A nicotine-induced positive feedback loop between HIF1A and YAP1 contributes to epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma

The human PDAC cell lines, Panc-1 and BxPC-3, were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell lines were authenticated by analysis of short tandem repeats, and were confirmed to be free of mycoplasma contamination at the beginning of this study. All cell lines were used for experiments within 30 passages from thawing, and were maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% FBS at 37 °C in a humidified atmosphere of 95% air and 5% CO₂.

A total of 173 sequential formalin-fixed paraffin-embedded (FFPE) PDAC tissue samples were available from cases who had received radical surgery at the Changhai Hospital (Shanghai, China). All samples with tumor tissues (TTs) and matched adjacent non-cancerous tissues (ANTs) were used to construct a tissue microarray (TMA). No patients had a history of chemotherapy or radiotherapy prior to surgery. Pathological diagnosis was assessed according to the 7th edition of the American Joint Committee on Cancer (AJCC) staging system [24]. Retrospective clinicopathologic data were also obtained from these patients. Participants were defined as “ever-smokers” if they had smoked more than 100 cigarettes during their lifetime. Accordingly, “never smokers” were determined if they smoked less than 100 cigarettes during their lifetime. The use of these specimens and patient information was approved by the Ethics Committee of at the Changhai Hospital (Shanghai, China) in accordance with recognized ethical guidelines of Declaration of Helsinki.

For IHC assessment of YAP1, HIF1A, E-cad, and Vim in human PDAC tissues, the DAKO Envision system (Dako, Carpinteria, California, USA) was used, as described previously [25]. Briefly, after paraffin-embedded sections of tumor tissues were heated, the sections were incubated with primary antibodies (Supplementary Table s1) overnight at 4 °C. A DAB substrate kit (Dako) was used to perform the chromogenic reaction. IHC staining scores for YAP1, HIF1A, and E-cad were obtained by multiplying the intensity (0, negative; 1, low; 2, medium; and 3, high) with the extent of staining (0, 0%; 1, 0–10%; 2, 10–50%; 3, 50–75%; 4, > 75%). The final scores were used to classify the samples into three grades: 0–3, weak staining (+); 4–6, medium staining (++); and 7–12, strong staining (+++). For subsequent analyses, weak and medium staining were grouped together as a low expression (score 0–6), and strong staining was considered as high expression (score 7–12). For Vim evaluation, the IHC staining score was classified by the area of positive-staining stroma into four categories: score 0 (negative, < 25%), score 1 (focal, 25–50%), score 2 (multifocal, 50–75%), or score 3 (diffused staining, 75–100%). Two pathologists, who were blinded to the clinical data, independently scored all sections. For evaluation of Ki67, the number of positive cells was calculated in three representative areas of high staining.

IF staining was performed on PDAC cell lines and tissue samples. Briefly, cells were fixed in 4% polyformaldehyde, washed in PBS, and blocked with 5% BSA in PBS. Then, these cells or frozen sections were incubated with primary antibodies against E-cad (1:100), Vim (1:200), and YAP1 (1:100) at 4 °C overnight, and subsequently incubated with fluorescent dye–labeled secondary antibodies at room temperature for one hour. DAPI (1:1000) was used to counterstain cell nuclei and images were captured with a confocal fluorescence microscope.

We treated Panc-1 cells with Nic (1.0 μM) or DMSO for 24 h. Total RNA was extracted from the cells and analyzed on an Arraystar Human LncRNA Microarray V4.0 Microarray, provided by Kangchen Biotech (Shanghai, China). Cells were prepared and analyzed in three independent biological replicates. Differences between groups were analyzed using the significance analysis of microarrays (SAM) algorithm.

The lentivirus-delivered shRNAs against YAP1 (Lv-shYAP1) and the negative control (Lv-shNC) were acquired from Shanghai GeneChem (Shanghai, China). The viral particles and the establishment of shYAP1 stable clones in Panc-1 cells were performed according to the manufacturer’s instructions [26]. The control clone (shNC) was constructed similarly. The transfection efficiency was confirmed by immunoblotting. Of the two stable cell lines, we selected the cell line exhibiting the most efficient knockdown of YAP1 for the xenograft tumor models.

The expression plasmids, pcDNA4-YAP1 and pcDNA3.0-HIF1A, were purchased from Addgene (Boston, USA). For knockdown studies, siRNA targeting human YAP1 (siYAP1) and HIF1A (siHIF1A) were synthesized. The targeting sequences are shown in Supplementary Table s2. Non-targeting siRNAs (siNCs) and negative control empty vectors (EVs) were used as controls for the siRNA and expression plasmids, respectively. Plasmids or siRNAs were transiently transfected into PDAC cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.

