Background: Mechanisms that redirect androgen receptor signaling to primarily support prostate tumor growth are poorly understood. Results: Prostate cancer cells were addicted to ELK1, which tethered AR to activate growth genes in hormone-dependent and castration-recurrent PC without ELK1 phosphorylation. Conclusion: ELK1 directs a critical arm of transcriptional growth signaling by AR that is preserved in CRPC. Significance: The ELK1-AR interaction offers a functionally tumor-selective drug target.

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.

Retinoic acid receptor alpha (RARα) selective compounds may guide the design of drugs that can be used in conjunction with hormonal adjuvant therapy in the treatment of breast cancer. Herein we report a modified synthesis of a known RARα antagonist, 2-fluoro-4-[[[8-bromo-2,2-dimethyl-4-(4-methylphenyl)chroman-6-yl]carbonyl]amino]benzoic acid and a synthesis of its unknown, desfluoro analog, 4-[[[8-bromo-2,2-dimethyl-4-(4-methylphenyl)chroman-6-yl]carbonyl]amino]benzoic acid. The modified route allows for facile reaction workups, increased yields, lower cost and incorporates a green alternative step. Structure–activity relationship studies determined through functional cell-based assays, demonstrated antagonism to RARα for both compounds. Molecular modeling within the RARα binding pocket was used to compare binding interactions of the desfluoro analog to a known RAR antagonist.

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.

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 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

Genetic or epigenetic changes must occur and be propagated for breast cancer cells to develop resistance to hormonal adjuvant therapies. This would require a basal level of cell cycling during hormone depletion or tamoxifen treatment. We have previously demonstrated that the retinoic acid receptor α1 (RARα1) apoprotein supports a basal level of proliferation in breast cancer cells that are either hormone depleted or treated with tamoxifen. This action of RARα1 was ligand-insensitive (reflecting a nonclassical mechanism) and involved the activation of growth supporting genes by the RARα1 apoprotein. As RARα1 is genetically redundant, we hypothesize that including targeted downregulation of RARα1 will improve the outcome of hormonal adjuvant therapy in breast cancer. Tissue array analysis indicated prevalence of RARα1 in clinical breast tumors covering a wide pathologic spectrum. We have synthesized a high affinity RARα-specific antagonist, AGN194301, as a means of selectively targeting RARα. AGN194301 did not affect the ability of RARα1 to support basal cell proliferation and did not alter the level of RARα1 protein. Preliminary SAR studies showed the positions at which the compound could be modified without abrogating its ability to bind to RARα. Further studies and strategies for attaining downregulation of RARα1 using modified versions of this compound will be discussed. Citation Format: Venkatesh V. Chari, Robert J. Trumbly, Marcela d'A Salazar, Manohar Ratnam. RARα1 as a novel drug target to improve conventional hormonal adjuvant therapy of breast cancer. [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 4757. doi:10.1158/1538-7445.AM2013-4757