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An ID2-dependent mechanism for VHL inactivation in cancer

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.

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The DYRK1-ID2-T27 pathway controls GSCs and HIF2αa, GSC#48 cells were transduced with lentiviruses expressing ID2-WT, ID2-T27A, or the empty vector. b, Cells were analyzed by in vitro LDA. Representative regression plot used to calculate gliomasphere frequency in panel c. c, The frequency of cells capable of forming gliomaspheres by in vitro LDA. Data in the histograms represent means of 3 biological replicates ± s.d.; **: p = 0.00163. d, The microphotographs show representative gliomasphere cultures of cells treated as in a. e, HIF2α mRNAs from cells treated as in a were analyzed by semi-quantitative RT-PCR. f, U87 cells stably expressing FLAG-ID2 or FLAG-ID2-T27A were analyzed by western blot using the indicated antibodies. Arrows point to specific bands. Asterisk indicates a non-specific band. g, GSC#34 cells were transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Cells were analyzed by western blot using the indicated antibodies. Arrow points to specific band. Asterisk indicates a non specific band. h, qRT-PCR from cells treated as in g. Data in the histograms represent means ± s.d. (n=9, triplicate experiments each performed in triplicate; ***: p = 8.44524E-07 for TGFA). i, GSC#31 was transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Expression of HIF2α, DYRK1B-V5 and α-tubulin was analyzed by western blot. j, mRNAs from experiment shown in Fig. 3a-c were analyzed by semi-quantitative RT-PCR for HIF2α. k, GSC#31 cells were transduced with lentiviruses expressing DYRK1B and ID2, ID2-T27A, or the empty vector. Cells were analyzed by LDA. Representative regression plot used to calculate gliomasphere frequency in Fig. 3c. l, GSC#31 cells were transduced with lentiviruses expressing DYRK1B or the empty vector in the absence or in the presence of undegradable HIF2α (HIF2α-TM). Cells were analyzed by in vitro LDA. Representative regression plot used to calculate the frequency of gliomaspheres in cultures from three independent infections (Vect-Vect = 13.55%; DYRK1B-Vect = 4.36%; DYRK1B-HIF2α-TM = 9.73%).
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Figure 4: The DYRK1-ID2-T27 pathway controls GSCs and HIF2αa, GSC#48 cells were transduced with lentiviruses expressing ID2-WT, ID2-T27A, or the empty vector. b, Cells were analyzed by in vitro LDA. Representative regression plot used to calculate gliomasphere frequency in panel c. c, The frequency of cells capable of forming gliomaspheres by in vitro LDA. Data in the histograms represent means of 3 biological replicates ± s.d.; **: p = 0.00163. d, The microphotographs show representative gliomasphere cultures of cells treated as in a. e, HIF2α mRNAs from cells treated as in a were analyzed by semi-quantitative RT-PCR. f, U87 cells stably expressing FLAG-ID2 or FLAG-ID2-T27A were analyzed by western blot using the indicated antibodies. Arrows point to specific bands. Asterisk indicates a non-specific band. g, GSC#34 cells were transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Cells were analyzed by western blot using the indicated antibodies. Arrow points to specific band. Asterisk indicates a non specific band. h, qRT-PCR from cells treated as in g. Data in the histograms represent means ± s.d. (n=9, triplicate experiments each performed in triplicate; ***: p = 8.44524E-07 for TGFA). i, GSC#31 was transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Expression of HIF2α, DYRK1B-V5 and α-tubulin was analyzed by western blot. j, mRNAs from experiment shown in Fig. 3a-c were analyzed by semi-quantitative RT-PCR for HIF2α. k, GSC#31 cells were transduced with lentiviruses expressing DYRK1B and ID2, ID2-T27A, or the empty vector. Cells were analyzed by LDA. Representative regression plot used to calculate gliomasphere frequency in Fig. 3c. l, GSC#31 cells were transduced with lentiviruses expressing DYRK1B or the empty vector in the absence or in the presence of undegradable HIF2α (HIF2α-TM). Cells were analyzed by in vitro LDA. Representative regression plot used to calculate the frequency of gliomaspheres in cultures from three independent infections (Vect-Vect = 13.55%; DYRK1B-Vect = 4.36%; DYRK1B-HIF2α-TM = 9.73%).

Mentions: We used human GSCs to interrogate the effects of DYRK1 and ID2-T27A on HIF2α and glioma stemness. Lentiviral transduction of the DYRK1-resistant ID2-T27A mutant in GSC#48 resulted in elevation of HIF2α and enhanced tumor sphere forming capacity as measured by limiting dilution assay (LDA, Extended Data Fig. 4a-d). ID2-T27A-induced accumulation of the HIF2α protein was independent of transcription (Extended Data Fig. 4e). When detectable, HIF1α mirrored HIF2α but with more limited changes (Extended Data Fig. 4f).


