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 (pā=ā0.004), and proved to be an independent prognosis factor for progression-free survival (pā=ā0.0003), 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.
Similar content being viewed by others
Introduction
Since the tumor suppressor gene VHL was identified as the key point of the Von Hippel-Lindau (VHL) disease in 19931, it has been clearly proved that hypoxia or gene mutation could lead to the inactivation of VHL and induce the loss function of VHL complex (VBC, including elongin B and C), which targeting hypoxia-inducible factors (HIFs) for ubiquitylation and proteasomal degradation2,3,4,5. As well studied, large scale of chromatin immunoprecipitation sequencing (Chromatin immunoprecipitation (ChIP)-seq) showed that over 800 genes could directly regulated by HIFs, such as vascular endothelial growth factor (VEGF)6,7, and HIFs could also regulate downstream gene expression by trans activating gene encoding microRNAs and enzymes in chromatin modification8,9,10. VHL-HIFs signal is a important role in the development of clear cell renal cell carcinoma (ccRCC), and the tyrosine kinase inhibitors (TKIs) that target the VHL substrate HIF signal have showed treating benefit in patients with advanced ccRCC. However, drug resistance still occurs during following treatment in most patients, seems identify additional VHL substrates is urgent to improve therapeutic outcome of ccRCC patients11. Qing Zhang et al.12 have found that Zinc fingers and homeoboxes 2 (ZHX2) is a novel VHL substrate transcription factor by using a genome-wide human cDNA library strategy; and they reported that depletion of ZHX2 could inhibit the proliferation of VHL-deficient ccRCC cell lines through impairing NF-ĪŗB pathway. Though, the detailed mechanism of ZHX2 in ccRCC is still not clear, and whether it plays a role in TKIs drug resistance is also unknown.
ZHX2 is a member of ZHX protein family which including ZHX1 and ZHX3, and these proteins contains two zinc-fingers and four or five homeodomains. Several studies report that ZHX proteins are ubiquitously expressed and primary found in the nucleus and functions as transcriptional repressors13,14,15. However, there were some conflicting data when deep investigating the detail function of ZHX2 in human malignant cancers. Lv et al.16 demonstrated that ZHX2 promoter region was hypermethylated in hepatocellular carcinoma (HCC) samples, suggesting it might functions as a tumor suppressor. On the other side, another immunohistochemical research reported that ZHX2 was highly expressed in tumor samples compared to adjacent tissues in HCC patients, and the prognostic analysis showed ZHX2 was associated with poorer outcomes17, seems it act as an oncogenic biomarker.
As renal cancer, the evidence accumulating from past decade suggests that the HIF2a is the key factor for progression, and the HIF1a might function as a tumor suppressor18,19. These phenomena are very different from other vascularized tumors. However, HIF2a inhibitor has limited treating response in ccRCC patients, so there must be some kind of extra signals to promote cancer proliferation20. ZHX2 as a novel VHL substrate transcription factor like HIF2a, there may be synergy function between two signaling pathways, and this may be responsible for TKI drug resistance. In this study, we further investigate the ZHX2 expression in ccRCC patients, and explore the detailed mechanism about ccRCC progression in ZHX2 reprogramming cell lines. Further, we try to find out whether it is related with sunitinib resistance.
Materials and methods
Tissue immunohistochemistry
Tissue microarrays (TMAs) that contain 358 ccRCC samples (including tumor and adjacent tissues) were kindly provided by Prof. Jin Zhang. The pathologic information of all samples were determined by two experienced pathologists, and the comprehensive clinicopathologic data included gender, age, tumor size, the TNM stage, and Fuhrman grade were determined by according to the standard classification systems21,22. Annual laboratory test and MRI or CT scan was used to detect metastasis during the follow-up examination. Deep investigation of the ZHX2 localization and protein expression were preformed by immunohistochemistry (IHC) analysis according to the standard streptavidin-peroxidase method (Zymed Laboratories Inc, San Francisco, CA, USA). The primary antibody against ZHX2 (Rabbit ZHX2 antibody, 112232, Genetex, CA, USA) was diluted into 1:50 and PBS was as negative control. The localization and expression of ZHX2 were assessed independently by two observers (Dr Zhu and Dr Ding). The expression level of ZHX2 was divided into two groups according to the IHC score, which was calculated by staining percentageĆintensity as our previous study23. This study was approved by the Ethical Review Boards of Zhong Shan Hospital Fudan University.
