Insulin Receptor and GPCR Crosstalk Stimulates YAP via PI3K and PKD in Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, with overall 5-year survival rate of only 7%. The incidence of this disease in the United States is estimated to increase to 53,070 new cases in 2016. It is anticipated that PDAC deaths will surpass those caused by breast cancer, moving the disease from the fourth to the third leading cause of cancer-related deaths in the United States (1). As the current therapies offer very limited survival benefits (2), novel strategies are urgently required to prevent and treat this aggressive disease.

G protein–coupled receptors (GPCR) and their cognate agonists are increasingly implicated in growth stimulation of multiple solid tumors, including small-cell lung cancer, colon, prostate, breast, and pancreas (3, 4). We showed that pancreatic cancer cell lines express multiple GPCRs (5), and a variety of GPCR agonists, including neurotensin, angiotensin II, and bradykinin, stimulate DNA synthesis in these cells (5–8). Furthermore, a broad-spectrum GPCR antagonist (9, 10) inhibited the growth of pancreatic cancer cells either in vitro or xenografted into nu/nu mice (11). Other studies demonstrated increased expression of GPCRs, including those for neurotensin and angiotensin II in pancreatic cancer tissues (12). A recent characterization of cancer genomes demonstrated frequent mutations in GPCRs and G proteins (13). GPCRs and their cognate agonists also dramatically synergize with insulin/IGF-1 in inducing mitogenic signaling (14). Accordingly, we identified positive crosstalk between insulin/IGFI receptors and GPCR signaling systems in pancreatic cancer cells, leading to mTORC1 and ERK activation, and synergistic stimulation of DNA synthesis and cell proliferation (15–17). These findings assume an added importance in view of the large number of epidemiologic studies linking long-standing type II diabetes mellitus (T2DM), obesity, and metabolic syndrome, characterized by peripheral insulin resistance and compensatory overproduction of insulin, with increased risk for developing pancreatic cancer (18, 19). Furthermore, myofibroblasts and macrophages in the tumor microenvironment release IGF-1 that also stimulates insulin/IGF-1 receptors in PDAC cells (20). Neurotensin, a GPCR agonist that acts as a potent mitogen for PDAC cells in combination with insulin (5–8), has been identified as an important gastrointestinal peptide in the pathogenesis of obesity in mice and humans (21). All these findings reinforce the notion that crosstalk between insulin/IGF-1 receptor and GPCR signaling pathways is a major driver of PDAC proliferation (16). Consequently, the identification of key downstream effectors in the signaling network that mediates crosstalk in PDAC cells is of importance for identifying novel targets for prevention and/or treatment of this devastating disease.

The transcriptional coactivators Yes-associated protein (YAP) and WW-domain–containing transcriptional coactivator with PDZ-binding motif (TAZ) are major downstream effectors of the Hippo pathway and novel sensors of insulin, GPCR, and Ras signaling (22–25). The YAP/TAZ pathway, originally identified in Drosophila, is attracting intense attention as a key regulator of development, organ size, tissue regeneration, and tumorigenesis. It is increasingly accepted that YAP/TAZ acts as a context-specific oncogene, and several studies indicate that YAP and TAZ are overactive in PDAC patient tumor samples (26–28). Here, we examined the impact of crosstalk between the insulin receptor and GPCR signaling pathways on the regulation of YAP localization, phosphorylation, and transcriptional activity in PDAC cells. On the basis of the results presented here, we propose that YAP activation is a central node of transcriptional convergence in the crosstalk between insulin receptor and GPCR signaling systems and of critical importance in the proliferative response induced by these agonists in PDAC cells.

The human pancreatic cancer cell lines PANC-1 and MiaPaCa-2 were obtained from the ATCC. These cell lines were chosen because they harbor mutations typical of human pancreatic cancer, including mutations in KRAS and TP53 (encoding the p53 protein) and deletion of CDKN2A (also known as p16 or p16^INK4a). These cell lines, authenticated by ATCC by short tandem repeat analysis, were used within 15 passages and cultured for less than 6 months after recovery from frozen stocks (no authentication was done by the authors). Cells were grown in DMEM with 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at 37°C in a humidified atmosphere containing 10% CO₂.

