Hallmarks of cancer are fundamental principles involved in cancer progression. We propose an additional generalized hallmark of malignant transformation corresponding to the differential expression of a family of mitochondrial ncRNAs (ncmtRNAs) that comprises sense and antisense members, all of which contain stem-loop structures. Normal proliferating cells express sense (SncmtRNA) and antisense (ASncmtRNA) transcripts. In contrast, the ASncmtRNAs are down-regulated in tumor cells regardless of tissue of origin. Here we show that knockdown of the low copy number of the ASncmtRNAs in several tumor cell lines induces cell death by apoptosis without affecting the viability of normal cells. In addition, knockdown of ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis (IAP) family. Down-regulation of survivin is at the translational level and is probably mediated by microRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs, as suggested by evidence presented here, in which the ASncmtRNAs are bound to Dicer and knockdown of the ASncmtRNAs reduces reporter luciferase activity in a vector carrying the 3′-UTR of survivin mRNA. Taken together, down-regulation of the ASncmtRNAs constitutes a vulnerability or Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy.

The family of long noncoding mitochondrial RNAs (ncmtRNAs), comprising sense (SncmtRNA), and antisense (ASncmtRNA-1 and ASncmtRNA-2) members, are differentially expressed according to cell proliferative status; SncmtRNA is expressed in all proliferating cells, while ASncmtRNAs are expressed in normal proliferating cells, but is downregulated in tumor cells. ASncmtRNA knockdown with an antisense oligonucleotide induces massive apoptosis in tumor cell lines, without affecting healthy cells. Apoptotic death is preceded by proliferation blockage, suggesting that these transcripts are involved in cell cycle regulation. Here, we show that ASncmtRNA knockdown induces cell death preceded by proliferative blockage in three different human breast cancer cell lines. This effect is mediated by downregulation of the key cell cycle progression factors cyclin B1, cyclin D1, CDK1, CDK4, and survivin, the latter also constituting an essential inhibitor of apoptosis, underlying additionally the onset of apoptosis. The treatment also induces an increase in the microRNA hsa-miR-4485-3p, whose sequence maps to ASncmtRNA-2 and transfection of MDA-MB-231 cells with a mimic of this miRNA induces cyclin B1 and D1 downregulation. Other miRNAs that are upregulated include nuclear-encoded hsa-miR-5096 and hsa-miR-3609, whose mimics downregulate CDK1. Our results suggest that ASncmtRNA targeting blocks tumor cell proliferation through reduction of essential cell cycle proteins, mediated by mitochondrial and nuclear miRNAs. This work adds to the elucidation of the molecular mechanisms behind cell cycle arrest preceding tumor cell apoptosis induced by ASncmtRNA knockdown. As proof-of-concept, we show that in vivo knockdown of ASncmtRNAs results in drastic inhibition of tumor growth in a xenograft model of MDA-MB-231 subcutaneous tumors, further supporting this approach for the development of new therapeutic strategies against breast cancer.

Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors

SARS-CoV-2 is a positive, single-stranded RNA coronavirus responsible for the COVID-19 pandemic. The emergence of new variants and the limited efficacy of current antivirals demonstrate the need for novel therapeutic strategies. Here, we present the design, screening, and evaluation of antisense oligonucleotides (ASOs) targeting the SARS-CoV-2 genomic and sub-genomic RNA. By employing a combination of luminescence-based reporter assays and quantitative reverse-transcription PCR (RT-qPCR) in human cells, we selected ASO-N1, directed against the nucleocapsid RNA, as the best candidate to be validated in vivo. Hamsters infected with SARS-CoV-2 were treated with an initial intranasal ASO dose, followed by daily systemic administration. ASO-treated animals showed significantly improved clinical signs, indicated by increased food consumption and reduced weight loss. ASO-N1 induced a sustained reduction in viral RNA and inflammatory cytokine expression in the nasal mucosa and efficiently decreased infectious viral titers in nasal swabs at day 3 post infection. Further optimization of the ASO-N1 sequence with a combination of RNase-H1-dependent and steric blockage chemistries synergistically increased the antiviral efficacy in vitro. Finally, the target site of ASO-N1 remained completely conserved across all major SARS-CoV-2 variants over the past 4 years, demonstrating the potential of nucleocapsid RNA-targeting ASO as a robust antiviral strategy against SARS-CoV-2.