Non-coding RNA biomarkers in pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, which is usually diagnosed at an advanced stage. The late disease diagnosis, the limited availability of effective therapeutic interventions and lack of robust diagnostic biomarkers, are some of the primary reasons for the dismal 5-year survival rates (∼8%) in patients with PDAC. The pancreatic cancer develops through accumulation of a series of genomic and epigenomic alterations which lead to the transformation of normal pancreatic epithelium into an invasive carcinoma - a process that can take up to 15-20 years to develop, from the occurrence of first initiating mutational event. These facts highlight a unique window of opportunity for the earlier detection of PDAC, which could allow timely disease interception and improvement in the overall survival outcomes in patients suffering from this fatal malignancy. Non-coding RNAs (ncRNAs) have been recognized to play a central role in PDAC pathogenesis and are emerging as attractive candidates for biomarker development in various cancers, including PDAC. More specifically, the ncRNAs play a pivotal role in PDAC biology as they affect tumor growth, migration, and invasion by regulating cellular processes including cell cycle, apoptosis, and epithelial-mesenchymal transition. In this review, we focus on three types of well-established ncRNAs - microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) - and discuss their potential as diagnostic, prognostic and predictive biomarkers in PDAC.

Keywords: Diagnostic biomarkers; Non-coding RNAs; Pancreatic ductal adenocarcinoma; Predictive biomarkers; Prognostic biomarkers.

Figure 1:

The evolution and progression of PDAC: The PDAC develops gradually over a period of time in which a series of genomic, epigenomic and morphological alterations are initiated in the normal cells. Initial genetic and epigenomic changes occur slowly during the transformation of normal epithelium through premalignant lesions (PanIN I- III); where the changes are not visible at the organ level making detection of premalignant lesions difficult. The premalignant cells continue to grow leading to tumor formation which gradually invades nearby lymph nodes, ultimately enter the systemic circulation and metastasize to distant organ sites. Tumor cells that reach the blood circulation also release their cellular contents (e.g. DNA, RNA, proteins etc.), which are interrogated for the development of blood-based liquid biopsy assays for the early disease detection. PanIN – pancreatic intraepithelial neoplasia; PDAC – pancreatic ductal adenocarcinoma; lncRNA – long non-coding RNA; circRNA – circular RNA; miRNA – microRNA; RBC – Red blood cell, WBC – White blood cell

Figure 2:

Overview of the biogenesis and functions of key ncRNAs: The biogenesis of miRNAs (left panel) involves the transcription of primary miRNA (Pri-miRNA) by RNA polymerase (II or III) from the miRNA gene. The Pri-miRNAs are long, RNA stem-loop structures, which are eventually processed by the DROSHA–DiGeorge syndrome critical region 8 (DGCR8) complex resulting in the cleavage of the Pri-miRNA and production of a smaller product called, the precursor miRNA (Pre-miRNA), which is approximately 60 nucleotides in length. The Pre-miRNA is exported to the cytoplasm from the nucleus by Exportin-5 protein. The Pre-miRNA is processed further in the cytoplasm by the ribonuclease DICER protein in conjunction with the RNA-binding protein transactivation response element RNA-binding protein (TRBP). The DICER-TRBP complex cleaves the Pre-miRNA to form a miRNA/miRNA duplex. One of the strands from this duplex is degraded while the other functional strand binds to the Argonaute 2 (AG02) protein and is incorporated into the RNA-induced silencing complex (RISC) involving DICER and TRBP. The miRNA strand guides the RISC complex to target mRNAs causing translational repression or degradation. The lncRNAs (middle panel) are transcribed by the RNA polymerase and are usually adenylated at the 3’ end and capped at 5’ end. Their expression is cell type- and cell state-specific and they can undergo alternative splicing leading to different isoforms. lncRNAs can regulate genes in many ways. For example, they can activate (enhancer RNAs) or inhibit the transcription of nearby genes by either directly interacting with the RNA polymerase or transcription factors. lncRNAs can also interact with DNA by virtue of their sequence complementarity to single stranded DNA mediating chromosomal looping. They can also induce changes in the chromatin structure and interact with nucleolar (paraspeckle) proteins forming paraspeckle assembly. Their other functions include acting as miRNA sponges, regulating mRNA stability, interaction with proteins or acting as a scaffold for proteins. The circRNAs (right panel) are also transcribed by the RNA polymerase similar to mRNAs however unlike mRNAs, circRNAs can be processed through alternative splicing of both, exons and introns of the pre-mRNA. The circular shape of circRNAs is the result of back-splicing which is described by lariat-driven circularization and intron-pairing-driven circularization. circRNAs can act as miRNA sponges due to the presence of multiple miRNA response elements in their sequence. Some circRNAs can also interact with proteins harboring RNA-binding sites. Additionally, they can also encode for and translate into various proteins.

