This study is conducted to unveil the genetic alterations and its regulators that drive ADM toward the direction of PanIN and subsequently PDAC. We have identified 45 genes in ADM stage, 7 genes in PanIN and 28 genes in PDAC interacted with each other. Overlap and crosstalk genes in ADM-PanIN-PDAC contain several bridging ADM gene modules (e.g. Hist2h2ab and smarca1), PanIN gene modules (e.g. Hdac11 and smarca2) and PDAC gene modules (e.g. Olfr239 and Hdac6). GO analysis showed that these genes mainly played roles in nucleosome assembly, chromatin organization and G-protein coupled receptor signaling pathway. The expression of cancer-related genes was affected by epigenetic dysregulation via DNA methylation, histone modification and regulated by small non-coding regulatory microRNAs (e.g. mmu-miR-335-5p) and lncRNAs (e.g. H19).
Overlap genes and most crosstalk genes belong to histone family. Histone is highly conserved protein with extensive cellular functions. It constitutes two functional part: core histones (H2A, H2B, H3 and H4) and linker histones (H1 and H5). In our study, most overlap genes are core histones, including HIST1H2AN, also known as H2AC22 (homology of human H2AC11), three members of Histone H4
(Hist1h4c, Hist1h4m and Hist4h4). Crosstalk genes including H2AFZ (synonym of H2A.Z) were annotated in ADM-PanIN-PDAC. Histones are found to be associated with the cancer-predisposing inflammation or even trans-differentiation. Histone H3 has the potential to be a biomarker for evaluating the severity of acute pancreatitis due to caerulein triggered extensive pancreatic acinar cell death in animal model. Histone H4 was found to bind with smooth muscle cells and triggered arterial tissue inflammation. Histone variant H2A.J was accumulated with aging in specific tissue and may contribute to chronic inflammation, aging-associated disease and cancers. Roles of H2AFZ have been well established in several types of cancer, but poorly explored in PDAC. It is upregulated in breast, liver, bladder and lung cancer and has oncogenic properties in prostate cancer.
Apart from the differential expression level of histone variants, modification of histones could also exert effects on carcinogenesis. We found several histone deacetylase (HDAC) including HDAC3 in ADM module-1, HDAC11 in PanIN module-4 and HDAC6 in PDAC module-9 were important to network of ADM-PanIN-PDAC. High expression of HDAC3 in the precursor lesions of prostate cancer were presented, indicating its critical role in the initiation stage of tumorigenesis. Aberrant expression of HDAC6 also plays critical roles in cell differentiation, apoptosis and cell cycle control. Hdac11 has been shown as a novel target in antitumor therapy. Loss of histone trimethyl transferase, H3K36, facilitated ADM formation through epigenetic dysregulation and led to extracellular matrix (ECM) production in PDAC.
Great numbers of genes predicted in ADM-PanIN-PDAC regulation are also identified and they have interactions with histones in deferent extent. Several genes annotated have been previously validated in PDAC. Absence of ATRX in adult mice with oncogenic KRAS mutation, a subgroup of SWI/SNF complex (Switch/Sucrose Non-Fermentable chromatin remodeling), resulted in increased ADM and even more progressive PanIN lesions. Deletion of ARID1A in pancreas, a SWI/SNF component, exaggerated ADM formation, diminished regeneration after injury and lead to intraductal papillary mucinous neoplasm and PanIN when cooperated with mutant KRAS. We believe another family member, ARID4A, predicted in ADM module, might play a role in PDAC initiation as well. Crosstalk gene, SMARCA1, was related with inflammation related disease and cancer cell proliferation, migration, growth, death and DNA damage[42, 43]. The first study investigating its role in tumor was done in 2018, revealing its repression in soft tissue sarcoma. Another two member in SMARCA class have been successfully linked to PDAC. SMARCA2, annotated in PanIN module, was correlated with poor survival of pancreatic cancer patients and associated with cancer growth or chemoresistance. SMARCA4-deficient mice in cooperation with oncogenic Kras promoted PDAC precursor lesions. Given that SMARCA1 may act upstream of p53 which is a very common mutant gene in PDAC, by regulating the expression of p53 gene through Wnt pathway, and its putative prognostic biomarker for PDAC, researches targeting its potential roles in PDAC initiation is merited.
