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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Gastroenterology. 2019 Nov 29;158(5):1433–1449.e27. doi: 10.1053/j.gastro.2019.11.279

Diphenylbutylpiperidine Antipsychotic Drugs Inhibit Prolactin Receptor Signaling to Reduce Growth of Pancreatic Ductal Adenocarcinoma in Mice

Prasad Dandawate 1, Gaurav Kaushik 2, Chandrayee Ghosh 1, David Standing 1, Afreen Asif Ali Sayed 1, Sonali Choudhury 1, Dharmalingam Subramaniam 1, Ann Manzardo 3, Tuhina Banerjee 4, Santimukul Santra 4, Prabhu Ramamoorthy 1, Merlin Butler 3, Subhash B Padhye 1,5, Joaquina Baranda 6, Anup Kasi 6, Weijing Sun 6, Ossama Tawfik 7, Domenico Coppola 8, Mokenge Malafa 8, Shahid Umar 2, Michael J Soares 7,9,10,11, Subhrajit Saha 12, Scott J Weir 1,13, Animesh Dhar 1, Roy A Jensen 1,7, Sufi Mary Thomas 1,14, Shrikant Anant 1,2,5,*
PMCID: PMC7103550  NIHMSID: NIHMS1545537  PMID: 31786131

Abstract

Background & Aims

Prolactin (PRL) signaling is upregulated in hormone-responsive cancers. The PRL receptor (PRLR) is class I cytokine receptor that signals via the JAK-STAT and MAPK pathways to regulate cell proliferation, migration, stem-cell features, and apoptosis. Patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of PRL. We investigated whether PRLR signaling contributes to growth of pancreatic tumors in mice.

Methods

We used immunohistochemical analyses to compare levels of PRL and PRLR in multi-tumor tissue microarrays. We used structure-based virtual screening and fragment-based drug discovery to identify compounds likely to bind PRLR and interfere with its signaling. Human pancreatic cell lines (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2), with or without knockdown of PRLR (CRISPR or small-hairpin RNA), were incubated with PRL or penfluridol and analyzed in proliferation, spheroid formation. C57BL/6 mice were given injections of UNKC-6141 cells, with or without knockdown of PRLR, into pancreas, and tumor development was monitored for 4 weeks, with some mice receiving penfluridol treatment for 21 days. Human pancreatic tumor tissues were implanted into interscapular fat pads of NSG mice and mice were given injections of penfluridol daily for 28 days. Nude mice were given injections of Panc-1 cells, xenograft tumors were grown for 2 weeks, and mice were then given intraperitoneal penfluridol for 21 days. Tumors were collected from mice and analyzed by histology, immunohistochemistry, and immunoblots.

Results

Levels of PRLR were increased in PDAC compared with non-tumor pancreatic tissues. Incubation of pancreatic cell lines with PRL activated signaling via JAK2-STAT3 and ERK, as well as formation of pancospheres and cell migration; these activities were not observed in cells with PRLR knock down. Pancreatic cancer cells with PRLR knockdown formed significantly smaller tumors in mice. We identified several diphenylbutylpiperidine-class antipsychotic drugs as agents that decreased PRL-induced JAK2 signaling; incubation of pancreatic cancer cells with these compounds reduced their proliferation and formation of spheres. Injections of 1 of these compounds, penfluridol, slowed growth of xenograft tumors in the different mouse models, reducing proliferation and inducing autophagy of the tumor cells.

Conclusions

Levels of PRLR are increased in PDAC, and exposure to PRL increases proliferation and migration of pancreatic cancer cells. Antipsychotic drugs such as penfluridol block JAK2 signaling in pancreatic cancer cells to reduce their proliferation, induce autophagy, and slow growth of xenograft tumors in mice. These drugs might be tested in patients with PDAC.

Keywords: Dopamine receptor, molecular modeling, Combination Therapy, Gemcitabine

INTRODUCTION

Prolactin signaling has an established role in maintaining pregnancy, mammary gland development, immune regulation, adipocyte control, reproduction and islet cell differentiation1, 2 Moreover, the pathway is implicated in multiple endocrine driven tumors3. More recently, the pathway has been observed to play a role in other tumor types including colon and hepatocellular cancers4, 5 PRLR belongs to the class I cytokine receptor superfamily and lacks intrinsic kinase activity, transducing signals including JAK-STAT and MAPK pathways through kinases that interact with its cytoplasmic tail6. The pathway is implicated in the promotion of cell proliferation, migration, stemness and inhibition of apoptosis7, as well as chemoresistance8, 9 In colon cancers, PRL induces JAK2-STAT3 signaling to affect stemness by upregulating the notch pathway5.

We now demonstrate that PRLR-based signaling is active in pancreatic ductal adenocarcinoma (PDAC). The reason for choosing PDAC is two-fold; first is that patients with PDAC have high plasma levels of PRL10. Second, PDAC remains a leading cause of cancer-related deaths in the United States11. Poor prognosis, drug resistance, and high recurrence rates characterize PDAC after surgery12. Here, we show that PRL induces both JAK2-STAT3, and ERK signaling in the cells. PRLR knockdown reduced the signaling, and tumor growth in the orthotopic PDAC model, suggesting that PRLR may be an effective therapeutic target. To identify a small molecule inhibitor, we developed an in silico model for the JAK2 interacting domain, and subsequently identified potential interacting diphenylbutyl-piperidine (penfluridol, pimozide, fluspirilene) and phenothiazine (promethazine) anti-psychotic compounds. Using penfluridol as proof of principle, we show that it is effective in targeting PRLR signaling and suppressing tumor growth in the orthotopic PDAC model. Since penfluridol was initially identified as a Dopamine receptor D2 (DRD2) antagonist, we also determined whether its effect is due also through DRD2. While DRD2 expression is also induced in PDAC, penfluridol effect on PDAC cells was not affected by DRD2 knockdown. This suggests that penfluridol effect is through suppressing PRLR signaling.

