RB1CC1 functions as a tumor-suppressing gene in renal cell carcinoma via suppression of PYK2 activity and disruption of TAZ-mediated PDL1 transcription activation

Abstract

Rb1-inducible coiled-coil 1 (RB1CC1) has been demonstrated to function as an inhibitor of proline-rich/Ca-activated tyrosine kinase 2 (PYK2) by binding to the kinase domain of PYK2, which promotes the proliferation, invasion, and migration of renal cell carcinoma (RCC) cells. Additionally, in breast cancer, PYK2 positively regulates the expression of transcriptional co-activator with PDZ-binding motif (TAZ) which in turn can enhance PDL1 levels in breast and lung cancer cells. The current study was performed to decipher the impact of RB1CC1 in the progression of RCC via regulation of the PYK2/TAZ/PDL1 signaling axis. Expression of RB1CC1 and PYK2 was quantified in clinical tissue samples from RCC patients. The relationship between TAZ and PYK2, TAZ and PDL1 was then validated. The cellular processes of doxorubicin (DOX)-induced human RCC cell lines including the abilities of proliferation, colony formation, sphere formation and apoptosis, as well as the tumorigenicity of transfected cells, were evaluated after the alteration of RB1CC1 expression. RB1CC1 exhibited decreased expression in RCC tissues and was positively correlated with patient survival. RB1CC1 could inhibit the activity of PYK2, which in turn stimulated the stability of TAZ protein by phosphorylating TAZ. Meanwhile, TAZ protein activated PDL1 transcription by binding to the promoter region of PDL1. RB1CC1 overexpression or PYK2 knockdown could help everolimus (EVE) to inhibit tumor proliferation and activate immune response. Taken together, RB1CC1 can potentially augment the response of RCC cells to immunotherapy by suppressing the PYK2/TAZ/PDL1 signaling axis.

Keywords: Programmed cell death ligand 1; Proline-rich/Ca-activated tyrosine kinase 2; Rb1-inducible coiled-coil 1; Renal cell carcinoma; Tafazzin.

Fig. 1

RB1CC1 is an effective RCC tumor suppressor gene. A, A heat map highlighting the top 10 differentially expressed genes in the GSE6344 dataset. B. The expression of RB1CC1 mRNA in RCC tumor and adjacent normal tissue samples in TCGA. C, Western blot analysis of RB1CC1 protein in the RCC tissues (n = 5) and adjacent normal tissues (n = 5). T represents the tumor, and P–T represents the adjacent normal tissues. D, mRNA expression of RB1CC1 in RCC tissues (n = 5) and adjacent normal tissues (n = 5). E, Kaplan–Meier analysis of the survival curve of patients with high or low RB1CC1 expression was conducted using the UALCAN database (http://ualcan.path.uab.edu/analysis.html). The red line indicates the RCC patients with high RB1CC1 expression (expression cutoff 25%), and the blue line indicates the RCC patients with low RB1CC1 expression (expression cutoff 75%). F, Western blot analysis of the RB1CC1 expression in the A498i and ACHNi cells. The antibody was shown in the figure. G, Cell viability of A498 and ACHN cells measured by CCK-8 assay. For DOX-inducible expressing cell lines A498 and ACHN, DOX (1 µg/mL) and DMSO (DOX lysing agent) were added, and cell viability was measured as shown in the figure. H, R A498 and ACHN colony-forming ability determined using colony formation assay. I—J, A498 and ACHN sphere-forming ability determined using sphere formation assay. Data between RCC tissues and adjacent normal tissues were compared with paired t test, while those between the other two groups were compared with unpaired t test. * p < 0.05

Fig. 2

PYK2 restored RB1CC1 inhibition of RCC cell lines. A. Western blot analysis of whole cell lysates of cells confirms DOX (1 µg/mL) induction and expression of RB1CC1 and PYK2 in A498i and ACHNi transfected with PYK2. B-C, The A498i and ACHNi cell viability measured by CCK-8 assay. D-E, A498i and ACHNi sphere-forming ability determined using sphere formation assay. F, A498i and ACHNi colony-forming ability determined using colony formation assay. *, & or # p < 0.05

