Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading aggressive cancers and is the third most common cause for cancer deaths worldwide [
1]. According to the GLOBOCAN 2020, PDAC caused 4,66,003 deaths with an incidence of 4,95,773 new cases per year [
2]. PDAC has a poor prognosis with a 5-year survival rate of less than 8.5%, despite extensive research on the cancer type [
3]. Intrinsic heterogeneity across PDAC patients may contribute to poor survival [
4].
Epigenetic changes, such as aberrant methylation of DNA, histone deactelyation, chromatin remodelling affect gene expression and are important drivers of carcinogenesis. DNA methylation plays a crucial role in disease outcome [
5]. Altered methylation status at the CpG islands at the gene promoters, limits the access to transcription factors, significantly affect gene expression [
6]. Hypermethylation at CpG islands with down-regulated expressions of tumour suppressor genes (TSG) and hypomethylation with increased expression of oncogenes, have been documented for many cancers. Epigenomics has become a promising paradigm for PDAC diagnosis and has identified pathways that can be targeted for therapy [
7]. Past researches have tried to identify altered methylation of cell-free DNA in PDAC as the potential blood-based diagnostic and prognostic biomarkers. However, limited validation studies across patient population and applicability of such biomarkers was seen till date [
8,
9]. Thus, it is important to study the differential methylation marks associated with PDAC development.
Past studies had shown that both genetic and epigenetic alterations contributed to PDAC initiation and progression [
7,
10]. PDAC diagnosis based on gene-mutation has only brought partial success in early diagnosis and overall patients’ survival improvement [
7]. Distinct epigenetic markers have been observed for specific subtypes of pancreatic cancers and for specific stages of PDAC [
11,
12]. Also, studies including pancreatic cancer (PanCa) samples from different populations have identified partially similar methylation marks. The inter-population epigenetic variation has been reported and this may be explained by varied ethnicity, demographic, and occupational factors [
13,
14]. Globally few studies have been done on investigating the changing epigenetic landscapes through progressive stages of PDAC [
15] and on different ethnicity. In this study, we have reported the altered genome-wide DNA methylation profile of PDAC across progressive stages, compared our results with the TCGA cohort and validated these results in additional 4 independent cohorts.
Discussions
Globally, Pancreatic Cancer, especially ductal adenocarcinoma, is one of the most prevalent cancer types. Late diagnosis, treatment failure, and loco-regional recurrences contribute to its poor prognosis. Along with genetic alterations, studies in the past decade showed that epigenetic alterations contribute to PDAC development and pathogenesis [
10,
11,
33,
34,
35]. Recent findings have shown abnormal DNA methylation can mark the spectrum of cancer progression, including the precancerous lesions, thus serving as biomarkers for diagnosis and prognosis [
9,
36,
37,
38]. Thus, a specific abnormal methylation profile can serve as a biomarker for that cancer type [
39]. Since methylation marks can be reversed to an unmethylated state, epigenetic marks can act as a lucrative therapeutic target.The effects of methylation on gene expression, can vary across different ethnic populations with varied risk factors [
33]. In this study, we identified DNA methylation changes in 91 genes (gDMs) associated with PDAC in the Indian population. Among them, differential methylation in 47gDMs (Additional file
1: Table S6) in PDAC concordant with that in the TCGA PAAD cohort. The differential methylation marks were associated with progressive cancer stages and prognosis, both in our cohort and the TCGA cohort. Thus, our results showed both common and unique differential methylation marks in PDAC patients in the Indian cohort.
Promoter hypo-methylation was observed
IRF4, PML, MX2, OAS2, HLA-A, and
SIGIRR are involved in the interferon signalling pathway (MCODE1, Table
1). In our study, we have observed hypo-methylation of MCODE1, suggesting activation of immune pathways in PDAC. A recent study showed that over-expression of
MX2 reduced cell proliferation, migration, and invasion via
ERK/P38/NF-κB signalling pathway in glioblastoma cells [
34]. Up-regulated expression of
OAS2 was previously reported in PanCa [
35]. Interferon induced cell killing is a potent anti-tumour immune response in cancer. Previously a study had shown that induction of Type I Interferon signalling made PanCa vulnerable to innate immune systems and showed better response with immune checkpoint therapy [
36]. The interferon signalling pathway inhibits cell proliferation and cell migration in PanCa [
37]. Up-regulated expression of these genes may thus signify the active anti-tumour immune response in PDAC.
