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Page 278 Schwarzenbach et al. Cancer Drug Resist 2019;2:271-96 I http://dx.doi.org/10.20517/cdr.2019.010

into cells in vitro. These findings supported previous observations from Strathdee et al. , 20 years ago.
[23]
DNMT activity and thus, DNA methylation are regulated by the epidermal growth factor receptor (EGFR) .
[64]
Granados et al. examined whether cisplatin induces EGFR mediated changes in DNA methylation that
[65]
are associated with the development of cisplatin resistance. Acute cisplatin treatment activated EGFR and
downstream signaling pathways, as well as induced an EGFR-mediated increase in DNMT activity. This led
to an increase in global DNA methylation in cisplatin resistant cells. During repeated cisplatin treatments,
EGFR inhibition re-sensitized the cells to cisplatin and inhibited increases in DNA methylation and DNMT
activity.

The ten-eleven translocation (TET) family of dioxygenases comprising TET1/2/3 is involved in DNA
demethylation. Using cisplatin-sensitive and cisplatin-resistant ovarian cancer cell models, Han et al. [20]
detected that TET1 was significantly upregulated in cisplatin-resistant ovarian cancer cells compared with
cisplatin-sensitive cells. Its ectopic cell expression promoted cisplatin resistance and decreased cytotoxicity
induced by cisplatin via active DNA demethylation of vimentin, resulting in partial EMT.

Applying Illumina human methylation arrays and Affymetrix arrays, Bonito et al. showed that CpG
[31]
sites within the homeobox transcription factor MSX1 gene had significantly lower levels of methylation in
high-grade serous epithelial ovarian cancer patients who recurred by 6 months than patients who recurred
after 12 months. In cisplatin-resistant ovarian cancer cell lines, MSX1 overexpression led to cisplatin
sensitization, increased apoptosis and increased cisplatin-induced expression of the cyclin-dependent
kinase (CDK) inhibitor p21. A further CDK inhibitor of cell cycle progression is p27, also known as KIP1.
Zhao et al. detected that the expression level of p27 was dramatically downregulated in chemo-resistant
[66]
cells, but treatment with the demethylating agent 5-aza-2'-deoxycytidine restored p27 expression in cisplatin
resistant cells and increased sensitivity to cisplatin. Overexpression of p27 arrested the cell in the S phase
and promoted an apoptotic response to cisplatin.

To analyze genome-wide DNA methylation profiles of cisplatin sensitive and resistant ovarian cancer cell
lines, Yu et al. applied methyl-Capture sequencing (MethylCap-seq), which combines precipitation of
[27]
methylated DNA by the recombinant methyl-CpG binding domain of MBD2 protein with next generation
sequencing (NGS). They found a lower global CpG methylation in resistant cells. Methylation-specific PCR
and bisulfite sequencing confirmed hypermethylation of protein tyrosine kinase 6, protein kinase Cε and the
antiapoptotic gene BCL2L1 in sensitive cells compared with resistant cells. Performing genome-wide analyses
of hypermethylated CpG islands in combination with real-time PCR, Kritsch et al. identified Tribbles 2
[67]
(TRIB2) as the most pronounced downregulated gene on mRNA level among 37 commonly epigenetically
silenced genes in cisplatin-resistant ovarian cancer cells. Its re-expression increased the sensitivity to cisplatin
and other DNA-damaging agents in these cells, whereas its knockdown increased the resistance to cisplatin
in sensitive cells. TRIB2, that belongs to the family of pseudokinase proteins and degrades the myeloid
transcription factor CCAAT enhancer binding protein α , induced a cisplatin-dependent cell cycle arrest
[68]
and apoptosis by acting on p21 and survivin expression. It seems to be involved in the signal transduction
from nucleotide excision repair of intrastrand cross links. In line with its downregulation in ovarian cancer
cells, tumors from cisplatin-resistant patients also expressed the lowest levels of TRIB2 .
[67]

Using an integrated approach of analyzing simultaneously gene expression levels and DNA methylation
profiles, Yang et al. analyzed mRNA expression on gene chip arrays and DNA methylation profiles
[18]
on methylation bead chips. Among 26 genes that were differentially expressed and methylated between
cisplatin-resistant and -sensitive ovarian cancer cells, 3-oxoacid CoA transferase 1 (OXCT1) was selected
for further investigations. OXCT1, that catalyzes the first and rate-determining step of ketolysis , was
[69]
hypermethylated at CpG sites of its promoter and downregulated in cisplatin-resistant cells. Treatment with
a DNMT inhibitor restored hypermethylation-mediated gene silencing of OXCT1 in cisplatin-resistant cells,