TMPRSS4 induces cancer cell invasion through pro-uPA processing

TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates cancer cell invasion, epithelial-mesenchymal transition, and metastasis and that increased TMPRSS4 expression correlates with colorectal cancer progression. We also demonstrated that TMPRSS4 upregulates urokinase-type plasminogen activator (uPA) gene expression to induce cancer cell invasion. However, it remains unknown how proteolytic activity of TMPRSS4 contributes to invasion. In this study, we report that TMPRSS4 directly converted inactive pro-uPA into the active form through its proteolytic activity. Analysis of conditioned medium from cells overexpressing TMPRSS4 demonstrated that the active TMPRSS4 protease domain is released from the cells and is associated with the plasma membrane. Furthermore, TMPRSS4 could increase pro-uPA-mediated invasion in a serine proteolytic activity-dependent manner. These observations suggest that TMPRSS4 is an upstream regulator of pro-uPA activation. This study provides valuable insights into the proteolytic function of TMPRSS4 as well as mechanisms for the control of invasion.     

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation,proteolytic activity,and cell surface localization

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.     

Knockdown of SLC34A2 inhibits cell proliferation,metastasis, and elevates chemosensitivity in glioma

Solute carrier 34 A2 (SLC34A2) is a member of SLC34 family that is a group of phosphate transporters. SLC34A2 has been reported to play critical roles in tumorigenesis and progression. However, the researches about the biological roles of SLC34A2 in glioma have not yet been reported. In this study, we analyzed the expression patterns of SLC34A2 in clinical glioma tumor tissues and cell lines. The results demonstrated that SLC34A2 was generally overexpressed in both glioma tissues and cell lines. To further investigate the roles of SLC34A2 in glioma, lentivirus containing specific SLC34A2 short hairpin RNA (sh-SLC34A2) was used to infect glioma cell lines U251 and U87 for the knockdown of SLC34A2. The following studies proved that SLC34A2 knockdown exhibited suppressive effects on cell proliferation and migration/invasion. SLC34A2 knockdown also inhibited epithelial-mesenchymal transition (EMT) phenotype, as evidenced by the increased E-cadherin expression, and the decreased N-cadherin and fibronectin expressions. Besides, knockdown of SLC34A2 enhanced the temozolomide (TMZ) sensitivity of U251 and U87 cells. In vivo tumorigenicity assay demonstrated that SLC34A2 knockdown inhibited tumor growth. Moreover, SLC34A2 knockdown suppressed the activation of epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway in U87 cells. GW2974 (EGFR inhibitor) increased SLC34A2 knockdown-inhibited cell proliferation, migration/invasion, as well as enhanced SLC34A2 knockdown-increased the TMZ sensitivity of glioma cells. These findings suggested that SLC34A2 might be a new potential therapeutic target for the therapy of glioma patients.     

新型冠状病毒相关TMPRSS2蛋白结构特征和抗原表位分析

采用生物信息学方法分析预测新型冠状病毒(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)跨膜蛋白酶丝氨酸2(transmembrane protease serine 2,TMPRSS2)的理化特性、结构特征和抗原表位,为抗SARS-CoV-2药物研...     

Hyper-Activation of Notch3 Amplifies the Proliferative Potential of Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.     

Increased Notch Signaling Enhances Radioresistance of Malignant Stromal Cells Induced by Glioma Stem/ Progenitor Cells

Background

Host malignant stromal cells induced by glioma stem/progenitor cells were revealed to be more radiation-resistant than the glioma stem/progenitor cells themselves after malignant transformation in nude mice. However, the mechanism underlying this phenomenon remains unclear.

Methods

Malignant stromal cells induced by glioma stem/progenitor cell 2 (GSC-induced host brain tumor cells, ihBTC2) were isolated and identified from the double color-coded orthotopic glioma nude mouse model. The survival fraction at 2 Gy (SF2) was used to evaluate the radiation resistance of ihBTC2, the human glioma stem/progenitor cell line SU3 and its radiation-resistant sub-strain SU3-5R and the rat C6 glioma cell line. The mRNA of Notch 1 and Hes1 from ihBTC2 cells were detected using qPCR before and after 4 Gy radiation. The expression of the Notch 1, pAkt and Bcl-2 proteins were investigated by Western blot. To confirm the role of the Notch pathway in the radiation resistance of ihBTC2, Notch signaling blocker gamma secretase inhibitors (GSIs) were used.

Results

The ihBTC2 cells had malignant phenotypes, such as infinite proliferation, hyperpentaploid karyotype, tumorigenesis in nude mice and expression of protein markers of oligodendroglia cells. The SF2 of ihBTC2 cells was significantly higher than that of any other cell line (P<0.05, n = 3). The expression of Notch 1 and Hes1 mRNAs from ihBTC2 cells was significantly increased after radiation. Moreover, the Notch 1, pAkt and Bcl-2 proteins were significantly increased after radiation (P<0.05, n = 3). Inhibition of Notch signaling markedly enhanced the radiosensitivity of ihBTC2 cells.