Fig. 3

RB1CC1 inhibited PYK2 activity. A, 293 T was co-transfected with PCDNA3.1-PYK2, PCDNA3.1-RB1CC1, and the cells were lysed 48 h later. Preimmune and anti-RB1CC1 were used for co-immunoprecipitation experiments, followed by Western blot analysis detection of immune complexes or extracts (WCL), corresponding proteins in Preimmune and anti-RB1CC1 with antibodies shown in the figure. B, Top: Schematic diagram of the secondary structure of RB1CC1 protein, Bottom: N-terminal-RB1CC1 & C-terminal-RB1CC1 protein secondary Schematic. C, Co-transfected plasmids in 293 T cells: pKH3-Pyk2, pSG5-flag-FIP200/RB1CC1 (FIP200), pSG5-flag-N-FIP200/RB1CC1(NT-FIP), pSG5-flag-C-FIP200/RB1CC1 (CT-FIP). After immunoprecipitation, the interaction between RB1CC1 C-terminal and PYK2 in 293 T cells was detected by Western blot analysis. The antibodies are shown in the figure. D, In the case of E4Y1 characterizing PYK2 activity, different concentrations of GST-CT-RB1 and GST were added to determine the activity of PYK2. E, The PYK2 protein phosphorylation affected by RB1CC1 C-terminal in 293 T cells was detected by western blot analysis, with the antibodies shown in the figure. Each set of data had three independent experiments and was analyzed by unpaired t test. The error bars were a representation of the standard deviation, * p < 0.05, * * p < 0.01, * * * p < 0.001

Fig. 4

PYK2 enhanced the stability of TAZ protein by increasing the TAZ phosphorylation. A, TAZ mRNA levels in RCC and adjacent normal tissues obtained from the TCGA database. The red box indicates the expression in tumor samples, and the gray box indicates the expression in normal samples. KIRC stands for kidney renal clear cell carcinoma, and KIRP stands for kidney renal papillary cell carcinoma. T indicates tumor, and N indicates normal. B, A Kaplan–Meier plot of the survival curve of RCC patients with high or low TAZ expression. The cutoff value was determined by the median TAZ expression value. C, Western blot analysis of TAZ expression in the A498shi and ACHNshi cells. D, A498shi and ACHNshi cell viability measured by CCK-8 assay. E, A498shi and ACHNshi colony-forming ability determined using colony formation assay. F, The correlation between TAZ and PYK2 mRNA expression in RCC tumors in TCGA data. G, Western blot analysis of expression of TAZ and PYK2 proteins. H, TAZ protein in the 293 T cells was co-precipitated with Myc antibody, and its protein tyrosine residue was phosphorylated and measured using Western blot analysis. Each set of data had three independent experiments and was analyzed by unpaired t test. The error bars were a representation of the standard deviation, * p < 0.05, * * p < 0.01, * * * p < 0.001

Fig. 5

PYK2 promoted TAZ-mediated transcription of PDL1 in RCC cells. A, The mRNA level of PDL1 in A498shi and ACHNshi cell lines detected using RT-qPCR. B. The expression of PDL1 protein in the A498shi and ACHNshi cells was detected by Western blot analysis. C, The expression of PDL1 and PYK2 protein in the A498shi and ACHNshi cells was detected by Western blot analysis. D, The binding of TAZ to PDL1 promoter regions detected using ChIP-PCR. E, The activity of PDL1 promoter activated by TAZ detected by dual luciferase assay. F, PDL1 promoter deletion mutation scanned TAZ to recognize the activated PDL1 promoter region. Each set of data had three independent experiments and was analyzed by unpaired t test. The error bars were a representation of the standard deviation, * p < 0.05, * * p < 0.01, * * * p < 0.001

Fig. 6

RB1CC1 inhibited PDL1 transcription in RCC. A, The mRNA level of PDL1 in the A498i and ACHNi cell lines detected using RT-qPCR. B, Western blot analysis of the PDL1 and TAZ protein expression in A498i and ACHNi cell lines. C, Tumor growth measured every five days in mice inoculated with A498i cells overexpressing RB1CC1. D, The size of these transplanted tumors. E. Tumor weight. F, Quantitative analysis of immunohistochemistry and TUNEL results in Figure C. n = 8 for mice upon each treatment. * p < 0.05, compared with mice treated with vehicle. # p < 0.05, compared with mice treated with EVE. & p < 0.05, compared with mice treated with DOX +

Fig. 7

PYK2 inhibitor promoted RCC immunotherapy. A, Western blot analysis of the PDL1 expression in the A498-PDL1 cell line. B, Growth curve of xenograft tumors of nude mice in different treatment groups (n = 8). C, Size of these transplanted tumors. D, The tumor weight statistics in Figure C. E, Quantitative analysis of immunohistochemistry and TUNEL results in Figure C. * p < 0.05, compared with mice treated with vehicle. # p < 0.05, compared with mice treated with EVE. & p < 0.05, compared with mice treated with DOX +

Fig. 8

Schematic of the molecular mechanism outlining how RB1CC1 inhibits the activation of PDL1 transcription induced by TAZ by inhibiting PYK2 activity, thereby promoting the effectiveness of immunotherapy for RCC patients clinically