**2.2. Differential expression of lncmtRNAs as a tool for cancer diagnostics**

containing probes complementary to sense or antisense ncmtRNAs, previously labeled at the 3′ end with digoxigenin-11-dUTP (Boehringer Mannheim, Germany) as described previously [36]. For detection, sections were incubated with a monoclonal anti-digoxigenin antibody conjugated to alkaline phosphatase, and after color development, positive signal correspond to a blue color,

**Figure 1.** Representative in situ hybridization assay showing the differential expression pattern of lncmtRNAs in tissues according to proliferative status. Upper panel shows absence of signal for both RNAs in non-proliferating tissues such as liver. Middle panel shows presence of strong punctuate signal, corresponding to nuclei in normal proliferating cervix epithelium. Lower panel shows a strong signal corresponding only to SncmtRNA and complete absence of signal corresponding to ASncmtRNA in a tumor tissue, exemplified by cervix carcinoma. H&E, hematoxylin-eosin staining.

Regarding subcellular localization, we found that in biopsies of normal and cancer tissues, nuclear localization of these transcripts was frequently observed. The extra-mitochondrial localization of these transcripts was confirmed by electron microscopy ISH. In normal cells, SncmtRNA and the ASncmtRNAs were found in the nucleus associated to chromatin. In tumor cells, SncmtRNA shows similar localization plus association with nucleoli, while the ASncmtRNAs are down-regulated. Although the meaning of the nuclear localization in normal proliferating cells of SncmtRNA and the ASncmtRNAs is unclear, the results suggest that these

representing the expression of the corresponding RNA (see **Figure 1**).

Magnification in upper panel, ×200. Magnification in cervix tissues, ×100.

184 Mitochondrial DNA - New Insights

As mentioned above, the ASncmtRNAs are downregulated in tumor cell lines and cells in tumor biopsies, independently of the tissular origin of the tumor analyzed. Therefore, this differential expression might be used for screening of cancer cells.

Cervical cancer is the fourth most common cancer in women worldwide. In 2012, this disease accounted for 528,000 new cases and 266,000 deaths among females [46]. Cervical cancer is of slow progression and, according to histopathological studies, there are at least three well-defined stages preceding cervical squamous carcinoma, known as cervical intraepithelial neoplasia (CIN). These stages (CIN1, CIN2 and CIN3) correspond to the progressive invasion of the cervical epithelium from the basal cell layer to the surface of the squamous epithelium [47]. Therefore, detection of premalignant lesions is key to preventing disease progression to advanced stages.

Therefore, we performed a study in order to evaluate and quantify the differential expression of non-coding mitochondrial RNAs during the progression of the disease. We found down-regulation of the antisense mitochondrial transcripts at early stages of cervical neoplasia (CIN1). Moreover, differential expression of ASncmtRNA v/s S-ncmtRNA showed significant difference, while, as expected, normal proliferating tissues did not display downregulation of ASncmtRNAs. Moreover, downregulation of ASncmtRNAs correlated with the over-expression of the tumor suppressor protein p16INK-4a [48, 49].

Bladder cancer (BC) is a significant cause of morbidity and mortality with a high recurrence rate. Early detection of bladder cancer is essential in order to remove the tumor, to preserve the organ and to avoid metastasis. The "gold standard" in the detection of BC is cystoscopy. This examination, however, is unpleasant, time consuming, expensive and may result in infections and urethral damage [50]. In a pilot study, we analyzed the differential expression of SncmtRNA and ASncmtRNAs in cells isolated from voided urine from patients with bladder cancer as a noninvasive diagnostic assay. For this purpose, we developed a test based on a multiprobe mixture labeled with different fluorophores, which takes about 1 hour to complete. We examined the expression of these transcripts in cells isolated from urine of 24 patients with BC and 15 healthy donors. The samples from BC patients revealed expression of SncmtRNA and downregulation of the ASncmtRNAs. Exfoliated cells recovered from the urine of healthy donors did not express these mitochondrial transcripts. The differential expression of the SncmtRNA and the ASncmtRNAs in cells isolated from voided urine can be explored as a new non-invasive diagnostic test for bladder cancer [51].

death of the highly aggressive and metastatic murine melanoma cell line B16F10 *in vitro*, con-

Long Noncoding Mitochondrial RNAs (LncmtRNAs) as Targets for Cancer Therapy

http://dx.doi.org/10.5772/intechopen.75453

187

We assessed the efficacy of the ASO treatment in vivo, using a B16F10 syngeneic model in C57BL6/J, where we applied a therapeutic approach similar to the clinical practice guidelines of melanoma: surgical resection of the lesion followed by systemic administration of ASO targeted to mASncmtRNAs (1560S). Remarkably, there was no visible sign of lung or liver metastasis at 120 days since the beginning of treatment with ASO, although one cannot discard the possibility of micro-metastasis [54]. In tail vein injection lung colonization assay, ASO treatment significantly reduced the number of metastatic nodules in the lungs, as well as their size [54]. Similar results were obtained in this model with a lentiviral delivery approach of therapeutic sequences. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. ASK treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls [55]. These results provide additional proof-of-concept for knockdown of ncmtRNAs for cancer therapy and altogether,

our results suggest that ASncmtRNAs could be potent targets for melanoma therapy.

