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

Abstract

We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

In vivo knockdown of antisense non‐coding mitochondrial RNAs by a lentiviral‐encoded shRNA inhibits melanoma tumor growth and lung colonization

“…As reported previously by our group, knockdown of ASncmtRNAs with a specific ASO induces apoptotic death of tumor cells, while leaving normal cells unaffected, suggesting this strategy for the development of an efficient therapeutic approach for cancer (Borgna et al., ; Lobos‐Gonzalez et al., ; Vidaurre et al., ). We also reported that, in the syngeneic in vivo B16F10 murine melanoma model, an ASO directed to the mouse ASncmtRNAs delays tumor growth and considerably reduces metastasis, resulting in enhanced survival (Lobos‐Gonzalez et al., ). Up to date, there are two FDA‐approved ASOs, for treatment of cytomegalovirus retinitis (fomivirsen) and familial hypercholesterolemia (mipomersen) (Aartsma‐Rus, ; McGowan et al., ; Vitravene Study Group, , ), and there are currently many ongoing clinical trials involving ASOs for treatment of different pathologies, including cancer (clinicaltrials.gov).…”

“…In addition, invasive properties of tumor cells are hindered by the in vitro treatment. At the molecular level, the observed effects on viability and invasiveness are mediated by a strong downregulation of survivin and factors associated with epithelial–mesenchymal transition, such as N‐cadherin and metallopeptidase‐9, among others (Borgna et al., ; Lobos‐Gonzalez et al., ; Vidaurre et al., ). These global effects on tumor cell viability and invasiveness exerted by ASK strongly suggest the potential of this approach for the development of an effective and safe therapeutic strategy against different types of cancer.…”

“…We translated these results to a preclinical scenario, using the highly aggressive B16F10 murine melanoma cell line in syngeneic C57BL6/J mice. Through different experimental approaches, we observed that injection of an ASO targeted to the murine ASncmtRNAs caused a significant delay in tumor growth and metastasis to the lungs and liver (Lobos‐Gonzalez et al., ). These results are very promising in light of a potential therapeutic strategy for cancer.…”

“…We reported a family of novel non-coding RNAs in human and mouse cells, derived from the mitochondrial genome (mtDNA) (Burzio et al, 2009;Lobos-Gonzalez et al, 2016;Villegas et al, 2000Villegas et al, , 2007. These transcripts, termed non-coding mitochondrial RNAs (ncmtRNAs), originate from the bidirectional transcription of the mtDNA.…”

It Is Imperative to Establish a Pellucid Definition of Chimeric RNA and to Clear Up a Lot of Confusion in the Relevant Research

“…A few 16S-rRNA-containing chimeras have also been reported in human and mouse cells [109,110,111,112,113,114,115,116,117], which are repeatedly detected and well-studied but in our opinion are technical artifacts [4]. They are frequently detected partly because in human and mouse cells the 16S rRNA is encoded by the mitochondrial genome that not only has hundreds or even thousands of copies in a single cell but also contains many reversely complementary regions [4].…”