Pharmacologic inhibition of ATR and ATM offers clinically important distinctions to enhancing platinum or radiation response in ovarian, endometrial, and cervical cancer cells

doi:10.1016/j.ygyno.2014.12.035

Gynecologic Oncology

Volume 136, Issue 3, March 2015, Pages 554-561

https://doi.org/10.1016/j.ygyno.2014.12.035 Get rights and content

Highlights

•
Pharmacologic inhibition of the ATR kinase, but not the ATM kinase, significantly enhances platinum drug efficacy in gynecologic cancer cells.
•
Inhibition of either ATR or ATM enhances IR efficacy in gynecologic cancer cells, with further enhancement achieved with co-inhibition.

Abstract

Objective

Significant reductions in gynecologic (GYN) cancer mortality and morbidity require treatments that prevent and reverse resistance to chemotherapy and radiation. The objective of this study was to determine if pharmacologic inhibition of key DNA damage response kinases in GYN cancers would enhance cell killing by platinum-based chemotherapy and radiation.

Methods

A panel of human ovarian, endometrial and cervical cancer cell lines were treated with platinum drugs or ionizing radiation (IR) along with small molecule pharmacological kinase inhibitors of Ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR).

Results

Pharmacologic inhibition of ATR significantly enhanced platinum drug response in all GYN cancer cell lines tested, whereas inhibition of ATM did not enhance the response to platinum drugs. Co-inhibition of ATM and ATR did not enhance platinum kill beyond that observed by inhibition of ATR alone. By contrast, inhibiting either ATR or ATM enhanced the response to IR in all GYN cancer cells, with further enhancement achieved with co-inhibition.

Conclusions

These studies highlight actionable mechanisms operative in GYN cancer cells with potential to maximize response of platinum agents and radiation in newly diagnosed as well as recurrent gynecologic cancers.

Section snippets

Background

Gynecologic (GYN) cancers, specifically cancers of the uterine cervix, uterine corpus, and ovary, represent a significant worldwide health problem with 1,085,948 new diagnoses and 493,713 deaths observed annually (globocan.iarc.fr). Primary cytoreductive surgery with platinum- and paclitaxel-based chemotherapy, either in the adjuvant or neoadjuvant setting, are the standards of care for ovarian cancer [1]. Surgical staging plays a central role in endometrial cancer management, with

Cell culture

A2780 and A2780-CP20 (Sigma, St. Louis, MO) and maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS, ATCC, Manassas, VA). A2780-CP20 cells were treated with 1 μM cisplatin (Sigma) every other passage to maintain resistance to cisplatin. OVCAR3 cells maintained in RPMI medium supplemented with 20% FBS and 0.01 mg/mL insulin (Sigma) or DMEM:F12 with 10% FBS and 0.01 mg/mL insulin. KLE cells were maintained in DMEM:F12 medium supplemented with 10% FBS. HELA, SIHA, and HEC1B cells

Analysis of DDR protein expression across representative cell line models of gynecologic cancer

Protein abundances of ATR, ATM, Chk1, Chk2, and p53 were analyzed by immunoblot in seven cell line models of GYN carcinoma, including three models of ovarian [A2780, A2780-CP20 (a syngeneic model of cisplatin resistance derived from A2780), and OVCAR3], two models of cervical (HELA and SIHA) and two models of endometrial (KLE and HEC1B) carcinoma (Supplementary Fig. 1). Total ATR levels were largely invariant in these cells, with the exception of OVCAR3 where a greater level of ATR abundance

Discussion

Here we show that pharmacological inhibition of ATR, but not ATM, sensitizes ovarian, endometrial and cervical cancer cells to cisplatin and carboplatin, and that the combination of either platinum agent with the ATRi was synergistic. We present data utilizing a syngeneic model of platinum-resistant ovarian cancer, A2780 (platinum-sensitive) versus A2780-CP20 (platinum-resistant), showing that inhibition of ATR produces marked enhancement in response when combined with cisplatin or carboplatin.

Funding

This study was funded by an award from the United States Army Medical Research and Materiel Command (W81XWH-11-2-0131, all authors but CJB) and CA148644 (CJB) from the National Cancer Institute (CJB).