Cell lines with Lentiviral transduction and siRNA transfection
The two ccRCC cell lines used in our study were 786-O and CAKI-1 (Purchased from American Type Culture Collection, ATCC, Manassas, VA, USA). 786-O was VHL-deficient cells while CAKI-1 was VHL-normal cells24. All cell lines were maintained in the recommended medium 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Australia), 1% GlutaMAX, 1% nonessential amino acids, and 1% sodium pyruvate. Cultures were maintained at 37āĀ°C with 5% CO2, and the medium was changed at least twice weekly.
The lentivirus expression human ZHX2 (LV-ZHX2), lentivirus silence VHL expression (LV-shVHL), and the control lentivirus (LV-NC) were purchased from GENECHEM GROUP (shanghai, China). LV-ZHX2 was used to transduce 786-O to increase ZHX2 expression, LV-shVHL and LV-ZHX2 were used to transduce CAKI-1 for lineage reprogramming. New stable cell lines were established and used in further research after cultivating in the medium containing puromycin (5āĪ¼g/mL, Sigma) for 7 days (change the medium every there days). The protein and RNA level of ZHX2 were tested by western blot and quantitative real-time PCR respectively (Detail information of lentivirus was collected in Supplementary Table S1).
The ZHX2, EPAS1 (HIF-2a) and respective negative control small interfering RNAs (siRNAs) (Purchased from GenePharma Company, shanghai China.) were transfected with lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturerās instructions.
Cell proliferation and drug experiments
The proliferation ability of new reprogrammed 786-O and CAKI-1 cell lines were determined by Sulforhodamine B (SRB) assay in 96-well plates according to the manufactureās instruction. Whatās more, the cells also plated into 6-well plates (1.5āĆā105 cell/well) and incubated overnight at 37āĀ°C, then cell counts by using electronic cell counter (Invitrogen) were taken every 24āh for 96āh post incubation for another verification.
Sunitinib and Chloroquine (CQ) were purchased from Selleck (Shanghai, China.), and dissolved in nuclease-free water. Sunitinib applied to cell lines at a final concentration of 5āĪ¼M/L, while CQ applied at 10āĪ¼M/L. The inhibition effect of Sunitinib and the combination effects of Sunitinib + CQ were determined by cell count ratios with a time manner.
Enzyme-linked immunosorbent assay
The cells were seeded in 6-well plates with 2āĆā105 cells/well for 24āh, and change the medium with 2āml serum-free medium for another 24āh. Then cell supernatant was collected and centrifuged to remove the debris. The human VEGF ProQuantum Immunoassay Kit (A35602 ThermoFisher, USA) was used to measure the secreted VEGF.
Wound healing assay
ccRCC cells were seeded in 6-well plate with 5āĆā105 cell/well and cultured until the cells were fully grown. Then scraped the cells in a straight line by using a 200āĪ¼l pipette tip to make a scratch, images were captured after washing with PBS twice and replaced with serum-free medium at 0āh, 24āh, and 48āh.
Transwell migration assay
The new reprogramming ccRCC cell lines were seeded in the transwell inserts (pore size: 8.0āĪ¼m; Corning, Lowell, MA, USA) respectively with appropriate number of cells (3āĆā104 cells/well). The serum-free medium was in the upper migration stoppers while medium with 10% FBS serum was in the bottom wells. After 24āh, the invaded cells of each cell lines on the lower side of membrane were fixed in 95% methanol and stained with crystal violet according to the manufactureās instructions.
