In clear cell renal cell carcinoma (ccRCC), inactivation of the tumor suppressor von Hippel-Lindau (VHL) occurs in the majority of the tumors and is causal for the pathogenesis of ccRCC. Recently a large-scale genomic sequencing study of ccRCC tumors revealed that enzymes that regulate histone H3 lysine 4 trimethylation (H3K4Me3), such as JARID1C/KDM5C/SMCX and MLL2, were mutated in ccRCC tumors, suggesting that H3K4Me3 might play an important role in regulating gene expression and tumorigenesis. In this study, we report that in VHL-deficient ccRCC cells the overall H3K4Me3 levels were significantly lower than that of VHL+/+ counterparts. Furthermore, this was HIF-dependent, as depletion of HIF subunits by shRNA in VHL-deficient ccRCC cells restored H3K4Me3 levels. In addition, we demonstrated that only loss of JARID1C, not JARID1A or JARID1B, abolished the difference of H3K4Me3 levels between VHL−/− and VHL+/+ cells, and JARID1C displayed HIF-dependent expression pattern. JARID1C in VHL−/− cells was responsible for the suppression of HIF-responsive genes IGFBP3, DNAJC12, COL6A1, GDF15, and DEP-1. Consistent with these findings, the H3K4Me3 levels at the promoters of IGFBP3, DNAJC12, COL6A1, and GDF15 were lower in VHL−/− cells than in VHL+/+ cells, and the differences disappeared after JARID1C depletion. Although HIF2α is an oncogene in ccRCC, some of its targets might have tumor suppressive activity. Consistent with this, knockdown of JARID1C in 786-O VHL−/− ccRCC cells significantly enhanced tumor growth in a xenograft model, suggesting that JARID1C is tumor suppressive and its mutations are tumor-promoting in ccRCC. Thus, VHL inactivation decreases H3K4Me3 levels through JARID1C, which alters gene expression and suppresses tumor growth.
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Somatic mutations or loss of expression of tumor suppressor VHL happen in the vast majority of clear cell Renal Cell Carcinoma, and it’s causal for kidney cancer development. Without VHL, constitutively active transcription factor HIF is strongly oncogenic and is essential for tumor growth. However, the contribution of individual HIF-responsive genes to tumor growth is not well understood. In this study we examined the contribution of important HIF-responsive genes such as VEGF, CCND1, ANGPTL4, EGLN3, ENO2, GLUT1 and IGFBP3 to tumor growth in a xenograft model using immune-compromised nude mice. We found that the suppression of VEGF or CCND1 impaired tumor growth, suggesting that they are tumor-promoting genes. We further discovered that the lack of ANGPTL4, EGLN3 or ENO2 expression did not change tumor growth. Surprisingly, depletion of GLUT1 or IGFBP3 significantly increased tumor growth, suggesting that they have tumor-inhibitory functions. Depletion of IGFBP3 did not lead to obvious activation of IGFIR. Unexpectedly, the depletion of IGFIR protein led to significant increase of IGFBP3 at both the protein and mRNA levels. Concomitantly, the tumor growth was greatly impaired, suggesting that IGFBP3 might suppress tumor growth in an IGFIR-independent manner. In summary, although the overall transcriptional activity of HIF is strongly tumor-promoting, the expression of each individual HIF-responsive gene could either enhance, reduce or do nothing to the kidney cancer tumor growth.
p53 acetylation is indispensable for its transcriptional activity and tumor suppressive function. However, the identity of reader protein(s) for p53 acetylation remains elusive. PBRM1, the second most highly mutated tumor suppressor gene in kidney cancer, encodes PBRM1. Here, we identify PBRM1 as a reader for p53 acetylation on lysine 382 (K382Ac) through its bromodomain 4 (BD4). Notably, mutations on key residues of BD4 disrupt recognition of p53 K382Ac. The mutation in BD4 also reduces p53 binding to promoters of target genes such as CDKN1A (p21). Consequently, the PBRM1 BD4 mutant fails to fully support p53 transcriptional activity and is defective as a tumor suppressor. We also find that expressions of PBRM1 and p21 correlate with each other in human kidney cancer samples. Our findings uncover a tumor suppressive mechanism of PBRM1 in kidney cancer and provide a mechanistic insight into the crosstalk between p53 and SWI/SNF complexes.
Clear cell renal cell carcinoma (ccRCC) is the major subtype of kidney cancer that is characterized by frequent inactivation of von Hippel-Lindau (VHL) gene in 80–90% of the tumors. Recent reports using massive parallel sequencing technologies have discovered more cancer driver genes. PBRM1 was found to be mutated in about 40% of ccRCC tumors, while BAP1 and SETD2 were mutated in about 10–15% of ccRCC tumors each. JARID1C and UTX, two histone H3 demethylases, were also found to harbor mutations in ccRCC, albeit at lower rates. ccRCC tumors display high degree of intra-tumoral heterogeneity, with some mutations present in everyone cancer cells (ubiquitous) while some other subclonal. The VHL mutations were always ubiquitous in the tumors, while PBRM1 mutations were also ubiquitous but to a lesser extent. On the contrary, the mutations in BAP1, SETD2, JARID1C, and UTX were all subclonal, meaning that they were present in a subset of cancer cells in a tumor. The prognosis value of PBRM1 mutations in ccRCC is still controversial, while BAP1 mutations were tightly linked to worse clinical outcome in multiple studies. The molecular functions of these newly identified cancer driver genes were discussed, and they were known reader, writer, or eraser of histone marks on histone H2 and H3 tails that are very close to each other, suggesting that these factors might functionally interact and affect common pathways. The studies on these newly identified tumor suppressors will shed light on ccRCC tumorigenesis and development, and will likely lead to development of novel therapeutic interventions for ccRCC patients.
