T790M mutation sensitizes non-small cell lung cancer cells to radiation via suppressing SPOCK1.

Background: Approximately 50% of patients harbor the T790M mutation after developing first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance. Evidence has showed the major treatment failure is local relapses and limited metastases. Several studies have demonstrated the value of radiotherapy in metastatic non-small cell lung cancer (NSCLC) with the EGFR T790M mutation after the development of TKI resistance. The aim of this study was to explore the role of radiation in T790M-mutant NSCLC and the value of early radiotherapy for NSCLC with T790M-mediated EGFR-TKI resistance. Methods: Gefitinib-resistant NSCLC cell lines were established via stepwise exposure to increasing concentrations of gefitinib (PC-9-GR). Droplet digital PCR was used to determine the relative T790M subclone abundance. In vitro and in vivo models were established using different mixtures of PC-9-GR and PC-9 cells. Differentially expressed genes were identified using RNA sequencing. Two research models were constructed (salvage and prophylactic radiotherapy) to determine the effects of early radiotherapy on gefitinib-resistant cells. Results: PC-9-GR cells exhibited higher radiosensitivity than PC-9 cells (sensitivity enhancement ratio = 1.5). Salvage radiation reduced the number of T790M-mutant subclones, and the relative T790M abundance was significantly lower than that without radiation at 90 days (10.94% vs. 21.54%). Prophylactic radiation prevented the development of T790M subclones. These results were also confirmed in vivo. qRT-PCR revealed threefold elevation of miR-1243 in PC-9-GR cells, and the increased radiosensitivity of PC-9-GR cells was inhibited when miR-1243 was knocked down. RNA sequencing revealed that SPOCK1 was downregulated in PC-9-GR cells. Interestingly, bioinformatic analysis showed that SPOCK1 was a target gene of miR-1243. SPOCK1 knockdown markedly increased the radiosensitivity of PC-9 cells. Conclusion: Gefitinib-resistant NSCLC with the T790M mutation had higher radiosensitivity than that without the mutation, possibly mediated by SPOCK1. Early radiotherapy can eliminate T790M subclones, providing evidence for the benefit of early local treatment in patients with TKI-resistant NSCLC.

SPOCK1, as a potential prognostic and therapeutic biomarker for lung adenocarcinoma, is associated with epithelial-mesenchymal transition and immune evasion.

BACKGROUND: The occurrence of epithelial-mesenchymal transition (EMT) and immune evasion is considered to contribute to poor prognosis in lung adenocarcinoma (LUAD). Therefore, this study aims to explore the key oncogenes that promote EMT and immune evasion and reveal the expression patterns, prognostic value, and potential biological functions. METHODS: Firstly, we identified gene modules associated with EMT and Tumor Immune Dysfunction and Exclusion (TIDE) through weighted gene co-expression network analysis (WGCNA). Next, we utilized differential analysis and machine learning to identify the key genes and validate them. Moreover, we analyzed the correlation between key genes and tumor microenvironment remodeling, drug sensitivity, as well as mutation frequency. Furthermore, we explored and validated their malignant biological characteristics through in vitro experiments and clinical samples. Finally, potential drugs for LUAD were screened based on CMap and validated through experiments. RESULTS: Firstly, WGCNA analysis revealed that red and green modules were highly correlated with EMT and TIDE. Among them, upregulated expression of SPOCK1 was observed in lung adenocarcinoma tissues and was associated with poor prognosis. Additionally, patients in the high SPOCK1 group showed more activation of malignant oncogenic pathways, higher infiltration of immunosuppressive components, and a higher frequency of mutations. The knockdown of SPOCK1 suppressed invasion and metastasis capabilities of lung adenocarcinoma cells, and the high expression of SPOCK1 was associated with low infiltration of CD8+ T cells. Therapeutic aspects, SPOCK1 can be a candidate indicator for drug sensitivity and CMap showed that VER-155008 was the drug candidate with the largest perturbation effect on the SPOCK1 expression profile. In vitro and in vivo experiments validated the cancer-inhibitory effect of VER-155008 in LUAD. CONCLUSION: This study revealed through comprehensive bioinformatics analysis and experimental analysis that SPOCK1 can promote EMT and immune escape in LUAD, and it may serve as a promising candidate prognostic biomarker and therapeutic target for LUAD.

