Plasma extracellular vesicles proteomics in meningioma patients.

Tumor derived Extracellular vesicles (EVs) in circulating system may contain tumor-specific markers, and EV detection in body fluids could become an important tool for early tumor diagnosis, prognosis assessment. Meningiomas are the most common benign intracranial tumors, few studies have revealed specific protein markers for meningiomas from patients' body fluids. In this study, using proximity labeling technology and non-tumor patient plasma as a control, we detected protein levels of EVs in plasma samples from meningioma patients before and after surgery. Through bioinformatics analysis, we discovered that the levels of EV count and protein count in meningioma patients were significantly higher than those in healthy controls, and were significantly decreased postoperatively. Among EV proteins in meningioma patients, the levels of MUC1, SIGLEC11, E-Cadherin, KIT, and TASCTD2 were found not only significantly elevated than those in healthy controls, but also significantly decreased after tumor resection. Moreover, using publicly available GEO databases, we verified that the mRNA level of MUC1, SIGLEC11, and CDH1 in meningiomas were significantly higher in comparison with normal dura mater tissues. Additionally, by analyzing human meningioma specimens collected in this study, we validated the protein levels of MUC1 and SIGLEC11 were significantly increased in WHO grade 2 meningiomas and were positively correlated with tumor proliferation levels. This study indicates that meningiomas secret EV proteins into circulating system, which may serve as specific markers for diagnosis, malignancy predicting and tumor recurrent assessment.

Adaptive resistance of tumor cells to anti-vascular endothelial growth factor therapy: A reversible phenomenon.

Vascular endothelial growth factor (VEGF) inhibition is an essential targeted strategy for malignant tumors, but its efficacy is severely constrained by drug resistance. The traditional view holds that the target of VEGF inhibition is endothelial cells, and thus compensatory angiogenesis is considered the main mechanism of drug resistance. In this study, we found that tumor cells themselves could develop acquired resistance to VEGF therapy, indicating an independent resistance mechanism apart from angiogenesis. Notably, this acquired resistance was temporary, disappearing completely four days after discontinuing exposure to the drug in vitro. Our findings suggest that tumor cells may also be targets of VEGF inhibition, and their response to treatment should not be overlooked in contributing to drug resistance.

TMEM52B Isoforms P18 and P20 Differentially Promote the Oncogenesis and Metastasis of Nasopharyngeal Carcinoma.

Transmembrane protein 52B (TMEM52B), a newly identified tumor-related gene, has been reported to regulate various tumors, yet its role in nasopharyngeal carcinoma (NPC) remains unclear. Transcriptomic analysis of NPC cell lines reveals frequent overexpression of TMEM52B, and immunohistochemical results show that TMEM52B is associated with advanced tumor stage, recurrence, and decreased survival time. Depleting TMEM52B inhibits the proliferation, migration, invasion, and oncogenesis of NPC cells in vivo. TMEM52B encodes two isoforms, TMEM52B-P18 and TMEM52B-P20, differing in their N-terminals. While both isoforms exhibit similar pro-oncogenic roles and contribute to drug resistance in NPC, TMEM52B-P20 differentially promotes metastasis. This functional discrepancy may be attributed to their distinct subcellular localization; TMEM52B-P18 is confined to the cytoplasm, while TMEM52B-P20 is found both at the cell membrane and in the cytoplasm. Mechanistically, cytoplasmic TMEM52B enhances AKT phosphorylation by interacting with phosphoglycerate kinase 1 (PGK1), fostering NPC growth and metastasis. Meanwhile, membrane-localized TMEM52B-P20 promotes E-cadherin ubiquitination and degradation by facilitating its interaction with the E3 ubiquitin ligase NEDD4, further driving NPC metastasis. In conclusion, the TMEM52B-P18 and TMEM52B-P20 isoforms promote the metastasis of NPC cells through different mechanisms. Drugs targeting these TMEM52B isoforms may offer therapeutic benefits to cancer patients with varying degrees of metastasis.

C12orf59 Promotes Esophageal Squamous Cell Carcinoma Progression via YAP-Mediated Epithelial-Mesenchymal Transition.

