Huy Q. Ta

Physical and functional interactions between Cas and c-Src induce tamoxifen resistance of breast cancer cells through pathways involving epidermal growth factor receptor and signal transducer and activator of transcription 5b.

High expression of the adaptor molecule Cas has been linked to resistance to the antiestrogen tamoxifen, both in tissue culture and in human tumors. The aim of this study was to elucidate the mechanism(s) by which overexpression of Cas confers resistance to tamoxifen. Cas overexpression in MCF-7 breast cancer cells was shown to alleviate both tamoxifen-mediated growth inhibition and induction of apoptosis. This enhancement of cell proliferation/survival occurred in the absence of detectable effects on estrogen receptor (ER) transcriptional activity under conditions where tamoxifen was present, indicating that Cas-dependent tamoxifen resistance is not the result of a switch to an ER-negative phenotype or enhanced responses to the partial agonist activity of tamoxifen. Instead, we present evidence, suggesting that Cas promotes tamoxifen resistance by deregulation of alternative cell proliferation pathways, particularly those mediated through enhanced c-Src protein tyrosine kinase activity arising from Cas/c-Src interactions. Overexpression of Cas was found to drive endogenous c-Src into complex with Cas, a process that has been shown previously to cause up-regulation of c-Src tyrosine kinase activity. MCF-7 cells overexpressing Cas exhibited increased phosphorylation of two c-Src substrates, Tyr845 in the kinase domain of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) 5b. Importantly, Cas-dependent protection from the antiproliferative effects of tamoxifen was reversed by the expression of dominant inhibitory variants of these substrates (Y845F EGFR and COOH-terminally truncated STAT5b). Based on these findings, we suggest that the Cas/c-Src/EGFR/STAT5 signaling axis is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

A Novel Association between p130Cas and Resistance to the Chemotherapeutic Drug Adriamycin in Human Breast Cancer Cells

Resistance to chemotherapy remains a major obstacle for the treatment of breast cancer. Understanding the molecular mechanism(s) of resistance is crucial for the development of new effective therapies to treat this disease. This study examines the putative role of p130(Cas) (Cas) in resistance to the cytotoxic agent Adriamycin. High expression of Cas in primary breast tumors is associated with the failure to respond to the antiestrogen tamoxifen and poor prognosis, highlighting the potential clinical importance of this molecule. Here, we show a novel association between Cas and resistance to Adriamycin. We show that Cas overexpression renders MCF-7 breast cancer cells less sensitive to the growth inhibitory and proapoptotic effects of Adriamycin. The catalytic activity of the nonreceptor tyrosine kinase c-Src, but not the epidermal growth factor receptor, is critical for Cas-mediated protection from Adriamycin-induced death. The phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) is elevated in Cas-overexpressing cells treated with Adriamycin, whereas expression of the proapoptotic protein Bak is decreased. Conversely, Cas depletion in the more resistant T47D and MDA-MB-231 cell lines increases sensitivity to Adriamycin. Based on these data, we propose that Cas activates growth and survival pathways regulated by c-Src, Akt, and ERK1/2 that lead to the inhibition of mitochondrial-mediated apoptosis in the presence of Adriamycin. Because Cas is frequently expressed at high levels in breast cancers, these findings raise the possibility of resensitizing Cas-overexpressing tumors to chemotherapy through perturbation of Cas signaling pathways.

Androgen receptor phosphorylation: biological context and functional consequences

The androgen receptor (AR) is a ligand-regulated transcription factor that belongs to the family of nuclear receptors. In addition to regulation by steroid, the AR is also regulated by post-translational modifications generated by signal transduction pathways. Thus, the AR functions not only as a transcription factor but also as a node that integrates multiple extracellular signals. The AR plays an important role in many diseases, including complete androgen insensitivity syndrome, spinal bulbar muscular atrophy, prostate and breast cancer, etc. In the case of prostate cancer, dependence on AR signaling has been exploited for therapeutic intervention for decades. However, the effectiveness of these therapies is limited in advanced disease due to restoration of AR signaling. Greater understanding of the molecular mechanisms involved in AR action will enable the development of improved therapeutics to treat the wide range of AR-dependent diseases. The AR is subject to regulation by a number of kinases through post-translational modifications on serine, threonine, and tyrosine residues. In this paper, we review the AR phosphorylation sites, the kinases responsible for these phosphorylations, as well as the biological context and the functional consequences of these phosphorylations. Finally, what is known about the state of AR phosphorylation in clinical samples is discussed.

