Cullin 3SPOP ubiquitin E3 ligase promotes the poly-ubiquitination and degradation of HDAC6

Abstract

The histone deacetylase 6 (HDAC6) plays critical roles in human tumorigenesis and metastasis. As such, HDAC6-selective inhibitors have entered clinical trials for cancer therapy. However, the upstream regulator(s), especially ubiquitin E3 ligase(s), responsible for controlling the protein stability of HDAC6 remains largely undefined. Here, we report that Cullin 3SPOP earmarks HDAC6 for poly-ubiquitination and degradation. We found that the proteasome inhibitor MG132, or the Cullin-based E3 ligases inhibitor MLN4924, but not the autophagosome-lysosome inhibitor bafilomycin A1, stabilized endogenous HDAC6 protein in multiple cancer cell lines. Furthermore, we demonstrated that Cullin 3-based ubiquitin E3 ligase(s) primarily reduced the stability of HDAC6. Importantly, we identified SPOP, an adaptor protein of Cullin 3 family E3 ligases, specifically interacted with HDAC6, and promoted its poly-ubiquitination and subsequent degradation in cells. Notably, cancer-derived SPOP mutants disrupted their binding with HDAC6 and thereby failed to promote HDAC6 degradation. More importantly, increased cellular proliferation and migration in SPOP-depleted HCT116 colon cancer cells could be partly reversed by additional depletion of HDAC6, suggesting that HDAC6 is a key downstream effector for SPOP tumor suppressor function. Together, our data identify the tumor suppressor SPOP as an upstream negative regulator for HDAC6 stability, and SPOP loss-of-function mutations might lead to elevated levels of the HDAC6 oncoprotein to facilitate tumorigenesis and metastasis in various human cancers.

Keywords: Cullin 3; HDAC6; SPOP; tumorigenesis; ubiquitination.

Figure 1. The ubiquitin proteasome system, but not autophagosome-lysosome, controls the protein stability of HDAC6 in cells

(A-B) Immunoblot (IB) analysis of whole cell lysates (WCLs) derived from HT29 cells treatment with 10 μM, 20 μM MG132 (A) or 1 μM, 2 μM MLN4924 (B) for 12 hours before harvesting. (C-D) IB analysis of WCLs derived from HCT116 cells treatment with 10 μM, 20 μM MG132 (C) or 1 μM, 2 μM MLN4924 (D) for 12 hours before harvesting. (E) IB analysis of WCLs derived from HeLa cells treatment with 10 μM or 20 μM MG132 before harvesting. (F) IB analysis of WCLs derived from HCT116 cells treatment with/without 100 nM Bafilomycin A1 (Baf-A1) at indicated time course before harvesting.

Figure 2. HDAC6 protein stability is negatively regulated by the Cullin 3 family E3 ligase

(A) IB analysis of WCLs and immunoprecipitates (IP) derived from 293T cells transfected with Flag-HDAC6 and Myc-Cullins constructs as indicated and treated with 10 μM MG 132 before harvesting. (B-C) IB analysis of WCLs derived from HT29 cells infected with the indicated lentiviral shCullin 3 (B) or shCullin 1 (C), respectively. (D-E) IB analysis of WCLs derived from HCT116 cells infected with the indicated lentiviral shRNAs against Cullin 3 (D) or Cullin 1 (E), respectively. (F-G) IB analysis of WCLs derived from HT29 cells stably infected with the indicated lentiviral shRNAs and treated with 100 μg/ml cycloheximide (CHX) for indicated times (F). Quantification of the band intensities of (F) using the ImageJ software (G). HDAC6 immunoblot bands were normalized to Vinculin, then normalized to the t = 0 time point.

