cisplatin resistance and opportunities for precision medicine

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Title: Cisplatin Resistance and Opportunities for PrecisionMedicine

Author: Lauren Amable

PII: S1043-6618(16)00002-5

DOI: http://dx.doi.org/doi:10.1016/j.phrs.2016.01.001

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To appear in: Pharmacological Research

Received date: 28-12-2015Accepted date: 1-1-2016

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1

Cisplatin Resistance and Opportunities for Precision Medicine

Lauren Amable, Ph.D.

Corresponding author:

Lauren Amable, Ph.D.

National Institute on Minority Health and Health Disparities

National Institutes of Health

9000 Rockville Pike

Bethesda, MD 20892

Email: [email protected]

Phone: (301) 451‐6629

Fax: (301) 480‐4490



2



3

Abstract

Cisplatin is one of the most commonly used chemotherapy drugs, treating a wide range of cancer types.

Unfortunately, many cancers initially respond to platinum treatment but when the tumor returns, drug

resistance frequently occurs. Resistance to cisplatin is attributed to three molecular mechanisms:

increased DNA repair, altered cellular accumulation, and increased drug inactivation. The use of precision

medicine to make informed decisions on a patient’s cisplatin resistance status and predicting the tumor

response would allow the clinician to tailor the chemotherapy program based on the biology of the

disease. In this review, key biomarkers of each molecular mechanism will be discussed along with the

current clinical research. Additionally, known polymorphisms for each biomarker will be discussed in

relation to their influence on cisplatin resistance.

Abbreviations

ABC, ATP‐binding cassette; ASE‐1, anti‐sense ERCC1; ATP7A, ATPase copper‐transporting, alpha

polypeptide; ATP7B, ATPase copper‐transporting, beta polypeptide; CAST, T‐cell receptor complex

subunit CD3

‐associated

signal

transducer;

CTR1,

copper

transporter

1;

CTR2,

copper

transporter

2;

ERCC1, excision repair cross‐complementation group 1 gene; GSH, glutathione; GST, glutathione‐s‐

transferase; MRP, multidrug resistance associated protein; MT, metallothionein; NER, nucleotide excision

repair; NSCLC, non‐small cell lung cancer; OCT, organic ionic transporter; PCNA, Proliferating cell nuclear

antigen; Pol, polymerase; SLC, solute carrier; TFIIH, transcription factor II H; UTR, untranslated region;

XPA, xeroderma pigmentosum group A; XPB, xeroderma pigmentosum group B; XPD, xeroderma

pigmentosum group

D;

XPE,

xeroderma

pigmentosum

group

E;

XPF,

xeroderma

pigmentosum

group

F;

XPG, xeroderma pigmentosum group G;

Keywords: cisplatin resistance, nucleotide excision repair, ERCC1, copper transporters, ABC transporters,

glutathione



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1.0 Introduction

Rosenberg and colleagues first discovered in E. coli that the byproducts from platinum electrode activity

resulted in the inhibition of cell division [1, 2]. Within 15 years, cisplatin was approved for the treatment

of cancer by the FDA. Cisplatin is one of the most widely used anticancer drugs in North America and

Europe [3], treating a variety of cancers including: testicular, ovarian, non‐small cell lung cancer (NSCLC),

head and neck cancer, bladder, gastric, and other malignancies [4]. The main issue with obtaining the

optimum cisplatin cancer treatment is the significant interpatient variability with outcome, efficacy, and

toxicity.

There are two problems associated with cisplatin usage in the clinic: toxicity and resistance. Cisplatin has

a numerous toxicities including renal damage, deafness, and peripheral neuropathy, thus the overall

efficacy of the drug could not be reached due to the side effects. This has led to the development of

cisplatin analogs that would be clinically effective but without the toxicity. Carboplatin and oxaliplatin,

figure 1, are the most popular analogs and reached FDA approval for usage. Interestingly, there is a

variation in the cancers treated by the cisplatin analogs. Carboplatin is not as effective in treating germ

cell

malignancies

compared

to

cisplatin.

Oxaliplatin

is

very

effective

for

the

treatment

of

colon

cancer,

a

cancer where cisplatin is not effective. Understanding the molecular basis for the difference between

these three compounds could provide new insights and unlock novel mechanisms into how cancer cells

counteract the effects of DNA‐damaging drugs. While the analogs show hope for a better response with

less toxicity the scope of this review will not cover cisplatin analog resistance. For a review on cisplatin

analog resistance, the author directs the reader to a recent review from Perego & Robert [5].

The second issue associated with cisplatin treatment is resistance to therapy. Initially the tumor responds

to cisplatin but then the tumor comes back and is frequently refractory to further platinum therapy. There

are two forms of resistance found in the clinic: innate and acquired. Innate resistance is resistance without



5

out any prior drug exposure. Acquired resistance is a result of drug exposure. The differences between

innate and acquired resistance are not clear but it is generally thought that each operates through

different signaling pathways. This review will only focus on resistance as a whole since discerning between

the two requires further studies.