Western blot analysis
Western blot procedure was performed according to the standard protocol and the protein lysates were obtained from cultured cells with different treatment. Proteins were separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane (Millipore, Temecula, CA, USA). After blocking with non-fat milk for nearly 1āh at room temperature, the membranes were incubated overnight at 4āĀ°C with following primary antibodies: ZHX2 (Genetex, CA, USA), ERK1/2, p-ERK, LC3, AKT, p-AKT, STAT3, p-STAT3, JNK, and GAPDH (Cell Signaling Technology, Boston, MA, USA). Then the membranes were washed three times and incubated with secondary antibody at room temperature for 2āh. Then immunoreactive bands were detected by using ECL system (Beyotime, Jiangsu, China) according to the manufactureās instruction, and the GAPDH were used as a loading control. The Image J 1.47V software (http://imagej.nih.gov/ij) was used for semi-quantification of the western blot experiments.
Flow cytometry for cell apoptosis
For cell apoptosis analysis, cells that harvested after siRNA interference assay were collected according to the standard protocol (FITC Apoptosis Detection Kit). The apoptosis cells were measured by FACS Calibur flow cytometer (Becton-Dickinson, Mountain View, CA). All experiments were repeated three times.
Quantitative real-time PCR
The QRT-PCR was performed according to the standard protocol, and the human Ī²-actin was used as normalized control and each measurement was performed in triplicate. The primer sequences are presented in Supplementary Table S2.
Chromatin immunoprecipitation and luciferase reporter assay
Chromatin immunoprecipitation for ZHX2 was performed according to the manufactureās protocol (SimpleChIP Plus Enzymatic Chromatin IP Kit9005, CST, Boston, Massachusetts, USA), and the binding enrichment of MEK1 (also named MAP2K1) promoter (Forward primer: 5ā²-ccctagattcctttgtgctgc-3ā²; Reverse primer: 5ā²-cggttcgggtcgaaggaa-3ā²) was normalized against that in the input samples. The ZHX2 binding site of MAP2K1 promoter was clone into luciferase reporter pGL3-MAP2K1, and pGL3-basic as negative control. The luciferase activity was determined by using a Dual-Luciferase Assay Kit (E1910, Promega Biotechnology, USA) according to its manufacturerās instructions.
Autophagy protein labeling experiment
RFP-LC3 single-label-adenovirus was purchased from HanBio Company (Shanghai China). The LV-NC and LV-ZHX2 786-O cells were transfected with RFP-LC3 adenovirus, and drug experiments performed after its stable expression. RFP intensity was observed under fluorescence microscope after 24āh treatment. Autophagy intensity was determined by counting the number of red fluorescence.
In vivo animal experiments
All animal experiments procedures were approved by the Animal Care and Use Committee of Zhongshan Hospital Fudan University (Shanghai, China). For subcutaneous tumor implantation, an equal number of 786-O cells with LV-ZHX2 or LV-NC (2āĆā106 cells) were resuspended in PBS and matrigel (BD, USA) at 1:1 ratio. Then injected into flank region of the six-week-old female athymic nude mice. Tumor growth was monitored and measured twice a week, and tumor weight was measured in the end of in vivo experiments. The samples were collected and saved in 4% paraformaldehyde for further Ki-67 staining.
Statistical analysis
Univariate and multivariate analysis were used to determine the independent prognosis factors. Overall survival and Progression-free survival curves were plotted by KaplanāMeier method and compared with the log-rank test. Significant difference between different treatments groups were tested by using a two-tailed, paired t-test analysis (Graphpad Software, Inc., CA, USA). All P values of <ā0.05 were assigned significance.
