Reversal of Aberrant Cancer Methylome and Transcriptome upon Direct Reprogramming of Lung Cancer Cells Dashayini Mahalingam 1, Chiou Mee Kong 1, Jason Lai 1, Ling Lee Tay 1, Henry Yang 2 & Xueying Wang 1 1 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 2 Cancer Science Institute of Singapore, National University of Singapore, Singapore SCIENTIFIC REPORTS | 2 : 592 | DOI: /srep00592 Presented by Teh Hui Xin, UTAR

Introduction Lung cancer  The leading cause of death by cancer amongst men, second amongst women  Neoplasia is widely thought to be driven by genomic instability which is due to the reversible and irreversible alterations. Epiginetic  regulate gene expression results in aberrant silencing of tumor suppressors or upregulation of oncogenes Direct reprogramming may have reversed the aberrant epigenetic alterations in cancer cells.

Objectives To reprogram non-small cell lung cancer (NSCLC) and characterize the reprogrammed NSCLC. To study the genome-wide analyses of DNA methylation and gene expression patterns of reprogrammed NSCLC.

Methodology Non-small cells lung cancer (H358, H460) Human embryonic lung fibroblasts (IMR90) Embryonic stem cells (H1, HES3) Transfection & infection Gene expression profiling Retroviral transduction using Yamanaka’s four factors Characterization Genome-wide DNA methylation profiling qPCR Methylation-specific PCR Bisulfite Sequencing Alkaline phosphatase staining Immunofluorescence staining Telomerase activity assay In vitro differentiation HumanHT-12 v4 Expression Beadchip (Illumina) Infinium Human Methylation27 Beadchip (Illumina) Gene set analysis AMP, Commonly upregulated genes in NSCLC, oncogenes, tumor suppressors

Results (A)Characterization of iPC (B) Direct reprogramming hypomethylates aberrantly methylated promoters (AMP) in NSCLC. (C) Downregulation of NSCLC biomarkers upon reprogramming (D) Effects on oncogenes and tumor suppressors.

H358 Adenocarcinoma H460 Large cell carcinoma IMR90 Normal lung fibroblast Normal lung fibroblast is more readily reprogrammed. H1 & HES3 Embryonic Stem Cells

Expressed pluripotency marker. Stain positive for Alkaline phosphatase, Tra1-60 & Nanog.

Real time PCR ↑ Sox2, Nanog, FGF4, OCT3/4 ↑ Telomerase Activity

iPC, Differentiated iPC, iPS & H1 clustered together iPC, Differentiated iPC & iPS, clustered together but not with H1 Deviation of iPS and iPC from H1 in methylation profile is not consequential Hierarchical Clustering (Gene Expression) Hierarchical Clustering (Methylation Array)

Gene Onthology of Hypomethylated Promoter in iPC Enrichment of developmental associated genes

iPC is able to differentiate into three germ layers in vitro - ↑ ectoderm markers- CDX2, PAX6 - ↑ mesoderm markers – Brachyury. MSX1 - ↑Endoderm markers – GATA4, FOXA2 Embryoid Body FormationqPCR

To investigate if reversible alterations in cancer cells were reverted upon reprogramming. list of known AMP in lung cancer cells through literature search (237 AMP) Interrogated by illumina Infinium human methylation 27k beadchip array. (217 AMP) over-represented among all methylated promoters in H358 & H460 but under represented among all methylated promoters in IMR AMP overlap between H358 & H AMP in H AMP in H460

-Hypomethylated upon Reprogramming -developmental associated genes (HOX and PAX gene clusters) -tumor supressors (APC, TIMP3, WRN) Overlapped Reprogramming Validated by Methylated Specific PCR Bisulfite genomic sequencing Aberrant DNA methylation in cancer was reversed by direct reprogramming

Methylation pattern Green – unmethylated Red- methylated Gene expression pattern yellow- down regulation Blue- upregulated Heat Map qPCR Concurred with array data, ↑ HOXA5, HOXA7, HOXD13 in iPC vs parental cancer ↑ RPRM – known to be heavily methylated in lung cancer and its low expression correlated with poor prognosis

gene commonly upregulated in NSCLC (UR) GEO database – 420 genes interrogated in the illumina Human HT12 array – 391 genes H358 vs IMR upregulated UR H360 vs IMR upregulated UR iPCH358 vs H downregulated UR iPCH460 vs H downregulated UR Over-represented for genes downregulated upon reprogramming Prognosis factors KRT19, S100p, KRT7, PPAP2C and AGR2

Downregulation of UR genes in iPC that were initially upregulated in parental cancer cells After reprogramming

Hypermethylation of UR → Down regulation of UR In bisulfite sequencing, KRT19 gene methylation scores 86% in iPCH358 96% in iPCH460 Validated by Methylated Specific PCR Reprogramming reverses the abberantly upregulated genes in NSCLC both epigenetically and transcriptionally.

