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Manohar Ratnam - One of the best experts on this subject based on the ideXlab platform.

  • abstract 2469 elucidating the structure function relationships in the interactions of the androgen receptor ELK1 and the platform antagonist kci807 in prostate cancer
    Cancer Research, 2021
    Co-Authors: Claire Soave, Rayna Rosati, Yanfang Huang, Manohar Ratnam
    Abstract:

    Prostate cancer (PC) is generally dependent on the androgen signaling axis for growth. Advanced PC is managed by androgen deprivation therapy (ADT). However, the tumors frequently progress by restoring AR signaling that is androgen-independent or hypersensitive to it, leading to castration resistant prostate cancer (CRPC). CRPC is also supported by hormone-independent actions of AR splice variants which lack the ligand binding domain. Additionally, ADT has many undesirable side effects on a variety of normal tissues that depend on androgen for non-growth related functions, including effects on the cardiovascular system, central nervous system, bone and muscle. Therefore, a more strategic therapy approach would be to disrupt a functional arm of AR-signaling that is critical for PC/CRPC growth but not for the essential physiological roles of AR in normal tissues. Previous studies in this laboratory have identified ELK1 as an AR tethering protein essential for activation of a critical set of androgen/AR growth genes in various PC model cell line models, both in vitro and in vivo, including those resistant to castration and enzalutamide. It was also established that the N-terminal A/B domain of AR binds to ELK1 by co-opting the two ERK docking sites on ELK1 to constitutively activate genes enriched for cell growth functions in PC cells. A lead candidate drug molecule (KCI807) was discovered that bound to AR, blocking its association with ELK1 and inhibiting PC/CRPC tumor growth. KCI807 binds to the A/B domain in AR or its splice variants and has a limited target gene set (a subset of AR target genes) that is primarily and highly enriched for functions in cell cycle progression and mitosis. To further our drug development efforts, we must identify and characterize the ELK1 docking site recognition sites in the AR A/B domain and the binding site of KCI807. We applied a mammalian two-hybrid assay to map the ELK1 interacting motifs in the A/B domain of AR using a series of deletion constructs. Overlapping deletion constructs were made systematically to cover the entire A/B domain. For each of these constructs additional double fusion constructs were used as controls. Expression of fusion proteins was monitored by western blot. Our results excluded a role for the amino-terminal half of the A/B domain in ELK1 interactions but did identify repressor motifs within this region. We identified two docking site recognition sites in the AR A/B domain for the two docking sites in ELK1 in a parallel configuration. The KCI807 binding site was located in the downstream site of AR. Further structural and functional studies of the mapped motifs in AR will reveal more about the biology of the AR-ELK1 interaction and directly aid in the design of next generation antagonists to selectively target ELK1-dependent growth signaling by AR in PC/CRPC tumors. Citation Format: Claire L. Soave, Rayna Rosati, Yanfang Huang, Manohar Ratnam. Elucidating the structure-function relationships in the interactions of the androgen receptor, ELK1 and the platform antagonist KCI807 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2469.

  • The ternary complex factor protein ELK1 is an independent prognosticator of disease recurrence in prostate cancer.
    The Prostate, 2019
    Co-Authors: Luke Pardy, Rayna Rosati, Claire Soave, Yanfang Huang, Seongho Kim, Manohar Ratnam
    Abstract:

    BACKGROUND Both hormone-sensitive and castration- and enzalutamide-resistant prostate cancers (PCa) depend on the ternary complex factor (TCF) protein ELK1 to serve as a tethering protein for the androgen receptor (AR) to activate a critical set of growth genes. The two sites in ELK1 required for AR binding are conserved in other members of the TCF subfamily, ELK3 and ELK4. Here we examine the potential utility of the three proteins as prognosticators of disease recurrence in PCa. METHODS Transcriptional activity assays; Retrospective analysis of PCa recurrence using data on 501 patients in The Cancer Genome Atlas (TCGA) database; Unpaired Wilcoxon rank-sum test and multiple comparison correction using the Holm's method; Spearman's correlations; Kaplan-Meier methods; Univariable and multivariable Cox regression analyses; LASSO-based penalized Cox regression models; Time-dependent area under the receiver operating characteristic (ROC) curve. RESULTS ELK4 but not ELK3 was coactivated by AR similar to ELK1. Tumor expression of neither ELK3 nor ELK4 was associated with disease-free survival (DFS). ELK1 was associated with higher clinical T-stage, pathology T-stage, Gleason score, prognostic grade, and positive lymph node status. ELK1 was a negative prognosticator of DFS, independent of ELK3, ELK4, clinical T-stage, pathology T-stage, prognostic grade, lymph node status, age, and race. Inclusion of ELK1 increased the abilities of the Oncotype DX and Prolaris gene panels to predict disease recurrence, correctly predicting disease recurrence in a unique subset of patients. CONCLUSIONS ELK1 is a strong, independent prognosticator of disease recurrence in PCa, underscoring its unique role in PCa growth. Inclusion of ELK1 may enhance the utility of currently used prognosticators for clinical decision making in prostate cancer.

