An integrated analysis of microRNAs regulating DNA damage response in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) remains a clinical challenge due to its aggressive phenotype and limited treatment options for the patients. Many TNBC patients show an inherent defect in the DNA repair capacity primarily by acquiring germline mutations in BRCA1 and BRCA2 genes leading to Homologous Recombination Deficiency (HRD). Epigenetic modifications such as BRCA1 promoter methylation and miRNA expression targeting DNA repair pathway genes have contributed to the HRD phenotype in TNBC. Hence, we aimed to identify microRNAs that are associated with HRD status in the TCGA-BRCA project. MATERIALS AND METHODS: We implemented a miRNA prediction strategy for identifying miRNAs targeting HR pathway genes using an in silico predicted and experimentally validated list from published literature for their association with genomic instability and factors affecting HRD. In silico analysis was performed to study miRNA expression patterns regulated by DNA methylation and TMB status in the TNBC patients from TCGA-BRCA project. Finally, we analysed selected miRNA expression with immune cell infiltration pattern in the TNBC patient cohort. RESULTS: Our study identified miRNAs associated with HRD, tumour mutation burden (TMB), and immune cell infiltration. Identified miRNA signatures were associated with the miR-17 ~ 92 cluster, miR-106b ~ 25 cluster, and miR-200b ~ 429 cluster. Pathway analysis of selected miRNAs suggested their association with altered immune cell infiltration in TNBC. CONCLUSION: Our study identified 6 'HRD associated miRNAs' such as miR-106b, miR-93, miR-17, miR-20a, miR-200b, and miR-429 as novel miRNA-based signatures associated with HR deficiency in TNBC.

MiR-4521 perturbs FOXM1-mediated DNA damage response in breast cancer.

Introduction: Forkhead (FOX) transcription factors are involved in cell cycle control, cellular differentiation, maintenance of tissues, and aging. Mutation or aberrant expression of FOX proteins is associated with developmental disorders and cancers. FOXM1, an oncogenic transcription factor, is a promoter of cell proliferation and accelerated development of breast adenocarcinomas, squamous carcinoma of the head, neck, and cervix, and nasopharyngeal carcinoma. High FOXM1 expression is correlated with chemoresistance in patients treated with doxorubicin and Epirubicin by enhancing the DNA repair in breast cancer cells. Method: miRNA-seq identified downregulation of miR-4521 in breast cancer cell lines. Stable miR-4521 overexpressing breast cancer cell lines (MCF-7, MDA-MB-468) were developed to identify miR-4521 target gene and function in breast cancer. Results: Here, we showed that FOXM1 is a direct target of miR-4521 in breast cancer. Overexpression of miR-4521 significantly downregulated FOXM1 expression in breast cancer cells. FOXM1 regulates cell cycle progression and DNA damage response in breast cancer. We showed that miR-4521 expression leads to increased ROS levels and DNA damage in breast cancer cells. FOXM1 plays a critical role in ROS scavenging and promotes stemness which contributes to drug resistance in breast cancer. We observed that breast cancer cells stably expressing miR-4521 lead to cell cycle arrest, impaired FOXM1 mediated DNA damage response leading to increased cell death in breast cancer cells. Additionally, miR-4521-mediated FOXM1 downregulation perturbs cell proliferation, invasion, cell cycle progression, and epithelial-to-mesenchymal progression (EMT) in breast cancer. Discussion: High FOXM1 expression has been associated with radio and chemoresistance contributing to poor patient survival in multiple cancers, including breast cancer. Our study showed that FOXM1 mediated DNA damage response could be targeted using miR-4521 mimics as a novel therapeutic for breast cancer.

Double C-2 like domain beta (DOC2B) induces calcium dependent oxidative stress to promote lipotoxicity and mitochondrial dysfunction for its tumor suppressive function.

Mitochondria are biosynthetic and bioenergetic organelles that regulate many biological processes, including metabolism, oxidative stress, and cell death. Cervical cancer (CC) cells show impairments in mitochondrial structure and function and are linked with cancer progression. DOC2B is a tumor suppressor with anti-proliferative, anti-migratory, anti-invasive, and anti-metastatic function in CC. For the first time, we demonstrated the role of the DOC2B-mitochondrial axis with tumor growth regulatory functions in CC. We used DOC2B overexpression and knockdown model systems to show that DOC2B is localized to mitochondria and induces Ca2+-mediated lipotoxicity. DOC2B expression induced mitochondrial morphological changes with the subsequent reduction in mitochondrial DNA copy number, mitochondrial mass, and mitochondrial membrane potential. Intracellular and mitochondrial Ca2+, intracellular O.-2, and ATP levels were substantially elevated in the presence of DOC2B. DOC2B manipulation reduced glucose uptake, lactate production, and mitochondrial complex-IV activity. The presence of DOC2B significantly reduced the proteins associated with mitochondrial structure and biogenesis with the concomitant activation of AMPK signaling. Augmented lipid peroxidation (LPO) in the presence of DOC2B was a Ca2+-dependent process. Our findings demonstrated that DOC2B promotes lipid accumulation, oxidative stress, and LPO through intracellular Ca2+ overload, which may contribute to mitochondrial dysfunction and tumor-suppressive properties of DOC2B. We propose that the DOC2B-Ca2+-oxidative stress-LPO-mitochondrial axis could be targeted for confining CC. Further, the induction of lipotoxicity in tumor cells by activating DOC2B could serve as a novel therapeutic approach in CC.

