ZHX2 drives cell growth and migration via activating MEK/ERK signal and induces Sunitinib resistance by regulating the autophagy in clear cell Renal Cell Carcinoma

Abstract

Zinc fingers and homeoboxes 2 (ZHX2) was found as a novel VHL substrate target, and acted as an oncogenic driver in ccRCC. However, the detailed mechanism of ZHX2 in ccRCC development remains elusive, and no research has focused on studying ZHX2 in drug resistance yet. A tissue microarray with 358 ccRCC samples was used to determine the expression of ZHX2 in ccRCC patients. VHL-deficient cell line 786-O and VHL-normal cell line CAKI-1 was used for lineage reprogramming by transfecting with lentivirus. The in vitro and in vivo experiments were performed with these new cell lines to determine the mechanism of ZHX2 in ccRCC development and drug resistance. Immunohistochemistry analysis showed that ZHX2 was not highly expressed in ccRCC tumor tissues, only 33.2% (119/358) patients have high ZHX2 expression. However, high ZHX2 was significantly associated with advanced Fuhrman grade (p = 0.004), and proved to be an independent prognosis factor for progression-free survival (p = 0.0003), while there is no significant correlation with overall survival. We further discovered that ZHX2 overexpression could increase VEGF secretion and transcriptional activate the MEK/ERK1/2 and promote its downstream targets. We also found ZHX2 overexpression induce Sunitinib resistance though activating autophagy and the combination treatment of Sunitinib and Chloroquine could significantly rescue the phenomenon. In summary, these results indicate that ZHX2 drivers cell growth, migration though increase VEGF expression, and transcriptional activate MEK/ERK1/2 signaling pathway, and could induce Sunitinib resistance by regulating self-protective autophagy, these may provide new insight in advanced ccRCC treatment.

Fig. 1. Overexpressed ZHX2 in ccRCC patients is correlated with worse clinical outcome.

a The representative IHC images of ZHX2 protein expression level in ccRCC samples. a, negative; b, low; c, moderate; d, strong. Bar 100μm. b The association between different ZHX2 expression level and OS in ccRCC patients. Kaplan–Meier and log-rank test analysis were used to compare the two groups (p = 0.118). c The association between ZHX2 expression level and PFS in ccRCC patients. Kaplan–Meier and log-rank test analysis were used to compare the two groups (p = 0.0003).

Fig. 2. ZHX2 koncking down inhibits growth of 786-O cells.

a The proliferation assay showed that ZHX2 knockdown significantly decreased cell proliferation in 786-O cells as well as EPAS1 in 72h and 96h. b The mRNA level of VEFGA in 786-O cells after ZHX2 or EPAS1 knocking down (tested in 72h). c, d Flow cytometry showed that the cell apoptosis ratio was significantly increased after ZHX2 or EPAS1 knockdown. All experiments were repeated double times or more. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 3. Elevated ZHX2 expression promotes the proliferation and migration of VHL-deficient ccRCC cells.

a, b The mRNA and protein expression of ZHX2 after LV-ZHX2 transfection in 786-O cell line. The ratio in western blot semi-quantification was normalized by GAPDH respectively. c The proliferation assay showed ZHX2 overexpression promoted 786-O cells proliferation with a time manner. d, e The migration ability was significantly increased in ZHX2 overexpression cells compare with negative control in 786-O cells. Bar 100μm. f, g The xenograft tumor model of two reprogrammed 786-O cell lines, and tumor volume was much bigger in LV-ZHX2 group. h, i Tube formation could be seen in LV-ZHX2 cells under fluorescence microscope. The quantitative measurement showed the reprogrammed 786-O with ZHX2 overexpression had more tube length than negative control (p < 0.01). j The mRNA expression of some HIFs downstream genes in 786-O with LV-ZHX2 cells and negative control. k ELASA assay showed ZHX2 overexpression could increase VEGF secretion in 786-O cells. All experiments were repeated double times or more. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 4. VHL silence and ZHX2 overexpression increase the cell proliferation and migration in VHL-normal ccRCC cells.

a The representative images CAKI-1 after lentivirus-shVHL and LV-ZHX2 transfection. sh-VHL in red, LV-ZHX2 in green, and double reprogrammed in yellow. b, c The mRNA and protein expression of ZHX2 in reprogrammed CAKI-1 cells respectively. The ratio in western blot semi-quantification was normalized by GAPDH respectively. d The proliferation assay showed ZHX2 overexpression could promote cell growth after 96h in CAKI-1 cells. e, f The migration ability was also increased in new CAKI-1 cells with VHL silencing and ZHX2 overexpression. Bar 100μm. g The mRNA expression of some HIFs downstream genes in LV-shVHL/ZHX2 group and other groups in new CAKI-1 cells. h ELASA assay showed reprogrammed CAKI-1 could increase VEGF secretion as well. All experiments were repeated three times. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 5. ZHX2 promotes ccRCC growth by transcriptional activates the MEK1/ERK1/2 signaling pathway.

a The heat map of total differential genes in 786-O cells with LV-ZHX2 and negative control. b KEGG enrichment analysis was performed to explore the related pathways according to the RNA-seq data. c, d The mRNA expressions of related downstream genes in MAPK/ERK1/2 pathway in 786-O/LV-ZHX2 cells. e, f The ChIP-qpcr and luciferase assays were showed ZHX2 could direct bind to the promoter of MAP2K1 in 786-O cells. g The predicted binding motif of ZHX2 in genome. h The western blot assay was used to test the protein level of MEK-ERK signal in reprogrammed 786-O and CAKI-1 cells. All experiments were repeated double times. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 6. ZHX2 could induce Sunitinib resistance by activating autophagy.

a, b The sunitinib treatment experiments in new reprogrammed 786-O and CAKI-1 respectively. c The autophagy related proteins LC3-I/II were tested by western blot in new reprogrammed 786-O and CAKI-1 respectively. The ratio in western blot semi-quantification was normalized by GAPDH respectively. d The combination treatment of sunitinib and CQ could significantly increase the inhibition rate in 786-O/LV-ZHX2 cells. e–g The single-label-adenovirus RFP-LC3 was used to detect the LC3 expression before and after sunitinib treatment respectively. The quantitative measurements of LC3 were performed under fluorescence microscope. h All experiments summarized in a schematic diagram. The solid arrow is the conclusion of this study, while the hollow arrow is the experimental guess. All experiments were repeated double times. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant.