HHS Public Access Author manuscript Author Manuscript
Cell Rep. Author manuscript; available in PMC 2017 March 14. Published in final edited form as: Cell Rep. 2017 February 21; 18(8): 1970–1981. doi:10.1016/j.celrep.2017.01.072.
Targeting DNA damage response in prostate cancer through inhibition of androgen receptor/CDC6-ATR-Chk1 signaling: Combined AR/CDC6 and Chk1/2 inhibition for PCa
Author Manuscript
Styliani Karanika1,4, Theodoros Karantanos1,4, Likun Li1,4, Jianxiang Wang1, Sanghee Park1, Guang Yang1, Xuemei Zuo1, Jian H. Song1, Sankar N. Maity1, Ganiraju C. Manyam2, Bradley Broom2, Ana M. Aparicio1, Gary Gallick1, Patricia Troncoso3, Paul G. Corn1, Nora Navone1, Wei Zhang1, Shuhua Li1, and Timothy C. Thompson.1,* 1Department
of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer
Center 2Bioinformatics 3Pathology
& Comp Biology, The University of Texas MD Anderson Cancer Center
Administration, The University of Texas MD Anderson Cancer Center
Graphical abstract
Author Manuscript *
Author Manuscript
Corresponding Author: Dr. T.C. Thompson, Department of Genitourinary Medical Oncology – Research, Unit 18-3, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Tel.: +1 713-792-9955; Fax: +1 713-792-9956; [email protected]. 4Co-first authors Lead contact: Timothy C. Thompson. Tel.: +1 713-792-9955; Fax: +1 713-792-9956; [email protected].
Author Contributions: T.C.T., S.K., T.K. and L.L. conceived and designed the study and wrote the paper. In vitro studies, including siRNA and drug treatments, Western blotting analysis, flow cytometric, DNA fragmentation experiments, protein stability assays, statistic and synergy analysis: L.L., S.K., T.K., J.W., X.Z., W. Z., S.L.; IHC and tissue microarray analysis: G.Y., S.K., T.K.; Xenograft model studies: S.P., S.K., T.K., J.W., J.S. and G.G. P.G.C. contributed to manuscript preparation. S.N.M, A.M.A., P.T. and N.N. contributed to pathological analysis of human samples and the establishment of PDX lines. B.B and G.C.M. performed bioinformatics analyses of RNASeq data. Conflict of Interest: The authors declare that they have no conflict of interest. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Karanika et al.
Page 2
Author Manuscript Author Manuscript
Summary
Author Manuscript
Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment of AR with enzalutamide (ENZ) and Chk1/2 inhibition with AZD7762 demonstrates synergy with regard to inhibition of AR/CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications since they introduce a therapeutic strategy for AR-positive metastatic castrationresistant PCa, regardless of p53 status, through targeting AR/CDC6-ATR-Chk1 signaling.
Keywords Androgen receptor; CDC6; Chk1; ATR; TOPBP1; DNA damage; prostate cancer; enzalutamide; AZD7762
Author Manuscript
Introduction Metastatic prostate cancer remains an incurable disease with variable prognosis (Wu et al., 2014). After an initial period of response to systemic hormone therapy, the disease inexorably progresses to a state known as metastatic castration-resistant prostate cancer (mCRPC) (Karanika et al., 2014). The therapeutic armamentarium for mCRPC is limited to chemotherapy and novel inhibitors of AR signaling, such as abiraterone acetate and
Cell Rep. Author manuscript; available in PMC 2017 March 14.
Karanika et al.
Page 3
Author Manuscript
enzalutamide (ENZ), which provide only moderate survival benefits (Ryan et al., 2013, Scher et al., 2012). DNA damage response (DDR) refers to coordinated cellular mechanisms that prevent DNA damage accumulation and maintain genomic integrity (Karanika et al., 2014) and plays a central role in PCa initiation, development and progression (Tapia-Laliena et al., 2014). AR signaling in PCa cells has been associated with numerous aspects of DDR pathways including regulation of ATM-Chk2 signaling for initiation of DDR (Ide et al., 2012), poly(ADP-ribose) polymerase function (Schiewer et al., 2012) and non-homologous end joining recombination (Polkinghorn et al., 2013). AR is reported to regulate TopBP1-ATRChk1 signaling (Li et al., 2014) whereas ENZ decreases CHEK1 expression in PCa cells (Li et al., submitted).