  • the amino terminal domain of the androgen receptor co opts extracellular signal regulated kinase erk docking sites in ELK1 protein to induce sustained gene activation that supports prostate cancer cell growth
    Journal of Biological Chemistry, 2016
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Selvakumar Dakshnamurthy, Thomas Mcfall, Janice Saxton, Benjamin L Kidder, Peter E Shaw, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8 m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.

  • abstract 2876 the androgen receptor utilizes protein kinase docking sites to constitutively activate ELK1
    Cancer Research, 2016
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Selvakumar Dakshnamurthy, Thomas Mcfall, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for growth in established prostate cancer cell lines. This process does not entail the transient hyper-activation of immediate early genes typical of activation of ELK1 through phosphorylation by ERK1/2. We compared the structural requirements for the association of AR and ELK1 with that for activation of ELK1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with ELK1 and to activate ELK1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with ELK1 was optimal in the absence of ERK1/2 and serum response factor (SRF), which are the known binding partners of ELK1. We identified two sites on ELK1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 307 to 330, overlapping the D box, which is one of two ERK docking sites. The other site on ELK1 mapped to amino acid residues 372 to 397, overlapping the FXFP motif, which comprises the downstream ERK docking site. The results suggest that AR utilizes ERK docking sites to associate with ELK1 and is exclusive of the interaction of ELK1 with ERK1/2 or SRF. A nuclear receptor could thus function as a transcription factor co-activator by binding at protein kinase docking sites. Citation Format: Rayna Rosati, Mugdha Patki, Selvakumar Dakshnamurthy, Venkatesh Chari, Thomas McFall, Manohar Ratnam. The androgen receptor utilizes protein kinase docking sites to constitutively activate ELK1. [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 2876.

  • abstract 1855 structural requirements for the association of the ets domain transcription factor ELK1 and the androgen receptor in enabling the growth of prostate cancer cells
    Cancer Research, 2015
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for cell growth in established prostate cancer cell lines. This role of ELK1 is independent of the ability of ELK1 to transiently activate immediate early genes in response to phosphorylation by ERK. We compared the structural requirements for the association of AR and ELK1 with that for activation of ELK1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with ELK1 and to activate ELK1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with ELK1 did not require ERK1/2 or serum response factor (SRF), which are the known binding partners of ELK1. We identified two sites on ELK1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 297 to 317, overlapping one of two ERK docking sites. The other site on ELK1 mapped to amino acid residues 387 to 397, adjacent to the downstream ERK docking site. The results suggest that the association of AR with ELK1 in situ is direct rather than through the ELK1 ternary complex and that the association may be disrupted by small molecules to selectively inhibit growth signaling in prostate cancer cells. Citation Format: Rayna Rosati, Venkatesh Chari, Mugdha Patki, Manohar Ratnam. Structural requirements for the association of the ETS domain transcription factor ELK1 and the androgen receptor in enabling the growth of prostate cancer cells. [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 1855. doi:10.1158/1538-7445.AM2015-1855

Takashi Kawahara - One of the best experts on this subject based on the ideXlab platform.

  • ELK1 promotes urothelial tumorigenesis in the presence of an activated androgen receptor
    American Journal of Cancer Research, 2018
    Co-Authors: Satoshi Inoue, Takashi Kawahara, Hiroki Ide, Taichi Mizushima, Guiyang Jiang, Hiroshi Miyamoto
    Abstract:

    We have recently demonstrated that ELK1, a transcription factor that triggers downstream targets including c-Fos proto-oncogene, promotes the growth of bladder cancer cells possessing a functional androgen receptor (AR). We here assessed the function of ELK1, as well as the efficacy of a selective α1A-adrenergic blocker silodosin that has been shown to inhibit ELK1 activity in bladder cancer cells, in urothelial tumorigenesis. The level of ELK1 expression in an immortalized normal urothelial cell line SVHUC stably expressing wild-type AR (SVHUC-AR) was considerably higher than that in AR-negative SVHUC-vector cells, which was induced further or reduced by dihydrotestosterone or silodosin treatment, respectively. In SVHUC-AR cells exposed to a chemical carcinogen 3-methylcholanthrene, silodosin significantly reduced the expression levels of oncogenes (e.g. c-Fos, Jun, Myc), as well as phospho-p38 MAPK and phospho-ERK proteins, and increased those of tumor suppressor genes (e.g. p53, PTEN, UGT1A). ELK1 suppression via ELK1-short hairpin RNA virus infection or silodosin treatment also resulted in significant inhibition in 3-methylcholanthrene-induced neoplastic transformation of SVHUC-AR cells, but not that of SVHUC-vector cells. In N-butyl-N-(4-hydroxybutyl)nitrosamine-treated male C57BL/6 mice, the incidence rate of bladder tumors was significantly (P = 0.007) lower in the silodosin group than in the control group. ELK1 thus appears to play a critical role in urothelial tumorigenesis, and silodosin prevents it presumably via down-regulation of ELK1. Moreover, ELK1 may require an activated AR for inducing neoplastic transformation of urothelial cells. Our findings may therefore offer a novel chemopreventive approach, via ELK1 inactivation using silodosin treatment, for bladder cancer.