Regulation of mitochondrial function by forkhead transcription factors.

Mitochondria play a central role in several important cellular processes such as energy production, apoptosis, fatty acid catabolism, calcium regulation, and cellular stress response. Multiple nuclear transcription factors have been reported for their role in the regulation of mitochondrial gene expression. More recently, the role of the forkhead family of transcription factors in various mitochondrial pathways has been reported. Among them, FOXO1, FOXO3a, FOXG1, and FOXM1 have been reported to localize to the mitochondria, of which the first two have been observed to bind to the mitochondrial D-loop. This suggests an important role for forkhead transcription factors in the direct regulation of the mitochondrial genome and function. Forkheads such as FOXO3a, FOXO1, and FOXM1 are involved in the cellular response to oxidative stress, hypoxia, and nutrient limitation. Several members of the forkhead family of transcription factors are also involved in the regulation of nuclear-encoded genes associated with the mitochondrial pathway of apoptosis, respiration, mitochondrial dynamics, and homeostasis.

Organ-on-Chip platforms to study tumor evolution and chemosensitivity.

Despite tremendous advancements in oncology research and therapeutics, cancer remains a primary cause of death worldwide. One of the significant factors in this critical challenge is a precise diagnosis and limited knowledge on how the tumor microenvironment (TME) behaves to the treatment and its role in chemo-resistance. Therefore, it is critical to understand the contribution of a heterogeneous TME in cancer drug response in individual patients for effective therapy management. Micro-physiological systems along with tissue engineering have facilitated the development of more physiologically relevant platforms, known as Organ-on-Chips (OoC). OoC platforms recapitulate the critical hallmarks of the TME in vitro and subsequently abet in sensitivity and efficacy testing of anti-cancer drugs before clinical trials. The OoC platforms incorporating conventional in vitro models enable researchers to control the cellular, molecular, chemical, and biophysical parameters of the TME in precise combinations while analyzing how they contribute to tumor progression and therapy response. This review discusses the application of OoC platforms integrated with conventional 2D cell lines, 3D organoids and spheroid models, and the organotypic tissue slices, including patient-derived and xenograft tumor slice cultures in cancer treatment responses. We summarize the relevance and drawbacks of conventional in vitro models in assessing cancer treatment response, challenges and limitations associated with OoC models, and future opportunities enabled by the OoC technologies towards developing personalized cancer diagnostics and therapeutics.

Expression analysis and function of mitochondrial genome-encoded microRNAs.

MicroRNAs (miRNAs) play a significant role in nuclear and mitochondrial anterograde and retrograde signaling. Most of the miRNAs found inside mitochondria are encoded in the nuclear genome, with a few mitochondrial genome-encoded non-coding RNAs having been reported. In this study, we have identified 13 mitochondrial genome-encoded microRNAs (mitomiRs), which were differentially expressed in breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231), non-malignant breast epithelial cell line (MCF-10A), and normal and breast cancer tissue specimens. We found that mitochondrial DNA (mtDNA) depletion and inhibition of mitochondrial transcription led to reduced expression of mitomiRs in breast cancer cells. MitomiRs physically interacted with Ago2, an RNA-induced silencing complex (RISC) protein, in the cytoplasm and inside mitochondria. MitomiRs regulate the expression of both nuclear and mitochondrial transcripts in breast cancer cells. We showed that mitomiR-5 targets the PPARGC1A gene and regulates mtDNA copy number in breast cancer cells. MitomiRs identified in the present study may be a promising tool for expression and functional analysis in patients with a defective mitochondrial phenotype, including cancer and metabolic syndromes. This article has an associated First Person interview with the first author of the paper.

Advances in mitochondrial medicine and translational research.

Current knowledge of mitochondrial biology and function has provided tools and technologies that helped a better understanding of the molecular etiology of complex mitochondrial disorders. Dual genetic control of this subcellular organelle function regulates various signaling mechanisms which are essential for metabolism, bioenergetics, fatty acid biosynthesis, and DNA replication & repair. Understanding nuclear mitochondrial crosstalk through advanced genomics as well as clinical perspectives is the overall basis of mitochondrial research and medicine, also the sole objective of Society for Mitochondrial Medicine and Research (SMRM) - India. The eighth virtual international conference on 'Advances in Mitochondrial Medicine and Translational Research' was organized at the Manipal School of Life Sciences, MAHE, Manipal, India, during 6 - 7 November 2020. The aim of the virtual conference was to highlight the recent advances and future perspectives that represent comprehensive clinical and fundamental research interests in the area of mitochondrial biology of human diseases. To systematically present the various findings in mitochondrial biology, the meeting was themed with specific aspects comprising (a) mitochondrial disorders: clinical & genomic perspectives, (b) mitochondria in cancer, (c) mitochondrial metabolism & disorders, and (d) mitochondrial diseases & therapy. This report provides an overview of the recent advancements in the area of mitochondrial biology and medicine that was discussed at the conference.

