MicroRNA Expression in Colonic Adenocarcinoma

Nicole C. Panarelli

MicroRNAs (miRNAs) are 18 to 25 nucleotide noncoding RNA sequences that regulate gene expression by destabilizing transcription and inhibiting translation of target messenger RNAs (mRNAs). They were discovered in Caenorhabditis elegans in 1993, and more than 1,000 human miRNA species have since been described (miRBase at http://www.mir-base.org).¹ MicroRNAs have been implicated in the regulation of up to 30% to 60% of human genes, including some that are involved in normal tissue development, inflammation and repair, and carcinogenesis.² Data from early human studies indicated that genes encoding miRNAs are often located in close proximity to cancer-associated genomic regions, such as frequently mutated genes and/or common breakpoints, suggesting that they may be altered in neoplasia. In fact, dysregulated miRNAs are detected in a variety of human malignancies. They are found in colon cancer tissue extracts, cell lines, xenografted tumors, and plasma and stool from colon cancer patients.³, ⁴, ⁵, ⁶ and ⁷ Increased or decreased miRNA expression in colon cancer is predictive of cancer-specific survival in some studies, and novel therapies aimed at abrogating their effects are currently under investigation. Thus, these molecules comprise a major class of potentially useful diagnostic and prognostic markers, as well as candidates for future targeted therapies. This chapter discusses current knowledge regarding the role of miRNAs in the development and progression of colonic adenocarcinoma.

MicroRNA SYNTHESIS, TRANSLATION, AND TRANSCRIPTION

Primary miRNAs (pri-miRNAs) are transcribed in the cell nucleus by RNA polymerase II and processed into hairpin-shaped 60- to 70-nucleotide precursor miRNAs (pre-miRNA) by the Drosha enzyme complex. Pre-miRNAs are then transported to the cytoplasm and further processed by the ribonuclease (RNAse) III enzyme, Dicer, yielding an asymmetric duplex composed of one strand that is loaded into the RNA-induced silencing complex (RISC) and a second strand that is degraded (Figure 13.1).⁸

MicroRNA molecules that bind RISC can potentially bind to either the 3′- or 5′-untranslated region (UTR) of hundreds of different mRNAs by sequence complementarity. The miRNA/RISC apparatus controls gene expression at the translational level by causing bound mRNAs to prematurely dissociate from ribosomes, thereby facilitating degradation of mRNA transcripts (Figure 13.1). MicroRNAs also regulate translation in a RISC-independent fashion. They competitively inhibit mRNAs from binding other molecules that normally regulate protein synthesis, processing, and export.⁹ This process is referred to as “decoy activity” and prevents physiologic interactions between translational cofactors and mRNA transcripts, resulting in abnormally increased or decreased levels of target gene products.¹⁰

MicroRNA-driven transcriptional regulation also occurs, although its mechanisms are less understood. Some data suggest that miRNAs play a role in epigenetic gene silencing. MicroRNA-dependent methylation of target genes can decrease mRNA transcripts in plants that lack Dicer
activity, and some miRNAs promote epigenetic methylation of cell cycle genes in mammalian cell lines.¹¹ MicroRNAs also bind directly to DNA within promoter regions, thereby blocking transcription.¹²

FIGURE 13.1: Primary microRNAs (pri-miRNAs) are transcribed as multimers composed of several hairpin loop structures with imperfect base pairing. These multimers are subsequently cleaved by the Drosha enzyme complex into single hairpin loops and exported to the cytoplasm. The Dicer enzyme complex processes the transcripts into asymmetric duplexes. One strand of nucleic acids is degraded and the other is loaded into the RNA-induced silencing complex (RISC), which represents the functional unit of miRNA-driven translation regulation. Each miRNA-RISC complex can bind to several different mRNA targets by sequence complementarity, thereby preventing ribosomal interaction with the open reading frame (ORF).

Early investigations identified up to 50 miRNA species that were differentially expressed in colonic adenocarcinoma compared to nonneoplastic colonic tissue (Table 13.1).¹³,¹⁴ Overall, data from several studies indicate that colon cancers commonly show upregulation of miR-31, miR-96, miR-133b, miR-135b, and miR-183.¹⁵ On the other hand, marked downregulation of some species, such as miR-143 and miR-145, suggests that some miRNAs act as tumor suppressors in the colon.¹⁶ The importance of these and additional miRNAs in in vitro and in vivo studies is discussed below.

ROLES OF MicroRNAs IN CARCINOGENIC PATHWAYS

Critical genetic and epigenetic events promote the pathogenesis of sporadic and inherited colon cancers. Genetic mutations and alterations in chromatin structure lead to abnormal protein production and dysregulation of cell signaling pathways related to differentiation, proliferation, and survival. MicroRNAs interact with these intracellular mechanisms and promote cancer development through either oncogenic or tumor suppressor effects (Table 13.2).