  • expression of phospho ELK1 and its prognostic significance in urothelial carcinoma of the upper urinary tract
    International Journal of Molecular Sciences, 2018
    Co-Authors: Satoshi Inoue, Takashi Kawahara, Hiroki Ide, Kazutoshi Fujita, Taichi Mizushima, Guiyang Jiang, Seiji Yamaguchi, Hiroaki Fushimi
    Abstract:

    Using preclinical models, we have recently found that ELK1, a transcriptional factor that activates downstream targets, including c-fos proto-oncogene, induces bladder cancer outgrowth. Here, we immunohistochemically determined the expression status of phospho-ELK1, an activated form of ELK1, in upper urinary tract urothelial carcinoma (UUTUC). Overall, phospho-ELK1 was positive in 47 (47.5%; 37 weak (1+) and 10 moderate (2+)) of 99 UUTUCs, which was significantly (P = 0.002) higher than in benign urothelium (21 (25.3%) of 83; 17 1+ and 4 2+) and was also associated with androgen receptor expression (P = 0.001). Thirteen (35.1%) of 37 non-muscle-invasive versus 34 (54.8%) of 62 muscle-invasive UUTUCs (P = 0.065) were immunoreactive for phospho-ELK1. Lymphovascular invasion was significantly (P = 0.014) more often seen in phospho-ELK1(2+) tumors (80.0%) than in phospho-ELK1(0/1+) tumors (36.0%). There were no statistically significant associations between phospho-ELK1 expression and tumor grade, presence of concurrent carcinoma in situ or hydronephrosis, or pN status. Kaplan-Meier and log-rank tests revealed that patients with phospho-ELK1(2+) tumor had marginally and significantly higher risks of disease progression (P = 0.055) and cancer-specific mortality (P = 0.008), respectively, compared to those with phospho-ELK1(0/1+) tumor. The current results thus support our previous observations in bladder cancer and further suggest that phospho-ELK1 overexpression serves as a predictor of poor prognosis in patients with UUTUC.

  • silodosin inhibits prostate cancer cell growth via ELK1 inactivation and enhances the cytotoxic activity of gemcitabine
    The Prostate, 2016
    Co-Authors: Takashi Kawahara, Hiroki Ide, Satoshi Inoue, Hasanain Khaleel Shareef, Ali Kadhim Aljarah, John D. Patterson
    Abstract:

    BACKGROUND Biological significance of ELK1, a transcriptional factor whose phosphorylation is necessary for c-fos proto-oncogene activation, in prostate cancer remains far from fully understood. In this study, we aim to investigate the role of ELK1 in tumor growth as well as the efficacy of a selective α1A-adrenergic blocker, silodosin, in ELK1 activity in prostate cancer cells. METHODS We first immunohistochemically determined the levels of phospho-ELK1 (p-ELK1) expression in radical prostatectomy specimens. We then assessed the effects of ELK1 knockdown via short hairpin RNA and silodosin on cell proliferation, migration, and invasion in prostate cancer lines. RESULTS The levels of p-ELK1 expression were significantly higher in carcinoma than in benign (P < 0.001) or high-grade prostatic intraepithelial neoplasia (HGPIN) (P = 0.002) as well as in HGPIN than in benign (P < 0.001). Kaplan–Meier and log-rank tests revealed that moderate-strong positivity of p-ELK1 in carcinomas tended to correlate with biochemical recurrence after radical prostatectomy (P = 0.098). In PC3 and DU145 expressing ELK1 (mRNA/protein) but no androgen receptor (AR), ELK1 silencing resulted in considerable decreases in the expression of c-fos as well as in cell migration/invasion and matrix metalloproteinase-2 expression, but not in cell viability. Silodosin treatment reduced the expression/activity of ELK1 in these cells as well as the viability of AR-positive LNCaP and C4-2 cells and the migration of both AR-positive and AR-negative cells, but not the viability of AR-negative or ELK1-negative cells. Interestingly, silodosin significantly increased sensitivity to gemcitabine, but not to cisplatin or docetaxel, even in AR-negative cells. CONCLUSIONS ELK1 is likely to be activated in prostate cancer cells and promote tumor progression. Furthermore, silodosin that inactivates ELK1 in prostate cancer cells not only inhibits their growth but also enhances the cytotoxic activity of gemcitabine. Thus, ELK1 inhibition has the potential of being a therapeutic approach for prostate cancer. Prostate 76:744–756, 2016. © 2016 Wiley Periodicals, Inc.

  • Silodosin inhibits the growth of bladder cancer cells and enhances the cytotoxic activity of cisplatin via ELK1 inactivation.
    American journal of cancer research, 2015
    Co-Authors: Takashi Kawahara, Hiroki Ide, Eiji Kashiwagi, John D. Patterson, Satoshi Inoue, Hasanain Khaleel Shareef, Ali Kadhim Aljarah, Yichun Zheng, Alexander S. Baras, Hiroshi Miyamoto
    Abstract:

