Cardamonin inhibited cell viability and tumorigenesis partially through blockade of testes-specific protease 50-mediated nuclear factor-kappaB signaling pathway activation

Previous studies have shown that testes-specific protease 50 (TSP50), a pro-oncogene overexpressed in many types of tumors, could promote cell proliferation, invasion, tumorigenesis, and tumor metastasis, suggesting that it is a potential cancer therapeutic target in drug discovery. Here, a luciferase assay system driven by the TSP50 gene promoter was used to screen the inhibitor of expression of TSP50. The study found that cardamonin, a flavone compound, could efficiently inhibit the expression of TSP50 in both mRNA and protein levels. Further results revealed that cardamonin also efficiently inhibited the viability of TSP50 high-expressing cancer cells by inducing G2/M-phase arrest and mitochondrial-dependent apoptosis. Surprisingly, knocking down the expression of TSP50 gene had the same effects as treatment with cardamonin. Moreover, it has been found that cardamonin had an inhibitory potency on TSP50 high-expressing tumor growth in vivo. In contrast, overexpression of TSP50 greatly decreased the cell sensitivity to the inhibitory effect of cardamonin and reversed the decreased tumor-inhibitory effect of cardamonin. Additionally, both TSP50 interference and treatment with cardamonin could suppress p65 nuclear translocation, and overexpression of TSP50 reversed the suppressive effect of cardamonin on p65 nuclear translocation. Taken together, these results suggest that cardamonin inhibited cell viability and tumorigenesis at least partially via blocking the activation of TSP50-mediated nuclear factor-kappaB signaling pathway, and cardamonin may be a promising anticancer drug candidate in the development of a novel agent for TSP50 high-expressing cancer cells.     

Testes-specific protease 50 (TSP50) promotes cell proliferation through the activation of the nuclear factor κB (NF-κB) signalling pathway

TSP50 (testes-specific protease 50) is a testis-specific expression protein, which is expressed abnormally at high levels in breast cancer tissues. This makes it an attractive molecular marker and a potential target for diagnosis and therapy; however, the biological function of TSP50 is still unclear. In the present study, we show that overexpression of TSP50 in CHO (Chinese-hamster ovary) cells markedly increased cell proliferation and colony formation. Mechanistic studies have revealed that TSP50 can enhance the level of TNFα (tumour necrosis factor α)- and PMA-induced NF-κB (nuclear factor κB)-responsive reporter activity, IκB (inhibitor of NF-κB) α degradation and p65 nuclear translocation. In addition, the knockdown of endogenous TSP50 in MDA-MB-231 cells greatly inhibited NF-κB activity. Co-immunoprecipitation studies demonstrated an interaction of TSP50 with the NF-κB-IκBα complex, but not with the IKK (IκB kinase) α/β-IKKγ complex, which suggested that TSP50, as a novel type of protease, promoted the degradation of IκBα proteins by binding to the NF-κB-IκBα complex. Our results also revealed that TSP50 can enhance the expression of NF-κB target genes involved in cell proliferation. Furthermore, overexpression of a dominant-negative IκB mutant that is resistant to proteasome-mediated degradation significantly reversed TSP50-induced cell proliferation, colony formation and tumour formation in nude mice. Taken together, the results of the present study suggest that TSP50 promotes cell proliferation, at least partially, through activation of the NF-κB signalling pathway.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.     

Differential methylation of TSP50 and mTSP50 genes in different types of human tissues and mouse spermatic cells

Earlier studies identified human TSP50 as a testis-specific gene that encoded a threonine protease. Most importantly, TSP50 could be a cancer/testis antigen since there was a high frequency of reactivation in breast cancer biopsies. It was also found to be negatively regulated by the p53 gene. To further characterize this gene, we recently examined the DNA methylation patterns of the TSP50 gene promoter in normal human testis, as well as breast tissue and a testicular embryonic carcinoma cell line (HTECCL). Bisulfite genomic sequencing results demonstrated that the promoter exhibited mixed DNA methylation patterns in normal human testis, mainly non-methylation versus slight methylation, which could be attributed to the different stages spermatic cells go through during spermatogenesis. In contrast, it was methylated to a much greater extent in both breast tissue and HTECCL. To find out whether DNA methylation status was related to spermatogenesis stages, we analyzed DNA methylation patterns of the mTSP50 (the mouse ortholog of TSP50) promoter in spermatocytes and spermatozoa isolated from sexually mature mice. The results clearly demonstrated that each group of cells exhibited its preferential DNA methylation pattern that apparently was consistent with the gene expression status observed before. Taken together, our findings suggested that DNA methylation might regulate the TSP50 and mTSP50 gene expressions in different types of tissues and spermatic cells.     

