Mammalian cells are continuously being bombarded by endogenous and exogenous forces that jeopardize the integrity of DNA. In response to DNA damage, a conserved network of signaling pathways known as the DNA damage response (DDR) is activated to maintain cell viability and genome stability (Rhind and Russell, 2000). Prostate cancer (PCa) remains one of the leading causes of death among men of all races (cdc.gov), as castration-resistant prostate cancer (CRPC) is currently incurable. Recently, the DDR has been a focus of PCa research since the androgen receptor (AR), a major driver of PCa, modulates the transcription of DDR genes and DNA repair (Polkinghorn et al., 2013; Goodwin et al., 2013; Jividen et al., 2018). We have previously shown that checkpoint kinase 2 (CHK2) negatively regulates androgen sensitivity and PCa cell growth (Ta et al., 2015).

CHK2 is a serine/threonine protein kinase that plays a crucial role in sensing DNA damage and initiating the DDR, which is comprised of cell cycle arrest, DNA repair, and apoptosis (Matsuoka et al., 1998). CHK2 consists of an amino-terminal SQ/TQ cluster domain (SCD) where threonine 68 serves as a substrate for phosphorylation by ataxia-telangectasia mutated (ATM) kinase Kim et al., 1999; a carboxy-terminal kinase domain (KD) and nuclear localization sequence Ahn et al., 2004; and a central forkhead-associated domain (FHA) that provides an interface for interactions with phosphorylated proteins (Li et al., 2002). Currently, there are approximately 24 CHK2 substrates in human cells that have been experimentally validated, including polo-like kinase 1 (PLK1), promyelocytic leukemia protein (PML), E2F1, p53, and cell division cycle 25C (CDC25C) (García-Limones et al., 2016). These studies show that one mechanism CHK2 utilizes to affect cellular function is through direct protein-protein interactions.

CHK2 association with PLK1 leads to its localization at centrosomes where it regulates mitotic entry (Tsvetkov et al., 2003). CHK2 autophosphorylation and activation are regulated by the tumor suppressor PML within nuclear matrix-associated structures called PML-nuclear bodies, which are nuclear matrix-associated structures (Yang et al., 2002). Binding to PML keeps CHK2 in an inactive state within these PML-nuclear bodies. In return, activated CHK2 can phosphorylate PML on S117 and induce PML-mediated apoptosis. CHK2 can also modify the transcription of apoptotic genes through direct binding and S364 phosphorylation of the E2F1 transcription factor in response to DNA damage, thereby stabilizing E2F1 and activating gene transcription (Stevens et al., 2003). CHK2 also regulates apoptosis through p53 phosphorylation, and promotion of p53-mediated cell death (Hirao et al., 2000). The interaction with the core domain of p53 induces an allosteric change in CHK2 which permits p53 S20 phosphorylation (Bartek and Lukas, 2003). Moreover, CHK2 modulates CDC25C localization by associating with and phosphorylating CDC25 on S216, which creates a binding site for 14-3-3 proteins (Peng et al., 1997). 14-3-3 proteins in turn sequester CDC25C in the cytoplasm and block the G2/M transition since cyclin dependent kinase 1 (CDK1) cannot be activated. Finally, our group has shown that CHK2 co-immunoprecipitated with AR in PCa cells and regulated growth, suggesting that AR may be a novel substrate of CHK2 (Ta et al., 2015). Thus, given the importance of CHK2 and AR to the DDR and prostate cancer growth, a full understanding of the functional consequences of the CHK2–AR interaction is required, with the hope of possible clinical applications towards CRPC.

Here, we uncovered novel molecular interactions between CHK2 and AR that provide mechanistic insight into our observation that CHK2 negatively regulates prostate cancer growth. We demonstrate that AR directly binds CHK2, and that this interaction increases with ionizing radiation (IR). The IR-induced increase in AR – CHK2 interaction requires AR phosphorylation on both S81 and S308 and CHK2 kinase activity, which is impaired in PCa associated CHK2 mutations. Furthermore, these CHK2 mutants exhibit diminished effects on prostate cancer cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, clonogenic survival, and resolution of DNA double strand breaks (DSBs) following IR, while altering the kinetics of IR-induced γH2AX foci.

PCa is the most frequently diagnosed cancer and the second leading cause of cancer death among American men, with approximately 88 men dying from PCa every day (pcf.org). While androgen deprivation therapy (ADT) is effective initially, most patients will relapse and develop incurable CRPC. Recently, there has been an emphasis on understanding the link between the DDR and AR, since radiation is a standard of care for locally advanced PCa where the AR is a major driver, and PARP inhibitors may be efficacious in CRPC patients with mutations in DDR genes (Polkinghorn et al., 2013; Goodwin et al., 2013; Ta et al., 2015; Mateo et al., 2015; Li et al., 2014; Yin et al., 2017a).

Our study identifies AR as a direct interacting protein with CHK2 in PCa cells. Several studies elucidated the role of DDR protein-AR interactions in modulating AR transcriptional activity. PARP-1 was recruited to AR binding sites, enhancing AR occupancy and transcriptional function (Schiewer et al., 2012). Tandem mass spectroscopy analysis identified Ku70 and Ku80 as direct AR-interacting proteins that positively regulate AR transactivation (Mayeur et al., 2005). Furthermore, BRCA1 physically interacted with the DNA-binding domain (DBD) of AR to enhance AR transactivation and induce androgen-mediated cell death through p21 expression (Yeh et al., 2000). In contrast, the association of the LBD of AR with hRad9, a crucial member of the checkpoint Rad family, suppressed AR transactivation by preventing the androgen-induced interaction between the n-terminus and c-terminus of AR (Wang et al., 2004). Other groups reported non-genomic effects as a result of DDR protein-AR interactions. Mediator of DNA damage checkpoint protein 1 (MDC1), an essential player in the Intra-S phase and G2/M checkpoints, physically associated with FL-AR and AR^V7 to negatively regulate PCa cell growth and migration (Wang et al., 2015). Yin and colleagues, on the other hand, showed that increased clonogenic survival following IR was a consequence of DNA-PKc directly complexing with both FL-AR and AR^V5-7, with radiation increasing these interactions and enzalutamide blocking the association with FL-AR but not AR^V5-7 (Yin et al., 2017b). These data support a model where AR is integrated in the DDR, interfacing at multiple points.

We show that the direct association of CHK2 and AR by Far western requires phosphorylation of AR on S308. Since proteins containing FHA domains bind phosphoproteins (Li et al., 2002), we hypothesize that AR interacts with CHK2 through the CHK2 FHA domain. In support of this, the Zhao lab determined that AR physically associated with the FHA domain of another critical DDR member, MDC1 (Wang et al., 2015). Expression of truncation mutants of different MDC1 domains in LNCaP cells led to the discovery that AR only co-immunoprecipitated with MDC1 mutants containing the FHA domain in the absence and presence of dihydrotestosterone. Their results indicated that the FHA domain of MDC1 mediated the interaction with AR.

