RNA as a Therapeutic Molecule

Cancer as a Genetic Disease of Protein-Coding Genes and Noncoding RNAs

Why the need for RNAs as therapeutic molecules? One of the most unexpected and fascinating discoveries in the past few years in molecular oncology is that in a specific tumor, abnormalities in both protein-coding genes (PCGs) and ncRNAs can be identified, and the interplay between them is causally involved in the initiation and progression of human cancers. The “classic” molecular oncology dogma was that cancer is a genetic disease involving tumor suppressor and oncogenic proteins. Recent findings that small noncoding RNAs called microRNAs (miRNAs) are involved in the pathogenesis of most cancers reveal a new layer of complexity in the molecular architecture of human cancers ( Table 55-1 ).

MicroRNAs represent a new class of small ncRNAs able to regulate gene expression. MicroRNAs are distinct from, but related to, small interfering RNAs (siRNAs), which have been identified in a variety of organisms (for reviews, see References ). These small 19- to 24-nucleotide (nt) RNAs are transcribed as long primary transcripts of several kilobases (kb) in length, named pri-miRNA. Pri-miRNAs are processed in the nucleus into precursor hairpin RNAs (70 to 100 nt in length) named pre-miRNA by the double-stranded RNA-specific ribonuclease Drosha. The hairpin RNAs are transported to the cytoplasm, via an exportin-5–dependent mechanism, where they are digested by a second double-stranded specific ribonuclease named Dicer. In animals, single-stranded miRNAs bind specific mRNA through a low complementary binding site located in the target mRNA, mainly at their 3′ UTR. By a mechanism that is not fully characterized, the bound mRNA remains untranslated, resulting in reduced levels of the corresponding protein—and/or the bound mRNA can be degraded, causing a decrease in the transcript and consequently, in the corresponding protein. The role of miRNAs was proven to be important in essential biologic processes for the eukaryotic cell such as pancreatic cell insulin secretion ( miR-375 ), adipocyte development ( miR-375 ), cell proliferation control ( miR ′ 125b and Iet-7), brain patterning ( miR-430 ), hematopoietic B-lymphocyte lineage fate ( miR-181 ), or B-lymphocyte survival ( miR-15a and miR-16-1 ).

MicroRNAs were found to be involved in the pathophysiology of all types of analyzed human cancers. Among the new paradigms of molecular oncology are the following:

• 1.

  Several genome-wide profiling techniques (for review, see References ), such as oligonucleotide miRNA microarray, bead-based flow cytometric technique, and quantitative reverse-transcriptase-polymerase chain reaction (qRT-PCR) for precursor and active miRNA or the miRAGE (serial analyses of gene expression for miRNAs), were performed on various cancer histotypes, including chronic lymphocytic leukemia (CLL), breast cancer, glioblastoma, thyroid papillary carcinoma, hepatocellular carcinoma, lung cancer, colon cancer, and endocrine and exocrine pancreatic tumors. From these studies it has become clear that in cancer cells the main alteration of the microRNome (defined as the full complement of microRNAs present in a genome) is represented by aberrant gene expression, consisting of abnormal levels of expression for mature and/or precursor miRNA sequences compared with the corresponding normal tissues.

• 2.

  Germline and somatic mutations in miRNAs or polymorphisms in the protein coding mRNAs targeted by miRNAs may also contribute to cancer predisposition, initiation, and progression. In somatic cells, miRNA alterations could initiate or contribute to tumorigenesis, whereas germline mutations could represent cancer-predisposing events.

• 3.

  MiRNA profiling achieved by various methods has allowed the identification of signatures associated with diagnosis, staging, progression, prognosis, and response to treatment of human tumors (for review, see Reference ). Therefore, miRNA “fingerprinting” represents a new addition to the diagnostic and prognostic tools to be used in medical oncology.

Other types of ncRNAs (such as ultraconserved genes [UCGs] and long intergenic noncoding RNAs [lincRNAs]) have also been linked to human cancers. One of the most intriguing characteristics of miRNAs is the near-complete conservation of orthologous genes. For example, the active molecules of the miR-16-1 / miR-15a cluster, shown to be an essential player in the initiation of CLL, are completely conserved in humans, mice, and rats and highly conserved in 9 of 10 primate species sequenced. Comparative sequence analysis represents an essential tool in the identification of genomic DNA regions with important biologic functions. Several of these highly conserved genomic sequences were considered not genic (not producing a transcript) and were called conserved nongenic sequences. A special subset of conserved sequences named ultraconserved regions (UCRs) include, by definition, intra- and intergenic sections of the human genome that are absolutely conserved (100% identical with no insertions or deletions) between orthologous regions of the human, rat, and mouse genomes. Because of the high degree of conservation, the UCRs have been demonstrated to have fundamental functional importance for the ontogeny and phylogeny of mammals and other vertebrates. Recently, it was proved that most UCRs are transcribed and that hundreds of UCGs are consistently altered in a significant percentage of analyzed leukemias and carcinomas. UCGs are frequently located at fragile sites and genomic regions involved in cancers. It has also been proven that the inhibition of an overexpressed UCG induces apoptosis in colon cancer cells, and that the expression of some UCGs may be regulated by miRNAs abnormally expressed in CLL. These new regulatory mechanisms support a model in which various types of noncoding genes are actively involved and cooperate with protein-coding genes in human tumorigenesis. Gathering all these notions together makes it clear that noncoding RNA genes, once seen as second-level genomic elements, are now at the center of attention in cancer research. Therefore, it is reasonable to attempt to expand the anticancer ammunition with RNA molecules capable of attenuating or completely abolishing the function of overexpressed ncRNAs—or, alternatively, to reexpress at physiologic levels the deleted or downregulated ncRNAs.

Main Types of Therapeutic RNA Molecules

Three different types of RNA molecules—the ribozymes, the siRNAs, and the anti-miRNA agents—have passed preclinical testing for efficiency in downregulating a target and now are entering clinical trials. At least two more types of RNA molecules recently added to the expanding list of anticancer ammunition—the miRNA-mimic agents and the 8-mer anti-miRNA LNA (locked nucleic acid) molecules—are going through preclinical tests. The antisense oligonucleotide (ASO) strategy was primarily developed using DNA molecules (for detailed discussion, see References ). Although the history of RNAs as therapeutic molecules is two decades long, too few clinical trials have yet been conducted on large numbers of cancer patients to allow any convincing conclusion to be drawn. The first hints are encouraging and support the development of new and larger clinical trials. Most of the clinical and preclinical data were gathered from patients with viral infections, such as human immunodeficiency virus type-1 (HIV) or chronic hepatitis C virus (HCV). For a glossary of terms used in RNA inhibition strategies, see Table 55-2 .