SPOP Expression Research Articles

SPOP Regulates Endometrial Stromal Cell Decidualization in Mice.

Ubiquitination is a regulatory mechanism that occurs after protein translation. To date, few studies have reported on ubiquitination during embryo implantation. We used real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blotting analyses to analyze the expression of speckle-type pox virus and zinc finger (POZ) protein (SPOP; an adapter of E3 ligases of ubiquitination) in mouse uteri during early pregnancy and pseudopregnancy using an artificially induced decidualization model and a steroid hormone-processing model. At the same time, we established an artificially induced decidualization in vitro model. We observed that SPOP regulates endometrial stromal cell decidualization in mice and that hormones regulate the expression of SPOP. This study suggests that ubiquitination may be involved in embryonic implantation.

SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing.

Trimethylation of histone H3K36 is a chromatin mark associated with active gene expression, which has been implicated in coupling transcription with mRNA splicing and DNA damage response. SETD2 is a major H3K36 trimethyltransferase, which has been implicated as a tumor suppressor in mammals. Here, we report the regulation of SETD2 protein stability by the proteasome system, and the identification of SPOP, a key subunit of the CUL3 ubiquitin E3 ligase complex, as a SETD2-interacting protein. We demonstrate that SPOP is critically involved in SETD2 stability control and that the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro. ChIP-Seq analysis and biochemical experiments demonstrate that modulation of SPOP expression confers differential H3K36me3 on SETD2 target genes, and induce H3K36me3-coupled alternative splicing events. Together, these findings establish a functional connection between oncogenic SPOP and tumor suppressive SETD2 in the dynamic regulation of gene expression on chromatin.

SPOP suppresses tumorigenesis by regulating Hedgehog/Gli2 signaling pathway in gastric cancer.

BackgroundRecent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including gastric cancer. Speckle-type POZ protein, SPOP, is an E3 ubiquitin ligase adaptor, and it is found to inhibit oncogenic signaling. However, the molecular mechanisms are largely unknown.MethodsIn this study, we characterized the expression of SPOP in 88 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical staining and Western blotting. The relationship between SPOP expression and clinical pathologic factors was analyzed. Transfected gastric cancer cell lines were used in cell viability, wound healing and colony formation assays. The interaction of SPOP with Gli2 and other related apoptotic proteins was assessed by immunoprecipitation, Western blotting, real-time PCR and dual luciferase reporter assays. Intracellular interaction of SPOP and Gli2 was visualized by immunofluorescent staining in gastric cancer cells.ResultsImmunohistochemical staining of SPOP can be detected in gastric cancer tissues but much less than adjacent gastric tissues (P < 0.01). High SPOP expression is negatively correlated with lymph node metastasis, poor histological differentiation, and tumor malignancy according to TNM staging. In vitro experiments revealed that over-expression of SPOP prevented tumor cells from proliferation, migration and colony formation in gastric cancer cell lines. Likewise, repression of SPOP promoted cell viability, migration, proliferation, and attenuated apoptosis. Mechanistic studies revealed that increasing SPOP accelerated Gli2 degradation but regardless of Gli2 synthesis. Furthermore, cytoplasmic Gli2 decreased markedly along with the abundant expression of SPOP in MKN45 cells.ConclusionsOur findings indicate that SPOP plays critical roles in suppressing gastric tumorigenesis through inhibiting Hh/Gli2 signaling pathway. It may provide an alternative strategy for developing therapeutic agents of gastric cancer in future.

Clinico-pathological significance of the molecular alterations of the SPOP gene in prostate cancer

AimsSpeckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor recently described to be mutated in prostate cancer (PCa). Hence, studying the gene expression profile and the presence of SPOP mutations in PCa and understanding its clinico-pathological significance as prognostic and therapeutic biomarker are important to further understand its role in PCa development. Patients and methodsA cohort of 265 paraffin-embedded PCa samples from patients with more than 5years of follow-up and treated with radical prostatectomy were collected at our institution for SPOP evaluation. RT-qPCR analysis was performed for expression studies while mutations were assessed by next generation sequencing. Relationship with prognosis was analysed using log-rank analysis and multivariable Cox regression. ResultsSPOP was found to be strongly down-regulated in PCa (median=0.24; range=0.04–9.98) and its expression was associated with both, biochemical (p=0.003) and clinical progression free survival (p=0.023), the very low SPOP expression levels being associated to the worst prognosis. Multivariate analysis demonstrated that low levels of SPOP independently predicted a worse prognosis for both, biochemical (Hazard ratio (HR)=0.5; confidence interval (CI) 95% [0.4–0.9], p=0.011) and clinical progression (HR=0.6; IC 95% [0.4–1], p=0.046). SPOP mutations were found in 10% of TMPRSS2-ERG (T2E)-negative cases. Log-rank tests showed that mutations were significantly associated with biochemical progression free survival (BPFS) (p=0.009) and also were significant in the multivariable analysis (HR=3.4; IC 95% [1.5–7.6], p=0.004). ConclusionsThe present study demonstrates that prognosis varies depending on SPOP expression and mutational status, hence, defining a new biotype of PCa associated with a worse prognosis.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Differential expression of speckled POZ protein, SPOP: putative regulation by miR-145.

