Subunits For Degradation Research Articles

Abstract 677: Bi-directional crosstalk between the HIF and AHR transcription factors in clear cell kidney cancer

Abstract Hypoxia is a key feature of the solid tumour microenvironment, in which it is associated with poor prognosis and resistance to therapy. Hypoxia-inducible factor (HIF) is the central mediator of cellular responses to hypoxia and drives a transcriptional program with central roles in tumorigenesis. Clear cell renal cell cancers (ccRCC) exhibit the most direct and profound upregulation of the HIF pathway - through bi-allelic inactivation of the VHL tumour suppressor, a ubiquitin ligase that targets HIF-alpha subunits for degradation under normal oxygenated conditions. HIF-alpha proteins are members of the bHLH-PAS family and interact with HIF-1beta (also known as ARNT) to form functional heterodimers binding chromatin. However, the aryl hydrocarbon receptor (AHR), another bHLH-PAS protein that dimerises with HIF-1beta, is also up-regulated in ccRCC, raising the possibility of crosstalk between these two transcriptional pathways. Previous studies support an antagonistic relationship between HIF and AHR, with evidence of a context-dependent and gene-specific crosstalk. Here, we investigate the pan-genomic and precise relationship between the HIF and AHR transcription factors in ccRCC. Using immunoprecipitation, we show direct competition for HIF-1beta binding between the HIF-alpha and AHR proteins in renal cancer cell lines. Pan-genomic analysis of HIF-1alpha, HIF-2alpha, HIF-1beta and AHR chromatin binding by ChIP-seq analysis reveals a widespread competitive interaction between HIF and AHR. In RCC4 cells, this interaction is bidirectional, with induction of HIF leading to a reduction in AHR binding, while induction of AHR reduces binding of both HIF-1alpha and HIF-2alpha to chromatin. However, this competition is diminished at sites bound by both HIF and AHR, indicating a cooperative interaction between the two transcription factors in cis. Thus, while the HIF-alpha and AHR proteins compete for the common binding partner HIF-1beta, they appear to form a cooperative interaction when bound to neighbouring sites on chromatin. Linking these findings to changes in gene expression by RNA-seq analysis, we show the global downstream effects of the competition between HIF and AHR, but also reveal a degree of overlap and cooperativity between their transcriptional programs. Furthermore, we provide evidence of this antagonistic relationship in ccRCC tumours (TCGA database), in which we observe a negative correlation between HIF and AHR target gene expression. These findings shed light on the complex crosstalk between the HIF and AHR transcriptional pathways, which encompasses both a widespread inhibitory interaction and specific cooperative events and highlight a role for AHR in modifying the HIF transcriptional output in ccRCC. Citation Format: Véronique N. Lafleur, Silvia Halim, Peter J. Ratcliffe, David R. Mole. Bi-directional crosstalk between the HIF and AHR transcription factors in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 677.

Genetic deletion of the long isoform of the von Hippel–Lindau tumour suppressor gene product alters microtubule dynamics

The von Hippel-Lindau tumour suppressor protein (pVHL) controls distinct cellular responses ranging from targeting hypoxia inducible factor α (HIFα) subunits for degradation and promotion of chromosomal stability to the regulation of microtubule dynamics. pVHL is produced in mammalian cells as a long and a short isoform, derived from alternate translational initiation sites in a single Vhl mRNA. However, it is unclear whether these isoforms have different cell biological activities that may represent different tumour suppressor activities of pVHL. Through a knock-in strategy to mutate the first translational initiation site from methionine to leucine (M1L) we have genetically deleted the pVHL long protein isoform in mice, allowing dissection of isoform-specific functions of pVHL. Vhl(M1L/M1L) mice exhibit no obvious phenotypic abnormalities. While numerous pVHL-mediated activities, including degradation of HIFα transcription factors, are unaffected, microtubule dynamics are altered in primary cells derived from Vhl(M1L/M1L) mice to an extent similar to that seen following complete loss of pVHL function. We conclude that the microtubule-regulating function and the HIFα-regulating function of pVHL are separable activities mediated by different protein isoforms.

PRAJA1 is a ubiquitin ligase for the polycomb repressive complex 2 proteins

Methylation of lysine 27 on histone H3 by the polycomb repressive complex 2 (PRC2) leads to transcriptional repression of genes which are critical to development. PRC2 core complex is composed of the histone methyltransferase EZH2, EED, and SUZ12. Knockdown of any of the PRC2 core subunits results in a concomitant loss of the other subunits which is mediated by the ubiquitin (Ub)–proteasome system (UPS). Inhibition of cellular methyltransferases by 3-deazaneplanocin A (DZNep) also leads to dissociation of the PRC2 complex and rapid degradation of its subunits. Interestingly, the expression of several Ub ligases was induced following DZNep treatment, suggesting that PRC2 might repress the Ub ligase(s) that target its subunits for degradation. Here we confirm that individual PRC2 subunits are ubiquitinated and rapidly degraded by the proteasome. One of the DZNep-induced Ub ligases, PRAJA1, can target PRC2 subunits for proteasomal degradation. PRAJA1 directly ubiquitinates individual PRC2 subunits in a cell free system, which leads to their proteasomal degradation. Expression of PRAJA1 but not of an inactive RING finger mutant of the protein, enhanced the degradation of individual PRC2 subunits in cells. Taken together, our results suggest a role for PRAJA1 in regulating the level of PRC2 by targeting its free subunits for Ub-mediated proteasomal degradation.

Downregulation of integrins by von Hippel-Lindau (VHL) tumor suppressor protein is independent of VHL-directed hypoxia-inducible factor alpha degradation

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in the majority of clear-cell renal cell carcinomas (RCCs). It was previously shown that VHL decreased the abundance of integrins alpha2, alpha5, and beta1, which is consistent with VHL-associated changes in cell-cell and cell - extracellular matrix adhesions. We investigated the mechanism by which VHL downregulates integrins. Although VHL can target hypoxia-inducible factor alpha (HIFalpha) subunits for degradation, VHL-dependent reduction of integrins was independent of O2 concentration and HIFalpha levels. VHL reduced the half-lives of integrins, and this activity was blocked by proteasomal inhibition. Although ectopically expressed FLAG-VHL retained HIFalpha degradation activity, it neither downregulated integrins nor promoted adherens and tight intercellular junctions, in contrast to expressed wild-type VHL. Moreover, integrins co-immunoprecipitated with wild-type VHL, but not FLAG-VHL. These data indicate that the downregulation of integrins by VHL is distinct from the regulation of HIFalpha subunits by VHL, and suggests that the loss of this activity contributes to VHL-associated RCC development through disruption of adherens and tight junctions.