MATERIALS AND METHODS

Please refer to the online Supplementary Materials for detailed additional Methods.

Cells and culture condition

Human PDAC cell lines AsPC-1, BxPC-3, Panc-1, MiaPaCa-2 (all obtained from American Type Culture Collection), Panc 2.15, Panc 3.014, Panc 5.04, Panc 8.13, Panc 10.05 are gifts from John Hopkins University. UNKC-6141 cell line is a gift from Dr. Surinder Batra. The cell lines were grown in RPMI 1640 or DMEM with 4.5 g/L glucose, L-glutamine and Sodium Pyruvate (Corning, Tewksbury, MA) containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 1% antibiotic-antimycotic solution (Corning, Tewksbury, MA) at 37°C in a humidified atmosphere of 5% CO 2. HPNE cells were kindly provided by Dr. Anirban Maitra, and grown in DMEM with 4.5 g/L glucose, L-glutamine and Sodium Pyruvate (Corning, Tewksbury, MA) with 5% FBS, 1X N2, 10 ng/ml bFGF and 50 μg/ml Gentamycin. All the cell lines used in this study were within 20 passages after receipt or resuscitation (~3 months of non-continuous culturing).

Proliferation and apoptosis assays

Pancreatic cancer cells were plated in 96-well plates, allowed them to grow for 24 hours, and treated with increasing doses of respective compounds. Cell proliferation was measured by enzymatic hexosaminidase assay as described previously1. For apoptosis, the Apo-one Homogeneous Caspase-3/7 Assay kit was used to calculate caspase 3/7 activity (Promega Corporation, Madison, WI).

Clonogenicity assay

Briefly, 350 viable pancreatic cancer cells per well were seeded in 6 well dishes and treated with Penfluridol in 10% FBS containing RPMI1640/DMEM for 48 h, then medium with or without compound was removed, and the cells were incubated for an additional 10 days in fresh complete medium to form colonies. The colonies were fixed with formalin and stained using crystal violet.

Animal Studies

PDAC orthotopic model in mice

Five-week-old male C57BL/6 mice purchased from Charles River Laboratory were maintained with standard mouse chow ad libidum and used in protocols approved by the University’s Animal Studies Committee. Animals were injected with 1 × 106 UNKC-614113, empty vector, and UNKC-6141 KD cells in the pancreas and allowed to grow for 4 weeks. In another experiment, one week following implantation, penfluridol (5 mg/kg body weight) was administered intraperitoneally daily for 21 days. At the end of treatment, the animals were euthanized, and the tumors were removed, weighed and used for histology (hematoxylin & eosin), immunohistochemistry, and western blot studies.

PDAC patient-derived xenograft (PDX) model

For the PDX model, we obtained PDX-carrying NSG mice from the Jackson Laboratory (TM01212). The cryopreserved tumor tissue obtained from pancreatic cancer patients was cut into small pieces. The animals were anesthetized and then the tissue was implanted subcutaneously into the interscapular fat pad by making a shallow incision in the dorsal region. Once tumors were established, we started to treat the mice with penfluridol 5 mg/kg intraperitoneally for 28 days. Tumor size was measured weekly. At the end of treatment, the animals were euthanized, and the tumors were removed and weighed.

Panc-1 xenograft tumors in mice

Five-week-old male athymic nude mice were procured from Charles River Laboratory and maintained with water and standard mouse chow ad libidum. All mice used in the study are protocols approved by the University’s Animal Studies Committee. In brief, 1 × 106 Panc-1 cells were injected in the left and right flank of the mice and allowed to grow a xenograft for a week. One week following injection a palpable tumor was observed. Subsequently, penfluridol (5 mg/kg) was administered intraperitoneally for 35 days. Tumor volumes were measured weekly. At the end of treatment, the animals were euthanized, and the tumors were removed, weighed and use for histology (hematoxylin & eosin), immunohistochemistry, and western blot studies.

Statistical analysis

All values are expressed as the mean ± SEM. Data were analyzed using an unpaired 2-tailed t-test. A P value of less than 0.05 was considered statistically significant.

RESULTS

PRLR is upregulated in PDAC and other cancers

To investigate the role of PRLR in cancers, we first assessed expression in normal tissues and cancers using RNA-seq data generated by the Genotype-Tissue Expression (GTEx) project. PRLR mRNA expression is low in the normal pancreas and prostate compared to other organs (Supplementary Figure 1A). However, PRLR mRNA levels are high in all cancers (Figure 1A). We next analyzed PRLR gene expression using GeneAnalytics, using a set of PRL-signaling genes (Supplementary Figure 1B)14. There were 142 PRL signaling genes that matched to 919 entities, which represented 381 cell types, 81 anatomical compartments, 35 organs/tissues and 422 hits from high-throughput compound screening studies. Cerebrum and pancreatic islets exhibited the highest match scores ranging from 3.9–3.4 in normal tissues, while breast cancer (Score: 36.33) and PDAC (Score: 21.35) were the highest amongst cancers (Supplementary Figures 1C and D, Figure 1B). To confirm PRLR increase in cancers, we performed immunohistochemical analyses of multi-tumor tissue microarrays. We observed upregulation of PRLR expression in various cancers including the pancreas (Figure 1C and Supplementary Figure 1E). More importantly, the protein is also intracellular raising the question of its role inside the cell. Moreover, an increase in gene copy number correlated with poor survival in cancer patients in “PanCan” TCGA database (p < 0.0001, Figure 1D). These data suggest that PRLR is upregulated in multiple tumor types, especially PDAC, a cancer with high mortality and poor five-year survival rates15.

