Androgen receptor splicing variant 7 (ARV7) inhibits docetaxel sensitivity by inactivating the spindle assembly checkpoint

Abstract

The clinical efficacy of docetaxel (DTX) in prostate cancer treatment is barely satisfactory due to diverse responses of the patients, including the development of resistance. Recently, aberrant androgen receptor (AR) signaling, including expression of the constitutively active ARV7, was reported to contribute to DTX resistance. However, the underlying molecular mechanism remains largely unknown. Of note, previous studies have highlighted that ARV7, unlike its parental AR, potentially favors the expression of some genes involved in cell cycle progression. Since DTX mainly targets microtubule dynamics and mitosis, we wanted to test whether ARV7 plays a specific role in mitotic regulation and whether this activity is involved in DTX resistance. In the present study, we found that ARV7 mediates DTX sensitivity through inactivating the spindle assembly checkpoint (SAC) and promoting mitotic slippage. By shifting the balance to the slippage pathway, ARV7-expressing cells are more likely to escape from mitotic death induced by acute DTX treatment. Furthermore, we also identified E2 enzyme UBE2C as the primary downstream effector of ARV7 in promoting the SAC inactivation and premature degradation of cyclin B1. Moreover, we showed that combination treatment of DTX and an inhibitor of mitotic exit can exert synergistic effect in high ARV7-expressing prostate cancer cells. In sum, our work identified a novel role of ARV7 in promoting DTX resistance and offering a potential path to combat DTX resistance related to abnormal activation of the AR signaling and mitotic dysregulation. The clinical efficacy of docetaxel (DTX) in prostate cancer treatment is barely satisfactory due to diverse responses of the patients, including the development of resistance. Recently, aberrant androgen receptor (AR) signaling, including expression of the constitutively active ARV7, was reported to contribute to DTX resistance. However, the underlying molecular mechanism remains largely unknown. Of note, previous studies have highlighted that ARV7, unlike its parental AR, potentially favors the expression of some genes involved in cell cycle progression. Since DTX mainly targets microtubule dynamics and mitosis, we wanted to test whether ARV7 plays a specific role in mitotic regulation and whether this activity is involved in DTX resistance. In the present study, we found that ARV7 mediates DTX sensitivity through inactivating the spindle assembly checkpoint (SAC) and promoting mitotic slippage. By shifting the balance to the slippage pathway, ARV7-expressing cells are more likely to escape from mitotic death induced by acute DTX treatment. Furthermore, we also identified E2 enzyme UBE2C as the primary downstream effector of ARV7 in promoting the SAC inactivation and premature degradation of cyclin B1. Moreover, we showed that combination treatment of DTX and an inhibitor of mitotic exit can exert synergistic effect in high ARV7-expressing prostate cancer cells. In sum, our work identified a novel role of ARV7 in promoting DTX resistance and offering a potential path to combat DTX resistance related to abnormal activation of the AR signaling and mitotic dysregulation. Despite considerable effort has been made in recent years, prostate cancer (PCa) remains to be one of the most common causes of cancer-dependent deaths in men worldwide (1Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2019.CA Cancer J. Clin. 2019; 69: 7-34Crossref PubMed Scopus (11535) Google Scholar). Since PCa cells rely on androgen receptor (AR) signaling for growth and survival, androgen-deprivation therapy (ADT) is considered as the major treatment option for PCa patients. However, although most of the patients initially respond to ADT well, castration-resistant prostate cancer (CRPC) eventually occurs after several years and then progresses to metastatic diseases, leading to the treatment failure (2Karantanos T. Corn P.G. Thompson T.C. Prostate cancer progression after androgen deprivation therapy: Mechanisms of castrate resistance and novel therapeutic approaches.Oncogene. 