Author Manuscript
One of the main types of DNA damage istan and Lim, 2000 DNA strand breakage, which activates a cascade of intracellular events that promote cell-cycle arrest and DDR ensuring genomic integrity which can be particularly critical for cell survival in patients with aggressive malignancies accumulating a myriad of genetic errors (Karanika et al., 2014). DNA strand breaks activate ATR via upstream mediators such as TopBP1 leading to Chk1mediated checkpoint activation and cell-cycle arrest. Cells are thereby able to repair DNA damage alleviating replication stress and genomic instability. Chk1 pathway inhibition results in DNA damage accumulation and thus increased ATM auto-phosphorylation, which mediates apoptosis of cells with incompletely replicated DNA (d'Adda di Fagagna, 2008, Sarmento et al., 2015). These findings demonstrate that ATR-Chk1 signaling is crucial for the prevention of DNA damage-induced cell death associated with increased ATM phosphorylation/activation.
Author Manuscript Author Manuscript
CDC6 is an essential regulator of DNA replication in eukaryotic cells and its bestcharacterized function is pre-replicative complex assembly at origins of replication during G1 phase (Borlado and Mendez, 2008). Furthermore, CDC6 overexpression during G2 phase blocks mitotic entry by activating Chk1 (Clay-Farrace et al., 2003, Yoshida et al., 2010) which inhibits G2/M progression. CDC6 is required for Chk1 activation upon replication inhibition (Oehlmann et al., 2004) and human CDC6 interacts with ATR promoting activation of the replication checkpoint (Yoshida et al., 2010). Consequently, CDC6 decreases genomic instability which is vital for cancer cell survival. CDC6 can also manifest oncogenic activities via regulation of DNA replication and repression of tumor suppressors (Gonzalez et al., 2006). Targeting ATR-Chk1 signaling increases the sensitivity to treatment with DNA-damaging agents (Bartucci et al., 2012) making this approach particularly attractive for development of cancer therapies. Notably, Chk1 knockdown increases the sensitivity of PCa stem cells to radiotherapy through increased DNA damage (Fokas et al., 2012). AZD7762, a Chk1/2 inhibitor, synergizes with DNA-damaging agents and radiation to induce apoptosis via DNA double strand breaks mediated by ATM activation in many cell types (Mitchell et al., 2010, Sausville et al., 2014). Furthermore, Brooks et al found that Chk1 inhibition can selectively induce apoptosis in melanoma cells in proportion to the level of endogenous DNA damage related to replicative stress without further induction of DNA damage by chemotherapy (Brooks et al., 2013).
Cell Rep. Author manuscript; available in PMC 2017 March 14.
Karanika et al.
Page 4
Author Manuscript
Taking the results of these studies into account, a potential approach to treatment of mCRPC is inhibition of more than one level of a specific DDR signaling cascade with the goal of completely abolishing a specific signaling pathway. This approach would take advantage of the fact that cancer cells eventually accumulate more DNA damage than normal cells do, eluding adverse effects of chemotherapy. Targeting the ATR-Chk1 pathway at multiple levels to inhibit the repair of DNA damage induced by replication stress in cancer cells may represent an effective strategy for more complete DDR pathway inhibition. Because CDC6 is an androgen receptor (AR) target gene (Jin and Fondell, 2009, Bai et al., 2005), and is also involved with ATR-Chk1 signaling this is a particularly intriguing strategy for AR-positive PCa. In addition, this approach may be effective under conditions of wild-type p53 which is involved in multiple DDR pathways and can mitigate the response to some DNA-damaging agents (Ma et al., 2012).
Author Manuscript
The aim of the present study was to determine the role of CDC6 in regulation of ATR-Chk1 signaling and to test combined inhibition of AR and Chk1 signaling as a therapeutic approach for AR-positive mCRPC. Through this study we also aimed to introduce a therapeutic approach that effectively targets a DDR pathway that promotes sufficient DNA damage accumulation in PCa cells to induce cell death, regardless of p53 status.
Results CDC6 is induced during PCa progression and is positively correlated with AR expression
Author Manuscript
We first evaluated the CDC6 expression and phosphorylation, and analyzed its correlation with AR expression in normal human prostates, primary and metastatic prostate tumor specimens. In our immunohistochemistry (IHC) analysis, we found that AR expression (P=0.0057), and CDC6 expression (P
Cell Rep. Author manuscript; available in PMC 2017 March 14. Published in final edited form as: Cell Rep. 2017 February 21; 18(8): 1970–1981. doi:10.1016/j.celrep.2017.01.072.