Figure 1. PRLR is upregulated in PDAC.

Figure 1.

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(A) PRLR mRNA levels are high in multiple cancers in the Cancer Genome Atlas (TCGA) database. (B) PRLR pathway gene-set analyzed by GeneAnalytics, shows PRLR pathway genes are upregulated in various cancer types (BRCA: breast, PRAD: prostate adenocarcinoma, COAD: colon adenocarcinoma, LUAD: lung adenocarcinoma, ESCA: esophageal carcinoma, PAAD: pancreatic adenocarcinoma, AML: acute myeloid leukemia). (C) Immunohistochemistry of a multiple organ tumor microarray (TMA) shows that PRLR is differentially upregulated in several cancers (T) when compared to normal tissue (N). (D) Kaplan Meier plot shows that high PRLR gene copy number correlates with low survival in cancer patients (p < 0.00001). TCGA Pan-cancer (PANCAN) cohort. (E) Real-time PCR analyses shows PRLR expression is higher in PDAC when compared to normal samples (p = 0.0403). (F) Composite score of PDAC TMA shows significantly higher expression of PRLR in tumors compared to normal pancreas (p < 0.001). (G) PRLR mRNA is upregulated early in PDAC progression, generated from TCGA datasets.

In normal pancreatic tissue, PRLR expression is low and restricted to islets (Supplementary Figure 1A and F). In contrast, PRLR mRNA levels were significantly higher in cancer tissues compared to paired normal (Figure 1E), which was confirmed by immunohistochemistry (Figure 1F and Supplementary Figure 1F–K). Interrogation of the TCGA database also demonstrated higher levels of PRLR mRNA in pancreatic cancers (Supplementary Figure 1L). The high level of PRLR expression was observed early in the tumorigenesis process (Supplementary Figure 1G). We also assessed PRL levels. In normal tissues, PRL mRNA expression is observed in breast, ovary, pancreas, and prostate (Supplementary Figure 1M). However, the transcript is expressed in multiple cancers (Supplementary Figure 1N). In addition, Kaplan Meir analyses of data from TCGA PanCan database demonstrated significantly lower survival (p < 0.0001) in patients with higher PRL copy number (Supplementary Figure 1O). Mining of previously reported microarray data also suggested significantly higher PRL levels in primary PDACs (n = 145, p = 0.0042) compared to normal pancreatic tissue (n = 46) (Supplementary Figure 1P). In normal pancreas, PRL expression is seen within the β-cells, while surrounding acinar cells showed lower expression. However, PRL expression is upregulates in PDAC tissues irrespective of the cancer stage (Supplementary Figure 1Q). Similarly, we also observed higher PRL protein levels in established cell lines and primary patient-derived PDAC cells when compared to immortalized pancreatic ductal epithelial cell line HPNE (Supplementary Figure 1R).

Dopamine plays a predominant role in regulation of prolactin release16 and dopamine receptor D2 (DRD2) is present in PDAC tissues17. To understand the role of DRD2, we first analyzed RNA-seq data generated by the Genotype-Tissue Expression (GTEx) project. DRD2 mRNA expression is low in the normal tissues, while it is increased across several cancers (Supplementary Figure 1S and T). TCGA dataset also demonstrated DRD2 overexpression in PDAC patients (p < 0.01, Supplementary Figure 1U). In addition, Kaplan Meir analyses of data from TCGA PanCan dataset demonstrated a somewhat lower survival (p = 0.0912) in patients with higher DRD2 expression (Supplementary Figure 1V). To confirm the upregulation of DRD2 in PDAC, we utilized the same TMA as that used to probe for PRLR. In normal tissue, DRD2 levels were high in in islet cells (Supplementary Figure 1W). However, there was a significant increase in DRD2 expression in PDAC tissues (p < 0.01, Supplementary Figure 1W–Y).

PRLR signals through JAK2-STAT3-ERK-AKT phosphorylation

Alternative spliced transcript variants of PRLR have been identified that encode different membrane-bound and soluble isoforms18. Of these, the full length 110 kDa protein19 is expressed at higher levels in PDAC cells compared to HPNE cells (Figure 2A). PRLR protein is present in both the nucleus and cytoplasm in PDAC cells (Supplementary Figure 2A). On binding to PRLR, PRL activates JAK2-STAT3, and JAK2-MAPK/AKT signaling pathways 6, 20, 21. In PDAC cells, recombinant PRL increased phosphorylation of JAK2, STAT3, ERK and AKT in both, a dose and time dependent manner (Figure 2B and C).

Figure 2. PRLR signals through JAK2-STAT3-ERK-AKT phosphorylation.

Figure 2.

