DOT1L regulates MTDH-mediated angiogenesis in triple-negative breast cancer: intermediacy of NF-κB-HIF1α axis.

DOT1L, a specific H3K79 methyltransferase, has a tumour-promoting role in various cancers, including triple-negative breast cancer (TNBC). However, the molecular mechanism by which the deregulated DOT1L promotes cancer progression is unclear. Herein, we show that a significantly higher basal level of DOTL1 strongly correlates with MTDH, an oncogene, in clinical TNBC patient cohorts and mediates TNBC progression by enhancing MTDH-induced angiogenesis. In parallel, severe combined immunodeficiency mice-bearing MDA-MB-231 cells with MTDH-Wt or MTDHΔ7 (spliced isoform of MTDH) overexpression constructs showed enhanced blood vessel formations at the tumour site in comparison with control groups. Selective inhibition of DOT1L by EPZ004777, a specific DOT1L inhibitor, or siDOT1L, significantly impaired MTDH-induced proliferation, invasion and angiogenic markers expression in TNBC cells. ChIP assay revealed that Dot1L promotes MTDH-Wt/Δ7 transcription by increasing H3K79me3 levels on its promoter. Dot1L depletion reversed this effect. Mechanistically, DOT1L-induced MTDH caused enhanced nuclear factor kappa B (NF-κB) occupancy on the hypoxia-inducible factor1α (HIF1α) promoter and increased its transcription, leading to elevated levels of proangiogenic mediators in TNBC cells. Moreover, the condition media obtained from MDA-MB-231 cells stably expressing either MTDH-Wt or MTDHΔ7 treated with EPZ004777 or Bay-11-7082 (NF-κB inhibitor) or FM19G11 (HIF1α inhibitor) significantly inhibited MTDH-induced tube formation in human umbilical vein endothelial cells, rat aortic ring sprouting and vessel formations by chick chorioallantoic membrane assay mimicking physiological angiogenic vasculature. Collectively, our findings reveal a novel epigenetic regulation of MTDH by DOTL1, which drives angiogenesis, and that the therapeutic disruption of the DOT1L-MTDH-NF-κB-HIF1α axis may have usefulness in the management of TNBC.

Mitochondria-targeted esculetin mitigates atherosclerosis in the setting of aging via the modulation of SIRT1-mediated vascular cell senescence and mitochondrial function in Apoe-/- mice.

BACKGROUND AND AIMS: Age is a dominant and independent risk factor for the development of atherosclerosis, a major cardiovascular disease, and if left untreated leads to myocardial infarction and death. Mitochondria-targeted anti-oxidants are evolving as a new class of compounds that can alter the pathophysiology of age-related diseases, including atherosclerosis, where mitochondrial dysfunction plays a critical role in disease progression. METHODS: We recently synthesized an alkyl TPP + -tagged esculetin (mitochondria-targeted esculetin or Mito-Esc). Apoe-/- mice were chronically (14 months) administered with Mito-Esc to investigate its efficacy in the mitigation of atherosclerosis in the setting of aging. We monitored BP, and performed various biochemical assays, histopathology, immunohistochemistry, inflammatory factors, qPCR, and Western blotting. Simultaneously, human aortic endothelial cells (HAECs) were used as a model system to study the mechanistic aspects. RESULTS: A chronic low-dose administration of Mito-Esc to Apoe-/- mice greatly prevented alterations in lipid profile, blood pressure, and atherosclerotic plaque formation in the setting of aging. Mito-Esc administration significantly reduced vascular senescence and pro-inflammatory cytokines levels and prevented dysregulation of mitochondrial biogenesis markers in aortic tissue. Further, Mito-Esc treatment prevented replicative and stress-induced premature senescence (SIPS) in HAEC. Importantly, Mito-Esc treatment delayed endothelial cell senescence by increasing human telomerase reverse transcriptase (hTERT) levels via SIRT1 activation. Moreover, Mito-Esc treatment by altering miR-19b and miR-30c via a SIRT1 activation significantly inhibited the increase in PAI-1 levels in HAEC as well as in the serum of Apoe-/- mice. In addition, Mito-Esc treatment improved mitochondrial function in late passage (aged) HAECs by enhancing the oxygen consumption rate (OCR). Furthermore, Mito-Esc administration counteracted the decline in GSH and nitrite levels in Apoe-/- mice and in HAECs. CONCLUSIONS: Overall, Mito-Esc alleviates atherosclerosis in the setting of aging by delaying vascular senescence and pro-inflammatory processes, and by improving mitochondrial biogenesis and function.

A functional and self-assembling octyl-phosphonium-tagged esculetin as an effective siRNA delivery agent.

