Reversal of Aberrant Cancer Methylome and Transcriptome upon Direct Reprogramming of Lung Cancer...
-
Upload
vicente-hoddinott -
Category
Documents
-
view
214 -
download
1
Transcript of Reversal of Aberrant Cancer Methylome and Transcriptome upon Direct Reprogramming of Lung Cancer...
Reversal of Aberrant Cancer Reversal of Aberrant Cancer MethylomeMethylomeand Transcriptome upon Directand Transcriptome upon DirectReprogramming of Lung Reprogramming of Lung Cancer CellsCancer 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: 10.1038/srep00592
Presented by Teh Hui Xin, UTAR
IntroductionIntroductionLung 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.
ObjectivesObjectivesTo 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.
MethodoloMethodologygy 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
CharacterizationGenome-wide DNA
methylation profiling
qPCR
Methylation-specific PCR
Bisulfite Sequencing
Alkaline phosphatase stainingImmunofluorescence stainingTelomerase activity assayIn vitro differentiation
HumanHT-12 v4 Expression Beadchip
(Illumina)
Infinium Human Methylation27 Beadchip
(Illumina)
Gene set analysisAMP, Commonly
upregulated genes in NSCLC, oncogenes, tumor suppressors
ResultsResults
(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.
H358Adenocarcino
ma
H460Large cell carcinoma
IMR90Normal lung
fibroblast
Normal lung fibroblast is more readily reprogrammed.
H1 & HES3Embryonic Stem Cells
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)
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 Formation
qPCR
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 IMR90.
84 AMP overlap between H358 & H460
105 AMP in H35894 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 – unmethylatedRed- methylated
Gene expression patternyellow- down regulationBlue- 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 IMR90- 110 upregulated UR
H360 vs IMR90- 59 upregulated UR
iPCH358 vs H358- 52 downregulated UR
iPCH460 vs H460- 25 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 IMR90 - 495 oncogenes from database (Memorial Sloan-Kettering Cancer Centre Database)
H358 vs IMR90- 42 oncogenes upregulated
H460 vs IMR90- 29 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 H358Hypomethylation → 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.
DiscussionDiscussionTranscriptome 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.
DiscussionDiscussion
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.
DiscussionDiscussion
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.
DiscussionDiscussion
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.
DiscussionDiscussion