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(A) Western blot analysis shows that PRLR is upregulated in PDAC cell lines. Breast cancer cell line T47D is used as positive control. Different isoforms of PRLR (110 (a), 80 (b), 50 (c) and 40 (d) kDa) are shown. (B) Western blot analyses of PDAC cell lysates following treatment with 100 and 200 ng/ml PRL for 30 min in serum free media shows increased phosphorylation of ERK1/2, AKT, JAK2 and STAT3 proteins. (C) Western blot of lysates following treatment of PDAC cells with 200 ng/ml PRL for up to 4 h in serum free media shows a time-dependent increase in ERK1/2 and STAT3 phosphorylation. (D) PRL affects pancosphere formation. PDAC cells were grown in in ultra-low binding plates and treated with increasing dose of PRL. There was increased spheroid numbers after PRL (200 ng/ml) treatment in MiaPaCa-2 (p = 0.0010) and Panc-1 (p = 0.0161) cells. (E) PRL (200 ng/ml) treatment induces cell migration over a period of 12 h in MiaPaCa-2 (p = 0.0056) and Panc-1 (p = 0.0305) cells. (F) PRL (200 ng/ml) affects migration in scratch plate assay (p = 0.0015).

Previous reports suggest that PRL induces proliferation, migration and spheroid formation5, 22, 23 Although, PRL did not affect cell proliferation (Supplementary Figure 2A), it did increase the size and number of PDAC spheroids in a dose-dependent manner (Figure 2D and Supplementary Figure 2C). The significance of this finding is that tumor-derived spheroids are uniquely enriched for cells with stem cell-related characteristics24. Another important point about cells with higher spheroid forming capacity is that they are also highly motile 25 Our data demonstrate that PRL induces cell migration as assessed by a trans-well chamber (Figure 2E and Supplementary Figure 2D) and scratch closure assays (Figure 2F and Supplementary Figure 2E).

PRLR knockdown affects PDAC growth

To demonstrate that PRLR signaling is important for PDAC growth, we sought to knockout the gene using the CRISPR/Cas9 system. Although testing multiple clones were tested for each cell line, a complete knockout was not successful. We chose one clone each for MiaPaCa-2 and UNKC-6141 cells, named M64 and U518, respectively to further knockdown the expression using PRLR specific shRNA. We observed maximum suppression of PRLR expression when we combined CRISPR/Cas9 and specific shRNA (Figure 3A). However, all the clones (CRISPR alone, shRNA alone and the combination) demonstrated lack of JAK2, STAT3, ERK and AKT phosphorylation, in response to PRL (Figure 3B and Supplementary Figure 3A). CRISPR and shRNA PRLR knockdown also reduced PDAC proliferation compared to controls (Supplementary Figure 3B). Moreover, there was a significant reduction in the colony (2D) and spheroid sizes, as compared to controls (Figure 3C–E). In addition, PRLR knockdown impaired cell migration (Figure 3F and Supplementary Figure 3C). Taken together, these data suggest that PRLR signaling is important for PDAC growth and migration. This is in line with previous studies showing that antagonizing PRLR results in reduced ovarian tumor growth in mice23. To determine the effect of PRLR knockdown in vivo, we used a syngeneic orthotopic model. We injected control UNKC-6141 cells, or its CRISPR clone U518 cells expressing scrambled or PRLR specific shRNA into the pancreas of C57BL/6 mice. UNKC-6141 control cells formed large tumors compared to the PRLR knockdown clones (Figure 3G and Supplementary Figure 3D). Similarly, the U518 clone expressing a combination of CRISPR and PRLR shRNA (U518-sh) formed smaller tumors (Figure 3I and Supplementary Figure 3D). More importantly, mice with PRLR knockdown tumors had higher survival rates, than mice with tumors expressing wild-type PRLR (Figure 3H). In addition, PRLR knockdown tumors have fewer PCNA-positive nuclei suggesting reduced cell proliferation (Supplementary Figure 3E and F). Further, western blot analyses of extracts from the residual PDAC tumors lacking PRLR showed decreased STAT3, ERK, and AKT phosphorylation when compared to control tumors (Supplementary Figure 3G). These data demonstrate that PRLR is essential for pancreatic tumor formation and growth.

Figure 3. PRLR knockdown affects PDAC growth.

Figure 3.

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(A) Western blot shows PRLR knockdown following CRISPR-Cas9 and shRNA in MiaPaCa-2 (human PDAC) and UNKC-6141 (mouse PDAC) cells. M63 and M64, and U518 and U525 are CRISPR/Cas9 knockdown clones of MiaPaca-2 and UNKC-6141, respectively. These cells were further subjected to PRLR-specific (sh) and scrambled (Scr) shRNAs. (B) Western blot analyses of MiaPaCa-2 cells treated with PRL (200 ng/ml, 30 mins) shows no induction in phosphorylation of STAT3, AKT and ERK1/2 in PRLR knocked down cells. (C) Clonogenicity assay. PRLR knocked down MiaPaCa-2 and UNKC-6141 cells shows comparatively lower colony forming ability than their respective control cells (p < 0.01). (D) PRLR knock down significant reduced pancosphere formation. (E) PRLR knock down showed reduced spheroid size and number as compared to their respective control cells (p < 0.01). (F) PRLR knocked down reduced migration ability in scratch plate assay (p < 0.05). (G) Mice carrying UNKC-6141 tumors in the pancreas expressing PRLR had significant lower weight compared to those lacking PRLR (U518, n = 10) (p < 0.01). (H) Kaplan-Meier plot shows that animals (n = 10) harboring PRLR knocked down PDAC cells (U518-sh) had a significantly higher survival rate as compared to controls (p < 0.01).