TSP50 promotes the Warburg effect and hepatocyte proliferation via regulating PKM2 acetylation

Metabolic reprogramming is a hallmark of malignancy. Testes-specific protease 50 (TSP50), a newly identified oncogene, has been shown to play an important role in tumorigenesis. However, its role in tumor cell metabolism remains unclear. To investigate this issue, LC–MS/MS was employed to identify TSP50-binding proteins and pyruvate kinase M2 isoform (PKM2), a known key enzyme of aerobic glycolysis, was identified as a novel binding partner of TSP50. Further studies suggested that TSP50 promoted aerobic glycolysis in HCC cells by maintaining low pyruvate kinase activity of the PKM2. Mechanistically, TSP50 promoted the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our findings indicate that TSP50-mediated low PKM2 pyruvate kinase activity is an important determinant for Warburg effect in HCC cells and provide a mechanistic link between TSP50 and tumor metabolism.Subject terms: Cancer metabolism, Oncogenes, Tumour biomarkers

Gao et al. shows that testes-specific protease 50 (TSP50) binds to PKM2 and promotes the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our study reveals a link between an oncogene and a key enzyme in HCC glycolysis, which provides new ideas for human HCCs treatment with TSP50 as the target.     

Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients.     

The catalytic triad of testes-specific protease 50 (TSP50) is essential for its function in cell proliferation

Testes-specific protease 50 (TSP50) is a novelly identified pro-oncogene and it shares a similar enzymatic structure with many serine proteases. Our previous results suggested that TSP50 could promote tumorigenesis through degradation of IκBα protein and activating NF-κB signaling, and the threonine mutation in its catalytic triad could depress TSP50-mediated cell proliferation. However, whether the two other residues in the catalytic triad of TSP50 play a role in maintaining protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. Here, we constructed and characterized three catalytic triad mutants of TSP50 and found that all the mutants could significantly depress TSP50-induced cell proliferation and colony formation in vitro and tumor formation in vivo, and the aspartic acid at position 206 in the catalytic triad played a more crucial role than threonine and histidine in this process. Mechanistic studies revealed that the mutants in the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. Furthermore, our data indicated that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Most importantly, we demonstrated that TSP50 could interact with IκBα protein and cleave it directly as a new protease in vitro.     

Protumoral TSP50 Regulates Macrophage Activities and Polarization via Production of TNF-α and IL-1β, and Activation of the NF-κB Signaling Pathway

Testes-specific protease 50 (TSP50) is abnormally overexpressed in many kinds of cancers and promotes cell proliferation and migration. However, whether TSP50 can influence the tumor microenvironment, especially the function of immune cells in the microenvironment, remains largely unknown. We demonstrated that exposure to the conditioned medium from TSP50-overexpressing cells, or co-culture with TSP50-overexpressing cells, enhanced the cytokine production and phagocytic activities of macrophages, and induced M2b polarization. Further investigation showed that production of TNF-α and IL-1β was strongly induced by TSP50 in TSP50-overexpressing cells. TSP50-induced TNF-α and IL-1β were main factors that mediated the effects of TSP50-overexpressing cells on macrophages. The NF-κB pathway could be activated in macrophages upon the treatment of conditioned medium of TSP50-overexpressing cells and its activation is necessary for the observed effects on macrophages. Taken together, our results suggested that oncogenic TSP50 expressed in cells could activate surrounding macrophages and induce M2b polarization, partly through inducing TNF-α/ IL-1β secretion and subsequent NF-κB pathway activation. This implies a potential mechanism by which oncogene TSP50 regulates tumor microenvironment to support tumor development.     

