Speckle-type POZ Protein Expression Research Articles

SPOP expression is associated with tumor-infiltrating lymphocytes in pancreatic cancer.

Speckle Type POZ Protein (SPOP), despite its tumor type-dependent role in tumorigenesis, primarily as a tumor suppressor gene is associated with a variety of different cancers. However, its function in pancreatic cancer remains uncertain. SPOP expression and the association between its expression and patient prognosis and immune function were evaluated using The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), The Tumor Immune Estimation Resource 2.0 (TIMER2.0) database, cBioportal, and various bioinformatic databases. Enrichment analysis of SPOP and the association between SPOP expression with clinical stage and grade were analyzed using the R software package. Then immunohistochemistry (IHC) was used to estimate the correlation between SPOP and tumor-infiltrating lymphocytes (TILs) in patients with pancreatic cancer. As part of our study, we assessed that SPOP was anomalously expressed in kinds of cancers, associated with clinical stage and outcomes. Meanwhile, SPOP also played a crucial role in the tumor microenvironment (TME). The expression level of SPOP was significantly correlated to tumor-infiltrating immune cells (TICs) in pancreatic cancer. Our study uncovered the potential corrections in SPOP with TICs, suggesting that SPOP may act as a biomarker for immunotherapy in pancreatic cancer.

Overexpression of SERPINA3 suppresses tumor progression by modulating SPOP/NF‑κB in lung cancer.

The pathogenesis mechanism of lung cancer is very complex, with high incidence and mortality. Serpin family A member 3 (SERPINA3) expression levels were reduced in the sera of patients with lung cancer and may be a candidate diagnostic and prognostic survival biomarker in lung cancer, as previously reported. However, the detailed biological functions of SERPINA3 in the pathogenesis of lung cancer remain unknown. In the present study, it was aimed to explore the effects of SERPINA3 on the occurrence of lung cancer. SERPINA3 expression was assessed using bioinformatics database analysis and experimental detection. Then, the biological effects of SERPINA3 were investigated in a cell culture system and a xenograft model of human lung cancer. The potential regulatory mechanism of SERPINA3 in lung cancer was explored by data‑independent acquisition mass spectrometry (DIA‑MS) detection and further validated by western blotting (WB). The results indicated that SERPINA3 expression levels were significantly downregulated in lung cancer tissues and cell lines. At the cellular level, it was revealed that overexpressed SERPINA3 inhibited cell growth, proliferation, migration and invasion and promoted the apoptosis of lung cancer cells. Moreover, overexpressed SERPINA3 enhanced the sensitivity of lung cancer cells to osimertinib. Invivo, a xenograft model of human lung cancer was established with BALB/c nude mice. After the injection of A549 cells, the tumor growth of the tumor‑bearing mice in the SERPINA3‑overexpressing group increased more slowly, and the tumor volume was smaller than that in the empty‑vector group. Mechanistically, a total of 65 differentially expressed proteins were identified. It was found that the speckle‑type POZ protein (SPOP) was significantly upregulated in SERPINA3‑overexpressing H157 cells using DIA‑MS detection and analysis. WB validation showed that SPOP expression increased, and NF‑kappaB (NF‑κB) p65 was inhibited in cell lines and tumor tissues of mice when SERPINA3 was overexpressed. The present findings suggest that SERPINA3 is involved in the development of lung cancer and has an antineoplastic role in lung cancer.

Clinical significance of SPOP and APC gene alterations in colorectal cancer in Indian population.