Confluent cultures of PANC-1 or MiaPaCa-2 cells, grown on 35-mm tissue culture dishes, were washed twice with DMEM and incubated in serum-free medium for 4 hours and then treated as described in individual experiments. The cultures were then directly lysed in 2× SDS-PAGE sample buffer [200 mmol/L Tris-HCl (pH 6.8), 2 mmol/L EDTA, 0.1 mol/L Na₃VO₄, 6% SDS, 10% glycerol, and 4% 2-mercaptoethanol], followed by SDS-PAGE on 4% to 15% gels and transfer to Immobilon-P membranes (Millipore). For detection of proteins, membranes were blocked using 5% nonfat dried milk in PBS, pH 7.2, and then incubated overnight with the desired antibodies diluted in PBS containing 0.1% Tween. Primary antibodies bound to immunoreactive bands were visualized by enhanced chemiluminescence detection with horseradish peroxidase (HRP)-conjugated anti-mouse, anti-rabbit antibody, and a FUJI LAS-4000 mini luminescent image analyzer. Quantification of the bands was performed using the FUJI Multi Gauge V3.0 analysis program.

Immunofluorescence of PANC-1 and MiaPaCa-2 cells was performed by fixing the cultures with 4% paraformaldehyde followed by permeabilization with 0.4% Triton X-100. After extensive PBS washing, fixed cells were incubated for 2 hours at 25°C in blocking buffer (BB), consisting of PBS supplemented with 5% BSA, and then stained at 4°C overnight with a YAP mouse mAb (1:200) diluted in BB. Subsequently, the cells were washed with PBS at 25°C and stained at 25°C for 60 minutes with Alexa Fluor 488– conjugated goat-anti mouse diluted in BB (1:100) and washed again with PBS. Nuclei were stained using a Hoechst 33342 stain (1:10,000).

PANC-1 and MiaPaCa-2 cells were transfected with the plasmid containing a cDNA encoding a GFP-tagged AKT pleckstrin homology domain (pcDNA3-AKT-PH-GFP was a gift from Craig Montell (University of California, Santa Barbara, CA), Addgene plasmid #18836) by using Lipofectamine 3000 as suggested by the manufacturer. Analysis of the cells was performed 24 hours after transfection.

Immunolabeled and GFP-tagged AKT-PH samples were imaged and captured with an epifluorescence Zeiss Axioskop and a Zeiss (Achroplan 40/.75W objective). The selected cells displayed in the appropriate figures were representative of 90% of the population. For YAP localization, the average fluorescence intensity in the nucleus and just outside the nucleus (cytoplasm) was measured to determine the nuclear/cytoplasmic ratios. To determine membrane/cytoplasmic ratios of GFP-tagged AKT-PH, the average fluorescence intensity in the membrane and inside the cytoplasm was measured. All image analyses were performed using ImageJ software.

Silencer select siRNAs were purchased from Life Technologies and designed to target human YAP (#4392420, siRNA_id: s20367), TAZ (#4390771, siRNA_id: n269384) and PKD1 (#4392420, siRNA_id: s1111), PKD2 (#4390824, siRNA_id: s24645), and PKD3 (#4390824, siRNA_id: s233). Cells were transfected using the reverse transfection method. Either silencer select nontargeting negative control (10 nmol/L) or a target siRNA (10 nmol/L) was mixed with Lipofectamine RNAiMAX according to the manufacturer's protocol and added to 35-mm tissue culture plates. PANC-1 and MiaPaCa-2 cells were then plated on top of the siRNA/Lipofectamine RNAiMAX complex at a density of 10⁵ cells per well in DMEM containing 10% FBS. Three to 4 days after transfection, cells were used for experiments.