Epithelial-to-mesenchymal transition, inflammation, subsequent collagen production, and reduced proteinase expression cooperatively contribute to cyclosporin-A-induced gingival overgrowth development.

Drug-induced gingival overgrowth (DIGO), induced by certain immunosuppressive drugs, antihypertensive agents, and antiepileptic drugs, may contribute to the formation of deeper periodontal pockets and intractableness in periodontitis. To date, multiple factors such as enhanced matrix production, inflammation, and reduced matrix degradation might be involved in the pathogenesis of DIGO. We have previously reported that SPOCK-1, a heparan sulfate proteoglycan, could affect gingival thickening by promoting epithelial-to-mesenchymal transition (EMT) in gingival keratinocytes. However, few studies have investigated whether a combination of these factors enhances the DIGO phenotype in animal models. Therefore, we investigated whether SPOCK-1, periodontal inflammation, and cyclosporin-A (CsA) could cooperatively promote gingival overgrowth. We first confirmed that Spock-1 overexpressing (Spock1-Tg) mice showed significantly thicker gingiva and greater alveolar bone loss than WT mice in response to ligature-induced experimental periodontitis. DIGO was induced by the combination of CsA administration and experimental periodontitis was significantly enhanced in Spock1-Tg mice compared to that in WT mice. Ligature-induced alveolar bone loss in CsA-treated Spock1-Tg mice was also significantly greater than that in CsA-treated WT mice, while being accompanied by an increase in Rankl and Col1a1 levels and a reduction in matrix metalloprotease expression. Lastly, SPOCK-1 promoted RANKL-induced osteoclast differentiation in both human peripheral blood mononuclear cells and murine macrophages, while peritoneal macrophages from Spock1-Tg mice showed less TNFα and IL-1ß secretion than WT mice in response to Escherichia coli lipopolysaccharide. These results suggest that EMT, periodontal inflammation, and subsequent enhanced collagen production and reduced proteinase production contribute to CsA-induced DIGO pathogenesis.

Corrigendum: SPOCK1 promotes the development of hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fonc.2022.819883.].

SPOCK1 Overexpression Suggests Poor Prognosis of Ovarian Cancer.

PURPOSE: Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) has been found in a variety of malignant tumors and is associated with a poor prognosis. We aimed to explore the role of SPOCK1 in ovarian cancer. METHODS: Ovarian cancer cell lines SKOV3 and SW626 were transfected with SPOCK1 overexpressing or empty vector using electroporation. Cells were studied by immunostaining and an automated Western blotting system. BrdU uptake and wound healing assays assessed cell proliferation and migration. SPOCK1 expression in human ovarian cancer tissues and in blood samples were studied by immunostaining and ELISA. Survival of patients with tumors exhibiting low and high SPOCK1 expression was analyzed using online tools. RESULTS: Both transfected cell lines synthesized different SPOCK1 variants; SKOV3 cells also secreted the proteoglycan. SPOCK1 overexpression stimulated DNA synthesis and cell migration involving p21CIP1. Ovarian cancer patients had increased SPOCK1 serum levels compared to healthy controls. Tumor cells of tissues also displayed abundant SPOCK1. Moreover, SPOCK1 levels were higher in untreated ovarian cancer serum and tissue samples and lower in recipients of chemotherapy. According to in silico analyses, high SPOCK1 expression was correlated with shorter survival. CONCLUSION: Our findings suggest SPOCK1 may be a viable anti-tumor therapeutic target and could be used for monitoring ovarian cancer.

Proteoglycan SPOCK1 as a Poor Prognostic Marker Promotes Malignant Progression of Clear Cell Renal Cell Carcinoma via Triggering the Snail/Slug-MMP-2 Axis-Mediated Epithelial-to-Mesenchymal Transition.

Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) has been reported to play an oncogenic role in certain cancer types; however, the role of SPOCK1 in the progression of clear cell renal cell carcinoma (ccRCC) remains elusive. Here, higher SPOCK1 transcript and protein levels were observed in ccRCC tissues compared to normal tissues and correlated with advanced clinical stages, larger tumor sizes, and lymph node and distal metastases. Knockdown and overexpression of SPOCK1 in ccRCC cells led to decreased and increased cell clonogenic and migratory/invasive abilities in vitro as well as lower and higher tumor growth and invasion in vivo, respectively. Mechanistically, the gene set enrichment analysis (GSEA) database was used to identify the gene set of epithelial-to-mesenchymal transition (EMT) pathways enriched in ccRCC samples with high SPOCK1 expression. Further mechanistic investigations revealed that SPOCK1 triggered the Snail/Slug-matrix metalloproteinase (MMP)-2 axis to promote EMT and cell motility. Clinical ccRCC samples revealed SPOCK1 to be an independent prognostic factor for overall survival (OS), and positive correlations of SPOCK1 with MMP-2 and mesenchymal-related gene expression levels were found. We observed that patients with SPOCK1high/MMP2high tumors had the shortest OS times compared to others. In conclusion, our findings reveal that SPOCK1 can serve as a useful biomarker for predicting ccRCC progression and prognosis, and as a promising target for treating ccRCC.

SPOCK1 and POSTN are valuable prognostic biomarkers and correlate with tumor immune infiltrates in colorectal cancer.

BACKGROUND: Immune cells and stromal cells in the tumor microenvironment play a vital role in the progression of colorectal cancer (CRC). The study aimed to screen valuable prognostic biomarkers in CRC based on stromal and immune scores. METHOD: The ESTIMATE algorithm was used to calculate the immune and stromal scores of CRC samples in TCGA. Then samples were divided into high and low score groups based on the median value of the scores. Differentially expressed genes (DEGs) associated with immune and stromal scores were screened. WGCNA and univariate COX regression analysis were performed to further identify key prognostic genes. Analysis of scRNA-seq for CRC was used for verifying the main source of the key genes. The prognostic value of they was validated based on The Gene Expression Profiling Interactive Analysis and GSE17536 dataset. TIMER and CIBERSORT algorithms were applied to analyze the correlations among key genes and tumor-infiltrating immune cells. Several pairs of colon cancer tissue were used to be proven. RESULT: 1314 upregulated and 4 downregulated genes were identified, which were significantly enriched in immune-related biological processes and pathways. Among these DEGs, SPOCK1 and POSTN were identified as key prognostic genes and mainly expressed in cancer-associated fibroblasts for CRC. High expression of SPCOK1 and POSTN was associated with advanced clinical stage, T stage, N stage, and poor prognosis of CRC. The results from CIBERSORT and TIMER revealed that SPOCK1 and POSTN were associated with tumor-infiltrating immune cells, especially macrophages and neutrophils. Meanwhile, in several pairs of human colorectal tissue samples, SPOK1 and POSTN were found to be significantly overexpressed in colorectal tissue compared with para-cancer tissue, and macrophage surface markers CD68 (co-expressed by M1 and M2 macrophages) and CD206 (M2-specific macrophage expression) were also overexpressed in cancer tissue. Besides, SPOCK1 and POSTN expression were positively correlated with the expression of immune checkpoints. CONCLUSION: Collectively, our results indicate that SPOCK1 and POSTN associated with CAF may be novel prognostic biomarkers in CRC and correlate with immune infiltrates.

Knockdown of Long Noncoding RNA Urothelial Carcinoma-Associated 1 Represses Gallbladder Cancer Advancement by Regulating SPOCK1 Expression Through Sponging miR-613.