Speckle-Type Poz Protein (SPOP) involved in the regulation of proteasome-mediated degradation of several oncoproteins, resulting in cancer initiation and progression. Mutations in Adenomatous Polyposis Coli (APC) gene is reported in most sporadic and hereditary colorectal cancer (CRC). Identifying the cellular changes involved in carcinogenesis when APC is mutated is an important issue that needs attention. The tumor suppressive function of SPOP and APC has long been a major focus in the research field of colorectal cancer. However, the clinical significance of SPOP and APC gene alteration in CRC has not been established to date. Mutational analysis was performed by single-strand conformational polymorphism followed by Sanger sequencing, methylation status by methylation-specific PCR, and protein expression by immunohistochemistry on 142 tumor tissues along with their adjacent non-cancerous specimens. The overall survival (OS) and recurrence free survival (RFS) were estimated by Kaplan-Meier Curve. Mutation rates of APC and SPOP gene were 2.8% and 11.9% while that of promoter hypermethylation were 37% and 47%, respectively. The grade of differentiation and Lymph node metastasis were significantly correlated with APC methylation pattern (p ≤ 0.05). The down regulation of APC was more often seen in colonic cancer compared to rectal cancer (p = 0.07) and more commonly in T3-4 depth of invasion (p = 0.07) and in patients without lymphovascular and perineural invasion (p = 0.007, p = 0.08 respectively). The median overall survival and recurrence free survival (RFS) was 67 & 36months while 3-yr and 5-yr OS and RFS were 61.1% & 56.4% and 49.2% & 44.8%, respectively. APC promoter methylation had a better overall survival (p = 0.035) while loss of SPOP expression had a worse survival (p = 0.09). Our findings reveal high percentage of SPOP gene mutations in CRC. A significant link is found between promoter hyper methylationand protein expression in all mutant cases of APC and SPOP, suggesting that both genes may be associated in the development of colorectal cancer in people of Indian decent. Hypermethylation of APC gene and loss of SPOP expression have shown an association with disease prognosis and could be further studied looking at its potential role in planning adjuvant treatment in CRC patients.

Abstract P2-16-03: The Speckle-type POZ protein (SPOP) inhibits breast cancer malignancy by destabilizing oncogene TWIST1

Abstract Epithelial-mesenchymal transition (EMT) inducing transcription factor TWIST1 plays a vital role in cancer metastasis. How the tumor-suppressive E3 ligase, Speckle-type POZ protein (SPOP), regulates TWIST1 in breast cancer remains unknown. In this study, we report that SPOP physically interacts with, ubiquitinates, and destabilizes TWIST1. SPOP promotes K63-and K48-linked ubiquitination of TWIST1, predominantly at K73, thereby suppressing cancer cell migration and invasion. Silencing SPOP significantly enhances EMT, which accelerates breast cancer cell migration and invasiveness in vitro and lung metastasis in vivo. Clinically, SPOP is negatively correlated with the levels of TWIST1 in highly invasive breast carcinomas. Reduced SPOP expression, along with elevated TWIST1 levels, is associated with poor prognosis in advanced breast cancer patients, particularly those with metastatic triple-negative breast cancer (TNBC). Taken together, we have disclosed a new mechanism linking SPOP to TWIST1 degradation. Thus SPOP may serve as a prognostic marker and a potential therapeutic target for advanced TNBC patients. Citation Format: Chunli Wei, Yun Liu, Xiaoyan Liu, Jingliang Cheng, Jiewen Fu, Xiuli Xiao, Robb E Moses, Xiaotao Li, Junjiang Fu. The Speckle-type POZ protein (SPOP) inhibits breast cancer malignancy by destabilizing oncogene TWIST1 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-03.

The speckle-type POZ protein (SPOP) inhibits breast cancer malignancy by destabilizing TWIST1

Epithelial-mesenchymal transition (EMT) inducing transcription factor TWIST1 plays a vital role in cancer metastasis. How the tumor-suppressive E3 ligase, speckle-type POZ protein (SPOP), regulates TWIST1 in breast cancer remains unknown. In this study, we report that SPOP physically interacts with, ubiquitinates, and destabilizes TWIST1. SPOP promotes K63-and K48-linked ubiquitination of TWIST1, predominantly at K73, thereby suppressing cancer cell migration and invasion. Silencing SPOP significantly enhances EMT, which accelerates breast cancer cell migration and invasiveness in vitro and lung metastasis in vivo. Clinically, SPOP is negatively correlated with the levels of TWIST1 in highly invasive breast carcinomas. Reduced SPOP expression, along with elevated TWIST1 levels, is associated with poor prognosis in advanced breast cancer patients, particularly those with metastatic triple-negative breast cancer (TNBC). Taken together, we have disclosed a new mechanism linking SPOP to TWIST1 degradation. Thus SPOP may serve as a prognostic marker and a potential therapeutic target for advanced TNBC patients.

SPOP Inhibition of Endometrial Carcinoma and Its Clinicopathological Relationship.