Hyper-methylation and down-regulation of TSG, DNA repair genes are observed in tumours, which aid in cancer progression and metastasis [
38]. In current study, we observed hyper-methylation of (1) TSGs including
LOC645323, FOXE, and
TCERG1L, (2) transcription regulator genes (
BHLHE23, GSC2, FOXE1, HOXA10 and
TWIST1), (3) Ion transporters—
KCNA3,
KCNA6, CACNB2 and (4) Immune regulators including
HLA-A, IRF4. Previous studies have reported hyper-methylation of these transcription regulators in multiple cancers including PanCa [
39,
40,
41] and reduced expression of these genes was associated with a worse prognosis of PAAD cancer in the TCGA cohort.
FOXE1 is one of the most frequentlyhyper-methylated TSGs in PanCa [
40,
41]. Hyper-methylation of another previously reported TSG
TCERG1L was observed in PDAC compared to normal tissues [
12,
39].
FOXE1,
TWIST1 and other hyper-methylated genes involved in cell cycle regulation were observed in PDAC cancers, thus suggesting that their down-regulation may encourage uncontrolled cell proliferation in PDAC. Deregulations of ion transporters (Calcium, Potassium and Sodium) were observed in many cancers including PanCa’s and often represented as biomarkers [
26]. Ion transporters can help in angiogenesis and cancer metastasis [
42]. We observed hyper-methylation of potassium (
KCNA3, and
KCNA6) and calcium (
CACNB2) ion channels in PDAC. Studies on animal models showed that epigenetic modification of
KCNA3 gene limits T-cell activation [
43,
44]. The Ca
2+ and K
+ ion channels thus can inhibit T-cell activation and proliferation upon antigen recognition by epigenetic modulation, creating an immunosuppressive tumour microenvironment in PDAC, which is again associated with poor prognosis and recurrence. Hyper-methylation of potassium channels may thus alter the immune context in PDAC and limit anti-tumour immunity [
52,
57]. One of the recent finding documented epigenetic dysregulation (hyper-methylation) of Ca
2+ ion transporters in PDAC [
45]. Additionally, hyper-methylation of HLA-A also suggested suppression of antigen presentation and antitumor activity.
In this current study, we observed hyper-methylation of two genes—NPY and FAIM2 in Indian PanCa samples, which were also observed in the TCGA cohort and in the PanCa cell lines in the CCLE database.Hyper-methylation of both NPY and FAIM2 was correlated with reduced expression and was associated with poor survival, both in the TCGA and the Indian patient cohorts. The significant poorer survival also observed when FAIM2 and NPY act jointly. Our data strongly represents that the NPY and FAIM2 cumulatively contributed to significant poorer survival in the Indian Cohort. The survival curve using NPY and FAIM2 status couldn’t be derived from our validation cohorts based on the status that approximately > 80% of samples showed down-regulation of both the genes making the cohort not suitable for Kaplan–Meier survival analysis. In IHC study, validation cohort 4 we also reclaimed down-regulation of protein expression for both genes in PanCa samples with respect to normal counterparts. The result is consistent with the gene expression and promoter methylation. All our patient cohorts including discovery and validation cohorts have been revealed that hyper-methylation and down-regulation properties in mRNA and finally in protein levels of NPY and FAIM2. Considering CPTAC database it has been further proved that both Npy and Faim2 protein levels were also shown down-regulated in PanCa. All our data and from publicly available data sets strongly suggests and support that NPY and FAIM2 are the novel potential frequently hyper-methylated genes and trigger the PanCa progression and development in our patient cohort (Indian PanCa patient population). But surprisingly it has not been proven in other previously reported PanCa methylation studies around the globe. The cumulative down-regulation of NPY and FAIM2 also correlates with poor prognosis in the TCGA cohort data. Cumulatively, under expression of both FAIM2 and NPY have shown strong negative correlation between diabetes and hyper-methylation of both genes which are significant co-morbidity factors in the Indian patient population.
To our knowledge, hyper-methylation of
FAIM2 was previously observed in ductal carcinoma of breast cancer [
46], where the authors have shown an association of DNA methylation with progressive stages. Fas-apoptotic inhibitory molecule 2 is a member of the transmembrane BAX inhibitor motif-containing (TMBIM) family which comprised of 6 anti-apoptotic proteins.
