Activation of VIPR1 suppresses hepatocellular carcinoma progression by regulating arginine and pyrimidine metabolism

Background and aims: Vasoactive intestinal polypeptide type-I receptor (VIPR1) overexpression has been reported in numerous types of malignancies and utilized to develop novel target therapeutics and radiolabeled VIP analogue-based tumor imaging technology, but its role in liver carcinogenesis has not been explored. In the current study, we investigated the role of the VIP/VIPR1 signaling in controlling hepatocellular carcinoma (HCC) progression. Approach and results: By analyzing clinical samples, we found the expression level of VIPR1 was downregulated in human HCC tissues, which was correlated with advanced clinical stages, tumor growth, recurrence, and poor outcomes of HCC clinically. In vitro and in vivo studies revealed that activation of VIPR1 by VIP markedly inhibited HCC growth and metastasis. Intriguingly, transcriptome sequencing analyses revealed that activation of VIPR1 by VIP regulated arginine biosynthesis. Mechanistical studies in cultured HCC cells demonstrated that VIP treatment partially restored the expression of arginine anabolic key enzyme argininosuccinate synthase (ASS1), and to some extent, inhibited de novo pyrimidine synthetic pathway by downregulating the activation of CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase). VIP treatment upregulated ASS1 and subsequently suppressed CAD phosphorylation in an mTOR/p70S6K signaling dependent manner. Clinically, we found human HCC samples were associated with downregulation of ASS1 but upregulation of CAD phosphorylation, and that VIPR1 levels positively correlated with ASS1 levels and serum levels of urea, the end product of the urea cycle and arginine metabolism in HCC. Conclusions: Loss of VIPR1 expression in HCC facilitates CAD phosphorylation and tumor progression, and restoration of VIPR1 and treatment with the VIPR1 agonist may be a promising approach for HCC treatment.

Heatmap shows the difference of arginine metabolism-related genes expression in normal liver,VIPR1 high and VIPR1 low HCC of TCGA cohort.

Human Cohort Study
A 107-patient cohort diagnosed with hepatocellular carcinoma (HCC) was enrolled in our study for investigating the significance of VIPR1 in HCC clinical prognosis. All HCC patients received surgical resection in Xiangya Hospital during 2005-2019. Human samples were obtained under informed consent. The clinicopathological characteristics, including age, gender, pathology, tumor size, tumor multiplicity, cirrhosis, vascular invasion and differentiational status, were available for all patients. Diagnosis of HCC and non-tumor adjacent liver tissues were confirmed based on histological findings by independent pathologists. The assessment of HCC tumor burden after surgery and treatment was guided by RECIST version 1.1 Criteria. Frozen specimens at -80°C of non-tumor adjacent liver tissue (liver tissue close to the tumorous region) and HCC tissues were used to extract total RNA and protein for analyses. Serum samples of HCC patients were collected before surgery and were used to measure urea concentration by using the Urea Assay kit (Abcam, ab83362). Paraffin-embedded surgical tissues (non-tumor liver and HCC tissues) were used for immunohistochemistry analyses. The information for all HCC cohorts enrolled in this study are listed in Table S1-Table S6.

RNA isolation and quantitative reverse transcription PCR (RT-qPCR)
Total RNA was extracted from liver tissues and cell samples using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instruction. One microgram (1μg) RNA was reverse-transcribed into cDNA by using High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). RT-qPCR was performed by SYBR Green Realtime PCR master mix. The mRNA levels were measured by QuantStudio TM 6 Real-Time PCR System (278861830; Thermo Fisher Scientific). The expression levels of target genes were normalized to GAPDH (for human genes) and 18S rRNA (For mouse genes) expression. Comparative Ct (2-ΔΔCt) method was performed to quantify the mRNA expression level. All primers used for RT-qPCR are listed in Table S7.

Western Blotting
Liver tissues or cell lysates were homogenized in RIPA lysis buffer containing cocktail of protease inhibitors (Santa Cruz, CA) according to the manufacturer's instruction at 4°C and centrifuged at 10,000 g for 10 minutes. Protein extracts were quantified using BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA), mixed with loading buffer, and then were subjected to 4-12% Bis-Tris protein gels (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes (Thermo

Cell counting kit-8 (CCK-8) assay
In vitro cell proliferation and viability were determined by colorimetric method by using Cell counting kit-8 (CCK-8) assay (Cat # DJDB4000X, VitaScientific) according to the manufacturer's instruction. Cell viability was presented as absorbance value at 450nm. Cell proliferation was monitored at 4 different timepoints during consecutive 3 days. Similar method was previously described in [1].

Colony formation assay and Wound healing assay
These two assays were used to determine HCC cell proliferation and migration potential in vitro, which were performed as described previously [1,2]. All experiments were performed in triplicate. The MHCC97H and HCCLM3 cells were seeded in 6-well plates and were cultured in the routine condition. 200 uL tips were used for the wound healing when the cell confluency was above 90%. Then the cells were washed with PBS and cultured with a low concentration of serum. Photos were taken at two timepoints (0h, 24h) and statistically analyzed by using ImageJ software (National Institutes of Health, Bethesda, MD).