Relative transcript expression levels of CTGF, CYR61, and CXCL5 were determined by qRT-PCR using a TaqMan Gene Expression Assay. Briefly, total RNA was extracted from cells by using a PureLink RNA Mini Kit. Reverse transcription was performed with the High-Capacity cDNA Reverse Transcription Kit using 1 μg of total input RNA. The synthesized cDNA samples were used as templates for the real-time PCR analysis. All reactions were performed using the Applied Biosystems StepOne system, and the amplifications were done using the TaqMan Fast Advanced Master Mix. Gene-specific Homo sapiens oligonucleotide primers for CTGF (assay ID: Hs01026927_g1), CYR61 (assay ID: Hs00998500_g1), CXCL5 (assay ID: Hs01099660_g1), and 18s (assay ID: Hs03928990_g1) were from Life Technologies.

PANC-1 cells were transfected using the reverse transfection method with either 10 nmol/L nontargeted siRNA or a mixture of 5 nmol/L YAP and 5 nmol/L TAZ siRNA and grown in 3.5-cm tissue culture plates for 5 days in DMEM and 10% FBS. The cultures were then washed twice with DMEM and stimulated with either neurotensin, insulin, or a combination of both in serum-free DMEM. After 18 hours of incubation, the cultures were pulse labeled for 6 hours with [³H]-thymidine (0.25 Ci/mL), fixed with 5% trichloroacetic acid and washed twice with ethanol. Acid-insoluble pools corresponding to DNA were solubilized in 0.1 N NaOH with 1% SDS and counted in a liquid scintillation counter.

Cells (4 × 10⁴) were plated on 35-mm tissue culture dishes in DMEM containing 10% FBS. After 24 hours of incubation at 37°C, cultures were incubated with DMEM containing 1% FBS in the absence or presence of insulin and neurotensin or the combination of both agonists. After 4 days, cell count was determined from a minimum of 3 dishes per condition using a Coulter counter, after cell clumps were disaggregated by passing the cell suspension 10 times through a 19-gauge and, subsequently, a 21-gauge needle.

For cell colony formation, 10³ MiaPaCa-2 or PANC-1 cells were plated into 60-mm tissue culture dishes in DMEM containing 10% FBS. After 24 hours of incubation at 37°C, cultures were incubated with DMEM containing 1% FBS either in the absence or presence of insulin, neurotensin, or the combination of both drugs. A colony consisted of at least 50 cells. Cell colony numbers from 3 dishes per condition were determined after 8 days of incubation.

The one-way ANOVA followed by a Bonferroni t test (SigmaPlot software) was used to analyze our data. Data were considered significant if P < 0.05 and was presented as average ± SEM.

DMEM (#11995-073), FBS, goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488, silencer select siRNAs, and all qRT-PCR reagents were obtained from Invitrogen. Neurotensin and insulin were purchased from Sigma Chemical. The kinase inhibitors A66, CRT0066101, kb NB 142-70, and KU 0063794 were purchased from R&D Systems. Primary antibodies used were YAP (63.7: sc-101199 final dilution 1:200 for immunofluorescence, 1:400 for Western blotting) and GAPDH (sc-365062; final dilution 1:400) from Santa Cruz Biotechnology, Inc., phospho-YAP Ser¹²⁷ (D9W2I #13008; final dilution 1:1,000) and phospho-YAP Ser³⁹⁷ (D1E7Y #13619; final dilution 1:1,000), phosphor-PKD Ser⁹¹⁰ (#2051; final dilution 1:1,000), PKD (#2052; final dilution 1:1,000) and PKD3 (#5655; final dilution 1:1,000) from Cell Signaling Technology. HRP-conjugated anti-rabbit IgG and anti-mouse IgG were from GE Healthcare Bio-Sciences Corp. All other reagents were of the highest grade available.

Because cell density regulates YAP localization through the Hippo pathway in a variety of cell types, we initially examined the localization of endogenous YAP in PANC-1 and MiaPaCa-2 cells after various times of plating. At low cell density (2–4 days after plating), YAP was predominantly localized in the nuclei of these PDAC cells (Fig. 1A). In contrast, YAP was confined to the cytoplasm (therefore inactive) in confluent cultures of PDAC cells, that is, 6 to 7 days after plating.