    Silodosin, a selective α1A-adrenergic blocker prescribed for the symptomatic treatment of benign prostatic hyperplasia, was previously shown to decrease the expression of ELK1, a c-fos proto-oncogene regulator and a well-described downstream target of the PKC/Raf-1/ERK pathway, in human prostate smooth muscle cells. PKC/Raf-1/ERK activation has also been implicated in drug resistance. In the current study, we assessed the effects of silodosin on ELK1 expression/activity in bladder cancer cells as well as on their proliferation in the presence or absence of chemotherapeutic drugs, including cisplatin and gemcitabine. In bladder cancer cell lines, silodosin reduced the expression of ELK1 (mRNA/protein) and its downstream target, c-fos gene, as well as the transcriptional activity of ELK1. While silodosin alone (up to 10 μM) insignificantly affected the growth of bladder cancer cells cultured in androgen depleted conditions or those expressing ELK1-short hairpin RNA, it considerably inhibited the viability of androgen receptor (AR)-positive/ELK1-positive cells in the presence of androgens. Silodosin also inhibited the migration of ELK1-positive cells with or without a functional AR, but not that of ELK1 knockdown cells. Interestingly, silodosin treatment or ELK1 silencing resulted in increases in drug sensitivity to cisplatin, but not to gemcitabine, even in AR-negative cells or AR-positive cells cultured in an androgen-depleted condition. In addition, silodosin decreased the expression of NF-κB, a key regulator of chemoresistance, and its transcriptional activity. Moreover, immunohistochemistry in bladder cancer specimens from patients who received neoadjuvant chemotherapy revealed that phospho-ELK1 positivity strongly correlated with chemoresistance. Silodosin was thus found to not only inhibit cell viability and migration but also enhance the cytotoxic activity of cisplatin in bladder cancer lines via inactivating ELK1. Our results suggest that combined treatment with silodosin is useful for overcoming chemoresistance in patients with ELK1-positive urothelial carcinoma receiving cisplatin.

  • ELK1 is up regulated by androgen in bladder cancer cells and promotes tumor progression
    Oncotarget, 2015
    Co-Authors: Takashi Kawahara, Hiroki Ide, Eiji Kashiwagi, Hasanain Khaleel Shareef, Ali Kadhim Aljarah, George J Netto
    Abstract:

    Little is known about biological significance of ELK1, a transcriptional factor that activates downstream targets including c-fos proto-oncogene, in bladder cancer. Recent preclinical evidence also suggests the involvement of androgen receptor (AR) signaling in bladder cancer progression. In this study, we aim to investigate the functions of ELK1 in bladder cancer growth and their regulation by AR signals. Immunohistochemistry in bladder tumor specimens showed that the levels of phospho-ELK1 (p-ELK1) expression were significantly elevated in urothelial neoplasms, compared with non-neoplastic urothelium tissues, and were also correlated with AR positivity. Patients with p-ELK1-positive non-muscle-invasive and muscle-invasive tumors had significantly higher risks for tumor recurrence and progression, respectively. In AR-positive bladder cancer cell lines, dihydrotestosterone treatment increased ELK1 expression (mRNA, protein) and its nuclear translocation, ELK1 transcriptional activity, and c-fos expression, which was restored by an anti-androgen hydroxyflutamide. ELK1 silencing via short hairpin RNA (shRNA) resulted in decreases in cell viability/colony formation, and cell migration/invasion as well as an increase in apoptosis. Importantly, ELK1 appears to require activated AR to regulate bladder cancer cell proliferation, but not cell migration. Androgen also failed to significantly induce AR transactivation in ELK1-knockdown cells. In accordance with our in vitro findings, ELK1-shRNA expression considerably retarded tumor formation as well as its growth in xenograft-bearing male mice. Our results suggest that ELK1 plays an important role in bladder tumorigenesis and cancer progression, which is further induced by AR activation. Accordingly, ELK1 inhibition, together with AR inactivation, has the potential of being a therapeutic approach for bladder cancer.

Mugdha Patki - One of the best experts on this subject based on the ideXlab platform.

  • the amino terminal domain of the androgen receptor co opts extracellular signal regulated kinase erk docking sites in ELK1 protein to induce sustained gene activation that supports prostate cancer cell growth
    Journal of Biological Chemistry, 2016
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Selvakumar Dakshnamurthy, Thomas Mcfall, Janice Saxton, Benjamin L Kidder, Peter E Shaw, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8 m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.

  • abstract 2876 the androgen receptor utilizes protein kinase docking sites to constitutively activate ELK1
    Cancer Research, 2016
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Selvakumar Dakshnamurthy, Thomas Mcfall, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for growth in established prostate cancer cell lines. This process does not entail the transient hyper-activation of immediate early genes typical of activation of ELK1 through phosphorylation by ERK1/2. We compared the structural requirements for the association of AR and ELK1 with that for activation of ELK1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with ELK1 and to activate ELK1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with ELK1 was optimal in the absence of ERK1/2 and serum response factor (SRF), which are the known binding partners of ELK1. We identified two sites on ELK1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 307 to 330, overlapping the D box, which is one of two ERK docking sites. The other site on ELK1 mapped to amino acid residues 372 to 397, overlapping the FXFP motif, which comprises the downstream ERK docking site. The results suggest that AR utilizes ERK docking sites to associate with ELK1 and is exclusive of the interaction of ELK1 with ERK1/2 or SRF. A nuclear receptor could thus function as a transcription factor co-activator by binding at protein kinase docking sites. Citation Format: Rayna Rosati, Mugdha Patki, Selvakumar Dakshnamurthy, Venkatesh Chari, Thomas McFall, Manohar Ratnam. The androgen receptor utilizes protein kinase docking sites to constitutively activate ELK1. [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 2876.