Background: Gallbladder cancer (GBC) is the most common biliary tract malignancy. Long noncoding RNA urothelial carcinoma-associated 1 (UCA1) and MicroRNA-613 (miR-613) have been reported to be involved in the progression of various cancers. However, the regulatory mechanism between UCA1 and miR-613 in GBC is unclear. Materials and Methods: The expression levels of UCA1, miR-613, and secreted protein/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) mRNA were detected using quantitative real-time polymerase chain reaction. Cell proliferation, migration, invasion, and apoptosis were determined with MTT, transwell, or flow cytometry assays. The levels of SPOCK1 protein, Bax, cleaved-casp-3, and Bcl-2 were determined by Western blot analysis. The relationship between miR-613 and UCA1 or SPOCK1 was verified through dual-luciferase reporter and/or RNA immunoprecipitation assays. Xenograft assay was performed to verify the role of UCA1 in vivo. Results: UCA1 and SPOCK1 were upregulated, whereas miR-613 was downregulated in GBC tissues and cells. UCA1 silencing decreased tumor growth in vivo and impeded proliferation, migration, invasion, and induced apoptosis of GBC cells in vitro. Notably, UCA1 acted as a sponge for miR-613, which targeted SPOCK1 in GBC cells. Moreover, UCA1 enhancement reversed the repressive impact of miR-613 mimic on the malignancy of GBC cells. UCA1 regulated SPOCK1 expression through adsorbing miR-613. Furthermore, SPOCK1 elevation overturned UCA1 silencing mediated the malignant behaviors of GBC cells. Conclusion: UCA1 knockdown suppressed GBC progression through downregulating SPOCK1 via sponging miR-613, providing an evidence for UCA1 as a target for GBC treatment.

Circ_0061395 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1182 sponge.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in liver cancer, with a high rate of metastasis and recurrence. Circular RNA_0061395 (circ_0061395) has been shown to be involved in the advance of HCC. However, the interaction between circ_0061395 and microRNA (miRNA) in HCC has not been studied. Quantitative real-time polymerase-chain reaction (qRT-PCR) was used to detect the expression of related genes in liver cancer tissues and cells. The stability of circ_0061395 was verified by RNase R digestion. Through detection of cell malignant behavior and apoptosis, the capping experiment was carried out to verify the regulatory relationship between miR-1182 and circ_0061395 or SPARC/osteonectin, CWCV and Kazal-like domains proteoglycan 1 (SPOCK1). The expression of related proteins was detected by western blot. The interaction of miR-1182 with circ_0061395 or SPOCK1 has been notarized by Dual-luciferase reporter analysis and RNA immunoprecipitation (RIP) assay. Xenotransplantation experiments using BALB/C nude mice were used to confirm the function of circ_0061395 in vivo. Circ_0061395 and SPOCK1 were significantly expressed in liver cancer tissues and cells. Silencing circ_0061395 reduced the proliferation, migration, invasion, tube formation and tumor spheroid formation rate of Huh-7 and SNU-387 cells. MiR-1182 was a target of circ_0061395. Silencing circ_0061395 inhibited the malignant behavior of HCC cells by releasing miR-1182. In addition, SPOCK1 was the target of miR-1182. Overexpression of SPOCK1 partially restored the inhibitory effect of miR-1182 on cell proliferation. Animal experiments confirmed the anti-tumor effect of silence circ_0061395. Circ_0061395 induced the changes of the expression of SPOCK1 by regulating miR-1182, thereby mediating the process of HCC, and at least partially promoting the development of HCC cells, providing a novel targeted therapy for HCC.

Assessment of the prognostic value of SPOCK1 in clear cell renal cell carcinoma: a bioinformatics analysis.

