**5.2. Restoration of tumour suppressor miRNAs**

The restoration of tumour suppressor miRNAs, as a therapeutic strategy, includes viral delivery or synthetic miRNA mimics. Elevation of the expression levels of miRNAs can restore tumour inhibitory functions in cancer cells. Adeno-associated virus delivery of miRNAs or miRNA antagonists has the advantage of being efficient and because the virus does not integrate into the genome, non-mutagenic. In Myc-induced liver tumours, intravenous injection of adeno-associated virus 8 (AAV8)-expressing miR-26 resulted in the suppression of tumourigenicity by inducing tumour-apoptosis, without signs of toxicity [191]. These findings indicate a possibility strategy for the treatment of liver cancer, however, before this approach achieve widespread clinical use, the delivery and safety of different treatments needs to be improved.

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 133

chemotherapy [188]. In a non small cell lung cancer cell line, the down-regulation of miR-186\* which increased the expression of its direct target, Caspase-10, has been indicated the cause of the apoptosis induced by the chemopreventive agent curcumin [189]. Thus, the effect of anti-cancer drugs that modulate cell proliferation apoptosis on miRNA expression profiles was explored and could help for predicting apoptosis resistance. As a result, the knowledge of potential miRNAs implicated in apoptosis resistance could avoid unnecessary morbidity and may represent a novel class of biomarkers for facilitating personalized

Some cancers are originated in cells with intrinsic self-renewal activity or in differentiated cells in which self-renewal is activated by oncogenic mechanisms; hence, the study of normal self-renewal is important to improve our understanding of these mechanisms. Cancers express a spectrum of aberrantly differentiated cells, ranging from those that appear well differentiated to those that appear undifferentiated, and these phenotypes are commonly evident in the same tumour. This suggests that the transformation process can induce defects throughout the multistep differentiation process. Recent data suggest that cancers arise from rare self-renewing stem cells that are biologically distinct from their more numerous differentiated progeny. A small number of cells identied as cancer stem cells (CSCs) from solid tumours usually express organ-specic markers, contribute to chemotherapy resistance and are able to generate a new tumour in immunodecient mice. Moreover, there is growing evidence that pathways regulating normal stem cell self-renewal

Currently, there are two theories on the origin of cancer: the classic clonal evolution theory or stochastic model, by which malignant transformation results from random mutations and subsequent clonal selection of cancer cells with similar potential to regenerate the tumour growth [198,199]; and the CSC hypothesis, which considers the tumour to be formed by a small population of cells with stem cell-like properties. The features in common with stem cells are: indefinite self-replication, asymmetric cell division, and resistance to toxic agents, owing, in part, to elevated expression of ABC transporters [199,200,201]. In addition, they are also characterized by genetic instability (chromosomal and microsatellite), changes of chromatin, transcription and epigenetics, mobilization of cellular resources, and modified microenvironment interactions (tumour cells, stromal cells, extracellular, endothelium) [202]. Both paradigms of tumour propagation are likely to exist in human cancer but only the CSC model is hierarchical. It is important to note that the two models are not mutually

exclusive, as CSCs undergo clonal evolution, as shown for leukaemia stem cells [203].

The theory of cancer stem cells is not new, having started wonderings in the 19th century when comparing cancerous and embryonic tissue in the microscope and certain similarities were observed, annotating the idea that tumours arising from embryonic-like cells. This theory continued to evolve, and the isolation of four different tumour subpopulations from a single breast cancer in a mouse was reported in the decade of the 80s[204]. Tumour

treatment.

**6. Cancer stem cells and apoptosis** 

and differentiation are also present in cancer cells and CSCs [197].

Another strategy to increase the expression of a tumour-suppressor miRNA in cancer could be overcome by miRNA mimics, which are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs [192]. Introduction of synthetic miRNA mimics with tumour-suppressor function in cancer cells have been implicated to induce cell death and block proliferation in several studies [30,147,178,184,186]. In prostate and AMl cell lines mimics of miR-15a and miR-29 respectively, induced apoptosis by repression of anti-apoptotic genes Mcl-1 and Bcl-2 [184].

Multiple miRNAs have been found to inhibit the apoptotic pathway following their over expression during cancer development. Reduced expression of miR-15, miR-16, and let-7 has been observed in different types of cancers and as one consequence anti-apoptotic genes and apoptotic signalling pathways have been activated in these cancer cells [30,187,193]. As well, transfection of anti-miR-24 oligonucleotides has been proved to induce apoptosis in several cell lines [194]. It has been reported that miR-195, miR-24-2 and miR-365-2 act as negative regulators of the anti-apoptotic proto-oncogene Bcl2. The overexpression of these miRNAs caused an increase in apoptosis and also augmented the apoptotic effect of etoposide in breast cancer MCF7 cells [195]