Here we report that AR phosphorylation on S81 and S308 is required for the IR induction of AR – CHK2 binding by co-IP. Interestingly neither of these phosphorylation sites were altered by IR. AR S81 and S308 can both be phosphorylated by CDK1, which is downstream of canonical CHK2 signaling (Chen et al., 2012; Koryakina et al., 2015), and was the motivation for examining these sites in response to IR. The prediction is that IR would lead to a decrease in S81 and S308 phosphorylation. However, our previous studies demonstrated that S81 is predominantly phosphorylated by CDK9, and thus is more indicative of AR transcriptional activity (Gordon et al., 2010). We also found that S308 phosphorylation was restricted to late G2 and M phase of the cell cycle (Koryakina et al., 2015). CDK9 phosphorylation of S81 and the restriction of S308 phosphorylation to G2/M likely accounts for the lack of significant change in these phosphorylation sites in response to IR. The disconnect between these sites being required for the IR induced increase in AR – CHK2 association but not being regulated by IR suggests that the hormone induced activation state of the AR is a critical determinant in the IR induced increase in AR – CHK2 association. We observed differences when testing the AR – CHK2 association by Far western and co-IP. The direct protein-protein interaction is only impacted by loss of S308 phosphorylation, however, basal co-IP of AR and CHK2 is not affected by AR phosphorylation. Only the IR induced AR – CHK2 association is impacted by loss of AR phosphorylation on S81 and S308. This suggests that additional proteins present in cells may assist and participate in the AR – CHK2 interaction.

We found that CHK2 variants with diminished kinase activity impaired the IR-induced increase in AR – CHK2 interaction but did not completely block the interaction. As with AR phosphorylation, there were differences when the CHK2 mutant association with AR was examined by Far western or co-IP suggesting a more complicated dynamic of AR – CHK2 association in cells. The PCa associated CHK2 mutants also exhibited a reduced inhibition of cell growth. CHK2-depleted cells re-expressing CHK2 variants exhibited an approximate 2–3-fold reduction in growth inhibition in response to hormone when compared to cells re-expressing wtCHK2. Moreover, the fold change in growth suppression between wtCHK2 and CHK2 variants was greater in androgen-dependent LNCaP cells than in castration-resistant C4-2 and Rv1 cells, suggesting that in hormone sensitive PCa CHK2 variants may play a larger role in regulating growth. This raises the possibility that the CHK2 mutants found in PCa with diminished AR binding are selected for, decreasing CHK2 suppression of AR activity and PCa cell growth. Berge and colleagues discovered numerous CHK2 splice variants in breast cancer tissue, where all variants were co-expressed with wtCHK2 (Berge et al., 2010). Furthermore, several of these variants reduced kinase activity when simultaneously expressed with wtCHK2 and displayed a dominant negative effect on wtCHK2. The impact of the CHK2 variants found in PCa on wtCHK2 function has not yet been fully explored.

In our experiments examining AR – CHK2 binding, ERK was used as a positive control for a protein that interacts with CHK2 (Dai et al., 2011). Interestingly, we observed a significant increase in CHK2–ERK association with IR. This is consistent with IR increasing CHK2 T68 phosphorylation, which is required for the CHK2–ERK interaction. These data point to a potentially larger role for CHK2 beyond canonical DDR and cell cycle checkpoint signaling; consistent with this notion CHK2 has been implicated in diverse cellular processes (Zannini et al., 2014; Choi et al., 2014; Choudhuri et al., 2007). MEK inhibition is effective in lung tumors with ATM mutations where CHK2 is inactive (Smida et al., 2016) providing further support that CHK2 negatively regulates ERK. Negative regulation of both the AR and ERK by CHK2 suggests the hypothesis that CHK2 may serve as a general negative regulator of mitogenic signals in response to IR.

Multiple studies indicate that the AR is a critical regulator of genes in the DDR (Polkinghorn et al., 2013; Goodwin et al., 2013). Reports by others demonstrate that androgen and IR increased DNAPK, XRCC2, and XRCC3 (Goodwin et al., 2013). This concept was supported by more global analysis of transcripts demonstrating androgen regulation of DDR genes (Polkinghorn et al., 2013). Another global analysis identified a different set of DDR genes are regulated by AR (Jividen et al., 2018). Our test by RT-qPCR of ten different DDR genes found only a modest induction by IR or androgen treatment in two DDR genes, DNAPK and RAD54. The discrepancy in the DDR gene subsets regulated by the AR in these studies and ours may be system dependent. However, it also indicates that the dogma of AR regulating DDR genes needs further examination. Our data indicate that CHK2 knockdown increases DNAPK and RAD54 transcript levels is consistent with our earlier observation that CHK2 knockdown led to the increase in the transcripts of canonical AR target genes (Ta et al., 2015). This leads to the hypothesis that CHK2 binding to the AR suppresses AR transcription of DDR genes enabling cells to turn off the DDR following DNA repair.

We observed radiation resistance in CHK2 knockdown cells. We also observed an increase in γH2AX signal when CHK2 was knocked down, and a decrease in DNA breaks as measured by the comet assay under similar conditions. However, when 53BP1 signal was measured to assess DSB repair we found no significant differences when CHK2 was knocked down. These conflicting results of γH2AX and 53BP1 signal may be explained by phosphorylation of H2AX in response to transcription induced DNA breaks (Singh et al., 2015; Bunch et al., 2015; Ji et al., 2019). These incongruous results may also be due to competing effects of CHK2 as both a regulator of the cell cycle and apoptosis (Zannini et al., 2014).

The data reported herein along with our previous work (Ta et al., 2015) indicate that CHK2 acts as a tumor suppressor in PCa, either through loss of expression or mutation. This raises the concern that CHK2 antagonists in clinical development may paradoxically lead to enhanced PCa growth and resistance to IR. However, it is important to note that we have predominately used an RNAi/overexpression approach. Our RNAi approach is more similar to the CHK2 variants in PCa that have reduced kinase activity. It is important to consider that pharmacologic inhibition is different than inhibition by RNAi (Jensen et al., 2016). A pharmacologic approach that provides a sudden and complete inhibition of CHK2 kinase activity may impact PCa differently than our RNAi approach, especially when combined with IR or AR antagonists. However, our work and that in the literature also suggests that approaches downstream of CHK2 may be more straightforward than targeting CHK2.

In this study, we presented data that provides mechanistic insight into our observation that CHK2 negatively regulates PCa growth. We demonstrated that AR directly bound CHK2, and that IR elevated the AR – CHK2 interaction. Not only did these AR – CHK2 protein complexes require AR phosphorylation, but CHK2 kinase activity was also necessary. Furthermore, these CHK2 mutants found in PCa exhibited a diminished effect on restricting prostate cancer cell growth. We observed that knockdown of CHK2 led to an increase in PCa cell survival and resolution of DNA DSBs in response to IR. This suggests that the deregulation of CHK2 in PCa can confer resistance to radiation. In a previous study, we showed that CHK2 knockdown hypersensitized PCa cells to castrate levels of androgen and increased AR transcriptional activity on both androgen-activated and androgen-repressed genes (Ta et al., 2015). As part of a feedback loop, AR transcriptionally represses CHK2 levels. Thus, these data along with our previously published results suggest the following model (Figure 7). Wild-type CHK2 maintains homeostasis through mediating the balance between DNA repair and cell death, antagonizes the AR through direct binding and inhibition of transcription of AR target genes while AR transcriptionally represses CHK2. This feedback loop enables AR repression of CHK2 to turn off the DDR, while CHK2 negatively regulates AR to dampen growth during repair of damaged DNA. CHK2 mutation or loss of expression in PCa leads to increased AR transcriptional activity, possibly of DDR genes, and survival in response to DNA damage, all leading to a more aggressive cancer. Collectively, the work provides a foundation for the continued study of AR – CHK2 interactions and functional consequences to benefit PCa therapies.