  • abstract 1855 structural requirements for the association of the ets domain transcription factor ELK1 and the androgen receptor in enabling the growth of prostate cancer cells
    Cancer Research, 2015
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for cell growth in established prostate cancer cell lines. This role of ELK1 is independent of the ability of ELK1 to transiently activate immediate early genes in response to phosphorylation by ERK. We compared the structural requirements for the association of AR and ELK1 with that for activation of ELK1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with ELK1 and to activate ELK1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with ELK1 did not require ERK1/2 or serum response factor (SRF), which are the known binding partners of ELK1. We identified two sites on ELK1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 297 to 317, overlapping one of two ERK docking sites. The other site on ELK1 mapped to amino acid residues 387 to 397, adjacent to the downstream ERK docking site. The results suggest that the association of AR with ELK1 in situ is direct rather than through the ELK1 ternary complex and that the association may be disrupted by small molecules to selectively inhibit growth signaling in prostate cancer cells. Citation Format: Rayna Rosati, Venkatesh Chari, Mugdha Patki, Manohar Ratnam. Structural requirements for the association of the ETS domain transcription factor ELK1 and the androgen receptor in enabling the growth of prostate cancer cells. [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 1855. doi:10.1158/1538-7445.AM2015-1855

  • abstract 2116 mapping peptides critical for association of the androgen receptor with ELK1
    Cancer Research, 2014
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam
    Abstract:

    Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Both early stage and advanced prostate tumors are generally dependent on the androgen receptor (AR) for growth. Indeed a major clinical approach in management of the disease is to disrupt the AR signaling in the tumors by androgen ablation and the use of anti-androgen drugs. However, these options present major drawbacks including limited efficacy in advanced disease and many undesirable side effects on non-target tissues. Therefore a more tactical therapy approach would be one that could disrupt a functional arm of AR signaling that is critical for prostate tumor growth but not for the essential physiological roles of AR in normal adult tissues. A considerable amount of evidence suggests that in prostate cancer, androgen/AR signaling is redirected to support tumor growth through the association of AR with critical growth promoting genes via tethering proteins. Our previous studies have identified ELK1, a genetically redundant DNA binding transcription factor, as an AR tethering protein essential for AR-dependent growth in established models of both hormone-dependent prostate cancer and castration recurrent prostate cancer. ELK1 recruits AR to chromatin sites to upregulate a major subset of genes that is strongly and primarily enriched for cell growth functions. Further studies in the lab demonstrate that the A/B domain of the AR alone is capable of co-activation of ELK1, suggesting that ELK1 may also mediate the hormone-independent growth supporting function of AR splice variants. Peptide or small molecule inhibitors of the ELK1-AR interaction may therefore selectively target the growth supporting function of AR in the spectrum of prostate tumors, obviating the need for androgen ablation. Toward this end we undertook to map peptide segment(s) of ELK1and AR that are essential for the ELK1-AR synergy. In this study we used deletional and mutational analysis together with a promotor-reporter assay and a mammalian two-hybrid assay. The results demonstrate that the ability to recruit AR resides largely in the C-terminal activation domain of ELK1 (amino acids 307-428) and that residues within segments 367-428 and 307-337 are essential for this interaction. They also demonstrate that the recruitment of AR by ELK1 does not require ELK1 phosphorylation. Critical sites within the AR-A/B domain required for its association with ELK1 reside in the N-terminal region that is distal to known sites of co-regulator binding. This information will guide the development of peptide inhibitors of ELK1-dependent growth induction by androgen/AR and set the stage for future development of small molecule inhibitors. Citation Format: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam. Mapping peptides critical for association of the androgen receptor with ELK1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2116. doi:10.1158/1538-7445.AM2014-2116

  • the ets domain transcription factor ELK1 directs a critical component of growth signaling by the androgen receptor in prostate cancer cells
    Journal of Biological Chemistry, 2013
    Co-Authors: Mugdha Patki, Venkatesh Chari, Suneethi Sivakumaran, Mesfin Gonit, Robert J Trumbly, Manohar Ratnam
    Abstract:

    The androgen receptor (AR) is essential for diverse aspects of prostate development and function. Molecular mechanisms by which prostate cancer (PC) cells redirect AR signaling to genes that primarily support growth are unclear. A systematic search for critical AR-tethering proteins led to ELK1, an ETS transcription factor of the ternary complex factor subfamily. Although genetically redundant, ELK1 was obligatory for AR-dependent growth and clonogenic survival in both hormone-dependent PC and castration-recurrent PC cells but not for AR-negative cell growth. AR required ELK1 to up-regulate a major subset of its target genes that was strongly and primarily enriched for cell growth functions. AR functioned as a coactivator of ELK1 by association through its A/B domain, bypassing the classical mechanism of ELK1 activation by phosphorylation and without inducing ternary complex target genes. The ELK1-AR synergy per se was ligand-independent, although it required ligand for nuclear localization of AR as targeting the AR A/B domain to the nucleus recapitulated the action of hormone; accordingly, Casodex was a poor antagonist of the synergy. ELK3, the closest substitute for ELK1 in structure/function and genome recognition, did not interact with AR. ELK1 thus directs selective and sustained gene induction that is a substantial and critical component of growth signaling by AR in PC cells. The ELK1-AR interaction offers a functionally tumor-selective drug target.