Total RNA was prepared from fresh PDAC tissues and cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. qRT-PCR was conducted in a 96-well real time PCR system (Bio-Rad Inc., Hercules, CA) using a SYBR® Premix Ex Taq kit (Takara Bio Inc., Shiga, Japan). GAPDH was used to normalize gene expression. The primers are shown in Supplementary Table s2. Relative gene expression was calculated from the qRT-PCR data using the 2 ^−ΔΔCT method.

Total protein was extracted from cells and tissue using RIPA buffer containing a mixture of protease inhibitors. Equal amounts of total protein (30 μg) were subjected to SDS- polyacrylamide gel separation, followed by overnight incubation at 4 °C with primary antibodies against: YAP1, pYAP1, HIF1A, E-cad, Vim, N-cad, Claudin-1, MST1, LATS1, CHRNA3, CHRNA5, CHRNA7, and GAPDH. Anti-mouse IgG and anti-rabbit IgG HRP-conjugated secondary antibodies were used. Protein expression levels were quantified by normalizing to the GAPDH band using image J software (National Institutes of Health, Bethesda, MD, USA).

The HREs in the YAP1 promoter were predicted using the JASPAR database (http://jaspar. genereg.net/). Genomic DNA was extracted from Panc-1 cells following the manufacturer’s instructions, and the different truncated mutant YAP1 regulatory regions were amplified by PCR. The primers used to amplify the truncated YAP1 promoter regions are shown in Supplementary Table s2. YAP1 promoter region fragments were inserted into the pGL3 vector (Promega, Madison, WI), as described previously [26]. The YAP1 promoter reporter plasmids were co-transfected with HIF1A, or empty vector (EV) into cells. For the HIF1A transcriptional activity assay, pGL4-HREs-luciferase plasmid (4 μg) and pRL-TK plasmid (4 μg) were incubated with PDAC cells. These cells were also incubated with 1.0 μm Nic or DMSO. After 24 h, luciferase activity was detected using the Dual-luciferase reporter assay system (Promega, Madison, WI).

For ChIP assay, PDAC cells were treated with 1.0 μm Nic or DMSO for 12 h, as described previously [27]. Briefly, protein-DNA complexes were produced by adding 1% formaldehyde to the cells, and the chromatin was sheared by sonication to a mean fragment size of 300–500 bp. Cells were immunoprecipitated overnight with an anti-HIF1A antibody or rabbit IgG, and the associated genomic DNA was assessed by PCR and agarose gel electrophoresis. The specific primers for putative HREs in the YAP1 promoter are shown in Supplementary Table s2.

To evaluate cell proliferation rates, MTT assay was used. Briefly, cells were seeded in 96-well plates at 2 × 10³ cells/well and incubated with DMSO or 1.0 μm Nic. Sample absorbance at 490 nm was evaluated on a microplate spectrophotometer (Thermo, Spectronic, Madison, WI, USA). For the cell scratch–wound assays, cells were cultured in six-well plates until confluent, and horizontal streaks were created in the cells using the a 20-μL pipette tip. Then, cells were washed and incubated with DMSO or 1.0 μm Nic. An inverted microscope was used to measure the migratory distance at 0 h and 24 h, and cell migration was assessed by measuring gap sizes in multiple fields. For transwell assays, cells (1.0 × 10 [5]/ml) were placed in the top side of transwell chambers (8 μm pore size membranes, Millipore) with matrigel for invasion. Vehicle (DMSO) or nicotine was added into the upper well for 24 h. The invaded cells were fixed, stained and counted in five random fields.

All animal studies were performed following the Institutional Animal Care and Use Committee of Shanghai Jiaotong University (Shanghai, China). Six-week-old male BALB/c mice were obtained from Shanghai SLAC Laboratory Animal Center (Shanghai, China). All animals were maintained in a barrier facility in high-efficiency particulate air–filtered racks. Logarithmic phase Panc-1 cells (5.0 × 10 [6]/100 μL) transfected with Lv-shYAP1 or control vector were inoculated subcutaneously into the dorsal flank of mice. Tumor volume was evaluated by the following formula: volume (mm³) = length × width × height × 0.52. When tumor volume reached 75–125 mm³, mice were randomized into three groups, and that day was defined as day 1. Nicotine or DMSO was administered intraperitoneally thrice weekly for 3 weeks. On day 22, all mice were euthanized, and the tumors were excised and weighed.

