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SPOP targets oncogenic protein ZBTB3 for destruction to suppress endometrial cancer.

ABSTRACT: Dysregulation of the ubiquitin-proteasome pathway is closely associated with cancer initiation and progression. SPOP is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. Several whole genome/exome sequencing studies on endometrial cancers (ECs) revealed that the SPOP gene is frequently mutated. However, how SPOP mutations contribute to EC remains poorly understood. In this study, transcription factor ZBTB3 was identified as a proteolytic substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes two Ser/Thr (S/T)-rich degrons located in ZBTB3 and triggers the degradation of ZBTB3 via the ubiquitin-proteasome pathway. By contrast, EC-associated SPOP mutants are defective in regulating ZBTB3 stability. SPOP inactivation promotes endometrial cell proliferation, migration, and invasion partly through ZBTB3 accumulation. Sonic hedgehog (SHH) was found to be a transcriptional target of ZBTB3. SPOP inactivation leads to ZBTB3-dependent SHH upregulation in EC cells. RUSKI-43, a small molecule inhibitor of SHH, suppresses cell proliferation, migration, and invasion in SPOP-depleted or EC-associated SPOP mutant-overexpressed EC cells. Our data indicate that pharmacological inhibition of SHH represents a possible treatment strategy for SPOP-mutated ECs.

SUBMITTER: Jin X

PROVIDER: S-EPMC6943363 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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SPOP targets oncogenic protein ZBTB3 for destruction to suppress endometrial cancer.

Jin Xiaofeng X Wang Jian J Li Qian Q Zhuang Hui H Yang Jianye J Lin Zihan Z Lin Ting T Lv Zeheng Z Shen Liliang L Yan Chunhong C Zheng Jingfei J Zhu Jie J Gong Zhaohui Z Wang Chenji C Gao Kun K

American journal of cancer research 20191201 12

Dysregulation of the ubiquitin-proteasome pathway is closely associated with cancer initiation and progression. SPOP is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. Several whole genome/exome sequencing studies on endometrial cancers (ECs) revealed that the <i>SPOP</i> gene is frequently mutated. However, how SPOP mutations contribute to EC remains poorly understood. In this study, transcription factor ZBTB3 was identified as a proteolytic substrate for the SPOP-CUL3-RBX1 ...[more]

PMID: 31911863

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Project description:BackgroundDistinct subsets of cancer stem cells (CSCs) drive the initiation and progression of malignant tumors via enhanced self-renewal and development of treatment/apoptosis resistance. Endometrial CSC-selective drugs have not been successfully developed because most endometrial cell lines do not contain a sufficient proportion of stable CSCs. Here, we aimed to identify endometrial CSC-containing cell lines and to search for endometrial CSC-selective drugs.MethodsWe first assessed the presence of CSCs by identifying side populations (SPs) in several endometrial cancer cell lines. We then characterized cell viability, colony-formation, transwell invasion and xenotransplantion capability using the isolated SP cells. We also conducted real-time RT-PCR, immunoblot and immunofluorescence analyses of the cells' expression of CSC-associated markers. Focusing on 14 putative CSC-selective drugs, we characterized their effects on the proliferation and apoptosis of endometrial cancer cell lines, examining cell viability and annexin V staining. We further examined the inhibitory effects of the selected drugs, focusing on proliferation, invasion, expression of CSC-associated markers and tumor formation.ResultsWe focused on HHUA cells, an endometrial cancer cell line derived from a well-differentiated endometrial adenocarcinoma. HHUA cells contained a sufficient proportion of stable CSCs with an SP phenotype (HHUA-SP). HHUA-SP showed greater proliferation, colony-formation, and invasive capabilities compared with the main population of HHUA cells (HHUA-MP). HHUA-SP generated larger tumors with higher expression of proliferation-related markers, Ki67, c-MYC and phosphorylated ERK compared with HHUA-MP when transplanted into immunodeficient mice. Among the 14 candidate drugs, sorafenib, an inhibitor of RAF pathways and multiple kinase receptors, inhibited cell proliferation and invasion in both HHUA-SP and -MP, but more profoundly in HHUA-SP. In vivo treatment with sorafenib for 4 weeks reduced the weights of HHUA-SP-derived tumors and decreased the expression of Ki67, ZEB1, and RAF1.ConclusionsOur results suggest that HHUA is a useful cell line for discovery and identification of endometrial CSC-selective drugs, and that sorafenib may be an effective anti-endometrial cancer drug targeting endometrial CSCs.

Project description:BackgroundAbhydrolase domain containing 5 (ABHD5) functions as a tumor suppressor in colorectal and prostate cancers. The aim of this study was to investigate the roles of ABHD5 in endometrial cancer.Materials and methodsABHD5 expression was detected in clinical samples by immunohistochemical staining. Cell proliferation and invasion were evaluated with the Cell Counting Kit-8 and Transwell assay, respectively. Western blotting was performed to analyze protein expression. Glucose uptake was assessed by 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose. Lactate production was detected by a lactate assay kit.ResultsIn the present study, ABHD5 was overexpressed in endometrial cancer tissues, and its expression was closely correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis. In addition, we observed that the knockdown of ABHD5 inhibited cell proliferation, invasion, glucose uptake and lactate production in HEC-1A cells, which expressed high levels of ABHD5. Conversely, the opposite effects were observed when ABHD5 was ectopically expressed in Ishikawa cells, which had low levels of ABHD5. Furthermore, the changes in glycolysis regulators (enolase 1 [ENO1], glucose transporter 1 [GLUT1] and lactate dehydrogenase A [LDHA]) and epithelial-to-mesenchymal transition-related proteins (E-cadherin and Snail) in HEC-1A cells with ABHD5 knockdown were consistent with the effects of ABHD5 on glycolysis and cell invasion. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was increased, while the phosphorylated AKT (p-AKT) was decreased when ABHD5 was downregulated. Notably, treatment with the allosteric AKT inhibitor MK-2206 completely abolished the effects caused by ABHD5 overexpression in Ishikawa cells. Finally, ABHD5 knockdown potently suppressed tumor growth in vivo.ConclusionOverall, these results suggest that ABHD5 may play an oncogenic role in endometrial cancer via the AKT pathway.

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SPOP targets oncogenic protein ZBTB3 for destruction to suppress endometrial cancer.

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SPOP targets oncogenic protein ZBTB3 for destruction to suppress endometrial cancer.

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