Objective Endometrial carcinoma (EC) ranks first in the incidence of female genital malignancies in developed countries. SPOP (speckle-type POZ protein) has changed in EC with a statistically high frequency. This research may play a crucial role in the initiation and progression of EC, ultimately leading to fresh therapeutic targets. Explore the expression of SPOP in EC; observe its effect on the proliferation, invasion, and migration of EC cells after upregulating the expression of SPOP through RNA activation. Methods The expression levels of SPOP protein in 150 EC tissues and 45 normal endometrial tissues were detected by immunohistochemistry and Western blotting. Analyze the relationship between SPOP expression and clinicopathological characteristics. The differences of the proliferation, migration, and invasion abilities between before and after transfection were analyzed using CCK-8 and Transwell assays. Results The results of immunohistochemistry and Western blotting showed the expression level of SPOP in EC tissue significantly reduced or even missed compared with normal endometrial tissue. The results of CCK-8 showed that the growth of EC significantly slowed down after the upregulating of SPOP expression. The results of the Transwell assay showed the migration and invasion abilities of EC cells were weakened after the level of SPOP was upregulated. Conclusions The expression level of SPOP in EC tissues is lower and related to the clinicopathological features compared with normal endometrial tissues. After upregulating the SPOP expression by RNA activation in EC cell lines, the abilities of proliferation, migration, and invasion of cells were significantly inhibited.

SPOP-mediated ubiquitination and degradation of PDK1 suppresses AKT kinase activity and oncogenic functions

Background3-phosphoinositide-dependent protein kinase-1 (PDK1) acts as a master kinase of protein kinase A, G, and C family (AGC) kinase to predominantly govern cell survival, proliferation, and metabolic homeostasis. Although the regulations to PDK1 downstream substrates such as protein kinase B (AKT) and ribosomal protein S6 kinase beta (S6K) have been well established, the upstream regulators of PDK1, especially its degrader, has not been defined yet.MethodA clustered regularly interspaced short palindromic repeats (CRISPR)-based E3 ligase screening approach was employed to identify the E3 ubiquitin ligase for degrading PDK1. Western blotting, immunoprecipitation assays and immunofluorescence (IF) staining were performed to detect the interaction or location of PDK1 with speckle-type POZ protein (SPOP). Immunohistochemistry (IHC) staining was used to study the expression of PDK1 and SPOP in prostate cancer tissues. In vivo and in vitro ubiquitination assays were performed to measure the ubiquitination conjugation of PDK1 by SPOP. In vitro kinase assays and mass spectrometry approach were carried out to identify casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)-mediated PDK1 phosphorylation. The biological effects of PDK1 mutations and correlation with SPOP mutations were performed with colony formation, soft agar assays and in vivo xenograft mouse models.ResultsWe identified that PDK1 underwent SPOP-mediated ubiquitination and subsequent proteasome-dependent degradation. Specifically, SPOP directly bound PDK1 by the consensus degron in a CK1/GSK3β-mediated phosphorylation dependent manner. Pathologically, prostate cancer patients associated mutations of SPOP impaired PDK1 degradation and thus activated the AKT kinase, resulting in tumor malignancies. Meanwhile, mutations that occurred around or within the PDK1 degron, by either blocking SPOP to bind the degron or inhibiting CK1 or GSK3β-mediated PDK1 phosphorylation, could markedly evade SPOP-mediated PDK1 degradation, and played potently oncogenic roles via activating the AKT kinase.ConclusionsOur results not only reveal a physiological regulation of PDK1 by E3 ligase SPOP, but also highlight the oncogenic roles of loss-of-function mutations of SPOP or gain-of-function mutations of PDK1 in tumorigenesis through activating the AKT kinase.

A Natural Compound Screen for SPOP Downregulated Breast Cancer

Since breast cancer is the second leading cause of cancer deaths among American women there is a compelling need to uncover novel and superior treatment regimens for women suffering from this disease. Interestingly, several studies have indicated that up to 70% of breast cancer patients have downregulation of the E3 ubiquitin ligase Speckle Type POZ Protein (SPOP) thus indicating that SPOP is a critical tumor suppressor gene in breast cancer. Our lab has previously shown that SPOP targets GLI3 for ubiquitination to promote SHH signaling in prostate cancer patients, a finding that we hypothesized to be true for breast cancer as well since SHH signaling is often found to be hyper-activated in advanced breast cancer. We further hypothesized that SPOP downregulated breast cancer cells therefore will likely have a differential response to targeted therapy and that SHH targeted drugs could prove to be beneficial for SPOP downregulated breast cancer patients. First of all, to study the effect of downregulated SPOP, a lentivirus carrying an shRNA against SPOP was generated and infected into MCF-7 cells. Proliferation assays were utilized to confirm that downregulated SPOP plays a role in the increased proliferation of breast cancer cells. Next, quantitative PCR was performed to determine the effect of downregulated SPOP on the expression of GLI3-dependent SHH target genes. Finally, a natural compound library was utilized to find a novel therapeutic strategy for SPOP downregulated breast cancer. Our findings confirmed that SPOP knockdown increases proliferation of MCF-7 cells in vitro. Furthermore, quantitative PCR confirmed that GLI3 target genes are upregulated in MCF-7 cells when SPOP expression is low. Finally, our natural compound library screen identified novel therapeutic compounds that specifically target SPOP downregulated MCF-7 cells in a manner that involves disruption of GLI3-dependent SHH signaling. These findings therefore provide critical insight into novel treatment strategies for patients suffering from SPOP downregulated breast cancer.