Figure 7

Download asset Open asset

Source data are available via the Open Science Framework (https://osf.io/2bx5q/).

1. 1. Gioeli D

   (2020) Open Science Framework

   AR_CHK2_Gioeli.

   https://osf.io/2bx5q/

1. 1. Ahn J
   2. Urist M
   3. Prives C

   (2004) The Chk2 protein kinase

   DNA Repair 3:1039–1047.

   https://doi.org/10.1016/j.dnarep.2004.03.033

   • PubMed
   • Google Scholar
2. 1. Bartek J
   2. Lukas J

   (2003) Chk1 and Chk2 kinases in checkpoint control and Cancer

   Cancer Cell 3:421–429.

   https://doi.org/10.1016/S1535-6108(03)00110-7

   • PubMed
   • Google Scholar
3. 1. Berge EO
   2. Staalesen V
   3. Straume AH
   4. Lillehaug JR
   5. Lønning PE

   (2010) Chk2 splice variants express a dominant-negative effect on the wild-type Chk2 kinase activity

   Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1803:386–395.

   https://doi.org/10.1016/j.bbamcr.2010.01.005

   • Google Scholar
4. 1. Bonner WM
   2. Redon CE
   3. Dickey JS
   4. Nakamura AJ
   5. Sedelnikova OA
   6. Solier S
   7. Pommier Y

   (2008) γH2AX and cancer

   Nature Reviews Cancer 8:957–967.

   https://doi.org/10.1038/nrc2523

   • Google Scholar
5. 1. Bunch H
   2. Lawney BP
   3. Lin YF
   4. Asaithamby A
   5. Murshid A
   6. Wang YE
   7. Chen BP
   8. Calderwood SK

   (2015) Transcriptional elongation requires DNA break-induced signalling

   Nature Communications 6:10191.

   https://doi.org/10.1038/ncomms10191

   • PubMed
   • Google Scholar
6. 1. Bustin SA

   (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

   Journal of Molecular Endocrinology 25:169–193.

   https://doi.org/10.1677/jme.0.0250169

   • PubMed
   • Google Scholar
7. 1. Chatterjee P

   (2019) Supraphysiological androgens suppress prostate Cancer growth through androgen receptor-mediated DNA damage

   The Journal of Clinical Investigation 130:JCI127613.

   https://doi.org/10.1172/JCI127613

   • Google Scholar
8. 1. Chen S
   2. Xu Y
   3. Yuan X
   4. Bubley GJ
   5. Balk SP

   (2006) Androgen receptor phosphorylation and stabilization in prostate Cancer by cyclin-dependent kinase 1

   PNAS 103:15969–15974.

   https://doi.org/10.1073/pnas.0604193103

   • PubMed
   • Google Scholar
9. 1. Chen S
   2. Gulla S
   3. Cai C
   4. Balk SP

   (2012) Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation

   Journal of Biological Chemistry 287:8571–8583.

   https://doi.org/10.1074/jbc.M111.325290

   • PubMed
   • Google Scholar
10. 1. Choi HH
    2. Choi HK
    3. Jung SY
    4. Hyle J
    5. Kim BJ
    6. Yoon K
    7. Cho EJ
    8. Youn HD
    9. Lahti JM
    10. Qin J
    11. Kim ST

    (2014) CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110)

    Oncogene 33:108–115.

    https://doi.org/10.1038/onc.2012.535

    • PubMed
    • Google Scholar
11. 1. Choudhuri T
    2. Verma SC
    3. Lan K
    4. Murakami M
    5. Robertson ES

    (2007) The ATM/ATR signaling effector Chk2 is targeted by Epstein-Barr virus nuclear antigen 3C to release the G2/M cell cycle block

    Journal of Virology 81:6718–6730.

    https://doi.org/10.1128/JVI.00053-07

    • PubMed
    • Google Scholar
12. 1. Collins AR

    (2004) The comet assay for DNA damage and repair: principles, applications, and limitations

    Molecular Biotechnology 26:249–261.

    https://doi.org/10.1385/MB:26:3:249

    • PubMed
    • Google Scholar
13. 1. Dai B
    2. Zhao XF
    3. Mazan-Mamczarz K
    4. Hagner P
    5. Corl S
    6. Bahassi elM
    7. Lu S
    8. Stambrook PJ
    9. Shapiro P
    10. Gartenhaus RB

    (2011) Functional and molecular interactions between ERK and CHK2 in diffuse large B-cell lymphoma

    Nature Communications 2:402.

    https://doi.org/10.1038/ncomms1404

    • PubMed
    • Google Scholar
14. 1. García-Limones C
    2. Lara-Chica M
    3. Jiménez-Jiménez C
    4. Pérez M
    5. Moreno P
    6. Muñoz E
    7. Calzado MA

    (2016) CHK2 stability is regulated by the E3 ubiquitin ligase SIAH2

    Oncogene 35:4289–4301.

    https://doi.org/10.1038/onc.2015.495

    • PubMed
    • Google Scholar
15. 1. Gioeli D
    2. Ficarro SB
    3. Kwiek JJ
    4. Aaronson D
    5. Hancock M
    6. Catling AD
    7. White FM
    8. Christian RE
    9. Settlage RE
    10. Shabanowitz J
    11. Hunt DF
    12. Weber MJ

    (2002) Androgen receptor phosphorylation regulation and identification of the phosphorylation sites

    The Journal of Biological Chemistry 277:29304–29314.

    https://doi.org/10.1074/jbc.M204131200

    • PubMed
    • Google Scholar
16. 1. Goodwin JF
    2. Schiewer MJ
    3. Dean JL
    4. Schrecengost RS
    5. de Leeuw R
    6. Han S
    7. Ma T
    8. Den RB
    9. Dicker AP
    10. Feng FY
    11. Knudsen KE

    (2013) A hormone-DNA repair circuit governs the response to genotoxic insult

    Cancer Discovery 3:1254–1271.

    https://doi.org/10.1158/2159-8290.CD-13-0108

    • PubMed
    • Google Scholar
17. 1. Gordon V
    2. Bhadel S
    3. Wunderlich W
    4. Zhang J
    5. Ficarro SB
    6. Mollah SA
    7. Shabanowitz J
    8. Hunt DF
    9. Xenarios I
    10. Hahn WC
    11. Conaway M
    12. Carey MF
    13. Gioeli D

    (2010) CDK9 regulates AR promoter selectivity and cell growth through serine 81 phosphorylation

    Molecular Endocrinology 24:2267–2280.

    https://doi.org/10.1210/me.2010-0238

    • PubMed
    • Google Scholar
18. 1. Gordon MA
    2. D'Amato NC
    3. Gu H
    4. Babbs B
    5. Wulfkuhle J
    6. Petricoin EF
    7. Gallagher I
    8. Dong T
    9. Torkko K
    10. Liu B
    11. Elias A
    12. Richer JK

    (2017) Synergy between androgen receptor antagonism and inhibition of mTOR and HER2 in breast Cancer