FAIM2 can suppress cell death by regulating Calcium ion homeostasis in the endoplasmic reticulum [
32]. Thus hyper-methylation and downregulation of
FAIM2 can help the tumour to evade apoptosis. However in current scenario, nothing is known about the effects of
FAIM2 in PanCa.
The role of
FAIM2 in obesity has already been documented which is a prime risk factor in PDAC development. It has also been reported by Kang et al. 2016, that
FAIM2 acts as an novel biomarker in SCLC therefore emphasizing
FAIM2's role as cancer biomarkers [
47,
48,
49,
50]. Another hyper-methylated gene in PDAC was a
NPY, which was included in MCODE2 (Table
1) along with
ADCY3, GALR1, OPRK1. NPY stimulates cell proliferation and has been implicated as growth-promoting factor in various malignancies, but little is known about the effects of
NPY on PanCa.
NPY promoter showed frequently hyper-methylated in colorectal, and rectal cancers, surprisingly, in prostate and cholangiocarcinoma,
NPY overexpression was observed but no correlation with hypo-methylation has been found [
28,
29,
51,
52]. A physiological function of
NPY is to regulate food intake and increase fat storage which is a risk factor for PanCa [
28,
29,
51,
52,
53]. In addition, in our study showed that
NPY downregulation (
p-value: 0.041) and high alcohol consumption were associated with poor OS. All previous findings, including our data strongly support that in PDAC, hyper-methylation of
NPY promoter might be correlated with inactivation of gene expression and might promote carcinogenesis.
Considering PDAC aggressive nature and its associated high mortality rate, the urgency of novel therapeutic strategies to combat is of priority. Advances in the methylation landscape are crucial to understand the development of epigenetically targeted biomarker. Our novel findings in this ongoing study can direct that a combination of epigenetic drugs along with existing targeted therapies will be a determinant targeted therapeutic approach that emphasizes the importance of our 450 K methylome study in PDAC patients across our nation (India) [
54,
55,
56]. Epigenetic and genetic signatures of PDAC partially vary across the globe based on expression frequency and alternated driver behaviors. According to our previous study, Saha et al.,2020 [
57] explained
KRAS hotspot mutation was observed in low frequency in the Indian PDAC population whereas other parts of the world, like USA/Canada/European countries and Australia based studies documented
KRAS as a high-frequency gene around 90% in the same disease. This partially variable profile may be contributed by the patient pool led by combined factors such as ethnicity, environmental, lifestyle and occupational risk factors [
57]. Like the previous findings, it has also been observed in our methylome data. In our current study, correlation between sex, smoking and alcohol consumption has been documented which is specific to our demographic and ethnic beliefs. To gain a clear understanding of the epigenetic landscapes of PanCa, our study strongly suggests a novel architecture of epigenetic landmarks and insight into potential epigenetic outcome on PDAC in the Indian patient population.
Acknowledgements
Authors also acknowledged the National Institute of Biomedical Genomics (NIBMG) for instrumental support for performing experiments on an outsourcing basis under the Core Technologies Research Initiative (Co-TeRi) Initiative, National Institute of Biomedical Genomics (NIBMG), Kalyani, West Bengal carried out the Illumina 450 K bead chip experiment, directed by Prof. Arindam Maitra. Authors are also thankful to Dr. Dheeraj Anchlia, Dr. Sahid Khondaker, Dr. Abhisekh Mohata, Dr. Jitesh Midya, Dr. Vinu Shankar, Dr. Shuchismita Chakraborty, Dr. Debtanu Halder and Dr. Nilanjan Ghosh for collecting samples from respective hospitals. Authors specially thank Mr. Gourab Saha, Miss. Suchandra Pal and Mr. Sanjoy Kr. Dey from NS laboratory for collecting clinical specimens and DNA and RNA isolation. Authors are also thankful to Dr. Sudipto Saha, from Bose Institute (BI), Kolkata for support in IPA pathway analysis. Mr. Sandip Ghosh, and Dr. Biswarup Basu, from Chittaranjan National Cancer Research Institute (CNCRI), helped in IHC staining and scoring. The authors thank Dr. Meenakshi Munshi, from Department of Biotechnology, GOI for supporting and processing the fund. Associate Prof. Aniruddha Chatterjee would like to thank the Rutherford Discovery Fellowship (Royal Society of NZ) for supporting his current position. Authors are also thankful to Prof. Bidyut Roy for strategic input and proofreading of the manuscript. N.S. also thanks to Master. Saraswan Sikdar.
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