Hypoxia-induced SPOP attenuates the mobility of trophoblast cells through inhibition of the PI3K/AKT/GSK3β pathway.

Placental hypoxia has been implicated in pregnancy pathologies such as pre-eclampsia and intrauterine growth restriction. However, the underlying mechanism by which the trophoblasts respond to hypoxia remains unclear. Speckle-type POZ protein (SPOP), an E3 ubiquitin ligase adapter, was previously reported to play important roles in various physiological and pathological processes. This study aims to investigate the expression and biological functions of SPOP after exposure to cobalt chloride (CoCl2 )-mimicked hypoxia conditions using human trophoblast-derived choriocarcinoma cell lines and extravillous cytotrophoblast. These data showed that SPOP protein was directly induced by CoCl2 -mimicked hypoxia and regulated by HIF-1α at the posttranscription level. CoCl2 treatment could dramatically influence the localization of SPOP in trophoblasts, especially the accumulation of SPOP into the nucleus. In addition, both CoCl2 -mimicked hypoxia and induction of endogenous SPOP expression by lentivirus transfection attenuated the migration and invasion abilities of trophoblasts. Furthermore, we demonstrated that SPOP was involved in CoCl2 -induced the inhibition of the PI3K/AKT/GSK3β pathway in placental trophoblasts. Taken together, these data indicate that accumulation of HIF-1α augments the expression of SPOP in trophoblasts, which impairs trophoblastic mobility by targeting the PI3K/AKT/GSK3β pathway. This potentially leads to insufficient uterine spiral artery remodeling and suboptimal placental perfusion, and thus the development of pregnancy-related complication.

Alzheimer Gene BIN1 may Simultaneously Influence Dementia Risk and Androgen Deprivation Therapy Dosage in Prostate Cancer.

Androgen deprivation therapy (ADT) is extensively used in prostate cancer. Yet the risk of impaired cognition or Alzheimer disease (AD) in men with prostate cancer receiving ADT is uncertain. Some studies of prostate cancer and ADT suggest that the risk of AD is not increased. But other studies have found an increased risk of AD and cognitive impairment. As the uncertainty about ADT and dementia might relate to the genetics of prostate cancer and AD, the authors used the Cancer Genome Atlas (TCGA) to examine the relationship in men with prostate cancer between genes implicated in AD and genes implicated in prostate cancer. The authors examined the genomics of 492 prostate cancer cases in the Genomic Data Commons (GDC) TCGA Prostate Cancer (PRAD) data set. To access and analyze the data, 2 web-based interfaces were used: (1) the UCSC Xena browser, a web-based visual integration and exploration tool for TCGA data, including clinical and phenotypic annotations; and (2) cBioportal, a web-based interface that enables integrative analysis of complex cancer genomics and clinical profiles. Co-occurrence analysis indicates that alterations in the prostate cancer gene Speckle-type POZ protein (SPOP) significantly co-occur with alterations in the AD gene BIN1 (P<0.001). The presence of somatic mutations (deleterious and missense/in frame) in SPOP deranges BIN1 gene expression. SPOP/BIN1 RNA gene expression in 492 prostate cancer specimens is significantly correlated (P<0.001). Increased expression of SPOP in 492 prostate cancers is associated with reduced survival (P=0.00275). Men receiving pharmacologic therapy had a tumor with a significantly higher Gleason score (P=0.023). Gleason score and BIN1 RNA gene expression, unit log2 (fragments per kilobase of transcript per million mapped reads upper quartile [FPKM-UQ]+1), in 499 prostate cancer specimens were significantly inversely correlated (P<0.001). BIN1 forms part of a network that interacts with the MYC oncogene, activated at the earliest phases of prostate cancer and in its position on chr8q24 linked to disease aggressiveness. Dynamic regulation of the BIN1-Tau interaction is involved in AD. BIN1 loss in AD allows phosphorylated tau to be mis-sorted to synapses, which likely alters the integrity of the postsynapse, alongside reducing the functionally important release of physiological forms of tau. Alzheimer symptoms are usually preceded by a preclinical phase that may be 16 years long. The authors suggest that the ADT dosage reflects the severity of a process that is already underway. The severity is determined by the genetics of the tumor itself, at least in part by BIN1. ADT is not causing new cases of AD. The oncologist treats higher-grade prostate cancer with more ADT, which serves as a surrogate marker for disease severity. Our analysis of TCGA data does not support the idea that ADT causes AD or dementia.

