**5. miRNA-based therapy**

128 Apoptosis and Medicine

*4.2.3. Smac-mimicking IAP antagonist* 

the gene, and resulting in the degradation of the target mRNA. The specificity in the AS ODN approach is based on the fact that any sequence of approximately 13 bases in RNA and 17 bases in DNA is estimated to be represented only once in the human genome. AS ODNs targeting survivin expression in human lung adenocarcinoma cell lines decreased survivin protein levels in a dose-dependent manner, induced apoptosis, stimulated higher levels of caspase-3 activation, and increased the sensitivity of cells to chemotherapeutics. XIAP AS ODNs effectively down-regulated both specific mRNA and protein levels in human non-small cell lung cancer growth both in vitro and in vivo. XIAP AS ODNs effectively induced apoptosis on their own and sensitized the tumor cells to the cytotoxic effects of several chemotherapeutics, including Taxol, etoposide and doxorubicin [26]. Furthermore, the administration of XIAP AS ODNs in a xenograft model of human non-

small cell lung cancer results in a significant down-regulation of XIAP protein [124].

AEG35156 is a 19-mer oligonucleotide targeting XIAP. Its sequence was designed to achieve maximal stability and potency and to minimize immunostimulation through avoiding CpG motifs. Phase I studies in patients with refractory malignancies established safety [136]. A phase I/II study of AEG35156 in combination with idarubicin and high-dose cytarabine in patients with relapsed or refractory AML demonstrated a dose-dependent knock-down of XIAP mRNA and protein and a promising response rate [137]. The molecule may rapidly enter randomized studies in AML while being also tested in lymphomas. The dose-limiting toxicity of an antisense oligonucleotide designed to inhibit surviving mRNA expression (LY2181308) was headache and the compound demonstrated some biological efficacy in decreasing survivin expression [138]. A phase II study has opened in solid tumours. The locked nucleic acid strategy was used to design other survivin-targeting antisense oligonucleotides, including SPC3042 [139] and EZN-3042 [140], which are currently being evaluated in Phase I clinical trials as single agents and in combination with cytotoxic drugs.

Smac mimetics may be a useful therapeutic target as over-expression of Smac may potentiate apoptosis by neutralizing the caspase-inhibitory function of IAPs. Following the discovery that an IAP-binding motif consisting of four NH2-terminal amino acid residues was sufficient to bind to the BIR3 domain of XIAP, Smac-peptide mimetics were constructed which were capable of competing with caspase-9, displacing it from the BIR3 domain of XIAP. The three members of the IAP family, XIAP, cIAP1, and cIAP2, are structurally homologous (XIAP amino acid sequence identity to cIAP1 and cIAP2 of 36% and 39%, respectively, the amino acid sequence identity between cIAP1 and cIAP2 is 70%). In particular, the BIR3 IBM region is well conserved among the three IAPs. The XIAP BIR3 residues involved in van der Waals contacts (Val298, Lys299, and Trp310) and hydrogen bonds (Gly306, Leu307, and Trp323) with the inhibitory compounds are conserved. Minor exceptions are Leu292, replaced by Val in the cIAPs, Glu314 substituted by Asp in both cIAPs, and Gln319, which is Glu325 in cIAP1, and Gln311 in cIAP2. Finally, residues Thr308 and Asp309 that were found relevant for Smac-mimetics interaction with XIAP BIR3, are

replaced by Arg314/Arg300 and Cys315/ Cys307 in cIAP1/cIAP2, respectively [127].

The miRNAs, is a class of endogenous, small, non-coding RNAs of 18–25 nucleotides in length, that negatively regulates gene expression by degradation of mRNA or suppression of mRNA translation. Mature miRNA products are formed in from a longer primary miRNA (pri-miRNA) transcript through sequential processing by the ribonucleases Drosha and Dicer1 [143,144]. miRNAs are known to repress thousands of target genes because only partial complementarity to the target mRNA is required. Thus, one miRNA may be simultaneously targeting a complexity of mRNAs as well as the expression of a single mRNA may be regulated by many miRNAs [143].

