B7-H3 Promotes Proliferation and Migration of Colorectal Cancer Cells by Regulating PYCR1 in the Proline Metabolism

Research Article

Austin J Cancer Clin Res. 2021; 8(3): 1096.

B7-H3 Promotes Proliferation and Migration of Colorectal Cancer Cells by Regulating PYCR1 in the Proline Metabolism

Jin Y1,2, Jiang X2, Wu R2, Liu H2, Zhang T1, Xu X1, Zhang L3, Wang X1,2, Hua D1,2# and Yong Mao1,2*

1Department of Oncology, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China

2Wuxi Medical College, Jiangnan University, Wuxi, Jiangsu, China

3Zhongshan Hospital Affiliated to Xiamen University, Xiamen University, Xiamen, Fujian, China

#Contributed Equally to this Work

*Corresponding author: Yong Mao, Department of Oncology, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China

Received: August 09, 2021; Accepted: September 06, 2021; Published: September 13, 2021


Proline metabolism plays an essential role in tumor development; however, its underlying mechanism in CRC remains elusive. B7-H3, an immune checkpoint member of the B7 immunoregulatory family, is aberrantly overexpressed in a wide variety of malignancies and is associated with a poor prognosis. In this study, we found that overexpression of B7-H3 effectively enhanced proline consumption rate and reduced glutamate production, while knockout of B7-H3 had inverse effects. Moreover, we also identified that B7-H3 increased proline consumption and decreased glutamate production by promoting the expression of Pyrroline- 5-carboxylate reductase 1 (PYCR1) in CRC cells and that PYCR1 is a crucial mediator of B7-H3-induced CRC proliferation and migration. Overexpression of PYCR1 or treatment of cells with PYCR1 inhibitors could reverse B7-H3-induced proline metabolism and B7-H3-induced tumor cell proliferation and migration. Furthermore, we confirmed the positive correlation between B7-H3 and PYCR1 expression in tumor tissues of CRC patients. Collectively, the present study highlighted a previously unrecognized mechanism of B7-H3-mediated rewiring of proline metabolism through increased expression of PYCR1 in CRC cells. These findings suggested that B7-H3 may serve as a novel prognostic factor and a promising therapeutic target for CRC.

Keywords: B7-H3; PYCR1; Proline metabolism; Colorectal cancer; Correlation; Prognosis


CRC: Colorectal Cancer; B7-H3: B7 Homolog 3; PYCR1: Pyrroline-5-Carboxylate Reductase 1


Colorectal Cancer (CRC) represents the third most frequently diagnosed malignant neoplasm of the gastrointestinal tract and the fourth leading cause of cancer-associated mortality amongst all malignancies worldwide. Recurrence and metastasis remain the primary cause of death in CRC patients and represent a clinical challenge. Therefore, it is highly desirable to understand the molecular mechanisms underlying CRC progression and identify novel prognostic biomarkers and therapeutics for CRC.

B7 Homolog 3 (B7-H3), an essential immune checkpoint member of the B7 and CD28 families, shares 20%~27% amino acid homology with other members of the B7 family [1]. B7-H3 is identified to be highly expressed in various tumor tissues and tumor cell lines but low in normal tissues and cells, and its expression in cancer is closely associated with poor prognosis of patients. Limet et al. proposed that immunoregulatory protein B7-H3 affects glucose metabolism in cancer cells mediated by HIF-1A [2]. Liu et al. revealed that B7- H3 was highly expressed in CRC cells and facilitated the migration and invasion of CRC cells [3]. Although these studies have revealed multiple functions of B7-H3 in CRC, the function of B7-H3 in CRC metabolism and the mechanism of promoting tumor progression remain elusive.