Advances in Mitochondrial Medicine and Translational Research.

Current knowledge of mitochondrial biology and function has provided with tools and technologies that helped a better understanding of the molecular etiology of complex mitochondrial disorders. Dual genetic control of this subcellular organelle function regulates various signaling mechanisms which are essential for metabolism, bioenergetics, fatty acid biosynthesis, and DNA replication & repair. Understanding nuclear mitochondrial crosstalk through advanced genomics as well as clinical perspectives is the overall basis of mitochondrial research and medicine, also the sole objective of Society for Mitochondrial Medicine and Research (SMRM) - India. The eighth virtual international conference on 'Advances in Mitochondrial Medicine and Translational Research' was organized at the Manipal School of Life Sciences, MAHE, Manipal, India, during 6 - 7 November 2020. The aim of the virtual conference was to highlight the recent advances and future perspectives that represent comprehensive clinical and fundamental research interests in the area of mitochondrial biology of human diseases. To systematically present the various findings in mitochondrial biology, the meeting was themed with specific aspects comprising (a) mitochondrial disorders: clinical & genomic perspectives, (b) mitochondria in cancer, (c) mitochondrial metabolism & disorders, and (d) mitochondrial diseases & therapy. This report provides an overview of the recent advancements in the area of mitochondrial biology and medicine that was discussed at the conference.

Molecular Insights into the Antistress Potentials of Brazilian Green Propolis Extract and Its Constituent Artepillin C.

Propolis, also known as bee-glue, is a resinous substance produced by honeybees from materials collected from plants they visit. It contains mixtures of wax and bee enzymes and is used by bees as a building material in their hives and by humans for different purposes in traditional healthcare practices. Although the composition of propolis has been shown to depend on its geographic location, climatic zone, and local flora; two largely studied types of propolis: (i) New Zealand and (ii) Brazilian green propolis have been shown to possess Caffeic Acid Phenethyl Ester (CAPE) and Artepillin C (ARC) as the main bioactive constituents, respectively. We have earlier reported that CAPE and ARC possess anticancer activities, mediated by abrogation of mortalin-p53 complex and reactivation of p53 tumor suppressor function. Like CAPE, Artepillin C (ARC) and the supercritical extract of green propolis (GPSE) showed potent anticancer activity. In this study, we recruited low doses of GPSE and ARC (that did not affect either cancer cell proliferation or migration) to investigate their antistress potential using in vitro cell based assays. We report that both GPSE and ARC have the capability to disaggregate metal- and heat-induced aggregated proteins. Metal-induced aggregation of GFP was reduced by fourfold in GPSE- as well as ARC-treated cells. Similarly, whereas heat-induced misfolding of luciferase protein showed 80% loss of activity, the cells treated with either GPSE or ARC showed 60-80% recovery. Furthermore, we demonstrate their pro-hypoxia (marked by the upregulation of HIF-1α) and neuro-differentiation (marked by differentiation morphology and upregulation of expression of GFAP, ß-tubulin III, and MAP2). Both GPSE and ARC also offered significant protection against oxidative stress and, hence, may be useful in the treatment of old age-related brain pathologies.

Mitochondrial DNA copy number variation - A potential biomarker for early onset preeclampsia.

OBJECTIVES: Oxidative stress has been hypothesized as a central component of both placental and endothelial dysfunction, leading to PE. This oxidative stress leading to mitochondrial dysfunction may be due to variations in mtDNA copy numbers as an adaptive response. In the present study we aimed to analyse mtDNA copy numbers in the placenta obtained after delivery from the women with PE as compared to the controls. STUDY DESIGN: It was a prospective case control study. A total of 32 placental samples were analyzed (Cases: 17; Controls: 15). Samples were collected ex vivo, after childbirth. MtDNA content was determined useing real-time quantitative PCR qRT-PCR) using TaqMan probes designed for two genes: MT-ND1 and a mitochondrial gene encoding for the NADH dehydrogenase 1 protein. RESULTS: We found that the median (IQR) mtDNA copy number was higher in PE cases 24.32 (9.260-33.51) as compared with controls 20.32 (13.33-26.22). On subgroup analysis, the median (IQR) mtDNA copy number was higher in early onset PE 28.06 (20.80-36.87) as compared to late onset PE 9.215 (4.150-56.45) as well as the controls 20.32 (13.33-26.22). CONCLUSION: Our findings support a higher mtDNA copy number in early onset PE as compared to late onset PE and control population. Although, mtDNA may only be increased in very severe cases of early onset preeclampsia. Future research may be directed to ascertain if mtDNA copy numbers can be a novel biomarker to predict or prognosticate early onset preeclampsia.