Polybromo‐1 ( PBRM 1) is an important tumor suppressor in kidney cancer. It contains six tandem bromodomains ( BD s), which are specialized structures that recognize acetyl‐lysine residues. While BD 2 has been found to bind acetylated histone H3 lysine 14 (H3K14ac), it is not known whether other BD s collaborate with BD 2 to generate strong binding to H3K14ac, and the importance of H3K14ac recognition for the molecular and tumor suppressor function of PBRM 1 is also unknown. We discovered that full‐length PBRM 1, but not its individual BD s, strongly binds H3K14ac. BD s 2, 4, and 5 were found to collaborate to facilitate strong binding to H3K14ac. Quantitative measurement of the interactions between purified BD proteins and H3K14ac or nonacetylated peptides confirmed the tight and specific association of the former. Interestingly, while the structural integrity of BD 4 was found to be required for H3K14ac recognition, the conserved acetyl‐lysine binding site of BD 4 was not. Furthermore, simultaneous point mutations in BD s 2, 4, and 5 prevented recognition of H3K14ac, altered promoter binding and gene expression, and caused PBRM 1 to relocalize to the cytoplasm. In contrast, tumor‐derived point mutations in BD 2 alone lowered PBRM 1's affinity to H3K14ac and also disrupted promoter binding and gene expression without altering cellular localization. Finally, overexpression of PBRM 1 variants containing point mutations in BD s 2, 4, and 5 or BD 2 alone failed to suppress tumor growth in a xenograft model. Taken together, our study demonstrates that BD s 2, 4, and 5 of PBRM 1 collaborate to generate high affinity to H3K14ac and tether PBRM 1 to chromatin. Mutations in BD 2 alone weaken these interactions, and this is sufficient to abolish its molecular and tumor suppressor functions.
Recent studies have shown that intratumoral heterogeneity (ITH) is prevalent in clear cell renal cell carcinoma (ccRCC), based on DNA sequencing and chromosome aberration analysis of multiple regions from the same tumor. VHL mutations were found to be universal throughout individual tumors when it occurred (ubiquitous), while the mutations in other tumor suppressor genes tended to be detected only in parts of the tumors (subclonal). ITH has been studied mostly by DNA sequencing in limited numbers of samples, either by whole genome sequencing or by targeted sequencing. It is not known whether immunohistochemistry (IHC) can be used as a tool to study ITH. To address this question, we examined the protein expression of PBRM1, and PBRM1-related proteins such as ARID1A, SETD2, BRG1, and BRM. Altogether, 160 ccRCC (40 per stage) were used to generate a tissue microarray (TMA), with four foci from each tumor included. Loss of expression was defined as 0–5% of tumor cells with positive nuclear staining in an individual focus. We found that 49/160 (31%), 81/160 (51%), 23/160 (14%), 24/160 (15%), and 61/160 (38%) of ccRCC showed loss of expression of PBRM1, ARID1A, SETD2, BRG1, and BRM, respectively, and that IHC could successfully detect a high prevalence of ITH. Phylogenetic trees were constructed that reflected the ITH. Striking co-losses among proteins were also observed. For instance, ARID1A loss almost always accompanied PBRM1 loss, whereas BRM loss accompanied loss of BRG1, PBRM1 or ARID1A. SETD2 loss frequently occurred with loss of one or more of the other four proteins. Finally, in order to learn the impact of combined losses, we compared the tumor growth after cells acquired losses of ARID1A, PBRM1, or both in a xenograft model. The results suggest that ARID1A loss has a greater tumor-promoting effect than PBRM1 loss, indicating that xenograft analysis is a useful tool to investigate how these losses impact on tumor behavior, either alone or in combination.
The association between BDNF gene functional Val66Met polymorphism rs6265 and the schizophrenia is far from being consistent. In addition to the heterogeneous in schizophrenia per se leading to the inconsistent results, the interaction among multi-genes is probably playing the main role in the pathogenesis of schizophrenia, but not a single gene. Neurotrophic tyrosine kinase receptor 2 (NTRK2) is the high-affinity receptor of BDNF, and was reported to be associated with mood disorders, though no literature reported the association with schizophrenia. Thus, in the present study, total 402 patients with paranoid schizophrenia (the most common subtype of schizophrenia) and matched 406 healthy controls were recruited to investigate the role of rs6265 in BDNF, three polymorphisms in NTRK2 gene (rs1387923, rs2769605 and rs1565445) and their interaction in the susceptibility to paranoid schizophrenia in a Chinese Han population. We did not observe significant differences in allele and genotype frequencies between patients and healthy controls for all four polymorphisms separately. The haplotype analysis also showed no association between haplotype of NTRK2 genes (rs1387923, rs2769605, and rs1565445) and paranoid schizophrenia. However, we found the association between the interaction of BDNF and NTRK2 with paranoid schizophrenia by using the MDR method followed by conventional statistical analysis. The best gene-gene interaction model was a three-locus model (BDNF rs6265, NTRK2 rs1387923 and NTRK2 rs2769605), in which one low-risk and three high-risk four-locus genotype combinations were identified. Our findings implied that single polymorphism of rs6265 rs1387923, rs2769605, and rs1565445 in BDNF and NTRK2 were not associated with the development of paranoid schizophrenia in a Han population, however, the interaction of BDNF and NTRK2 genes polymorphisms (BDNF-rs6265, NTRK2-rs1387923 and NTRK2-rs2769605) may be involved in the susceptibility to paranoid schizophrenia.
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