Proline, a non-essential amino acid with low molecular weight and highly soluble in water, plays a protective role against oxidative stress and redox homeostasis [4]. Proline is synthesized from glutamate by two enzymes: Δ1-pyrroline-5-carboxylate synthetase (P5CS) and P5C reductase (P5CR) [5]. In the human, P5CRs is also called as PYCR, and three human PYCR isoenzymes have been identified: Pyrroline-5- carboxylate reductase 1 (PYCR1), Pyrroline-5-carboxylate reductase 2 (PYCR2), and Pyrroline-5-carboxylate reductase-like (PYCRL). P5CRs are localized in mitochondria, where the bulk of metabolic reactions take place to support cellular functions [6]. Nisebita et al. revealed that proline consumption and expression of proline synthase were closely associated with clonogenic and tumorigenic potential; besides, inhibition of proline synthase diminished clonogenic and tumorigenic potential [7]. Elia et al. demonstrated that proline catabolism supported the growth of breast cancer cells, which was related to tumor metastasis in vivo [8]. Moreover, Liu et al. found that MYC promoted mRNA expression and protein levels of key enzymes involved in proline synthesis, which can promote cell growth and energy production [9]. Together, these studies indicated that proline plays a crucial role in tumor cell growth.

Although multiple studies have suggested that B7-H3 promotes tumor cell growth and proliferation in CRC, the relationship between B7-H3 and proline metabolism and the exact mechanism of B7-H3 remains largely unknown in CRC. In this study, we found that B7- H3 exhibits a positive relationship with PYCR1, and B7-H3 enhances proline metabolism by up-regulating the expression of PYCR1, which in turn promotes the proliferation and metastasis of CRC cells. The present study’s findings reveal a previously unrecognized mechanism by which B7-H3 is involved in proline metabolism and tumor progression in CRC.

Materials and Methods

Cell lentivirus infection, cell transfection, and cell culture

The two human CRC cell lines, SW480 and Caco-2 cell lines with different expression levels of B7-H3 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). The stable B7-H3-overexpressing SW480 (SW480-B7) cells were generated by transfection with overexpression plasmid, and the stable knockdown of B7-H3 in Caco-2 cells were generated with a B7-H3 shRNA (Caco-2-shB7). SW480-NC and Caco-2-shNC were generated by transfection with a lentivirus carrying a negative control.

For PYCR1 gene knockdown, human PYCR1 siRNA-1, human PYCR1 siRNA-2, human PYCR1 siRNA-3, and their control siRNAs were procured from GenePharma Co., Ltd (Suzhou, China). SW480-B7 and Caco-2-shB7 cells were transfected with siRNAs using Lipofectamine 8000TM Transfection Reagent (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s instructions. The signaling pathway inhibitors used included PI3K inhibitor (LY294002), AKT inhibitor (MK2206). Transfection efficiency was determined by Real-time quantitative PCR (qRT-PCR) and Western blot assay.

Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; HyClone, SH30022.01) supplemented with 10% fetal bovine serum (FBS; Clark Bioscience, Houston, TX, United States ) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO2.

RNA isolation and RT-qPCR assays

Total RNA was extracted from the cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For reverse transcription, cDNA was synthesized using the PrimeScript RT-PCR kit (Takara, Japan). The expression level of B7-H3 was determined using the QuantinovATM SYBR Green PCR kit (Qiagen, Hilden, Germany) on an ABI 7500 Realtime PCR system (Applied Biosystems, Foster City, CA, United States). β-actin was used as endogenous control and the relative expression level of B7- H3 as fold change was calculated using the 2-ΔΔCt method. Primer sequences used in this experiment were listed in Table 1. All reactions were performed in triplicate and were repeated thrice. The qRTPCR cycle profile was performed at 95˚C for 2min to activate DNA polymerase, followed by 40 cycles of denaturation at 95˚C for 5sec and annealing at 60˚C for 10sec.