In the RenCa cell line, corresponding to murine renal adenocarcinoma, we showed that ASK in vitro induces apoptosis mediated by downregulation of survivin, Bcl-2 and BclxL, the latter 2 members of another family of anti-apoptotic factors [56]. ASK also induces detrimental effects on metastatic potential, such as downregulation of N-cadherin, P-cadherin and MMP9, further strengthening the potential of this strategy for renal cell carcinoma (RCC) therapy. Remarkably, our in vivo studies in a subcutaneous syngeneic model of RenCa cells in Balb/C mice show complete reversal of tumor growth [57]. Moreover, in an orthotopic assay of murine RCC induced by injection of RenCa cells into the subcapsular region of the kidney showed that all the control mice contained tumors of different size. In contrast, only one mouse treated with the therapeutic ASO exhibited a small tumor. Histological analysis of each lung showed that control mice contained several and large metastatic nodules. In contrast, only two lungs of mice treated with therapeutical ASO contained metastatic nodules, which were significantly fewer and smaller. Finally, direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules [57].

These pre-clinical results with the RenCa and B16F10 murine models establish proof-of-concept that the ASncmtRNAs constitute a potent and selective target to develop a treatment for different types of cancer and positions this approach as an attractive strategy ready for clinical testing.

In this respect, the USA Food and Drug Administration (FDA) approved an oligonucleotide directed to the human ASncmtRNAs Andes-1537) as IND for a Phase I Clinical Trial. This study, currently under way and close to completion at UCSF, California, USA, is a first-in-human, open-label, dose escalation and expansion, 2-part study to determine the safety, tolerability, and maximum tolerated dose of Andes-1537 for Injection in patients with advanced unresectable solid tumors that are refractory to standard therapy or for which no standard therapy is available (NCT02508441). The result of this trial will be very important in order to continue with the

next phase to assess the antitumoral efficacy of this therapy in human cancer patients.

comitantly with survivin downregulation [54].

#### **2.3. Targeting antisense noncoding mitochondrial RNA: From bench to clinic**

As mentioned before, we postulated that the ASncmtRNAs might function as a unique mitochondria-encoded tumor suppressor. Therefore, we tested whether ASncmtRNA knockdown (ASK for short) induces alteration of cancer cell function. We found, in several tumor cell lines, that knockdown of the low copy number of the ASncmtRNAs with antisense oligonucleotides (ASO) induces massive cancer cell death by apoptosis without affecting the viability of normal cells. Apoptosis is triggered or potentiated by a drastic reduction in levels of survivin, a member of the inhibitor of apoptosis (IAP) family that is overexpressed in virtually all human cancers. Down-regulation of survivin is at the post-transcriptional level and probably mediated by microRNAs generated by Dicer from the ASncmtRNAs after ASO-induced RNase H processing [52]. It is important to highlight that the ASO treatment is efficient in inducing knockdown of the ASncmtRNAs, despite the fact that it is well known that oligonucleotides are not able to enter mitochondria in vivo [53]. In consequence, the obvious question is how are these transcripts targeted by ASOs? We have demonstrated that in normal human kidneys, renal cell carcinoma, mouse testis and the murine melanoma cell line B16F10, SncmtRNA and the ASncmtRNAs exit the mitochondria and are found localized in the cytoplasm and in the nucleus [42]. Consequently, our results suggest that the functional role of these molecules lies outside the organelle. Besides cell viability, ASK also drastically reduces proliferative index, anchorage-independent growth capacity, migration and invasion [52]. Taken together, our results allow us to propose that downregulation of the ASncmtRNAs constitutes an Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy.

In consequence, the ultimate challenge is to translate these results to an in vivo preclinical scenario with immunocompetent mice. For this purpose, we first characterized the murine noncoding mitochondrial RNAs (mncmtRNAs), which display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Most remarkable however is the identical expression pattern of these transcripts in both species. The mASncmtRNAs, expressed in normal proliferating cells, are downregulated in mouse tumor cells. ASK with ASO targeted to the mASncmtRNAs induces apoptotic cell death of the highly aggressive and metastatic murine melanoma cell line B16F10 *in vitro*, concomitantly with survivin downregulation [54].

the organ and to avoid metastasis. The "gold standard" in the detection of BC is cystoscopy. This examination, however, is unpleasant, time consuming, expensive and may result in infections and urethral damage [50]. In a pilot study, we analyzed the differential expression of SncmtRNA and ASncmtRNAs in cells isolated from voided urine from patients with bladder cancer as a noninvasive diagnostic assay. For this purpose, we developed a test based on a multiprobe mixture labeled with different fluorophores, which takes about 1 hour to complete. We examined the expression of these transcripts in cells isolated from urine of 24 patients with BC and 15 healthy donors. The samples from BC patients revealed expression of SncmtRNA and downregulation of the ASncmtRNAs. Exfoliated cells recovered from the urine of healthy donors did not express these mitochondrial transcripts. The differential expression of the SncmtRNA and the ASncmtRNAs in cells isolated from voided urine can be explored as a new