Results
Overexpression of ZHX2 predicts worse outcome in ccRCC patients
The protein expression of ZHX2 was detected by IHC in 358 ccRCC patients, the characteristic information was showed in Table 1. As showed in Fig. 1a, ZHX2 was mainly localized in nucleus and we divided the patients into two groups according to the ZHX2 expression level (Fig. 1a, b represent low expression group while c, d represent high group). Only 33.2% (119/358) patients showed a high expression of ZHX2 (Table 1), but high expression of ZHX2 was significantly associated with advanced Fuhrman grade (pā=ā0.004) and metastasis after surgery (pā=ā0.009). The prognostic analysis showed that the patients with high ZHX2 have worse progression-free survival (PFS) while there was no significant relation with overall survival (OS) (Fig. 1b, c). We also searched the OS data in TCGA database and the result showed no correlation as well (Supplementary Fig. S1a). Univarivate and multivariate Cox regression analysis showed the higher expression of ZHX2 had independent predictive value for PFS in ccRCC patients. (HRā=ā0.567, pā=ā0.035, Supplementary Table S3āS4), which means ZHX2 might act as a tumor promoter that contributed to occurrence and recurrence of ccRCC.
ZHX2 is involved in ccRCC cell proliferation and migration
As reported, ZHX2 depletion could inhibit the VHL-deficient ccRCC cell growth. We used the siRNA to knockdown the ZHX2 expression in 786-O cells as well as EPAS1 (Encode HIF-2a), and both genes silenced could inhibit the cell proliferation (Fig. 2a). We also detected the VEGF expression, one of the most important downstream targets of VHL-HIF signal, and the VEGF level was significantly decreased in si-EPAS1 cells while no significant reduction was observed in si-ZHX2 cells (Fig. 2b). Apoptosis analysis showed that either EPAS1 or ZHX2 knockdown could induce cell apoptosis (Fig. 2cāf), and wound healing assay showed both can inhibit cell migration (Supplementary Fig. S1b, c), which means that ZHX2 may have similar function with HIF-2a in ccRCC proliferation.
Further, we used lentivirus LV-ZHX2 to overexpress ZHX2 expression in 786-O cells (VHL-deficient RCC cell line). Both mRNA and protein level of ZHX2 were effectively up-regulated in new cell line (Fig. 3a, b). Proliferation assay showed 786-O with LV-ZHX2 could significantly promote cell growth after 96āh compared with negative control (Fig. 3c). Wound healing assay and Transwell assay also showed ZHX2 overexpression could increase the migration ability in 786-O cells compared with control group (Fig. 3d, e, Supplementary Fig. S2a, b). Furthermore, the xenograft tumor models were established by using 786-O with LV-ZHX2 and LV-NC respectively, as showed in Fig. 3f, average tumor volume was much bigger in LV-ZHX2 group (pā<ā0.01, Fig. 3g), and the tumor weight was showed heavier in LV-ZHX2 group (pā<ā0.01, Supplementary Fig. S2c) either. Besides, we performed IHC staining of Ki-67 in mouse tumors as well (Supplementary Fig. S3). These results indicated that the ZHX2 had strong promotive effect on cell proliferation and migration. Interestingly, tube formation could be seen under fluorescence microscope in 786-O/LV-ZHX2 cells (Fig. 3h, i), and the immunofluorescence staining of CD31 was more expressed in reprogrammed 786-O cells (Supplementary Fig. S4). We also detected some HIFās downstream targets and found the mRNA expression of EGFR, VEGF, and TGFB1 were significantly increased compared with control group (Fig. 3j). Enzyme-linked immunosorbent assay (ELISA) assay was used to measure the secreted VEGF level in different cell lines, and ZHX2 overexpression cells may secret more VEGF to promote its self-tube formation and proliferation (Fig. 3k). At the same time, we used lentivirus silence VHL expression (LV-shVHL) and LV-ZHX2 to reprogram the CAKI-1 cells (VHL-normal RCC cell line) (Fig. 4a). VHL silence and ZHX2 overexpression in CAKI-1 could increase cell proliferation and migration as well (Fig. 4bāf, Supplementary Fig. S2c, d). The mRNA expression of EGFR, VEGF, and TGFB1 were up-reguated in reprogram CAKI-1 cells as well as the secret VEGF (Fig. 4g, h). These phenomena are consistent with the previous results that ZHX2 and HIFs may have some same mechanism in the process of promoting cancer.