Protein extraction and Western blot analysis

Total protein was extracted with RIPA lysis buffer supplemented with protease inhibitor, a phosphatase inhibitor, and 100mmol/L PMSF (Keygen Biotech, China) on ice for 25min. Protein quantification was performed using the BCA Protein Analysis kit (Beyotime Institute of Biotechnology, Shanghai, China) and adjusted to the same concentration of protein. An equivalent amount of the protein was separated on 10% SDS-PAGE and then transferred to a Polyvinylidene Fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk for 1h at room temperature. Then, membranes were incubated overnight with the following primary antibodies: mouse anti-human B7-H3 mAb (1:1000, ptoteintech), rabbit anti-human PYCR1 pAb (1:1000, ptoteintech) and mouse anti-human β-actin mAb (Beyotime, Nantong, China). After three washes with TBST for 10 minutes each, membranes were incubated with the corresponding goat versus mouse or goat versus rabbit secondary antibodies at room temperature for 1 hour. After washing with TBST, the membrane was treated with enhanced chemiluminescence (ECL) detection reagent (ABSIN, Shanghai, China). The immunoreactive bands were visualized using the ChemidocTM XRS+ detection system and the Image LabTM software (Bio-Rad, Hercules, CA, USA). Quantity One (Bio-Rad, Hercules, CA, USA) was used for quantitative analysis.

Proline and glutamic acid yield determination

Cells were seeded onto a six-well plate and cultured in a serumfree medium. After 24 hours of culture, the supernatant of the cells was collected in a sterile tube. The precipitate was removed by centrifugation and stored at -20˚C until further use. The production of proline and glutamate in the culture medium was determined by the Proline (PRO) assay kit (ml107611, mlbio) and human glutamate (Glu) assay kit (ml038308, mlbio), and the absorbance was measured at 520 nm, and 450 nm with a microplate analyzer, respectively, and these values were normalized to the total protein concentration.

Colony formation, EDU and proliferation assays

For the colony formation assay, 800 cells per well were seeded into 6-well plates. Following 2-3 weeks of incubation, colonies were washed with PBS, fixed with anhydrous methanol, and stained with 0.5% crystal violet for 30 min, dried and observed under a microscope.

Cell proliferation ability was assessed using the Cell Counting Kit- 8 (CCK8 kit; SolarBio, China) assay according to the manufacturer’s protocol. In brief, cells were seeded into 96-well plates at a density of 2000 cells per well. After a 24h culture, CCK8 reagent was added to each well and incubated for 90min. Finally, the absorbance at 450nm wavelength was measured at 0, 24, 48, 72, and 96 hours, respectively, after each medium replacement. Each experiment was repeated three times independently, and a growth curve was plotted.

EdU labeling was performed to assess cells’ proliferation capacity using the EdU Proliferation Kit (RIBOBIO) according to the manufacturer’s instructions. Briefly, 8000 cells per well were seeded in a 96-well plate and incubated for 24h. Then, cells were fixed with anhydrous methanol for 30min and treated with 0.5% Triton X-100 for 10min for permeability. Then, the Apollo reaction cocktail was added and incubated at room temperature for 30min, followed by staining with Hoechst 33342 for 30min. After a brief wash with PBS, EdU‐stained cells were visualized under Nikon Inverted Research Microscope at 10X and 20X magnification.

Invasion and migration assays

Transwell assay was used to assess cell invasion ability. Briefly, 5x104 cells per well were seeded into 24-well plates, and 100μL serum-free medium was added to the upper membrane, and 10% FBS -containing medium was added to the lower chamber. After 24 hours, the cells remaining on the upper membrane were carefully wiped off with a cotton swab, while the cells that had invaded through the membrane were fixed with anhydrous methanol and stained with 0.5% crystal violet for 20min at room temperature. Cells were then counted in five randomly selected fields (at ×100 magnification) under a microscope. All experiments were repeated independently three times.

For migration assay, 5×104 cells were seeded to the upper chamber of each well (coated with 20μL Matrigel) then, FBS-containing complete medium was added to the lower chamber. After incubation for 24h, cells that migrated to the lower membrane of the chamber were fixed with anhydrous methanol and stained with 0.5% crystal violet for 20min at room temperature. Cells were then counted in five randomly selected fields (at ×100 magnification) under an inverted microscope. All experiments were repeated independently three times.

Cells were cultured in a 6-well plate at a density of 1x105 per well. After incubation for 24 hours, the monolayers were scratched with a 10-10‐μL tip. Following washing with PBS, cellular migration toward the scratched area was captured under a microscope at 0, 24, and 48 hours, respectively.

Patients and tissue sample

The medical records of patients who were diagnosed and underwent CRC surgical resection at the Affiliated Hospital of Jiangnan University between June 2008 and December 2011 were retrieved. A total of 206 formalin-fixed paraffin-embedded tissue samples from CRC patients were included. These patients did not receive preoperative radiotherapy or chemotherapy, and the clinical data and pathological tissue were intact. This study was approved by the Medical Ethics Committee of the Affiliated Hospital of Jiangnan University, and written informed consent was obtained from each patient. To obtain patient survival data, all patients were followed over the telephone until October 31, 2017, with a median followup time of 79 months (range 6-114 months). Two experienced pathologists examined hematoxylin and eosin (H&E) stained slides to confirm tumor tissue and adjacent normal tissue. The corresponding spots on the tissue block were marked for a correct tissue core punch, which was cut into 4μm thick continuous sections and affixed to antidewaxing glass slides.


The tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. Antigen retrieval was performed by heating the tissue sections at 100°C in sodium citrate buffer in a microwave oven for 30 minutes. The endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 minutes. Subsequently, the non-specific protein was blocked by incubation with 5% skim milk at room temperature for 30min, followed by incubation with the following primary antibodies: mouse anti-human B7-H3 monoclonal antibody (1:200, Santa Cruz, Dallas, TX, United States) and rabbit antihuman PYCR1 Polyclonal antibody (1:100, proteintech) overnight at 4oC. Then, sections were washed with PBS and incubated at room temperature with secondary antibody (1:1; GK600710, Genetech, Shanghai, China) for one hour. The immunostaining was carried out by staining with 3, 3’-diaminobenzidine chromogen (DAB substrate; 1:1; GK600710, Genetech, Shanghai, China) and counterstained with hematoxylin for 60 s at room temperature, dehydrated and mounted, and the sections were examined under a microscope.

Evaluation of immunochemistry staining

The sections were examined in a blinded manner by two independent pathologists. Both the intensity and extent of immunological staining of B7-H3 and PYCR1 were analyzed semiquantitatively and scored according to the percentage of positively stained tumor cells and the staining intensity. The percentage of positively stained cells was scored as follows: ≤5% positive cells (score of 0), 6% to 25% positive cells (score of 1), 26% to 50% positive cells (score of 2), 51 % to 75% positive cells (score of 3) and ≥76 % positive cells (score of 4). The staining intensity was scored as follows: 0 (no staining), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The immunoreactivity score for each specimen was multiplied by the percentage of positively stained cell score and the intensity score. According to the total score, we defined 0~3 as low expression and 4~12 as high expression.

Statistical analysis

Statistical analysis was performed with IBM SPSS Statistics software (22.0; IBM, Chicago, IL USA. The association between the clinicopathological data and the protein expression levels of PYCR1 and B7-H3 was assessed by the chi-square test. The overall survival was calculated using the Kaplan-Meier curve and compared using the log-rank test. The proportional hazard model method was used to perform the univariate and multivariate regression analyses and compared using Cox proportional risk regression analysis. A twotailed p-value of <0.05 was considered statistically significant.


B7-H3 promotes proline metabolism in CRC cells

In order to determine the expression of B7-H3 in CRC cells, the expression of B7-H3 was analyzed in six intestinal cancer cells by Western blot assay, including HCT-8, Caco-2, SW480, Lovo, DLD1, and HCT-116 (Figure 1A). The results revealed that the expression of B7-H3 was markedly higher in Caco-2 cells and lower in SW480 cells. Therefore, four stable transgenic strains expressing high and low B7-H3 were established (SW480-NC, SW480-B7; Caco-2-shNC, Caco-2-shB7) (Figure 1B). The consumption of proline in SW480-B7 cells with overexpression of B7-H3 was significantly higher compared with the control group (Figure 1C), and the production of glutamate was lower (Figure 1E). Similarly, Caco-2 cells with B7-H3 knockdown significantly reduced proline consumption (Figure 1D), and significantly increased glutamate production compared with the control group (Figure 1F).