The short isoform of PRLR suppresses the pentose phosphate pathway and nucleotide synthesis through the NEK9-Hippo axis in pancreatic cancer

Prolactin binding to the prolactin receptor exerts pleiotropic biological effects in vertebrates. The prolactin receptor (PRLR) has multiple isoforms due to alternative splicing. The biological roles and related signaling of the long isoform (PRLR-LF) have been fully elucidated. However, little is known about the short isoform (PRLR-SF), particularly in cancer development and metabolic reprogramming, a core hallmark of cancer. Here, we reveal the role and underlying mechanism of PRLR-SF in pancreatic ductal adenocarcinoma (PDAC). Methods: A human PDAC tissue array was used to investigate the clinical relevance of PRLR in PDAC. The in vivo implications of PRLR-SF in PDAC were examined in a subcutaneous xenograft model and an orthotopic xenograft model. Immunohistochemistry was performed on tumor tissue obtained from genetically engineered KPC (KrasG12D/+; Trp53R172H/+; Pdx1-Cre) mice with spontaneous tumors. 13C-labeled metabolite measures, LC-MS, EdU incorporation assays and seahorse analyses were used to identify the effects of PRLR-SF on the pentose phosphate pathway and glycolysis. We identified the molecular mechanisms by immunofluorescence, coimmunoprecipitation, proximity ligation assays, chromatin immunoprecipitation and promoter luciferase activity. Public databases (TCGA, GEO and GTEx) were used to analyze the expression and survival correlations of the related genes. Results: We demonstrated that PRLR-SF is predominantly expressed in spontaneously forming pancreatic tumors of genetically engineered KPC mice and human PDAC cell lines. PRLR-SF inhibits the proliferation of PDAC cells (AsPC-1 and BxPC-3) in vitro and tumor growth in vivo. We showed that PRLR-SF reduces the expression of genes in the pentose phosphate pathway (PPP) and nucleotide biosynthesis by activating Hippo signaling. TEAD1, a downstream transcription factor of Hippo signaling, directly regulates the expression of G6PD and TKT, which are PPP rate-limiting enzymes. Moreover, NEK9 directly interacts with PRLR-SF and is the intermediator between PRLR and the Hippo pathway. The PRLR expression level is negatively correlated with overall survival and TNM stage in PDAC patients. Additionally, pregnancy and lactation increase the ratio of PRLR-SF:PRLR-LF in the pancreas of wild-type mice and subcutaneous PDAC xenograft tumors. Conclusion: Our characterization of the relationship between PRLR-SF signaling, the NEK9-Hippo pathway, PPP and nucleotide synthesis explains a mechanism for the correlation between PRLR-SF and metabolic reprogramming in PDAC progression. Strategies to alter this pathway might be developed for the treatment or prevention of pancreatic cancer.

2 For orthotopic xenograft model in genetic overexpression study, 10 nude mice were averagely divided into two groups randomly. A total of 2 × 10 6 Ctrl or PRLR-SF AsPC-1 cells or BxPC-3 cells in 25 μL PBS were injected into the head or body of pancreas. After 4 weeks, mice were sacrificed, and the whole pancreas coherent with tumor and spleen was obtained and photographed, then tumor was isolated and weighted.
For survival analysis of nude mice orthotopically implanted with AsPC-1 cells, the treatments were same as orthotopic xenograft model. Survival days were recorded once the mouse was dead.

PRL stimulation and PRLR inhibition assay
For PRL stimulation in cell proliferation assay, cells were cultured in media containing 2% FBS and treated with Vehicle or reconstitute PRL (final concentration: 0.5μg/ml) for 96 hrs. PRL was supplemented every 24 hrs as the same final concentration.