ZHX2 promote ccRCC growth by transcriptional activate the MEK1/ERK1/2 signaling pathway
To identify the more detail mechanism of ZHX2 in ccRCC cell proliferation and migration, RNA-sequence analysis was performed. As demonstrated in Fig. 5a, the heat-map showed the overall change of mRNA expression pattern, the upregulated genes indicated in red while downregulated genes in green. The enrichment of KEGG analysis showed several cancer-related pathways were activated in ZHX2 overexpression cells, especially MAPK signaling pathway (Fig. 5b). Then we detected the specific genesā mRNA expression in MAPK pathway, and results showed MAP2K1, ERK1, and ERK2 were significantly increased in 786-O/LV-ZHX2 cells compared with negative control. Also the downstream genes of ERK pathway that c-MET and STAT3 were upreguated as well (Fig. 5c, d). These results suggest that MAP2K1-ERK1/2 pathway was transcriptionally upregulated upon ZHX2 overexpression. Whatās more, we used ChIP-qPCR to determine whether ZHX2 could specifically bind to the promoter of MAP2K1 (which encode MEK1 protein and the direct upstream of ERK). The ChIP and Luciferase assays showed ZHX2 as a transcription factor can specifically target to the promoter of MAP2K1 (Fig. 5e, f). In the meantime, the ChIP-sequence also predicted the binging motifs of ZHX2 on genome (Fig. 5g). We examined the protein level of ERK pathway later, ERK with its phosphorylation and downstream STAT3 with its phosphorylation were increased as well as JNK, and no obvious change was showed in AKT (Fig. 5h), and the semi-quantification of the western blot were showed in Supplementary Fig. S5a. Similar, we test the MAPK-related protein expression in mouse tissue as well (Supplementary Fig. S5b). Taking together, our findings indicated that ZHX2 could direct activate the MEK1-ERK1/2 pathway in ccRCC cell lines.
ZHX2 could induce sunitinib resistance by activating autophagy
As previous work has reported, MAPK-ERK pathway may be involved in tyrosine kinase drugs resistance25,26. Then we tested the inhibition rate of sunitinib in reprogramed ccRCC cell lines (Supplementary Fig. S6a), and choose 5āĪ¼mol/L for further investigation. As showed in Fig. 6a, b, the inhibitory effect of sunitinib was significantly decreased in the cell lines with overexpression of ZHX2. In this process, we found that many vesicles were turned up after treating with sunitinib in LV-ZHX2 cells, while there was no such change in control group (Supplementary Fig. S6c). So we wondered if autophagy was involved in this situation, and autophagy related proteins LC3-I/II were tested after sunitinib treatment in cell lines by western blot. The ratio of LC3-II/LC3-I was increased in ZHX2 overexpression cells, which meant autophagy was over-activated (Fig. 6c). The combination treatment of sunitinib and autophagy inhibitor CQ could significantly increase the inhibition rate in LV-ZHX2 cells (Fig. 6d). For further verification, we used RFP-LC3 single-label-adenovirus to detect the expression of LC3 after sunitinib treatment. Compared with the control group, the adenovirus labeled red light was significantly increased in LV-ZHX2 cells (Fig. 6e, g), although sunitinib treatment can also increase LC3 expression in normal ccRCC cells. Therefore, we put forward the assumption that ZHX2 overexpression could activate the MEK-ERK signaling pathway and reduce the inhibition effect of sunitinib by activating self-protective autophagy. So we proposed the following schematic diagram to help illustrate these mechanisms in which ZHX2 participates in ccRCC development (Fig. 6h) and ultimately serves as a potential therapeutic target.
Discussion
ZHX2 was found as a novel VHL substrate transcription factor, and VHL could regulate its protein stability when itās hydroxylated, just like HIFs. Although previous research has well illustrated VHL in the regulation of ZHX2 in ccRCCās development, little is known about the downstream signaling pathway of ZHX2 as a transcription factor. In this study, we focused on the biological function of ZHX2 in ccRCC and the potential molecular downstream targets. Firstly, we tested the localization and expression of ZHX2 and only 33.2% of ccRCC patients have high expression in nuclear. We observed that high ZHX2 expression significantly correlated with worse outcome, and ZHX2 could activate VEGFA like HIF-2a did, and it markedly promoted cell proliferation, migration by transcriptional activating MEK1/ERK1/2 signaling. In this process, the expression of downstream MEK1 target genes, such as ERK1/2, STAT3, and JNK were upregulated, which may responsible for the tumor angiogenesis and metastasis of ccRCC.
