microRNA regulators of apoptosis in cancer

This brief review summarizes our current knowledge on the microRNAs that regulate apoptosis machinery and are potentially involved in the dysregulation or deregulation of apoptosis, a well known hallmark of cancer. microRNAs are critical regulators of the most important cellular processes, including apoptosis. Expression of microRNAs is found to be dysregulated in many malignancies, leading to apoptosis inhibition in cancer, or resistance to current therapies. To date, there are over 80 microRNAs directly involved in apoptosis regulation or dysregulation that can impact cancer detection, initiation, progression, invasion, metastasis or resistance to anti-cancer therapy. Development of microRNA-based therapeutic strategies is now taking shape in the clinic. Thus, these microRNAs represent potential targets or tools for cancer therapy in the future.


Introduction:
Apoptosis is an evolutionary conserved physiological form of cell death required for removal of harmful and unuseful cells [1][2][3][4] . Apoptosis is not only necessary for the development and maintenance of the cellular and organismal homeostasis [1][2][3][4] , but it also serves as a defense mechanism, removing cells infected by viruses 5 . Signaling pathways that lead to cell death by apoptosis have as central effectors the proteolytic enzymes names caspases [1][2][3][4]6 . Caspases, which are cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases, are important not only in executing apoptosis, but are also involved in executing other forms of cell death (caspasedependent cell death) and in inflammation [1][2][3][4]7 . General consensus established two pathways of apoptosis: the extrinsic pathway, which is mediated by death receptors, and the intrinsic pathway, which is mediated by mitochondria [1][2][3][4] . Apoptosis dysregulation is a hallmark of cancer and many studies focused on targeting these mechanisms as an anti-cancer strategy [1][2][3][4] . A schematic representation of the apoptotic pathways is presented in Figure 1.
Over the last decade, microRNAs have emerged as critical regulators of normal and cancer cell structure and functions. microRNAas are involved in the regulation and control of most, if not all, cellular responses and fate. Some of these fundamental cellular processes that are linked to cancer and tumorigenesis are apoptosis, cell proliferation, stress response, differentiation and development. [8][9][10][11][12][13] In the last decade, a significant number of studies revealed and demonstrated Most of the components of these pathways can be targeted/regulated by microRNAs, including, but not limited to the ligands, death receptors, p53, Bcl-2 family members (Bim, Bax, Bcl-2, Bcl-xL, Mcl-1), XIAP and caspases. without doubt the involvement of the microRNAs and other non-coding RNAs in tumorigenesis, invasion, metastasis and susceptibility to anticancer therapies [8][9][10][11][12][13] . Moreover, regulation of apoptosis by microRNAs is one of the important mechanisms that can lead to apoptosis inhibition in cancer or resistance to current cancer therapies 8,9,13 .
MicroRNAs are small, 19-22 nucleotides long, non-coding RNAs, that negatively regulate gene expression, by translation inhibition and stepping up the degradation of the specific target mRNAs 8,9 . There have been more than few thousands microRNAs identified until now, which have been found to regulate more than 60% of the genes from human genome 14 . A single microRNA can regulate many (maybe even hundreds of) transcripts and a single transcript can have binding sites for (or can be regulated by) several microRNAs of the same or different sequence 8,9,14 . Currently, the major methods used to discover and verify the microRNAs and other non-coding RNAs are RNAseq and qPCR. While qPCR remains the standard for verification, RNAseq is starting to be widely employed in the microRNA identification 15 .
This brief review summarizes the most important microRNAs involved in apoptosis regulation or dysregulation in cancer. Table 1) Dysregulated microRNA expression has been observed in cancer tumorigenesis and this process is at least in part explained by epigenetic silencing of microRNA genes 12 . In the recent years, significant progress was made in the identification and characterization of the microRNAs involved in targeting genes critical for apoptosis process, including genes involved in apoptotic pathways or the regulatory pathways of apoptosis. The expression of many of these microRNAs is found to be dysregulated in cancers 12 , inducing an altered expression of the target genes and resulting in dysregulated apoptosis.