In subsequent experiments, we used confluent PDAC cells to examine whether crosstalk between insulin receptor and GPCR signaling systems reactivates YAP by regulating its localization, phosphorylation, and transcriptional coactivator activity. Cultures of PANC-1 cells were transferred to serum-free medium for 16 hours and then stimulated with 10 ng/mL insulin, the GPCR agonist neurotensin at 5 nmol/L or their combination to elicit positive crosstalk. In unstimulated cells and in line with the results shown in Fig. 1A, YAP was detected mainly in the cytoplasm of PANC-1 cells plated at high cell density. Stimulation of these cells with either insulin or neurotensin induced a significant translocation of YAP to the nucleus. Challenging with a combination of insulin and neurotensin promoted striking YAP nuclear localization (Fig. 1B and C). Quantification of multiple individual cells corroborated that exposure of PANC-1 cells to the combination of insulin and neurotensin was more effective in promoting YAP nuclear localization than each of the agonists added singly (Fig. 1B and C; right). Similar results were obtained when YAP localization was determined in MiaPaCa-2 cells exposed to insulin, neurotensin, or their combination (Fig. 1D).

The phosphorylation of YAP at Ser¹²⁷ plays a major role in restricting its cellular localization to the cytoplasm. Consequently, we determined whether insulin and neurotensin regulate YAP phosphorylation at Ser¹²⁷ and Ser³⁹⁷, highly conserved residues located within a consensus sequence phosphorylated by the Hippo kinases Lats1/2 (HXRXXS). PANC-1 cells were stimulated with neurotensin, insulin, or a combination of these agonists for 30 or 60 minutes and lysed. Cell lysates were analyzed by Western blotting with antibodies that detect the phosphorylated state of YAP at these residues. As shown in Fig. 2A and B, the combination of neurotensin and insulin induced a rapid decrease in YAP phosphorylation at Ser¹²⁷ and Ser³⁹⁷ in PANC-1 cells. Similar results were obtained when we used MiaPaCa-2 cells instead of PANC-1 cells (Fig. 2C and D). The results in Figs. 1 and 2 indicate that crosstalk between insulin and GPCR signaling pathways rapidly regulates YAP localization and phosphorylation in PDAC cells.

We next determined whether the rapid nuclear import and dephosphorylation of YAP in response to crosstalk between insulin receptor and GPCR signaling systems promotes its coupling to the transcription factors of the TEA domain–containing transcription factors (TEAD1–4) and thereby stimulate the expression of YAP/TEAD–regulated genes, including connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61). Indeed, CTGF is one of the best characterized direct target genes of YAP that contains three putative YAP-TEAD–binding sites (GGAATG) in its promoter region. As shown in Fig. 3A and B, stimulation of either PANC-1 or MiaPaCa-2 cells for 30 or 60 minutes with a combination of neurotensin and insulin induced a marked increase in the levels of CTGF and CYR61 transcripts, as determined by qRT-PCR. The combination of insulin and neurotensin was significantly more effective in promoting CTGF and CYR61 expression than each of the agonists added singly (Fig. 3A and B).

CXCL5, a chemokine produced by pancreatic cancer cells (29), has been proposed to promote infiltration by polymorphonuclear myeloid-derived suppressor cells into cancer tissues. Recently, CXCL5 has been identified as a novel YAP/TEAD–regulated gene in prostate cancer cells (30). We determined whether CXCL5 mRNA levels are also rapidly enhanced by insulin and neurotensin in PDAC cells. As shown in Fig. 3A and B, stimulation of PANC-1 or MiaPaCa-2 cells with a combination of neurotensin and insulin markedly increased the levels of CXCL5 mRNA in these cells.