C12orf59 is a novel gene widely expressed in diverse normal human tissues. Aberrant expression of C12orf59, which is involved in tumor progression, has been reported in a few types of cancer. However, its expression and biological function in esophageal squamous cell carcinoma (ESCC) remain largely unclear. Here, we found that the mRNA and protein levels of C12orf59 were prominently higher in both tumor tissues and most ESCC cell lines. Functionally, C12orf59 overexpression promoted ESCC cell proliferation, migration and invasion, whereas C12orf59 depletion worked oppositely. Mechanistically, C12orf59 exerted its oncogenic function through the induction of epithelial-mesenchymal transition (EMT) of ESCC cells, which relied on Yes-associated protein (YAP) dephosphorylation and nuclear translocation. Constitutively active YAP further facilitated cell migration, invasion and EMT induced by enforced C12orf59 overexpression. On the contrary, increased cell motility and EMT caused by enforced C12orf59 overexpression were dramatically repressed upon YAP inactivation by verteporfin. Thus, we conclude that YAP activation driven by C12orf59 contributes to the malignancy of ESCC through EMT and that targeting drugs for C12orf59 combined with YAP inhibitor may be a potential therapeutic strategy for ESCC.

The Clinical Relevance and Tumor Promoting Function of C19orf10 in Kidney Renal Clear Cell Carcinoma.

Kidney renal clear cell carcinoma (KIRC) is the most common primary renal neoplasms. Currently, there are few molecular indicators and therapeutic targets that can be used in diagnostic and prognostic assessment. In this study, we identified the C19orf10 expression in KIRC specimens and explored the diagnostic and prognostic value of C19orf10 in KIRC using TCGA and CPTAC database. Loss-of- and gain-of- function of C19orf10 was performed to investigate the roles of C19orf10 on KIRC cell viability, proliferation, migration and invasion via CCK-8, Edu incorporation and Transwell assays respectively. C19orf10 was overexpressed in KIRC tissues and the elevated C19orf10 expression was closely associated with clinicopathological characteristics of KIRC including histological grade, TNM stage, metastatic status. Silencing C19orf10 significantly suppressed the viability, proliferation, migration and invasion ability, while overexpression of C19orf10 promoted the progression and malignant phenotype in KIRC cells. Furthermore, C19orf10 exerted its carcinogenic function by regulating ZO-1 and PTEN/Akt signaling pathway. Moreover, the Kaplan-Meier survival analysis, Cox regression analysis and receiver operating curve analysis showed that patients with C19orf10 overexpression have poor survival time. C19orf10 could discriminate KIRC patients with high-risk from low-risk. Taken together, C19orf10 contributes to KIRC development via ZO-1 and PTEN/Akt signaling pathway and C19orf10 could serve as a potential diagnostic and prognostic candidate and therapeutic target of KIRC.

PAPP-A functions as a tumor suppressor and is downregulated in renal cell carcinoma.

Pregnancy-associated plasma protein A (PAPP-A) is a proteolytic enzyme produced by the placenta. The expression and role of PAPP-A in renal cell carcinoma (RCC) remain elusive. The aim of this study was to investigate the role and the molecular mechanisms of PAPP-A in RCC. Initially, we evaluated the expression of PAPP-A in samples from patients with RCC and cell lines by quantitative PCR, western blot and immunohistochemical staining, and examined the role of PAPP-A in RCC cells by cell viability, colony formation and Transwell assays. Next, we investigated the molecular mechanisms regulating the tumor suppressor function of PAPP-A. Our results demonstrated that PAPP-A is expressed at low levels in RCC tissues and cells. Clinical data analysis revealed a significant correlation between PAPP-A expression and RCC-related death (P < 0.0115). Overexpression of PAPP-A inhibited viability, proliferation, migration and invasion of RCC cells. Furthermore, PAPP-A overexpression significantly increased phosphorylation of c-Jun N-terminal kinase and decreased the expression of cyclin D1, phosphorylated glycogen synthase kinase-3ß and ß-catenin. This study is the first to report that downregulation of PAPP-A is associated with poor prognosis in patients with RCC. In conclusion, PAPP-A may serve as a novel prognostic marker and potentially as a therapeutic target in patients with RCC.

mTORC2/Rac1 Pathway Predisposes Cancer Aggressiveness in IDH1-Mutated Glioma.

Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in lower grade gliomas. The neomorphic enzyme activity of IDH mutants leads to tumor formation through epigenetic alteration, dysfunction of dioxygenases, and metabolic reprogramming. However, it remains elusive as to how IDH mutants regulate the pathways associated with oncogenic transformation and aggressiveness. In the present study, by using unbiased transcriptomic profiling, we showed that IDH1 mutations result in substantial changes in the gene sets that govern cellular motility, chemotaxis, and invasion. Mechanistically, rapamycin-insensitive companion of mammalian target of rapamycin (Rictor)/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling plays an essential role in the motility and proliferation of IDH1-mutated cells by prompting cytoskeleton reorganization, lamellipodia formation, and enhanced endocytosis. Targeting the Rictor/Rac1 pathway suppresses IDH1-mutated cells by limiting endocytosis and cell proliferation. Overall, our findings indicate a novel metabolic reprogramming mechanism of IDH1-mutated cells by exploiting metabolites from the extracellular milieu. Targeting the Rictor/Rac1 pathway could be an alternative therapeutic strategy for IDH1-mutated malignancies.

