Fibroblast growth factor inhibition by molecular-targeted agents mitigates immunosuppressive tissue microenvironment in hepatocellular carcinoma.

BACKGROUND & AIMS: Combination immunotherapy refers to the use of immune checkpoint inhibitors (ICI) and molecular-targeted agents (MTA), which have recently been approved for the treatment of advanced hepatocellular carcinoma (HCC). Owing to its relatively low antitumor effect (up to 30%), sequential therapy following ICIs treatment is required in patients with HCC. This study aimed to determine the impact of MTAs on the tumor immune microenvironment (TIME). METHODS: We established immune syngeneic orthotopic HCC mouse models using Hep-55.1C and Hep-53.4, and treated them with MTAs (lenvatinib, sorafenib, regorafenib, cabozantinib, and DC101 as anti-vascular endothelial growth factor receptor-2 antibodies, and AZD4547 as a fibroblast growth factor receptor (FGFR)-1/2/3/4 inhibitor) for 2 weeks. Subsequently, alterations in the TIME caused by MTAs were evaluated using immunohistochemistry (antibodies for CD3, CD8, Foxp3, Granzyme B, Arginase-1, NK1.1, F4/80, CD11c, PD-1, and PD-L1). We conducted RNA-seq analysis using lenvatinib- and AZD4547-treated tumors. To confirm the clinical relevance of these findings, we analyzed the transcriptome data of human HCC cells (MHCC-97H) treated with various concentrations of lenvatinib for 24 h using RNA-seq data from the Gene Expression Omnibus database. RESULTS: The number of Foxp3- and F4/80-positive cells in the TIME was decreased in many MTAs. Cabozantinib increased the numbers in NK1.1-, Granzyme B, and CD11c-positive cells. Lenvatinib and AZD4547 increased the number of CD8, Granzyme B, and PD-L1-positive cells. Gene ontology enrichment analysis revealed that lipid metabolism-related genes were downregulated by lenvatinib and AZD4547. In total, 161 genes downregulated by FGFR inhibition in rodent models overlapped with those downregulated by lenvatinib in human HCC cells. CONCLUSIONS: In this study, we showed that cabozantinib activated the innate immune system, and lenvatinib and AZD4547, which commonly inhibit FGFR signaling, altered TIME to a hot immune state by downregulating lipid metabolism-related genes. These findings support the therapeutic use of combination immunotherapies.

Integrated multi-omics analysis for lung adenocarcinoma in Xuanwei, China.

BACKGROUND: Xuanwei lung cancer (XWLC) is well-known for its high incidence and mortality. However, the molecular mechanism is still unclear. METHODS: We performed a comprehensive transcriptomic, proteomic, and phosphoproteomic characterization of tumors and matched normal adjacent tissues from three XWLC patients with lung adenocarcinoma (LUAD). RESULTS: Integrated transcriptome and proteome analysis revealed dysregulated molecules and pathways in tumors and identified enhanced metabolic-disease coupling. Non-coding RNAs were widely involved in post-transcriptional regulatory mechanisms to coordinate the progress of LUAD and partially explained the molecular differences between RNA and protein expression patterns. Phosphoproteome provided evidence support for new phosphate sites, reporting the potential roles of core kinase family members and key kinase pathways involved in metabolism, immunity, and homeostasis. In addition, by comparing with the previous LUAD researches, we emphasized the higher degree of oxidative phosphorylation in Xuanwei LUAD and pointed that VIPR1 deficiency aggravated metabolic dysfunction. CONCLUSION: Our integrated multi-omics analysis provided a powerful resource for a systematic understanding of the molecular structure of XWLC and proposed therapeutic opportunities based on redox metabolism.

Research Note: Association of VIPR-1 gene polymorphism with growth traits in meat type Japanese quail (Coturnix Japonica).