Background: Clear cell renal cell carcinoma (ccRCC) is one of the most common urological malignancies, and once metastasis occurs, it often has a poor prognosis and lacks effective treatment. Therefore, there is an urgent need to screen some new biomarkers and explore their molecular mechanisms to improve the early clinical diagnosis and targeted therapy of ccRCC. SPOCK1 (SPARC/osteonectin, CWCV and Kazal-like domains proteoglycan 1) is a conserved multi-domain proteoglycan that plays an important role in the development of multiple cancer types; however, its prognostic value in ccRCC has not been investigated. The study of the prognostic value of SPOCK1 in ccRCC is a good complement to the study of ccRCC biomarkers. Methods: Databases of this study included Oncomine, Kaplan-Meier Plotter, GEPIA, GeneMANIA, cBioPortal, and TIMER. Student's t-test was used to analyze the differences in SPOCK1 expression in ccRCC tissues compared with tumor-adjacent normal tissues. Kaplan-Meier curves for survival analysis were used to assess the correlation between the expression of SPOCK1 and the prognostic outcomes. Correlation module drew the expression scatterplots between SPOCK1 and immune cell infiltration in ccRCC, together with the Spearman's rho value and estimated statistical significance. Results: The SPOCK1 mRNA expression was significantly higher in ccRCC tissues (mean expression ± SD: 920.2±195.2) than in normal tissues (mean expression ± SD: 358.4±29.1, P=0.008), and high SPOCK1 expression significantly and positively correlated with the pathological stage of ccRCC patients (F value =10.2, P<0.001). Higher expression of SPOCK1 was also associated with significantly shorter overall survival (OS) and disease-free survival (DFS) in ccRCC patients (GEPIA: P=0.046, P<0.001, respectively; Kaplan-Meier Plotter: P=0.002, P=0.0022, respectively). The function of SPOCK1 is mainly related to tumor development and extracellular matrix remodeling, and it may participate in the epithelial-mesenchymal transition process. SPOCK1 expression significantly and positively correlated with infiltration of several immune cells in ccRCC, including cancer-associated fibroblasts (CAFs) (Rho =0.333, P=2.16×10-13), tumor-associated macrophages (TAMs) (Rho =0.18, P=1.02×10-4), and tumor-associated neutrophils (TANs) (Rho =0.165, P=3.83×10-4). Conversely, there was a significant and negative correlation between SPOCK1 expression and infiltration of CD4+ T cells (Rho =-0.113, P=0.015). Conclusions: SPOCK1 may be a potential prognostic biomarker in ccRCC.

SPOCK1 silencing decreases 5-FU resistance through PRRX1 in colorectal cancer.

SPOCK1 is an extracellular proteoglycan and involved in tumor growth and metastasis in various cancers. 5-fluorouracil (5-FU) is commonly used for the treatment of colorectal cancer (CRC) in patients who receive concurrent chemoradiotherapy. However, the relationship between development of resistance to 5-FU and SPOCK1 remain unclear. In this study, we established two 5-fluorouracil (5-FU)-resistant CRC cell lines, HCT116/FU and LOVO/FU, and found that SPOCK1 is upregulated in 5-FU-resistance CRC cells compared with its parental cell line. knockdown of SPOCK1 in 5-FU-resistant CRC cells increases their sensitivity to 5-FU. In contrast, transient transfection of SPOCK1 enhanced HCT116 and LOVO cell resistance to 5-FU and reduced cell apoptosis. Mechanistically, SPOCK1 promoted 5-FU resistance by regulating PRRX1 expression and the downstream apoptosis signaling pathway. Taken together, our results revealed for the first time that SPOCK1 plays a crucial role in the resistance of CRC cells to 5-FU and indicated that targeting SPOCK1 may be a promising therapeutic strategy to overcome 5-FU resistance in CRC.

SPOCK1 with unexpected function. The start of a new career.

SPOCK1, 2, and 3 are considered matricellular proteoglycans without a structural role. Their functions are only partly elucidated. SPOCK1 was detected in the brain as a member of the neural synapses, then in the neuromuscular junctions. It plays a role in the regulation of the blood-brain barrier. Its best-characterized activity was its oncogenic potential discovered in 2012. Its deleterious effect on tumor progression was detected on 36 different types of tumors by the end of 2020. However, its mode of action is still not completely understood. Furthermore, even less was discovered about its physiological function. The fact that it was found to localize in the mitochondria and interfered with the lipid metabolism indicated that the full discovery of SPOCK1 is still waiting for us.

