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RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Zhang CS, Liu Q, Li M, Lin SY, Peng Y, Wu D, Li TY, Fu Q, Jia W, Wang X, Ma T, Zong Y, Cui J, Pu C, Lian G, Guo H, Ye Z, Lin SC - Cell Res. (2015)

Bottom Line: Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα.Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα.Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

No MeSH data available.


Related in: MedlinePlus

RHOBTB3 suppresses tumorigenesis. (A) Expression of RHOBTB3 in human renal cancer samples in public data sets summarized by the Oncomine Platform. (B) Xenografts derived from RHOBTB3−/− MEFs are significantly larger compared with those derived from control MEFs. Ras V12/E1A H133-transformed RHOBTB3−/− MEFs and control WT MEFs (1 × 106) were injected intradermally into each flank of nude mice. Tumors were allowed to develop for 35 days. Tumor volumes were then determined by direct measurement using a caliper and calculated by the formula: (widest diameter × smallest diameter2)/2. Data were presented as mean ± SEM, n = 5 for each group, P < 0.0001 (Student's t-test). (C) The protein levels of HIF1α and its targets are upregulated in xenografts derived from RHOBTB3−/− MEFs. Xenografts derived from RHOBTB3−/− and WT MEFs as described in B were homogenized and analyzed by immunoblotting with the indicated antibodies. (D) Xenografts derived from RHOBTB3 knocked down HeLa cells are significantly larger compared with those derived from control cells. Suspensions of HeLa cells expressing siGFP or siRHOBTB3 (1 × 106 each) were injected intradermally into each flank of nude mice. Tumors were allowed to develop for 37 days and their volumes were calculated as described in Figure 7B. The values of tumor volumes are presented as mean ± SEM, n = 12 for each group, P < 0.0001 (Student's t-test). (E) Protein levels of HIF1α and its target genes are upregulated in xenografts of RHOBTB3 knocked down HeLa cells. Xenografts derived from HeLa-siGFP or HeLa-siRHOBTB3 cells were homogenized and analyzed by immunoblotting with antibodies indicated. (F) Simplified model depicting that RHOBTB3 and LIMD1 promote the formation of the HIF1α degradation complex. In this scheme, RHOBTB3 and LIMD1 form a heterodimer, which interacts with PHD2 and VHL, and recruits HIFα, forming a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex that promotes the hydroxylation, ubiquitination and degradation of HIFα. Hypoxia loosens the interaction between RHOBTB3-LIMD1 and HIFα-VHL-PHD2, allowing for the accumulation of HIFα in cells under hypoxia. Notably, RHOBTB3 can directly promote PHD2-mediated hydroxylation of HIFα, whereas LIMD1 cannot.
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fig7: RHOBTB3 suppresses tumorigenesis. (A) Expression of RHOBTB3 in human renal cancer samples in public data sets summarized by the Oncomine Platform. (B) Xenografts derived from RHOBTB3−/− MEFs are significantly larger compared with those derived from control MEFs. Ras V12/E1A H133-transformed RHOBTB3−/− MEFs and control WT MEFs (1 × 106) were injected intradermally into each flank of nude mice. Tumors were allowed to develop for 35 days. Tumor volumes were then determined by direct measurement using a caliper and calculated by the formula: (widest diameter × smallest diameter2)/2. Data were presented as mean ± SEM, n = 5 for each group, P < 0.0001 (Student's t-test). (C) The protein levels of HIF1α and its targets are upregulated in xenografts derived from RHOBTB3−/− MEFs. Xenografts derived from RHOBTB3−/− and WT MEFs as described in B were homogenized and analyzed by immunoblotting with the indicated antibodies. (D) Xenografts derived from RHOBTB3 knocked down HeLa cells are significantly larger compared with those derived from control cells. Suspensions of HeLa cells expressing siGFP or siRHOBTB3 (1 × 106 each) were injected intradermally into each flank of nude mice. Tumors were allowed to develop for 37 days and their volumes were calculated as described in Figure 7B. The values of tumor volumes are presented as mean ± SEM, n = 12 for each group, P < 0.0001 (Student's t-test). (E) Protein levels of HIF1α and its target genes are upregulated in xenografts of RHOBTB3 knocked down HeLa cells. Xenografts derived from HeLa-siGFP or HeLa-siRHOBTB3 cells were homogenized and analyzed by immunoblotting with antibodies indicated. (F) Simplified model depicting that RHOBTB3 and LIMD1 promote the formation of the HIF1α degradation complex. In this scheme, RHOBTB3 and LIMD1 form a heterodimer, which interacts with PHD2 and VHL, and recruits HIFα, forming a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex that promotes the hydroxylation, ubiquitination and degradation of HIFα. Hypoxia loosens the interaction between RHOBTB3-LIMD1 and HIFα-VHL-PHD2, allowing for the accumulation of HIFα in cells under hypoxia. Notably, RHOBTB3 can directly promote PHD2-mediated hydroxylation of HIFα, whereas LIMD1 cannot.

