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An ID2-dependent mechanism for VHL inactivation in cancer

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ABSTRACT

Mechanisms that maintain cancer stem cells are crucial to tumor progression. The ID2 protein underpins cancer hallmarks including the cancer stem cell state. HIFα transcription factors, most notably HIF2α, are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. We report that DYRK1A and DYRK1B kinases phosphorylate ID2 on Threonine-27 (T27). Hypoxia down regulates this phosphorylation via inactivation of DYRK1, whose activity is stimulated in normoxia by the oxygen sensing prolyl hydroxylase PHD1. ID2 binds to the VHL ubiquitin ligase complex, displaces VHL-associated Cullin-2, and impairs HIF2α ubiquitylation and degradation. Phosphorylation of ID2-T27 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma ID2 positively modulates HIF2α activity. Conversely, elevated expression of DYRK1 phosphorylates ID2- T27, leading to HIF2α destabilization, loss of glioma stemness, inhibition of tumor growth, and a more favorable outcome for patients with glioblastoma.

No MeSH data available.


Model for the regulation of HIFα stability by the DYRK1 kinase-ID2 pathwayIn cellular contexts that favor HIFα protein instability (normal oxygen levels, but also low ID2 expression and high DYRK1 expression) prolyl hydroxylases (PHD1) is active and positively regulates DYRK1 kinases. Active, tyrosine phosphorylated DYRK1 kinases keep ID2 under functional constraint by phosphorylation of Thr-27. The VCB-Cul2 ubiquitin ligase complex efficiently ubiquitylates HIFα (left). With decreasing oxygenation and PHD1 inactivation but also in the presence of downregulation of DYRK1, elevated expression of ID2, or ID2-T27A mutation, the un-phosphorylated/un-phosphorylatable pool of ID2 exerts an inhibitory function towards the VCB-Cul2 complex by binding directly VHL and Elongin C proteins and displacing Cul2. This results in HIFα accumulation (right). The transcriptional activation of the ID2 gene, a HIFα target, by HIF2α generates a feed-forward ID2-HIF2α loop that amplifies the effects.
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Figure 10: Model for the regulation of HIFα stability by the DYRK1 kinase-ID2 pathwayIn cellular contexts that favor HIFα protein instability (normal oxygen levels, but also low ID2 expression and high DYRK1 expression) prolyl hydroxylases (PHD1) is active and positively regulates DYRK1 kinases. Active, tyrosine phosphorylated DYRK1 kinases keep ID2 under functional constraint by phosphorylation of Thr-27. The VCB-Cul2 ubiquitin ligase complex efficiently ubiquitylates HIFα (left). With decreasing oxygenation and PHD1 inactivation but also in the presence of downregulation of DYRK1, elevated expression of ID2, or ID2-T27A mutation, the un-phosphorylated/un-phosphorylatable pool of ID2 exerts an inhibitory function towards the VCB-Cul2 complex by binding directly VHL and Elongin C proteins and displacing Cul2. This results in HIFα accumulation (right). The transcriptional activation of the ID2 gene, a HIFα target, by HIF2α generates a feed-forward ID2-HIF2α loop that amplifies the effects.

Mentions: Here we report a novel mechanism of functional inactivation of the VHL ubiquitin ligase that is independent of genetic mutations of the VHL gene. This mechanism is centered on the ability of active ID2 to disrupt the VCB-Cul2 complex, leading to HIF2α stabilization. We also unravel a hypoxia-directed cascade of events that by overriding the restraining effect of DYRK1-mediated phosphorylation of ID2 on T27, culminates with ID2 activation and HIF2α stabilization. The transcriptional activation of the ID2 gene, a HIFα target27, by HIF2α generates a feed-forward ID2-HIF2α loop that further supports cancer stem cells and glioma aggressiveness (Extended Data Fig. 10). By showing that PHD1-mediated prolyl hydoxylation enhances the enzymatic activity of DYRK1 kinases towards ID2, our findings provide a clue to the mechanism of DYRK1 inhibition in hypoxia. Thus, inhibition of DYRK1 kinases is an oxygen-sensing signal that disables VBC-Cul2. The gene coding for DYRK1A is gained in Down's syndrome, a disease characterized by impaired neural proliferation during development28-30, reduced self-renewal and premature withdrawal from cell cycle31. These findings are consistent with the notion that activation of DYRK1A and DYRK1B inhibits proliferation and activates a cellular quiescence program32-37. It is plausible that DYRK1-activating signals are negative regulators of self-renewal of both normal and cancer stem cells. By phosphorylating ID2 on T27 DYRK1 weakens the core of the stemness machinery centered on the ID2-HIF2α pathway. We suggest that together with the control of ID2 activity, regulation of Sonic Hedgehog/Gli signaling and CREB-mediated transcription by DYRK kinases cooperate to restrain stem cell functions and tumorigenesis in the nervous system38-40. The glioma inhibitory effect of DYRK1 in the mouse and the predictive value of DYRK1 expression on GBM patient survival support the tumor suppressor function of DYRK1 kinases in vivo. Whereas there is consistency for the negative role of DYRK1 towards stemness and cell proliferation31,35, the broad spectrum of DYRK kinase substrates may account for the pro-tumorigenic functions of DYRK1 reported by other studies41. It has been proposed that loss of DYRK1 and accumulation of ID2 and HIF2α drive tumor progression of other cancer types, beside GBM. The mechanism reported here might operate broadly during the transition of cancer cells towards the more aggressive stem-like state that drives maintenance and progression of solid tumors.


