Up-regulation of MELK by E2F1 promotes the proliferation in cervical cancer cells

Cervical cancer is a common gynecologic cancer and a frequent cause of death. In this study, we investigated the role of MELK (maternal embryonic leucine zipper kinase) in cervical cancer. We found that HPV 18 E6/E7 promoted MELK expression by activating E2F1. MELK knockdown blocked cancer cells growth. Furthermore, we used MELK-8A to inhibit the kinase activity of MELK and caused the G2/M phase arrest of cancer cells. Under the treatment of inhibitors, Hela cells formed multipolar spindles and eventually underwent apoptosis. We also found that MELK is involved in protein translation and folding during cell division through the MELK interactome and the temporal proteomic analysis under inhibition with MELK-8A. Altogether, these results suggest that MELK may play a vital role in cancer cell proliferation and indicate a potential therapeutic target for cervical cancer.     

Activation of G‐protein coupled estrogen receptor inhibits the proliferation of cervical cancer cells via sustained activation of ERK1/2

Cervical cancer is one of the most common gynaecological women cancer and suggested to be modulated by estrogenic signals. G protein‐coupled receptor (GPER), a seven‐transmembrane G protein‐coupled receptor, has been reported to regulate the cell proliferation of various cancers. But there is no study investigating the effects of GPER on the progression of cervical cancer. In the present study, we revealed for the first time that GPER was also highly expressed in various human cervical cancer cells. Activation of GPER via its specific agonist G‐1 induced G2/M cell cycle arrest and down regulation of cyclin B via a time dependent manner. Furthermore, G‐1 treatment induced sustained activation of extracellular‐signal‐regulated kinases (ERK)1/2 via epidermal growth factor receptor (EGFR) signals. Both inhibitors of ERK1/2 and EGFR significantly abolished G‐1‐induced suppression of cell proliferation and down regulation of cyclin B. Generally, our study revealed that GPER is highly expressed in human cervical cancer cells and its activation inhibits cell proliferation via EGFR/ERK1/2 signals. It suggested that G‐1 can be considered as a potential new pharmacological tool to reduce the growth of cervical cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

GBP3 promotes glioma cell proliferation via SQSTM1/p62-ERK1/2 axis

Guanylate binding proteins (GBPs) are interferon-inducible large GTPases and play a crucial role in cell-autonomous immunity. However, the biology function of GBPs in cancer remains elusive. GBP3 is specifically expressed in adult brain. Here we show that GBP3 is highly elevated in human glioma tumors and glioma cell lines. Overexpression of GBP3 dramatically increased glioma cell proliferation whereas silencing GBP3 by RNA interference produced opposite effects. We further showed that GBP3 expression was able to induce sequestosome-1(SQSTM1, also named p62) expression and activate extracellular signal-regulated kinase (ERK1/2). The SQSTM1-ERK1/2 signaling cascade was essential for GBP3-promoted cell growth because depletion of SQSTM1 markedly reduced the phosphorylated ERK1/2 levels and GBP3-mediated cell growth, and inhibition of mitogen-activated protein kinase/ERK kinase abolished GBP3-induced glioma cell proliferation. Consistently, GBP3 overexpression significantly promoted glioma tumor growth in vivo and its expression was inversely correlated with the survival rate of glioma patients. Taken together, these results for the first time suggest that GBP3 contributes to the proliferation of glioma cells via regulating SQSTM1-ERK1/2 pathway, and GBP3 might represent as a new potential therapeutic target against glioma.     