Expression and Potential Prognostic Value of SOX9, MCL-1 and SPOCK1 in Gastric Adenocarcinoma.

Gastric cancer is a common malignancy and remains one of the leading causes of cancer-related deaths, though its incidence is in decline in most developed countries. One of the major challenges of treating gastric cancer is tumor heterogeneity, which portends a high degree of prognostic variance and the necessity for different treatment modalities. Tumor heterogeneity is at least in part due to divergent differentiation of tumor cells to clones harboring different molecular alterations. Here we studied the expression of emerging prognostic markers SOX9, MCL-1, and SPOCK1 (Testican-1) in a cohort of gastric cancer by immunohistochemistry and investigated how individual biomarkers and their combinations predict disease prognosis. We found frequent expression of SPOCK1 (in both nuclei and cytoplasm), MCL-1 and SOX9 in gastric cancer. In univariate analysis, nuclear SPOCK1 expression and pathologic TNM stage were negative prognostic markers in this cohort. In multivariate analysis, SOX9 expression stood out as a predictor of poor prognosis. Further subgroup analysis suggested prognostic value of SOX9 expression in poorly differentiated gastric adenocarcinoma. MCL-1 showed no prognostic role in this cohort.

SPOCK1 Promotes the Development of Hepatocellular Carcinoma.

The extracellular matrix proteoglycan SPOCK1 is increasingly recognized as a contributor to the development and progression of cancers. Here, we study how SPOCK1, which is present in non-tumorous hepatocytes at low concentrations, promotes the development and progression of malignant hepatocellular tumors. Although SPOCK1 is an extracellular matrix proteoglycan, its concentration increases in the cytoplasm of hepatocytes starting with very low expression in the normal cells and then appearing in much higher quantities in cells of cirrhotic human liver and hepatocellular carcinoma. This observation is similar to that observed after diethylnitrosamine induction of mouse hepatocarcinogenesis. Furthermore, syndecan-1, the major proteoglycan of the liver, and SPOCK1 are in inverse correlation in the course of these events. In hepatoma cell lines, the cytoplasmic SPOCK1 colocalized with mitochondrial markers, such as MitoTracker and TOMM20, a characteristic protein of the outer membrane of the mitochondrion and could be detected in the cell nucleus. SPOCK1 downregulation of hepatoma cell lines by siRNA inhibited cell proliferation, upregulated p21 and p27, and interfered with pAkt and CDK4 expression. A tyrosine kinase array revealed that inhibition of SPOCK1 in the liver cancer cells altered MAPK signaling and downregulated several members of the Sarc family, all related to the aggressivity of the hepatoma cell lines. These studies support the idea that SPOCK1 enhancement in the liver is an active contributor to human and rodent hepatocarcinogenesis and cancer progression. However, its mitochondrial localization raises the possibility that it has a currently unidentified physiological function in normal hepatocytes.

LncRNA THEMIS2-211, a tumor-originated circulating exosomal biomarker, promotes the growth and metastasis of hepatocellular carcinoma by functioning as a competing endogenous RNA.

Hepatocellular carcinoma (HCC) is a major challenge for human health. Finding reliable diagnostic biomarkers and therapeutic targets for HCC is highly desired in the clinic. Currently, circulating exosomal lncRNA is a promising biomarker for the diagnosis of cancer and lncRNA is also a potential target in cancer therapy. Here, the diagnostic value of a panel based on exosomal lncRNA THEMIS2-211 and PRKACA-202, superior to that of AFP, was identified for diagnosing human HCC. Besides, the performance of exosomal lncRNA THEMIS2-211 alone exceeds that of AFP in diagnosing early-stage HCC patients (stage I). Furthermore, lncRNA THEMIS2-211 is highly expressed in HCC tissues and correlated with the poor prognosis of HCC patients. LncRNA THEMIS2-211 is upregulated and localized in the cytoplasm of HCC cells. LncRNA THEMIS2-211 exerts its biological function as an oncogene that promotes the proliferation, migration, invasion, EMT of HCC cells by physically interacting with miR-940 and therefore promoting SPOCK1 expressions. Rescue assays show the regulation of SPOCK1 by lncRNA THEMIS2-211 dependents on miR-940. The discovery of lncRNA THEMIS2-211 further illuminates the molecular pathogenesis of HCC and the THEMIS2-211/miR-940/SPOCK1 axis may act as a potential therapeutic target for HCC.