MicroRNA­124 negatively regulates chloride intracellular channel 1 to suppress the migration and invasion of liver cancer cells.

The dysregulation of microRNAs (miRNAs) is associated with the development and progression of a variety of cancers, including liver cancer. Aberrant expression of miRNA (miR)­124 has been demonstrated in liver cancer, but its functional mechanism in liver cancer is still largely unknown. Metastasis of liver cancer is one of the most common causes of mortality. The present study showed that miR­124 inhibited the proliferation, migration and invasion of liver cancer cells. Furthermore, chloride intracellular channel 1 (CLIC1) was identified as a novel target of miR­124 in liver cancer cells. Overexpression of miR­124 reduced CLIC1 expression at both the protein and mRNA levels in liver cancer cells. Downregulation of CLIC1 decreased the migration and invasion of liver cancer cells without affecting cell proliferation. Taken together, these results showed that CLIC1 is a critical target for miR­124­mediated inhibitory effects on cell migration and invasion. Thus, miR­124 or suppression of CLIC1 may have diagnostic value and therapeutic potential for the treatment of human liver cancer.

Combination of PARP inhibitor and temozolomide to suppress chordoma progression.

Chordoma, a malignant bone cancer, is highly resistant to conventional therapeutic approaches; this greatly limits radio- and chemotherapeutic options and disease management. In the present study, we investigated three patient-derived chordoma cell lines to elucidate the molecular mechanism of resistance to therapeutics. An in vitro high-throughput chemical screening assay and an in vivo xenograft model were used to identify novel chemosensitizers for chordoma. We found that patient-derived chordoma cell lines recapitulated disease phenotypes, which were highlighted by robust resistance to medical therapy manifested as lack of DNA damage accumulation. Mechanistically, the PARP DNA repair pathway was found to play a central role in this resistance. Chemical screening confirmed that PARP inhibitors could strikingly enhance temozolomide (TMZ) therapy in chordoma cells. Combining the FDA-approved PARP inhibitor, olaparib, with chemotherapeutics not only potentiated DNA damage accumulation, cell cycle arrest, and apoptosis in vitro but also suppressed chordoma xenograft expansion in vivo. We conclude that combining PARP inhibition with TMZ could be an effective therapeutic approach for the clinical management of chordoma. KEY MESSAGES: The PARP DNA repair pathway enhances chemoresistance in chordoma cells. Combining PARP inhibitors with genotoxic agents induces chordoma cell cytotoxicity. PARP inhibitor combining with temozolomide suppresses growth of chordoma in vivo.

Targeting IDH1-Mutated Malignancies with NRF2 Blockade.

BACKGROUND: Neomorphic IDH1 mutations disrupt the redox balance by promoting reactive oxygen species (ROS) production. However, the mechanism by which IDH1-mutant cells maintain ROS homeostasis remains elusive. It is also not known whether reprogrammed ROS homeostasis establishes targetable vulnerability in IDH1-mutated cancers. METHODS: We investigated ROS homeostasis in wild-type (GSC827, GSC923, GSC627, and GSC711) and IDH1-mutated cells (IDH1R132C- and IDH1R132H-transduced U87, U251; MGG152, and TS603 cells). We analyzed the stability and transcriptional activity of NRF2 in IDH1-mutated cells. The oxidative DNA damage was analyzed using NRF2-targeting small interfering RNA. Moreover, we evaluated the effect of the NRF2 inhibitor brusatol in an IDH1-mutated subcutaneous xenograft nude mouse model (control group, n = 5; brusatol-treated group, n = 6). All statistical tests were two-sided. RESULTS: We showed that IDH1-mutated cells develop a dependency on the NRF2 antioxidative pathway. Genetic or pharmacologic blockade of NRF2 not only disrupted ROS homeostasis (mean [SD] ROS levels increased by 317 [42.1]%, P = .001, in IDH1R132C and by 286. 5 [48.7]%, P = .003, in IDH1R132H cells) but also enhanced oxidative DNA damage and decreased proliferation of IDH1-mutated cells. Brusatol selectively suppressed IDH1-mutated cancer progression in vivo (mean [SD] final tumor volume was 761.6 [391.6] mm3 in the control and 246.2 [215] mm3 in the brusatol-treated group, P = .02). CONCLUSIONS: IDH1 mutation reprograms ROS homeostasis in cancer cells, which leads to dependency on the NRF2 antioxidant pathway for ROS scavenging. NRF2 blockade might be a novel therapeutic approach to treat malignancies with IDH1 mutation.