In the clinic, the definition of whether a patient is sensitive or resistant to cisplatin is generally as follows.

If a patient is more than two years from the last platinum dose, the patient is considered sensitive. There

is a greater than 70% likelihood that the patient will respond to treatment with platinum‐based therapy

[6]. The percentage of patients who will respond to cisplatin decreases with the shortening of the disease

free period. Patients who have disease recurrence within the first months after the recent platinum dose

will have a low likelihood of treatment response with cisplatin and are considered to have platinum

resistant disease.

When cisplatin is transported into the cell, cisplatin has several fates, figure 2. Frist, cisplatin can be

exported from the cell using a transmembrane transporter system. Second, cisplatin can be chemically

neutralized

by

binding

sulfhydryl

groups

in

proteins

such

as

glutathione

or

metallothioneins.

Finally,

cisplatin nonspecifically reacts with a variety of subcellular components: proteins, RNA, and DNA. RNA is

most sensitive to react with cisplatin, followed by DNA, and then protein. The primary and widely accepted

mechanism of action for cisplatin is the binding to cellular DNA, resulting in DNA‐platinum adducts. This

prevents the cell from replicating its DNA until the damage is repaired. If the cell cannot repair the DNA

or the damage is too severe, then the cell dies.

Resistance to cisplatin occurs by the following molecular mechanisms: altered cellular accumulation of

drug, altered DNA repair, and cytosolic inactivation of drug. The processes of resistance were studied in

L1210 mouse leukemia cells and human ovarian cancer cells [7‐10]. The observations were similar in both



6

cell models: all three components contributed to cisplatin resistance. There were differences of each

mechanism regarding the relative contributions. At low levels of cisplatin resistance, about 10‐15 fold

above baseline, the primary mechanism of resistance was DNA repair. Intermediate levels of resistance,

up to 40‐50 fold over baseline, was due to reduced cellular cisplatin accumulation. At very high levels of

resistance, cytosolic inactivation of cisplatin was the primary mechanism. However, in many cell lines it

has been observed that more than one mechanism can be in play here.

The goal of precision medicine is to generate better responses in the clinic. Making an informed decision

on predicting the tumor response to cisplatin as well as the type of resistance that is occurring allows for

tailoring the chemotherapy program based on the biology of the disease. Here in this review, we will

comprehensively discuss the mechanisms of cisplatin resistance‐ altered DNA repair, altered cellular

accumulation, and drug inactivation. For each mechanism, the most promising biomarkers identified so

far will be discussed and are summarized in table 1. Polymorphisms of each biomarker that correlate with

cisplatin resistance from current clinical studies will also be presented, and are summarized in table 2.

2.0 Altered DNA Repair

Once inside the cell, cisplatin binds to DNA and forms adducts. The primary form of DNA damage are N7‐

d(GpG) and N7‐d(ApG) intrastrand DNA‐platinum adducts. These bulky adducts result in substantial

kinking of the DNA (12), which is recognized and repaired by the nucleotide excision repair (NER)

pathway, shown

in

figure

3.

In

this

pathway,

which

requires

more

than

30

proteins,

the

DNA

‐platinum

adduct is first recognized by XPE and XPC‐DDB1/2. The transcription factor II H (TFIIH) complex verifies

the damage and assembles the pre‐incision complex: RPA, XPA, and XPG. The DNA is then unwound by

the XPB and XPD helicases. ERCC1‐XPF and XPG endonucleases create an excision several bases upstream



7

and downstream from the DNA‐platinum adduct. This releases the oligonucleotide containing the adduct.

The gap is filled in by the DNA repair synthesis complex containing RPA, RFC, PCNA, and Pol

/. In the final step, the DNA is ligated by DNA ligase I, thus completing the DNA repair. The balance of

DNA damage and DNA repair dictates death versus survival after cisplatin therapy [11]. Changes in the

ability to repair the DNA adducts results in changes in cisplatin sensitivity.

2.1 ERCC1

ERCC1 is one of the most highly studied biomarkers for cisplatin resistance to date. The DNA damage

excision step is catalyzed by the ERCC1‐XPF dimer is the rate limiting step in the NER pathway. High ERCC1

levels are associated with increased removal of DNA‐platinum adducts and resulting in increased

resistance to cisplatin [12]. There is a linear correlation of ERCC1 expression and cisplatin sensitivity, with

resistant cells expressing more ERCC1 compared to sensitive. It was first demonstrated in ovarian cancer

that ERCC1 levels are increased in cancer tissue in comparison to normal [13]. Even higher levels of ERCC1

mRNA are found in patients with clinically resistant cancer. Lower levels are found in patients that are

clinically sensitive

to

platinum

therapy.