H358 & H460 vs IMR oncogenes from database (Memorial Sloan- Kettering Cancer Centre Database) H358 vs IMR oncogenes upregulated H460 vs IMR oncogenes upregulated iPCH358 vs H358 – 25 oncogenes downregulated iPCH460 vs H460 – 14 oncogenes downregulated EFNA1, CXCL1, CXCL2 – prognosis factors, downregulated upon reprogramming ID1- oncogenes that promotes lung cancer proliferation, downregulated upon reprogramming. iPCH358 vs H358 Hypomethylation → Upregulation of oncogenes Hypermethylation → Downregulation of oncogenes iPCH460 vs H460 – not significant

873 tumor suppressors from database (Memorial Sloan-Kettering Cancer Centre Database). H358 vs IMR90 – 87 tumor suppressors downregulated H460 vs IMR90 – 74 tumor suppressors downregulated iPCH358 vs H358 – 21 upregulated iPCH460 vs H460 – 6 upregulated

↑ CADM1 & PLAGL1 in both iPCH358 & iPCH460 Total percentage of upregulated tumor suppressors in reprogrammed H358 & H460 are low Tumor suppressors were probably need to be maintained at low levels for cell proliferations and survivals tumor suppressors hypermethylated in H358 were hypomethylated iPCH358 but not in H460 & iPCH460. Dysregulation of oncogenes and tumor suppressors in NSCLC were reversed upon reprogramming and were partially explainable by intricate DNA methylation pattern.

Discussion Transcriptome of iPS, iPC and differentiated iPC were indistinguishable with each other. The reversible changes that account for tumorigenesis such as aberrant hypermthylation of promoters as well as abnormal upregulation of genes in NSCLC have been assessed. The fate of oncogene and tumor suppressors followed by reprogramming have been investigated. Previous study has reported reprogramming could reverse hypermethylated promoters- tumor suppressor gene p16 in hTERT immortalized human lung fibroblast (WI-38). Direct reprogramming were able to perturb the epigenetics of lung cancer cells by causing the reversal of AMPs, resulted in active gene transcription. Following reprogramming, the iPCs no longer harbor the same aberrant DNA methylation mark, and may no longer exhibit malignancy.

The markers that are found to be aberrantly upregulated in H358 & H460 were downregulated upon reprogramming. Supposing that these prognostic factors are pertinent in cancer progression, direct reprogramming may result in loss of malignancy. Prognostic factors as well as DNA methylation markers that are crucial for NSCLC progression seem to be reversed upon direct reprogramming. In vitro differentiated iPC cells did not have aberant dysregulation of these genes as well as DNA methylation markers. Direct reprogramming of cancer cells resulted in the reversion to normal DNA methylation and gene expression regulation. Discussion

Effects on oncogenes and tumor suppressors Oncogene / Proangiogenic factors (EFNA1, CSCL1, CXCL2, ID1) which promote tumorigenesis were reverse to the normal expression levels in iPC and remained so in differentiated iPC. Tumor suppressors (CADM1 & PLAGL1) were upregulated in the NSCLC upon reprogramming. Regulation of these genes in H358 were explainable by DNA methylation but not in H460 (?) The mechanism behind aberrant dysregulation of tumor suppressors and oncogenes are more robust and may include other mechanisms such as gene deletions and gene amplification. Discussion

This study has revealed a better understanding of cancer. Direct reprogramming is a new tool to study and understand cancer cells, which may result in paradigm shifts. By globally resetting the epigenetic state of lung cancer through direct reprogramming, this study provides evidence that cells may become reticent by reversing aberrant epigenetic changes in NSCLC which in turn affects the gene regulation. Discussion

Future study : Will the directed differentiated of these iPC to different comitment lineage will result in malignant manifestation phenotyically and epigenetically ? To elucidate the indirect roles of Yamanaka’s factors in the delicate regulation of epigenetics in a cancer cells, for examples, hypomethylation or hypermethylation at specific loci. Better understanding of this mechasnism would contribute a more sophisticated and effective treatment of cancer than currently tested non-specific DNA methylation inhibitors or DNA demethylating agents. Discussion

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