Herein, we document a self-assembling octyl-TPP tagged esculetin (Mito-Esc) as functionally active and as a novel small molecule siRNA delivery vector. While Mito-Esc itself induces selective breast cancer cell death, the amphiphilic nature of Mito-Esc delivers therapeutic siRNAs intracellularly without the need for any excipient to exacerbate the anti-proliferative effects.

p53 Antibodies as a Diagnostic Marker for Cancer: A Meta-Analysis.

Importance: The protein p53 is an unequivocal tumor suppressor that is altered in half of all cancers. The immune system produces systemic p53 autoantibodies (p53 Abs) in many cancer patients. Objective: This systemic review and meta-analysis focuses on the prognostic value of p53 Abs expressed in the serum of patients with solid tumors. Data Sources: All the clinical investigations were searched on PubMed from the first study dated 1993 until May 2021 (date of submission of the manuscript). Study Selection: Studies were included that met the following criteria: (1) participants with cancer; (2) outcome results expressed in relation to the presence of a p53 antibody; (3) a primary outcome (disease-free survival, overall survival or progression-free survival) expressed as hazard ratio (HR). The following exclusion criteria were used: (1) insufficient data available to evaluate outcomes; (2) animal studies; (3) studies with less than 10 participants. As a result, 12 studies were included in the analysis. Data Extraction and Synthesis: PRISMA guidelines were used for abstracting and assessing data quality and validity by three independent observers. The summary estimates were generated using a fixed-effect model (Mantel-Haenszel method) or a random-effect model (DerSimonian-Laird method), depending on the absence or presence of heterogeneity (I2). Main Outcome(s) and Measure(s): The primary study outcome was to determine the prognostic value of p53 Abs from a large population of patients with solid tumors, as determined before data collection. Results: In total, 12 clinical studies involving 2094 patients were included in the meta-analysis, and it was determined that p53 Abs expression in the serum significantly correlated with poorer survival outcomes of cancer patients (95% CI 1.48 [1.24, 1.77]; p < 0.00001). Conclusions and Relevance: This is the first meta-analysis proving the diagnostic utility of p53-Abs for cancer patients in predicting poorer outcomes. The serum-p53 value (s-p53-value) may be useful for future theranostics.

AR-V7 in Metastatic Prostate Cancer: A Strategy beyond Redemption.

Metastatic prostate cancer is the most common cancer in males and the fifth cause of cancer mortality worldwide. Despite the major progress in this field, leading to the approval of novel anti-androgens, the prognosis is still poor. A significant number of patients acquire an androgen receptor splice variant 7 (AR-V7), which is constitutively activated and lacks the ligand-binding domain (LBD) while maintaining the nuclear localization signal and DNA-binding domain (DBD). This conformational change, even in the absence of the ligand, allows its retention within the nucleus, where it acts as a transcription factor repressing crucial tumor suppressor genes. AR-V7 is an important oncogenic driver and plays a role as an early diagnostic and prognostic marker, as well as a therapeutic target for antagonists such as niclosamide and TAS3681. Anti-AR-V7 drugs have shown promise in recent clinical investigations on this subset of patients. This mini-review focuses on the relevance of AR-V7 in the clinical manifestations of castration-resistant prostate cancer (CRPC) and summarizes redemptive therapeutic strategies.

sp3 -Rich Glycyrrhetinic Acid Analogues Using Late-Stage Functionalization as Potential Breast Tumor Regressing Agents.

Late-stage functionalization (LSF) aids drug discovery efforts by introducing functional groups onto C-H bonds on pre-existing skeletons. We adopted the LSF strategy to synthesize analogues of the abundantly available triterpenoid, glycyrrhetinic acid (GA), by introducing aryl groups in the A-ring, expanding the A-ring and selectively activating one methyl group of the gem-dimethyl groups. Intriguingly, two compounds were found to preferentially accumulate in the mitochondrial compartment of MDA-MB-231 breast cancer cells, to cause depolarization of mitochondrial membrane potential and to induce antiproliferative and anti-invasive effects through enhanced mitochondrial superoxide production with parallel depletion of GSH levels. Furthermore, intraperitoneal administration of these two compounds, in comparison with GA, greatly regressed breast tumor growth and metastasis in a SCID mouse model bearing labeled MDA-MB-231 cells.

A novel metadherinΔ7 splice variant enhances triple negative breast cancer aggressiveness by modulating mitochondrial function via NFĸB-SIRT3 axis.