2013; 32: 5501-5511Crossref PubMed Scopus (449) Google Scholar). Notably, most CRPC cells are still dependent on active AR signaling for survival, which is primarily due to aberrant alterations of the AR pathway including gain-of-function mutations, overexpression of the AR gene, stimulation by other signaling pathways as well as expression of constitutively active AR-splicing variants (3Dai C. Heemers H. Sharifi N. Androgen signaling in prostate cancer.Cold Spring Harb. Perspect. Med. 2017; 7a030452Crossref PubMed Scopus (128) Google Scholar). Recently, expression of AR-splicing variants (ARVs) emerges as one crucial mechanism accounts for abnormal activation of the AR pathway (4Wadosky K.M. Koochekpour S. Androgen receptor splice variants and prostate cancer: From bench to bedside.Oncotarget. 2017; 8: 18550-18576Crossref PubMed Scopus (62) Google Scholar). In general, ARVs are characterized as shortened forms of AR, which lack the C-terminal ligand-binding domain (LBD). Among all the identified variants, androgen receptor splicing variant 7 (ARV7) draws the most attention as it is the best studied ARV so far and also determined to be the most abundant form in cell lines, xenografts, and clinical samples (5Nakazawa M. Antonarakis E.S. Luo J. Androgen receptor splice variants in the era of enzalutamide and abiraterone.Horm. Cancer. 2014; 5: 265-273Crossref PubMed Scopus (89) Google Scholar). Accumulating studies have indicated that ARV7, no matter whether androgen is available, can constitutively localize to the nucleus and play a crucial role in PCa progression (6Ciccarese C. Santoni M. Brunelli M. Buti S. Modena A. Nabissi M. Artibani W. Martignoni G. Montironi R. Tortora G. Massari F. AR-V7 and prostate cancer: The watershed for treatment selection?.Cancer Treat. Rev. 2016; 43: 27-35Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). ARV7 is rarely detectable in primary PCa, but its level is dramatically increased in response to ADT (7Bastos D.A. Antonarakis E.S. CTC-derived AR-V7 detection as a prognostic and predictive biomarker in advanced prostate cancer.Expert Rev. Mol. Diagn. 2018; 18: 155-163Crossref PubMed Scopus (33) Google Scholar). In one recently conducted sample analysis, ARV7 was found to present in over 75% of patients who received ADT and correlate with worse PSA responses and overall survival (8Sharp A. Coleman I. Yuan W. Sprenger C. Dolling D. Rodrigues D.N. Russo J.W. Figueiredo I. Bertan C. Seed G. Riisnaes R. Uo T. Neeb A. Welti J. Morrissey C. et al.Androgen receptor splice variant-7 expression emerges with castration resistance in prostate cancer.J. Clin. Invest. 2019; 129: 192-208Crossref PubMed Scopus (143) Google Scholar). It is well known that ARV7 is capable of activating canonical AR downstream targets, mediating androgen-independent cell growth and resistance to AR-targeting therapy. Nevertheless, many studies have highlighted that ARV7 might also control a transcriptional program that is largely different from the one regulated by its parental AR (9Wang Q. Li W. Zhang Y. Yuan X. Xu K. Yu J. Chen Z. Beroukhim R. Wang H. Lupien M. Wu T. Regan M.M. Meyer C.A. Carroll J.S. Manrai A.K. et al.Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer.Cell. 2009; 138: 245-256Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 10Lu J. Lonergan P.E. Nacusi L.P. Wang L. Schmidt L.J. Sun Z. Van der Steen T. Boorjian S.A. Kosari F. Vasmatzis G. Klee G.G. Balk S.P. Huang H. Wang C. Tindall D.J. The cistrome and gene signature of androgen receptor splice variants in castration resistant prostate cancer cells.J. Urol. 2015; 193: 690-698Crossref PubMed Scopus (42) Google Scholar, 11Shao C. Yu B. Liu Y. Androgen receptor splicing variant 7: Beyond being a constitutively active variant.Life Sci. 2019; 234: 116768Crossref PubMed Scopus (7) Google Scholar). Specifically, expression of ARV7, but not AR, was positively correlated with the level of some cell cycle genes such as ubiquitin-conjugating enzymes 2C (UBE2C), raising the question that what are the roles of these unique regulations in PCa progression (12Hu R. Dunn T.A. Wei S. Isharwal S. Veltri R.W. Humphreys E. Han M. Partin A.W. Vessella R.L. Isaacs W.B. Bova G.S. Luo J. Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer.Cancer Res. 2009; 69: 16-22Crossref PubMed Scopus (763) Google Scholar). Docetaxel (DTX) has been the first-line drug for treating metastatic CRPC patients since 2004 although its effect is significantly impaired by the acquired drug resistance. Mechanistically, DTX can inhibit microtubule depolymerization, consequently activating the spindle assembly checkpoint (SAC) to arrest cells in mitosis and triggering apoptosis (13Pienta K.J. Preclinical mechanisms of action of docetaxel and docetaxel combinations in prostate cancer.Semin. Oncol. 2001; 28: 3-7Crossref PubMed Google Scholar). Unattached kinetochores lead to the formation of the mitotic checkpoint complex (MCC) and suppression of Cdc20, rendering it unable to activate the anaphase-promoting complex/cyclosome (APC/C). Thus, as the E3 ligase for both securin and cyclin B1, the APC/C is able to control the sister-chromatid separation and mitotic exit (14Lara-Gonzalez P. Westhorpe F.G. Taylor S.S. The spindle assembly checkpoint.Curr. Biol. 2012; 22: R966-R980Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 15Yu H. Regulation of APC-Cdc20 by the spindle checkpoint.Curr. Opin. Cell Biol. 2002; 14: 706-714Crossref PubMed Scopus (284) Google Scholar, 16Han J.S. Holland A.J. Fachinetti D. Kulukian A. Cetin B. Cleveland D.W. Catalytic assembly of the mitotic checkpoint inhibitor BubR1-Cdc20 by a Mad2-induced functional switch in Cdc20.Mol. Cell. 2013; 51: 92-104Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). However, some cancer cells can prematurely degrade cyclin B1 before reaching the apoptotic signal intensity required for initiating mitotic cell death. By doing so, cells are able to exit mitosis without proper chromosome segregation and cytokinesis, through a process defined as mitotic slippage (17Cheng B. Crasta K. Consequences of mitotic slippage for antimicrotubule drug therapy.Endocr. Relat. Cancer. 2017; 24: T97-T106Crossref PubMed Scopus (36) Google Scholar, 18Brito D.A. Rieder C.L. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint.Curr. Biol. 2006; 16: 1194-1200Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). It is postulated that mitotic slippage can facilitate cancer cells to escape from taxol-induced cell death, leading to drug resistance (19Shi J. Orth J.D. Mitchison T. Cell type variation in responses to that and Res. PubMed Scopus Google Scholar, Chen X. of sensitivity to Biol. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the SAC is regulated is crucial for the potential mechanism for DTX resistance. previous studies have indicated that expression of ARV7 can DTX resistance in PCa K. K. F. Wang X. M. H. G.S. to docetaxel in prostate cancer is with androgen receptor activation and of Sci. S. A. 2016; PubMed Scopus Google Scholar, G. Liu X. Li J. E. X. Y. X. Y. Zhang H. Androgen receptor splice variants AR by 2015; PubMed Scopus (62) Google Scholar, Antonarakis E.S. A. G. S. D. J. A. K. S. Y. L. et of AR-V7 and in cells with to therapy in men with metastatic prostate cancer in Cancer Res. 2019; PubMed Scopus Google Scholar). Of note, ARV7 was to distinct pathways of and also the of other ARVs progression Y. Zhang G. Wang X. Y. S. Li D. T. Zhang H. P. Y. and androgen receptor splice variants mediates castration Cancer Res. 2017; PubMed Scopus Google Scholar, M. L. Sun S. M. A. Vessella R.L. E. P. Androgen receptor splice variants sensitivity in prostate cancer.Cancer Res. 2014; PubMed Scopus Google Scholar). However, the molecular mechanism of ARV7 its the of PCa cells DTX treatment remains largely In this study, we to that ARV7 the survival of PCa cells DTX treatment by inactivating the SAC and promoting mitotic offering a novel mechanism to ARV7 to DTX