The speckle POZ protein, SPOP, is an adaptor of the Cul3-based ubiquitination process, and has been implicated in the carcinogenesis process. Despite recent elucidation of biological functions, regulation of SPOP gene expression has not been reported. In this study, the mRNA levels of the mouse SPOP (mSPOP) gene were first shown to vary noticeably in different tissues. However, the SPOP protein was detected in high abundance only in Purkinje cells of the cerebellum and seminiferous tubule of the testis, echoing previous reports of involvement of ubiquitination in neuron cells and in spermatogenesis. In other mouse tissues and human cancer cell lines analysed, only low SPOP protein levels were detected. The 3'-untranslated regions of both the mSPOP and human SPOP transcripts harbor a conserved putative miR-145 binding site (BS). In some tissues and cell lines, miR-145 and SPOP protein levels were in an inverse relationship suggesting miR-145 regulation. Luciferase assays of deletion and point mutation constructs of the miR-145 BS, and miR-145 induction by serum starvation that resulted in reduced endogenous SPOP levels provided further evidence that miR-145 is likely involved in post-transcriptional regulation of SPOP expression in selected tissues, and possibly with the participation of other miRNA species.

SPOP suppresses tumorigenesis by regulating hedgehog/Gli2 signaling pathway in gastric cancer

Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including gastric cancer. Speckle-type POZ protein, SPOP, is an E3 ubiquitin ligase adaptor, and it is found to inhibit oncogenic signaling. However, the molecular mechanisms are largely unknown. In this study, we characterized the expression of SPOP in 88 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical staining and Western blotting. The relationship between SPOP expression and clinical pathologic factors was analyzed. Transfected gastric cancer cell lines were used in cell viability, wound healing and colony formation assays. The interaction of SPOP with Gli2 and other related apoptotic proteins was assessed by immunoprecipitation, Western blotting, real-time PCR and dual luciferase reporter assays. Intracellular interaction of SPOP and Gli2 was visualized by immunofluorescent staining in gastric cancer cells. Immunohistochemical staining of SPOP can be detected in gastric cancer tissues but much less than adjacent gastric tissues (P < 0.01). High SPOP expression is negatively correlated with lymph node metastasis, poor histological differentiation, and tumor malignancy according to TNM staging. In vitro experiments revealed that over-expression of SPOP prevented tumor cells from proliferation, migration and colony formation in gastric cancer cell lines. Likewise, repression of SPOP promoted cell viability, migration, proliferation, and attenuated apoptosis. Mechanistic studies revealed that increasing SPOP accelerated Gli2 degradation but regardless of Gli2 synthesis. Furthermore, cytoplasmic Gli2 decreased markedly along with the abundant expression of SPOP in MKN45 cells. Our findings indicate that SPOP plays critical roles in suppressing gastric tumorigenesis through inhibiting Hh/Gli2 signaling pathway. It may provide an alternative strategy for developing therapeutic agents of gastric cancer in future.

Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors

Gli2 and Gli3 are primary transcriptional regulators that mediate hedgehog (Hh) signaling. Mechanisms that stabilize and destabilize Gli2 and Gli3 are essential for the proteins to promptly respond to Hh signaling or to be inactivated following the activation. In this study, we show that loss of suppressor of fused (Sufu; an inhibitory effector for Gli proteins) results in destabilization of Gli2 and Gli3 full-length activators but not of their C-terminally processed repressors, whereas overexpression of Sufu stabilizes them. By contrast, RNAi knockdown of Spop (a substrate-binding adaptor for the cullin3-based ubiquitin E3 ligase) in Sufu mutant mouse embryonic fibroblasts (MEFs) can restore the levels of Gli2 and Gli3 full-length proteins, but not those of their repressors, whereas introducing Sufu into the MEFs stabilizes Gli2 and Gli3 full-length proteins and rescues Gli3 processing. Consistent with these findings, forced Spop expression promotes Gli2 and Gli3 degradation and Gli3 processing. The functions of Sufu and Spop oppose each other through their competitive binding to the N- and C-terminal regions of Gli3 or the C-terminal region of Gli2. More importantly, the Gli3 repressor expressed by a Gli3 mutant allele (Gli3(Delta699)) can mostly rescue the ventralized neural tube phenotypes of Sufu mutant embryos, indicating that the Gli3 repressor can function independently of Sufu. Our study provides a new insight into the regulation of Gli2 and Gli3 stability and processing by Sufu and Spop, and reveals the unexpected Sufu-independent Gli3 repressor function.

Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer.

We constructed a large-scale functional network model in Drosophila melanogaster built around two key transcription factors involved in the process of embryonic segmentation. Analysis of the model allowed the identification of a new role for the ubiquitin E3 ligase complex factor SPOP. In Drosophila, the gene encoding SPOP is a target of segmentation transcription factors. Drosophila SPOP mediates degradation of the Jun kinase phosphatase Puckered, thereby inducing tumor necrosis factor (TNF)/Eiger-dependent apoptosis. In humans, we found that SPOP plays a conserved role in TNF-mediated JNK signaling and was highly expressed in 99% of clear cell renal cell carcinomas (RCCs), the most prevalent form of kidney cancer. SPOP expression distinguished histological subtypes of RCC and facilitated identification of clear cell RCC as the primary tumor for metastatic lesions.