METTL16 promotes cell proliferation by up-regulating cyclin D1 expression in gastric cancer

N6-methyladenosine (m6A) is a well-known modification of RNA. However, as a key m6A methyltransferase, METTL16 has not been thoroughly studied in gastric cancer (GC). Here, the biological role of METTL16 in GC and its underlying mechanism was studied. Immunohistochemistry was used to detect the expression of METTL16 and relationship between METTL16 level and prognosis of GC was analysed. CCK8, colony formation assay, EdU assay and xenograft mouse model were used to study the effect of METTL16. Regulatory mechanism of METTL16 in the progression of GC was studied through flow cytometry analysis, RNA degradation assay, methyltransferase inhibition assay, RT-qPCR and Western blotting. METTL16 was highly expressed in GC cells and tissues and was associated with prognosis. In vitro and in vivo experiments confirmed that METTL16 promoted proliferation of GC cells and tumour growth. Furthermore, down-regulation of METTL16 inhibited proliferation by G1/S blocking. Significantly, we identified cyclin D1 as a downstream effector of METTL16. Knock-down METTL16 decreased the overall level of m6A and the stability of cyclin D1 mRNA in GC cells. Meanwhile, inhibition of methyltransferase activity reduced the level of cyclin D1. METTL16-mediated m6A methylation promotes proliferation of GC cells through enhancing cyclin D1 expression.     

Differences in degradation lead to asynchronous expression of cyclin E1 and cyclin E2 in cancer cells

Cyclin E1 is expressed at the G₁/S phase transition of the cell cycle to drive the initiation of DNA replication and is degraded during S/G₂M. Deregulation of its periodic degradation is observed in cancer and is associated with increased proliferation and genomic instability. We identify that in cancer cells, unlike normal cells, the closely related protein cyclin E2 is expressed predominantly in S phase, concurrent with DNA replication. This occurs at least in part because the ubiquitin ligase component that is responsible for cyclin E1 downregulation in S phase, Fbw7, fails to effectively target cyclin E2 for proteosomal degradation. The distinct cell cycle expression of the two E-type cyclins in cancer cells has implications for their roles in genomic instability and proliferation and may explain their associations with different signatures of disease.     

The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer

The deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination of FASN, MDM2, MDMX or Aurora A. Aberrant expression of USP2a has been linked to progression of human tumors, particularly prostate cancer. However, little is known about the role of USP2a or its mechanism of action in bladder cancer. Here, we provide evidence that USP2a is an oncoprotein in bladder cancer cells. Enforced expression of USP2a caused enhanced proliferation, invasion, migration and resistance to several chemotherapeutic reagents, while USP2a loss resulted in slower proliferation, greater chemosensitivity and reduced migratory/invasive capability compared with control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal human bladder epithelial cells. USP2a bound to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation of cyclin A1 by a block in degradation. Enforced expression of wild type USP2a, but not an inactive USP2a mutant, resulted in cyclin A1 accumulation and increased cell proliferation. We conclude that USP2a impairs ubiquitination and stabilizes an important cell cycle regulator, cyclin A1, raising the possibility of USP2a targeting as a therapeutic strategy against bladder tumors in combination with chemotherapy.     

Long non-coding RNA POU6F2-AS2 promotes cell proliferation and drug resistance in colon cancer by regulating miR-377/BRD4

The aim of this study was to explore the molecular mechanism of lncRNA POU6F2-AS2 in proliferation and drug resistance of colon cancer. Total paired 70 colon cancer and adjacent normal tissues were collected from colon cancer patients. Colon cancer and normal colonic epithelial cells were purchased. POU6F2-AS2 was up- or down-expressed by vectors. LC50 of all cell lines before and after transfection with these plasmids was detected. qRT-PCR was used to detect the expression of POU6F2-AS2, miR-377 and BRD4 before or after transfection. In situ hybridization was also undertaken to detect the level of POU6F2-AS2. Different concentrations of 5-Fu (0, 1, 2.5, 5, 10, 20, 40 and 80 μg/mL) were used for 5-FU insensitivity assay. CCK-8 and crystal violet staining assay were used for detecting cell proliferation, and flow cytometry was used for identifying cell cycle distribution and apoptosis. In order to detect the fragmented DNA in apoptotic cells, TUNEL assay was used. RNA pull-down assay and luciferase reporter assay were used to verify the binding site. Rescue assay confirmed the subtractive effect of miR-377 inhibitors. POU6F2-AS2 was highly expressed in colon cancer, which was associated with clinical pathology. Up-regulated POU6F2-AS2 promoted cell proliferation and cell cycle of colon cancer cells. Overexpression of POU6F2-AS2 inhibited the expression of miR-377 and then up-regulated the expression of BRD4. Up-regulated BRD4 ultimately promoted cell proliferation and cell survival Down-regulated POU6F2-AS2 showed enhanced sensitivity of 5-FU. POU6F2-AS2 promoted cell proliferation and drug resistance in colon cancer by regulating miR-377/BRD4 gene.     