Figure 3:

Mechanistic role of the miRNAs in PDAC: miRNAs can act both as tumor suppressors and oncogenes by regulating different key downstream gene targets that mediate cellular growth signaling pathways. For example, miR-217 which is often found downregulated in PDAC, acts as a tumor suppressor and targets KRAS oncogene which endows proliferation, survival and invasion properties onto cancer cells through the activation of several downstream effector pathways such as the PI3K/AKT and the RAF/ERK pathway. On the other hand, miR-21, which is usually found upregulated in PDAC, targets the tumor suppressor genes PTEN and PDCD4. miR-21 mediated inhibition of PTEN leads into activated downstream signaling of PI3K/AKT pathway. PDCD4, a tumor suppressor gene involved in apoptosis, invasion is inhibited by miR-21 and is potentially involved in PI3K/AKT signaling pathway. miRNAs can also induce transcriptional repression or activation by modulating chromatin structure through the targeting of epigenetic regulatory genes. For example, miR-342-3p can act as a tumor suppressor by inhibiting cancer cell proliferation and invasion through targeting DNMT1.

Figure 4:

Functional roles of lncRNAs and circRNAs in PDAC: The lncRNAs and circRNAs implicated in PDAC are shown. The left half of the circle shows the lncRNAs and their downstream targets, cellular phenotype and target genes or pathways affected. For example, HOTAIR interacts with EZH2 and binds to the promoter region of miR-34a. This binding leads to chromatin modification due to hypermethylation of miR-34a promoter repressing its expression. The lncRNA ENST00000480739 acts as an enhancer for OS9, increasing the expression of OS9. OS9 potentially binds to the ubiquitination complex comprised of prolyl hydroxylase (PHD) and other proteins which lead to the degradation of HIF-1α protein changing the hypoxic condition in the cells. The lncRNA PVT1 interacts with the SMAD2/3 proteins of the TGF-β/SMAD pathway, activating the expression of vimentin and inducing EMT changes in pancreatic cancer cells. The lncRNA MALAT1 interacts with EZH2 which in turn leads to the hypermethylation of E-cadherin promoter inhibiting its expression and increasing invasive potential of pancreatic cancer cells. The lncRNA HOTTIP regulate the expression of HOXA9, activating the Wnt-β-catenin pathway and increasing cancer stem cell properties of the pancreatic cancer cells. CircRNAs and their target miRNAs are shown in the right half of the circle. For example, circLDLRAD3 sequester miR-137-3p. In the absence of miR-137-3p, pleiotrophin (PTN) is expressed and leads to increased proliferation and invasion of pancreatic cancer cells. Circ0030235 inhibits the expression of miR-1253 and miR-1294 and leads to increased cell progression. Circ0007534 acts as a sponge for miR-625 and miR-892b and affects apoptosis. Circ-PDE8A targets miR-338 activating the MET/AKT pathway and increase invasive potential of pancreatic cancer cells. Circ-IARS increases the expression of RhoA by inhibiting miR-122 increasing the permeability and invasive potential of pancreatic cancer cells.