Multiple databases, including GEPIA (http://​gepia.​cancer-pku.​cn/​) [28], StarBase 3.0 (http://​starbase.​sysu.​edu.​cn/​) [29], and KM plotter (http://​kmplot.​com/​analysis), were queried for gene expression in PDAC tissues. Data are shown as mean ± SD. Differences between groups were evaluated using unpaired t-test for two groups or the chi square test. Survival analyses were performed using the Kaplan-Meier method with the log-rank test and univariate and multivariate Cox regression. All statistical analyses were performed using the PASW Statistics 19.0 software program (SPSS, Chicago, IL, USA). A two tailed p-value of P < 0.05 was considered to be statistically significant. In the graphed data *, **, and *** denote p values of P < 0.05, P < 0.01, and P < 0.001, respectively.

Our previous research indicated that nicotine induces an EMT process in PDAC cells [30]. Here we examined whether there is an association between smoking status and expression of EMT markers in human PDAC tissues. Using IHC assays, we found that expression levels of E-cad were significantly lower, while expression levels of Vim were higher, in PDAC tumor tissue (TT) samples from patients who were “ever smokers” than from patients who were “never smokers” (Supplementary Fig. s1a and b). A similar difference was also observed in adjacent non-tumor (ANT) pancreas samples (Supplementary Fig. s1c and d). These results were further confirmed by IF assays in human PDAC tissues (Supplementary Fig. s1e and f). Treatment of Panc-1 cells with 1.0 μM Nic in vitro also significantly increased Vim expression, as demonstrated by IF analysis (Supplementary Fig. s1g). Additionally, phase contrast microscopy revealed EMT-like morphological changes in Panc-1 cells treated with 1.0 μM Nic (Supplementary Fig. s1h). These data indicate that smoking/nicotine exposure can stimulate EMT processes in human PDAC cells and tissues.

To investigate gene expression altered by nicotine exposure in Nic-treated PDAC cells, we screened differential mRNA expression profiles of Panc-1 cells treated with nicotine or DMSO by high-throughput microarray. Following 1.0 μM Nic treatment for 24 h, 118 mRNAs were found to be dysregulated (fold change ≥2.0, data not shown). The top 25 upregulated and downregulated mRNAs, are illustrated in a heat map (Fig. 1a). The YAP1 gene, which has been reported to enhance PDAC progression, was upregulated 5.31-fold in nicotine-treated cells. Subsequently, we evaluated the effects of treatment with nicotine on YAP1 expression in Panc-1 and BxPC-3 cells, and found that YAP1 mRNA (Fig. 1b) and protein (Fig. 1c) were significantly upregulated by exposure to nicotine in a dose-dependent manner, as compared to DMSO-treated cells. We knocked down YAP1 in two PDAC cell lines with two YAP1-targeting siRNAs. The siYAP1#2 siRNA exhibited higher knockdown efficiency (Supplementary Fig. s2a-b), and was used to perform subsequent siRNA-mediated YAP1 knockdown experiments. We also overexpressed YAP1 in two PDAC cell lines (Supplementary Fig. s2c-d). Cell proliferation, migration, and invasion induced by nicotine was significantly attenuated in YAP1-silenced PDAC cells, while YAP1 overexpression strengthened these effects (Supplementary Fig. s2e-j). In addition, treatment with nicotine led to an enhanced EMT process, which was attenuated by YAP1 knockdown and strengthened by YAP1 overexpression (Fig. 1d-g). We further used IHC to assess the expression levels of YAP1, E-cad, and Vim in human PDAC tissue samples stratified according to smoking status. We found that “ever smokers” with PDAC had higher expression levels of YAP1 in both TT and ANT pancreas samples, compared with “never smokers” (Supplementary Fig. s3 a-b). In addition, expression of YAP1 was inversely correlated with E-cad in cancer specimens (Spearman’s r = − 0.238, P < 0.001), but was positively associated with Vim expression (Spearman’s r = 0.219, P < 0.001; Fig. 1h-i). Furthermore, we discovered that YAP1 was positively correlated with the majority of EMT markers in PDAC tissues from the cancer genome atlas (TCGA) dataset (Supplementary Fig. s4 a-h). Based on these data, we hypothesized that YAP1 may play an important role in regulating nicotine-induced EMT in PDAC.