Another crosstalk gene CHD1, a chromatin remodeling factor which specifically binds to methylated histone H3 lysine 4 residue (H3K4me3), is involved in nuclear shuttling in pancreatic cancer cell. Similarly, BPTF (bromodomain PHD transcription factor) could also bind with H3K4me3 to stabilize NURF (nucleosome remodeling factor) complex on chromatin, resulting in transcriptional regulation. It was recently reported that BPTF was expressed at intermediate levels in PDAC-derived cell lines and responsible for cancer cell proliferation and PDAC initiation and maintenance by interacting with c-MYC. FRK played a role in pancreatic cancer cells migration and proliferation, and was suggested to be critical therapeutic targets of pancreatic cancer. CLIP3 was shown as anti-inflammatory regulator involved in TNF-α signaling and also attributed to injury recovery, while it was a novel target in cancer predicted in the crosstalk gene network. CECR2 functions with SMARCA1 in CERF complex (CECR2-containing remodeling factor) to regulate cell differentiation and development. Interestingly, SMARCA1, SMARCA2 and CLIP3 were not only overexpressed in human PDAC, but also significant prognostic factors for overall survival prediction. It highlights necessities to explore their roles in ADM-PanIN-PDAC and independently confirmed our findings.
We, for the first time showed that Olfactory receptors (OR/OLFR) superfamily genes were relatively independent modules involved in PDAC formation. Emerging data has been shown that ORs were related with cell invasiveness and could work as putative drivers of cancer. Olfactory receptors (OR/OLFR) superfamily genes belongs to G protein-coupled receptors (GPCR) which is involved in inflammatory response and cancer development via NF-kB signaling. Our findings on the relation of 22 ORs with ADM-PanIN-PDAC concededly support the notion that olfactory transduction pathway is associated with elevated pancreas cancer risk. Another OR member, prostate-specific G-protein-coupled receptor (PSGR/OR51E2), was upregulated in prostate cancer and could allegedly induce prostatitis at early age of mouse and promote prostatic intraepithelial neoplasia. MiRNA-374a and miRNA-410, putative regulators of cancer-related genes, were inversely correlated with PSGR overexpression. Lately, overexpression of Olfr544, Olfr543 and Olfr1349 at mRNA level were found to play a role in regulating glucagon secretion in pancreatic cells. ORs are linked to H2AFZ and histone superfamily through 4 lncRNAs (Gm9866, 4833422C13Rik, BC016548 and 2810407A14Rik). Exploring the relationship between the spectrum of OR and cancer is invaluable for kindling new therapeutic target.
It is well known that long noncoding RNAs (lncRNAs) and small noncoding RNAs (miRNA) facilitate tumor initiation and progression through regulating tumor suppressor genes or oncogenes. Our analysis revealed a complex network among miRNAs, lncRNAs and their targeted genes during PDAC tumorigenesis. Consistently, one of our strongest candidates, Gas5, was recently shown to regulate pancreatic cancer metastasis via PTEN. MiR-143, reported to be suppressed in colorectal neoplasia, was predicted to regulate ADM-PanIN-PDAC via targeting ACTR10. MiR-193b, validated as a tumor suppressor in various cancer types, was found to regulate CLIP3 and interact with oncogenic lncRNA (MIR31HG) in our network. Another tumor suppressor Meg3, predicted to interact with Hist1h2bn in our study, was found to be involved in pancreatic cancer. H19, identified as oncogenic lncRNA, played a role in pancreatic cancer invasion and metastasis. In line with previous findings, we found H19 as the top lncRNA in crosstalk with ADM, PanIN and PDAC. miR-335-5p, miR-7646-5p and miR-669n target both Hist4h4 and Hist1h4m. For now, only one study pointed out that MiR-335-5p may attenuate pancreatic cancer development trough modulating the downstream oncogene. Biological functions of these lncRNA/miRNAs in PDAC identified in our network for the first time remain to be clarified. Some other tumor suppressors were also annotated in our network.