The convergence of DNA damage checkpoint pathways and androgen receptor signaling in prostate cancer

It is increasingly clear that castration-resistant prostate cancer (PCa) is dependent on the androgen receptor (AR). This has led to the use of anti-androgen therapies that reduce endogenous steroid hormone production as well as the use of AR antagonists. However, the AR does not act in isolation and integrates with a milieu of cell-signaling proteins to affect cell biology. It is well established that cancer is a genetic disease resulting from the accumulation of mutations and chromosomal translocations that enables cancer cells to survive, proliferate, and disseminate. To maintain genomic integrity, there exists conserved checkpoint signaling pathways to facilitate cell cycle delay, DNA repair, and/or apoptosis in response to DNA damage. The AR interacts with, affects, and is affected by these DNA damage-response proteins. This review will focus on the connections between checkpoint signaling and the AR in PCa. We will describe what is known about how components of checkpoint signaling regulate AR activity and what questions still face the field.

Checkpoint Kinase 2 Negatively Regulates Androgen Sensitivity and Prostate Cancer Cell Growth

Prostate cancer is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated prostate cancer initially respond to androgen deprivation therapy (ADT), but virtually all patients will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating prostate cancer cell growth led to our discovery that checkpoint kinase 2 (CHK2) knockdown dramatically increased prostate cancer growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects prostate cancer proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. In addition, we provide evidence that CHK2 physically associates with the AR and that cell-cycle inhibition increased this association. Finally, IHC analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and prostate cancer growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation.

Abstract 3747: Translating the functional interactions of checkpoint kinase 2 and the androgen receptor into more effective therapies for the treatment of prostate cancer

Prostate cancer remains the most diagnosed cancer among men in the United States behind skin cancer, and advanced prostate cancer is the third leading cause of cancer-related deaths, with a 5-year survival rate of 26%. Radiation is the standard of care for the treatment of prostate cancer at the early and late stages. Checkpoint kinase 2 (CHK2) is a serine/threonine protein kinase whose main function is regulating the DNA damage response (DDR) induced by ionizing radiation. The androgen receptor (AR) is a major driver of prostate cancer, even at the castration-resistant stage of the disease. The development of the second-generation anti-androgen enzalutamide, which is a selective AR antagonist, highlights the enduring importance of the AR. We have previously demonstrated that CHK2 is a critical negative regulator of prostate cancer cell growth, androgen sensitivity, and AR transcriptional activity. We have now uncovered novel molecular interactions between CHK2 and AR that provide mechanistic insight into our observation that CHK2 regulates prostate cancer growth. The AR directly interacts with CHK2, and that interaction increases with radiation. We found that the interaction of CHK2 and AR occurs at sites of DNA damage. The binding of CHK2 with AR can be disrupted with CHK2 kinase inhibitors suggesting that the kinase activity of CHK2 is required. This was verified using kinase-impaired CHK2 variants, including the K373E variant associated with 4.2% of prostate cancer. Furthermore, the radiation-induced increase in CHK2-AR interactions requires AR phosphorylation on both serine 81 and serine 308. Interestingly, CHK2-depletion in LNCaP cells increases ionizing radiation induced AR expression and DNA damage. Together, these data provide the rationale for targeting the CHK2-AR signaling axis to improve the effectiveness of prostate cancer therapies. The combination of CHK2 or CDK1 inhibitors with androgen deprivation therapy (ADT) and radiation shows an additive effect on the repression of tumor cell growth. Nearly every patient with disseminated prostate cancer will relapse following ADT and develop incurable castration-resistant prostate cancer. We have uncovered the molecular details of a signaling axis involving CHK2 and AR that, when perturbed in combination with ADT and/or ionizing radiation, effectively inhibits prostate cancer cell growth. This may enable resensitization of castration-resistant prostate cancer to the currently approved treatment options. Citation Format: Huy Q. Ta, Natalia Dworak, Rosalie Sleppy, Jeffery A. Allende, Daniel Gioeli. Translating the functional interactions of checkpoint kinase 2 and the androgen receptor into more effective therapies for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3747.

Abstract 979: Identification of a novel long noncoding RNA within the LCK gene locus that regulates prostate cancer cell growth