resistance. of we the expression level of ARV7 in several PCa cell to whether ARV7 as we our overexpression of ARV7 in and both of which level of ARV7, not the growth of these cells and In we not ARV7 cells and control cells in cell cycle However, in ARV7-expressing cells showed to form control in both cell of ARV7 significantly the growth of which high level ARV7 not Moreover, of ARV7 dramatically the cell cycle as more cells in with ARV7 the formation of cells these that ARV7 the of PCa cells and might play an role in cell cycle progression. we the to whether ARV7 level DTX we found that cells with ARV7 more resistant to different of DTX cells with control Moreover, as in DTX ARV7 cells induced level of apoptosis control which was indicated by in cells the other after we ARV7 in response to DTX treatment was However, degradation of AR and ARV7 is not a major to DTX as we not in the degradation of both AR and ARV7 after DTX treatment Of note, to ARV7-expressing cells able to form more control cells as indicated in the level of ARV7, we not of some apoptotic which reported to be crucial for mitotic death, including and and M. G. A. mitosis for therapy: cell death the 2018; PubMed Scopus Google Scholar, C. M. Taylor S. dynamics mitotic slippage and death in 2016; 7: PubMed Scopus Google Scholar). In sum, these that ARV7 expression level is related to sensitivity to DTX in but the apoptosis are not with other to the cell after microtubule the mitotic death pathway a death and the mitotic slippage pathway cyclin B1, leading to mitotic Since we of the apoptotic pathway after ARV7 expression we that cells with ARV7 have different cell cycle progression DTX treatment. we found that after we cells from DTX cells with ARV7 showed increased degradation of cyclin B1 with control the level of mitotic in ARV7 and ARV7 showed cells control that ARV7 can exit mitotic arrest more B and we also in cells the other DTX cells showed to exit mitosis we ARV7 in cells cells mitotic slippage can exit mitosis without and we to whether ARV7 regulates this process after DTX treatment. the in after different to of ARV7 more cells with more and with and this we also the of cells The showed that DTX ARV7 more cells control and that ARV7 mitotic slippage DTX treatment. In to slippage from mitotic the be and the has to be to of the we to level in the cells more resistant to DTX treatment and exit from mitotic arrest more and has identified that to be by the APC/C before from the we the level of the ARV7 expression level in PCa cells. in level of was significantly in ARV7-expressing cells after DTX Moreover, the and crucial including and impaired in ARV7 that ARV7 the of the In we ARV7 in the level of was the and the DTX arrest and treatment and these that ARV7 the SAC through promoting the of from the DTX treatment. we on to ARV7 regulates mitotic slippage and the SAC activity by some of its potential the of previous analysis, we found that ARV7 expression is positively correlated with many genes involved in mitotic microtubule and all of which are related to the effect of Among targets with high UBE2C is one of the genes considered to be involved in mitotic exit Of note, UBE2C is also to be the E2 enzyme required for the APC/C to degrade cyclin B1 and also crucial for the SAC inactivation C. C. M. Wu J. Q. enzyme potential cancer J. Cell Biol. 2014; PubMed Scopus Google Scholar, M. by the anaphase-promoting complex spindle checkpoint PubMed Scopus Google Scholar). on these we that UBE2C might be the downstream of ARV7 to the mitotic slippage with the role of UBE2C in mitotic we found that UBE2C cells showed increased mitotic cells showed degradation of cyclin B1 and after from DTX that it is more for cells with UBE2C to from mitotic arrest and In cells in control DTX treatment not that UBE2C plays a crucial role in mitotic slippage. In of these we also found that after UBE2C the level of after DTX was the and and we whether UBE2C sensitivity to and UBE2C cells more to DTX treatment control cells and DTX to apoptosis level in In sum, these