The role of ZHX2 in different tumorigenesis is controversial. Kawata et al.13 reported it worked as a transcriptional repressor, and the changes of ZHX2 could regulate several oncogenes expression in human disease16. Similarly, Nagel et al.15 demonstrated some mechanisms that chromosomal rearrangement are related to decrease the expression of ZHX2 in Hodgkin lymphoma, including enhancing binding sites inactivation, upstream genes MSX1 and XBP1 reduction, and MSX1-corepressor H1C overexpression. In summary, their research proved that ZHX2 was involved in this regulative network and functioned as a tumor suppressor in B-cells malignant tumor as well. Yue et al.27 also discovered that ZHX2 inhibited the growth of HCC cell lines by transcriptional represses of two key cell cycle regulators, Cyclin A and Cyclin E27. Later, Wu et al.28 reported ZHX2 could protect hepatocytes from lipid deposition disorder in non-alcoholic fatty liver disease to retard cell growth and its-related HCC progression. All these researches illustrated that in the process of disease and tumorigenesis, ZHX2 usually played the role of tumor suppressor gene.
Nevertheless, Zhang et al.12 proposed that ZHX2 was a novel VHL substrate transcription factor and functioned as an oncogenic driver in the development of ccRCC. In his research, higher ZHX2 was found to express in most of ccRCC samples as well as HIF2a than those of the paired normal tissues. Depletion of ZHX2 could significantly inhibit ccRCC proliferation in vitro and in vivo. Here, we found that ZHX2 knocking down by siRNA could increase cell apoptosis and inhibit cell migration as well. Further, we used lentivirus to overexpress the ZHX2 in ccRCC cell lines to investigate the detail mechanism. Our study demonstrated that ZHX2 had some same functions as HIFs, and can activate VEGF signal. More importantly, RNA-seq analysis showed that ZHX2 could activate some other signals, especially MAPK/ERK pathway. ERK1/2 is a subfamily of MAPK signaling pathway, and it is regarded as a vital role to promoter cell survival and proliferation by entering into the nucleus after phosphorylation29. STAT3 is one of the important downstream molecules in ERK pathway, activated ERK1/2 can enhance transcription activity by regulating the phosphorylation of STAT3 in nucleus30,31. Our result showed that the ZHX2 overexpression increased the phosphorylation of ERK while both STAT3 and phosphorylated STAT3 were increased as well. STAT3 also has been recognized as a critical factor in cell growth and metastasis of various human malignant tumors32,33. The studies show that VEGF is the key factor of tumor angiogenesis, which is involved in the formation of tumor blood vessels and provides conditions for tumor invasion and metastasis. STAT3 is proved to be one of the regulatory factors of VEGF and it can mediate VEGF expression in two different ways. On the one hand, activated STAT3 can promote the transcription of HIF and regulate the expression of VEGF34; on the other hand, activated STAT3 can promote the transcription activity of VEGF by directly binding to its promoter35. In our in vitro study, ZHX2 silencing could part decrease VEGF expression and ZHX2 overexpression could significantly activate VEGF. The result may be because ZHX2 is upstream of VEGF or ZHX2 regulate VEGF through MEK/ERK/STAT3 signal.