In

another

study

comparing

the

six

histologic

types

of

ovarian

cancer, there is an upregulation of NER genes that correlates with cisplatin resistance. Clear cell tumors

are known to be the most chemoresistant to cisplatin, and they displayed the highest levels of ERCC1 [14].

The evaluation of ERCC1’s potential role as a cisplatin resistance biomarker has been explored in other

cancers. High ERCC1 levels that correlate with increased resistance to cisplatin have been observed in:

ovarian [15, 16], NSCLC [15, 17‐23], nasopharyngeal [24, 25], esophageal [26], cervical [27, 28], head and

neck squamous

carcinoma

[29,

30],

liver

[31],

osteosarcoma

[32],

lung

adenocarcinoma

[33],

advanced

biliary tract adenocarcinoma [34], mesothelioma [35], pulmonary adenocarcinoma [36], and gastric [11].

Thus, the expression of ERCC1 makes an attractive biomarker for cisplatin resistance since the increased

expression has been observed in a variety of cancer types.



8

There are two polymorphisms of ERCC1 that appear to have clinical significance with sensitivity to cisplatin

treatment. The first polymorphism, rs11615, is located in codon 118, that codes for the same amino acid‐

asparagine, was first described by Reed and colleagues [37, 38]. A point to note for this polymorphism:

there is a discrepancy in the literature on whether the change is from C to T or T to C. The reader should

take caution in evaluating studies, as these alleles are switched in the analyses. Here this review the

rs11615 polymorphism is from C to T. The rs11615, or N118N, polymorphism was originally thought to

result in reduced levels of ERCC1 mRNA and protein, as the codon is an infrequently used codon [37‐39].

A clinical molecular correlative study in ovarian cancer was confirmatory [40] but in another recent study

it was shown to not change the expression or function of ERCC1 but rather may be linked to other

causative variants [41]. There are conflicting data as to whether or not this polymorphism determines

sensitivity to cisplatin. In ovarian cancer, the T allele was associated with an increased response to

cisplatin therapy [40]. This was also observed in colorectal cancer [42], pancreatic cancer [43],

osteosarcoma [44] and NSCLC [45]. Yet in another study, C allele was associated with a higher response

rate

to

cisplatin,

progression

free

survival,

and

overall

survival

[46,

47].

Thus

two

opposite

results

have

been observed.

The second ERCC1 polymorphism relating to cisplatin sensitivity is C8092A. This polymorphism was first

identified in gliomas and is located in the 3’ UTR of ERCC1 [48]. The A allele is thought to result in

decreased mRNA stability of ERCC1. This polymorphism results in an A substitution in two additional

genes, nucleolar

protein

ASE

‐1 and

t‐cell

receptor

complex

subunit

CD3

‐associated

signal

transducer

(CAST) [48]. Thus the exact role of this polymorphism is not fully understood as these genes may have an

effect that has not been evaluated. Studies in the C8092A polymorphism are few and are additionally

associated with conflicting data. The clinical implication of the C8092A ERCC1 polymorphism has been



9

studied in lung [49] and esophageal cancer [50] and both studies demonstrated the A polymorphism

results in increased cisplatin response. There is also conflicting data as to the A allele and clinical resistance

to platinum‐based therapy. In a nasopharyngeal cancer study, the A allele of C8092A was associated with

an increased risk of disease progression with patients on cisplatin‐based chemotherapy [51]. In a

malignant pleural mesothelioma study, the A polymorphism of C8092A was associated with a shorter

progression free survival [35]. Yet, in a meta‐analysis of 39 NSCLC studies, there was no relationship of

survival and sensitivity to treatment with platinum‐based chemotherapy [52].

2.2 Other NER genes

While the majority of the studies have focused on ERCC1, there are several studies that suggest other NER

genes are involved in cisplatin resistance. Dabholkar et al., showed that other NER genes are additionally

upregulated in patients who responded to cisplatin therapy [53]. XPA, which is part of the pre‐incision

complex, and XPB, a helicase, displayed increased expression in cisplatin resistant ovarian cancer tumors

[53, 54]. However, this has not been explored further in other clinical studies. Neither XPA nor XPB

polymorphisms

have

been

discovered

that

correlate

with

cisplatin

resistance.

It is logical to think that XPF would be an additional cisplatin resistance marker since it is dimerizes with

ERCC1 to catalyze the incision of the damaged DNA strand. However, XPF has been overlooked in many

studies as to whether or not it is a valid biomarker for cisplatin resistance. In ovarian and colon cancer cell

lines, the increased protein expression of XPF was correlated with increased cisplatin resistance [55].