A novel FoxM1-PSMB4 axis contributes to proliferation and progression of cervical cancer

The abnormally high activity of the proteasome system is closely related to the occurrence and development of various tumors. PSMB4 is a non-catalytic subunit for the proteasome assembly. Although the reports from genetic screening have demonstrated it’s a driver gene for cell growth in several types of solid tumor, its expression pattern and regulatory mechanisms in malignant diseases are still elusive. Here, we found that PSMB4 is overexpressed in cervical cancer tissues. And knockdown of PSMB4 significantly inhibited cervical cancer cell proliferation. The mechanistic study revealed that FoxM1, a master regulator of cell division, binds directly to the promoter region of PSMB4 and regulates the PSMB4 expression in the mRNA level. In addition, the data analysis from TCGA showed a positive correlation between FxoM1 and PSMB4 in cervical cancer. Furthermore, the loss of functional and rescue experiments confirmed that PSMB4 is required for FoxM1-driven cervical cancer cell proliferation. Collectively, our study explains the phenomenon of dysregulated expression of PSMB4 in cervical cancer tissues and verifies its driver effect on cancer cell proliferation. More importantly, it highlights a FoxM1-PSMB4 axis could be a potential target for the treatment of cervical cancer.     

CP-31398 inhibits the progression of cervical cancer through reversing the epithelial mesenchymal transition via the downregulation of PAX2s

CP-31398, a styrylquinazoline, emerges from a screen for therapeutic agents that restore the wild-type DNA-binding conformation of mutant p53 to suppress tumors in vivo, but its effects on cervical cancer (CC) remain unknown. Hence, this study aimed to explore the effects CP-31398 has on the CC cells and to investigate whether it is associated with paired box 2 (PAX2) expression. CC cells were treated with different concentrations of CP-31398 (1, 2, 4, 6, 8, and 10 μg/ml) to determine the optimum concentration using fluorometric microculture cytotoxicity assay. After constructing the sh-PAX2 vector, CC cells were transfected with sh-PAX2 or treated with CP-31398. The effects of CP-31398 or PAX2 silencing on CC cell proliferation, apoptosis, invasion, and migration were evaluated. Epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, vimentin, N-cadherin, snail, and twist in CC cells were detected. Tumor formation experiment in nude mice was performed to observe tumor growth. The optimum concentration of CP-31398 was 2 μg/ml. PAX2 was overexpressed in CC cells. CC cells treated with CP-31398 or treated with sh-PAX2 inhibited proliferation, invasion, and migration but promoted apoptosis with decreased PAX2 expression. The EMT process in CC cells was also reversed after treatment with CP-31398 or sh-PAX2. Moreover, the tumor formation experiment in nude mice revealed the inhibitory activity of CP-31398 in CC tumor in nude mice by suppressing PAX2. Our results provide evidence that CP-31398 could inhibit EMT and promote apoptosis of CC cells to curb CC tumor growth by downregulating PAX2.     

