Shama Prasada Kabekkodu

A comprehensive review on the role of PIWI-interacting RNA (piRNA) in gynecological cancers

The ester derivative Palmitoylcarnitine abrogates cervical cancer cell survival by enhancing lipotoxicity and mitochondrial dysfunction

Abstract Background In cervical cancer (CC), Double C2 Like Domain Beta (DOC2B) functions as a metastatic suppressor. The present study aims to determine whether ectopic expression of DOC2B causes global metabolomic changes in extracellular vesicles (EVs) and corresponds with its tumor suppressive properties. Methods Using a retroviral method, we first ectopically expressed DOC2B in SiHa cells, which do not normally express DOC2B. Results We observed that ectopically expressed DOC2B significantly altered the global metabolite profile of EVs. Metabolomics identified significant enrichment of palmitoylcarnitine (PC) in EVs upon ectopic expression of DOC2B. We identified that SiHa and HeLa cells exhibited greater cytotoxicity to PC than gingival fibroblast, HaCaT, Cal27, and MCF7. PC treatment reduced the growth, proliferation, and migration of SiHa and HeLa cells, via increasing apoptosis and decreasing S-Phase cells. PC treatment resulted in morphological alterations, decreased length and number of filopodia, and expression of proteins related to cell cycle progression, proliferation, and the epithelial-to-mesenchymal transition. Further, PC treatment caused mitochondrial morphological changes, increased mitochondrial membrane potential, and decreased mtDNA content. The decreased GSH activity, glucose consumption rate, and lactate production upon PC treatment suggest that PC can induce metabolic reprogramming in CC cells. Increased oxidative stress, calcium overload, lipid droplet accumulation, mitochondrial lipotoxicity, and mitophagy suggest that PC can cause mitochondrial dysfunction. N-acetyl cysteine (NAC) treatment reversed the cytotoxic effect of PC, via decreasing lipid peroxidation rate and increasing GSH activity. PC treatment enhanced the cytotoxic effect of cisplatin in CC. Conclusion DOC2B restoration or the use of PC may be employed as a novel therapeutic approach for CC.

Biological functions of extracellular vesicle double C2-like domain beta in cervical cancer

Abstract Double C-2 Like Domain Beta (DOC2B) located at 17q13.3 prevents metastasis by senescence induction and epithelial to mesenchymal transition inhibition in cervical cancer (CC). The extracellular vesicle (EV) mediated trafficking of DOC2B and its impact on tumor suppressive activity are not investigated in CC. Using a retroviral method, we first ectopically expressed DOC2B in SiHa, which do not normally express DOC2B. DOC2B-SiHa and vector-SiHa EVs were co-incubated separately with recipient cell and subjected to various cellular and biochemical experiments. For the first time, we demonstrated that DOC2B localizes to EVs, and its transfer to EV may require intracellular calcium. Co-culture of SiHa and HeLa cells with DOC2B-SiHa derived EVs induced morphological changes and suppressed their growth and migration, possibly by induction of G0/G1 to S phase arrest and anoikis. DOC2B-SiHa EVs elevated intracellular reactive oxygen species (ROS) and calcium levels and promoted lipid droplet accumulation and lipid peroxidation rate in recipient cells. DOC2B-SiHa EVs reduced active AKT1 and ERK1/2 levels and EMT marker expression and enhanced cellular senescence and cytotoxic effects of cisplatin. Re-expression of DOC2B significantly altered the global metabolite profile of EVs.  Finally, we demonstrated that intracellular calcium chelation significantly reduces DOC2B localization to EVs and impacts its tumor-suppressive properties. Altogether, EV-mediated DOC2B transfer may reduce the aggressive behavior of CC cells.

Analysis of miR-497/195 cluster identifies new therapeutic targets in cervical cancer

Abstract Objective miR-497/195, located at 17p13.1, is a highly conserved miRNA cluster whose abnormal expression is a key regulator of carcinogenesis. We performed a comprehensive analysis of the miR-497/195 cluster to determine its prognostic utility and role in cervical cancer (CC) using publicly available datasets. Results In silico analysis and validation revealed that this cluster is downregulated in CC. A total of 60 target genes of miR-497/195 cluster were identified as differentially expressed between normal and CC samples. ShinyGO, STRING, CytoHubba, Timer 2.0, HPA, and HCMBD were used for functional enrichment, PPIN network construction, hub gene identification, immune infiltration correlation, histopathological expression, and determination of the metastatic potential of miR-497/195 cluster and their target genes. PPIN analysis identified CCNE1, CCNE2, ANLN, RACGAP1, KIF23, CHEK1, CDC25A, E2F7, CDK1, and CEP55 as the top 10 hub genes (HGs). Furthermore, the upregulation of RECK, ATD5, and BCL2, downregulation of OSBPL3, RCAN3, and HIST1H3H effected overall survival of CC patients. We identified 6 targets (TFAP2A, CLSPN, RASEF, HIST1H3H, AKT3, and ITPR1) of miR-497/195 cluster to influence metastasis. In addition, 8 druggable genes and 38 potential drugs were also identified. Our study identified miR-497/195 cluster target genes and pathways that could be used for prognostic and therapeutic applications in CC.

