SPOP promotes ubiquitination and degradation of LATS1 to enhance kidney cancer progression

Abstract

Background: Emerging evidence has demonstrated that SPOP functions as an oncoprotein in kidney cancer to promote tumorigenesis by ubiquitination-mediated degradation of multiple regulators of cellular proliferation and apoptosis. However, the detailed molecular mechanism underlying the oncogenic role of SPOP in kidney tumorigenesis remains elusive.

Methods: Multiple approaches such as Co-IP, Transfection, RT-PCR, Western blotting, and animal studies were utilized to explore the role of SPOP in kidney cancer.

Findings: Here we identified LATS1, a critical component of the Hippo tumour suppressor pathway, as a novel ubiquitin substrate of SPOP. We found that LATS1 interacted with Cullin3, and depletion of Cullin 3 upregulated the abundance of LATS1 largely via prolonging LATS1 protein half-life. Mechanistically, SPOP specifically interacted with LATS1, and promoted the poly-ubiquitination and subsequent degradation of LATS1 in a degron-dependent manner. As such, over-expression of SPOP promoted cell proliferation partly through regulating cell cycle distribution in kidney cancer cells. Furthermore, SPOP also promoted kidney cancer cell invasion via degrading LATS1.

Interpretation: Our study provides evidence for a novel mechanism of SPOP in kidney cancer progression in part through promoting degradation of the LATS1 tumour suppressor.

Keywords: Degradation; Growth; Kidney; LATS1; SPOP; Ubiquitination.

Fig. 1

Cullin3^SPOP is the physiological E3 ubiquitin ligase for LATS1. (a) IB analysis of WCLs derived from 293T, 786-O and A498 cells treated with 10μM MG132 for 10 hr. (b) IB analysis of immunoprecipitates (IPs) and WCLs derived from 293T kidney cells transfected with indicated constructs. Cells were treated with MG132 (10 μM) before harvesting. (c) IB analysis of WCLs derived from A498 cells transfected with Cullin3 siRNA. (d) IB analysis of WCLs derived from 786-O cells after the specified duration of 100 μg/ml cycloheximide (CHX) transfected with Cullin3 siRNA. (e) The abundance of LATS1 protein in (d) was quantified and plotted. (f) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated constructs. Cells were treated with MG132 (10μM) before harvesting. (g) IB analysis of WCLs derived from 293T kidney cells transfected with increasing doses of plasmid encoding SPOP. (h) IB analysis of WCLs derived from 786-O cells transfected with increasing doses of plasmid encoding SPOP. (i) IB analysis of WCLs derived from 293T kidney cells or A498 cells transfected with indicated constructs. Where indicated, cells were treated with 10μM MG132 before harvesting. (j) IB analysis of WCLs and products of ubiquitination derived from 293T cells transfected with indicated constructs.

Fig. 2

Cullin3^SPOP E3 ubiquitin ligase negatively regulates the protein stability of LATS1. (a) IB analysis of WCLs derived from A498 or 786-O cells infected with the indicated lentiviral shRNA vectors. (b) IB analysis of WCLs derived from 293T or A498 cells transfected with HA-tagged SPOP plasmid. (c) Quantitative real-time PCR (qRT-PCR) analysis to detect LATS1 and SPOP mRNA levels derived from 786-O or A498 cells after depletion of SPOP. Data are shown as mean ±SD of three independent experiments. *P<0.05, Student's t test. (d) IB analysis of WCLs derived from 293T cells after the specified duration of cycloheximide (CHX) transfected with Myc-tagged SPOP plasmid. (e) The abundance of LATS1 protein in (d) was quantified and plotted. (f) IB analysis of WCLs derived from A498 cells after the specified duration of 100μg/ml cycloheximide (CHX) infected with indicated lentiviral shRNA vectors. (g) The abundance of LATS1 protein in (f) was quantified and plotted.

Fig. 3

SPOP-mediated ubiquitination and degradation of LATS1 depends on the degron motif. (a) Amino acid sequence alignment of LATS1 with the SPOP–binding motif (degron) in known substrates of SPOP. (b) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (c) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 h before harvesting. (d) IB analysis of WCLs derived from 293T cells after the specified duration of 100μg/ml cycloheximide (CHX) transfected with indicated Flag-tagged LATS1 plasmids. (e) The abundance of LATS1 protein in (d) was quantified and plotted. (f) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 hr before harvesting. (g) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 h before harvesting. (h) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (i) IB analysis of WCLs derived from 293T cells after the specified duration of 100μg/ml cycloheximide (CHX) transfected with indicated Flag-tagged LATS1 plasmids. (j) The abundance of LATS1 protein in (i) was quantified and plotted.

Fig. 4

CKΙδ promotes the interaction and degradation of LATS1 by SPOP. (a) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (b) IB analysis of WCLs derived from 293T, A498 and 786-O cells incubated with CKΙ inhibitor IC261 (50μM) or D4476 (20μM) before harvesting. (c) IB analysis of WCLs derived from kidney derived 293T cells transfected with CKΙδ siRNA. (d) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T kidney cells transfected with indicated plasmids and treated with 10μM MG132 for 10 hr before harvesting. (e) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (f) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (g) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 h before harvesting.

Fig. 5

SPOP promote the proliferation and invasion of kidney cancer cells. (a) The CTG assay was used to detect the proliferation capability of A498 kidney cancer cells infected with SPOP lentiviral shRNA. Data are shown as mean ±SD of three independent experiments. *P<0.05. (b) The CTG assay was used to detect the proliferation capability of A498 and 786-O kidney cancer cells transfected with SPOP plasmids. Data are shown as mean ±SD of three independent experiments. *P<0.05. (c) Transwell chambers assay was performed to detect the invasion capability of 786-O kidney cancer cells transfected with the indicated plasmids. (d) The quantification of invasion cells in (c). Data are shown as mean ±SD of three independent experiments. **P<0.01. (e) Transwell chambers assay was performed to detect the invasion capability of 786-O kidney cancer cells infected with the indicated lentiviral shRNA or transfected with the indicated siRNA. (f) The quantification of invasion cells in (e). Data are shown as mean ±SD of three independent experiments. *P<0.05, **P<0.01. (g) Transwell chambers assay was performed to detect the invasion capability of 786-O kidney cancer cells transfected with the indicated plasmids. (h) The quantification of invasion cells in (g). Data are shown as mean ±SD of three independent experiments. *P<0.05, **P<0.01. ns: no significant difference.

Fig. 6

SPOP promotes tumorigenesis. (a) 786-O-pcDNA3.1, 786-O-SPOP polyclonal stable kidney cancer cell lines were injected subcutaneously into the BALB/c-nu/nu mice. 40 days later, mice were anesthetic and taken a picture. (b) The tumours were dissected and taken a picture. (c) The weights of the dissected tumours in (b). (d) In vivo tumour growth was measured over the indicated time period. (e) The body weights of the BALB/c-nu/nu mice are measured over the indicated time period. (f) Immunohistochemistry staining of SPOP and LATS1 in tissue sections of xenografted tumours in BALB/c-nu/nu mice injected with the indicated cell lines. Scale bars, 20 μm. (g) IB analysis of the LATS1 protein levels in the dissected tumours. (h) Statistical analyses of the correlation between SPOP and LATS1 expression in cytoplasm of the kidney cancer tissue microarray. (i) Representative images of SPOP and LATS1 IHC from 89 cases of kidney cancer.