There have

only

been

two

clinical

studies

that

have

examined

XPF

[56,

57].

Both

studies

examined

head

and neck cancer and increased XPF expression was correlated with cisplatin resistance. Vaezi and

colleagues went on further to examine XPF polymorphisms, however the four polymorphisms they



10

identified showed marginal association with treatment failure [57]. Further studies, examining the role of

XPF in other cancers are needed.

The helicase XPD was additionally identified to have a strong correlation between increased expression

and cisplatin resistance in NSCLC and glioma cell lines [58, 59]. The expression of XPD has not been

evaluated in clinical samples. The majority of the clinical studies have examined the effects of XPD

polymorphisms on cisplatin resistance. Two polymorphisms in XPD have been identified, Asp312Asn and

Lys751Gln, both result in decreased DNA repair capacity. Both polymorphisms were found to be potentials

markers for clinical outcome in osteosarcomas and lung cancers treated with cisplatin [44, 60]. The

Asp312Asn polymorphism was associated with a better survival in osteosarcoma patients treated with

cisplatin [61]. The Lys751Gln polymorphism was associated with longer progression free survival in

pancreatic and NSCLC [62, 63].

3.0 Altered accumulation of cisplatin

The

second

mechanism

of

cisplatin

resistance

is

altered

cellular

accumulation

of

cisplatin.

It

has

long

been

noted that cisplatin resistant cells tend to exhibit decreased levels of cisplatin [64]. Tissue platinum

concentrations are correlated with percent reduction of the tumor, meaning reduced tissue platinum

concentrations are associated with resistance [65]. Altered accumulation of cisplatin is the result of two

independent cellular pathways: decreased uptake or increased export.

3.1 Decreased

cellular

uptake

of

cisplatin

Cisplatin has a simple chemistry, the core is a single platinum metal bound to two amino groups and two

chlorides, figure 1. At physiologic pH, the chlorides of cisplatin are replaced with –OH molecules, resulting

in a neutral charge. This it makes it possible for diffusion across the cellular membrane, flowing from the



11

high concentration of cisplatin outside to the lower concentration inside the cell. Thus, cisplatin uptake

was first thought to be via passive diffusion as uptake was not saturated against time or drug

concentrations, up to 3 mM [66, 67]. However, it was discovered that cisplatin mostly enters the cell by

membrane transporters. This would explain why it has been observed that low levels of transporters

correlate with decreased levels of cellular cisplatin. Cisplatin uptake is performed by the copper

transporters CTR1 and CTR2, as well as the organic cationic transporter (OCT) family [68].

3.1.1 Copper transporters CTR1 and CTR2

Copper transporter protein 1 (CTR1) was shown to be one of the primary cisplatin transporters. It was first

discovered in yeast, noting that knocking down CTR1 resulted in reduced uptake in cisplatin [69]. CTR1

primarily transports copper, which is important in a variety of biological functions within a cell.

Interestingly, resistance to cisplatin is accompanied by resistance to copper and cisplatin resistant cells

display reduced levels of copper [70]. Cisplatin resistant cells show decreased levels of CTR1 mRNA and

decreased cisplatin uptake [71, 72]. In the clinic, CTR1 has been evaluated in two cancer types, ovarian

and

lung,

both

resulting

in

the

same

observation.

In

ovarian

cancer

patients

treated

with

cisplatin

chemotherapy, high levels of CTR1 mRNA expression was correlated with increased disease free survival

[73]. In NSCLC, the same pattern has emerged, high CTR1 protein levels were associated with a favorable

cisplatin response [74, 75].

Only one study has examined the relationship of CTR1 polymorphisms with cisplatin resistance. Xu et al.

found two

polymorphisms

in

CTR1

that

correlate

with

platinum

resistance

and

survival:

rs7851395

and

rs12686377 [76]. These two polymorphisms are located in the intron region of CTR1 and are hypothesized

to play a role in the epigenetic regulation of CTR1. Currently, it is not known what effect these

polymorphisms have on the function of CTR1, as this article was the first to describe them.



12

Cisplatin is also transported by another copper transporter, CTR2. While CTR2 is in the same family as

CTR1, they only share a 33% homology on the protein level [77]. CTR2 is found on the cellular membrane

like CTR1, but it is also found on intracellular organelle membranes and may have alternative cellular

functions [78]. The opposite effect has been observed with CTR2 in terms of its correlation with cisplatin

resistance. Unlike CTR1, knockdown of CTR2 in cells results in increased uptake, increased cytotoxicity,

and increased sensitivity to cisplatin [79]. In two clinical studies, lower levels of CTR2 are associated with

a better outcome to cisplatin therapy [80, 81]. Interestingly, it has been suggested that the CTR1/CTR2

ratio may be a useful biomarker for identifying tumors which may be more sensitive to cisplatin than on

one of the transporters alone [81]. No CTR2 polymorphisms have been identified to influence cisplatin

sensitivity.

