providing another potential microenvironment-dependent tumor-promoting mechanism (53). Taken together, this literature suggests that functional outcomes of the HGF–MET–Id1 axis are environmentally and cell type dependent.

Finally, we cannot exclude the possibility that non-nicotine components of cigarette smoke may be responsible for reduced survival in patients with pancreatic cancer. Indeed, multiple investigations implicating cigarette smoke in the promotion of cancer cell growth and metastasis describe mechanisms that might not be fully dependent on nicotine (54–56). However, the isolation of nicotine-specific effects in pancreatic cancer is especially relevant given the current social climate regarding electronic cigarettes (e-cigarettes). Widely touted as a healthier alternative to smoking despite the induction of comparable serum nicotine levels, electronic cigarettes remain clinically concerning (10). Further, recent investigations indicate a latency time of approximately 10 years between the initiating mutation and the “birth” of the pancreatic cancer–initiating cell, with metastatic potential likely requiring an additional 5 years (57). These data suggest a 15-year latency time prior to diagnosis. Given current epidemiologic evidence that 80% to 85% of patients diagnosed with pancreatic cancer present with advanced lesions (58), we must be aware of such tumor promoting “healthier alternatives” to smoking that might be influencing tumor progression. Taken together with data presented here regarding nicotine-induced tumor-promoting effects through direct and paracrine signaling mechanisms, a robust case is established against the systemic administration of nicotine with respect to progression of established as well as potentially undiagnosed malignancies.

Disclosure of Potential Conflicts of Interest

T. George reports receiving a commercial research grant from Bristol-Meyers Squibb. No potential conflicts of interest were disclosed by the other authors.

Authors' Contributions

Conception and design: D. Delitto, G.A. Sarosi, K.E. Behrns, S.J. Hughes, J.G. Trevino

Development of methodology: D. Delitto, S. Han, G.A. Sarosi, R.M. Thomas, S.J. Hughes, J.G. Trevino

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Delitto, B.S. Black, A.E. Knowlton, A.C. Vlada, K.E. Behrns, C. Liu, S.J. Hughes, J.G. Trevino

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Delitto, A.C. Vlada, G.A. Sarosi, K.E. Behrns, R.M. Thomas, X. Lu, T.J. George, S.J. Hughes, S.M. Wallet, J.G. Trevino

Writing, review, and/or revision of the manuscript: D. Delitto, A.C. Vlada, G.A. Sarosi, K.E. Behrns, R.M. Thomas, X. Lu, T.J. George, S.J. Hughes, S.M. Wallet, J.G. Trevino

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Delitto, D. Zhang, S. Han, A.E. Knowlton, S.M. Wallet, J.G. Trevino

Study supervision: D. Delitto, S.J. Hughes, J.G. Trevino

Grant Support

This study was supported by grant NCI 5T32CA106493-09 and the Cracchiolo Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • Received May 30, 2015.
  • Revision received November 17, 2015.
  • Accepted November 26, 2015.

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Clinical Cancer Research: 22 (7)
April 2016
Volume 22, Issue 7

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Nicotine Reduces Survival via Augmentation of Paracrine HGF–MET Signaling in the Pancreatic Cancer Microenvironment
Daniel Delitto, Dongyu Zhang, Song Han, Brian S. Black, Andrea E. Knowlton, Adrian C. Vlada, George A. Sarosi, Kevin E. Behrns, Ryan M. Thomas, Xiaomin Lu, Chen Liu, Thomas J. George, Steven J. Hughes, Shannon M. Wallet and Jose G. Trevino
Clin Cancer Res April 1 2016 (22) (7) 1787-1799; DOI: 10.1158/1078-0432.CCR-15-1256
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