Competing interests

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army, Department of the Air Force, Department of the Navy, or the Department of Defense. The authors have no financial disclosures.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors thank Darcy Franicola for her assistance in generating the IR clonogenic plots.

References (41)

A. Ciccia et al.
The DNA damage response: making it safe to play with knives
Mol Cell
(2010)
W.P. Roos et al.
DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis
Cancer Lett
(2013)
J. Smith et al.
The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer
Adv Cancer Res
(2010)
S. Arora et al.
RNAi screening of the kinome identifies modulators of cisplatin response in ovarian cancer cells
Gynecol Oncol
(2010)
J.M. Furgason et al.
Targeting DNA repair mechanisms in cancer
Pharmacol Ther
(2013)
N.A. Wallace et al.
Manipulation of cellular DNA damage repair machinery facilitates propagation of human papillomaviruses
Semin Cancer Biol
(2014)
J.S. Skilling et al.
p53 gene mutation analysis and antisense-mediated growth inhibition of human ovarian carcinoma cell lines
Gynecol Oncol
(1996)
A. Nowosielska et al.
Spontaneous and cisplatin-induced recombination in Escherichia coli
DNA Repair (Amst)
(2004)
A. Nowosielska et al.
Cisplatin induces DNA double-strand break formation in Escherichia coli dam mutants
DNA Repair (Amst)
(2005)
H.C. Reinhardt et al.
p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage
Cancer Cell
(2007)

R.R. Barakat

Principles and practice of gynecologic oncology

(2013)

R. Siegel et al.

Cancer statistics, 2014

CA Cancer J Clin

(2014)

H. Zhao et al.

ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1

Mol Cell Biol

(2001)

Q. Liu et al.

Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint

Genes Dev

(2000)

Z. Chen et al.

Selective Chk1 inhibitors differentially sensitize p53-deficient cancer cells to cancer therapeutics

Int J Cancer

(2006)

A.M. Huehls et al.

Identification of DNA repair pathways that affect the survival of ovarian cancer cells treated with a poly(ADP-ribose) polymerase inhibitor in a novel drug combination

Mol Pharmacol

(2012)

R. Sultana et al.

Ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase inhibition is synthetically lethal in XRCC1 deficient ovarian cancer cells

PLoS One

(2013)

T. Abdel-Fatah et al.

Clinicopathological and functional significance of XRCC1 expression in ovarian cancer

Int J Cancer

(2013)

C.J. Huntoon et al.

ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status

Cancer Res

(2013)

A. Peasland et al.

Identification and evaluation of a potent novel ATR inhibitor, NU6027, in breast and ovarian cancer cell lines

Br J Cancer

(2011)

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¹: These authors contributed equally to this work.

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Pharmacologic inhibition of ATR and ATM offers clinically important distinctions to enhancing platinum or radiation response in ovarian, endometrial, and cervical cancer cells

Highlights

Abstract

Objective

Methods

Results

Conclusions

Section snippets

Background

Cell culture

Analysis of DDR protein expression across representative cell line models of gynecologic cancer

Discussion

Funding

Competing interests

Conflict of interest statement

Acknowledgments

Mol Cell

Cancer Lett

Adv Cancer Res

Gynecol Oncol

Pharmacol Ther

Semin Cancer Biol

Gynecol Oncol

DNA Repair (Amst)

DNA Repair (Amst)

Cancer Cell

Principles and practice of gynecologic oncology

Cancer statistics, 2014

CA Cancer J Clin

ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1

Mol Cell Biol

Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint

Genes Dev

Selective Chk1 inhibitors differentially sensitize p53-deficient cancer cells to cancer therapeutics

Int J Cancer

Identification of DNA repair pathways that affect the survival of ovarian cancer cells treated with a poly(ADP-ribose) polymerase inhibitor in a novel drug combination

Mol Pharmacol

Ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase inhibition is synthetically lethal in XRCC1 deficient ovarian cancer cells

PLoS One

Clinicopathological and functional significance of XRCC1 expression in ovarian cancer

Int J Cancer

ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status

Cancer Res

Identification and evaluation of a potent novel ATR inhibitor, NU6027, in breast and ovarian cancer cell lines

Br J Cancer