**2.3. Targeting antisense noncoding mitochondrial RNA: From bench to clinic**

As mentioned before, we postulated that the ASncmtRNAs might function as a unique mitochondria-encoded tumor suppressor. Therefore, we tested whether ASncmtRNA knockdown (ASK for short) induces alteration of cancer cell function. We found, in several tumor cell lines, that knockdown of the low copy number of the ASncmtRNAs with antisense oligonucleotides (ASO) induces massive cancer cell death by apoptosis without affecting the viability of normal cells. Apoptosis is triggered or potentiated by a drastic reduction in levels of survivin, a member of the inhibitor of apoptosis (IAP) family that is overexpressed in virtually all human cancers. Down-regulation of survivin is at the post-transcriptional level and probably mediated by microRNAs generated by Dicer from the ASncmtRNAs after ASO-induced RNase H processing [52]. It is important to highlight that the ASO treatment is efficient in inducing knockdown of the ASncmtRNAs, despite the fact that it is well known that oligonucleotides are not able to enter mitochondria in vivo [53]. In consequence, the obvious question is how are these transcripts targeted by ASOs? We have demonstrated that in normal human kidneys, renal cell carcinoma, mouse testis and the murine melanoma cell line B16F10, SncmtRNA and the ASncmtRNAs exit the mitochondria and are found localized in the cytoplasm and in the nucleus [42]. Consequently, our results suggest that the functional role of these molecules lies outside the organelle. Besides cell viability, ASK also drastically reduces proliferative index, anchorage-independent growth capacity, migration and invasion [52]. Taken together, our results allow us to propose that downregulation of the ASncmtRNAs constitutes an Achilles' heel of cancer cells, suggesting that the ASncmtRNAs

In consequence, the ultimate challenge is to translate these results to an in vivo preclinical scenario with immunocompetent mice. For this purpose, we first characterized the murine noncoding mitochondrial RNAs (mncmtRNAs), which display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Most remarkable however is the identical expression pattern of these transcripts in both species. The mASncmtRNAs, expressed in normal proliferating cells, are downregulated in mouse tumor cells. ASK with ASO targeted to the mASncmtRNAs induces apoptotic cell

non-invasive diagnostic test for bladder cancer [51].

186 Mitochondrial DNA - New Insights

are promising targets for cancer therapy.

We assessed the efficacy of the ASO treatment in vivo, using a B16F10 syngeneic model in C57BL6/J, where we applied a therapeutic approach similar to the clinical practice guidelines of melanoma: surgical resection of the lesion followed by systemic administration of ASO targeted to mASncmtRNAs (1560S). Remarkably, there was no visible sign of lung or liver metastasis at 120 days since the beginning of treatment with ASO, although one cannot discard the possibility of micro-metastasis [54]. In tail vein injection lung colonization assay, ASO treatment significantly reduced the number of metastatic nodules in the lungs, as well as their size [54]. Similar results were obtained in this model with a lentiviral delivery approach of therapeutic sequences. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. ASK treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls [55]. These results provide additional proof-of-concept for knockdown of ncmtRNAs for cancer therapy and altogether, our results suggest that ASncmtRNAs could be potent targets for melanoma therapy.

In the RenCa cell line, corresponding to murine renal adenocarcinoma, we showed that ASK in vitro induces apoptosis mediated by downregulation of survivin, Bcl-2 and BclxL, the latter 2 members of another family of anti-apoptotic factors [56]. ASK also induces detrimental effects on metastatic potential, such as downregulation of N-cadherin, P-cadherin and MMP9, further strengthening the potential of this strategy for renal cell carcinoma (RCC) therapy. Remarkably, our in vivo studies in a subcutaneous syngeneic model of RenCa cells in Balb/C mice show complete reversal of tumor growth [57]. Moreover, in an orthotopic assay of murine RCC induced by injection of RenCa cells into the subcapsular region of the kidney showed that all the control mice contained tumors of different size. In contrast, only one mouse treated with the therapeutic ASO exhibited a small tumor. Histological analysis of each lung showed that control mice contained several and large metastatic nodules. In contrast, only two lungs of mice treated with therapeutical ASO contained metastatic nodules, which were significantly fewer and smaller. Finally, direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules [57].

These pre-clinical results with the RenCa and B16F10 murine models establish proof-of-concept that the ASncmtRNAs constitute a potent and selective target to develop a treatment for different types of cancer and positions this approach as an attractive strategy ready for clinical testing.

In this respect, the USA Food and Drug Administration (FDA) approved an oligonucleotide directed to the human ASncmtRNAs Andes-1537) as IND for a Phase I Clinical Trial. This study, currently under way and close to completion at UCSF, California, USA, is a first-in-human, open-label, dose escalation and expansion, 2-part study to determine the safety, tolerability, and maximum tolerated dose of Andes-1537 for Injection in patients with advanced unresectable solid tumors that are refractory to standard therapy or for which no standard therapy is available (NCT02508441). The result of this trial will be very important in order to continue with the next phase to assess the antitumoral efficacy of this therapy in human cancer patients.
