USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells

The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer.     

Flap endonuclease 1 Facilitated Hepatocellular Carcinoma Progression by Enhancing USP7/MDM2-mediated P53 Inactivation

Overexpression of Flap endonuclease 1 (FEN1) has been previously implicated in hepatocellular carcinoma (HCC), while its expression features and mechanisms remain unclear. In the current study, differential expression genes (DEGs) were screened in HCC tissues and normal liver tissues in 4 Gene Expression Omnibus (GEO) datasets. FEN1, one of the hub co-overexpressed genes, was further determined overexpressed in HCC tissues in TCGA, local HCC cohorts, and hepatocarcinogenesis model. In addition, high expression of FEN1 indicated poor prognosis of HCC patients. Loss-of-function and gain-of-function assays demonstrated that FEN1 enhanced the proliferation, cell cycle phage transition, migration/ invasion, therapy resistance, xenograft growth, and epithelial-mesenchymal transition (EMT) process of HCC cells. Mechanically, FEN1 could inactivate P53 signaling by preventing the ubiquitination and degradation of mouse double minute 2 (MDM2) via recruiting ubiquitin-specific protease 7 (USP7). Interfering USP7 with {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 significantly reversed the malignant phenotypes activated by FEN1. In conclusion, this study suggests FEN1 as a robust prognostic biomarker and potential target for HCC.     

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.     

The proteins DLK1 and DLK2 modulate NOTCH1-dependent proliferation and oncogenic potential of human SK-MEL-2 melanoma cells

NOTCH receptors regulate cell proliferation and survival in several types of cancer cells. Depending on the cellular context, NOTCH1 can function as an oncogene or as a tumor suppressor gene. DLK1 is also involved in the regulation of cell growth and cancer, but nothing is known about the role of DLK2 in these processes. Recently, the proteins DLK1 and DLK2 have been reported to interact with NOTCH1 and to inhibit NOTCH1 activation and signaling in different cell lines. In this work, we focused on the role of DLK proteins in the control of melanoma cell growth, where NOTCH1 is known to exert an oncogenic effect. We found that human DLK proteins inhibit NOTCH signaling in SK-MEL-2 metastatic melanoma cells. Moreover, the proliferation rate of these cells was dependent upon the level of NOTCH activation and signaling as regulated by DLK proteins. In particular, high levels of NOTCH inhibition resulted in a decrease, whereas lower levels of NOTCH inhibition led to an increase in melanoma cell proliferation rates, both in vitro and in vivo. Finally, our data revealed additive NOTCH-mediated effects of DLK proteins and the γ-secretase inhibitor DAPT on cell proliferation. The data presented in this work suggest that a fine regulation of NOTCH signaling plays an important role in the control of metastatic melanoma cell proliferation. Our results open the way to new research on the role of DLK proteins as potential therapeutic tools for the treatment of human melanoma.     

Synthesis and biological evaluation of thiazole derivatives as novel USP7 inhibitors

Herpesvirus-associated Ubiquitin-Specific Protease (HAUSP, also called USP7) interacts with and stabilizes Mdm2, and represents one of the first examples that deubiquitinases oncogenic proteins. USP7 has been regarded as a potential drug target for cancer therapy. Inhibitors of USP7 have been recently shown to suppress tumor cell growth in vitro and in vivo. Based on leading USP7 inhibitors P5091 and P22077, we designed and synthesized a series of thiazole derivatives. The results of in vitro assays showed that the thiazole compounds exhibited low micromolar inhibition activity against both USP7 enzyme and cancer cell lines. The compounds induced cell death in a p53-dependent and p53-independent manner. Taken together, this study may provide thiazole compounds as a new class of USP7 inhibitors.     

Lidocaine inhibits melanoma cell proliferation by regulating ERK phosphorylation

The melanoma is responsible for the majority of all skin cancer–related deaths worldwide. Evidence suggests that local anesthetics provide some benefit in the treatment of cancer via inhibition of cellular proliferation, invasion and migration. However, the potential antiproliferative effects of local anesthetics in the treatment of melanoma remain to be elucidated. In this study, we investigated the antiproliferative effects and underlying mechanism of the commonly used local anesthetic (lidocaine) on melanoma cells. A375 melanoma cells were treated by lidocaine or vemurafenib. Cell Counting Kit-8, histological staining, flow cytometric analysis, immunohistochemical staining, and Western blot analyses were carried out to test the effects of lidocaine and vemurafenib on A375 cells. BALB/C-nu/nu mice intraperitoneally injected with A375 cells were treated by lidocaine, and then tumor volume and weight were calculated. Lidocaine exhibited vemurafenib-like effects totally. Lidocaine inhibited A375 melanoma cell proliferation in a dose- and time-dependent manner and colony formation also showed a dose-dependent inhibition. Lidocaine treatment resulted in the arrest of cell-cycle progression in the G1 phase and inhibited Ki-67 expression in a dose-dependent manner. This effect was associated with inhibited extracellular signal–regulated kinase (ERK) phosphorylation. In vivo experiments revealed that intravenous injections of lidocaine suppressed tumor volume and weight. Lidocaine inhibits melanoma cell proliferation in a dose- and time-dependent manner via a mechanism that may involve inhibition of the ERK signaling pathway. Thus, lidocaine may provide some benefit for the treatment of melanoma.     

Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth

FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R.

Previously, using virtual screening and functional testing, we identified a lead compound (INT2–31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo.

INT2–31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2–31 significantly inhibited cell proliferation and viability (range 0.05–10 μM). More importantly, 15 mg/kg of INT2–31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo.

Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2–31) has potential anti-neoplastic therapeutic effects in human melanoma.     

Inhibition of Proliferation of Non-small Cell Lung Cancer Cells by a bFGF Antagonist Peptide

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality worldwide. Basic fibroblast growth factor (bFGF) is up-regulated in NSCLC patients and plays an important role in tumor growth. In this paper, we attempt to evaluate the therapeutic potential of bFGF binding peptide (named as P7) using as a potent bFGF antagonist via exploration of its anti-proliferation effect on NSCLC cells. Our experiments showed that P7 peptide inhibited bFGF-stimulated proliferation of NSCLC cell lines including A549, H1299, and H460. The inhibitory mechanism of P7 involved cell cycle arrest at the G0/G1phase caused by suppression of cyclin D1, blockage of the activation of Erk1/2, P38, Akt, and inhibition of bFGF internalization. Strategies using bFGF antagonist peptides with potent anti-proliferation property may have therapeutic potential in NSCLC.     

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd³⁺) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca²⁺ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.     

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.     

Ubiquitin-specific protease 2 regulates Ang Ⅱ–induced cardiac fibroblasts activation by up-regulating cyclin D1 and stabilizing β-catenin in vitro

Cardiac fibrosis, featuring abnormally elevated extracellular matrix accumulation, decreases tissue compliance, impairs cardiac function and accelerates heart failure. Mounting evidence suggests that the ubiquitin proteasome pathway is involved in cardiac fibrosis. In the present study, ubiquitin-specific protease 2 (USP2) was identified as a novel therapeutic target in cardiac fibrosis. Indeed, USP2 expression was increased in angiotensin II–induced primary cardiac fibroblasts (CFs) from neonatal rats. In addition, USP2 inhibition suppressed CFs proliferation, collagen synthesis and cell cycle progression. Furthermore, USP2 interacted with β-catenin, thereby regulating its deubiquitination and stabilization in CFs. To sum up, these findings revealed that USP2 has a therapeutic potential for the treatment of cardiac fibrosis.     

Anti-Proliferation Effect of a Short Peptide on bFGF-Induced Human Gastric Cancer Cells

Gastric cancer is the second cause of the common cancer deaths worldwide. Basic fibroblast growth factor (bFGF) was up-regulated in patients with gastric cancer and was regarded as a potential therapeutic target. We have previously obtained a high-affinity bFGF-binding peptide (named P7) from the phage display random heptapeptide library. In this study, we first proved that P7 peptides inhibited proliferation of gastric cancer cell lines including SGC-7901, AGS, and BGC-823 cells stimulated with bFGF. Further investigations with SGC-7901 cells revealed that the mechanisms of P7 peptides inhibiting bFGF-stimulated cell proliferation in vitro involved cell cycle arrest at the G0/G1 phase, blockade of the activation of Erk and P38 cascades, and suppression of bFGF internalization. The results suggested that the bFGF-binding peptide may have therapeutic potential in gastric cancer therapy.     

Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.     

Protein kinase D1 stimulates proliferation and enhances tumorigenesis of MCF-7 human breast cancer cells through a MEK/ERK-dependent signaling pathway

Protein kinase D1, PKD1, is a novel serine/threonine kinase whose altered expression and dysregulation in many tumors as well as its activation by several mitogens suggest that this protein could regulate proliferation and tumorigenesis. Nevertheless, the precise signaling pathways used are still unclear and the potential direct role of PKD1 in tumor development and progression has not been yet investigated. In order to clarify the role of PKD1 in cell proliferation and tumorigenesis, we studied the effects of PKD1 overexpression in a human adenocarcinoma breast cancer cell line, MCF-7 cells. We demonstrated that overexpression of PKD1 specifically promotes MCF-7 cell proliferation through accelerating G0/G1 to S phase transition of the cell cycle. Moreover, inhibition of endogenous PKD1 significantly reduced cell proliferation. Taken together, these results clearly strengthen the regulatory role of PKD1 in cell growth. We also demonstrated that overexpression of PKD1 specifically diminished serum- and anchorage-dependence for proliferation and survival in vitro and allowed MCF-7 cells to form tumors in vivo. Thus, all these data highlight the central role of PKD1 in biological processes which are hallmarks of malignant transformation. Analysis of two major signaling pathways implicated in MCF-7 cell proliferation showed that PKD1 overexpression significantly increased ERK1/2 phosphorylation state without affecting Akt phosphorylation. Moreover, PKD1 overexpression-stimulated cell proliferation and anchorage-independent growth were totally impaired by inhibition of the MEK/ERK kinase cascade. However, neither of these effects was affected by blocking the PI 3-kinase/Akt signaling pathway. Thus, the MEK/ERK signaling appears to be a determining pathway mediating the biological effects of PKD1 in MCF-7 cells. Taken together, all these data demonstrate that PKD1 overexpression increases the aggressiveness of MCF-7 breast cancer cells through enhancing their oncogenic properties and would, therefore, define PKD1 as a potentially new promising anti-tumor therapeutic target.     