    Molecular Cancer Therapeutics 16:1389–1400.

    https://doi.org/10.1158/1535-7163.MCT-17-0111

    • Google Scholar
19. 1. Haffner MC
    2. Aryee MJ
    3. Toubaji A
    4. Esopi DM
    5. Albadine R
    6. Gurel B
    7. Isaacs WB
    8. Bova GS
    9. Liu W
    10. Xu J
    11. Meeker AK
    12. Netto G
    13. De Marzo AM
    14. Nelson WG
    15. Yegnasubramanian S

    (2010) Androgen-induced TOP2B-mediated double-strand breaks and prostate Cancer gene rearrangements

    Nature Genetics 42:668–675.

    https://doi.org/10.1038/ng.613

    • PubMed
    • Google Scholar
20. 1. Higashiguchi M
    2. Nagatomo I
    3. Kijima T
    4. Morimura O
    5. Miyake K
    6. Minami T
    7. Koyama S
    8. Hirata H
    9. Iwahori K
    10. Takimoto T
    11. Takeda Y
    12. Kida H
    13. Kumanogoh A

    (2016) Clarifying the biological significance of the CHK2 K373E somatic mutation discovered in the Cancer genome atlas database

    FEBS Letters 590:4275–4286.

    https://doi.org/10.1002/1873-3468.12449

    • PubMed
    • Google Scholar
21. 1. Hirao A
    2. Kong YY
    3. Matsuoka S
    4. Wakeham A
    5. Ruland J
    6. Yoshida H
    7. Liu D
    8. Elledge SJ
    9. Mak TW

    (2000) DNA damage-induced activation of p53 by the checkpoint kinase Chk2

    Science 287:1824–1827.

    https://doi.org/10.1126/science.287.5459.1824

    • PubMed
    • Google Scholar
22. 1. Jensen KJ
    2. Moyer CB
    3. Janes KA

    (2016) Network architecture predisposes an enzyme to either pharmacologic or genetic targeting

    Cell Systems 2:112–121.

    https://doi.org/10.1016/j.cels.2016.01.012

    • PubMed
    • Google Scholar
23. 1. Ji JH
    2. Min S
    3. Chae S
    4. Ha GH
    5. Kim Y
    6. Park YJ
    7. Lee CW
    8. Cho H

    (2019) De novo phosphorylation of H2AX by WSTF regulates transcription-coupled homologous recombination repair

    Nucleic Acids Research 47:6299–6314.

    https://doi.org/10.1093/nar/gkz309

    • PubMed
    • Google Scholar
24. 1. Jividen K
    2. Kedzierska KZ
    3. Yang CS
    4. Szlachta K
    5. Ratan A
    6. Paschal BM

    (2018) Genomic analysis of DNA repair genes and androgen signaling in prostate Cancer

    BMC Cancer 18:960.

    https://doi.org/10.1186/s12885-018-4848-x

    • PubMed
    • Google Scholar
25. 1. Kim ST
    2. Lim DS
    3. Canman CE
    4. Kastan MB

    (1999) Substrate specificities and identification of putative substrates of ATM kinase family members

    Journal of Biological Chemistry 274:37538–37543.

    https://doi.org/10.1074/jbc.274.53.37538

    • PubMed
    • Google Scholar
26. 1. Koryakina Y
    2. Knudsen KE
    3. Gioeli D

    (2015) Cell-cycle-dependent regulation of androgen receptor function

    Endocrine-Related Cancer 22:249–264.

    https://doi.org/10.1530/ERC-14-0549

    • PubMed
    • Google Scholar
27. 1. Li J
    2. Williams BL
    3. Haire LF
    4. Goldberg M
    5. Wilker E
    6. Durocher D
    7. Yaffe MB
    8. Jackson SP
    9. Smerdon SJ

    (2002) Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2

    Molecular Cell 9:1045–1054.

    https://doi.org/10.1016/S1097-2765(02)00527-0

    • PubMed
    • Google Scholar
28. 1. Li L
    2. Chang W
    3. Yang G
    4. Ren C
    5. Park S
    6. Karantanos T
    7. Karanika S
    8. Wang J
    9. Yin J
    10. Shah PK
    11. Takahiro H
    12. Dobashi M
    13. Zhang W
    14. Efstathiou E
    15. Maity SN
    16. Aparicio AM
    17. Li Ning Tapia EM
    18. Troncoso P
    19. Broom B
    20. Xiao L
    21. Lee HS
    22. Lee JS
    23. Corn PG
    24. Navone N
    25. Thompson TC

    (2014) Targeting poly(ADP-ribose) polymerase and the c-Myb-regulated DNA damage response pathway in castration-resistant prostate Cancer

    Science Signaling 7:ra47.

    https://doi.org/10.1126/scisignal.2005070

    • PubMed
    • Google Scholar
29. 1. Mateo J
    2. Carreira S
    3. Sandhu S
    4. Miranda S
    5. Mossop H
    6. Perez-Lopez R
    7. Nava Rodrigues D
    8. Robinson D
    9. Omlin A
    10. Tunariu N
    11. Boysen G
    12. Porta N
    13. Flohr P
    14. Gillman A
    15. Figueiredo I
    16. Paulding C
    17. Seed G
    18. Jain S
    19. Ralph C
    20. Protheroe A
    21. Hussain S
    22. Jones R
    23. Elliott T
    24. McGovern U
    25. Bianchini D
    26. Goodall J
    27. Zafeiriou Z
    28. Williamson CT
    29. Ferraldeschi R
    30. Riisnaes R
    31. Ebbs B
    32. Fowler G
    33. Roda D
    34. Yuan W
    35. Wu YM
    36. Cao X
    37. Brough R
    38. Pemberton H
    39. A'Hern R
    40. Swain A
    41. Kunju LP
    42. Eeles R
    43. Attard G
    44. Lord CJ
    45. Ashworth A
    46. Rubin MA
    47. Knudsen KE
    48. Feng FY
    49. Chinnaiyan AM
    50. Hall E
    51. de Bono JS

    (2015) DNA-Repair defects and olaparib in metastatic prostate Cancer

    New England Journal of Medicine 373:1697–1708.

    https://doi.org/10.1056/NEJMoa1506859

    • PubMed
    • Google Scholar
30. 1. Matsuoka S
    2. Huang M
    3. Elledge SJ

    (1998) Linkage of ATM to cell cycle regulation by the Chk2 protein kinase

    Science 282:1893–1897.

    https://doi.org/10.1126/science.282.5395.1893

    • PubMed
    • Google Scholar
31. 1. Mayeur GL
    2. Kung WJ
    3. Martinez A
    4. Izumiya C
    5. Chen DJ
    6. Kung HJ

    (2005) Ku is a novel transcriptional recycling coactivator of the androgen receptor in prostate Cancer cells

    Journal of Biological Chemistry 280:10827–10833.

    https://doi.org/10.1074/jbc.M413336200

    • PubMed
    • Google Scholar
32. 1. Peng CY
    2. Graves PR
    3. Thoma RS
    4. Wu Z
    5. Shaw AS
    6. Piwnica-Worms H

    (1997) Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216

    Science 277:1501–1505.