Circ_0005615 promotes cervical cancer cell growth and metastasis by modulating the miR-138-5p/KDM2A axis

Cervical cancer (CC) is a highly fatal gynecological malignancy due to its high metastasis and recurrence rate. Circular RNA (circRNA) has been regarded as a regulator of CC. However, the underlying molecular mechanism of circ_0005615 in CC remains unclear. The levels of circ_0005615, miR-138-5p, and lysine demethylase 2A (KDM2A) were measured using qRT-PCR or western blot. Cell proliferation was assessed by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, and colony formation experiments. Cell invasion and migration were tested by transwell assay and wound healing assay. Flow cytometry and Caspase-Glo 3/7 Assay kit were used to analyze cell apoptosis. The expression of proliferation-related and apoptosis-related markers was detected by western blot. The binding relationships among circ_0005615, miR-138-5p, and KDM2A were verified by dual-luciferase reporter assay or RNA immunoprecipitation assay. Xenograft assay was applied to detect the effect of circ_0005615 in vivo. Circ_0005615 and KDM2A were upregulated, while miR-138-5p was downregulated in CC tissues and cells. Circ_0005615 knockdown retarded cell proliferation, migration, and invasion, while promoting apoptosis. Besides, circ_0005615 sponged miR-138-5p, and miR-138-5p could target KDM2A. miR-138-5p inhibitor reversed the regulation of circ_0005615 knockdown on CC cell growth and metastasis, and KDM2A overexpression also abolished the inhibitory effect of miR-138-5p on CC cell growth and metastasis. In addition, we also discovered that circ_0005615 silencing inhibited CC tumor growth in vivo. Circ_0005615 acted as a tumor promoter in CC by regulating the miR-138-5p/KDM2A pathway.     

Long noncoding RNA POU3F3 promotes cancer cell proliferation in prostate carcinoma by upregulating rho-associated protein kinase 1

Long noncoding RNAs (lncRNAs) POU3F3 is overexpressed in esophageal squamous-cell carcinomas, while its role in other human cancers is unclear. In this study we found that POU3F3 and rho-associated protein kinase 1 (ROCK1) were both increased in tumor tissues than in adjacent healthy tissues of patients with prostate carcinoma. Expression levels of POU3F3 increased with increase in the diameter of tumor but were not significantly affected by lymph node metastasis or distant metastasis. Expression levels of POU3F3 and ROCK1 were positive correlated in tumor tissues but not in adjacent healthy tissues. POU3F3 and ROCK1 overexpression promoted, while ROCK1 knockdown inhibited the proliferation of prostate carcinoma cells. ROCK1 knockdown reduced the enhancing effect of POU3F3 overexpression on cancer cell proliferation. POU3F3 overexpression led to ROCK1 overexpression in prostate carcinoma cells, while ROCK1 overexpression did not significantly affect POU3F3 expression. Therefore, lncRNA POU3F3 may promote cancer cell proliferation in prostate carcinoma by upregulating ROCK1.     

miR-503 expression is downregulated in cervical cancer and suppresses tumor growth by targeting AKT2

Previous studies have reported that microRNAs function as key regulators in tumor development and progression. This study aims to investigate the functional effects of miR-503 expression in cervical cancer (CC) progression. We detected the expression of miR-503 in CC tissues and cell lines using quantitative real-time polymerase chain reaction. Synthesized miR-503 mimics or inhibitors were used to upregulate or downregulate the expression of miR-503 in HeLa or SiHa cells. Cell Counting Kit-8 and colony formation assay were used to detect the ability of cell proliferation. Furthermore, luciferase assay and Western blot were applied to confirm the target of miR-503 in CC cells. Here, we demonstrated that miR-503 expression was significantly downregulated in CC tissues, compared with adjacent normal tissues. miR-503 expression was significantly associated with tumor size and International Federation of Gynecology and Obstetrics stage. Furthermore, increasing miR-503 expression in CC cells dramatically inhibited cell proliferation, colony formation ability of CC. However, reducing miR-503 had reverse effects on these malignant behaviors. Moreover, we demonstrated that miR-503 inhibited cell proliferation by targeting AKT2 3′-untranslated region and affected its expression. Overexpression of AKT2 rescued the effects induced by miR-503 on cell proliferation. Therefore, our results indicated that miR-503 may serve as a tumor suppressor in CC and provide a potential value for CC treatment.