Co-occurrent Alterations of Alzheimer's Genes and Prostate Cancer Genes in Prostate Cancer.

Androgen deprivation therapy (ADT) is extensively employed in treatment of prostate cancer. Some studies have found increased risk of Alzheimer's disease and cognitive impairment in patients treated with ADT. Since the uncertainty about ADT and dementia might relate to the genetics of prostate cancer and Alzheimer's disease, we used the Cancer Genome Atlas (TCGA) to examine the relationship between genes implicated in Alzheimer's disease and genes implicated in prostate cancer in men with prostate cancer. The genomics of 492 prostate cancer cases in the Genomic Data Commons TCGA Prostate Cancer data set were examined. Alterations (mutation, amplification or deletion) in prostate cancer gene speckle-type POZ protein (SPOP) significantly co-occurred with alterations in Alzheimer's disease gene bridging integrator-1 (BIN1). Alterations in prostate cancer gene spectrin alpha 1 (SPTA1) significantly co-occurred with alterations in Alzheimer's disease gene CD2-associated protein (CD2AP) (p<0.001). The presence of somatic mutations (deleterious and missense/in frame) in SPOP disturbs BIN1 gene expression. SPOP and BIN1 RNA expression in 492 prostate cancer specimens was significantly positively correlated (p<0.001). Increased expression of SPOP in 492 cases of prostate cancer was associated with reduced survival (p=0.00275). BIN1 forms part of a network that interacts with the MYC oncogene, which is activated at the earliest phases of prostate cancer and is linked to disease aggressiveness. Men receiving ADT had tumor with a significantly higher Gleason score (p=0.023). Gleason score and BIN1 RNA expression in 499 prostate cancer specimens were significantly correlated (p<0.001). The severity of prostate cancer is determined by the genetics of the tumor itself, possibly at least in part by the interactions of SPOP/BIN1, MYC/BIN1 and SPTA1/CD2AP. Oncologists treats higher grade prostate cancer with more ADT, which serves as a surrogate marker for disease severity. A weakness of our study is that we did not examine Alzheimer's disease or dementia at all in patients with cancer, only co-occurrence of genetic alterations. Nevertheless, our analysis of TCGA data does not support the idea that ADT causes Alzheimer's disease or dementia.

SPOP Regulates The Biological Mechanism Of Ovarian Cancer Cells Through The Hh Signaling Pathway.

BackgroundOvarian cancer is characterized by high metastatic potential and high mortality. More than 80% of primary ovarian malignancies are epithelial ovarian cancers. There is increasing evidence that Speckle-type POZ protein (SPOP) is highly correlated with the development of various types of cancer. However, the effects of SPOP on epithelial ovarian cancer and the associated molecular mechanisms remain unclear.Materials and methodsWe compared SPOP expression between epithelial ovarian cancer tissues and normal ovarian tissues by using immunohistochemical staining. To determine the role of SPOP in epithelial ovarian cancer cells, we overexpressed or knocked down SPOP in the epithelial ovarian cancer cell line OVCAR-3 using lentiviral vectors.ResultsOur results from the present study indicated that SPOP expression was significantly downregulated in human epithelial ovarian cancer and was associated with the FIGO stage and the histopathologic grading of the tumor. The overexpression and knockdown experiments revealed that SPOP inhibited proliferation while promoting apoptosis in ovarian cancer cells. Inhibition of SPOP mis-activated the Hedgehog (Hh) signaling pathway, thereby inhibiting apoptosis in ovarian cancer cells.ConclusionSPOP suppresses proliferation and promotes apoptosis in human ovarian cancer cells by inhibiting the Hh signaling pathway, offering the possibility of new approaches for the treatment of ovarian cancer.

The association of speckle-type POZ protein with lymph node metastasis and prognosis in cancer patients: A meta-analysis.