Rayna Rosati - One of the best experts on this subject based on the ideXlab platform.

  • abstract 2469 elucidating the structure function relationships in the interactions of the androgen receptor ELK1 and the platform antagonist kci807 in prostate cancer
    Cancer Research, 2021
    Co-Authors: Claire Soave, Rayna Rosati, Yanfang Huang, Manohar Ratnam
    Abstract:

    Prostate cancer (PC) is generally dependent on the androgen signaling axis for growth. Advanced PC is managed by androgen deprivation therapy (ADT). However, the tumors frequently progress by restoring AR signaling that is androgen-independent or hypersensitive to it, leading to castration resistant prostate cancer (CRPC). CRPC is also supported by hormone-independent actions of AR splice variants which lack the ligand binding domain. Additionally, ADT has many undesirable side effects on a variety of normal tissues that depend on androgen for non-growth related functions, including effects on the cardiovascular system, central nervous system, bone and muscle. Therefore, a more strategic therapy approach would be to disrupt a functional arm of AR-signaling that is critical for PC/CRPC growth but not for the essential physiological roles of AR in normal tissues. Previous studies in this laboratory have identified ELK1 as an AR tethering protein essential for activation of a critical set of androgen/AR growth genes in various PC model cell line models, both in vitro and in vivo, including those resistant to castration and enzalutamide. It was also established that the N-terminal A/B domain of AR binds to ELK1 by co-opting the two ERK docking sites on ELK1 to constitutively activate genes enriched for cell growth functions in PC cells. A lead candidate drug molecule (KCI807) was discovered that bound to AR, blocking its association with ELK1 and inhibiting PC/CRPC tumor growth. KCI807 binds to the A/B domain in AR or its splice variants and has a limited target gene set (a subset of AR target genes) that is primarily and highly enriched for functions in cell cycle progression and mitosis. To further our drug development efforts, we must identify and characterize the ELK1 docking site recognition sites in the AR A/B domain and the binding site of KCI807. We applied a mammalian two-hybrid assay to map the ELK1 interacting motifs in the A/B domain of AR using a series of deletion constructs. Overlapping deletion constructs were made systematically to cover the entire A/B domain. For each of these constructs additional double fusion constructs were used as controls. Expression of fusion proteins was monitored by western blot. Our results excluded a role for the amino-terminal half of the A/B domain in ELK1 interactions but did identify repressor motifs within this region. We identified two docking site recognition sites in the AR A/B domain for the two docking sites in ELK1 in a parallel configuration. The KCI807 binding site was located in the downstream site of AR. Further structural and functional studies of the mapped motifs in AR will reveal more about the biology of the AR-ELK1 interaction and directly aid in the design of next generation antagonists to selectively target ELK1-dependent growth signaling by AR in PC/CRPC tumors. Citation Format: Claire L. Soave, Rayna Rosati, Yanfang Huang, Manohar Ratnam. Elucidating the structure-function relationships in the interactions of the androgen receptor, ELK1 and the platform antagonist KCI807 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2469.

  • The ternary complex factor protein ELK1 is an independent prognosticator of disease recurrence in prostate cancer.
    The Prostate, 2019
    Co-Authors: Luke Pardy, Rayna Rosati, Claire Soave, Yanfang Huang, Seongho Kim, Manohar Ratnam
    Abstract:

    BACKGROUND Both hormone-sensitive and castration- and enzalutamide-resistant prostate cancers (PCa) depend on the ternary complex factor (TCF) protein ELK1 to serve as a tethering protein for the androgen receptor (AR) to activate a critical set of growth genes. The two sites in ELK1 required for AR binding are conserved in other members of the TCF subfamily, ELK3 and ELK4. Here we examine the potential utility of the three proteins as prognosticators of disease recurrence in PCa. METHODS Transcriptional activity assays; Retrospective analysis of PCa recurrence using data on 501 patients in The Cancer Genome Atlas (TCGA) database; Unpaired Wilcoxon rank-sum test and multiple comparison correction using the Holm's method; Spearman's correlations; Kaplan-Meier methods; Univariable and multivariable Cox regression analyses; LASSO-based penalized Cox regression models; Time-dependent area under the receiver operating characteristic (ROC) curve. RESULTS ELK4 but not ELK3 was coactivated by AR similar to ELK1. Tumor expression of neither ELK3 nor ELK4 was associated with disease-free survival (DFS). ELK1 was associated with higher clinical T-stage, pathology T-stage, Gleason score, prognostic grade, and positive lymph node status. ELK1 was a negative prognosticator of DFS, independent of ELK3, ELK4, clinical T-stage, pathology T-stage, prognostic grade, lymph node status, age, and race. Inclusion of ELK1 increased the abilities of the Oncotype DX and Prolaris gene panels to predict disease recurrence, correctly predicting disease recurrence in a unique subset of patients. CONCLUSIONS ELK1 is a strong, independent prognosticator of disease recurrence in PCa, underscoring its unique role in PCa growth. Inclusion of ELK1 may enhance the utility of currently used prognosticators for clinical decision making in prostate cancer.