Conclusions

In an orthotopic glioma model, the malignant transformation of host stromal cells was induced by glioma stem/progenitor cells. IhBTC2 cells are more radiation-resistant than the glioma stem/progenitor cells, which may be mediated by activation of the Notch signaling pathway.     

Notch1 Is a 5-Fluorouracil Resistant and Poor Survival Marker in Human Esophagus Squamous Cell Carcinomas

Notch signaling involves the processes that govern cell proliferation, cell fate decision, cell differentiation and stem cell maintenance. Due to its fundamental role in stem cells, it has been speculated during the recent years that Notch family may have critical functions in cancer stem cells or cancer cells with a stem cell phenotype, therefore playing an important role in the process of oncogenesis. In this study, expression of Notch family in KYSE70, KYSE140 and KYSE450 squamous esophageal cancer cell lines and virus transformed squamous esophageal epithelial cell line Het-1A was examined by quantitative RT-PCR. Compared to the Het-1A cells, higher levels of Nocth1 and Notch3 expression in the cancer cell lines were identified. Due to the finding that NOTCH3 mainly mediates squamous cell differentiation, NOTCH1 expression was further studied in these cell lines. By Western blot analyses, the KYSE70 cell line which derived from a poorly differentiated tumor highly expressed Notch1, and the Notch1 expression in this cell line was hypoxia inducible, while the KYSE450 cell line which derived from a well differentiated tumor was always negative for Notch1, even in hypoxia. Additional studies demonstrated that the KYSE70 cell line was more 5-FU resistant than the KYSE450 cell line and such 5-FU resistance is correlated to Notch1 expression verified by Notch1 knockdown experiments. In clinical samples, Notch1 protein expression was detected in the basal cells of human esophagus epithelia, and its expression in squamous cell carcinomas was significantly associated with higher pathological grade and shorter overall survival. We conclude that Notch1 expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro and is significantly associated with a poor survival in human esophageal squamous cell carcinomas.     

Hypoxic remodelling of Ca2+ signalling in proliferating human arterial smooth muscle

Since Notch signaling plays a critical role in stem cells and oncogenesis, we hypothesized that Notch signaling might play roles in cancer stem cells and cancer cells with a stem cell phenotype. In this study, we accessed potential functions of the Notch pathway in the formation of cancer stem cells using human glioma. Using RT-PCR, we found that most human astrogliomas of different grades expressed moderate to high level of Notch receptors and ligands. mRNA of Hes5 but not Hes1, both of which are major downstream molecules of the Notch pathway, was also detected. In human glioma cell lines BT325, U251, SHG-44, and U87, mRNA encoding different types of Notch receptors were detected, but active form of Notch1 (NIC) was only detected in SHG-44 and U87 by Western blot. Interestingly, proliferation of these two glioma cell lines appeared faster than that of the other two lines in which NIC was not detected. We have over-expressed NIC of Notch1 in SHG-44 cells by constitutive transfection to evaluate the effects of Notch signaling on glioma cells. Our results showed that over-expression of NIC in SHG-44 cells promoted the growth and the colony-forming activity of SHG-44 cells. Interestingly, over-expression of NIC increased the formation neurosphere-like colonies in the presence of growth factors. These colonies expressed nestin, and could be induced to cells expressing neuron-, astrocyte-, or oligodendrocyte-specific markers, consistent with phenotypes of neural stem cells. These data suggest that Notch signaling promote the formation of cancer stem cell-like cells in human glioma. Xue-Ping Zhang, Gang Zheng and Lian Zou are contributed equally to this study.     

The Different Role of Notch1 and Notch2 in Astrocytic Gliomas

It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway–EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.     

Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-κB activation

The Notch signaling regulator Numblike (Numbl) is expressed in the brain, but little is known regarding its role in the pathophysiology of glial cells. In this paper, we report that Numbl expression was down-regulated in high-grade human glioma tissue samples and glioblastoma cell lines. To investigate the role of Numbl in glioma migration and invasion, we generated human glioma cell lines in which Numbl was either overexpressed or depleted. Overexpression of Numbl suppressed, while elimination of Numbl promoted, the migration and invasion of glioma cells. Numbl inhibited glioma migration and invasion by dampening NF-κB activity. Furthermore, Numbl interacted directly with tumor necrosis factor receptor-associated factor 5 (TRAF5), which signals upstream and is required for the activation of NF-κB, and committed it to proteasomal degradation by promoting K48-linked polyubiquitination of TRAF5. In conclusion, our data suggest that Numbl negative regulates glioma cell migration and invasion by abrogating TRAF5-induced activation of NF-κB.     