Targeting NAD+/PARP DNA Repair Pathway as a Novel Therapeutic Approach to SDHB-Mutated Cluster I Pheochromocytoma and Paraganglioma.

Purpose: Cluster I pheochromocytomas and paragangliomas (PCPGs) tend to develop malignant transformation, tumor recurrence, and multiplicity. Transcriptomic profiling suggests that cluster I PCPGs and other related tumors exhibit distinctive changes in the tricarboxylic acid (TCA) cycle, the hypoxia signaling pathway, mitochondrial electron transport chain, and methylation status, suggesting that therapeutic regimen might be optimized by targeting these signature molecular pathways.Experimental Design: In the present study, we investigated the molecular signatures in clinical specimens from cluster I PCPGs in comparison with cluster II PCPGs that are related to kinase signaling and often present as benign tumors.Results: We found that cluster I PCPGs develop a dependency to mitochondrial complex I, evidenced by the upregulation of complex I components and enhanced NADH dehydrogenation. Alteration in mitochondrial function resulted in strengthened NAD+ metabolism, here considered as a key mechanism of chemoresistance, particularly, of succinate dehydrogenase subunit B (SDHB)-mutated cluster I PCPGs via the PARP1/BER DNA repair pathway. Combining a PARP inhibitor with temozolomide, a conventional chemotherapeutic agent, not only improved cytotoxicity but also reduced metastatic lesions, with prolonged overall survival of mice with SDHB knockdown PCPG allograft.Conclusions: In summary, our findings provide novel insights into an effective strategy for targeting cluster I PCPGs, especially those with SDHB mutations. Clin Cancer Res; 24(14); 3423-32. ©2018 AACR.

Double-barreled gun: Combination of PARP inhibitor with conventional chemotherapy.

DNA repair pathways are evolutionarily conserved molecular mechanisms that maintain the integrity of genomic DNA. In cancer therapies, the integrity and activity of DNA repair pathways predict therapy resistance and disease outcome. Members of the poly (ADP-ribose) polymerase (PARP) family initiate and organize the biologic process of DNA repair, which counteracts many types of chemotherapies. Since the first development in approximately 3 decades ago, PARP inhibitors have greatly changed the concept of cancer therapy, leading to encouraging improvements in tumor suppression and disease outcomes. Here we summaries both pre-clinical and clinical findings of PARP inhibitors applications, particularly for combination therapies.

Evaluating In Vitro DNA Damage Using Comet Assay.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair.

Mutations in isocitrate dehydrogenase (IDH) are the most prevalent genetic abnormalities in lower grade gliomas. The presence of these mutations in glioma is prognostic for better clinical outcomes with longer patient survival. In the present study, we found that defects in oxidative metabolism and 2-HG production confer chemosensitization in IDH1-mutated glioma cells. In addition, temozolomide (TMZ) treatment induced greater DNA damage and apoptotic changes in mutant glioma cells. The PARP1-associated DNA repair pathway was extensively compromised in mutant cells due to decreased NAD+ availability. Targeting the PARP DNA repair pathway extensively sensitized IDH1-mutated glioma cells to TMZ. Our findings demonstrate a novel molecular mechanism that defines chemosensitivity in IDH-mutated gliomas. Targeting PARP-associated DNA repair may represent a novel therapeutic strategy for gliomas. Cancer Res; 77(7); 1709-18. ©2017 AACR.

MicroRNA-223 functions as an oncogene in human colorectal cancer cells.

Aberrant microRNA (miRNA) expression has been frequently observed in colorectal cancer (CRC), the third most common human cancer in the world. However, the roles of miRNAs in CRC remain poorly understood. The present study explored, identified and characterized the miRNAs that correlate with CRC progression. Deregulated level of microRNA-223 (miR-223) was screened out by miRNA microarray in colorectal tumor tissues and further confirmed by quantitative real-time PCR in a large cohort of CRC samples (n=90). After silencing miR-223 by artificial anti­miR-223 (miR-223-inhibitor), WST-1 and colony formation assays were employed to assess cell proliferation, and cell migration and invasion were evaluated by wound healing test and Transwell assays, respectively. Compared with adjacent non-tumor tissues, 22 miRNAs were screened out in CRC tissues with significance (>2-fold), of which 13 were upregulated and 9 were downregulated. miR-223 is one of the noticeably upregulated miRNAs. Large cohort CRC sample analyses showed that a higher level of miR-223 correlated with a higher clinical stage. Reducing miR-223 expression resulted in a decreased cell proliferation, migration and invasion in CRC cells. miR-223 functions as an oncomiR in CRC, therefore serving as a potential diagnostic and therapeutic target for the treatment of CRC.