Mentions: Finally, we explored whether regulation of HIFα by RHOBTB3 has any relevance to tumorigenesis. A search in three independent public data sets of kidney cancer: Yusenko Renal (GEO data set GSE11151)74, Beroukhim Renal (GEO data set GSE14994)75 and Gumz Renal (GEO data set GSE6344)76, summarized by The Oncomine Platform (Life Technologies, Ann Arbor, MI, USA) reveal that the mRNA levels of RHOBTB3 are significantly decreased in clear cell renal cell carcinoma, papillary renal cell carcinoma, hereditary clear cell renal cell carcinoma and non-hereditary clear cell renal cell carcinoma subtypes (Figure 7A). None of the data sets deposited in Oncomine show a significant upregulation of RHOBTB3 in kidney cancer (Figure 7A). These results suggest a potential role of RHOBTB3 in suppressing tumorigenesis. To test this hypothesis, we performed xenograft experiment implanting subcutaneously Ras V12/E1A H133-transformed RHOBTB3−/− MEFs and, as a control, WT MEFs, into nude mice. Xenografts derived from RHOBTB3−/− MEFs were significantly larger in both volume and weight when compared with those derived from control MEFs (Figure 7B and Supplementary information, Figure S7A). In addition, these xenografts had elevated levels of HIF1α and its targets including GLUT1, HK2, LDHA, PHD2 and carbonic anhydrase IX (CA9) (Figure 7C and Supplementary information, Figure S7B). Moreover, sections from RHOBTB3 xenografts showed enhanced staining for downstream targets of HIF (Supplementary information, Figure S7C). Furthermore, the xenografts derived from RHOBTB3-deficient (siRHOBTB3) HeLa cells showed increased growth rates, tumor volumes, tumor weights and deregulated HIF signaling (Figure 7D, 7E and Supplementary information, Figure S7D-S7F), whereas HeLa cells overexpressing RHOBTB3 were unable to form xenograft tumors (Supplementary information, Figure S7G). We also detected higher proliferation rates in RHOBTB3−/− MEFs compared with WT MEFs, which could be reduced by the knockdown of HIF1α, suggesting that RHOBTB3 suppresses cell proliferation, at least in part, through HIFs (Supplementary information, Figure S7H). Thus, it is reasonable to conclude that RHOBTB3 suppresses tumorigenesis through downregulating HIFα levels.


RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Zhang CS, Liu Q, Li M, Lin SY, Peng Y, Wu D, Li TY, Fu Q, Jia W, Wang X, Ma T, Zong Y, Cui J, Pu C, Lian G, Guo H, Ye Z, Lin SC - Cell Res. (2015)