APC/β-Catenin/Wnt Signaling

Most colonic adenocarcinomas are derived from preexisting adenomas that progress via chromosomal instability, aneuploidy, and loss of heterozygosity.¹⁷ Approximately 80% of sporadic tumors harbor molecular abnormalities in adenomatous polyposis coli (APC) or other elements
of this pathway that promote constitutive activation of Wnt-mediated signaling.¹⁸ In the absence of functional APC, Wnt signaling leads to accumulation of β-catenin in the cytoplasm, which is translocated to the nucleus where it facilitates transcription of protooncogenes, such as CMYC and cyclin D1.¹⁸ Biallelic APC inactivation usually results from mutations, but may also occur due to downregulation by miRNAs. For example, both miR-135a and miR-135b are complementary
to the 3’ UTR of APC mRNA. Colon cancers with high miR-135a and miR-135b levels also contain fewer APC mRNA transcripts, suggesting that miR-135a and miR-135b regulate Wnt signaling by promoting decay of APC mRNA.¹⁹

Table 13.1 Summary of the Literature Regarding Dysregulation of MicroRNAs in Colon Cancer

Study	Most Significantly Overexpressed miRNAs in Colorectal Cancer	Most Significantly Underexpressed miRNAs in Colorectal Cancer
Bandres et al.¹⁵	miR-31, miR-96, miR-133b, miR-135b, miR-183	miR-145
Guo et al.²⁸	miR-93, miR-92, miR-520h, miR-508, miR-505, miR-449, miR-429, miR-384, miR-373, miR-34c, miR-326, miR-25, miR-224, miR-210, miR-200a, miR-19b, miR-19a, miR-18a, miR-183, miR-182, miR-181b, miR-181a, miR-181c, miR-17-5p, miR-148a, miR-141, miR-130b, miR-128a, miR-106b, miR-106a, miR-let-7d	miR-96, miR-485-5p, miR-422b, miR-342, miR-214, miR-199a, miR-195, miR-150, miR-145, miR-143, miR-133a, miR-126, miR-125b, miR-100
Michael et al.¹⁶		miR-143, miR-145
Sarver et al.⁴⁵	miR-135b, miR-96, miR-182, miR-182,^* miR-183	miR-1, miR-133a, miR-30a-3p, miR-30a-5p, miR-20b, miR-363
Schepeler et al.³⁹	miR-20a, miR-510, miR-92, miR-513	miR-145, miR-455, miR-484, miR-101
Schetter et al.³⁴	miR-20a, miR-21, miR-106a, miR-181b, miR-203
Slaby et al.³³	miR-31, miR-21	miR-145, miR-143
Volinia et al.⁷	miR-24-1, miR-29b-2, miR-20a, miR-10a, mir-32, miR-203, miR-106a, miR-17-5p, miR-30c, miR-223, miR-126^*, miR-128b, miR-21, miR-24-2, miR-99b, miR-155, miR-213, miR-150, miR-107, miR-191, miR-221	miR-9-3
Wang et al.¹⁴	miR-106b, miR-135b, miR-18a, miR-18b, miR-196b, miR-19a, miR-224, miR335, miR-424, miR-20a^*, miR-301b, miR-734a	miR-378, miR-378^*
Xi et al.⁴⁰	miR-15b, miR-181b, miR-191, miR-200c

Table 13.2 Regulatory MicroRNA Targets and Function in Colon Cancer

Regulatory miRNA	Status in Colon Cancer	Regulatory Function	Net Effect
miR-135a, b	Increased	Inhibits APC translation	Promotes Wnt signaling
miR-143	Decreased	Inhibits KRAS translation	Decreased response to EGFR-targeted therapy
miR-126	Decreased	Suppresses p85Β regulatory subunit of PI3K	Stimulates cell proliferation through the AKT pathway
miR-21	Increased	Inhibits PDCD4 translation	Increases cell growth and division through TGF-β pathway
miR-101	Decreased	Inhibits COX2 translation	Promotes tumor angiogenesis
miR-155	Increased	Inhibits MLH1, MSH2, and MSH6 transcription	Inhibits DNA mismatch repair mechanisms

Ras/Raf/MAPK and PI3K/AKT/mTOR Pathways

Epidermal growth factor receptor (EGFR) is a cell surface tyrosine kinase receptor that is overexpressed in a subset of colonic adenocarcinomas.²⁰ Increased expression usually results from EGFR amplification, but can reflect other poorly characterized alterations. Activation of EGFR-mediated signaling promotes carcinogenesis and cancer progression by stimulating the KRAS/BRAF/MAPK and PI3K/AKT/mTOR pathways, both of which are modulated by miRNAs.²¹

KRAS mutations are found in 30% to 40% of colon cancers, and their detection is extremely important from a clinical standpoint. The presence of KRAS mutations is strongly predictive of resistance to EGFR inhibitors, such as cetuximab and similar agents.²² Several miRNA species show different expression levels among KRAS wild-type tumors compared to KRAS-mutated colon cancers. Colon cancers with KRAS mutations typically show increased miR-92a, miR-127-3p, and miR-486-5p and decreased miR-378, miR-143, miR-let-7a, and miR-18a.²³ MicroRNA-92a is a member of the miR-17-92 cluster that promotes cancer cell proliferation, suppresses apoptosis, and facilitates angiogenesis. KRAS is also a likely target for miR-143. Transfection of colon cancer cell lines with anti-miR-143 oligonucleotides increases KRAS transcript levels, whereas KRAS transcripts decrease in cell lines transfected with pre-miR-143.⁴,²⁴ These findings suggest that miR-143 downregulation promotes cancer cell growth by disinhibiting KRAS translation. Similarly, transfection of both miR-let-7a and miR-18a into colon cancer cell lines reduces KRAS

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