Identification of anti-psychotics as PRLR interacting compounds

Previous clinical studies with PRLR-specific peptide or antibody antagonists showed poor clinical efficacy, in part because they target the extracellular domain26. We chose a different approach, wherein we targeted the intracellular domain of PRLR responsible for downstream JAK2 activation. However, the intracellular domain of PRLR has not been crystallized27. Hence, we used homology-modeling algorithms (iTASSER, SWISS modeler, RaptorX, and Phyre2) to predict the structure and evaluated by Ramachandran plot (Supplementary Figure 4A and B). Next, we performed structure-based virtual screening of compounds in iTASSER and iDOCK servers and identified two classes of compounds with potentially binding capacity (Figure 4A). Subsequently, using a “Fragment-based drug discovery” approach 28, 29, we identified six compounds with similarity to the two classes of predicted compounds (Figure 4B and Supplementary Figure 4C). We confirmed that the compounds can interact with the PRLR-JAK2 domain by molecular docking (Figure 4C and Supplementary Figure 4D). However, only one compound penfluridol showed interaction within the JAK2 binding domain, while the others had allosteric interactions outside the JAK2 binding domain (Supplementary Figure 4D). Since penfluridol is an antipsychotic compound, we selected additional antipsychotic drugs belonging to chemical classes of butyrophenones (haloperidol and pipamperone), Diphenylbutylpiperidines (fluspirilene and pimozide) and Phenothiazines (promethazine and fluphenazine) (Figure 4B and Supplementary Figure 4E). Of these, only promethazine and fluspirilene showed interaction within the JAK2 binding domain, while the others had allosteric interactions outside the JAK2 binding domain (Supplementary Figure 4E). Moreover, promethazine, penfluridol and fluphenazine showed lower binding energies (Supplementary Figure 4F), indicating tight binding to the JAK2 binding site. Nevertheless, we tested all the compounds for antiproliferative activity and observed that the anti-psychotic compounds penfluridol, pimozide, fluspirilene and promethazine were effective in suppressing proliferation (Figure 4D and Supplementary Figure 4G–L). Since penfluridol was the first anti-psychotic that we identified, we chose this compound for proof of principle experiments. In addition to inhibiting PDAC cell proliferation, penfluridol suppressed colony and spheroid formation (Figure 4E–H and Supplementary Figure 4M). No other compound showed any effect on spheroid formation (Supplementary Figure 4N and O). Moreover, Penfluridol suppressed cell proliferation even in the presence of exogenous PRL (Supplementary Figure 4P). Penfluridol induced autophagy at 48 h as evidenced by an increase in the expression autophagy related proteins (Figure 4I). Similarly, Fluspirilene, Promethazine, Fluphenazine and Pimozide also increased levels of LC3II and p62 protein expression indication upregulation of autophagy (Supplementary Figure 4Q). However, Penfluridol did not induce caspase activity except at a higher concentrations (Supplementary Figure 4R). In order to confirm the specificity of the inhibitor, we treated PRLR knockdown cells with penfluridol. PRLR knockdown cells were resistant to penfluridol treatment with a shift in the IC50 from 2–2.1 μM to 3.2–3.8 μM in MiaPaCa-2 and from 5.8–6 μM to 7.4–7.6 μM in UNKC-6141 cell lines (Supplementary Figure 4S and T). We also determined the effect of combining penfluridol with gemcitabine. There was synergistic activity observed in both MiaPaCa-2 and Panc-1 cell lines, with the best combination observed at a dose combination of 2.5 μM penfluridol and 5.0 μM Gemcitabine (Supplementary Figure 4U–W).

Figure 4. In-silico molecular modeling identifies penfluridol as a PRLR interacting compound.

Figure 4.

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(A) The JAK2 binding region within the cytoplasmic domain of PRLR was subjected to homology modeling using I-TASSER and subsequently used for predicting potential interacting compounds with I-DOCK and I-TASSER. The predicted structure and their common features are shown in red boxes. (B) Fragment-based drug discovery was used to identify compounds targeting PRLR based on common structural features in showed red boxes of panel A. The panel shows the most potent antipsychotic drugs identified including penfluridol, pimozide, fluspirilene and fluphenazine. (C) Molecular docking of penfluridol (PEN) performed with the JAK2 binding site (green) using Autodock vina software. (D) Antipsychotic drugs inhibit PDAC cell proliferation. Cells were incubated with increasing concentration (0–40 μM) of compound for 72 h, resulting in a dose and time-dependent decrease in cell proliferation when compared with untreated controls. (E) Penfluridol inhibits colony formation. Cells were incubated with 4 μM penfluridol for 48 h and allowed to grow into colonies for 10 d. Incubation with penfluridol inhibits clonogenicity. (F) Penfluridol inhibits number of colonies as compared to control in MiaPaCa-2 (p = 0.0004) and Panc-1 (p = 0.0005) cells. (G) Penfluridol inhibits spheroid formation assay. Penfluridol suppressed the number of pancospheres in both primary and secondary spheroids cultures (p < 0.001). (H) Cells were grown in spheroid growth media in ultra-low adherent plates and treated with Pen (4 μM) for 5 d. Penfluridol significantly decreased pancosphere formation. (I) Penfluridol induces autophagy. Lysates from MiaPaCa-2 or Panc-1 cells incubated with 4 μM penfluridol were analyzed by western blotting for LC3B, p62, ATG-5, −7, and −12 and Beclin-1. Penfluridol treated cells shows increased levels of all these proteins.

