Genetic Mutations for Predicting Pancreatic Ductal Adenocarcinoma Susceptibility
Pancreatic ductal adenocarcinoma (PDAC) is the deadliest malignancy representing about 90% of all pancreatic cancers. PDAC progresses asymptomatically until death and is refractory to most treatment methods. Surgery is the most effective treatment. Nevertheless, it is well known that only 15-20% of PDAC patients are surgical candidates and radiology has its limitations. The survival is limited with 0.9 being the case-fatality ratio. This is mainly attributed to delayed diagnosis, lack of early biomarkers, and metastasis formation. Researchers projected that PDAC could be the 2nd cause of cancer mortality by 2030.
Genomic landscape of Pancreatic Ductal Adenocarcinoma (PDAC)
The very recent genomic studies revealed a hypermutated background of Pancreatic ductal adenocarcinoma.
Genetic mutations and altered molecular pathways in pancreatic cancer have been described for decades. Recent studies showed about 60 gene alterations per tumor in PDAC, most of which are point mutations. However, most PDAC genome studies confirmed the mutation of four genes in particular – KRAS, TP53, SMAD4, and CDKN2A.
More than 95% of PDACs carry mutated KRAS alleles. The KRAS gene, located on chromosome 12, is a member of the RAS gene family and encodes KRAS protein. When bound to GTP, KRAS regulates cell proliferation, cell signaling, differentiation, and apoptosis. In PDAC, KRAS mutations are found in codons 12,13, 16, 60, and 61. These point mutations arise due to a single amino acid substitution from G to C.
CDKN2A, the causative gene of FAMMM, is a tumor suppressor gene located at chromosome 9p21. It encodes two proteins – p16 (p16/INK4A) and p14ARF- both of which suppress PDAC onset by arresting cell cycle at G1 and G1/S – G2/M phases respectively. Numerous studies indicated that CDKN2A is silenced in 90-95% of PDAC cases, by homozygous deletion, allelic mutation, or hypermethylation of promoter genes.
TP53 (also known as p53) is the tumor suppressor gene that induces apoptosis by activating target genes in response to DNA damage or oxidative stress. It also boosts the expression of cyclin-dependent kinase inhibitor CDKN1A, thereby stopping cell cycle progression. TP53 is mutated in 75% of Pancreatic ductal adenocarcinoma (PDAC) cases, making it a potent biomarker for prognosis and therapy prediction.
SMAD4 is an important transcription factor in the transforming growth factor β (TGFβ) signaling pathway. Also referred to as pancreatic cancer deletion gene 4 (DPC4), SMAD4 is silenced in approximately 50-60% of advanced PDAC cases. Along with TGFβ1, it acts as a tumor suppressor and regulates the pancreatic cell cycle and apoptosis. Numerous studies reported that cancer patients with biallelic deletion of SMAD4 had a higher incidence of metastasis than those with wild SMAD4.
Aberrant expression and genetic alteration of lncRNAs in pancreatic cancer
Most of the human transcriptome is non-coding; the transcripts referred to as non-coding RNAs (ncRNAs) have limited to no translation capacity. Examples include transfer RNA(tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs), exosomal RNAs (exRNAs), small Cajal body-specific RNAs (scaRNAs), and long noncoding RNAs (lncRNAs). Of these, miRNAs and lncRNAs are the most extensively investigated ncRNAs in molecular carcinogenic studies.
Studies at both transcript and genomic levels indicated that lncRNAs can act both as oncogenes or tumor suppressors. But in the majority of the cases, they are up-regulated for their expression in normal tissues. Recently, it has been found that three lncRNAs – HOTTIP, HOTAIR, and H19 – play vital roles in cancerization by changing the chromatin states of the genome. Zhang. J et al showed increased expression of HOTAIR in pancreatic cancer patients. Later, K Kim et al. confirmed the pro-oncogenic nature of HOTAIR in pancreatic cancer.
Accumulating evidence indicates that ncRNAs play a significant role in the carcinogenesis and prognosis of PDAC. More recently, numerous integrative genomic studies have revealed the role of lncRNA in apoptosis, cell proliferation, metastasis, and relapse of pancreatic cancers. Since any dysregulation of lncRNA expression can lead to cancer, these RNA transcripts of ≥200 bp are considered a potential prognostic cancer biomarker in PDAC. Some studies showed that the dysregulation of Small Nucleolar RNA Host Gene 15 (SNHG15), a recently identified lncRNA on chromosome 7p13, is associated with the progression and metastasis of pancreatic cancer.
lncRNA polymorphisms and pancreatic cancer susceptibility
Genetic alterations including single nucleotide polymorphisms (SNPs) affect susceptibility to various cancers. However, a large number of these SNPs are located within the non-coding regions of the transcriptome. Research in this field confirmed the polymorphic nature of lncRNAs. Studies have also reported that polymorphisms in long noncoding RNA may regulate their expression and function, leading to tumor susceptibility. Previous studies have identified two PDAC risk loci in lncRNAs – LINC00673 rs11655237 SNP and rs6971499 (LINC-PINT) SNP. Cancer-risk SNPs have also been found in other cancer types such as breast cancer, ovarian cancer, lung cancer, glioma, colorectal cancer, and more.