Speckle-type POZ protein (SPOP) has recently been reported as a prognostic tumor biomarker. However, the predictive value of SPOP remains controversial in human cancers. The current meta-analysis was performed to obtain a comprehensive evaluation of the relationship between SPOP expression and prognosis of cancer patients. Embase, Pubmed, Web of Science, and Chinese Biomedical Literature database were systematically searched up to January 2, 2019. The pooled hazard ratios (HRs) and/or pooled odds ratios (ORs) with 95% confidence intervals (CIs) were used to quantitatively assess the relationship of SPOP expression with prognosis and lymph node metastasis (LNM). A total of 9 studies with 928 patients were included in this meta-analysis. The results showed that low SPOP expression was significantly related to poor overall survival (high/low: HR = 0.55; 95% CI: 0.38-0.79, P = .001), especially for digestive system cancers (high/low: HR = 0.46; 95% CI: 0.27-0.78, P = .003). However, SPOP expression did not affect progression-free survival in cancer patients (high/low: HR = 2.07; 95% CI: 0.16-26.70, P = .578). Additionally, the association between SPOP overexpression and LNM was positive in patients with clear cell renal cell carcinoma (ccRCC) (OR = 5.26; 95% CI: 1.66-16.68, P = .005) but negative in cancer patients without ccRCC (OR = 0.36; 95% CI: 0.21-0.62, P < .001). Decreased SPOP expression could predict poor prognosis of cancer patients, suggesting that SPOP protein may be a useful prognostic biomarker in cancer patients.

P1.04-53 A High PD-L1 Expression in Non-Small Cell Lung Cancer Correlates with Expression of SPOP and CD8 Tumor-Infiltrating Lymphocytes

Immunotherapy that targets PD 1 pathway has emerged as a novel treatment modality for malignant diseases. Clinical trials have reported durable responses and long-term remissions using PD-1/PD-L1. However, despite promising clinical results, checkpoint blockade therapies are only successful in a subset of patients. Thus, it is crucial to more fully understand the mechanisms behind immunotherapies. Recently, it has been reported that PD-L1 expression can be regulated at both transcriptional and post-transcriptional levels, including HIF-1, STAT3, NF-κB, AP-1, cyclin-dependent kinase 4 (CDK4) and speckle-type POZ protein (SPOP). In this study, we focused on the regulation of PD-L1 expression in lung cancer, and we investigated the association between the expression of PD-L1 and biomarkers in regard to clinical outcomes. We investigated CDK4, CDK6 and SPOP expression of lung cancer, and CD4 and CD8 expression of tumor-infiltrating lymphocytes (TILs) by immunohistochemistry of 52 patients with non-small cell lung cancers who had undertaken an operation or chemotherapy from May 2008 to December 2018 and analyzed PD-L1 expression (negative:<1%, low expression:1-49%, high expression: ≥50%). The staining intensity of CDK4, CDK6 and SPOP was scored as 0 (negative), 1 (weak), 2 (medium), and 3 (strong). Extent of staining was scored as 0 (0-10%), 1 (11-25%), 2 (26-50%), 3 (51-75%), and 4 (76-100%) according to the percentages of the positive staining areas in relation to the whole cancer area. For CD4+ and CD8+ cells, a number of stained cells were counted semi-quantitatively in high-powered fields (400x). A number of CD8+ cells in tumor tissue were scored as 0 (0-5 cells/HPF), 1 (6-10 cells/HPF), 2 (11-25 cells/HPF), 3 (26-50 cells/HPF), and 4 (51- cells/HPF). The observed protein expression levels and TILs counts were analyzed for correlation to PD-L1 expression and clinicopathological parameters. PD-L1 expression was observed in 33 (64%) patients of lung cancer, being low in 19 (37%) and high in 14 (27%). The positive rate of expression of SPOP was 42% in tumors with negative expression of DP-L1, 53% low expression and 14% high expression. That of CDK4, CDK6 and CD8 in tumor tissue was 79%, 79%, 57% and 53%, 63%, 43%, and 41%, 41%, 92%, respectively. The positive rate of SPOP of CDK4 positive group was significantly higher than that of CDK4 negative group (50% vs 7%, p=0.005). The positive rate of SPOP of high PD-L1 expression group was significantly lower than negative and low expression groups (14% vs 47%, p=0.03). CD8+ cell counts showed positive relation to PD-L1 expression. In previous report, CDK4/6 inhibitors treatment decreased SPOP protein abundance and elevated PD-L1 protein. Our data supported those findings that PD-L1 expression is regulated by CDK4 and SPOP. Then combination treatment with CDK4/6 inhibitors and PD-1/PD-L1 immune checkpoint blockade may have potential to enhance therapeutic efficacy for cancers.