Curcumin increased the expression of c-FLIP in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) disease is a chronic neuroinflammatory disease, which is associated with HTLV-1 infection. There is no effective and satisfactory treatment of HAM/TSP. It has been shown that curcumin exhibits modulatory effects on apoptosis and cytotoxicity-related molecules in HAM/TSP patients. In the present study, we examined the effect of curcumin on the gene expression of caspase-8, caspase-10, and anti-apoptotic protein c-FLIP, in HAM/TSP patients. Furthermore, we compared the expression of these molecules between HAM/TSP and asymptomatic carriers. Real-time PCR was performed to examine the mRNA expression of caspase-8, caspase-10, and c-FLIP in studied groups. The mRNA expression of caspase-8 and caspase-10 was similar before and after curcumin treatment in HAM/TSP patients (P > 0.05). The mRNA expression of c-FLIPL and c-FLIPs was higher after curcumin treatment compared with before treatment and significant differences were observed between the two groups (P = 0.004 and P = 0.044, respectively). The mRNA expression levels of caspase-8, caspase-10, c-FLIPL, and c-FLIPs were not statistically significant between HAM/TSP patients and asymptomatic carriers (P < 0.05). In conclusion, our results showed that curcumin increased the expression of c-FLIP in HAM/TSP patients which might suggest that, this molecule is involved in the apoptosis of HTLV-1-infected cells. Further studies with large sample size could be useful to clarify the role of this supplement in HAM/TSP patients.     

CT23 knockdown attenuating malignant behaviors of hepatocellular carcinoma cell is associated with upregulation of metallothionein 1

The cancer-testis antigen 23 (CT23) gene has been reported in association with the pathogenesis and progress of hepatocellular carcinoma (HCC). However, the alterations of gene expression profiling induced by CT23 knockdown in HCC cells remains largely unknown. In this study, the RNA interfering (RNAi) method was used to silence CT23 expression in BEL-7404 cells. Microarray analysis was performed on mRNA extracted from the CT23 knockdown cells and the control cells to determine the alterations of gene expression profiles. The result showed a total of 1051 genes expressed differentially (two-fold change), including 470 genes upregulated and 581 gene downregulated in the CT23 knockdown cells. A bioinformatic analysis showed that the functional differentially expressed genes (DEGs) were linked to cell proliferation, migration, and apoptosis, and metallothionein 1 (MT1) attained the maximum enrichment scores in functional annotation, classification, and pathway analysis of DEGs. Furthermore, Western blot analysis and cell behaviors assays verified that CT23 modulates cell proliferation, migration, and apoptosis by regulating MT1 expression in HCC cells and non-neoplastic hepatocytes. In summary, downregulated CT23 gene in BEL-7404 cells might change the expressions of carcinogenesis and progression related genes in HCC by upregulating MT1 expression, which would provide insight into searching for a novel therapeutic target for HCC.     

RPS27a promotes proliferation,regulates cell cycle progression and inhibits apoptosis of leukemia cells

Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.     

NUSAP1 knockdown inhibits cell growth and metastasis of non-small-cell lung cancer through regulating BTG2/PI3K/Akt signaling

Non-small-cell lung cancer (NSCLC) is the most common malignancy along with high mortality rate worldwide. Recently, nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in the malignant progression of several cancers. However, in NSCLC, the biological function of NUSAP1 and its molecular mechanism have not been reported. Here, our findings indicated that the NUSAP1 messenger RNA expression level was remarkably upregulated in NSCLC tissues compared with that of adjacent normal tissues. We also found that NUSAP1 gene expression was notably upregulated in NSCLC cell lines (A549, 95-D, H358, and H1299) compared with that of normal human bronchial epithelial cell line (16HBE). Subsequently, the biological function of NUSAP1 was investigated in A549 and H358 cells transfected with NUSAP1 small interfering RNA (siRNA), respectively. Results showed that NUSAP1 knockdown inhibited NSCLC cell proliferation, and promoted cell apoptosis. Furthermore, the number of cell migration and invasion was significantly suppressed by NUSAP1 knockdown. In addition, our results indicated that NUSAP1 knockdown increased the gene expression of B-cell translocation gene 2 (BTG2), but decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT). BTG2 siRNA partly abrogates the effect of NUSAP1 knockdown on BTG2 gene expression. Fumonisin B1 (FB1), a AKT activator, reversed the effect of NUSAP1 knockdown on the biological function in NSCLC. Taken together, NUSAP1 knockdown promotes NSCLC cell apoptosis, and inhibits cell proliferation, cell migration, and invasion, which is associated with regulating BTG2/PI3K/Akt signal pathway. Our findings suggest that NUSAP1 is a promising molecular target for NSCLC treatment.     

Knockdown of Lymphoid Enhancer factor 1 Inhibits Colon Cancer Progression In Vitro and In Vivo

Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.     

Screening of single-chain variable fragments against TSP50 from a phage display antibody library and their expression as soluble proteins

A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.