Sunitinib is still one of the standard treatments in advanced ccRCC patients, researchers searched the GEO database and microarray data in sunitinib-resistant RCC tumors and demonstrated that MAPK/ERK pathway was excessive activating in sunitinib-resistance RCC tumors25. The mechanism of sunitinib resistance is still unknown, as a new transcription factor of VHL, ZHX2 may regulate VEGF signal and activate MAPK/ERK signal pathway, so we speculate that ZHX2 may be related to sunitinib resistance. Sunitinib inhibition test confirmed that the inhibition effect of sunitinib on the ccRCC with high expression of ZHX2 was obviously decreased and this result can be rescued by combination treatment with CQ. Meanwhile, RFP-LC3 single-label-adenovirus was used to detect autophagy related protein expression. These results indicated that ZHX2 overexpression might induce sunitinib resistance by self-protective autophagy, and the more detail mechanism investigation is still needed.
In summary, we test and verify the role of ZHX2 in ccRCC development, and ZHX2 facilitates the proliferation and migration in ccRCC cell lines by activating the MEK1/ERK1/2 signaling pathway. Our study also provides a new perspective on the involvement of ZHX2 in sunitinib resistance. These discoveries may give a new insight for ccRCC treatment.
Change history
14 April 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41419-021-03678-9
References
Latif, F. et al. Identification of the von Hippel-Lindau disease tumor suppressor gene. Science 260, 1317ā1320 (1993).
Epstein, A. C. et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107, 43ā54 (2001).
Jaakkola, P. et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468ā472 (2001).
min, J. H. et al. Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science 296, 1886ā1889 (2002).
Tedesco, L. et al. von Hippel-Lindau mutants in renal cell carcinoma are regulated by increased expression of RSUME. Cell Death Dis. 10, 266 (2019).
Wizigmann-Voos, S., Breier, G., Risau, W. & Plate, K. H. Up-regulation of vascular endothelial growth factor and its receptors in von Hippel-Lindau disease-associated and sporadic hemangioblastomas. Cancer Res. 55, 1358ā1364 (1995).
Xia, X. et al. Integrative analysis of HIF binding and transactivation reveals its role in maintaining histone methylation homeostasis. Proc. Natl Acad. Sci. USA 106, 4260ā4265 (2009).
Crosby, M. E., Devlin, C. M., Glazer, P. M., Calin, G. A. & Ivan, M. Emerging roles of microRNAs in the molecular responses to hypoxia. Curr. Pharm. Des. 15, 3861ā3866 (2009).
Wu, M. Z. et al. Interplay between HDAC3 and WDR5 is essential for hypoxia-induced epithelial-mesenchymal transition. Mol. Cell 43, 811ā822 (2011).
Gossage, L., Eisen, T. & Maher, E. R. VHL, the story of a tumour suppressor gene. Nat. Rev. Cancer 15, 55ā64 (2015).
Escudier, B., Szczylik, C., Porta, C. & Gore, M. Treatment selection in metastatic renal cell carcinoma: expert consensus. Nat. Rev. Clin. Oncol. 9, 327ā337 (2012).
Zhang, J. et al. VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma. Science 361, 290ā295 (2018).
Kawata, H. et al. Zinc-fingers and homeoboxes (ZHX) 2, a novel member of the ZHX family, functions as a transcriptional repressor. Biochem. J. 373, 747ā757 (2003).
Yamada, K. et al. Analysis of zinc-fingers and homeoboxes (ZHX)-1-interacting proteins: molecular cloning and characterization of a member of the ZHX family, ZHX3. Biochem. J. 373, 167ā178 (2003).
Nagel, S. et al. Transcriptional deregulation of homeobox gene ZHX2 in Hodgkin lymphoma. Leuk. Res. 36, 646ā655 (2012).
Lv, Z. et al. Promoter hypermethylation of a novel gene, ZHX2, in hepatocellular carcinoma. Am. J. Clin. Pathol. 125, 740ā746 (2006).
Hu, S. et al. Expression of zinc-fingers and homeoboxes 2 in hepatocellular carcinogenesis: a tissue microarray and clinicopathological analysis. Neoplasma 54, 207ā211 (2007).
Raval, R. R. et al. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol. Cell. Biol. 25, 5675ā5686 (2005).
Keith, B., Johnson, R. S. & Simon, M. C. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nat. Rev. Cancer 12, 9ā22 (2011).