Targeting DNA damage response in prostate cancer through inhibition of androgen receptor/CDC6-ATR-Chk1 signaling: Combined AR/CDC6 and Chk1/2 inhibition for PCa
Author Manuscript
Styliani Karanika1,4, Theodoros Karantanos1,4, Likun Li1,4, Jianxiang Wang1, Sanghee Park1, Guang Yang1, Xuemei Zuo1, Jian H. Song1, Sankar N. Maity1, Ganiraju C. Manyam2, Bradley Broom2, Ana M. Aparicio1, Gary Gallick1, Patricia Troncoso3, Paul G. Corn1, Nora Navone1, Wei Zhang1, Shuhua Li1, and Timothy C. Thompson.1,* 1Department
of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer
Center 2Bioinformatics 3Pathology
& Comp Biology, The University of Texas MD Anderson Cancer Center
Administration, The University of Texas MD Anderson Cancer Center
Graphical abstract
Author Manuscript *
Author Manuscript
Corresponding Author: Dr. T.C. Thompson, Department of Genitourinary Medical Oncology – Research, Unit 18-3, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Tel.: +1 713-792-9955; Fax: +1 713-792-9956; [email protected]. 4Co-first authors Lead contact: Timothy C. Thompson. Tel.: +1 713-792-9955; Fax: +1 713-792-9956; [email protected].
Author Contributions: T.C.T., S.K., T.K. and L.L. conceived and designed the study and wrote the paper. In vitro studies, including siRNA and drug treatments, Western blotting analysis, flow cytometric, DNA fragmentation experiments, protein stability assays, statistic and synergy analysis: L.L., S.K., T.K., J.W., X.Z., W. Z., S.L.; IHC and tissue microarray analysis: G.Y., S.K., T.K.; Xenograft model studies: S.P., S.K., T.K., J.W., J.S. and G.G. P.G.C. contributed to manuscript preparation. S.N.M, A.M.A., P.T. and N.N. contributed to pathological analysis of human samples and the establishment of PDX lines. B.B and G.C.M. performed bioinformatics analyses of RNASeq data. Conflict of Interest: The authors declare that they have no conflict of interest. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Karanika et al.
Page 2
Author Manuscript Author Manuscript
Summary
Author Manuscript
Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment of AR with enzalutamide (ENZ) and Chk1/2 inhibition with AZD7762 demonstrates synergy with regard to inhibition of AR/CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications since they introduce a therapeutic strategy for AR-positive metastatic castrationresistant PCa, regardless of p53 status, through targeting AR/CDC6-ATR-Chk1 signaling.
Keywords Androgen receptor; CDC6; Chk1; ATR; TOPBP1; DNA damage; prostate cancer; enzalutamide; AZD7762
Author Manuscript
Introduction Metastatic prostate cancer remains an incurable disease with variable prognosis (Wu et al., 2014). After an initial period of response to systemic hormone therapy, the disease inexorably progresses to a state known as metastatic castration-resistant prostate cancer (mCRPC) (Karanika et al., 2014). The therapeutic armamentarium for mCRPC is limited to chemotherapy and novel inhibitors of AR signaling, such as abiraterone acetate and
Cell Rep. Author manuscript; available in PMC 2017 March 14.
Karanika et al.
Page 3
Author Manuscript
enzalutamide (ENZ), which provide only moderate survival benefits (Ryan et al., 2013, Scher et al., 2012). DNA damage response (DDR) refers to coordinated cellular mechanisms that prevent DNA damage accumulation and maintain genomic integrity (Karanika et al., 2014) and plays a central role in PCa initiation, development and progression (Tapia-Laliena et al., 2014). AR signaling in PCa cells has been associated with numerous aspects of DDR pathways including regulation of ATM-Chk2 signaling for initiation of DDR (Ide et al., 2012), poly(ADP-ribose) polymerase function (Schiewer et al., 2012) and non-homologous end joining recombination (Polkinghorn et al., 2013). AR is reported to regulate TopBP1-ATRChk1 signaling (Li et al., 2014) whereas ENZ decreases CHEK1 expression in PCa cells (Li et al., submitted).
