Transcript April - UTAR Research Portal

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
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
1
SCIENTIFIC REPORTS | 2 : 592 | DOI: 10.1038/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
Retroviral transduction using
Yamanaka’s four factors
Characterization
HumanHT-12 v4 Expression
Beadchip (Illumina)
Alkaline phosphatase staining
Immunofluorescence staining
Telomerase activity assay
In vitro differentiation
Genome-wide DNA
methylation profiling
Gene expression profiling
Infinium Human Methylation27
Beadchip (Illumina)
Gene set analysis
AMP, Commonly
upregulated genes in
NSCLC, oncogenes, tumor
suppressors
qPCR
Methylation-specific PCR
Bisulfite Sequencing
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.
IMR90
Normal lung
fibroblast
H358
Adenocarcinoma
H460
Large cell
carcinoma
H1 & HES3
Embryonic Stem
Cells
Normal lung
fibroblast is more
readily
reprogrammed.
Expressed
pluripotency
marker.
 Stain positive
for Alkaline
phosphatase,
Tra1-60 &
Nanog.

Real time PCR
↑ Sox2, Nanog,
FGF4, OCT3/4
↑ Telomerase Activity
Hierarchical Clustering
(Gene Expression)
iPC, Differentiated iPC, iPS & H1 clustered
together
Hierarchical Clustering
(Methylation Array)
iPC, Differentiated iPC & iPS, clustered
together but not with H1
Deviation of iPS and iPC from H1 in
methylation profile is not consequential
Gene Onthology of
Hypomethylated Promoter in iPC
•Enrichment of developmental
associated genes
Embryoid Body Formation
qPCR
iPC is able to differentiate into three germ layers in vitro
- ↑ ectoderm markers- CDX2, PAX6
- ↑ mesoderm markers – Brachyury. MSX1
- ↑Endoderm markers – GATA4, FOXA2
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)
84 AMP overlap between
H358 & H460
105 AMP in H358
94 AMP in H460
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.
Reprogramming
Overlapped
-Hypomethylated upon
Reprogramming
-developmental associated genes
(HOX and PAX gene clusters)
-tumor supressors (APC, TIMP3, WRN)
Validated by Methylated Specific PCR
Bisulfite
genomic
sequencing
Aberrant DNA
methylation in cancer
was reversed by
direct reprogramming
Methylation pattern
Green – unmethylated
Red- methylated
Heat Map
Gene expression pattern
yellow- down regulation
Blue- upregulated
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
After
reprogramming
Downregulation of UR genes in iPC that were initially upregulated in parental
cancer cells
Hypermethylation of UR →
Down regulation of UR
Validated by Methylated Specific PCR
In bisulfite sequencing, KRT19 gene
methylation scores
86% in iPCH358
96% in iPCH460
Reprogramming reverses the
abberantly upregulated genes in
NSCLC both epigenetically and
transcriptionally.
H358 & H460 vs IMR90 - 495 oncogenes
from database (Memorial SloanKettering Cancer Centre Database)
H358 vs IMR90- 42 oncogenes
upregulated
H460 vs IMR90- 29 oncogenes
upregulated
iPCH358 vs H358 – 25 oncogenes
downregulated
iPCH358 vs H358
Hypomethylation →
Upregulation of
oncogenes
Hypermethylation
→ Downregulation
of oncogenes
iPCH460 vs H460 – 14 oncogenes
downregulated
iPCH460 vs H460 –
EFNA1, CXCL1, CXCL2 – prognosis
not significant
factors, downregulated upon
reprogramming
ID1- oncogenes that promotes lung
cancer proliferation, downregulated
upon reprogramming.
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.
Discussion
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.
Thank You