    https://doi.org/10.1126/science.277.5331.1501

    • PubMed
    • Google Scholar
33. 1. Polkinghorn WR
    2. Parker JS
    3. Lee MX
    4. Kass EM
    5. Spratt DE
    6. Iaquinta PJ
    7. Arora VK
    8. Yen WF
    9. Cai L
    10. Zheng D
    11. Carver BS
    12. Chen Y
    13. Watson PA
    14. Shah NP
    15. Fujisawa S
    16. Goglia AG
    17. Gopalan A
    18. Hieronymus H
    19. Wongvipat J
    20. Scardino PT
    21. Zelefsky MJ
    22. Jasin M
    23. Chaudhuri J
    24. Powell SN
    25. Sawyers CL

    (2013) Androgen receptor signaling regulates DNA repair in prostate cancers

    Cancer Discovery 3:1245–1253.

    https://doi.org/10.1158/2159-8290.CD-13-0172

    • PubMed
    • Google Scholar
34. 1. Prickett TD
    2. Ninomiya-Tsuji J
    3. Broglie P
    4. Muratore-Schroeder TL
    5. Shabanowitz J
    6. Hunt DF
    7. Brautigan DL

    (2008) TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappaB

    Journal of Biological Chemistry 283:19245–19254.

    https://doi.org/10.1074/jbc.M800943200

    • PubMed
    • Google Scholar
35. 1. Rhind N
    2. Russell P

    (2000)

    Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathways

    Journal of Cell Science 113:3889–3896.

    • PubMed
    • Google Scholar
36. 1. Schiewer MJ
    2. Goodwin JF
    3. Han S
    4. Brenner JC
    5. Augello MA
    6. Dean JL
    7. Liu F
    8. Planck JL
    9. Ravindranathan P
    10. Chinnaiyan AM
    11. McCue P
    12. Gomella LG
    13. Raj GV
    14. Dicker AP
    15. Brody JR
    16. Pascal JM
    17. Centenera MM
    18. Butler LM
    19. Tilley WD
    20. Feng FY
    21. Knudsen KE

    (2012) Dual roles of PARP-1 promote Cancer growth and progression

    Cancer Discovery 2:1134–1149.

    https://doi.org/10.1158/2159-8290.CD-12-0120

    • PubMed
    • Google Scholar
37. 1. Singh I
    2. Ozturk N
    3. Cordero J
    4. Mehta A
    5. Hasan D
    6. Cosentino C
    7. Sebastian C
    8. Krüger M
    9. Looso M
    10. Carraro G
    11. Bellusci S
    12. Seeger W
    13. Braun T
    14. Mostoslavsky R
    15. Barreto G

    (2015) High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

    Cell Research 25:837–850.

    https://doi.org/10.1038/cr.2015.67

    • PubMed
    • Google Scholar
38. 1. Smida M
    2. Fece de la Cruz F
    3. Kerzendorfer C
    4. Uras IZ
    5. Mair B
    6. Mazouzi A
    7. Suchankova T
    8. Konopka T
    9. Katz AM
    10. Paz K
    11. Nagy-Bojarszky K
    12. Muellner MK
    13. Bago-Horvath Z
    14. Haura EB
    15. Loizou JI
    16. Nijman SM

    (2016) MEK inhibitors block growth of lung tumours with mutations in ataxia-telangiectasia mutated

    Nature Communications 7:13701.

    https://doi.org/10.1038/ncomms13701

    • PubMed
    • Google Scholar
39. 1. Stevens C
    2. Smith L
    3. La Thangue NB

    (2003) Chk2 activates E2F-1 in response to DNA damage

    Nature Cell Biology 5:401–409.

    https://doi.org/10.1038/ncb974

    • PubMed
    • Google Scholar
40. 1. Ta HQ
    2. Ivey ML
    3. Frierson HF
    4. Conaway MR
    5. Dziegielewski J
    6. Larner JM
    7. Gioeli D

    (2015) Checkpoint kinase 2 negatively regulates androgen sensitivity and prostate Cancer cell growth

    Cancer Research 75:5093–5105.

    https://doi.org/10.1158/0008-5472.CAN-15-0224

    • PubMed
    • Google Scholar
41. 1. Tsvetkov L
    2. Xu X
    3. Li J
    4. Stern DF

    (2003) Polo-like kinase 1 and Chk2 interact and co-localize to centrosomes and the midbody

    Journal of Biological Chemistry 278:8468–8475.

    https://doi.org/10.1074/jbc.M211202200

    • PubMed
    • Google Scholar
42. 1. Vignard J
    2. Mirey G
    3. Salles B

    (2013) Ionizing-radiation induced DNA double-strand breaks: a direct and indirect lighting up

    Radiotherapy and Oncology 108:362–369.

    https://doi.org/10.1016/j.radonc.2013.06.013

    • PubMed
    • Google Scholar
43. 1. Wang L
    2. Hsu CL
    3. Ni J
    4. Wang PH
    5. Yeh S
    6. Keng P
    7. Chang C

    (2004) Human checkpoint protein hRad9 functions as a negative coregulator to repress androgen receptor transactivation in prostate Cancer cells

    Molecular and Cellular Biology 24:2202–2213.

    https://doi.org/10.1128/MCB.24.5.2202-2213.2004

    • PubMed
    • Google Scholar
44. 1. Wang C
    2. Sun H
    3. Zou R
    4. Zhou T
    5. Wang S
    6. Sun S
    7. Tong C
    8. Luo H
    9. Li Y
    10. Li Z
    11. Wang E
    12. Chen Y
    13. Cao L
    14. Li F
    15. Zhao Y

    (2015) MDC1 functionally identified as an androgen receptor co-activator participates in suppression of prostate Cancer

    Nucleic Acids Research 43:4893–4908.

    https://doi.org/10.1093/nar/gkv394

    • PubMed
    • Google Scholar
45. 1. Wasielewski M
    2. Hanifi-Moghaddam P
    3. Hollestelle A
    4. Merajver SD
    5. van den Ouweland A
    6. Klijn JG
    7. Ethier SP
    8. Schutte M

    (2009) Deleterious CHEK2 1100delc and L303X mutants identified among 38 human breast Cancer cell lines

    Breast Cancer Research and Treatment 113:285–291.

    https://doi.org/10.1007/s10549-008-9942-3

    • PubMed
    • Google Scholar
46. 1. Whitworth H
    2. Bhadel S
    3. Ivey M
    4. Conaway M
    5. Spencer A
    6. Hernan R
    7. Holemon H
    8. Gioeli D

    (2012) Identification of kinases regulating prostate Cancer cell growth using an RNAi phenotypic screen

    PLOS ONE 7:e38950.

    https://doi.org/10.1371/journal.pone.0038950

    • PubMed
    • Google Scholar
47. 1. Wong ML
    2. Medrano JF

    (2005) Real-time PCR for mRNA quantitation

    BioTechniques 39:75–85.

    https://doi.org/10.2144/05391RV01

    • PubMed
    • Google Scholar
48. 1. Wu Y
    2. Li Q
    3. Chen XZ

    (2007) Detecting protein-protein interactions by far western blotting

    Nature Protocols 2:3278–3284.

    https://doi.org/10.1038/nprot.2007.459

    • PubMed
    • Google Scholar
49. 1. Yang S
    2. Kuo C
    3. Bisi JE
    4. Kim MK