Fifteen nude mice challenged with subcutaneous pancreatic xenograft tumors were randomized into three groups (Fig. 2a). The animal experiments demonstrated that YAP1 knockdown inhibited nicotine-enhanced tumor growth, and reduced expression of Ki67 and YAP1 (P < 0.01; Fig. 2b-d). Immunoblot and IF assays on the xenograft tumors revealed that YAP1 knockdown significantly decreased nicotine-promoted EMT processes (Fig. 2e-f). As an important transcriptional co-activator, YAP1 can change the expression of various target genes, including CTGF, CDX2, CYR61, and CDC20 [17]. We found that knockdown of YAP1 significantly inhibited nicotine-stimulated upregulation of CTGF, CDX2, CYR61, and CDC20 (Fig. 2g). These data demonstrate that YAP1 may mediate the tumor-promoting role of nicotine in PDAC in vivo.

We examined the expression of YAP1 in human PDAC (n = 173) in a TMA-based IHC study. The clinicopathologic characteristics of the TMAs are shown in Supplementary Table s3. YAP1 protein expression levels in PDAC specimens were much higher than those in ANT specimens (Fig. 3a-b and Supplementary Fig. s5a). qRT-PCR analysis of the mRNA expression levels of YAP1 in paired normal pancreatic tissue and PDAC specimens (n = 12) confirmed this finding, which was also confirmed in gene expression analysis from the TCGA (n = 179; Fig. 3c-d). We then evaluated the association between YAP1 expression and clinicopathological features in PDAC cases. Although there were no significant associations between YAP1 expression and sex, age, tumor size, T category, and neural invasion (data not shown), chi-square analyses confirmed that higher expression of YAP1 was positively associated with lymph mode invasion (Fig. 3e). Analysis of the data from TCGA validated these findings (Fig. 3f). Additionally, Kaplan-Meier survival analysis from our data and from the TCGA dataset showed that the survival time for patients with low expression levels of YAP1 was significantly longer than patients with high expression levels of YAP1 (Fig. 3g-h). Univariate and multivariate Cox analyses confirmed that YAP1 is an independent predictor of unfavorable OS in PDAC (hazard ratio [HR] = 1.51, 95% confidence interval [CI]: 1.04–2.20; Table 1). These data suggest that YAP1 plays critical roles in PDAC development and progression.

Nuclear localization is essential for the activity of YAP1. We determined whether nicotine treatment affects the subcellular localization of YAP1 in PDAC cells. IHC assessment indicated that YAP1 was localized in both the cytoplasm and nucleus, and mainly in the nucleus (Supplementary Fig. s5b-c). To explore the clinical significance of cytoplasmic and nuclear expression of YAP1, we evaluated associations of cytoplasmic and nuclear YAP1 expression with OS, and we found significantly shorter OS time (P < 0.001; Supplementary Fig. s5d) in patients with nuclear, but not cytoplasmic, expression of YAP1 (Supplementary Fig. s5e). Likewise, IF assessment confirmed that nicotine treatment promoted YAP1 nuclear translocation in both BxPC-3 and PANC-1 cells (Fig. 4a). Western blot analysis showed that the expression of YAP1 serine 127 phosphorylation (p-YAP1) was downregulated upon nicotine treatment in a dose-dependent manner (Fig. 4b-c and Supplementary Fig. s6a-c). Furthermore, we evaluated the levels of YAP1 in the cytoplasm and nucleus, and found that nicotine stimulated high YAP1 expression in nucleus and low expression in the cytoplasm, while p-YAP1 was exclusively expressed in the cytoplasm (Fig. 4d-e). The activated kinase cascade MST1/LATS1 is a key mediator that prohibits YAP activity by direct phosphorylation of YAP1, sequestering p-YAP1 in the cytoplasm [31]. We found that nicotine treatment inhibited MST1/LATS1, and increased the protein expression of phosphorylated MST1 and LATS1 in both PDAC cell lines (Fig. 4f and Supplementary Fig. s6d-e). Together, these data indicate that nicotine affects the intranuclear and cytoplasmic distribution of YAP1.

