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. 2017 Apr;10(2):190-196.
doi: 10.1016/j.tranon.2017.01.007. Epub 2017 Feb 6.

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib

Chen Wang  1 , Nicholas Jette  1 , Daniel Moussienko  1 , D Gwyn Bebb  2 , Susan P Lees-Miller  3
Affiliations

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib

Chen Wang et al. Transl Oncol. 2017 Apr.

Abstract

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor.

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Figures

Figure 1
Figure 1
Predicted rates of ATM and p53 alteration in CRC. Redrawn from c-Bioportal , , accessed November 2016. From left to right: MCL, cutaneous squamous cell carcinoma, liver hepatocellular carcinoma, colorectal adenocarcinoma, stomach adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, cholangiocarcinoma, esophageal carcinoma, prostate adenocarcinoma, and lung adenocarcinoma.
Figure 2
Figure 2
The colorectal cell line SK-CO-1 lacks ATM protein expression. (A) Extracts from human CRC cell lines were generated by NETN lysis, and 50-μg aliquots of total protein were analyzed by SDS-PAGE and immunoblotting to antibodies as shown on the right-hand side. Results are representative of three separate experiments. (B) Quantitation of ATM protein levels in the CRC cell lines, normalized to SMC1, from three separate experiments. ATM protein expression (normalized to SMC1) in LS123 cells was set as one.
Figure 3
Figure 3
Analyses of DNA damage response proteins in colorectal cell lines by immunoblotting. Human CRC cell lines (HCT116, SNU-C1, LS123, SK-CO-1, T84, and LoVo) and ATM-proficient and ATM-deficient human lymphoblastoid cells (BT and L3, respectively) were treated with 2 Gy IR and harvested 1 hour later. Whole cell lysates were prepared and analyzed by immunoblotting as in Figure 2. The figure shows results from one experiment run under identical conditions on two separate gels (indicated by the white line). For each gel, exposures were for the same time under the same conditions.
Figure 4
Figure 4
The ATM-deficient SK-CO-1 CRC cell line is sensitive to olaparib. Cells were counted, seeded onto 6-cm dishes, and allowed to adhere overnight. The PARP inhibitor olaparib or DMSO (vehicle control) was then added at the indicated doses, and the cells were allowed to grow for 14 days undisturbed. The number of colonies remaining on each plate was counted after fixing and staining with crystal violet solution. SK-CO-1 cells, which carry two copies of mutated ATM, are indicated by circles, and LoVo cells that express WT-ATM are indicated by squares. Results show the average of three separate experiments with each treatment carried out in triplicate. Error bars show standard deviation. **P < 0.001.
Figure 5
Figure 5
shRNA depletion of ATM in HCT116 cells. (A) ATM was depleted in HCT116 cells using shRNA as described in Materials and Methods. shRNA to GFP was used as a control. Immunoblotting of shRNA control (shGFP) and shRNA ATM is shown. BT and L3 cells were used as ATM-proficient and ATM-deficient controls, respectively. (B) Clonogenic assays with olaparib at 0, 1, 2, or 3 μM with HCT116 p53 +/+ cells plus and minus shRNA depletion of ATM. HCT116 parental cells are represented by squares, HCT116 shGFP cells by circles, and HCT116 shATM cells by triangles. (C) As in panel B but with HCT116 p53−/− cells. Statistical significance of olaparib sensitivity in shATM depleted cells compared with shGFP control cells is indicated by the asterisks where * = P < 0.05 and ** = P < 0.001.
Figure 6
Figure 6
Clonogenic assays in HCT116p53+/+ and HCT116p53−/− cells treated with the PARP inhibitor olaparib and ATM inhibitor KU55933. (A) HCT116 p53+/+ cells were treated with either DMSO (circles) or 2.5 (squares), 5 (triangles), or 7.5 μM (diamonds) of the ATM inhibitor KU55933 in addition to treatment with olaparib at 0, 1, 2, or 3 μM. (B) As in panel B but with HCT116 p53−/− cells. Statistical significance of olaparib sensitivity in shATM depleted cells compared with shGFP control cells is indicated by the asterisks where * = P < 0.05 and ** = P < 0.001.
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