Penfluridol binds to PRLR

Given that penfluridol affects cell viability, we further characterized its interaction with PRLR. In silico modeling demonstrated penfluridol binding to the JAK2 domain of PRLR (Figure 5A). To validate the in silico observation, we first performed surface plasmon resonance (SPR) analyses with penfluridol and a PRLR peptide encoding the JAK2 binding site. Penfluridol binding to PRLR peptide on the surface of gold nanoparticles altered the local refractive index in a dose-dependent manner resulting in a shift of the SPR spectrum (Figure 5B). We confirmed the direct binding of penfluridol with the PRLR peptide using the Magnetic relaxometry, a novel method where multivalent magnetic nanosensors are generated with the peptide30, 31. Detection of the interaction between the peptide nanosensor and the compound is achieved by changes in the solution’s water relaxation times (ΔT2, measured in milliseconds), using a magnetic relaxometer. Initial studies with 0.5 μM penfluridol demonstrated maximum interaction at 60 min (Supplementary Figure 5A and B). Using this time point, we determined the effect of increasing doses of penfluridol and observed a dose-dependent increase in the compound binding (Figure 5C).

Figure 5. Penfluridol binds to PRLR.

Figure 5.

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(A) Molecular docking shows penfluridol binds to JAK2 binding site of PRLR. Cartoon format of binding model (upper panel) and ball and stick format of model (lower panel) (Protein: Blue, Penfluridol: Pink, JAK-2 binding site: Green, Interacting amino acid: Yellow) are shown. (B) Dose dependent (10 nM-20 μM) increase in penfluridol binding to PRLR calculated by surface plasmon resonance (SPR). (C) Dose dependent (10 nM-20 μM) increase in penfluridol binding to PRLR calculated by the magnetic resonance technique. (D) Drug Affinity Responsive Target Stability (DARTS). MiaPACa-2 cells were treated with increasing concentration of penfluridol (0–20 μM) and then with pronase. Resulting lysates were subjected to western blot analyses. Penfluridol protected PRLR against pronase-induced cell lysis demonstrating PEN-PRLR interaction. (E) Cellular thermal shift assay (CETSA). MiaPACa-2 cells were treated with different concentration of penfluridol and subjected to differential temperature treatment for 3 mins. Western blot shows Penfluridol protected PRLR against thermal denaturation, suggesting compound interaction with receptor. (F) Penfluridol inhibits PRL-induced ERK and STAT3 phosphorylation. Western blot analyses of cells lysates following penfluridol treatment show reduced ERK and STAT3 phosphorylation in response to PEN. (G) Western blot analysis PDAC cell lysates following penfluridol treatment shows that the compound does not affect PRLR protein levels, while it inhibits STAT3 phosphorylation.

We next validated the binding by treating PDAC cell lines with penfluridol and subjecting the cell to drug affinity responsive target stability (DARTS) assay and cellular thermal shift assay (CETSA). Here the target protein is subject to various lysis (DARTS) or denaturation (CETSA) conditions with the assumption that a change in conformation following compound binding will result in an increase in its stability. We observed significant protection from pronase digestion (Figure 5D) and temperature (Figure 5E). On the other hand, penfluridol treatment did not change the stability of STAT3 confirming the specificity of penfluridol binding to PRLR (Supplementary Figure 5C).

We next determined the effect of penfluridol treatment on JAK2-STAT3 signaling pathway. Pretreatment with penfluridol decreased PRL-induced ERK and STAT3 phosphorylation (Figure 5F and G). However, there was no effect on PRLR levels, suggesting that compound does not induce degradation of the protein. Further, we also found that antipsychotic drugs including fluphenazine, fluspirilene, pimozide and promethazine inhibited PRL-induced STAT3 and ERK phosphorylation in MiaPaCa-2 cell line (Supplementary Figure 5D).

Penfluridol induced suppression of PDAC growth is not mediated through DRD2 receptor

DRD2 is present in the normal pancreas and is upregulated in PDAC17. Penfluridol exerts its antipsychotic activity by blocking dopamine receptor 2 (DRD2)32, 33. Molecular docking studies show that penfluridol binds DRD2 protein, stabilizing the interaction by forming a hydrogen bond with ASP114 (Figure 6A and B). Previously, another DRD2 antagonist risperidone was also shown to bind to ASP114, suggesting that binding to this site is the potential mechanism of action of penfluridol34. To understand the role of DRD2 in penfluridol inhibition of PDAC cell growth, we first determined the DRD2 expression levels. Western blot analysis of PDAC cell lines showed increase in DRD2 protein expression compared to HPNE cells (Figure 6C). Although the major band indicating DRD2 is found at 52 kDa, there are multiple weak bands observed between 85 and 100 kDa suggestive of DRD2 isoforms35. We next knocked down DRD2 using specific siRNA in two PDAC lines, Panc-1 and Panc 01728, resulting in reduction in both DRD2 mRNA and protein (p < 0.01, Figure 6D and E). However, penfluridol treatment did not demonstrate any significant difference in antiproliferative activity following DRD2 knockdown, although at one concentration (1.5 μM) there was a small but insignificant effect (Figure 6F). This suggests that DRD2 is not a potential target for penfluridol in suppressing PDAC growth.