    (2002) PML-dependent apoptosis after DNA damage is regulated by the checkpoint kinase hCds1/Chk2

    Nature Cell Biology 4:865–870.

    https://doi.org/10.1038/ncb869

    • PubMed
    • Google Scholar
50. 1. Yeh S
    2. Hu YC
    3. Rahman M
    4. Lin HK
    5. Hsu CL
    6. Ting HJ
    7. Kang HY
    8. Chang C

    (2000) Increase of androgen-induced cell death and androgen receptor transactivation by BRCA1 in prostate Cancer cells

    PNAS 97:11256–11261.

    https://doi.org/10.1073/pnas.190353897

    • PubMed
    • Google Scholar
51. 1. Yin Y
    2. Shen Q
    3. Zhang P
    4. Tao R
    5. Chang W
    6. Li R
    7. Xie G
    8. Liu W
    9. Zhang L
    10. Kapoor P
    11. Song S
    12. Ajani J
    13. Mills GB
    14. Chen J
    15. Tao K
    16. Peng G

    (2017a)

    Chk1 inhibition potentiates the therapeutic efficacy of PARP inhibitor BMN673 in gastric Cancer

    American Journal of Cancer Research 7:473–483.

    • PubMed
    • Google Scholar
52. 1. Yin Y
    2. Li R
    3. Xu K
    4. Ding S
    5. Li J
    6. Baek G
    7. Ramanand SG
    8. Ding S
    9. Liu Z
    10. Gao Y
    11. Kanchwala MS
    12. Li X
    13. Hutchinson R
    14. Liu X
    15. Woldu SL
    16. Xing C
    17. Desai NB
    18. Feng FY
    19. Burma S
    20. de Bono JS
    21. Dehm SM
    22. Mani RS
    23. Chen BPC
    24. Raj GV

    (2017b) Androgen receptor variants mediate DNA repair after prostate Cancer irradiation

    Cancer Research 77:4745–4754.

    https://doi.org/10.1158/0008-5472.CAN-17-0164

    • PubMed
    • Google Scholar
53. 1. Zannini L
    2. Delia D
    3. Buscemi G

    (2014) CHK2 kinase in the DNA damage response and beyond

    Journal of Molecular Cell Biology 6:442–457.

    https://doi.org/10.1093/jmcb/mju045

    • PubMed
    • Google Scholar
54. 1. Zong H
    2. Chi Y
    3. Wang Y
    4. Yang Y
    5. Zhang L
    6. Chen H
    7. Jiang J
    8. Li Z
    9. Hong Y
    10. Wang H
    11. Yun X
    12. Gu J

    (2007) Cyclin D3/CDK11p58 complex is involved in the repression of androgen receptor

    Molecular and Cellular Biology 27:7125–7142.

    https://doi.org/10.1128/MCB.01753-06

    • PubMed
    • Google Scholar

Acceptance summary:

The manuscript presents novel and compelling data on the interaction between CHK2 and Androgen Receptor in prostate cancer cells, by which CHK2 suppresses prostate cancer cell growth. These data explain how CDK2 mutations in prostate cancer lead to increased androgen receptor activity and tumor cell survival in response to DNA damage induction.

Decision letter after peer review:

Thank you for submitting your article "Checkpoint kinase 2 regulates prostate cancer cell growth through physical interactions with the androgen receptor" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Wilbert Zwart as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jonathan Cooper as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Kavita Shah (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

In this study, Gioeli and colleagues build on their 2015 Cancer Research paper showing that AR transcriptionally represses CHK2 and its negative effects on PCa growth and androgen sensitivity, by investigating mechanisms underlying this CHK2/AR cross-talk in the specific context of radiation-induced signaling and DNA damage response. They propose that their findings indicate that wild-type CHK2 can sequester AR and prevent its transcriptional enhancement of DNA repair genes, which in turn confers PCa radioresistance and survival under DNA damage.

Overall this is an interesting and conceptually novel study. The authors convincingly show that AR and CHK2 interact physically, that IR transiently stabilizes this interaction, and that AR activity is required for this interaction. However, a number of issues need to be addressed in a revised manuscript:

Essential revisions:

1) Concerns were raised on the experimental design and interpretation of data in Figure 8 and Supplementary Figure 2. Assuming the model in Figure 9 is correct, then under IR treatment, CHK2 depletion should significantly decrease unrepaired DNA breaks by increasing the frequency/efficacy of DNA repair through AR activity. This should manifest, over time, in fewer unresolved γH2AX foci and lower %tail DNA/tail moments relative to the pLKO controls, indicative of radioresistance. Although the AR/CHK2 interaction peaks at 1 hour, this is not the optimal time frame in which to evaluate the levels of unrepaired DNA breaks which would compromise PCa growth and survival by triggering tumor-inhibitory responses to radiation. γH2AX typically appear as punctate foci 6-12 hours following DNA damaging stresses. The diffuse staining in Figure 8B indicates the timepoints of detection are prior to formation of DDR complexes at DSB sites or potentially technical issues (typically optimal detection of this epitope requires stringent washing and antibody incubation in a detergent-based buffer to minimize the degree of background seen here). Either issue calls the accuracy of foci quantitation into question.

2) The alkaline comet assay detects not just DNA double strand breaks but also DNA nicks and abasic sites (through initiation of base excision repair) which will abound following IR treatment. To assess whether these have been repaired properly or not, the assay should be repeated at later timepoints (12-18 hours) when the changes in %tail DNA and tail moments will better represent efficiency and extent of repair. The authors may find a far more striking difference between their control and CHK2-depleted cells, if they repeat the γH2Ax experiment at 12-18 hours following IR when the punctate foci (indicative of repair efficiency or lack thereof) will become more apparent. The difference in comet tail DNA metrics will also most clearly reflect unrepaired genotoxic DNA intermediates at a longer time point. The data, as they stand, do not support their model. Similarly, the relatively small changes in DNA PKc and RAD54 transcripts in Figure 7 do not convince of their assertion that CHK2 depletion increases PCa survival through AR-mediated transcription of DNA repair genes. Such transcript-level changes alone in these factors cannot be extrapolated to their functional effects on DNA repair. The increase in clonogenic survival 14 days post-IR in CHK2-depleted cells is fairly modest and does not necessarily support that CHK2 inhibits radioresistance (Figure 8A). As the authors show in Figure 6, LNCaP cells are generally more growth inhibited by wt CHK2 so the differences in Figure 8A may not reflect radioresistance but just a general growth advantage upon CHK2 loss.

3) It is unclear what PCa cell growth actually refers to in this study – is the reduction in cell numbers due to a proliferation arrest or cell death? The known CHK2 interactomes could affect either (or both) pathway. Some textual and/or experimental elaboration of this point is warranted, considering this is a central premise of the study.

4) The findings from this study taken in context with the previous 2015 study would suggest that the CHK2/AR interaction is mutually inhibitory, as the earlier study suggested that CHK2 is transcriptionally repressed by AR. However, this mutual repression and the notion of negative feedback regulation from CHK2 to AR is not discussed here, for instance in the model in Figure 9. This aspect should be addressed, at least textually, to provide a complete picture of this crosstalk in PCa.