Lentivirus infection
The lentiviral vector GV358 (Ubi-MCS-3FLAG-SV40-EGFP-IRES-Puromycin) containing human VIPR1 (NM_004624) and its Control vector, and lentiviral vector GV260 (Ubi-MCS-firefly_Luciferase-IRES-Puromycin) for bioluminescent imaging analysis were constructed from Shanghai GeneChem, China. Cell infection was performed according to the manufacturer's instruction. To select stably transduced cell clones, the infected cells were cultured with puromycin (2.5ug/mL) for 2 weeks; RT-qPCR was then performed to determine the overall infection efficiency.

Urea concentration measurement
Urea Assay Kit (ab83362, Abcam) was used to measure the urea concentration in HCC serum samples and cell culture medium according to the manufacturer's instruction. The samples should be diluted by 1xPBS to a proper concentration range for analysis. The standards added to 96-well plates were used for plotting standard curve. Urea in samples was acted on by enzymes to form a product; the reaction mixture was incubated at 37℃ for 1h, and measured the absorbance by using microplate reader (ODmax=570nm). Absorbance is directly proportional to the urea concentrationin the system.

Immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining
Paraffin-embedded tissues were used for immunohistochemistry staining. 4-μm-thick paraffin sections were stained with hematoxylin and eosin (H&E) for histological analysis. For IHC staining, heat-induced epitope retrieval was carried in 3% citrate buffer, endogenous peroxidases were blocked with 3% H2O2 for 20 minutes, and non-specific binding was blocked by using 3% normal goat serum for 1 hour at room temperature. Slides were then incubated with primary antibody overnight at 4°C. After PBS washing, slides were subsequently incubated with anti-mouse or anti-rabbit secondary antibodies (SignalStain® Boost IHC Detection Reagent, Cell Signaling Technology, Danvers, MA, USA) for 1 hour at room temperature. Staining results were developed and visualized by Vectastain Elite ABC Staining Kit DAB peroxidase substrate kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer's instruction, and then slides were counterstained with hematoxylin. The analysis of positive area was determined with ImageJ software (National Institutes of Health, Bethesda, MD).

Immunofluorescent staining for adherent cells
Cell adherent sections were fixed in 4% paraformaldehyde (4% PFA, pH7.4) for 10 minutes and washed with PBS three times. Slides were blocked in blocking solution (3% bovine serum albumin in PBS) for 1 hour at room temperature and incubated with primary antibodies (listed in Table S8) overnight at 4°C. After PBS washing, slides were incubated with the fluorescence conjugated antibodies (listed in Table S8) for 1 hour at room temperature. Nuclear staining was obtained by incubation with 1 mg/mL 4', 6'-diamino-2-phenylindole (DAPI) for 5 min at room temperature. Images were acquired using LSM 710 confocal microscope (Zeiss, Thornwood, NY, USA).The images were analyzed by the con-focal microscopic system from Zeiss (Thornwood, NY).

HCC tumor cell derived xenograft (CDX) model
The method for building HCC cell derived xenograft (CDX) model was discussed in Methods and materials and figure legends of the main text. Tumor size was measured every 3 days. Tumor volume was calculated by using the following formula: volume =1/2× (length×width 2 ). All mice were sacrificed at day 21. Tumor tissues were isolated and embedded in paraffin for Ki-67 (AY0064, ABZOOM) staining and analysis.

In vivo bioluminescent imaging on metastatic model
Bioluminescence imaging (BLI) method was utilized to visualize and quantify bioluminescent signal of metastatic HCC in mouse model (OD-Prkdcem26Cd52Il2rgem26Cd22/Nju (NOD/SCID IL2rg -/mice, or NCG mice; Nanjing Biomedical Research Institute of Nanjing University). Luciferase Vector GV260 (Ubi-MCS-firefly_Luciferase-IRES-Puromycin) labeled HCCLM3 cells was injected via tail vein (More details are mentioned in main text). The mice were anaesthetized under 2% isoflurane gas, then were intraperitoneally injected with D-Luciferin (Sodium Salt D 40901ES01, YEASEN, China), and were imaged 10-15 minutes after the injection in an IVIS imaging system (IVIS® In Vivo Imaging Systems, Spectrum BL; PerkinElmer, MA) according to the manufacturer's instruction under anesthesia. BLI signal is quantified in regions of interest (ROIs). Images were analyzed by using Living Image 4.3.1 software (PerkinElmer, MA). Metastatic lesions were measured once a week after model establishment, over the next 4 weeks. After sacrificing the mice, spleen, liver and lung tissues were isolated and measured to quantify the metastases. The targeted organs were collected and fixed in formalin, and embedded in paraffin for H&E staining.