Regulation and tumor‐suppressive function of the miR‐379/miR‐656 (C14MC) cluster in cervical cancer

Cervical cancer (CC) is a key contributor to cancer‐related mortality in several countries. The identification of molecular markers and the underlying mechanism may help improve CC management. We studied the regulation and biological function of the chromosome 14 microRNA cluster (C14MC; miR‐379/miR‐656) in CC. Most C14MC members exhibited considerably lower expression in CC tissues and cell lines in The Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma patient cohorts. Bisulfite Sanger sequencing revealed hypermethylation of the C14MC promoter in CC tissues and cell lines. 5‐aza‐2 deoxy cytidine treatment reactivated expression of the C14MC members. We demonstrated that C14MC is a methylation‐regulated miRNA cluster via artificial methylation and luciferase reporter assays. C14MC downregulation correlated with poor overall survival and may promote metastasis. C14MC activation via the lentiviral‐based CRISPRa approach inhibited growth, proliferation, migration, and invasion; enhanced G2/M arrest; and induced senescence. Post‐transcriptional regulatory network analysis of C14MC transcriptomic data revealed enrichment of key cancer‐related pathways, such as metabolism, the cell cycle, and phosphatidylinositol 3‐kinase (PI3K)– AKT signaling. Reduced cell proliferation, growth, migration, invasion, and senescence correlated with the downregulation of active AKT , MYC , and cyclin E1 ( CCNE1 ) and the overexpression of p16 , p21 , and p27 . We showed that C14MC miRNA activation increases reactive oxygen species (ROS) levels, intracellular Ca 2+ levels, and lipid peroxidation rates, and inhibits epithelial–mesenchymal transition (EMT). C14MC targets pyruvate dehydrogenase kinase‐3 ( PDK3 ) according to the luciferase reporter assay. PDK3 is overexpressed in CC and is inversely correlated with C14MC. Both miR‐494‐mimic transfection and C14MC activation inhibited PDK3 expression. Reduced glucose uptake and lactate production, and upregulation of PDK3 upon C14MC activation suggest the potential role of these proteins in metabolic reprogramming. Finally, we showed that C14MC activation may inhibit EMT signaling. Thus, C14MC is a tumor‐suppressive and methylation‐regulated miRNA cluster in CC. Reactivation of C14MC can be useful in the management of CC.

Molecular landscape of recurrent cervical cancer

Cervical cancer (CC) is a major gynecological problem in developing and underdeveloped countries. Despite the significant advancement in early detection and treatment modalities, several patients recur. Moreover, the molecular mechanisms responsible for CC recurrence remains obscure. The patients with CC recurrence often show poor prognosis and significantly high mortality rates. The clinical management of recurrent CC depends on treatment history, site, and extent of the recurrence. Owing to poor prognosis and limited treatment options, recurrent CC often presents a challenge to the clinicians. Several in vitro, in vivo, and patient studies have led to the identification of the critical molecular changes responsible for CC recurrence. Both aberrant genetic and epigenetic modifications leading to altered cell signaling pathways have been reported to impact CC recurrence. Researchers are currently trying to dissect the molecular pathways in CC and translate these findings for better management of disease. This article attempts to review the existing knowledge of disease relapse, accompanying challenges, and associated molecular players in CC.

Analysis of Nuclear Encoded Mitochondrial Gene Networks in Cervical Cancer

Cervical cancer (CC) is one of the most common female cancers in many developing and underdeveloped countries. High incidence, late presentation, and mortality suggested the need for molecular markers. Mitochondrial defects due to abnormal expression of nuclear-encoded mitochondrial genes (NEMG) have been reported during cancer progression. Nevertheless, the application of NEMG for the prognosis of CC is still elusive. Herein, we aimed to investigate the associations between NEMG and CC prognosis. The differentially expressed genes (DEG) in the TCGA-CESC dataset and NEMGs were retrieved from TACCO and Mitocarta2.0 databases, respectively. The impact of methylation on NEMG expression were predicted using DNMIVD and UALCAN tools. HCMDB tool was used to predict genes having metastatic potential. The prognostic models were constructed using DNMIVD, TACCO, GEPIA2, and SurvExpress. The functional enrichment analysis (FEA) was performed using clusterProfiler. The protein-protein interaction network (PPIN) was constructed to identify the hub genes (HG) using String and CytoHubba tools. Independent validation of the HG was performed using Oncomine and Human Protein Atlas databases. The druggable genes were predicted using DGIdb. Among the 52 differentially expressed NEMG, 15 were regulated by DNA methylation. The expression level of 16, 10, and 7 has the potential for CC staging, prediction of metastasis, and prognosis. Moreover, 1 driver gene and 16 druggable genes were also identified. The FEA identified the enrichment of cancer-related pathways, including AMPK and carbon metabolism in cancer. The combined expression of 10 HG has been shown to affect patient survival. Our findings suggest that the abnormal expression of NEMGs may play a critical role in CC development and progression. The genes identified in our study may serve as a prognostic indicator and therapeutic target in CC..

