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Reversal of Aberrant Cancer Methylome and Transcriptome upon Direct Reprogramming of Lung Cancer Cells

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* * * * * * * Introduction Lung cancer The leading cause of death by cancer amongst men, ... Objectives To reprogram non-small cell lung cancer (NSCLC) ... – PowerPoint PPT presentation

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Title: Reversal of Aberrant Cancer Methylome and Transcriptome upon Direct Reprogramming of Lung Cancer Cells


1
Reversal of Aberrant Cancer Methylomeand
Transcriptome upon DirectReprogramming 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
10.1038/srep00592
Presented by Teh Hui Xin, UTAR
2
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.

3
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.

4
Methodology
Non-small cells lung cancer (H358, H460) Human
embryonic lung fibroblasts (IMR90) Embryonic stem
cells (H1, HES3)
Transfection infection
Retroviral transduction using Yamanakas four
factors
Genome-wide DNA methylation profiling
Gene expression profiling
Characterization
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
Methylation-specific PCR
qPCR
Bisulfite Sequencing
5
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.

6
Characterization
H358 Adenocarcinoma
H460 Large cell carcinoma
IMR90 Normal lung fibroblast
H1 HES3 Embryonic Stem Cells
Normal lung fibroblast is more readily
reprogrammed.
7
Characterization
  • Expressed pluripotency marker.
  • Stain positive for Alkaline phosphatase, Tra1-60
    Nanog.

8
Characterization
  • Real time PCR
  • ? Sox2, Nanog, FGF4, OCT3/4
  • ? Telomerase Activity

9
Characterization
Hierarchical Clustering (Methylation Array)
  • 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
10
Characterization
Gene Onthology of Hypomethylated Promoter in iPC
  • Enrichment of developmental associated genes

11
Characterization
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

12
AMPs upon direct reprogramming
  • 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.
105 AMP in H358 94 AMP in H460
13
Reprogramming
Validated by Methylated Specific PCR
Aberrant DNA methylation in cancer was reversed
by direct reprogramming
Bisulfite genomic sequencing
14
AMPs upon 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
15
Commonly Upregulated Genes were Reversed
  • 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

16
Commonly Upregulated Genes were Reversed
After reprogramming
  • Downregulation of UR genes in iPC that were
    initially upregulated in parental cancer cells

17
Commonly Upregulated Genes were Reversed
  • 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.
18
Oncogenes
  • 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 H358
  • Hypomethylation ? Upregulation of oncogenes
  • Hypermethylation ? Downregulation of oncogenes

iPCH460 vs H460 not significant
19
Tumor Suppressors
  • 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

20
Tumor Suppressors
  • ?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.

21
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.

22
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.

23
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.

24
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.

25
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 Yamanakas
    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.

26
Thank You
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