Figure 6. Penfluridol-induced suppression of PDAC growth is not mediated through DRD2 receptor.

Figure 6.

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(A) Molecular docking of penfluridol (PEN) performed with DRD2 protein using Autodock vina software. (B) Table showing docking scores of molecular docking of penfluridol with DRD2. (C) Western blot analysis shows that DRD2 is upregulated in PDAC cell lines. (D) RT-PCR analysis showing knockdown of DRD2 expression induced by siRNA in Panc-1 and Panc 01728 cell lines. (E) Western blot analysis showing siRNA-mediated knockdown of DRD2 expression. (F) Penfluridol does not have any differential effect in proliferation of PDAC cells when DRD2 is knocked down.

Penfluridol suppresses PDAC growth in vivo

To determine the effect of penfluridol on tumor growth, we injected UNKC-6141 and KPCC cells into the pancreas of immunocompetent C57BL/6 mice and waited one week for tumors to develop. Subsequently, we injected penfluridol (5 mg/kg, i.p. daily), which reduced the weights of orthotopic tumors by more than 50% as compared to control (Supplementary Figure 6A–C). Next, we performed subcutaneous-xenograft models with patient derived xenografts (PDX) and Panc-1 xenografts in immunodeficient NSG and athymic nude mice, respectively. Penfluridol treatment significantly reduced tumor weight and volume in both xenograft tumors (Figure 6A–D, Supplementary Figure 6D and E). Immunohistochemistry also demonstrated significantly lower PCNA positive cells in penfluridol treated tumors (Supplementary Figure 6F and G). Western blot analyses suggested that penfluridol induced autophagy-related proteins p62, ATG-5, ATG-7, ATG-12, LC3B, and beclin-1 (Figure 6G). These data suggest that antipsychotics such as penfluridol significantly induce autophagy resulting in suppression of PDAC tumor growth.

DISCUSSION

This is the first demonstration for PRLR role in PDAC, a deadly cancer with abysmal five-year survival rate. PRL can be produced by both the anterior pituitary gland and other tissues including mammary glands, adipocytes, pancreatic β-cells and immune cells to produce autocrine effects36. In normal tissue, PRL signaling is important for pancreatic β-cell proliferation37. Previous studies have focused on PRLR activity in hormone response cancers. Although PRLR in PDAC has not been demonstrated, elevated serum PRL levels has been reported10 suggesting a role for this signaling pathway in PDAC. Moreover, we demonstrate using Gene Analytics that 22% of all published reports on PDAC pertain to molecules associated with PRLR signaling. Probing the TCGA database also showed higher PRLR expression in PDAC, which we validated using a cDNA panel and tissue microarray. These results indicate that PRLR has a role in PDAC pathogenesis.

PRL binds to its cognate receptor (PRLR) and activates downstream signaling pathways including JAK-STAT and MAPK. Although previous reports suggested that PRLR induces JAK2-mediated STAT5 phosphorylation in the breast38 and ovarian23 cancer cells, we did not observe STAT5 activation. Interestingly, it was shown that STAT5 and STAT3 mediate opposing effects, and activation of both STAT3 and STAT5 at the same time results in reduced proliferation of breast cancer cells39. In our study, we observed dose-dependent phosphorylation of STAT3. In addition, we observed that PRL did not affect PDAC cell proliferation. However, PRL did increase stemness and migration in PDAC, which we also previously observed in colon cancer cells5. Similar results have been reported for ovarian cancer, where PRL failed to induce proliferation23. Moreover, PRL has been shown to suppress cell death and enhance survival in various cancers3.

PDAC patients reported elevated psychological distress compared to those with other cancers40. Indeed, under conditions of stress, there is increased production of pituitary PRL and higher circulating PRL levels41. PRL plays key roles in stress response by suppressing the hypothalamic–pituitary–adrenal axis and weakening the immune system42. While the evidence for psychological stress causing cancer may be weak, those under stress may cultivate certain unwanted behaviors, including smoking, overeating, and decreased exercise, resulting in increased cancer risk. In addition, studies have demonstrated that mice subjected to stress have significantly larger tumors, angiogenesis and increased metastasis43. Moreover, antidepressants, antihypertensive agents, and drugs which increase bowel motility are known to induce hyperprolactinemia by acting through suppressing DRD2 activity44. A recent study also demonstrated that stress accelerates PDAC45 in a mouse model by inducing catecholamines, which was suppressed by the nonselective β-adrenergic receptor blocker, propranolol. In fact, previous studies have demonstrated β-adrenergic receptor agonists can induce PRL secretion from pituitary cells and pre-adipocytes, which can be reversed by an antagonist such as propranolol46. Taken together, these data raise the question of whether inhibition of aggressive PDAC behavior may be due in part to the β-blocker reduction of PRL levels.

To get further insights into the role of PRLR in PDAC, we first used the CRISPR/Cas9 system to knockout the gene. However, after repeated testing, we were unable to obtain a complete knockout. We considered problems such as short guide RNA design, delivery methods, off-target effects and homology-directed repair47. To overcome the shortcomings with guide RNA design, we did generate multiple guide RNAs, but the results were the same. To negate the potential for off-target effects, we designed scrambled controls. Finally, we tried multiple ways to deliver the guide RNAs including transfection reagents and lentiviral systems. Since, none of these worked, we believe that it is not the system but rather the increased copy number at the gene loci or the potential presence of pseudogenes. However, subsequent knockdown using specific shRNA mitigated PRLR function in the cells, resulting in reduced proliferation, clonogenicity, migration and pancosphere formation capacity.