Metastatic suppression by DOC2B is mediated by inhibition of epithelial-mesenchymal transition and induction of senescence

AbstractSenescence induction and epithelial-mesenchymal transition (EMT) events are the opposite sides of the spectrum of cancer phenotypes. The key molecules involved in these processes may get influenced or altered by genetic and epigenetic changes during tumor progression. Double C2-like domain beta (DOC2B), an intracellular vesicle trafficking protein of the double C2 protein family, plays a critical role in exocytosis, neurotransmitter release, and intracellular vesicle trafficking. DOC2B is repressed by DNA promoter hypermethylation and functions as a tumor growth regulator in cervical cancer. To date, the molecular mechanisms of DOC2B in cervical cancer progression and metastasis is elusive. Herein, the biological functions and molecular mechanisms regulated by DOC2B and its impact on senescence and EMT are described. DOC2B inhibition promotes proliferation, growth, and migration by relieving G0/G1-S arrest, actin remodeling, and anoikis resistance in Cal27 cells. It enhanced tumor growth and liver metastasis in nude mice with the concomitant increase in metastasis-associated CD55 and CD61 expression. Inhibition of EMT and promotion of senescence by DOC2B is a calcium-dependent process and accompanied by calcium-mediated interaction between DOC2B and CDH1. In addition, we have identified several EMT and senescence regulators as targets of DOC2B. We show that DOC2B may act as a metastatic suppressor by inhibiting EMT through induction of senescence via DOC2B-calcium-EMT-senescence axis. Graphical abstract

Integrated bioinformatic analysis of miR-15a/16-1 cluster network in cervical cancer

The miR-15a/16-1 cluster is abnormally expressed in cervical cancer (CC) tissues and plays a vital role in cervical carcinogenesis. We aimed to evaluate the miR-15a/16-1 expression in healthy and cancerous cervical tissues, identify the associated networks, and to test its prognostic significance. miR-15a/16-1-MC expressions were analyzed in TCGA-CESC datasets by UALCAN, GEPIA2, and Datasetviewer. miR-15a/16-1 validated targets were extracted from mirTarBase and in silico functional analysis of the target genes were performed using WebGestalt. The interaction networks were constructed by the miRNet, STRING, and NetworkAnalyst tools. The prognostic significance and metastatic potential of the target genes were predicted using UALCAN and HCMDB. The FDA approved drugs to target miR-15a/16-1 and target gene network in CC were performed using DGIdb, STITCH and PanDrugs. TCGA-CESC and GEO data analysis suggested significant overexpression of miR-15a/16-1 in CC samples. The Kaplan-Meier survival analysis showed that miR-15a and its four target genes (BCL2, CCNE1, NUP50, and RBPJ) influence the overall survival of CC patients. Among the 66 differentially expressed target genes, 12 of them are linked to head, neck, or lung metastasis. Functional enrichment analysis predicted the association of this cluster with p53 signaling, human papillomavirus infection, PI3-AKT signaling pathway, and pathways in cancer. Drug-gene interaction analysis showed 52 potential FDA approved drugs to interact with the miR-15a/16-1 target genes. Nine of the 52 drugs are currently used as a chemotherapeutic agent for the treatment of CC patients. The present study shows that miR-15a/16-1 expression can be used as a clinical marker and target for therapy in CC.

Targeted drug delivery in cervical cancer: Current perspectives

Cervical cancer is preventable yet one of the most prevalent cancers among women around the globe. Though regular screening has resulted in the decline in incidence, the disease claims a high number of lives every year, especially in the developing countries. Owing to rather aggressive and non-specific nature of the conventional chemotherapeutics, there is a growing need for newer treatment modalities. The advent of nanotechnology has assisted in this through the use of nanocarriers for targeted drug delivery. A number of nanocarriers are continuously being developed and studied for their application in drug delivery. The present review summarises the different drug delivery approaches and nanocarriers that can be useful, their advantages and limitation.

A comprehensive review on the functional role of miRNA clusters in cervical cancer.

Cervical cancer (CC) poses a significant health threat in women globally. MicroRNA clusters (MCs), comprising multiple miRNA-encoding genes, are pivotal in gene regulation. Various factors, including circular RNA and DNA methylation, govern MC expression. Dysregulated MC expression correlates strongly with CC development via promoting the acquisition of cancer hallmarks. Certain MCs show promise for diagnosis, prognosis and therapy selection due to their distinct expression patterns in normal, premalignant and tumor tissues. This review explains the regulation and biological functions of MCs and highlights the clinical relevance of abnormal MC expression in CC.