The miRNAs are involved in normal processes, including cellular development, differentiation, proliferation, apoptosis, and stem cell self-renewal [28].The aberrant expression or alteration of miRNAs contributes to a range of human pathologies, including cancer. Furthermore, the deregulation of miRNA causes evasion of apoptosis which involved tumourigenesis and drug resistance [122]. During tumour initiation and progression, the functionality of aberrant miRNAs may act as oncogenes (OncomiRs) or tumour suppressors (TSmiRs), a numbers of them are strongly related to the apoptosis phenomenon. Therefore, manipulation of miRNA expression levels which target genes and pathways that are involved in apoptosis could be a potential therapeutic strategy for developing efficient therapies against cancer. In addition, given that cancer cells often exhibit a distinctive pattern of miRNA expression, unique profiles of altered miRNAs expression could be useful as molecular biomarkers for tumour diagnosis, prognosis of disease-specific outcomes, and evaluation of tumour aggressiveness. Based on this, several anticancer therapies focusing on restoring miRNA activities and repairing gene regulatory networks or drug sensitivity are being developed.

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 131

Although several in vitro and in vivo technologies have been developed to inhibit the oncomiRs, it is still a long and arduous way to go for substantial applications of miRNAs in cancer treatments. To date, many OncomiRs seem to have a role in apoptosis (Table 5), therefore some have been regarded as hallmarks in tumour progressions and hot targets in cancer therapy. Thus, miR-21 is functionally considered oncogenic because it is overexpressed in various tumours [157,158]. In fact, antisense inhibition of miR-21 leads to the induction of programmed cell death in neuroepithelial cells, through activation of caspases [159]. This apoptosis induction was also confirmed in breast cancer, colon cancer, pancreas cancer, lung cancer, liver cancer, prostate cancer, stomach cancer and oral squamous cell carcinoma (OSCC) [31,160,161,162,163]. So far, multiple targets of miR-21 have been identified and mapped to anti-apoptotic signalling pathways which suggest a promising miRNA treatment for cancer [31,164]. Other examples have been reported; in breast cancer cells, the overexpresion of the anti-apoptotic Bcl-2 is restored by the silencing of miR-15a and miR-16 through the use of specific inhibitors [165]. As the anti-apoptotic Bcl-2 is frequently overexpressed in a number of human cancers, such as Hodgkin's lymphoma, cell lymphoma and breast, miR-15 and miR-16, it could be used for therapy of cancer-associated phenotypes.

**miRNA Function Gene target Cancer Type Reference(s)** 

Hepatocarcinoma, melanoma, glioblastoma, lung, prostate cancer, leukemia, gastric carcinoma

Non-small cell lung cancer, breast, prostate, gastric, hepatocellular cancer, colorectal cancer,

glioblastoma and leukemia

Lymphoma, lung, breast, stomach, colon and pancreatic cancer

Lymphocytic leukaemia, cholangiocarcinomahepatoc arcinoma, colon, breast, and

non-small-cell lung cancer and neuroblastoma

Leukemia, gastric cancer cells and prostate cancer

ovarian and breast cancer.

lung cancer

Bcl-2, SIRT1 Prostate cancer

Bcl-Xl Lung, colon, stomach,

[153,166,167,168,

[31,157,164,165,1 71,172,173,174,17

[148,149,178,179]

[180,181,182,183]

[30,184,185]

190]

[186,187,188,189,

169,170]

5]

[176,177]

**miR-221, miR-222** 

**miR-17-92 cluster** 

**miR-29a, miR-29b and miR-29c** 

**miR-34 family** 

**miR-15a, miR-16-1,** 

**Let 7** Tumour

OncomiRs PTEN

**miR-21** OncomiRs PTEN

Tumour suppressor

Tumour suppressor

Tumour suppressor

suppressor

**Table 5.** Key microRNAs involved in apoptosis

p27kip1 Bim PUMA

PDCD4 Bcl-2 Fas L

CDC42 and p85a (upregulating p53), Bcl-2

Bcl-2, Mcl1, PDCD6IP

OncomiRs PTEN, BIM, p21

There are two strategies of molecular therapy targeted at miRNA, one by the inhibition of oncogenic miRNAs and the other the over-expression of tumour suppressor miRNAs.