Given the aggressive role of PRLR in PDAC growth, we conclude that it is an excellent therapeutic target. Previously, two PRLR antagonists, peptide G129R and monoclonal antibody LFA102, have been reported. G129R is a novel antagonist peptide of PRL that inhibited PRL/PRLR signaling and reduced tumor growth in an orthotopic mouse model of ovarian cancer23. However, this has not progressed towards clinical trials possibly due to stability issues on systemic delivery of the peptide. A second inhibitor, LFA102 is a monoclonal antibody but clinical trials in breast and prostate cancer failed48. However, this might be for a couple of reasons. One is the potential unanticipated compensatory changes of downstream signaling pathways or upregulation of other compensatory tumorigenic signaling pathways in response to PRLR inhibition. A second reason could be that the receptor is present intracellularly in cancer cells. Hence, targeting the extracellular domain may not be an effective strategy. Therefore, we believe that targeting the intracellular domain may be a better approach. We have observed that multiple anti-psychotic agents can bind PRLR and affect cell viability. As proof of principle, we have taken penfluridol in our subsequent studies. We demonstrate that penfluridol targets the JAK2 binding site in PRLR, thereby suppressing JAK2-STAT3 and ERK/AKT signaling (Supplementary Figure 7). Penfluridol is an FDA-approved, long lasting and orally bioavailable drug used for the treatment of schizophrenia49. Recent reports have demonstrated that penfluridol has anticancer properties against breast50, 51 and PDAC52 but the mechanism of action is not yet fully described. We have now determined that this inhibition is through suppression of PRL signaling. In addition, we have also observed that fluspirilene and promethazine have similar activity. Since these are FDA-approved antipsychotics, further research into these compounds is warranted to determine clinical efficacy. Previous studies have identified that DRD2, also a target for these anti-psychotics are upregulated in PDAC and may play a role in growth of the tumor17. However, in our current studies, penfluridol did not appear to affect cells where DRD2 expression was suppressed. This is similar to another study where thioridazine, also a DRD2 antagonist inhibits proliferation of breast cancer cells but is independent of DRD2 receptor expression53. This suggests that the anti-psychotics function to inhibit PDAC growth exclusively through PRLR. Finally, growth hormone receptor can also activate JAK2-mediated signaling54. It would be interesting to determine whether these compounds also interfere with GHR-JAK2 interaction, or whether the compound has a unique activity against PRLR. These are part of the future directions of the project.

Supplementary Material

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Figure 7. Penfluridol suppresses PDAC growth in vivo.

Figure 7.

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(A) Penfluridol treatment reduced pancreatic cancer patient derived xenograft tumor tissue (PDX) grown subcutaneously in the flanks of the NSG mice when compared to control (p < 0.05). (B) Penfluridol treatment resulted in significantly lower PDX tumor volumes when compared to controls (p < 0.05). (C) Penfluridol treatment reduced Panc-1 tumor xenografts (p < 0.01) as compared to control. (D) Penfluridol treatment resulted in significantly lower Panc-1 xenograft tumor weight when compared to control (p < 0.01). (E) Western blot analyses of tissue lysates from penfluridol treated animals show significantly higher levels of autophagy protein markers.

What you need to know.

BACKGROUND AND CONTEXT

Prolactin signaling is upregulated in hormone-responsive cancers, and patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of prolactin. The prolactin receptor (PRLR) signals via the JAK–STAT and MAPK pathways to regulate cell proliferation, migration, stem-cell features, and apoptosis.

NEW FINDINGS

Levels of PRLR are increased in PDAC, and exposure of pancreatic cancer cells to PRL increases their proliferation and migration. Antipsychotic drugs such as penfluridol block PRLR signaling in pancreatic cancer cells and reduced their proliferation, induced autophagy, and slowed growth of xenograft tumors in mice.

LIMITATIONS

This study was performed in cell lines and mice. Studies in humans are needed.

IMPACT

Antipsychotic drugs such as penfluridol block JAK2 signaling in pancreatic cancer cells, slow tumor growth in mice, and might be tested in patients with PDAC.

LAY SUMMARY.

We identified a receptor on pancreatic cells that promotes their proliferation, movement, and formation of tumors in mice. We discovered a class of drugs that blocks this receptor to slow tumor growth in mice and might be tested in patients with pancreatic cancer.

Acknowledgments

Funding

This work was supported by a pilot grant from the NCI-designated University of Kansas Cancer Center (P30CA168524; SA), and NIH Grants R01CA182872 and R01CA190291, SA), Kansas IDeA Networks of Biomedical Research Excellence (K-INBRE, P20GM103418, PD) and University of Kansas Medical Center Biomedical Sciences Training Program (BRTP, PD). S. Anant is an Eminent Scientist of the Kansas Biosciences Authority. We also thank the Thomas O’Sullivan Foundation and the Rod Rogers Foundation for providing financial support. The Flow Cytometry Core Laboratory is funded, in part, by NIH grants NCRR P20 RR016443 and P30CA168524.

Footnotes

Conflict of Interest

Authors declare no conflict of interests.

The authors declare no potential conflicts of interest

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Supplementary Materials

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