SPOP inhibits mice pancreatic stellate cell activation by promoting FADD degradation in cerulein-induced chronic pancreatitis

Pancreatic stellate cells (PSCs) have been recognized as key mediators of pancreatic fibrosis, a characteristic feature of chronic pancreatitis (CP). As a cullin-based E3 ubiquitin ligase, speckle-type POZ protein (SPOP) has been identified to participate in tumorigenesis and organ development. However, its biological role in CP remains unknown. Therefore, this study sought to investigate the changed expression of SPOP in CP and to examine the effect on mice PSCs activation of SPOP. We found that SPOP was downregulated in the pancreatic tissues of cerulein-induced CP mice. siRNA-mediated knockdown of SPOP led to significant promotion in primary PSCs activity by activating the nuclear factor-kappaB (NF-κB)/interleukin-6 (IL-6) signaling pathway. In addition, we examined the effects of Fas-associated death domain (FADD), a proven SPOP substrate that activates NF-κB, on the regulation of PSCs activation. We found that FADD was downregulated by SPOP via interaction-mediated degradation, and was upregulated during PSCs activation. The promotion of PSCs activation in knocking down SPOP with siSPOP-1 were counteracted by knocking down FADD. The results suggest that the SPOP-induced inhibition of PSCs activation partially depended on FADD. These results highlight the importance of SPOP in CP and provide a potential target for therapeutic intervention.

MiR-373 promotes proliferation and metastasis of oral squamous cell carcinoma by targeting SPOP.

To explore the biological function of microRNA-373 (miR-373) in regulating the progression of oral squamous cell carcinoma (OSCC) and the related mechanism. 50 patients who were diagnosed as OSCC in the Department of Stomatology of the Stomatological Hospital of Chongqing Medical University were enrolled as the cancer group. 20 healthy oral mucosa specimens were obtained as the control group. The miR-373 level in both OSCC clinical samples and cell lines was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The binding relationship between speckle-type POZ protein (SPOP) and miR-373 was detected through online prediction software and luciferase reporter assay. MiR-373 was upregulated in OSCC samples and cell lines. It could negatively regulate the protein expression of SPOP. However, it did not affect the mRNA expression of SPOP. The up-regulation of miR-373 promoted proliferation, invasion, and migration ability of the OSCC cells. However, the effects of miR-373 were abolished by the over-expression of SPOP in cells. Up-regulated miR-373 promotes proliferation, invasion, and migration of OSCC by targeting SPOP. MiR-373/SPOP axis could be a potential therapeutic target for OSCC.

Understanding the molecular link between SPOP gene expression and breast cancer stem cell

Purpose: Breast cancer stem cells are responsible for tumor initiation, abnormal cellular proliferation and metastasis. The activation of breast cancer stem cells is regulated by many genes and the expression of tumor suppressors has a control over it. Speckle-type POZ protein (SPOP) is similar to tumor suppressor that their intact presence inhibits tumor development and any mutation in this gene is responsible for many cancer types. However the exact mechanism of SPOP in controlling cancer stem cells still has not been revealed. Materials and Methods: NOD SCID mouse was used for initiating breast tumor by injecting MDA-MB-231 cell type. The expression of ALDH1 and SPOP are analyzed using Immunohistochemistry and Western blotting. Results: The mice injected with tumor cells develop a primary tumor at four weeks and it develops advanced stage of breast cancer at nine weeks. Tumor formation and associated cellular complexity are observed through histological evidence. Also, the expression of ALDH1 shows overexpression pattern as tumor progress to the next level. Interestingly, the authors observed the expression of SPOP shows an abundant expression pattern in the initial stage of breast cancer and noticed its downregulated expression as tumor adverse. Conclusion: The results demonstrate that the highest expression of SPOP had a control over cancer stem cells in the initial stages of breast cancer.