3.1.2 Organic cation transporters

The solute carrier (SLC) transporter family, specifically the SLC22 family members, also transport cisplatin

into

cells.

Members

of

this

family,

OCT1,

OCT2,

and

OCT3,

have

been

shown

to

uptake

platinum‐

compounds into cells, but vary in the expression and substrate for each transporter [68]. OCT1 has been

indicated to transport cisplatin, however the evidence is weak [68]. OCT1 does transport the cisplatin

analogs carboplatin and oxaliplatin. OCT3 is known to primarily transport oxaliplatin.

Cisplatin is primarily transported by OCT2, or SLC22A2, and this transporter is found in the kidney. OCT2

transfection into

cells

results

in

increased

cellular

levels

of

cisplatin

[82].

There

is

not

a lot

of

clinical

studies

examining OCT2’s role in cisplatin resistance, primarily due to the fact it is expressed in the kidney. In the

NCI‐60 panel of cancer cell lines, OCT2 was the most frequently expressed gene but its expression in

clinical ovarian cancer specimens was low and did not correlate with the treatment outcome of a



13

platinum‐based regimen [83]. In one gastric cancer study, higher levels of OCT2 were observed in

responders to cisplatin‐based neoadjuvant therapy in comparison to non‐responders [84]. The majority

of OCT2 polymorphism studies have primarily focused on the effect on nephrotoxicity. There is one study

in lung cancer that evaluated the OCT2 polymorphisms. The polymorphisms rs195854 and rs186941 were

associated with increased response to cisplatin [85].

3.2 Increased cellular export of cisplatin

The export of cisplatin has been suggested to occur via passive efflux, however the issue with studies to

examine this phenomena are performed with high concentration of cisplatin. Thus it is thought that export

of cisplatin from cells occurs via membrane transporters. There are two major pathways in which cisplatin

is removed from the cell: removal by P‐type ATPase transporters or removal by ATP‐binding cassette

transporters.

3.2.1 P‐type ATPase transporters

Cisplatin

is

exported

by

ATP7A

and

ATP7B,

which

belong

to

the

transporter

family

of

P‐

type

ATPases

which

use ATP to export. These transporters are associated with removing excessive copper from cells. Under

normal conditions within the cell, ATP7A and ATP7B are found in the trans‐Golgi network and are

trafficked to the cell membrane to remove copper. As mentioned earlier, copper levels are lower in

cisplatin resistant cells which is additionally regulated by ATP7A and ATP7B. Defects in these transporters

are associated with diseases with excessive copper accumulation: ATP7A is defective in Menkes disease

while ATP7B

is

defective

in

Wilson’s

disease

[86].

ATP7A is found is most tissues, aside from the liver. Increased ATP7A expression is found in cancer cells

but not in normal tissue [87]. Increased expression of ATP7A in cells renders the cells resistant to cisplatin



14

but interestingly this was not due to altered cisplatin export [88, 89]. There are only a few clinical studies

evaluating the role of ATP7A and cisplatin resistance. In NSCLC and ovarian cancer, increased ATP7A

expression is associated with a poorer response to cisplatin [87, 89]. ATP7A levels are additionally higher

in NSCLC tumors that are resistant to cisplatin [90]. No ATP7A polymorphisms have been identified with

sensitivity to cisplatin, though studies are very limited in examining ATP7A polymorphisms in general [91].

ATP7B is found mostly in the liver, kidney and brain. Similar observations found with ATP7A in terms of

cisplatin resistance have also been observed with ATP7B. ATP7B was first proposed to be a biomarker of

cisplatin resistance, as transfection of ATP7B into cells resulted in an increase in cisplatin resistance

accompanied by reduced cisplatin accumulation [92]. Increased expression of ATP7B is associated with

poorly differentiated tumors and are poor responders to cisplatin therapy in a variety of cancers including:

gastric, hepatocellular, esophageal, oral, breast, endometrial, lung, and ovarian [93‐101]. Currently, there

are no identified ATP7B polymorphisms that are associated with cisplatin resistance.

3.2.2

ATP‐

binding

cassette

(ABC)

transporters

Multidrug resistance‐associated proteins (MRPs), belong to the ABCC subfamily of ABC (ATP binding

cassette) transporters and been implicated in cisplatin resistance [102]. MRPs are membrane transporters

responsible for the efflux of glutathione‐platinum conjugates, in an ATP‐dependent fashion. MRP1 was

first explored as a cisplatin transporter as it was found that cisplatin resistant cells displaying increased

levels of glutathione concurrently had increased levels of MRP1 [103]. Reports from other groups

suggested that

MRP1

alone

was

not

enough

to

confer

cisplatin

resistance

and

there

is

no

relationship

between MRP1 and cisplatin accumulation and cytotoxicity [104‐106]. There are no polymorphisms of

MDR1 associated with cisplatin response as well. Thus, MRP1 is generally not thought to play a role in

cisplatin resistance.