Metadherin (MTDH) expression inversely correlates with prognosis of several cancers including mammary carcinomas. In this work, we identified a novel splice variant of MTDH with exon7 skipping (MTDHΔ7) and its levels were found significantly high in triple negative breast cancer (TNBC) cells and in patients diagnosed with TNBC. Selective overexpression of MTDHΔ7 in MDA-MB-231 and BT-549 cells enhanced proliferation, invasion, and epithelial-to-mesenchymal (EMT) transition markers in comparison to its wildtype counterpart. In contrast, knockdown of MTDHΔ7 induced antiproliferative/antiinvasive effects. Mechanistically, MTDH-NFĸB-p65 complex activated SIRT3 transcription by binding to its promoter that in turn enhanced MnSOD levels and promoted EMT in TNBC cells. Intriguingly, mitochondrial OCR through Complex-I and -IV, and glycolytic rate (ECAR) were significantly high in MDA-MB-231 cells stably expressing MTDHΔ7. While depletion of SIRT3 inhibited MTDH-Wt/Δ7-induced OCR and ECAR, knockdown of MnSOD inhibited only ECAR. In addition, MTDH-Wt/Δ7-mediated pro-proliferative/-invasive effects were greatly obviated with either siSIRT3 or siMnSOD in these cells. The functional relevance of MTDHΔ7 was further proved under in vivo conditions in an orthotopic mouse model of breast cancer. Mice bearing labeled MDA-MB-231 cells stably expressing MTDHΔ7 showed significantly more tumor growth and metastatic ability to various organs in comparison to MTDH-Wt bearing mice. Taken together, MTDHΔ7 promotes TNBC aggressiveness through enhanced mitochondrial biogenesis/function, which perhaps serves as a biomarker.

Metformin regulates mitochondrial biogenesis and senescence through AMPK mediated H3K79 methylation: Relevance in age-associated vascular dysfunction.

Endothelial senescence in conjunction with mitochondrial dysfunction orchestrates age-associated cardiovascular disorders. In this study we investigated the causal link between these two processes and studied the molecular mechanisms by which metformin acts to coordinate the delay of endothelial senescence via enhancing mitochondrial biogenesis/function. AMPK activators metformin and AICAR delayed endothelial senescence via SIRT1-mediated upregulation of DOT1L, leading to increased trimethylation of H3K79 (H3K79me3). Treatment of cells with either siAMPK or siSIRT1 repressed DOT1L-mediated enhancement of H3K79me3. Moreover, the increase in SIRT3 expression and mitochondrial biogenesis/function by AMPK activators was H3K79me-dependent as H3K79N mutant or siDOT1L abrogated these effects. This was confirmed by the enrichment of H3K79me3 in the SIRT3 promoter with AMPK activation. Intriguingly, enhanced PGC-1α expression by SIRT3 via AMPK activation was responsible for increased hTERT expression and delayed endothelial senescence. In contrast, SIRT3 knockdown caused increased oxidative stress and premature senescence, possibly by depleting hTERT expression. Furthermore, a chronic low dose administration of metformin significantly attenuated vascular aging and inhibited age-associated atherosclerotic plaque formation in ApoE-/- mice. Overall, the results of this study show a novel regulation of mitochondrial biogenesis/function, and cellular senescence by H3K79me acting through SIRT3, thus providing a molecular basis for metformin-mediated age-delaying effects.

AMPK inhibits MTDH expression via GSK3ß and SIRT1 activation: potential role in triple negative breast cancer cell proliferation.

Recent studies have highlighted the involvement of metadherin (MTDH), an oncogenic protein, in promoting cancer progression, metastasis and chemoresistance in many cancers including mammary carcinomas. However, the molecular regulation of MTDH is still not completely understood. In this study we document that AMP activated protein kinase (AMPK) activation-induced anti-proliferative effects are, in part, mediated by inhibiting MTDH expression in MDA-MB-231 and BT-549 triple negative breast cancer (TNBC) cells. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, caused growth arrest, inhibition of migration and invasion of TNBC cells. Intriguingly, AICAR or metformin treatment resulted in significant downregulation of MTDH expression via inhibiting c-Myc expression. In contrast, treatment of cells with compound C, an inhibitor of AMPK, increased both c-Myc and MTDH expressions in TNBC cells. Also, AMPK activation caused increased glycogen synthase kinase 3ß (GSK3ß) activity by inhibiting the inactive phosphorylation at Ser9, on the one hand, and activation of sirtuin1 (SIRT1) by inhibiting Ser47 phosphorylation, as evidenced by deacetylation of p53, on the other hand. Moreover, AMPK-induced GSK3ß and SIRT1 activities were found to be responsible for inhibiting c-Myc-mediated upregulation of MTDH, as LiCl (an inhibitor of GSK3ß) and EX-527 (an inhibitor of SIRT1) reversed AICAR-mediated downregulation of c-Myc and MTDH expressions. Similar results were observed with siSIRT1 treatment. Furthermore, AICAR and EX-527 treatments caused increased cell death under MTDH-depleted conditions. Finally, we uncovered a novel regulation of MTDH expression and showed that AMPK activation by inducing GSK3ß and SIRT1 downregulates MTDH expression via inhibiting c-Myc in TNBC cells.