Speckle-type POZ protein functions as a tumor suppressor in non-small cell lung cancer due to DNA methylation

BackgroundTumor suppressor epigenetic silencing plays an important role in non-small cell lung cancer (NSCLC) development and progression. Previously, the expression of speckle-type POZ protein (SPOP) has been found to be significantly inhibited in NSCLC. Our research aimed to investigate the molecular mechanisms, clinical significance and epigenetic alteration of SPOP in NSCLC.Materials and methodsBisulfite sequencing PCR and methylation-specific PCR were performed to test gene methylation. Chromatin immunoprecipitation (ChIP) was performed to detect transcription factor C/EBPα combinations and the promoter of the SPOP gene. Furthermore, we evaluated the effects of C/EBPα siRNA on SPOP expression, tumor cell migration and proliferation via MTT and Transwell assays in vitro and tumor growth in vivo. The relationship between the methylation status of the SPOP gene and clinicopathologic characteristics was investigated.ResultsHypermethylation was found in the CpG island of the SPOP gene promoter in NSCLC tissues, and this methylation was found to be correlated with SPOP expression. SPOP promoter methylation was associated with the pathology grade. The transcriptional activities were significantly inhibited by the hypermethylation of specific CpG sites within the SPOP gene promoter, while 5-aza-2′-deoxycytidine significantly increased SPOP gene expression. C/EBPα also played a key role in SPOP regulation. Five C/EBPα binding sites in the CpG island of the SPOP gene promoter were identified by ChIP. Inhibition of C/EBPα significantly reduced SPOP expression. SPOP mediated the C/EBPα-regulated suppression of invasion, migration and proliferation in vitro and tumor growth in vivo.ConclusionsSPOP function and expression in NSCLS were regulated by DNA methylation and C/EBPα transcriptional regulation combination effects, indicating that the SPOP promoter methylation status could be utilized as an epigenetic biomarker and that the C/EBPα-SPOP signaling pathway could be a potential therapeutic target in NSCLC.

Speckle-type POZ protein suppresses hepatocellular carcinoma cell migration and invasion via ubiquitin-dependent proteolysis of SUMO1/sentrin specific peptidase 7

Hepatocellular carcinoma (HCC) is associated with high metastatic potential and high mortality. Accumulating evidence has demonstrated that speckle-type POZ protein (SPOP) is a key adaptor molecule of ubiquitination. However, the molecular mechanism of SPOP-mediated ubiquitination in HCC metastasis remains obscure. In the present study, our results indicated that SPOP expression was significantly downregulated in HCC and was associated with tumor size, differentiation and metastasis. Cox regression model showed that low SPOP expression was a risk factor related to the prognosis of HCC patients. Loss- and gain-of-function assays demonstrated that SPOP inhibited HCC cell migration and invasion in vitro. Mechanisitically, co-immunoprecipitation and ubiquitination assays revealed that SPOP recognized and bound SENP7 and promoted its degradation via ubiquitin-dependent proteolysis. Analysis of immunohistochemistry showed that vimentin expression was correlated negatively with SPOP and positively with SENP7. These results implied that increased degradation of SENP7 by overexpression of SPOP decreased vimentin levels, which in turn attenuated HCC cell metastasis. Moreover, in vivo assays showed that SPOP overexpression also significantly suppressed liver and lung metastases. In summary, SPOP inhibits HCC cell metastasis via ubiquitin-dependent SENP7 proteolysis and may thus serve as a new opinion for the prevention of HCC metastasis.

SPOP suppresses osteosarcoma invasion via PI3K/AKT/NF-κB signaling pathway.

Speckle-type POZ protein (SPOP), is an E3 ubiquitin ligase adaptor that is frequently mutated in prostate and endometrial cancers. SPOP has been shown to be responsible for oncogene SRC-3 ubiquitination and proteolysisin prostate cancers. However, whether SPOP plays a role in osteosarcoma (OS) is unknown. In this study, we investigated the inhibitory effect of SPOP on invasion and migration of OS cells. Real-time PCR and Western blot were used to detect the expression of SPOP in human OS samples and cell lines. Short hairpin RNA (shRNA) was used to silencing the expression of SPOP. Small scale Real-time PCR screen was used to identify the matrix metalloproteases (MMP) family members responsible for the phenotype caused by SPOP depletion. Matrigel-coated invasion chambers were used to detect the invasion ability of SPOP in OS cells. We found that SPOP was down-regulated in clinic OS samples and cultured OS cells. Furthermore, we showed that silencing of SPOP promoted cell migratory and invasive ability of OS cells in vitro, whereas restored the expression of SPOP achieved the opposite effects. At the molecular level, we found that SPOP regulated the activity of "PI3K/Akt/NF-κB" signaling pathway in OS cells. Our results suggested that down-regulation of SPOP promoted OS cells migratory and invasive ability via modulating the "PI3K/Akt/NF-κB" signaling pathway. Thus, SPOP could be a promising drug target for the treatment of OS invasion.