Author Manuscript
One of the main types of DNA damage istan and Lim, 2000 DNA strand breakage, which activates a cascade of intracellular events that promote cell-cycle arrest and DDR ensuring genomic integrity which can be particularly critical for cell survival in patients with aggressive malignancies accumulating a myriad of genetic errors (Karanika et al., 2014). DNA strand breaks activate ATR via upstream mediators such as TopBP1 leading to Chk1mediated checkpoint activation and cell-cycle arrest. Cells are thereby able to repair DNA damage alleviating replication stress and genomic instability. Chk1 pathway inhibition results in DNA damage accumulation and thus increased ATM auto-phosphorylation, which mediates apoptosis of cells with incompletely replicated DNA (d'Adda di Fagagna, 2008, Sarmento et al., 2015). These findings demonstrate that ATR-Chk1 signaling is crucial for the prevention of DNA damage-induced cell death associated with increased ATM phosphorylation/activation.
Author Manuscript Author Manuscript
CDC6 is an essential regulator of DNA replication in eukaryotic cells and its bestcharacterized function is pre-replicative complex assembly at origins of replication during G1 phase (Borlado and Mendez, 2008). Furthermore, CDC6 overexpression during G2 phase blocks mitotic entry by activating Chk1 (Clay-Farrace et al., 2003, Yoshida et al., 2010) which inhibits G2/M progression. CDC6 is required for Chk1 activation upon replication inhibition (Oehlmann et al., 2004) and human CDC6 interacts with ATR promoting activation of the replication checkpoint (Yoshida et al., 2010). Consequently, CDC6 decreases genomic instability which is vital for cancer cell survival. CDC6 can also manifest oncogenic activities via regulation of DNA replication and repression of tumor suppressors (Gonzalez et al., 2006). Targeting ATR-Chk1 signaling increases the sensitivity to treatment with DNA-damaging agents (Bartucci et al., 2012) making this approach particularly attractive for development of cancer therapies. Notably, Chk1 knockdown increases the sensitivity of PCa stem cells to radiotherapy through increased DNA damage (Fokas et al., 2012). AZD7762, a Chk1/2 inhibitor, synergizes with DNA-damaging agents and radiation to induce apoptosis via DNA double strand breaks mediated by ATM activation in many cell types (Mitchell et al., 2010, Sausville et al., 2014). Furthermore, Brooks et al found that Chk1 inhibition can selectively induce apoptosis in melanoma cells in proportion to the level of endogenous DNA damage related to replicative stress without further induction of DNA damage by chemotherapy (Brooks et al., 2013).
Cell Rep. Author manuscript; available in PMC 2017 March 14.
Karanika et al.
Page 4
Author Manuscript
Taking the results of these studies into account, a potential approach to treatment of mCRPC is inhibition of more than one level of a specific DDR signaling cascade with the goal of completely abolishing a specific signaling pathway. This approach would take advantage of the fact that cancer cells eventually accumulate more DNA damage than normal cells do, eluding adverse effects of chemotherapy. Targeting the ATR-Chk1 pathway at multiple levels to inhibit the repair of DNA damage induced by replication stress in cancer cells may represent an effective strategy for more complete DDR pathway inhibition. Because CDC6 is an androgen receptor (AR) target gene (Jin and Fondell, 2009, Bai et al., 2005), and is also involved with ATR-Chk1 signaling this is a particularly intriguing strategy for AR-positive PCa. In addition, this approach may be effective under conditions of wild-type p53 which is involved in multiple DDR pathways and can mitigate the response to some DNA-damaging agents (Ma et al., 2012).
Author Manuscript
The aim of the present study was to determine the role of CDC6 in regulation of ATR-Chk1 signaling and to test combined inhibition of AR and Chk1 signaling as a therapeutic approach for AR-positive mCRPC. Through this study we also aimed to introduce a therapeutic approach that effectively targets a DDR pathway that promotes sufficient DNA damage accumulation in PCa cells to induce cell death, regardless of p53 status.
Results CDC6 is induced during PCa progression and is positively correlated with AR expression
Author Manuscript
We first evaluated the CDC6 expression and phosphorylation, and analyzed its correlation with AR expression in normal human prostates, primary and metastatic prostate tumor specimens. In our immunohistochemistry (IHC) analysis, we found that AR expression (P=0.0057), and CDC6 expression (P