15

MRP2, also known as cMOAT (canalicular multispecific organic anion transporter), is the most favored

MRP transporter contributing to cisplatin resistance. Overexpression of MRP2 is found in a variety of

cisplatin resistant cells lines [107‐109]. The expression of MRP2 is induced by cisplatin as well [110]. In the

clinic, there are different observations of correlating MRP2 expression with cisplatin sensitivity, which

may reflect the tissue specific nature of the transporter. Increased MRP2 expression is associated with

cisplatin resistance in colorectal, esophageal, and hepatocellular cancers [111‐113]. However, in ovarian

and lung cancer, MRP2 did not predict cisplatin response [100, 114, 115]. One polymorphism in MRP2, C‐

24T, has been correlated with increased response to platinum‐based chemotherapy in lung cancer [116,

117]. The C‐24T polymorphism is found in the promoter of MRP2 and its function is not currently known.

4.0 Cytosolic inactivation of cisplatin

Finally, the last resistance mechanism is cytosolic inactivation of cisplatin. This inactivation results in the

inability of cisplatin to react with DNA. Less damage is produced and the cancer cell survives the drug

treatment.

The

primary

form

of

inactivation

is

conjugation

of

cisplatin

with

glutathione

(GSH),

resulting

in

cellular export by the MRP transporters, discussed in the prior section. The secondary form of inactivation

is by binding to metallothioneins.

4.1 Inactivation by glutathione conjugation

Glutathione‐S‐transferases (GSTs) catalyze the conjugation of glutathione (GSH) to cisplatin. The

formation of

platinum

‐glutathione

conjugates

inactivates

the

drug

by

increasing

its

solubility,

leading

to

excretion. Inside the cell, glutathione acts as antioxidant. It maintains the redox environment by keeping

reduced sulfhydryl groups [118]. Depletion of cellular GSH in cisplatin resistant cells enhances the

cytotoxicity of cisplatin [119]. However, the cisplatin sensitivity is not restored to levels of the parental



16

cell lines. In ovarian cancer cells, increased levels of GSH were observed in platinum resistant cell lines

[120]. There are two families of GST enzymes involved in cisplatin detoxification‐ GSTP1 and GSTM.

4.1.1 Glutathione‐S‐transferase Pi 1

GSTP1, also called GST Pi 1, is expressed in different epithelial tissues. The cellular and clinical studies of

GSTP1 are inconclusive as to whether or not it is an indicator of cisplatin resistance. In colon, lung, and

glioblastoma cancer cell lines, the levels of GSTP1 are correlated between high GSTP1 expression and

cisplatin resistance [121]. In ovarian and head and neck carcinoma patient samples, there is a correlation

between high expression of GSTP1 and cisplatin resistance [122‐124]. Several studies in NSCLC have

demonstrated that low levels of GSTP1 are associated with increased sensitivity to cisplatin [125‐129].

However, in other clinical studies of ovarian and cervical cancer there was no association of GSTP1 levels

and response to cisplatin chemotherapy [130‐132].

GSTP1 has two polymorphisms: rs1695 and rs1138272. Similar to the expression data, GSTP1

polymorphism

data

is

conflicting

and

inconclusive.

The

rs1695

polymorphism

affects

the

ability

of

GSTP1

to conjugate GSH to cisplatin [133]. Studies in NSLC have yielded multiple responses for the GSTP1 rs1695

polymorphism: associated with a favorable response to cisplatin therapy [116, 134], associated with

reduced survival to cisplatin therapy [135], and no association with survival [136]. The rs1138272

polymorphism additionally has different responses to cisplatin therapy: one study found it was associated

with greater median survival [136] and another study correlated to the polymorphisms to a shorter event

free survival

and

shorter

overall

survival

in

osteosarcoma

patients

[61].



17

4.1.2 Glutathione‐S‐transferase Mu

GSTM, or GST Mu, is the other GST involved in the inactivation of cisplatin. GSTM is more known for the

detoxification of xenobiotics thus there is little research in evaluating GSTM in cisplatin resistance. There

are five GSTM genes: GSTM1, GSTM2, GSTM3, GSTM4, and GST5. Earlier studies showed that there was

no difference and no contribution by GSTMs to cisplatin resistance [137‐139]. However, recent data using

a paired cisplatin sensitive/resistant breast cancer cell line demonstrated decreased GSTM3 and GSTM4

levels were found in cisplatin resistant cells compared to sensitive cells [140, 141]. This has not been

investigated further as one would assume that decreased levels of GSTs would show decreased resistance

to cisplatin, like observed with GSTP1. However, pharmacologic inhibition of GSTM1 resulted in the

increased sensitivity of cells to cisplatin [142]. There are no clinical studies examining the relationship of

GSTM levels with cisplatin sensitivity.