Even with the potential importance of lncSNPs in pancreatic cancer, little has been investigated about the biological risk markers. The purpose of this study was to find new PDAC susceptible loci and to improve the risk stratification and treatment measures.
Materials and Methods
This multicentric study included the data from Genome-Wide Association Studies (GWAS). The cohort consisted of 9893 PDAC cases (defined by the standard PDAC diagnostic protocols) and 9969 controls (individuals who were generic blood donors or were hospitalized for non-cancerous cases). For each subject, sex, age (at diagnosis for PDAC cases; at recruitment for controls), and country were collected.
Identification of lncRNA and lncSNPs
NONCODE, a systematic database dedicated to non-coding RNAs (ncRNAs), was the source of the human lncRNA list. The database uses Coding-Non-Coding Index (CNCI), a software that discriminates coding from non-coding transcripts using intrinsic sequence features. lncRNASNP2, a comprehensive database for SNPs and lncRNA transcripts was used to identify all the human lncSNPs. lncRNASNP2 also provides information on lncRNA mutations, as well as their impacts.
Genotyping and statistical analysis
DNA extraction of the cohorts and genotyping was done for data filtering and statistical analysis. Five independent SNPs with the lowest P values of association with PDAC risk were screened during the logistic analysis. These were then genotyped in a cohort consisting of 2686 pancreatic ductal adenocarcinoma subjects and 2907 controls from the PANcreatic Disease ReseArch (PANDoRA) consortium. The researchers failed to observe deviation from Hardy-Weinberg equilibrium in the genotyped SNPs. The average call rate observed was 98%. Further to the replication analysis, a gene-based software analysis was also performed to test the relation between non-coding transcripts and PDAC risk.
To support the functional explanation, the researchers used genomic data from several databases such as Genotype-Tissue Expression Project (GTEx), LncRNASNP2, and miRBase. miRDB (version 6.0) was also used to predict lncRNA-miRNA interactions. To explore chromatin states, conservation, and gene regulation, HaploReg and RegulomedB were used in this study. Last but not the least, Chiara et al. used LDlink to study the nonrandom association of alleles of different loci aka linkage disequilibrium.
Results & Discussion
Chiara et al. followed a two-phase approach to investigate the genetic variability in all lncRNAs.
- Discovery phase comprising 7207 PDAC cases and 7062 controls from four GWAS.
- Validation phase comprising 2686 PDAC cases and 2907 controls from PANDoRA consortium.
The researchers identified 67 lncSNPS associated with PDAC risk (P<1 x 10-4); further filtering for residual linkage disequilibrium (LD) between known loci gave them the list of 5 variants to be tested in the second phase.
From the statistical point of view, the researchers observed an increase in the risk of developing PDAC in the allele of the rs7046076 variant. rs7046076 is found to be present in the region of SMC2 ((Structural Maintenance Of Chromosomes 2), risk loci associated with PDAC. It was also observed that the presence of the rs7046076 C allele results in the loss of miRNA-lncRNA binding. Meaning, it could be involved in the deregulation of the genes on the 9p21.3 risk locus, thus increasing PDAC risk.
The regulatory role of the gene NONHSAG053086.2 on DAAM1 (Dishevelled Associated Activator Of Morphogenesis 1) was well established. The encoded protein of NONHSAG053086.2 promotes vesicular transport and facilitates cellular movement. Ang et al. demonstrated that loss of this protein could result in random cell migration. Additionally, a DAAM1-mediated migration and invasion of pancreatic cancer cells have been reported. Furthermore, the link between NONHSAG053086.2 and PDAC risk has been demonstrated. All these confirm the role of this locus and PDAC risk. Though the presence of several SNPs in putative regulatory regions was observed during Ensembl analysis, the researchers suggested follow-up studies to uncover the link between polymorphisms and pancreatic cancer tissues.
This study also selected several other SNPs belonging to risk loci that were reported in PDAC cases, either as significant at the genomic level or as suggestive ones.
One of the SNPs selected for genotyping was near EDNRA ((Endothelin Receptor Type A) at chromosome 4q31.22. Though PDAC risk was reported in this region, this study could not confirm the association.
Gene analysis with MAGMA showed that 3108 out of 11857 lncRNAs are involved in pancreatic ductal adenocarcinoma. This highlights why lncRNAs have attracted (and still do so) intense attention from genomic scientists for their aberrant expression in carcinogenesis.
A possible limitation of this study is that patients and controls were of Caucasian descent and therefore there might be some racial-based differences.
Only rs7663891 showed a statistically significant nominal p-value.
An additional limitation may be that instead of deriving functional explanations from experimental results, this study used databases.
Overall, this study has investigated genetic variability in all lncRNAs and reported pancreatic ductal adenocarcinoma (PDAC) risk loci. The study also pointed out the interaction between lncRNA and miRNA and their role in maintaining cellular homeostasis. Additionally, the role of lncRNA deregulation in tumor progression was suggested. These findings may open a new research avenue into the genetics and biology of PDAC and, ultimately, the risk stratification and implementation of prevention modalities.
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