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For PRLR inhibition in cell proliferation assay, cells were cultured in completed media and treated with vehicle or PRLR antibody (MAB1167, Novus, final concentration: 3μg/ml) for 96 hrs. PRLR antibody was supplemented every 24 hrs as the same final concentration.
For PRL stimulation in Western blotting assay, cells were cultured in FBS-free media for at least 2 hrs in advance. Then PRL (final concentration: 0.5μg/ml) was added into media lasting for 0, 5, 15, 30, 60 and 120 mins, respectively.
Cell proliferation, colony formation assay. For cell proliferation assay, cells were seeded at 2×10 3 cell/well in 96-well plates and cultured at 37 ℃. Then 10% (v/v) CCK-8 (Dojindo, Kumamoto, Japan) was added to the wells and the plates were incubated at 37 ℃ for 1 hr.
Absorbance was measured at 450 nm using a Power Wave XS microplate reader (Bio-Tek, Winooski, VT) to analyze cell viability. This experiment was performed as five biological replicates for each group and repeated twice.
For colony formation, cells were seeded at 2×10 4 cells/well in 6-well plates in culture medium and cultured at 37 ℃ for 2-3 weeks. Cells were stained with crystal violet until the colonies were visualized by naked eyes. The number of colonies was calculated with ImageJ. This experiment was repeated twice.
Histology and immunohistochemistry. Routinely, slides were deparaffinized and hydrated in xylene and gradient ethanol in order. For hematoxylin and eosin (H&E) staining, slides were stained in hematoxylin and eosin according to standard protocol. For immunohistochemistry, antigen retrieval was performed by boiling the slides in citrate sodium buffer for 15 mins. Next, endogenous peroxidase was blocked by incubating the slides in 0.3% (v/v) hydrogen peroxide methanol solution for 20 mins at 37 ℃. Then slides were blocked by 10% BSA at room temperature for 1 hr, and incubated with primary antibodies at 4℃ overnight. After HRPconjugated secondary antibodies incubation and DAB staining, slides were counterstained with hematoxylin.

Cell transfection and lentivirus constructs.
For usual cell transfection assay, cells were seeded at 60%-70% confluence in plates or dishes. The shRNA against PRLR and scrambled sequences were purchased from GenePharma (Shanghai, China), and overexpressed-PRLR plasmids were purchased from (GeneCopoeia, USA). Lentivirus particles were generated using a three-plasmidsystem (pPACKH1-GAG, pPACKH1-REV and pVSV-G) preserved by our lab. Lentivirus packaging was performed in 293T cells with co-transfection of shRNA or plasmids via Lipofectamine® 2000 Reagent according to the protocol. For lentivirus transfection assay, cells were infected with reconstructed lentivirus for 24 hrs. Then cells were treated with 1 μg/ml puromycin (Gibco, A1113802) for more than 7 days. The transfection efficiency was detected later by Western blot.
NADPH and NADP + /NAPDH ratio measurement. Intracellular NADPH and NADP + /NAPDH ratio were measured using cell lysates according to manufacturer's instruction provided by NADPH assay kit (Biovision, #K347). The values were normalized to the protein concentration.
Quantitative real-time PCR. Total RNA was extracted using RNAiso plus (Takara, Code No. 9108) and reverse transcribed with PrimeScript RT-PCR kit (TaKaRa,Code No. RR036A) according to the protocols. Real-time PCR was performed using 2x SYBR Green qPCR Master Mix (bimake, Cat#: B21202) on a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) as following thermal cycling settings: 1 initial cycle at 95 ℃ for 10 mins, followed by 40 cycles of 15 secs at 95 ℃ and 34 secs at 60 ℃. All the primers used for Real-time PCR were purchased from TsingKe and sequences were listed in supplementary Table. YAP-mutant rescue experiments. Mutant plasmids pcDNA3.1-YAP (S127A) and control plasmid pcDNA3.1 were kept in our laboratory. For rescue of PRL stimulation effects, AsPC-1 cells and BxPC-3 cells were treated with PRL as mentioned above for 24 hrs, then plasmids pcDNA3.1-YAP (S127A) transfection was performed as protocol with PRL treatment all the time.
After culturing for 48 hrs, treated cells were used for immunofluorescence assay, LC-MS analysis and EdU incorporation assay.