that UBE2C is a crucial for mitotic slippage and DTX that ARV7 mediates DTX sensitivity through the mitotic slippage pathway and we the we found that we cells with the APC/C specific inhibitor of ARV7 survival of PCa cells the treatment of DTX as as and Since can inhibit mitotic ARV7 might its effect on survival the mitotic arrest as in and we UBE2C the of ARV7 ARV7 not its to significantly cell survival but also to premature mitotic exit after DTX treatment. Furthermore, cells can resistance to DTX treatment after we UBE2C these our that ARV7 mediates DTX sensitivity through the mitotic exit and UBE2C regulation on these we that combination of the APC/C inhibitor and DTX have a synergistic effect in ARV7-expressing PCa cells. a although treatment with effect on the survival of with significantly increased the of a synergistic effect of these in ARV7-expressing PCa cells In combination treatment of cells with DTX and induced level of apoptosis drug treatment we also the ARV7-expressing cells treatment DTX and to inhibit formation of ARV7-expressing cells Thus, although we the combination for these due to the high of in the PCa cell lines, we able to the in the of ARV7-expressing PCa cells DTX has been as the first-line for it is is largely due to the of cells after mitotic arrest dramatically in different cell and cells from the Recently, that the cell after DTX treatment to be determined by and one mitotic cell death and the other mediating mitotic slippage. Mitotic slippage occurs in the presence of the SAC signaling, which is crucial for cells to escape from mitotic arrest M. G. A. mitosis for therapy: cell death the 2018; PubMed Scopus Google Scholar). However, the and the APC/C activity are PCa progression remains largely unknown. on previous ARV7 not as the major for CRPC progression but also to DTX resistance the mechanisms are The that unlike its parental AR, ARV7 might a of genes to the unique role of ARV7 in cell cycle regulation we found that of ARV7 in cells dramatically induced mitotic arrest mitotic progression and the SAC activity are for cell death, the potential ARV7 and these is of in to the role of ARV7 in DTX resistance. In this study, we are the to that ARV7 mediates DTX sensitivity through inactivating the SAC and promoting mitotic slippage the apoptotic with our previous have that cells have impaired activity of the SAC and expression of ARV7 G. Liu X. Li J. E. X. Y. X. Y. Zhang H. Androgen receptor splice variants AR by 2015; PubMed Scopus (62) Google Scholar, Y. D. Chen H. W. F. X. spindle checkpoint with expression in cell Oncol. Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, we also identified UBE2C as the downstream effector of ARV7 to the and the APC/C UBE2C was initially identified as a of androgen-independent (9Wang Q. Li W. Zhang Y. Yuan X. Xu K. Yu J. Chen Z. Beroukhim R. Wang H. Lupien M. Wu T. Regan M.M. Meyer C.A. Carroll J.S. Manrai A.K. et al.Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer.Cell. 2009; 138: 245-256Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). In some of the to be dependent on that ARV expression as an to therapy and the and ARV might play a crucial role in mediating Chen J. A. K. C.L. active androgen receptor splice variants in castration-resistant prostate cancer androgen Sci. S. A. PubMed Scopus Google Scholar). However, studies have that ARV7 is the major for mitotic genes and the of UBE2C was by AR-V7 B. Y. Zhang G. Xu D. Y. X. Z. X. Zhang H. Y. Androgen receptor splice variants activating the receptor in mediating resistance to 2014; 5: PubMed Scopus Google Scholar, R. C. S. M. C. J. Isaacs W.B. E. Luo J. transcriptional by the androgen receptor and its splice variants in castration-resistant prostate cancer.Cancer Res. 2012; PubMed Scopus Google Scholar). Thus, it is to the mechanism of UBE2C expression different in Of note, some have that cells UBE2C the mitotic spindle checkpoint and which to cancer progression L. of the E2 ubiquitin-conjugating enzyme causes chromosome and Cell Biol. PubMed Scopus Google Scholar, Z. Zhang H. J. enzyme role in and potential as a Biol. 2012; PubMed Scopus Google Scholar). that UBE2C plays a crucial role in the APC/C and was also indicated to be regulated by ARV7 in it is to the role of ARV7 in the of mitotic exit regulation J. G. D. E. H. P. J.D. J. A. et of an expression signature derived from cell cycle genes in patients with prostate cancer: Oncol. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. Z. S. Wang L. M. Tindall D.J. Li B. R. Wang L. Huang H. Androgen receptor splice variants to constitutively and growth of prostate Res. 2018; PubMed Scopus Google Scholar). In as our that the APC/C inhibitor can the of DTX in ARV7-expressing it is to a novel path to combat resistance of patients, with high ARV7 expression In general, our showed that ARV7 DTX resistance largely by promoting the SAC and mitotic slippage as the mitotic death is the major for DTX in cell However, the in DTX mitotic arrest as as it in cell due to and In other the effect of mitotic death in has been significantly by Thus, that is the some clinical to ARV7 to the DTX response of patients as the leading to the whether ARV7 to DTX efficacy Antonarakis E.S. A. G. S. D. J. A. K. S. Y. L. et of AR-V7 and in cells with to therapy in men with metastatic prostate cancer in Cancer Res. 2019; PubMed Scopus Google Scholar, E.S. C. B. Wang H. Chen Y. M. R. Luo J. Androgen receptor splice variant 7 and efficacy of in patients with metastatic castration-resistant prostate and in metastatic castration-resistant prostate and in metastatic castration-resistant prostate Oncol. 2015; PubMed Google Scholar, E.S. C. B. Wang H. Chen Y. M. R. Luo J. Androgen receptor splice variant 7 and efficacy of in patients with metastatic castration-resistant prostate Oncol. 2015; PubMed Scopus Google Scholar). It is postulated that the efficacy of mitotic in clinical therapy is determined by the chromosome and the with cells treatment M. G. A. mitosis for therapy: cell death the 2018; PubMed Scopus Google Scholar, D. A.J. of in cancer is due to chromosome on Med. 2014; Scopus Google Scholar, J. A.J. Mitotic progression 2017; PubMed Scopus Google Scholar). cells can apoptosis as the of cells are capable of some related to and which is as (17Cheng B. Crasta K. Consequences of mitotic slippage for antimicrotubule drug therapy.Endocr. Relat. Cancer. 2017; 24: T97-T106Crossref PubMed Scopus (36) Google Scholar, A. J. The The of Rev. 5: PubMed Scopus Google Scholar). Thus, as we are ARV7 with it is still to that ARV7 is a biomarker for DTX therapeutic Nevertheless, on the novel the regulation of mitotic we to the of ARV7 in cells in for and more specific targets for DTX resistance. DTX and from and from The APC/C inhibitor was from and in and cells from and cells by of and cells in with and in cells in the was to the The was from was from cells ARV7 cells with ARV7 by and cells as the The for ARV7 expression is in with the for ARV7 is the UBE2C UBE2C from and UBE2C cells as Cell with and inhibitor cell with in then was to sample for by with and for the from was for The ARV7 and from UBE2C and from Cell and the and from is the of and in for and to for and then with for with for cells with primary The cells with for in the cells with for and acquired by an in to different cells and was to the samples by cells in indicated as control for was and was for with and was a of The was to cells a the was the and to an by the was in the The more the cells are on the the the in Cell is to the in to cell The was to the Cell The Cell with and the of cells the cells drug in for with cells with and with The of in prostate cancer cells from to the samples ARV7 and its control of gene expression was the in the in was The level of indicated by was was considered The of that the of this are the and the which are not in the are from the The no potential of and B. Y. and C. S. of B. C. and H. L. of B. H. and C. S. and of the C. B. and Y. L. and C. Y. J. and Y. L. Y. L. and C. S. work was by the of and the B

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