Prostate cancer remains the second most common type of cancer and frequent cause of cancer-related mortality in American men. Even though many patients with metastatic prostate cancer will initially respond to androgen deprivation therapy, virtually all patients will relapse and develop lethal castration-resistant prostate cancer. Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and there is increasing evidence demonstrating that dysregulation of lncRNAs is associated with many human cancers, including cancers of the prostate, breast, and lung. We have discovered in a high-throughput RNAi screen identifying regulators of prostate cancer cell growth that knockdown of lymphocyte-specific protein tyrosine kinase (LCK) significantly decreases growth of prostate cancer cells in the presence and absence of androgen. Surprisingly, immunoprecipitation and western blot analyses show that LCK is not expressed at the protein level in prostate cancer cells. Rapid amplification of cDNA ends (RACE) and sequencing have revealed that a previously unannotated lncRNA lies within exon six and the 3’UTR of the LCK gene. While short hairpin RNAs (shRNAs) targeting the carboxy-terminus of the LCK gene decreases cell growth, expression of shRNAs specific for the amino-terminus has no effect on growth. Furthermore, only quantitative polymerase chain reaction (qPCR) primers directed toward the 3’ section of the LCK gene yield detectable levels of transcript. These data provide further validation for the existence of a lncRNA within the LCK gene locus. Remarkably, the lncRNA situated within the LCK gene is dramatically upregulated in response to androgen. Therefore, we have labeled this lncRNA “HULLK” for Hormone-upregulated lncRNA within LCK. Cellular fractionation and qPCR show that HULLK predominantly localizes to the cytoplasm. In addition to the effects on prostate cancer cell growth, we have data that alludes to the increase in transcript levels of several Src family members following depletion of HULLK. Thus, these studies indicate that the LCK gene contains a lncRNA involved in the regulation of prostate cancer cell growth and perhaps transcription. While additional analyses will be required to fully characterize HULLK, our data suggest that it may serve as a novel regulator of prostate cancer proliferation. Citation Format: Huy Q. Ta, Samuel R. Jackson, Hilary Whitworth, Shriti Bhadel, Daniel Gioeli. Identification of a novel long noncoding RNA within the LCK gene locus that regulates prostate cancer cell growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 979.

Abstract 5049: Checkpoint kinase 2 is a novel regulator of prostate cancer cell growth

Prostate cancer (PCa) is the second leading cause of cancer death in American men, and the cure for metastatic disease remains a significant challenge. While nearly all patients with disseminated PCa initially respond to androgen deprivation therapy (ADT), virtually every patient will relapse and develop incurable castration-resistant prostate cancer (CRPC). Androgen receptor (AR) signaling pathways continue to play a crucial role in CRPC progression. Previous studies have shown that signal transduction pathways can stimulate AR activation, suggesting that the ability of signaling cascades to influence AR function may have a significant role in CRPC progression, and that CRPC may not be effectively treated by ligand-directed therapy alone. A high-throughput RNAi screen identifying signaling pathways that regulate PCa cell growth led to our discovery that knockdown of Checkpoint Kinase 2 (CHK2) dramatically increased PCa proliferation in the presence and absence of androgen. Furthermore, CHK2 depletion hypersensitized cells to castrate androgen levels. These CHK2-mediated effects on growth were dependent on the downstream signaling proteins CDC25C and CDK1 and could be blocked by the AR antagonist MDV3100. Immunohistochemical analysis of CHK2 in patient samples demonstrated that reduced CHK2 expression significantly correlated with increased Gleason score indicating the clinical relevance of CHK2 in PCa. Moreover, CHK2 expression is lower in castration-resistant C4-2 and CWR22Rv1 cells compared to androgen-sensitive LNCaP cells consistent with loss of CHK2 expression during PCa progression. CHK2 depletion increased AR transcriptional activity on both androgen-activated and androgen-repressed genes, substantiating that CHK2 affects PCa growth through the AR. Remarkably, quantitative PCR, chromatin immunoprecipitation, and western blot analyses revealed that CHK2 is a novel AR-repressed gene, suggesting a negative feedback loop between CHK2 and the AR. Furthermore, we show that CHK2 physically associates with the AR, and that cellular stress, such as DNA damage and serum starvation, increases this association. Based on these data, we propose that CHK2 is a negative regulator of androgen sensitivity and PCa growth. Thus, alterations to CHK2 signaling may sensitize CRPC to ADT and radiation. Citation Format: Huy Q. Ta, Melissa L. Ivey, Henry F. Frierson, Mark R. Conaway, Jaroslaw Dziegielewski, James M. Larner, Daniel Gioeli. Checkpoint kinase 2 is a novel regulator of prostate cancer cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5049. doi:10.1158/1538-7445.AM2015-5049

Abstract 3577: The role of Checkpoint Kinase 2 in the regulation of androgen receptor signaling and prostate cancer cell growth during progression to castration resistance.