microRNA regulators of apoptosis in cancer (summary in
As an example, miR-125b targets p53 gene expression, decreasing p53-induced apoptosis in neuroblastoma cells 16 . p53 is not only a target of microRNAs, but can also regulate, similar to other transcription factors (Myc and E2Fs 11 ) the microRNAs expression. p53 directly regulates the expression of miR-34 family members (miR-34a, b, c), which mediates p53-induced apoptosis, and miR-34 downregualtion correlates with p53 mutation/downregulation and resistance to p53 induced apoptosis in pancreatic cancer 17 , lung cancer 18 , or neuroblastoma 19 . Noteworthy, other microRNAs are involved in either directly targeting the expression of death receptors or their ligands, or in modulating sensitivity to death ligands/ nonligand-induced apoptosis. For example, miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma 20 . Moreover, miR-221 and miR-222 are implicated in modulating the sensitivity to Apo2L/Trail-induced apoptosis in lung cancer cells, partly by direct targeting p27kit1 or Kit genes. These two microRNAs were also shown to directly target the estrogen receptor alpha (ER-alpha) and to confer resistance to Tamoxifen induced apoptosis in breast cancer 21,22 (see Table 1).
Most (if not all) Bcl-2 family members were shown to be direct targets of microRNAs, with altered expression in different types of cancers. For example, Mcl-1 (which is an anti-apoptotic member) is targeted by miR-29 and miR-113B (cholangiocarcinoma, lung cancer) 23,24 . Bim (a proapoptotic member) is targeted by miR-17-92 (B cell lymphoma) 25 and Bmf (a pro-apoptotic member) is a direct target of miR-221 (hepatocellular carcinoma) 26 . Many other microRNAs were shown to target these Bcl-2 family members, as well as other members of the family, such as Bax and Bcl-xL (see Table 1).
Caspases, the central effectors of apoptosis, are also confirmed targets of several microRNAs, including microRNA-224, involved in lung cancer pathogenesis, by targeting caspases 3 and 7 27 . Other microRNAs targeting caspases are summarized in Table 1.
Other genes involved in apoptosis regulation are also targeted by microRNAs, but are not discussed in this review. This long list includes the transcription factors such as Myc 28 and FOXOs 29 , phosphatases such as PTEN or PP2A, or kinases such as PI3K, Akt, Erk, Mek (and other kiases related to MAPK pathways) 30-32 .
Moreover, anti-cancer therapeutic strategies based on the regulation of microRNA activity have promise as anti-cancer treatments, since microRNAs have the ability to modulate fundamental cellular processes, such as apoptosis, cell proliferation or differentiation 8,9 . Systemic administration of microRNAs in mice (adeno viruses-mediated or by other strategies) successfully induced tumor-specific apoptosis. Noteworthy, no significant toxicity was observed 33 .

Conclusions
This brief review highlights our up-to-date basic knowledge on the microRNAs that regulate apoptosis machinery and are potentially involved in its dysregulation/deregulation, a well defined hallmark of cancer.
In cancer, many microRNAs can act as either oncogenes or tumor suppressors, and their action is context dependent 8,9 . Thus, many of the microRNAs mentioned in this article that regulate apoptosis can potentially be classified in one of these two categories.
microRNAs are involved in targeting expression of genes involved in apoptosis/cell survival, proliferation, differentiation, tumorigenesis, tumor invasion, angiogenesis and more. Expression of certain microRNAs is responsible for resistance of different malignancies to at least some of the current employed cancer treatments. Moreover, expression of specific microRNAs can predict patient outcomes. Regulation of apoptosis by microRNAs is one of the important mechanisms that can lead to apoptosis inhibition in cancer, or resistance to current therapies. Our current knowledge reveals over 80 microRNAs involved in apoptosis regulation or dysregulation, that can impact cancer initiation, progression, invasion, metastasis or resistance to anti-cancer therapy (Table 1). Moreover, circulating microRNA biomarkers are investigated for detection of tumor cells, diagnosis of different pathologies and for evaluation of treatment response.
microRNA abnormalities were identified in many types of malignancies and other diseases, and the development of miRNA-based gene therapy is now taking shape in clinical practice 8,9 .