To verify that the increase in the expression of these early response genes is mediated by stimulation of YAP/TAZ coactivator activity, we determined whether siRNA-mediated knockdown of YAP/TAZ prevents the increase in the expression of CTGF, CYR61, and CXCL5. Transient transfection of PANC-1 cells with siRNAs targeting YAP and TAZ prevented the increase in CTGF, CYR61, and CXCL5 mRNA levels induced by challenging these cells with insulin and neurotensin (Fig. 4A) and virtually extinguished the detection of YAP protein (Fig. 4B). Collectively, these results indicate that YAP/TAZ activation is a novel early point of transcriptional convergence in the crosstalk between GPCR and insulin signaling pathways in PDAC cells.

Subsequently, we determined whether the increase in the activity of YAP/TAZ is required for the stimulation of DNA synthesis and cell proliferation induced by insulin and neurotensin in PDAC cells. As shown in Fig. 4C, stimulation with insulin and neurotensin of control PANC-1 cells (either untreated cells or cells transiently transfected with nontargeting oligonucleotides) induced a marked increase in ³H-thymidine incorporation into DNA. Knockdown of YAP/TAZ sharply reduced the increase in DNA synthesis induced by the combination of insulin and neurotensin (from ∼5- to 1.8-fold). To substantiate the results obtained with ³H-thymidine incorporation, we next examined whether YAP plays a role in the proliferation of PDAC cells in response to long-term exposure to insulin and neurotensin. The combination of these stimuli promoted a marked increase in the number of PANC-1 (Fig. 4D) and MiaPaCa-2 (Fig. 4E) cells. Similarly, insulin and neurotensin enhanced the ability to form colonies by these cells (Fig. 4F and G). Knockdown of YAP/TAZ averted the increase in cell number (Fig. 4D and E) and colony formation (Fig. 4F and G) induced by insulin and neurotensin in these PDAC cells. Collectively, these results indicate that YAP/TAZ activity plays a critical role in mediating the growth-promoting crosstalk between insulin receptor and GPCR signaling pathways in PDAC cells.

We next explored the mechanisms by which crosstalk between insulin receptor and GPCR signaling stimulate YAP/TEAD activity in PDAC cells. A key pathway in insulin receptor signaling is PI3K leading to AKT/mTOR activation, but it is not known whether PI3Ks are a point of convergence in the crosstalk between insulin/IGR-1R and GPCR signaling systems in PDAC cells. To examine the effect of crosstalk between insulin receptor and GPCR signaling systems on PI3K activity in single cells, PANC-1 cells were transiently transfected with a plasmid encoding a fusion protein between GFP and the PH domain of AKT (AKT-PH-GFP), an in vivo reporter of phosphatidylinositol 3,4,5-trisphosphate (PIP₃) in the plasma membrane of individual PDAC cells. In most unstimulated PANC-1 and MiaPaCa-2 cells, the PIP₃ sensor displays little localization at the plasma membrane. Stimulation with insulin or neurotensin markedly stimulated PI3K activity, as judged by the increase in the translocation of the PIP₃ sensor to the plasma membrane of PANC-1 (Fig. 5A; quantification in Fig. 5B) and MiaPaCa-2 cells (Fig. 5C; quantification in Fig. 5D).