SPOCK1 promotes metastasis in pancreatic cancer via NF-κB-dependent epithelial-mesenchymal transition by interacting with IκB-α.

BACKGROUND: Sparc/osteonectin, cwcv and kazal-like domain proteoglycan 1 (SPOCK1) has been reported to function as an oncogene in a variety of cancer types. Increasing evidence suggests that SPOCK1 contributes to the metastatic cascade, including invasion, epithelial-mesenchymal transition (EMT) and micro-metastasis formation. As yet, however, the underlying mechanism is not clearly understood. Here, we evaluated the expression and clinicopathological significance of SPOCK1 in primary pancreatic cancer (PC) specimens and explored the mechanisms underlying SPOCK1-mediated PC cell growth and metastasis. METHODS: The clinical relevance of SPOCK1 was evaluated in 81 patients with PC. The effect of SPOCK1 on proliferation, cell cycle progression, EMT and metastasis was examined in vitro and in vivo. The molecular mechanisms involved in SPOCK1-mediated regulation of NF-κB-dependent EMT were assessed in PC cell lines. RESULTS: We found that SPOCK1 expression was increased in PC tissues and was associated with lymph node metastasis. Silencing or exogenous overexpression of SPOCK1 markedly altered the proliferation of PC cells through cell cycle transition. Overexpression of SPOCK1 promoted PC cell migration and invasion by regulating EMT progression. Moreover, we found that SPOCK1 contributes to EMT and metastasis by activating the NF-κB signalling pathway via direct interaction with IκBα. After NF-κB pathway inhibition by BAY11-7082, we found that PC cell motility and EMT induced by SPOCK1 were reversed. CONCLUSION: From our data we conclude that SPOCK1 promotes PC metastasis via NF-κB-dependent EMT by interacting with IκBα. This newly identified mechanism may provide novel clues for the (targeted) treatment of PC patients.

Multiomic analysis of the function of SPOCK1 across cancers: an integrated bioinformatics approach.

OBJECTIVE: To investigate SPARC (osteonectin), cwcv and kazal like domains proteoglycan 1 (SPOCK1) gene expression across The Cancer Genome Atlas (TCGA) cancers, both in cancer versus normal tissues and in different stages across the cancer types. METHODS: This integrated bioinformatics study used data from several bioinformatics databases (Cancer Cell Line Encyclopedia, Genotype-Tissue Expression, TCGA, Tumor Immune Estimation Resource [TIMER]) to define the expression pattern of the SPOCK1 gene. A survival analysis was undertaken across the cancers. The search tool for retrieval of interacting genes (STRING) database was used to identify proteins that interacted with SPOCK1. Gene Set Enrichment Analysis was conducted to determine pathway enrichment. The TIMER database was used to explore the correlation between SPOCK1 and immune cell infiltration. RESULTS: This multiomic analysis showed that the SPOCK1 gene was expressed differently between normal tissues and tumours in several cancers and that it was involved in cancer progression. The overexpression of the SPOCK1 gene was associated with poor clinical outcomes. Analysis of gene expression and tumour-infiltrating immune cells showed that SPOCK1 correlated with several immune cells across cancers. CONCLUSIONS: This research showed that SPOCK1 might serve as a new target for several cancer therapies in the future.

Long non-coding RNA TTN antisense RNA 1 facilitates hepatocellular carcinoma progression via regulating miR-139-5p/SPOCK1 axis.