  • the amino terminal domain of the androgen receptor co opts extracellular signal regulated kinase erk docking sites in ELK1 protein to induce sustained gene activation that supports prostate cancer cell growth
    Journal of Biological Chemistry, 2016
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Selvakumar Dakshnamurthy, Thomas Mcfall, Janice Saxton, Benjamin L Kidder, Peter E Shaw, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8 m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.

  • abstract 2876 the androgen receptor utilizes protein kinase docking sites to constitutively activate ELK1
    Cancer Research, 2016
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Selvakumar Dakshnamurthy, Thomas Mcfall, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for growth in established prostate cancer cell lines. This process does not entail the transient hyper-activation of immediate early genes typical of activation of ELK1 through phosphorylation by ERK1/2. We compared the structural requirements for the association of AR and ELK1 with that for activation of ELK1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with ELK1 and to activate ELK1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with ELK1 was optimal in the absence of ERK1/2 and serum response factor (SRF), which are the known binding partners of ELK1. We identified two sites on ELK1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 307 to 330, overlapping the D box, which is one of two ERK docking sites. The other site on ELK1 mapped to amino acid residues 372 to 397, overlapping the FXFP motif, which comprises the downstream ERK docking site. The results suggest that AR utilizes ERK docking sites to associate with ELK1 and is exclusive of the interaction of ELK1 with ERK1/2 or SRF. A nuclear receptor could thus function as a transcription factor co-activator by binding at protein kinase docking sites. Citation Format: Rayna Rosati, Mugdha Patki, Selvakumar Dakshnamurthy, Venkatesh Chari, Thomas McFall, Manohar Ratnam. The androgen receptor utilizes protein kinase docking sites to constitutively activate ELK1. [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 2876.

  • abstract 1855 structural requirements for the association of the ets domain transcription factor ELK1 and the androgen receptor in enabling the growth of prostate cancer cells
    Cancer Research, 2015
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for cell growth in established prostate cancer cell lines. This role of ELK1 is independent of the ability of ELK1 to transiently activate immediate early genes in response to phosphorylation by ERK. We compared the structural requirements for the association of AR and ELK1 with that for activation of ELK1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with ELK1 and to activate ELK1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with ELK1 did not require ERK1/2 or serum response factor (SRF), which are the known binding partners of ELK1. We identified two sites on ELK1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 297 to 317, overlapping one of two ERK docking sites. The other site on ELK1 mapped to amino acid residues 387 to 397, adjacent to the downstream ERK docking site. The results suggest that the association of AR with ELK1 in situ is direct rather than through the ELK1 ternary complex and that the association may be disrupted by small molecules to selectively inhibit growth signaling in prostate cancer cells. Citation Format: Rayna Rosati, Venkatesh Chari, Mugdha Patki, Manohar Ratnam. Structural requirements for the association of the ETS domain transcription factor ELK1 and the androgen receptor in enabling the growth of prostate cancer cells. [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 1855. doi:10.1158/1538-7445.AM2015-1855

Venkatesh Chari - One of the best experts on this subject based on the ideXlab platform.

  • the amino terminal domain of the androgen receptor co opts extracellular signal regulated kinase erk docking sites in ELK1 protein to induce sustained gene activation that supports prostate cancer cell growth
    Journal of Biological Chemistry, 2016
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Selvakumar Dakshnamurthy, Thomas Mcfall, Janice Saxton, Benjamin L Kidder, Peter E Shaw, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8 m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.

  • abstract 2876 the androgen receptor utilizes protein kinase docking sites to constitutively activate ELK1
    Cancer Research, 2016
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Selvakumar Dakshnamurthy, Thomas Mcfall, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for growth in established prostate cancer cell lines. This process does not entail the transient hyper-activation of immediate early genes typical of activation of ELK1 through phosphorylation by ERK1/2. We compared the structural requirements for the association of AR and ELK1 with that for activation of ELK1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with ELK1 and to activate ELK1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with ELK1 was optimal in the absence of ERK1/2 and serum response factor (SRF), which are the known binding partners of ELK1. We identified two sites on ELK1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 307 to 330, overlapping the D box, which is one of two ERK docking sites. The other site on ELK1 mapped to amino acid residues 372 to 397, overlapping the FXFP motif, which comprises the downstream ERK docking site. The results suggest that AR utilizes ERK docking sites to associate with ELK1 and is exclusive of the interaction of ELK1 with ERK1/2 or SRF. A nuclear receptor could thus function as a transcription factor co-activator by binding at protein kinase docking sites. Citation Format: Rayna Rosati, Mugdha Patki, Selvakumar Dakshnamurthy, Venkatesh Chari, Thomas McFall, Manohar Ratnam. The androgen receptor utilizes protein kinase docking sites to constitutively activate ELK1. [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 2876.