Subsequently, we determined the contribution of PI3K to the synergistic stimulation of YAP/TEAD–regulated genes CTGF, CYR61, and CXCL5 induced by insulin and neurotensin in PANC-1 and MiaPaCa-2 cells. We used A66, a highly selective inhibitor of the catalytic activity of the PI3Ks with preferential inhibitory activity against the p110α catalytic subunit of PI3K (31). Indeed, in cell-free assays, A66 at 10 μmol/L only inhibited PI3K isoforms when tested against a panel of 318 protein kinases (31). Treatment of intact PANC-1 with 10 μmol/L A66 prevented the increase in the localization of the PIP₃ sensor to the plasma membrane (Fig. 5E; quantification in Fig. 5F) or MiaPaCa-2 (Fig. 5G; quantification in Fig. 5H) cells induced by insulin and neurotensin. A66 impeded AKT-PH-GFP localization to the plasma membrane in a dose-dependent manner with inhibitory effects at 2.5 and 5 μmol/L (Supplementary Fig. S1). Accordingly, treatment of PANC-1 or MiaPaCa-2 cells with increasing concentrations of A66 prior to stimulation with insulin and neurotensin greatly attenuated AKT phosphorylation at Thr³⁰⁸, a site targeted by phosphoinositide-dependent protein kinase 1 (PDK1), a kinase activated by PIP₃ (Fig. 5I). Maximal inhibitory effect of A66 on AKT phosphorylation at Thr³⁰⁸ was achieved at 10 μmol/L in intact PANC-1 and MiaPaCa-2 cells (84% and 87% inhibition, respectively). Importantly, treatment with A66 at 10 μmol/L also blocked the increase in the expression of the YAP/TEAD–regulated genes CTGF, CYR61, and CXCL5 induced by insulin and neurotensin in PANC-1 (Fig. 5K) or MiaPaCa-2 (Fig. 5L). Inhibition of CTGF, CYR61, and CXCL5 expression was also observed when A66 was added at 2.5 or 5 μmol/L with maximal effect achieved at 10 μmol/L (Supplementary Fig. S1). These results imply that PI3K isoforms contribute to YAP activation in response to crosstalk between insulin/IGF1R and GPCR signaling pathways in PDAC cells.

As PI3K leads to mTORC1 and mTORC2 activation in PDAC cells, we determined whether mTOR plays a role in mediating the increase in the expression of CTGF, CYR61, and CXCL5. Treatment with the active-site mTOR inhibitor KU63794 (32) did not interfere with the increase in the expression of CTGF, CYR61, and CXCL5 in PANC-1 or MiaPaCa-2 cells (Fig. 5K and L). We verified that KU63794, at the concentration used, suppressed mTOR activation by insulin and neurotensin in PANC-1 and MiaPaCa-2 cells, as scored by inhibition of p70S6K phosphorylation at Thr³⁸⁹, a direct target of mTORC1 (Fig. 5J). In agreement with results in other cell types (33, 34), we conclude that a PI3K-dependent pathway acting upstream of mTOR leads to YAP/TEAD activation in response to insulin and neurotensin in PDAC cells.

We previously demonstrated that neurotensin increases PKD family (PKD1, PKD2, and PKD3) activity in PDAC cells (6, 35, 36) and induces proliferation of these cells in a PKD-dependent manner (37, 38). Here, we show that the combination of neurotensin and insulin potently stimulated PKD activity in PANC-1 (Fig. 6A) and MiaPaCa-2 (Fig. 6B), as scored by the increase in its autophosphorylation at Ser⁹¹⁰. To determine the role of PKD in PDAC cell signaling, we used two different PKD family inhibitors with different structure, to minimize possible off-target effects. Treatment of intact PANC-1 and MiaPaCa-2 cells with the PKD inhibitors CRT0066101 (2.5 μmol/L) or kb NB 142-70 (3.5 μmol/L) prevented PKD activation (scored by autophosphorylation at Ser⁹¹⁰) in these cells in response to challenge with insulin and neurotensin (Fig. 6A and B). Further studies showed that treatment with CRT0066101 counteracted PKD activation in a dose-dependent manner in PANC-1 and MiaPaCa-2 cells stimulated with insulin and neurotensin. Maximal inhibition was achieved at 2.5 μmol/L in these cells (Fig. 6C and D). Recently, we implicated the PKD family in the regulation of YAP/TEAD activity in intestinal epithelial cells (25), but the role of the PKDs in YAP activation in response to growth-promoting factors in PDAC cells has not been examined. Consequently, we next determined whether the PKDs contribute to mediate YAP activation in PDAC cells challenged with neurotensin and insulin. Exposure of intact PDAC cells to the structurally unrelated PKD family inhibitors CRT0066101 and kb NB 142-70 prevented the increase of CTGF, CYR61, and CXCL5 mRNA levels induced by insulin and neurotensin in PANC1 (Fig. 6E) and MiaPaCa-2 cells (Fig. 6F). These results imply that the PKDs contribute to the stimulation of YAP/TEAD transcriptional activity in response to crosstalk signaling in PDAC cells.