5) "Kinase-impaired CHK2 variants, including the K373E variant associated with 4.2% of PCa, blocked IR-induced CHK2-AR interactions". This is a strong statement considering the blot in Figure 3B doesn't appear representative of the quantitation shown – it seems the variants reduce binding but certainly don't seem to block it completely.

6) It seems IR increases the CHK2/AR interaction by increasing CHK2 phosphorylation and therefore its activity, which is an interesting finding. However, both the experimental design and Figure 4 (which describes this effect) are hard to understand. For example, the blots on the left vs. the respective quantitation on the right are confusing. The graphs look different (- or + radiation of the V5-CHK2 cells) but the blots look very similar. It would help if Figure 4 included a schematic of the experimental design.

7) Statistical analyses and quantifications should be provided for all figure and supplementary panels, when appropriate (e.g. Figure 5A, Figure 8C, Supplementary Figure 2, Figure 6—figure supplement 1).

8) Did the authors check whether CHK2 directly phosphorylates AR, and whether this phosphorylation is required for their binding?

9) Although the authors show that CHK2 binding with AR peaks at 1 hour, it would be ideal if the others would have tested additional early time points (such as 30 min, 45 min, 1.5h) to exactly determine the peaking of signal. Are these data available?

10) As ARS81 is phosphorylated by CDK1, does CDK1 inhibition (using small molecule inhibitors) impact IR induced association of CHK2 and AR?

11) Please check whether the ERK1/2 control blots in Figure 6B (C42) vs. 6C (22Rv1) have been accidentally duplicated.

Essential revisions:

1) Concerns were raised on the experimental design and interpretation of data in Figure 8 and Supplementary Figure 2. Assuming the model in Figure 9 is correct, then under IR treatment, CHK2 depletion should significantly decrease unrepaired DNA breaks by increasing the frequency/efficacy of DNA repair through AR activity. This should manifest, over time, in fewer unresolved γH2AX foci and lower %tail DNA/tail moments relative to the pLKO controls, indicative of radioresistance. Although the AR/CHK2 interaction peaks at 1 hour, this is not the optimal time frame in which to evaluate the levels of unrepaired DNA breaks which would compromise PCa growth and survival by triggering tumor-inhibitory responses to radiation. γH2AX typically appear as punctate foci 6-12 hours following DNA damaging stresses. The diffuse staining in Figure 8B indicates the timepoints of detection are prior to formation of DDR complexes at DSB sites or potentially technical issues (typically optimal detection of this epitope requires stringent washing and antibody incubation in a detergent-based buffer to minimize the degree of background seen here). Either issue calls the accuracy of foci quantitation into question.

We thank the reviewers for suggesting that we look at later times for DNA damage and DNA repair activity. When we examine DNA damage using the comet assay we do in fact observe statistically significantly less DNA breaks with CHK2 knockdown at 24hrs. The comet assay shows less % DNA in tail, shorter DNA tail length, and lower tail moment with CHK2 knockdown. Interestingly, we observe more γH2AX foci early and quicker resolution of the foci with CHK2 depletion, suggesting a more rapid resolution of the DNA damage when CHK2 is knocked down. These data are now included in the resubmitted manuscript.

With regards to the kinetics of DNA repair following irradiation, data in the literature demonstrate that DSB repair has short half-life of 10 to 60 minutes depending on the type of DNA repair utilized (PMIDs: 1677379, 7841046, 10728683, 12643795, 17088286). DSBs are also repaired faster in cancer cells (PMID: 3182330), DSB repair is fast when NHEJ is utilized (PMIDs: 21530420), and the CWR22Rv1 cells have a mutation in BRCA2 and the LNCaP lineage displays “BRCAness” (PMIDs: 28536297, 29465803, 30012171), which results in reliance on the more rapid NHEJ. Additionally, cells in G1 cannot use HR and will use NHEJ to repair DSBs and the majority of the PCa cells are in G1. Furthermore, NHEJ is utilized in G2 (PMID: 18155970). Thus, given the system that we are using, the data in the literature supports the importance of looking at early time points for DSBs and DNA repair foci.

In response to IR, γH2AX foci can be cleared in a little as 3 hours (PMIDs: 16546909, 29127285, 25786477), although as noted by the reviewers, foci can remain for much longer and importantly, the remaining low number of foci may reflect unresolved DSB repair that could compromise PCa cell growth/survival. This is reflected in our observation of a statistically significant reduction in DSBs in the CHK2 knockdown. We have also extended our time course to 24hrs and have now included data for both γH2AX and 53BP1 foci in the resubmitted manuscript.

We have rigorously optimized foci staining (no primary/no secondary controls) and do our antibody incubations in detergent containing buffer. We have added additional details to our Materials and methods for clarity. We have also developed an unbiased method for foci quantitation as described in Figure 6—figure supplement 1. We strongly believe the reported data are an accurate reflection of the γH2AX and 53BP1 foci in PCa cells in response to IR.

2) The alkaline comet assay detects not just DNA double strand breaks but also DNA nicks and abasic sites (through initiation of base excision repair) which will abound following IR treatment. To assess whether these have been repaired properly or not, the assay should be repeated at later timepoints (12-18 hours) when the changes in %tail DNA and tail moments will better represent efficiency and extent of repair. The authors may find a far more striking difference between their control and CHK2-depleted cells, if they repeat the γH2Ax experiment at 12-18 hours following IR when the punctate foci (indicative of repair efficiency or lack thereof) will become more apparent. The difference in comet tail DNA metrics will also most clearly reflect unrepaired genotoxic DNA intermediates at a longer time point. The data, as they stand, do not support their model. Similarly, the relatively small changes in DNA PKc and RAD54 transcripts in Figure 7 do not convince of their assertion that CHK2 depletion increases PCa survival through AR-mediated transcription of DNA repair genes. Such transcript-level changes alone in these factors cannot be extrapolated to their functional effects on DNA repair. The increase in clonogenic survival 14 days post-IR in CHK2-depleted cells is fairly modest and does not necessarily support that CHK2 inhibits radioresistance (Figure 8A). As the authors show in Figure 6, LNCaP cells are generally more growth inhibited by wt CHK2 so the differences in Figure 8A may not reflect radioresistance but just a general growth advantage upon CHK2 loss.

We are very appreciative of the suggestion to look at later time points for the comet assay. As mentioned above in our response to the first comment, when we look at 24hrs the comet assay shows less % DNA in tail, shorter DNA tail length, and lower tail moment with CHK2 knockdown. This data is now included in the manuscript.

The reviewers’ point that the changes in DNA PKc and RAD54 transcripts when CHK2 is knocked down may be insufficient to drive our observations of increased DNA repair and survival with CHK2 knockdown in PCa is valued. This has been a point of discussion in my laboratory since we first examined the level of DDR transcripts reported in PMIDs: 24027197 and 24027196 and only observed differences in DNA PKc and RAD54. Another study (PMID: 30305041) showed that a different set of DDR genes is regulated by AR. The discrepancy in the DDR gene subsets regulated by the AR in these studies may be system dependent, however, it also indicates that the dogma that AR regulates DDR gene expression needs further examination. We have added text in the Results and Discussion of the manuscript that we feel more completely addresses this issue.

3) It is unclear what PCa cell growth actually refers to in this study – is the reduction in cell numbers due to a proliferation arrest or cell death? The known CHK2 interactomes could affect either (or both) pathway. Some textual and/or experimental elaboration of this point is warranted, considering this is a central premise of the study.