An ID2-dependent mechanism for VHL inactivation in cancer
Model for the regulation of HIFα stability by the DYRK1 kinase-ID2 pathwayIn cellular contexts that favor HIFα protein instability (normal oxygen levels, but also low ID2 expression and high DYRK1 expression) prolyl hydroxylases (PHD1) is active and positively regulates DYRK1 kinases. Active, tyrosine phosphorylated DYRK1 kinases keep ID2 under functional constraint by phosphorylation of Thr-27. The VCB-Cul2 ubiquitin ligase complex efficiently ubiquitylates HIFα (left). With decreasing oxygenation and PHD1 inactivation but also in the presence of downregulation of DYRK1, elevated expression of ID2, or ID2-T27A mutation, the un-phosphorylated/un-phosphorylatable pool of ID2 exerts an inhibitory function towards the VCB-Cul2 complex by binding directly VHL and Elongin C proteins and displacing Cul2. This results in HIFα accumulation (right). The transcriptional activation of the ID2 gene, a HIFα target, by HIF2α generates a feed-forward ID2-HIF2α loop that amplifies the effects.
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Figure 10: Model for the regulation of HIFα stability by the DYRK1 kinase-ID2 pathwayIn cellular contexts that favor HIFα protein instability (normal oxygen levels, but also low ID2 expression and high DYRK1 expression) prolyl hydroxylases (PHD1) is active and positively regulates DYRK1 kinases. Active, tyrosine phosphorylated DYRK1 kinases keep ID2 under functional constraint by phosphorylation of Thr-27. The VCB-Cul2 ubiquitin ligase complex efficiently ubiquitylates HIFα (left). With decreasing oxygenation and PHD1 inactivation but also in the presence of downregulation of DYRK1, elevated expression of ID2, or ID2-T27A mutation, the un-phosphorylated/un-phosphorylatable pool of ID2 exerts an inhibitory function towards the VCB-Cul2 complex by binding directly VHL and Elongin C proteins and displacing Cul2. This results in HIFα accumulation (right). The transcriptional activation of the ID2 gene, a HIFα target, by HIF2α generates a feed-forward ID2-HIF2α loop that amplifies the effects.
Mentions: Here we report a novel mechanism of functional inactivation of the VHL ubiquitin ligase that is independent of genetic mutations of the VHL gene. This mechanism is centered on the ability of active ID2 to disrupt the VCB-Cul2 complex, leading to HIF2α stabilization. We also unravel a hypoxia-directed cascade of events that by overriding the restraining effect of DYRK1-mediated phosphorylation of ID2 on T27, culminates with ID2 activation and HIF2α stabilization. The transcriptional activation of the ID2 gene, a HIFα target27, by HIF2α generates a feed-forward ID2-HIF2α loop that further supports cancer stem cells and glioma aggressiveness (Extended Data Fig. 10). By showing that PHD1-mediated prolyl hydoxylation enhances the enzymatic activity of DYRK1 kinases towards ID2, our findings provide a clue to the mechanism of DYRK1 inhibition in hypoxia. Thus, inhibition of DYRK1 kinases is an oxygen-sensing signal that disables VBC-Cul2. The gene coding for DYRK1A is gained in Down's syndrome, a disease characterized by impaired neural proliferation during development28-30, reduced self-renewal and premature withdrawal from cell cycle31. These findings are consistent with the notion that activation of DYRK1A and DYRK1B inhibits proliferation and activates a cellular quiescence program32-37. It is plausible that DYRK1-activating signals are negative regulators of self-renewal of both normal and cancer stem cells. By phosphorylating ID2 on T27 DYRK1 weakens the core of the stemness machinery centered on the ID2-HIF2α pathway. We suggest that together with the control of ID2 activity, regulation of Sonic Hedgehog/Gli signaling and CREB-mediated transcription by DYRK kinases cooperate to restrain stem cell functions and tumorigenesis in the nervous system38-40. The glioma inhibitory effect of DYRK1 in the mouse and the predictive value of DYRK1 expression on GBM patient survival support the tumor suppressor function of DYRK1 kinases in vivo. Whereas there is consistency for the negative role of DYRK1 towards stemness and cell proliferation31,35, the broad spectrum of DYRK kinase substrates may account for the pro-tumorigenic functions of DYRK1 reported by other studies41. It has been proposed that loss of DYRK1 and accumulation of ID2 and HIF2α drive tumor progression of other cancer types, beside GBM. The mechanism reported here might operate broadly during the transition of cancer cells towards the more aggressive stem-like state that drives maintenance and progression of solid tumors.

View Article: PubMed Central - PubMed

ABSTRACT

Mechanisms that maintain cancer stem cells are crucial to tumor progression. The ID2 protein underpins cancer hallmarks including the cancer stem cell state. HIFα transcription factors, most notably HIF2α, are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. We report that DYRK1A and DYRK1B kinases phosphorylate ID2 on Threonine-27 (T27). Hypoxia down regulates this phosphorylation via inactivation of DYRK1, whose activity is stimulated in normoxia by the oxygen sensing prolyl hydroxylase PHD1. ID2 binds to the VHL ubiquitin ligase complex, displaces VHL-associated Cullin-2, and impairs HIF2α ubiquitylation and degradation. Phosphorylation of ID2-T27 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma ID2 positively modulates HIF2α activity. Conversely, elevated expression of DYRK1 phosphorylates ID2- T27, leading to HIF2α destabilization, loss of glioma stemness, inhibition of tumor growth, and a more favorable outcome for patients with glioblastoma.

No MeSH data available.