MicroRNA-124 suppresses growth of human hepatocellular carcinoma by targeting STAT3.

The aberrant expression of microRNAs is associated with development and progression of cancers. Down-regulation of miR-124 has been demonstrated in the hepatocellular carcinoma (HCC), but the underlying mechanism by which miR-124 suppresses tumorigenesis in HCC remains elusive. In this study, we found that miR-124 suppresses the tumor growth of HCC through targeting the signal transducers and activators of transcription 3 (STAT3). Overexpression of miR-124 suppressed proliferation and induced apoptosis in HepG-2 cells. Luciferase assay confirmed that miR-124 binding to the 3'-UTR region of STAT3 inhibited the expression of STAT3 and phosphorylated STAT3 proteins in HepG-2 cells. Knockdown of STAT3 by siRNA in HepG-2 cells mimicked the effect induced by miR-124. Overexpression of STAT3 in miR-124-transfected HepG-2 cells effectively rescued the inhibition of cell proliferation caused by miR-124. Furthermore, miR-124 suppressed xenograft tumor growth in nude mice implanted with HepG-2 cells by reducing STAT3 expression. Taken together, our findings show that miR-124 functions as tumor suppressor in HCC by targeting STAT3, and miR-124 may therefore serve as a biomarker for diagnosis and therapeutics in HCC.

MiR-124 suppresses growth of human colorectal cancer by inhibiting STAT3.

Emerging evidence indicate that microRNAs (miRNAs) may play important roles in cancer. Aberrant expression of miRNAs has been frequently identified in different human malignancies, including colorectal cancer (CRC). However, the mechanism by which deregulated miRNAs impact the development of CRC remains largely elusive. In this study, we show that miR-124 is significantly down-regulated in CRC compared to adjacent non-tumor colorectal tissues. MiR-124 suppresses the expression of STAT3 by directly binding to its 3'-untranslated region (3'-UTR). Overexpression of miR-124 led to increased apoptosis of CRC cells and reduced tumor growth in vitro and in vivo. Knocking down STAT3 expression by specific siRNA suppressed the growth of CRC cells in vitro and in vivo, resembling that of miR-124 overexpression. Moreover, overexpression of STAT3 in miR-124-transfected CRC cells effectively rescued the inhibition of cell proliferation caused by miR-124. These data suggest that miR-124 serves as a tumor suppressor by targeting STAT3, and call for the use of miR-124 as a potential therapeutic tool for CRC, where STAT3 is often hyper-activated.

Genome-wide analysis of miRNA signature differentially expressed in doxorubicin-resistant and parental human hepatocellular carcinoma cell lines.

Chemotherapy regiments have been widely used in the treatment of a variety of human malignancies including hepatocellular carcinoma (HCC). A major cause of failure in chemotherapy is drug resistance of cancer cells. Resistance to doxorubicin (DOX) is a common and representative obstacle to treat cancer effectively. Individual microRNA (miRNA) has been introduced in the evolution of DOX resistance in HCC in recent studies. However, a global and systematic assessment of the miRNA expression profiles contributing to DOX resistance is still lacking. In the present study, we applied high-throughput Illumina sequencing to comprehensively characterize miRNA expression profiles in both human HCC cell line (HepG2) and its DOX-resistant counterpart (HepG2/DOX). A total of 269 known miRNAs were significantly differentially expressed, of which 23 were up-regulated and 246 were down-regulated in HepG2/DOX cells, indicating that part of them might be involved in the development of DOX resistance. In addition, we have identified 9 and 13 novel miRNAs up- and down-expressed significantly in HepG2/DOX cells, respectively. miRNA profiling was then validated by quantitative real-time PCR for selected miRNAs, including 22 known miRNAs and 6 novel miRNAs. Furthermore, we predicted the putative target genes for the deregulated miRNAs in the samples. Function annotation implied that these selected miRNAs affected many target genes mainly involved in MAPK signaling pathway. This study provides us a general description of miRNA expression profiling, which is helpful to find potential miRNAs for adjunct treatment to overcome DOX resistance in future HCC chemotherapy.