For our earlier study when we first described that CHK2 functioned as a tumor suppressor gene and that CHK2 knockdown increased cell growth, we performed preliminary studies of the cell cycle distribution based on DNA content and apoptosis by western blotting of cleaved caspase and PARP. Normal growing PCa cells have low to not detectable levels of cleaved caspase and PARP, making it difficult to determine if there was a reduction in apoptosis with CHK2 knockdown. This would favor a model whereby CHK2 knockdown leads to increased proliferation. Preliminary cell cycle analysis by flow cytometry in LNCaP cells +/- CHK2 KD indicated an increase in S and G2/M, however, this would have to be explored further to be conclusive. We have not examined proliferation/death in the context of IR. Since DSBs induce apoptosis we expect the increased clonagenic survival with CHK2 knockdown is due to the increase in DSB repair (supported by the comet assay, see response to concerns #1 and 2) and corresponding reduction in DSB-induced apoptosis. We have added text to the manuscript addressing this point.

4) The findings from this study taken in context with the previous 2015 study would suggest that the CHK2/AR interaction is mutually inhibitory, as the earlier study suggested that CHK2 is transcriptionally repressed by AR. However, this mutual repression and the notion of negative feedback regulation from CHK2 to AR is not discussed here, for instance in the model in Figure 9. This aspect should be addressed, at least textually, to provide a complete picture of this crosstalk in PCa.

We have added text discussing the negative feedback model. In brief, we suggest that AR represses CHK2 to turn off the DDR, while CHK2 negatively regulates AR to dampen growth while damaged DNA is being repaired.

5) "Kinase-impaired CHK2 variants, including the K373E variant associated with 4.2% of PCa, blocked IR-induced CHK2-AR interactions". This is a strong statement considering the blot in Figure 3B doesn't appear representative of the quantitation shown – it seems the variants reduce binding but certainly don't seem to block it completely.

We agree that the CHK2 variants association with the AR is slightly enhanced with IR and not completely blocked. We have changed the wording to more accurately reflect the reduced binding in the presence of IR.

6) It seems IR increases the CHK2/AR interaction by increasing CHK2 phosphorylation and therefore its activity, which is an interesting finding. However, both the experimental design and Figure 4 (which describes this effect) are hard to understand. For example, the blots on the left vs. the respective quantitation on the right are confusing. The graphs look different (- or + radiation of the V5-CHK2 cells) but the blots look very similar. It would help if Figure 4 included a schematic of the experimental design.

We have completely revised the Far Western data reported in the paper, which is now shown in Figure 1. We now include a schematic of the technique, in Figure 1—figure supplement 1, and thank the reviewer for the suggestion. The data initially reported in Figures 4 and 5 have been removed for clarity and robustness.

7) Statistical analyses and quantifications should be provided for all figure and supplementary panels, when appropriate (e.g. Figure 5A, Figure 8C, Supplementary Figure 2, Figure 6—figure supplement 1).

We have now included a description of the statistical analysis for each figure in the legend.

8) Did the authors check whether CHK2 directly phosphorylates AR, and whether this phosphorylation is required for their binding?

We have examined whether CHK2 phosphorylates the AR on select sites, as AR phosphorylation has long been an interest of my laboratory. We have spent considerable effort in the intervening time since first submitting our manuscript (and in prior years) in an effort to answer this question. However, our experiments have not yet yielded robust reportable data.

9) Although the authors show that CHK2 binding with AR peaks at 1 hour, it would be ideal if the others would have tested additional early time points (such as 30 min, 45 min, 1.5h) to exactly determine the peaking of signal. Are these data available?

The data demonstrating CHK2 and AR peak binding at 1 hour was with endogenous proteins in LNCaP cells. To address the question raised by the reviewers we performed co-IP experiments at earlier time points following IR in LNCaP. In the process of conducting these experiments, we learned that the endogenous co-IP does not always work for reasons that are unclear, despite considerable effort on our part to determine reliable experimental conditions for a robust co-IP of endogenous CHK2 and AR. In contrast, the co-IP of exogenous proteins is robust. Therefore, we attempted to use the co-IP of exogenous CHK2 and AR to refine the kinetics of the interaction. However, exogenous expression of CHK2 leads to basal CHK2 T68 phosphorylation making it more difficult to define the kinetics of CHK2 – AR co-IP since the interaction is dependent on CHK2 T68 phosphorylation that is induced by IR. We could show data summarizing multiple replicates of the endogenous co-IP of CHK2 and AR, however, we feel this would be disingenuous without having defined the conditions to reliably perform the endogenous protein co-IP.

10) As ARS81 is phosphorylated by CDK1, does CDK1 inhibition (using small molecule inhibitors) impact IR induced association of CHK2 and AR?

We thank the reviewers for raising this interesting question. We have one replicate showing that inhibition of CDK1 with RO3306 reduces CHK2-AR interaction by co-IP. One challenge with this type of experiment is that RO3306 treatment results in complete cell cycle arrest at the G2/M transition. This is an inherent issue with all experiments that pharmacologically inhibit cell cycle regulators – it is difficult to separate the observations due to inhibition of the protein of interest from the fundamental alteration in the cell cycle that could indirectly effect the readout. For that reason, we did not pursue this approach further. The other challenge is that while CDK1 does phosphorylate the AR on S81, our data demonstrates that CDK9 is the predominate S81 kinase (PMID: 20980437). This raises the larger issue that the context of CDK1 phosphorylation of S81 may be functionally different then the CDK9 phosphorylation of S81 that happens during transcription of AR target genes; this question cannot be adequately addressed within the scope of this manuscript.

When considering that CHK2 may not be phosphorylating the AR (see response to concern # 8), we asked the fundamental question of whether CHK2 facilitated CDK1 – AR association, with subsequent plans to examine AR pS81. We used CHK2 knockdown to determine the impact on the co-IP of CDK1 and AR. However, when cells were transduced with CHK2 shRNAs, the co-IP experiments of endogenous CDK1 and AR were not consistent, similar to the lack of consistency of endogenous CHK2 – AR co-IPs (see response to concern #9). These technical challenges have limited our ability to further explore this interesting questions at this time.

11) Please check whether the ERK1/2 control blots in Figure 6B (C42) vs. 6C (22Rv1) have been accidentally duplicated.

Thank you for bringing this to attention. We have ensured that the correct corresponding blots are now shown.

Author details

1. Huy Q Ta

   Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, United States

   Contribution

   Data curation, Formal analysis, Visualization, Methodology, Writing-original draft

   Contributed equally with

   Natalia Dworak

   Competing interests

   No competing interests declared

2. Natalia Dworak

   Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, United States

   Contribution

   Data curation, Formal analysis, Visualization

   Contributed equally with

   Huy Q Ta

   Competing interests

   No competing interests declared

3. Melissa L Ivey

   Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

4. Devin G Roller

   Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, United States

   Contribution

   Data curation, Formal analysis, Writing - review and editing

   Competing interests

   No competing interests declared

5. Daniel Gioeli

   1. Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, United States
   2. Cancer Center Member, University of Virginia, Charlottesville, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   dgioeli@virginia.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2284-8152

• 978

  Page views

• 131

  Downloads

• 1

  Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.