Nicotine can regulate HIF1A expression in several types of cancer, and therefore we suspected that nicotine might affect YAP expression through a HIF1A-mediated mechanism. We confirmed that nicotine promoted the mRNA and protein expression of HIF1A in a dose-dependent manner (Fig. 5 a-b). After HIF1A knockdown or overexpression (Supplementary Fig. s7 a-d), we observed that HIF1A knockdown significantly inhibited, while overexpression of HIF1A enhanced, the proliferation and migration of PDAC cells in the presence of nicotine (Supplementary Fig. s7 e-l). Interestingly, nicotine stimulated total YAP1 expression, but inhibited phosphorylation of YAP1. Overexpression of HIF1A enhanced, whereas knockdown of HIF1A attenuated, nicotine-induced changes in YAP1 expression and phosphorylation in Panc-1 and BxPC3 cells (Fig. 5 c-d). Furthermore, overexpression of HIF1A enhanced, whereas knockdown of HIF1A attenuated, expression of YAP1 target genes (Fig. 5 e-h). Additional IHC analysis revealed that HIF1A staining scores were positively associated with YAP1 nuclear staining, but not cytoplasmic staining (Fig. 5 i). These results demonstrate that nicotine stimulates YAP1 nuclear translocation through a HIF1A-mediated mechanism.

To further analyze the mechanism by which YAP1 expression is regulated by HIF1A upon nicotine treatment, we surveyed the YAP1 promoter sequence and identified three potential HIF1A–binding sites (HRE1–3) upstream to the transcription start site (TSS; Fig. 6a). The full-length YAP1 promoter and deletion mutant reporter constructs were generated, and were then co-transfected with or without HIF1A expression vectors or NC vectors into Panc-1 cells. Dual luciferase assays confirmed that deletion of the region from − 800 to − 700 bp, covering the HRE3 site, significantly attenuated YAP1 promoter activity (Fig. 6b-c). To further evaluate HIF1A transcriptional activity following nicotine treatment, we co-transfected an HRE-luc plasmid along with HIF1A expression vectors or shRNA into PDAC cells. Nicotine stimulated YAP1 promoter activity in PDAC cells (Fig. 6d-g); overexpression of HIF1A enhanced, whereas knockdown of HIF1A attenuated, YAP1 promoter activity. To determine whether HIF1A directly binds to HREs on the YAP promoter, we performed ChIP assays. Immunoprecipitation with an anti-HIF1A antibody pulled down the predicted binding-site sequence of the YAP1 promoter, and nicotine treatment significantly enhanced these effects in both PDAC cells (Fig. 6h-i). These results indicate that, upon nicotine treatment, HIF1A binds to the promoter region of YAP1 and upregulates YAP1 transcription in PDAC cells.

Previous studies have determined that nicotine may promote cancer cell proliferation and metastasis through calcium channels or through CHRNA-3, 5, 7 in human PDAC [32, 33]. Western blot assessment confirmed that nicotine treatment activated CHRNA3, CHRNA5, and CHRNA7 (Fig. 7a). After the three receptors were silenced in Panc-1 and BxPC3 cells, respectively, we observed decreased YAP1 expression only in CHRNA7-sileneced cells following nicotine exposure (Fig. 7b-d). Additionally, we analyzed human PDAC samples from the TCGA database and found that YAP1 and HIF1A expression were significantly associated only with CHRNA7, but not with expression of CHRNA3 and CHRNA5, (Supplementary Fig. s8a-f). Furthermore, knockdown of CHRNA7 decreased the protein expression of HIF1A, Vim, and N-cad, but increased the expression of MST1, LATS1 and E-cad, in nicotine-treated PDAC cells (Fig. 7e). In addition, silencing of CHRNA7 led to the inhibition of PDAC cell proliferation, migration, and invasion (Fig. 7f-k). These results indicate that nicotine induces HIF1A and YAP1 expression through the CHRNA7 pathway.

Upregulation of HIF1A protein in tumor tissues can be achieved by promoting DNA transcription, enhancing mRNA translation, or inhibiting proteasomal degradation [34]. The upregulation or knockdown of YAP1 did not change the mRNA level of HIF1A in the absence or presence of nicotine (Fig. 8a-b). We found that YAP1 knockdown substantially attenuated, while overexpression of YAP1 enhanced, expression of HIF1A protein levels in both the absence and presence of nicotine (Fig. 8c-d). Since YAP1 expression had significant effects on HIF1A protein expression, but little effect on HIF1A mRNA levels, we evaluated the effects of YAP1 on HIF1A protein in Panc-1 cells treated with cycloheximide (CHX), an inhibitor of eukaryote protein synthesis. Knockdown of endogenous YAP1 inhibited synthesis of HIF1A protein at early time points (Fig. 8e). Moreover, treatment with the proteasome inhibitor MG132 (10 μM) abolished the effects of YAP1 knockdown or overexpression (Fig. 8f-g) on HIF1A protein. These results indicated that YAP1 regulates HIF1A by enhancing the stability of the HIF1A protein.