The endogenous cellular protease inhibitor SPINT2 controls SARS-CoV-2 viral infection and is associated to disease severity

COVID-19 outbreak is the biggest threat to human health in recent history. Currently, there are over 1.5 million related deaths and 75 million people infected around the world (as of 22/12/2020). The identification of virulence factors which determine disease susceptibility and severity in different cell types remains an essential challenge. The serine protease TMPRSS2 has been shown to be important for S protein priming and viral entry, however, little is known about its regulation. SPINT2 is a member of the family of Kunitz type serine protease inhibitors and has been shown to inhibit TMPRSS2. Here, we explored the existence of a co-regulation between SPINT2/TMPRSS2 and found a tightly regulated protease/inhibitor expression balance across tissues. We found that SPINT2 negatively correlates with SARS-CoV-2 expression in Calu-3 and Caco-2 cell lines and was down-regulated in secretory cells from COVID-19 patients. We validated our findings using Calu-3 cell lines and observed a strong increase in viral load after SPINT2 knockdown, while overexpression lead to a drastic reduction of the viral load. Additionally, we evaluated the expression of SPINT2 in datasets from comorbid diseases using bulk and scRNA-seq data. We observed its down-regulation in colon, kidney and liver tumors as well as in alpha pancreatic islets cells from diabetes Type 2 patients, which could have implications for the observed comorbidities in COVID-19 patients suffering from chronic diseases.     

Knockdown of RPL34 inhibits the proliferation and migration of glioma cells through the inactivation of JAK/STAT3 signaling pathway

Ribosomal protein L34 (RPL34), belonging to the L34E family of ribosomal proteins, was reported to be dysregulated in several types of cancers and plays important roles in tumor progression. However, the expression and roles of RPL34 in human glioma remain largely unknown. Thus, the objective of this study was to investigate the expression and role of RPL34 in glioma. We report here that RPL34 is highly expressed in human glioma tissues and cell lines. Knockdown of RPL34 markedly inhibited the proliferation, migration, and invasion, as well as prevented the epithelial-mesenchymal transition phenotype in glioma cells. Further, mechanistic analysis showed that knockdown of RPL34 significantly downregulated the levels of p-JAK and p-STAT3 in glioma cells. Taken together, our findings indicated that knockdown of RPL34 inhibits the proliferation and migration of glioma cells through the inactivation of JAK/STAT3 signaling pathway. Thus, RPL34 may serve as a potential therapeutic target for the treatment of glioma.     

Essential role of endocytosis of the type II transmembrane serine protease TMPRSS6 in regulating its functionality

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.     

前列腺癌中TMPRSS2-ETS基因融合及机制研究进展

超过50%的前列腺癌中存在跨膜丝氨酸蛋白酶2(TMPRSS2)和E26(ETS)转录因子间的基因融合,其中TMPRSS2-ERG最为常见。TMPRSS2-ERG基因融合造成的ERG过表达参与了前列腺的癌变。雄激素受体结合和遗传毒性胁迫共同诱导了染色体的靠近和TMPRSS2-ETS的基因融合。TMPRSS2-ERG基因融合可作为前列腺癌诊断的一种生物标志物,并可通过病人尿液检测来实现。文章对TMPRSS2-ETS基因融合的特征、融合及致癌及临床应用进行了综述。     

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.     

Regulation of ICAM-1 mRNA stability by cycloheximide: Role of serine/threonine phosphorylation and protein synthesis

Cycloheximide is a protein synthesis inhibitor that superinduces the expression of many genes by preventing the degradation of otherwise labile mRNAs. In some genes this depends on the presence of the AUUUA destabilizing multimers in the 3′UTR. We examined the effect of cycloheximide on the murine intercellular adhesion molecule-1 (ICAM-1; CD54) gene expression in several cell lines including A20 (B cell lymphoma), T28 (T cell hybridoma), P388D1 (monocytic cell), SVEC4-10 (lymphoid endothelial cell), and ICAM-1-transfected murine fibroblast L cells. Cycloheximide was indeed able to dramatically increase the accumulation of ICAM-1 mRNA in all the cell lines examined except T28, and this seemed to be due to the stablization of the ICAM-1 mRNA as indicated by the half-life analysis. To determine whether this effect is dependent on the 3′UTR containing the AUUUA sequences, L cells were transfected with either the full-length ICAM-1 cDNA or a truncated form lacking the AUUUA sequences in the 3′UTR (ICAM-1Δ3). There was no discernible difference in the effect of cycloheximide on ICAM-1 mRNA accumulation or half-life between the two types of transfected cells. The effect of cycloheximide on ICAM-1 mRNA was markedly suppressed by serine/threonine (ser/thr) kinase inhibitors, H-7 and staurosporine, whereas the ser/thr phosphatase inhibitor, okadaic acid, augmented the cycloheximide effect. Inhibitors of protein tyrosine kinases and phosphatases had no effect. Unexpectedly, the level of cell surface ICAM-1 as well as de novo synthesis of ICAM-1 in SVEC4-10 and the ICAM-1-transfected L cells were also upregulated by cycloheximide, whereas the overall protein synthesis in these cells was profoundly inhibited, suggesting that ICAM-1 protein synthesis in these cells escapes the translational inhibition by cycloheximide. These results suggest that the stabilization of ICAM-1 mRNA by cycloheximide is independent of its translational inhibition and that ser/thr phosphorylation of unidentified protein(s) seems to play a crucial role in this effect. © 1995 Wiley-Liss, Inc.