The majority of GSTM polymorphism studies have focused on the susceptibility to cancer and not so much

on the relationship with cisplatin resistance. GSTM2 and GSTM5 do not have any reported

polymorphisms.

Polymorphisms

for

GSTM3

and

GSTM4

have

not

been

evaluated

for

their

relationship

with cisplatin resistance. There are few studies with polymorphisms of GSTM1. Wheeler et al. showed

that the rs10431718 GSTM1 polymorphism was associated with the cisplatin IC50 [143]. In a NSCLC meta‐

analysis, the GSTM1 null genotype was associated with improved response to platinum therapy [144].

4.2 Inactivation by metallothionein binding

Finally, cisplatin

is

also

inactivated

by

binding

to

metallothionein

(MT)

proteins.

MT

proteins

are

cysteine

‐

rich, low molecular weight proteins that bind to metals such as copper, zinc, cadmium, and mercury. While

there are multiple MTs, mostly MTI and MTII have studied since they are ubiquitously expressed.

However, it is not always evident which MT is being examined. MTs function as regulators of cellular metal



18

homeostasis as well as detoxification of heavy metal exposure in cells. In terms of cisplatin resistance,

MTs serve as a heavy‐metal detoxifier of cisplatin in tumors. Overexpression of metallothionein has been

observed in several cell lines that are resistant to cisplatin [145‐147]. Additionally, the overexpression of

MTII confers resistance to cisplatin in cancer cells [148]. Cisplatin treatment also induces the expression

of MT [149]. In germ cell tumors, MT expression was higher in cell lines and tumors, but there was no

difference between patients who responded to cisplatin based therapy compared to non‐responders

[150]. In esophageal cancer, expression of MT was associated with a shorter survival rate after cisplatin

therapy [151]. This was additionally observed in ovarian cancer patients receiving cisplatin based therapy

[123]. There are no MT polymorphisms that are associated with cisplatin resistance.

5.0 Summary

Cisplatin is a clinical mainstay for the treatment of a variety of cancers. Unfortunately, many tumors

develop resistance and are refractory to treatment. Resistance stems from three overall mechanisms‐

increased DNA repair, altered drug cellular accumulation, and increased drug cytosolic inactivation. In this

review,

potential

biomarkers

and

their

known

polymorphisms

for

each

resistance

mechanism

were

examined and are summarized in tables 1 and 2. While ERCC1 represents the most promising biomarker

for cisplatin resistance, as it has been extensively studied in a variety of cancers, there are several

opportunities and areas ripe for further study. Other components of NER, CTR1 and CTR2, OCT2, ATP7A

and ATP7B, GSTs, and metallothioneins have the potential to be valid cisplatin biomarkers as well, and

would benefit from additional clinical studies.

While this list is comprehensive, there are several things to consider. Not all biomarkers were examined

in a multitude of cancer types, so some may be tumor specific. Many biomarkers displayed conflicting

evidence with their role in cisplatin resistance. Some of the conflicting data reflects the fact that many



19

studies maybe underpowered for the analyses, and would benefit from having larger sample sizes.

Because resistance to cisplatin is multilayered and multifactorial, different mechanisms are likely to be

activated depending on the cancer type and stage. It is highly likely that multiple resistance mechanisms

will be activated within a patient. While one biomarker may not be completely informative for all cancers,

a combination of biomarker expression and polymorphism screening may yield a comprehensive

approach to elucidate the resistance status of a patient.

The method by which patients are screened is critical. The type of biospecimen used in the evaluation

(blood, tissue, etc.), the expression type (DNA, mRNA, protein), and the method used to examine

expression (PCR‐based, IHC, etc.) will all need to be standardized for analysis of resistance. The definition

of what is considered high versus low expression, as well as the cutoff points between the categories, will

also require standardization. A recent paper described the challenge of biomarker based screening. In a

round robin analysis of three independent commercial labs, 18 tumor blocks were sent for testing of

ERCC1 status, and the results were inconsistent and unreliable [152]. Only 4 of 18 blocks tested were fully

concordant

with

ERCC1

status

between

all

three

labs.

Thus

further

evaluation

and

standardization

are

needed before these assays become clinical standard‐of ‐care.

Precision medicine serves two purposes for cisplatin resistance: to determine if resistance is occurring and

to determine the nature of the activated resistance mechanism(s). Screening the patient prior to initiation

of treatment, and during the course of treatment, allows for the improvement of cancer diagnosis by

predicting tumor

response.