As the second leading cause of cancer deaths in men, prostate cancer is a persistent, significant challenge for clinicians and researchers. Almost all patients with disseminated prostate cancer initially respond to androgen deprivation therapy. However, virtually every patient will relapse due to the growth of castration-resistant cancer cells and develop metastatic and lethal disease. Even with the recent development of new androgen ablation treatments such as Abiraterone and Enzalutamide, castration-resistant prostate cancer is still incurable. We hypothesize that compensatory signaling mechanisms that limit the effectiveness of androgen ablation can be overcome with therapeutic strategies targeting kinase cascades. In order to identify signaling pathways that regulate AR activity and prostate cancer cell growth we screened a panel of shRNAs targeting the human kinome in LNCaP prostate cancer cells grown in the presence and absence of androgen. We discovered that knockdown of Checkpoint Kinase 2 (CHK2) dramatically increased LNCaP prostate cancer cell proliferation. This observation is clinically relevant since CHK2 inactivating mutations arise in over 10% of prostate cancer patients and CHK2 expression decreases as prostate cancer progresses to a castration-resistant disease. Consistent with these clinical observations, CHK2 knockdown did not affect cell growth in the castration resistant C4-2 cell line, suggesting that the selection of the castration resistant C4-2 line from LNCaP involved the same effector as observed in clinical specimens. Consistent with this, CHK2 expression is lower in C4-2 cells compared to LNCaP. We determined that CHK2 knockdown increases androgen receptor (AR) transcriptional activity on both androgen activated and androgen repressed genes, providing evidence that CHK2 affects prostate cancer cell proliferation, at least in part, through the AR. These data suggest that CHK2 is a negative regulator of androgen sensitivity and prostate cancer cell growth and that CHK2 is lost during the progression to castration resistance. We are examining the role of CHK2 in regulating androgen-AR signaling, growth, and survival of prostate cancer cells; specifically we are identifying the crucial intracellular proteins that mediate CHK2 signaling and growth regulation of prostate cancer cells. Since CHK2 signaling is activated by DNA damage, such as that triggered by radiation therapy and brachytherapy, understanding how CHK2 signaling regulates the AR will provide critical insights into how we can combine current therapies with androgen blockade for greater clinical effectiveness. Citation Format: Huy Q. Ta, Melissa L. Ivey, Daniel Gioeli. The role of Checkpoint Kinase 2 in the regulation of androgen receptor signaling and prostate cancer cell growth during progression to castration resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3577. doi:10.1158/1538-7445.AM2013-3577

Abstract 3177: Cas overexpression in breast cancer confers resistance to chemotherapy by evading apoptosis and overcoming G0/G1 arrest

Breast cancer patients treated with chemotherapy often develop resistance, resulting in highly aggressive tumors that are insensitive to drugs. Elucidating the mechanisms by which cancer cells are able to escape drug-induced apoptosis is thus paramount for developing better approaches to treat and cure breast cancer. Adriamycin is an anthracycline that is regularly used for treatment of breast cancer. Upon exposure to adriamycin, sensitive cells undergo cell death. However, as is the case for many chemotherapeutic drugs, cancer cells frequently develop resistance to adriamycin. We have previously reported that MCF-7 breast cancer cells that overexpress the adaptor molecule p130Cas (Cas) are resistant to adriamycin, whereas MCF-7 cells with endogenous levels of Cas are sensitive to the drug (Ta et al., 2006 Cancer Research). This is consistent with reports showing that high expression of Cas correlates with poor relapse-free and overall survival (Van der Flier et al., 2000 JNCI). Our group has shown that Cas overexpression results in significantly less apoptosis in the presence of adriamycin, and that, the kinase activities of c-Src and PI3K are required for this protection from apoptosis. Furthermore, we show that there is a significant increase in AKT activation when Cas-overexpressing cells are treated with adriamycin. Interestingly, while MCF-7 cells expressing endogenous levels of Cas exhibit a G1 arrest upon adriamycin treatment, the more resistant Cas-overexpressing cells effectively transit through G1 and accumulate in S-phase. This suggests that overexpression of Cas may allow damaged cells to bypass the G0/G1 DNA damage checkpoint. Indeed, both MCF-7 cells expressing either endogenous or elevated levels of Cas show similar degrees of DNA damage when treated with adriamycin, as measured by phospho-histone H2AX. We saw a similar progression through the G0/G1 checkpoint in Cas-overexpressing MCF-7 cells under conditions of serum deprivation, suggesting that high Cas expression may have a relatively global effect on G0/G1 cell cycle checkpoints in response to distinct cellular insults. Based on these data, we hypothesize that Cas overexpression enables breast cancer cells to proceed through the G0/G1 cell cycle arrest induced by conditions of stress, such as DNA damage or nutrient deprivation. Future studies will focus on determining mechanisms by which Cas overexpression allows cancer cells to bypass these cell cycle checkpoints. By gaining a better understanding of how cancer cells are able to adapt and continue to proliferate in an unfavorable environment, and develop resistance to chemotherapy, this study will further the development of better strategies for targeting this aggressive cell population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3177.