RHOBTB3 suppresses tumorigenesis. (A) Expression of RHOBTB3 in human renal cancer samples in public data sets summarized by the Oncomine Platform. (B) Xenografts derived from RHOBTB3−/− MEFs are significantly larger compared with those derived from control MEFs. Ras V12/E1A H133-transformed RHOBTB3−/− MEFs and control WT MEFs (1 × 106) were injected intradermally into each flank of nude mice. Tumors were allowed to develop for 35 days. Tumor volumes were then determined by direct measurement using a caliper and calculated by the formula: (widest diameter × smallest diameter2)/2. Data were presented as mean ± SEM, n = 5 for each group, P < 0.0001 (Student's t-test). (C) The protein levels of HIF1α and its targets are upregulated in xenografts derived from RHOBTB3−/− MEFs. Xenografts derived from RHOBTB3−/− and WT MEFs as described in B were homogenized and analyzed by immunoblotting with the indicated antibodies. (D) Xenografts derived from RHOBTB3 knocked down HeLa cells are significantly larger compared with those derived from control cells. Suspensions of HeLa cells expressing siGFP or siRHOBTB3 (1 × 106 each) were injected intradermally into each flank of nude mice. Tumors were allowed to develop for 37 days and their volumes were calculated as described in Figure 7B. The values of tumor volumes are presented as mean ± SEM, n = 12 for each group, P < 0.0001 (Student's t-test). (E) Protein levels of HIF1α and its target genes are upregulated in xenografts of RHOBTB3 knocked down HeLa cells. Xenografts derived from HeLa-siGFP or HeLa-siRHOBTB3 cells were homogenized and analyzed by immunoblotting with antibodies indicated. (F) Simplified model depicting that RHOBTB3 and LIMD1 promote the formation of the HIF1α degradation complex. In this scheme, RHOBTB3 and LIMD1 form a heterodimer, which interacts with PHD2 and VHL, and recruits HIFα, forming a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex that promotes the hydroxylation, ubiquitination and degradation of HIFα. Hypoxia loosens the interaction between RHOBTB3-LIMD1 and HIFα-VHL-PHD2, allowing for the accumulation of HIFα in cells under hypoxia. Notably, RHOBTB3 can directly promote PHD2-mediated hydroxylation of HIFα, whereas LIMD1 cannot.
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fig7: RHOBTB3 suppresses tumorigenesis. (A) Expression of RHOBTB3 in human renal cancer samples in public data sets summarized by the Oncomine Platform. (B) Xenografts derived from RHOBTB3−/− MEFs are significantly larger compared with those derived from control MEFs. Ras V12/E1A H133-transformed RHOBTB3−/− MEFs and control WT MEFs (1 × 106) were injected intradermally into each flank of nude mice. Tumors were allowed to develop for 35 days. Tumor volumes were then determined by direct measurement using a caliper and calculated by the formula: (widest diameter × smallest diameter2)/2. Data were presented as mean ± SEM, n = 5 for each group, P < 0.0001 (Student's t-test). (C) The protein levels of HIF1α and its targets are upregulated in xenografts derived from RHOBTB3−/− MEFs. Xenografts derived from RHOBTB3−/− and WT MEFs as described in B were homogenized and analyzed by immunoblotting with the indicated antibodies. (D) Xenografts derived from RHOBTB3 knocked down HeLa cells are significantly larger compared with those derived from control cells. Suspensions of HeLa cells expressing siGFP or siRHOBTB3 (1 × 106 each) were injected intradermally into each flank of nude mice. Tumors were allowed to develop for 37 days and their volumes were calculated as described in Figure 7B. The values of tumor volumes are presented as mean ± SEM, n = 12 for each group, P < 0.0001 (Student's t-test). (E) Protein levels of HIF1α and its target genes are upregulated in xenografts of RHOBTB3 knocked down HeLa cells. Xenografts derived from HeLa-siGFP or HeLa-siRHOBTB3 cells were homogenized and analyzed by immunoblotting with antibodies indicated. (F) Simplified model depicting that RHOBTB3 and LIMD1 promote the formation of the HIF1α degradation complex. In this scheme, RHOBTB3 and LIMD1 form a heterodimer, which interacts with PHD2 and VHL, and recruits HIFα, forming a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex that promotes the hydroxylation, ubiquitination and degradation of HIFα. Hypoxia loosens the interaction between RHOBTB3-LIMD1 and HIFα-VHL-PHD2, allowing for the accumulation of HIFα in cells under hypoxia. Notably, RHOBTB3 can directly promote PHD2-mediated hydroxylation of HIFα, whereas LIMD1 cannot.
Mentions: Finally, we explored whether regulation of HIFα by RHOBTB3 has any relevance to tumorigenesis. A search in three independent public data sets of kidney cancer: Yusenko Renal (GEO data set GSE11151)74, Beroukhim Renal (GEO data set GSE14994)75 and Gumz Renal (GEO data set GSE6344)76, summarized by The Oncomine Platform (Life Technologies, Ann Arbor, MI, USA) reveal that the mRNA levels of RHOBTB3 are significantly decreased in clear cell renal cell carcinoma, papillary renal cell carcinoma, hereditary clear cell renal cell carcinoma and non-hereditary clear cell renal cell carcinoma subtypes (Figure 7A). None of the data sets deposited in Oncomine show a significant upregulation of RHOBTB3 in kidney cancer (Figure 7A). These results suggest a potential role of RHOBTB3 in suppressing tumorigenesis. To test this hypothesis, we performed xenograft experiment implanting subcutaneously Ras V12/E1A H133-transformed RHOBTB3−/− MEFs and, as a control, WT MEFs, into nude mice. Xenografts derived from RHOBTB3−/− MEFs were significantly larger in both volume and weight when compared with those derived from control MEFs (Figure 7B and Supplementary information, Figure S7A). In addition, these xenografts had elevated levels of HIF1α and its targets including GLUT1, HK2, LDHA, PHD2 and carbonic anhydrase IX (CA9) (Figure 7C and Supplementary information, Figure S7B). Moreover, sections from RHOBTB3 xenografts showed enhanced staining for downstream targets of HIF (Supplementary information, Figure S7C). Furthermore, the xenografts derived from RHOBTB3-deficient (siRHOBTB3) HeLa cells showed increased growth rates, tumor volumes, tumor weights and deregulated HIF signaling (Figure 7D, 7E and Supplementary information, Figure S7D-S7F), whereas HeLa cells overexpressing RHOBTB3 were unable to form xenograft tumors (Supplementary information, Figure S7G). We also detected higher proliferation rates in RHOBTB3−/− MEFs compared with WT MEFs, which could be reduced by the knockdown of HIF1α, suggesting that RHOBTB3 suppresses cell proliferation, at least in part, through HIFs (Supplementary information, Figure S7H). Thus, it is reasonable to conclude that RHOBTB3 suppresses tumorigenesis through downregulating HIFα levels.

Bottom Line: Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα.Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα.Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

No MeSH data available.


Related in: MedlinePlus