Personalizing

this

therapy

will

increase

the

efficacy

and

decrease

the

toxicity

of platinum‐based chemotherapy. While there is a long road ahead, several of the biomarkers listed here

may serve as a foundation for larger, prospective studies to determine which biomarker, or combination

of biomarkers, would result in the best prediction of cisplatin sensitivity and resistance in patients.



20

Conflict of interest.

The author has no conflict of interest.

Acknowledgements

The research was supported by the Intramural Research Program of the NIH, National Institute on

Minority Health and Health Disparities. This manuscript is dedicated to my former mentor, Dr. Eddie Reed.



21

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37

Figure legends

Figure 1. Cisplatin and analogs.

Figure 2. Cellular fate of cisplatin (Pt). Cisplatin crosses the cell membrane by passive diffusion or by

transmembrane transporters. CTR1, CT2, and OCT2 have been identified as transporters that import

cisplatin into the cell. Once inside the cell, cisplatin binds to DNA to cause DNA‐platinum adducts. The

damage is repaired by ERCC1 and members of the NER pathway. Cisplatin is also inactivated by

glutathione‐s‐transferase, which add a glutathione (GSH) to cisplatin. The conjugated cisplatin‐GSH is then

exported via the MRP2 transporters. Cisplatin is also exported by ATP7A and ATP7B. Inactivation of

cisplatin can also result from binding metallothionein proteins (MT).

Figure 3. Schematic of nucleotide excision repair (NER). DNA‐platinum adducts are removed by the NER

pathway. First the DNA‐platinum adduct is detected. Then the damage is verified and the pre‐incision

complex is set up containing RPA, XPA, and XPG. DNA is unwound by XPB and XPD. XPF‐ERCC1 and XPG

create

incisions

5’

and

3’

from

the

damaged

base.

The

oligonucleotide

containing

the

damaged

base

is

removed. The gap is filled in by DNA repair synthesis complex: RPA, RFC, PCNA, and Pol /. Finally, the

DNA is ligated.



38



39



40



41

Table 1. Proteins associated with cisplatin resistance.

Protein Relationship to

resistance

Cancer References

NER

ERCC1 Increased expression Ovarian, NSCLC,

Nasopharyngeal,

esophageal, cervical, head

and neck squamous

carcinoma, liver,

osteosarcoma, lung

adenocarcinoma, biliary

tract adenocarcinoma,

mesothelioma, pulmonary

adenocarcinoma, gastric

13‐16, 15, 17‐23, 24‐25,

26,

27‐

28,

29‐

30,

31,

32,

33, 34, 35, 36, 11

XPA Increased expression Ovarian cancer 53, 54

XPB Increased expression Ovarian cancer 53, 54

XPF

Increased expression

Ovarian

and

colon

cancer

cell lines; head and neck

carcinoma

55‐57

XPD Increased expression NSCLC and glioma cell lines 58, 59

Cellular Uptake

CTR1 Decreased expression Ovarian cancer, NSCLC 73‐75

CTR2 Increased expression Ovarian cancer 80, 81

OCT2 No change Ovarian cancer 83

Decreased expression Gastric cancer 84

Cellular Export

ATP7A Increased expression NSCLC, ovarian cancer 87, 89, 90

ATP7B Increased expression Gastric, hepatocellular,

esophageal, oral, breast,

endometrial, lung, ovarian

cancer

93‐101

MRP2 Increased expression Colorectal, esophageal,

hepatocellular cancer

111‐113

Drug Inactivation

GSTP1 Increased expression Ovarian cancer, head and

neck carcinoma,

NSCLC

122‐124

No change Ovarian, cervical cancer 130‐132

MT Increased expression Esophageal, ovarian cancer 151, 123



Table 2. Gene polymorphisms associated with cisplatin resistance.

Gene Polymorphism Response to Cisplatin Cancer Reference

NER

ERCC1 rs11615,

N118N

Increased response Ovarian cancer,

colorectal,

pancreatic,

osteosarcoma and

NSCLC

38,40, 42, 43, 44,

45

Decreased response NSCLC 46, 47

C8092A Increased response NSCLC,

esophageal

49, 50,

Decreased response Nasopharyngeal,

mesothelioma

51, 35

No relationship NSCLC 52

XPD Asp312Asn Increased response NSCLC,

osteosarcoma,

pancreatic cancer

54,60, 62, 63

Cellular uptake

CTR1 rs7851395,

rs12686377

Increased response NSCLC 76

OCT2 rs195854,

rs186941

Increased response NSCLC 85

Cellular Export

MRP2 Increased response NSCLC 116, 117

Drug

Inactivation

GSTM1 rs10431718 Increased response lymphoblastoid

cell lines

143

Null allele Increased response NSCLC 144

cisplatin resistance and opportunities for precision medicine

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