In consonance with the results obtained with chemical inhibitors in Fig. 6, transfection of a mixture of siRNAs targeting PKD1, PKD2, and PKD3 abrogated the increase in CTGF, CYR61, and CXCL5 expression in response to insulin and neurotensin stimulation in PANC1 (Fig. 7A) and MiaPaCa-2 cells (Fig. 7B). We verified that the mixture of siRNAs markedly decreased the expression of the PKDs in these cells (Fig. 7C and D). Collectively, our findings identify a novel function for PKDs in mediating YAP/TEAD transcriptional activity in response to insulin and neurotensin in PDAC cells (Fig. 7E).

Many studies have indicated that crosstalk between the insulin/IGF-1 receptor and GPCR signaling systems plays a critical role in the regulation of a variety of normal and abnormal processes, including cardiovascular and renal pathologies in obesity, metabolic syndrome, and T2DM. GPCR agonists also mediate autocrine/paracrine growth stimulation in a variety of cancer cells and dramatically synergize with insulin/IGF-1 in inducing growth-promoting signaling. Accordingly, our previous studies identified crosstalk mechanisms between insulin/IGF-1 receptor and GPCR signaling systems that stimulate DNA synthesis and cell proliferation in human PDAC cells (15, 17, 39). Consequently, we hypothesized that crosstalk between insulin/IGF-1 receptor and GPCR signaling systems is a mechanism for promoting the development of PDAC (16), the third leading cause of cancer-related mortality in the United States (1). However, a major gap in our current understanding is the identity of the key downstream transcriptional effectors in signaling crosstalk that drive PDAC development.

The highly conserved YAP/TAZ pathway is attracting intense interest as a key regulator in cancer cell biology. Preclinical studies indicated that YAP is a key downstream target of K-Ras signaling required for acinar-to-ductal metaplasia and subsequent PanIN progression into PDAC (40, 41). Furthermore, amplification and overexpression of Yap can substitute for mutant K-Ras expression in PDAC (26). These studies indicate that Yap acts downstream of K-Ras and that its hyperactivation can compensate for the need of K-Ras–mutant expression in PDAC (42). Several studies indicate that YAP and TAZ are active in PDAC patient tumor samples (26–28). However, the impact of crosstalk between insulin/IGF-1R and GPCR signaling pathways on YAP/TAZ activity had not been previously examined in PDAC cells or in any other cell type.

The phosphorylation of YAP is thought to restrict its activity, cellular localization, and stability. In the absence of phosphorylation, YAP localizes to the nucleus where it binds and activates the TEA-domain DNA-binding transcription factors (TEAD 1–4) thereby stimulating the expression of a variety of genes. Typical YAP/TEAD–regulated genes include CTGF, CYR61/CCN1, and the cytokine CXCL5. The products of these genes have a major impact on important cell processes, including proliferation, survival, and differentiation as well as in shaping the microenvironment (CTGF), promoting angiogenesis (CYR61), and mediating communication between cancer and immune cells (CXCL5). The results presented here demonstrate that stimulation of confluent cultures of PDAC cells with a combination of insulin and the GPCR agonist neurotensin induces rapid YAP nuclear localization, decreases YAP phosphorylation at Ser¹²⁷, and promotes the expression of YAP/TEAD–regulated genes, including CTGF, CYR61, and CXCL5. We conclude that activation of YAP/TEAD is a novel early point of transcriptional convergence in the crosstalk between GPCR and insulin receptor signaling pathways in PDAC cells.