Cho, H. et al. On-target efficacy of a HIF-2alpha antagonist in preclinical kidney cancer models. Nature 539, 107ā111 (2016).
Elmore, J. M., Kadesky, K. T., Koeneman, K. S. & Sagalowsky, A. I. Reassessment of the 1997 TNM classification system for renal cell carcinoma. Cancer 98, 2329ā2334 (2003).
Hong, S. K. et al. Application of simplified Fuhrman grading system in clear-cell renal cell carcinoma. BJU Int. 107, 409ā415 (2011).
Zhu, L. et al. LSD1 inhibition suppresses the growth of clear cell renal cell carcinoma via upregulating P21 signaling. Acta Pharm. Sin. B 9, 324ā334 (2019).
Shinojima, T. et al. Renal cancer cells lacking hypoxia inducible factor (HIF)-1alpha expression maintain vascular endothelial growth factor expression through HIF-2alpha. Carcinogenesis 28, 529ā536 (2007).
Gao, Y. et al. Adrenomedullin blockade suppresses sunitinib-resistant renal cell carcinoma growth by targeting the ERK/MAPK pathway. Oncotarget 7, 63374ā63387 (2016).
Cocco, E. et al. Resistance to TRK inhibition mediated by convergent MAPK pathway activation. Nat. Med. 25, 1422ā1427 (2019).
Yue, X. et al. Zinc fingers and homeoboxes 2 inhibits hepatocellular carcinoma cell proliferation and represses expression of Cyclins A and E. Gastroenterology 142, 1559ā1570. e1552 (2012).
Wu, Z. et al. Tumor suppressor ZHX2 inhibits NAFLD-HCC progression via blocking LPL-mediated lipid uptake. Cell Death Differ. https://doi.org/10.1038/s41418-019-0453-z (2019).
She, T. et al. Hyperglycemia suppresses cardiac stem cell homing to peri-infarcted myocardium via regulation of ERK1/2 and p38 MAPK activities. Int. J. Mol. Med. 30, 1313ā1320 (2012).
Vainchenker, W. & Constantinescu, S. N. JAK/STAT signaling in hematological malignancies. Oncogene 32, 2601ā2613 (2013).
Yu, H., Pardoll, D. & Jove, R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Cancer 9, 798ā809 (2009).
He, M. & Xue, Y. MicroRNA-148a suppresses proliferation and invasion potential of non-small cell lung carcinomas via regulation of STAT3. OncoTargets Ther. 10, 1353ā1361 (2017).
Yu, H., Lee, H., Herrmann, A., Buettner, R. & Jove, R. Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat. Rev. Cancer 14, 736ā746 (2014).
Niu, G. et al. Signal transducer and activator of transcription 3 is required for hypoxia-inducible factor-1alpha RNA expression in both tumor cells and tumor-associated myeloid cells. Mol. Cancer Res. 6, 1099ā1105 (2008).
Wei, D. et al. Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis. Oncogene 22, 319ā329 (2003).
Acknowledgements
This work was supported by Shanghai Science and Technology Development Foundation (17JC1400904).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisherās note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by B. Zhivotovsky
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the articleās Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the articleās Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Zhu, L., Ding, R., Yan, H. et al. 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. Cell Death Dis 11, 337 (2020). https://doi.org/10.1038/s41419-020-2541-x
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41419-020-2541-x
This article is cited by
-
ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression
Cell Death & Disease (2023)
-
Zinc fingers and homeoboxes 2 inhibition could suppress the proliferation of ovarian cancer cells by apoptosis pathway
Clinical and Translational Oncology (2023)
-
ZHX2 inhibits thyroid cancer metastasis through transcriptional inhibition of S100A14
Cancer Cell International (2022)
-
Sulfated alginate oligosaccharide exerts antitumor activity and autophagy induction by inactivating MEK1/ERK/mTOR signaling in a KSR1-dependent manner in osteosarcoma
Oncogenesis (2022)
-
Histone demethylase KDM4D inhibition suppresses renal cancer progression and angiogenesis through JAG1 signaling
Cell Death Discovery (2021)