Reportedly, long non-coding RNAs (lncRNAs) are implicated in hepatocellular carcinoma (HCC) progression, yet little is known concerning the biological functions of TTN antisense RNA 1 (TTN-AS1) in HCC. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was performed for detecting TTN-AS1, SPOCK1 mRNA, and miR-139-5p expressions in HCC cells and tissues. After TTN-AS1 was overexpressed or knocked down in HCC cells, CCK-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assays were carried out for examining cell multiplication. Transwell assays were conducted for evaluating HCC cell migration and invasion. Dual-luciferase reporter assay was employed for verifying the binding relationships between miR-139-5p and TTN-AS1, and between SPOCK1 3'UTR and miR-139-5p. Western blot was employed to measure SPOCK1, E-cadherin, N-cadherin, and Vimentin protein expressions. We demonstrated that, TTN-AS1 and SPOCK1 expression levels were remarkably enhanced in HCC cells and tissues, whereas miR-139-5p expression was observably reduced. Functional experiments suggested that TTN-AS1 knockdown markedly repressed HCC cell multiplication, migration, epithelial-mesenchymal transition (EMT), and invasion. In addition, TTN-AS1 interacted with miR-139-5p and decreased its expression. Moreover, SPOCK1 was a miR-139-5p target, and miR-139-5p inhibitors were able to reverse TTN-AS1 knockdown-induced inhibitory effect on SPOCK1 expression. SPOCK1 overexpression plasmid could counteract TTN-AS1 knockdown-induced inhibiting impact on HCC cell multiplication, migration, invasion, and EMT. In conclusion, TTN-AS1 expression level is remarkably enhanced in HCC, and TTN-AS1 can promote the multiplication, migration, invasion, and EMT of HCC cells via regulating miR-139-5p/SPOCK1 axis.

SPOCK1/SIX1axis promotes breast cancer progression by activating AKT/mTOR signaling.

SPOCK1 is highly expressed in many types of cancer and has been recognized as a promoter of cancer progression. Its regulatory mechanism in breast cancer (BC) remains unclear. This study aimed to explore the precise function of SPOCK1 in BC progression and to identify the mechanism by which SPOCK1 is involved in cell proliferation and epithelial-mesenchymal transition (EMT). Immunohistochemistry (IHC) experiments and database analysis showed that high expression of SPOCK1 was positively associated with histological grade, lymph node metastasis (LN) and poor clinical prognosis in BC. A series of in vitro and in vivo assays elucidated that altering the SPOCK1 level led to distinct changes in BC cell proliferation and metastasis. Investigations of potential mechanisms revealed that SPOCK1 interacted with SIX1 to enhance cell proliferation, cell cycle progression and EMT by activating the AKT/mTOR pathway, whereas inhibition of the AKT/mTOR pathway or depletion of SIX1 reversed the effects of SPOCK1 overexpression. Furthermore, SPOCK1 and SIX1 were highly expressed in BC and might indicate poor prognoses. Altogether, the SPOCK1/SIX1 axis promoted BC progression by activating the AKT/mTOR pathway to accelerate cell proliferation and promote metastasis in BC, so the SPOCK1/SIX1 axis might be a promising clinical therapeutic target for preventing BC progression.

SPOCK1: a multi-domain proteoglycan at the crossroads of extracellular matrix remodeling and cancer development.

The SPARC/osteonectin, CWCV and Kazal-like domains proteoglycan 1 (SPOCK1) is a highly conserved, multi-domain proteoglycan that regulates the dynamic equilibrium of extracellular matrix (ECM). Besides, SPOCK1 is one of the key regulatory genes in the tumor ECM dynamic homeostasis process, which activates many molecular signaling pathways (such as EMT process, Wnt/ß-catenin, PI3K/Akt, and mTOR/S6K signaling pathways). This activation leads to ECM remodeling and promotes cell proliferation and invasion, but inhibits cell apoptosis. Whereas there is immense information about SPOCK1's roles in different biological settings, there is need for further studies that interrogate this protein as a potential therapeutic target in cancer.