Figure 3. SPOP, but not other Cullin 3 family adaptor proteins, specifically interacts with and promotes HDAC6 poly-ubiquitination and degradation

(A) IB analysis of WCLs and IP derived from 293T cells transfected with HA-HDAC6 and Flag-tagged Cullin 3 family adaptor constructs as indicated and treated with 10 μM MG132 for 12 hours before harvesting. (B-C) IB analysis of WCL derived from 293 (B) or 293T (C) cells transfected with indicated constructs. (D) IB analysis of WCL derived from HT29 cells transfected with indicated constructs. (E-F) IB analysis of WCL derived from HCT116 (E) or DU145 (F) infected with the indicated lentiviral shRNAs against SPOP and subjected to puromycin selection for 72 hours before harvesting. (G-H) IB analysis of WCL derived from HeLa cells stably infected with the indicated lentiviral shRNAs against SPOP and treated with 100 μg/ml CHX for indicated times (G). Quantification of the band intensities of (G) using the ImageJ software (H). HDAC6 immunoblot bands were normalized to Vinculin, then normalized to the t = 0 time point. (I) IB of WCL and His pull-down from HCT116 cells transfected with the indicated constructs. Cells were treated with 30 μM MG132 for 6 hours and lysed with denature buffer.

Figure 4. Deletion of the degron motif in HDAC6 confers resistance to SPOP-mediated degradation

(A) Sequence comparison of HDAC6 with the putative SPOP binding motif (degron) with other known SPOP substrates, and a schematic illustration of HDAC6. NLS: Nuclear localization signal, NES: Nuclear export signal, CD: Catalytic domain, SET14: Cytoplasmic retention domain, UBP: Ubiquitin binding domain. (B) IB analysis of WCLs derived from 293T cells transfected with indicated constructs. (C) IB analysis of WCL and GST pull-down products derived from 293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 12 hours before harvesting. (D-E) IB analysis of WCL derived from 293T cells transfected with indicated Flag-HDAC6 plasmids and treated for indicated times with 100 μg/ml CHX before harvesting (D). Quantification of the band intensities of (D) using the ImageJ software (E). HDAC6 immunoblot bands were normalized to Vinculin, then normalized to the t = 0 time point. (F) IB of WCL and His pull-down from HCT116 cells transfected with the indicated constructs. Cells were treated with 30 μM MG132 for 6 hours and lysed with denature buffer.

Figure 5. Cancer-associated SPOP mutants fail to interact with and promote the degradation of HDAC6 in cells

(A) A schematic illustration of SPOP with MATH and BTB domain and cancer-associated mutations. (B) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from HeLa cells transfected with indicated plasmids and treated with 10 μM MG132 before harvesting. (C) IB analysis of WCLs and GST pull-down products derived from 293T cells transfected with indicated constructs. (D) IB analysis of WCL derived from melanoma WM2664 cells stably expressed with SPOP WT and SPOP mutants. (E) IB analysis of WCL derived from 293T cells transfected with indicated constructs. (F) IB of WCL and His pull-down from HCT116 cells transfected with the indicated constructs. Cells were treated with 30 μM MG132 for 6 hours and lysed with denature buffer.

Figure 6. Depletion of SPOP enhances the cellular proliferation and migration, which can be reversed partly by additional depletion of HDAC6 in colon cancer cells

(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HCT116 infected with the indicated lentiviral shRNAs against SPOP and HDAC6, and subjected to puromycin selection for 72 hours before harvesting. (B) Colony formation assay using HCT116 cells described in Figure 6A. (C) Quantification of colony number of colony formation assays described in Figure 6B. Data were shown as mean ± SD from three independent experiments. *p < 0.05. (D) In vitro scratch assay at 0 and 48 hours using HCT116 cells described in Figure 6A. (E) Quantification of gap closure of in vitro scratch assay described in Figure 6D. Data were shown as mean ± SD from three independent experiments. *p < 0.05. (F) Representative images of migrated HCT116 cells described in Figure 6A in migration assays. (G) Quantification of migrated cells described in Figure 6F. Data were shown as mean ± SD of three independent experiments. *p < 0.05.