An ID2-dependent mechanism for VHL inactivation in cancer
The DYRK1-ID2-T27 pathway controls GSCs and HIF2αa, GSC#48 cells were transduced with lentiviruses expressing ID2-WT, ID2-T27A, or the empty vector. b, Cells were analyzed by in vitro LDA. Representative regression plot used to calculate gliomasphere frequency in panel c. c, The frequency of cells capable of forming gliomaspheres by in vitro LDA. Data in the histograms represent means of 3 biological replicates ± s.d.; **: p = 0.00163. d, The microphotographs show representative gliomasphere cultures of cells treated as in a. e, HIF2α mRNAs from cells treated as in a were analyzed by semi-quantitative RT-PCR. f, U87 cells stably expressing FLAG-ID2 or FLAG-ID2-T27A were analyzed by western blot using the indicated antibodies. Arrows point to specific bands. Asterisk indicates a non-specific band. g, GSC#34 cells were transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Cells were analyzed by western blot using the indicated antibodies. Arrow points to specific band. Asterisk indicates a non specific band. h, qRT-PCR from cells treated as in g. Data in the histograms represent means ± s.d. (n=9, triplicate experiments each performed in triplicate; ***: p = 8.44524E-07 for TGFA). i, GSC#31 was transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Expression of HIF2α, DYRK1B-V5 and α-tubulin was analyzed by western blot. j, mRNAs from experiment shown in Fig. 3a-c were analyzed by semi-quantitative RT-PCR for HIF2α. k, GSC#31 cells were transduced with lentiviruses expressing DYRK1B and ID2, ID2-T27A, or the empty vector. Cells were analyzed by LDA. Representative regression plot used to calculate gliomasphere frequency in Fig. 3c. l, GSC#31 cells were transduced with lentiviruses expressing DYRK1B or the empty vector in the absence or in the presence of undegradable HIF2α (HIF2α-TM). Cells were analyzed by in vitro LDA. Representative regression plot used to calculate the frequency of gliomaspheres in cultures from three independent infections (Vect-Vect = 13.55%; DYRK1B-Vect = 4.36%; DYRK1B-HIF2α-TM = 9.73%).
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Figure 4: The DYRK1-ID2-T27 pathway controls GSCs and HIF2αa, GSC#48 cells were transduced with lentiviruses expressing ID2-WT, ID2-T27A, or the empty vector. b, Cells were analyzed by in vitro LDA. Representative regression plot used to calculate gliomasphere frequency in panel c. c, The frequency of cells capable of forming gliomaspheres by in vitro LDA. Data in the histograms represent means of 3 biological replicates ± s.d.; **: p = 0.00163. d, The microphotographs show representative gliomasphere cultures of cells treated as in a. e, HIF2α mRNAs from cells treated as in a were analyzed by semi-quantitative RT-PCR. f, U87 cells stably expressing FLAG-ID2 or FLAG-ID2-T27A were analyzed by western blot using the indicated antibodies. Arrows point to specific bands. Asterisk indicates a non-specific band. g, GSC#34 cells were transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Cells were analyzed by western blot using the indicated antibodies. Arrow points to specific band. Asterisk indicates a non specific band. h, qRT-PCR from cells treated as in g. Data in the histograms represent means ± s.d. (n=9, triplicate experiments each performed in triplicate; ***: p = 8.44524E-07 for TGFA). i, GSC#31 was transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Expression of HIF2α, DYRK1B-V5 and α-tubulin was analyzed by western blot. j, mRNAs from experiment shown in Fig. 3a-c were analyzed by semi-quantitative RT-PCR for HIF2α. k, GSC#31 cells were transduced with lentiviruses expressing DYRK1B and ID2, ID2-T27A, or the empty vector. Cells were analyzed by LDA. Representative regression plot used to calculate gliomasphere frequency in Fig. 3c. l, GSC#31 cells were transduced with lentiviruses expressing DYRK1B or the empty vector in the absence or in the presence of undegradable HIF2α (HIF2α-TM). Cells were analyzed by in vitro LDA. Representative regression plot used to calculate the frequency of gliomaspheres in cultures from three independent infections (Vect-Vect = 13.55%; DYRK1B-Vect = 4.36%; DYRK1B-HIF2α-TM = 9.73%).
Mentions: We used human GSCs to interrogate the effects of DYRK1 and ID2-T27A on HIF2α and glioma stemness. Lentiviral transduction of the DYRK1-resistant ID2-T27A mutant in GSC#48 resulted in elevation of HIF2α and enhanced tumor sphere forming capacity as measured by limiting dilution assay (LDA, Extended Data Fig. 4a-d). ID2-T27A-induced accumulation of the HIF2α protein was independent of transcription (Extended Data Fig. 4e). When detectable, HIF1α mirrored HIF2α but with more limited changes (Extended Data Fig. 4f).

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.


Related in: MedlinePlus