Let-7a下调k-Ras和c-Myc癌基因的表达抑制肾癌细胞增殖

目的：MicroRNA 是近年发现的一类单链小分子RNA,对它的研究已成为一个新的热点。最近的研究发现,1et-7a 在细胞内影响着基因的表达调控,在疾病发生中起着及极重要的作用,尤其是在肿瘤的发展过程中,let-7a扮演着不可替代的角色。本文主要研究let-7a 在肾癌细胞株中的表达情况及其调控的靶基因、抑制细胞增殖的机制,对探索肾癌的致病基因,寻求肾癌新的治疗途径有重要意义。方法：应用化学合成的let-7a 模拟物（mimics）用脂质体Lipofectamine 2000 在体外瞬时转染786-O 和Caki-1肾癌细胞株,转染48 小时后采用荧光定量RT-PCR的方法检测let-7a 及c-Myc、k-Ras mRNA的表达情况,Western blot 检测这两株肾癌细胞转染了let-7a mimics 后c-Myc 及k-Ras 蛋白的表达变化;转染let-7a mimics 后分别在24、48、72 小时三个时间点用CCK-8 试剂盒检测对肾癌细胞株增殖的影响。结果：786-O 和Caki-1 肾癌细胞株中let-7a 的表达量明显低于正常肾小管上皮细胞株HK-2（P〈0.05）; 转染了let-7amimics的786-O 和Caki-1 肾癌细胞株,RT-PCR 及Western blot 结果显示c-Myc、k-Ras 在基因及蛋白的表达水平明显下调（P〈0.05）;CCK-8 检测结果显示转染了let-7a mimics的肾癌细胞株细胞增殖能力明显明显受到抑制,与阴性对照组比较差异有统计学意义（P〈0.05）。结论：Let-7a 在在肿瘤细胞与正常细胞中存在明显差异,let-7a 通过调控c-Myc、k-Ras的表达能抑制肾癌细胞增殖。Let-7a mimics 可以抑制肾癌细胞的增殖,因此上调Let-7a 的表达有可能成为肾癌基因治疗的一种有效治疗手段。     

Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3 （GSK-3） might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3β may contribute to NF-κB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3β activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.     

Circ_0079593 facilitates proliferation,metastasis, glucose metabolism and inhibits apoptosis in melanoma by regulating the miR-516b/GRM3 axis

Many studies confirm that circular RNA (circRNA) plays an important regulatory role in the malignant progression of cancer, including melanoma. However, the role of a novel circRNA, circ_0079593, in melanoma is unclear. The expression levels of circ_0079593 and miR-516b were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was measured by cell counting kit-8 (CCK-8) assay, and cell migration and invasion were evaluated using transwell assay. Meanwhile, western blot (WB) analysis was employed to determine the levels of proliferation and metastasis-related proteins, as well as metabotropic glutamate receptor 3 (GRM3) protein. Furthermore, cell apoptosis was tested by detecting the cell apoptosis rate and Caspase-3 activity. The glucose consumption and lactate production of cells were measured to evaluate cell glucose metabolism. Moreover, dual-luciferase reporter assay and biotin-labeled RNA pull-down assay were used to confirm the interaction between miR-516b and circ_0079593 or GRM3. In addition, mice xenograft models were constructed to explore the effect of circ_0079593 on melanoma tumor growth in vivo. Our results discovered that circ_0079593 was highly expressed in melanoma, and its silencing suppressed melanoma cell proliferation, migration, invasion, glucose metabolism and promoted apoptosis. Moreover, we found that circ_0079593 could serve as a sponge of miR-516b, and miR-516b could target GRM3 in melanoma. The rescue experiments revealed that both miR-516b inhibitor and GRM3 overexpression could reverse the inhibition effect of circ_0079593 knockdown on melanoma progression. Additionally, in vivo experiments also revealed that circ_0079593 interference suppressed melanoma tumor growth. Our study concluded that circ_0079593 accelerated melanoma progression via upregulating GRM3 by sponging miR-516b, which suggested that circ_0079593 had the potential to be a new therapeutic biomarker for melanoma.