The Small Molecule Antagonist KCI807 Disrupts Association of the Amino-Terminal Domain of the Androgen Receptor with ELK1 by Modulating the Adjacent DNA Binding Domain.

The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.

Identification of ELK1 interacting peptide segments in the androgen receptor.

Prostate cancer (PCa) growth requires tethering of the androgen receptor (AR) to chromatin by the ETS domain transcription factor ELK1 to coactivate critical cell proliferation genes. Disruption of the ELK1-AR complex is a validated potential means of therapeutic intervention in PCa. AR associates with ELK1 by coopting its two ERK docking sites, through the amino-terminal domain (A/B domain) of AR. Using a mammalian two-hybrid assay, we have now functionally mapped amino acids within the peptide segments 358-457 and 514-557 in the A/B domain as required for association with ELK1. The mapping data were validated by GST (glutathione S-transferase)-pulldown and BRET (bioluminescence resonance energy transfer) assays. Comparison of the relative contributions of the interacting motifs/segments in ELK1 and AR to coactivation of ELK1 by AR suggested a parallel mode of binding of AR and ELK1 polypeptides. Growth of PCa cells was partially inhibited by deletion of the upstream segment in AR and nearly fully inhibited by deletion of the downstream segment. Our studies have identified two peptide segments in AR that mediate the functional association of AR with its two docking sites in ELK1. Identification of the ELK1 recognition sites in AR should enable further structural studies of the ELK1-AR interaction and rational design of small molecule drugs to disrupt this interaction.

ELK1-induced upregulation of long non-coding RNA MIR100HG predicts poor prognosis and promotes the progression of osteosarcoma by epigenetically silencing LATS1 and LATS2.

Osteosarcoma (OS) is the commonest malignant bone tumor in the world. High incidence of OS has gradually become a social problem. Recent years, numerous studies have revealed that long non-coding RNAs (lncRNAs) are crucial regulators in the tumor progression. As a member of lncRNA family, MIR100HG has been reported to be an oncogene in breast cancer and acute megakaryoblastic leukemia. Nevertheless, the specific role of MIR100HG in osteosarcoma is still unclear. In this study, we investigated the biological function and molecular mechanism of MIR100HG in the progression of osteosarcoma. At first, we measured the high expression of MIR100HG in OS tissues and cell lines by qRT-PCR. Kaplan-Meier method revealed that high expression of MIR100HG is a factor for the poor prognosis of OS patients (P = 0.004). To explore the effect of MIR100HG on the biological processes of OS, loss-of-function assays were conducted in OS cells. Functionally, MIR100HG knockdown suppressed cell proliferation, cell cycle progression while promoted cell apoptosis. Mechanistically, MIR100HG was upregulated by the transcription factor ELK1. The upregulation of MIR100HG led to the inactivation of Hippo pathway. Furthermore, we found that MIR100HG inactivated Hippo pathway in OS cells by epigenetically silencing LATS1 and LATS2. Rescue assays demonstrated that LATS1/2 involved in MIR100HG-mediated OS progression. In summary, our study indicated that ELK1-induced upregulation of MIR100HG promoted OS progression by epigenetically silencing LATS1 and LATS2 and inactivating Hippo pathway.