We reported the presence in human cells of a noncoding mitochondrial RNA that contains an inverted repeat (IR) of 815 nucleotides (nt) covalently linked to the 5 end of the mitochondrial 16S RNA (16S mtrRNA). The transcript contains a stem-loop structure and is expressed in human proliferating cells but not in resting cells. Here, we demonstrate that, in addition to this transcript, normal human proliferating cells in culture express 2 antisense mitochondrial transcripts. These transcripts also contain stem-loop structures but strikingly they are down-regulated in tumor cell lines and tumor cells present in 17 different tumor types. The differential expression of these transcripts distinguishes normal from tumor cells and might contribute a unique vision on cancer biology and diagnostics.differential expression in cancer ͉ RNAs with stem-loop structures R ecently, we described a novel human mitochondrial transcript of 2,374 nt that contains a long inverted repeat (IR) linked to the 5Ј end of the 16S mitochondrial rRNA (16S mtrRNA) (1, 2), which we designated noncoding mitochondrial RNA or ncmtRNA (2). The IR generates a stem-loop structure with an 820-bp doublestranded region and a 40-nt loop (2). In situ hybridization (ISH) showed that the ncmtRNA is overexpressed in several tumor cell lines but not in nondividing cells, suggesting that the ncmtRNA may play a role in cell proliferation (2).Because the results described before were obtained using tumor cell lines (2), we asked whether the ncmtRNA (from now on, sense ncmtRNA, SncmtRNA) is expressed in normal proliferating cells. Here we show that ISH of human umbilical vein endothelial cells (HUVEC), foreskin keratinocytes (HFK) (3), and human tonsil endothelial cells (HUTEC) (4) also express the SncmtRNA. However, and in striking contrast with tumor cell lines, ISH of normal proliferating cells revealed expression of antisense transcripts. These molecules were identified as 2 unique transcripts containing IRs linked to the 5Ј region of the antisense 16S mtRNA transcribed from the L-strand of the mtDNA (1, 2). We named these transcripts antisense ncmtRNA-1 (ASncmtRNA-1) and antisense ncmtRNA-2 (ASncmtRNA-2). Finally, we show that the antisense transcripts are also expressed in proliferating cells present in normal human tissues but are down-regulated in cells present in human tumors of different types and patients.

Previously, we reported the presence in mouse cells of a mitochondrial RNA which contains an inverted repeat (IR) of 121 nucleotides (nt) covalently linked to the 5′ end of the mitochondrial 16S RNA (16S mtrRNA). Here, we report the structure of an equivalent transcript of 2374 nt which is over-expressed in human proliferating cells but not in resting cells. The transcript contains a hairpin structure comprising an IR of 815 nt linked to the 5′ end of the 16S mtrRNA and forming a long double-stranded structure or stem and a loop of 40 nt. The stem is resistant to RNase A and can be detected and isolated after digestion with the enzyme. This novel transcript is a non-coding RNA (ncRNA) and several evidences suggest that the transcript is synthesized in mitochondria. The expression of this transcript can be induced in resting lymphocytes stimulated with phytohaemagglutinin (PHA). Moreover, aphidicolin treatment of DU145 cells reversibly blocks proliferation and expression of the transcript. If the drug is removed, the cells re-assume proliferation and over-express the ncmtRNA. These results suggest that the expression of the ncmtRNA correlates with the replicative state of the cell and it may play a role in cell proliferation.

Background: Down-regulation of the antisense mitochondrial ncRNAs (ASncmtRNAs) is a general vulnerability of cancer cells.Results: Knocking down the ASncmtRNAs induces apoptosis, down-regulation of survivin, and generation of microRNAs.Conclusion: Induction of apoptosis in cancer cells is potentiated by down-regulation of survivin.Significance: The ASncmtRNAs represent a new potential therapeutic target for cancer.

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.

Background: Antisense mitochondrial ncRNAs are down-regulated during oncogenesis by unknown mechanisms.Results: High risk HPV E2 oncogene induces down-regulation of the antisense transcripts. Additionally, E6 and E7 induce expression of a new sense mitochondrial ncRNA.Conclusion: HPV oncogenes modulate expression of mitochondrial ncRNAs.Significance: During non-viral oncogenesis, cellular factor(s), analogously to E2, could induce down-regulation of the antisense mitochondrial ncRNAs.

The ubiquitous localization of these mitochondrial transcripts in the nucleus suggests that they are new players in the mitochondrial-nuclear communication pathway or retrograde signaling. Down regulation of the ASncmtRNAs seems to be an important step on neoplastic transformation and cancer progression.

Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma.

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.