We next explored the mechanism(s) that mediate YAP activation in response to PDAC cell stimulation with insulin and neurotensin. Recently, p110α PI3K has been identified as a major signaling enzyme for PDAC development (43), and PI3K isoforms play a key role in insulin receptor signaling. Here, we show that neurotensin and insulin rapidly stimulated PI3K activity, as judged by the increase in the translocation of the PIP₃ sensor GFP-PH-AKT to the plasma membrane of PDAC cells. A66, a selective inhibitor of PI3K with preferential activity against p110α PI3K (31), prevented the accumulation of the PIP₃ sensor at the plasma membrane and AKT phosphorylation at Thr³⁰⁸ in a dose-dependent manner. At similar concentrations, A66 abrogated the increase in the expression of YAP/TEAD–regulated genes. We conclude that PI3K-mediated formation of PIP₃ contributes to YAP activation in response to crosstalk between insulin receptor and GPCR signaling pathways in PDAC cells (Fig. 7E).

PKDs have been identified as a major downstream signaling node in GPCR signaling (44, 45). PKDs are rapidly activated by GPCR agonists in PDAC cells (6, 35, 36) and are overexpressed in PDAC tissues (46). Induced PKD overexpression in PDAC cells promotes cell proliferation (37), invasion (38), and acinar-to-ductal transdifferentiation (47). Here, we examined whether PKDs are implicated in the regulation of YAP activity in PDAC cells. Using structurally unrelated PKD family inhibitors and siRNA-mediated knockdown of the PKDs, we identify a novel role of the PKDs in the stimulation of YAP/TEAD transcriptional activity in response to crosstalk signaling in PDAC cells (Fig. 7E).

Our findings have potential translational implications. Obesity and T2DM, which are prominent in the western world, have been linked to increased risk and worse clinical outcomes for developing PDAC (16, 18, 19). These diet-related metabolic disorders are multifaceted but characterized by peripheral insulin resistance and compensatory overproduction of insulin. In turn, neurotensin has been implicated in the pathogenesis of obesity (21). Therefore, our results showing that crosstalk between insulin and neurotensin stimulates YAP/TEAD via PI3K and PKD not only reveal a potential mechanism linking obesity, T2DM, and PDAC but also identify potential targets highly relevant in PDAC. A variety of pharmacologic inhibitors directed against the PI3K pathway are now available for clinical investigation, (48) and orally active PKD inhibitors, including CRT0066101 (46), are being identified. Our results imply that combinations of PI3K and PKD inhibitors could provide a novel strategy for targeting YAP and for disrupting growth-promoting crosstalk between insulin/IGF-1R and GPCR signaling in PDAC.

In conclusion, we show that the stimulation of PDAC cells with insulin and the GPCR agonist neurotensin induces rapid nuclear import and dephosphorylation of YAP and marked increase in the expression of YAP/TEAD–regulated genes through PI3K- and PKD-dependent pathways (Fig. 7E). We propose that YAP is a central node of transcriptional convergence in the crosstalk between insulin receptor and GPCR signaling systems and of major importance in the proliferative response induced by these growth-promoting agonists in PDAC cells.

No potential conflicts of interest were disclosed.

Conception and design: Q. Xu, J. Sinnett-Smith, E. Rozengurt

Development of methodology: F. Hao, Q. Xu, Y. Zhao, J. Sinnett-Smith, E. Rozengurt

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.V. Stevens, S.H. Young

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Hao, Q. Xu, Y. Zhao_, J.V. Stevens, S.H. Young, J. Sinnett-Smith, E. Rozengurt

Writing, review, and/or revision of the manuscript: F. Hao, S.H. Young, J. Sinnett-Smith, E. Rozengurt

Study supervision: E. Rozengurt

The authors thank the Imaging and Stem Cell Biology Core of the CURE: Digestive Diseases Research Center (P30DK41301) for their assistance.

This work was supported by NIH grants R01DK100405, P30DK41301, and P01CA163200 and by the Department of Veterans Affairs Merit Award 1I01BX001473 (to E. Rozengurt). Additional funding came from the Ronald S. Hirshberg Endowed Chair of Pancreatic Cancer Research (to E. Rozengurt). F. Hao and Q. Xu were recipients of grants from the China Scholarship Council.

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.