This study aimed to analyze the polymorphism of the vasoactive intestinal peptide receptor-1 (VIPR-1) gene and its association with growth traits in quail using the PCR-RFLP and sequencing techniques. Genomic DNA was extracted from blood samples of 36 female Savimalt (SV) quails and 49 female French Giant (FG) quails. Growth traits were measured and used for VIPR-1 gene analysis, as body weight (BW), tibia length (TL), chest width (CW), chest depth (CD), sternum length (SL), body length (BL), and tibia circumference (TC). The results showed that 2 SNPs (BsrD I and HpyCH4 IV) were detected in exon 4 to 5 and exon 6 to 7 of the VIPR-1 gene, respectively. The results of association showed that the BsrD I site was not significantly associated with growth traits at 3 or 5 wk of age in the SV strain (P < 0.05), while the BsrD I site was significantly associated with BL at 3 or 5 wk of age in FG (P < 0.05). The HpyCH4 IV site was significantly associated with TL, CW, CD, SL, and BL at 3 wk of age in the SV strain (P < 0.05), while the HpyCH4 IV site was significantly correlated with BW, CW, SL, and BL at 5 wk of age in SV (P < 0.05). The HpyCH4 IV site was significantly associated with TL and TC at 3 wk of age in FG (P < 0.05), while the HpyCH4 IV site was significantly associated with TC at 5 wk of age in FG (P < 0.05). Four haplotype combinations based on 2 SNPs showed significantly association with BW, CW, CD, SL, BL, and TC at 3 or 5 wk of age in SV (P < 0.05). There was not significant association between 3 haplotype combinations with growth trait at 3 or 5 wk of age in FG (P > 0.05). In conclusion, the VIPR-1 gene could be used as a molecular genetic marker to improve growth traits in quail.

Vasoactive Intestinal Peptide (VIP) Protects Nile Tilapia (Oreochromis niloticus) against Streptococcus agalatiae Infection.

Vasoactive intestinal peptide (VIP), a member of secretin/glucagon family, is involved in a variety of biological activities such as gut motility, immune responses, and carcinogenesis. In this study, the VIP precursor gene (On-VIP) and its receptor gene VIPR1 (On-VIPR1) were identified from Nile tilapia (Oreochromis niloticus), and the functions of On-VIP in the immunomodulation of Nile tilapia against bacterial infection were investigated and characterized. On-VIP and On-VIPR1 contain a 450 bp and a 1326 bp open reading frame encoding deduced protein of 149 and 441 amino acids, respectively. Simultaneously, the transcript of both On-VIP and On-VIPR1 were highly expressed in the intestine and sharply induced by Streptococcus agalatiae. Moreover, the positive signals of On-VIP and On-VIPR1 were detected in the longitudinal muscle layer and mucosal epithelium of intestine, respectively. Furthermore, both in vitro and in vivo experiments indicated several immune functions of On-VIP, including reduction of P65, P38, MyD88, STAT3, and AP1, upregulation of CREB and CBP, and suppression of inflammation. Additionally, in vivo experiments proved that On-VIP could protect Nile tilapia from bacterial infection and promote apoptosis and pyroptosis. These data lay a theoretical basis for further understanding of the mechanism of VIP guarding bony fish against bacterial infection.

Innate frequency-discrimination hyperacuity in Williams-Beuren syndrome mice.

Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of ∼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.

Enteric VIP-producing neurons maintain gut microbiota homeostasis through regulating epithelium fucosylation.

Interactions between the enteric nervous system (ENS) and intestinal epithelium are thought to play a vital role in intestinal homeostasis. How the ENS monitors the frontier with commensal and pathogenic microbes while maintaining epithelial function remains unclear. Here, by combining subdiaphragmatic vagotomy with transcriptomics, chemogenetic strategy, and coculture of enteric neuron-intestinal organoid, we show that enteric neurons expressing VIP shape the α1,2-fucosylation of intestinal epithelial cells (IECs). Mechanistically, neuropeptide VIP activates fut2 expression via the Erk1/2-c-Fos pathway through the VIPR1 receptor on IECs. We further demonstrate that perturbation of enteric neurons leads to gut dysbiosis through α1,2-fucosylation in the steady state and results in increased susceptibility to alcohol-associated liver disease (ALD). This was attributed to an imbalance between beneficial Bifidobacterium and opportunistic pathogenic Enterococcus faecalis in ALD. In addition, Bifidobacterium α1,2-fucosidase may promote Bifidobacterium adhesion to the mucosal surface, which restricts Enterococcus faecalis overgrowth and prevents ALD progression.

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.

Mechanism of VIPR1 gene regulating human lung adenocarcinoma H1299 cells.