  • abstract 1855 structural requirements for the association of the ets domain transcription factor ELK1 and the androgen receptor in enabling the growth of prostate cancer cells
    Cancer Research, 2015
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam
    Abstract:

    The ETS domain transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for cell growth in established prostate cancer cell lines. This role of ELK1 is independent of the ability of ELK1 to transiently activate immediate early genes in response to phosphorylation by ERK. We compared the structural requirements for the association of AR and ELK1 with that for activation of ELK1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with ELK1 and to activate ELK1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with ELK1 did not require ERK1/2 or serum response factor (SRF), which are the known binding partners of ELK1. We identified two sites on ELK1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 297 to 317, overlapping one of two ERK docking sites. The other site on ELK1 mapped to amino acid residues 387 to 397, adjacent to the downstream ERK docking site. The results suggest that the association of AR with ELK1 in situ is direct rather than through the ELK1 ternary complex and that the association may be disrupted by small molecules to selectively inhibit growth signaling in prostate cancer cells. Citation Format: Rayna Rosati, Venkatesh Chari, Mugdha Patki, Manohar Ratnam. Structural requirements for the association of the ETS domain transcription factor ELK1 and the androgen receptor in enabling the growth of prostate cancer cells. [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 1855. doi:10.1158/1538-7445.AM2015-1855

  • abstract 2116 mapping peptides critical for association of the androgen receptor with ELK1
    Cancer Research, 2014
    Co-Authors: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam
    Abstract:

    Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Both early stage and advanced prostate tumors are generally dependent on the androgen receptor (AR) for growth. Indeed a major clinical approach in management of the disease is to disrupt the AR signaling in the tumors by androgen ablation and the use of anti-androgen drugs. However, these options present major drawbacks including limited efficacy in advanced disease and many undesirable side effects on non-target tissues. Therefore a more tactical therapy approach would be one that could disrupt a functional arm of AR signaling that is critical for prostate tumor growth but not for the essential physiological roles of AR in normal adult tissues. A considerable amount of evidence suggests that in prostate cancer, androgen/AR signaling is redirected to support tumor growth through the association of AR with critical growth promoting genes via tethering proteins. Our previous studies have identified ELK1, a genetically redundant DNA binding transcription factor, as an AR tethering protein essential for AR-dependent growth in established models of both hormone-dependent prostate cancer and castration recurrent prostate cancer. ELK1 recruits AR to chromatin sites to upregulate a major subset of genes that is strongly and primarily enriched for cell growth functions. Further studies in the lab demonstrate that the A/B domain of the AR alone is capable of co-activation of ELK1, suggesting that ELK1 may also mediate the hormone-independent growth supporting function of AR splice variants. Peptide or small molecule inhibitors of the ELK1-AR interaction may therefore selectively target the growth supporting function of AR in the spectrum of prostate tumors, obviating the need for androgen ablation. Toward this end we undertook to map peptide segment(s) of ELK1and AR that are essential for the ELK1-AR synergy. In this study we used deletional and mutational analysis together with a promotor-reporter assay and a mammalian two-hybrid assay. The results demonstrate that the ability to recruit AR resides largely in the C-terminal activation domain of ELK1 (amino acids 307-428) and that residues within segments 367-428 and 307-337 are essential for this interaction. They also demonstrate that the recruitment of AR by ELK1 does not require ELK1 phosphorylation. Critical sites within the AR-A/B domain required for its association with ELK1 reside in the N-terminal region that is distal to known sites of co-regulator binding. This information will guide the development of peptide inhibitors of ELK1-dependent growth induction by androgen/AR and set the stage for future development of small molecule inhibitors. Citation Format: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam. Mapping peptides critical for association of the androgen receptor with ELK1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2116. doi:10.1158/1538-7445.AM2014-2116

  • the ets domain transcription factor ELK1 directs a critical component of growth signaling by the androgen receptor in prostate cancer cells
    Journal of Biological Chemistry, 2013
    Co-Authors: Mugdha Patki, Venkatesh Chari, Suneethi Sivakumaran, Mesfin Gonit, Robert J Trumbly, Manohar Ratnam
    Abstract:

    The androgen receptor (AR) is essential for diverse aspects of prostate development and function. Molecular mechanisms by which prostate cancer (PC) cells redirect AR signaling to genes that primarily support growth are unclear. A systematic search for critical AR-tethering proteins led to ELK1, an ETS transcription factor of the ternary complex factor subfamily. Although genetically redundant, ELK1 was obligatory for AR-dependent growth and clonogenic survival in both hormone-dependent PC and castration-recurrent PC cells but not for AR-negative cell growth. AR required ELK1 to up-regulate a major subset of its target genes that was strongly and primarily enriched for cell growth functions. AR functioned as a coactivator of ELK1 by association through its A/B domain, bypassing the classical mechanism of ELK1 activation by phosphorylation and without inducing ternary complex target genes. The ELK1-AR synergy per se was ligand-independent, although it required ligand for nuclear localization of AR as targeting the AR A/B domain to the nucleus recapitulated the action of hormone; accordingly, Casodex was a poor antagonist of the synergy. ELK3, the closest substitute for ELK1 in structure/function and genome recognition, did not interact with AR. ELK1 thus directs selective and sustained gene induction that is a substantial and critical component of growth signaling by AR in PC cells. The ELK1-AR interaction offers a functionally tumor-selective drug target.