GPR176 Promotes Cancer Progression by Interacting with G Protein GNAS to Restrain Cell Mitophagy in Colorectal Cancer

Abstract GPR176 belongs to the G protein‐coupled receptor superfamily, which responds to external stimuli and regulates cancer progression, but its role in colorectal cancer (CRC) remains unclear. In the present study, expression analyses of GPR176 are performed in patients with colorectal cancer. Genetic mouse models of CRC coupled with Gpr176‐deficiency are investigated, and in vivo and in vitro treatments are conducted. A positive correlation between GPR176 upregulation and the proliferation and poor overall survival of CRC is demonstrated. GPR176 is confirmed to activate the cAMP/PKA signaling pathway and modulate mitophagy, promoting CRC oncogenesis and development. Mechanistically, the G protein GNAS is recruited intracellularly to transduce and amplify extracellular signals from GPR176. A homolog model tool confirmed that GPR176 recruits GNAS intracellularly via its transmembrane helix 3‐intracellular loop 2 domain. The GPR176/GNAS complex inhibits mitophagy via the cAMP/PKA/BNIP3L axis, thereby promoting the tumorigenesis and progression of CRC.

Human CRC cell lines (SW480,SW620, RKO, HT29, DLD-1, HCT116, and LoVo) and normal epithelial cells of the colon (FHC) were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in their corresponding medium, incubated in a 5% CO2 humidified incubator at 37°C.

RNA preparation and qRT-PCR
Total RNA of cells and tissues was obtained and then reverse transcribed into cDNA using the PrimeScript RT reagent kit (TaKara, Dalian, China). The SYBR Premix Ex Taq Kit (TaKaRa) was used to perform qRT-PCR on an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems). Primer sequences for qRT-PCR are listed in Supplementary Table 2.

Western blot and immunohistochemistry
For western blot assay, protein lysates were extracted from tissues and cultured cells using RIPA buffer (Beyotime, China). A BCA kit (Beyotime) was used to quantify the protein concentration. Equal amounts of protein were separated using SDS-PAGE.
For IHC, tumor samples were embedded in paraffin and sectioned into 4 mm slices.
The slices were first incubated with primary antibodies against the target antigens.
Secondary antibodies were used to bind to the primary antibody and detected using the detection system. Antibodies against phospho-IMMT (Ser528) and phospho-MIC19 (Thr11) were generated using the synthetic phospho-peptides [2]. All antibodies used in this experiment are listed in (supplementary table 2).

Generation of GPR176 -CKO mice
GPR176 conditional knockout mice (GPR176 -CKO ) with the deletion of GPR176 exons 1-4 in intestinal cells were constructed using Cre recombinase expressed by the intestinal-cell-specific villin 1 (Vil1) promote. The GPR176 FL/FL mice were first generated by breeding the floxed allele into homozygosity and then crossed with Vil1-Cre mice to obtain GPR176 -CKO mice. All the mice were on the C57BL/6 background.

Animal models
All animal experiments were approved by the Animal Care and Use Committee of

Electron microscopy
Samples, including tumor tissues and CRC cells, were fixed in 4% formaldehyde and 1% glutaraldehyde. They were then processed for transmission electron microscopy using a Hitachi H-500 electron microscope (FEI, USA).

MitoTracker Red
To assess the mitochondrial membrane potential in CRC cells, a MitoTracker Red kit (Invitrogen) was used (final concentration 100 nM). Briefly, cells were grown on coverslips inside a Petri dish. When the cells reached the desired confluency, they were fixed with growth medium containing 3% formaldehyde at 37°C for 15 min.
After fixation, the cells were rinsed three times in PBS and stained with a solution containing MitoTracker Red for 20 min at 37°C. All cells were then observed under a fluorescence microscope (Leica Microsystems CMS Gmbh Ernst-Leitz-Str, 17-37) and analyzed using Leica Application Suite X, 3.6.20104.0.

Enzyme-linked immunosorbent assay
Human cAMP enzyme-linked immunosorbent assay kits (E-EL-0056c) were purchased from ElabScience (Wuhan, China). Briefly, 50 μl of the standard or test samples was added to the plate. Then, 50 μl of biotinylated antibody was immediately added to each well and incubated at 37°C for 45 min. After washing three times, 100 μl of HRP-conjugated working solution was added to each well and incubated at 37°C for 30 min, followed by the addition of 90 μl substrate solution and incubation at 37°C for 15 min. Next, the terminating solution was added, and absorbance values were read at 450 nm.

Co-immunoprecipitation assay
The Co-IP assay was performed as described previously [4]. The corresponding antibodies were used to capture the protein complexes. The precipitate was then subjected to western blotting or mass spectrum analysis.

Immunofluorescence assay (IF)
IF was performed as previously described [5]. First, the test cells were grown to the appropriate density and then washed with PBS. Fixed cells in PBS containing 10% formalin for 10 minutes. The cells were rinsed in PBS-Tween 20 for 2 × 2 min, followed by serum blocking and incubation in a primary antibody dilution buffer for 1 h at 25℃. The cells were then incubated with a secondary antibody for 20-30 minutes at 25℃. After rinsing in PBS-Tween 20 for 3 × 2 min, the procedures were performed using the second antibody. After the above steps were completed, samples were stained with DAPI and analyzed with a confocal microscope.

Homology modeling
We generated GPR176 and GNAS homology models using the Advanced Homology Modeling tool based on the Maestro platform in Schrodinger (2015. v4). The GPR176 model was based on PBD:5G53 and GNAS was based on 1SVS. The potential binding locations were established based on the homology models. Mutant plasmids were then constructed using OBiO Technology (Shanghai, China).