The vasoactive intestinal peptide receptor-1(VIPR1) has prominent growth effects on a number of common neoplasms. However, there were contradictions in the effect cross different cancers. We aimed to explore the effect of VIPR1 overexpression on a human lung adenocarcinoma cell line H1299. GEO dataset was used to screen differentially expressed genes in lung adenocarcinoma tissues. The expression of VIPR1 mRNA was determined in the cancer Genome Atlas (TCGA). Immunohistochemical analysis was performed to determine VIPR1 protein expression in lung adenocarcinoma and corresponding adjacent tissues (n = 22). Fluorescence real-time quantitative PCR detected the expression of VIPR1 in human normal lung epithelial cell line BEAS-2B and lung adenocarcinoma cell line H1299. Overexpression strategies were employed to assess functions of VIPR1 expression on several malignant phenotypes in H1299. The expression of VIPR1 was lower in lung adenocarcinoma tissues than that in adjacent tissues. Compared with the normal lung epithelial cells BEAS-2B, VIPR1 was down-regulated in lung cancer cells H1299 (P < 0.05). After the overexpression of VIPR1, we found that VIPR1 significantly inhibited growth, migration, and invasion of H1299 cells (P < 0.05). Our findings point out the tumor suppressor roles of VIPR1 in human LUAD pathogenesis.

A Molecular Dynamics Study of Vasoactive Intestinal Peptide Receptor 1 and the Basis of Its Therapeutic Antagonism.

Vasoactive intestinal peptide receptor 1 (VPAC1) is a member of a secretin-like subfamily of G protein-coupled receptors. Its endogenous neuropeptide (VIP), secreted by neurons and immune cells, modulates various physiological functions such as exocrine and endocrine secretions, immune response, smooth muscles relaxation, vasodilation, and fetal development. As a drug target, VPAC1 has been selected for therapy of inflammatory diseases but drug discovery is still hampered by lack of its crystal structure. In this study we presented the homology model of this receptor constructed with the well-known web service GPCRM. The VPAC1 model is composed of extracellular and transmembrane domains that form a complex with an endogenous hormone VIP. Using the homology model of VPAC1 the mechanism of action of potential drug candidates for VPAC1 was described. Only two series of small-molecule antagonists of confirmed biological activity for VPAC1 have been described thus far. Molecular docking and a series of molecular dynamics simulations were performed to elucidate their binding to VPAC1 and resulting antagonist effect. The presented work provides the basis for the possible binding mode of VPAC1 antagonists and determinants of their molecular recognition in the context of other class B GPCRs. Until the crystal structure of VPAC1 will be released, the presented homology model of VPAC1 can serve as a scaffold for drug discovery studies and is available from the author upon request.

Promoter methylation and H3K27 deacetylation regulate the transcription of VIPR1 in hepatocellular carcinoma.

Vasoactive intestinal peptide receptor 1 (VIPR1) is observed to express differently in human malignancies. Here, we aim to reveal clinical significance and transcriptional regulation mechanism of VIPR1 in hepatocellular carcinoma (HCC). Using immunohistochemistry, pyrosequencing, quantitative real-time PCR (qPCR), decitabine (DAC)/4-phenylbutyricacid (PBA) treatment and chromatin immunoprecipitation (ChIP), we found the low expression of VIPR1 was correlated with poor histological differentiation and poor survival. The promoter region of VIPR1 was methylated and DNA methylation inhibited VIPR1 gene transcription. Deacetylation of H3K27 in the promoter of VIPR1 inhibited the transcription of VIPR1 in HCC. In conclusion, low expression of VIPR1 had an adverse prognostic impact on HCC, and such expression is at least partially mediated by epigenetic modification.

Association of VIPR-1 gene polymorphisms and haplotypes with egg production in laying quails.

The laying quail is a worldwide breed which exhibits high economic value. In our current study, the vasoactive intestinal peptide receptor-1 (VIPR-1) was selected as the candidate gene for identifying traits of egg production. A single nucleotide polymorphism (SNP) detection was performed in 443 individual quails, including 196 quails from the H line, 202 quails from the L line, and 45 wild quails. The SNPs were genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Two mutations (G373T, A313G) were detected in all the tested quail populations. The associated analysis showed that the SNP genotypes of the VIPR-1 gene were significantly linked with the egg weight of G373T and A313G in 398 quails. The quails with the genotype GG always exhibited the largest egg weight for the two mutations in the H and L lines. Linkage disequilibrium (LD) analysis indicated that G373T and A313G loci showed the weakest LD. Seven main diplotypes from the four main reconstructed haplotypes were observed, indicating a significant association of diplotypes with egg weight. Quails with the h1h2 (GGGT) diplotype always exhibited the smallest egg weight and largest egg number at 20 weeks of age. The overall results suggest that the alterations in quails may be linked with potential major loci or genes affecting reproductive traits.