Abstract LB-211: The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in prostate cancer
Authors: Ben-Salem Salma , Senapati Dhirodatta , Mudduluru Giridhar , Su Yixue , Venkadakrishnan Varadha Balaji , Heemers Hannelore V. 
Published: August 2018

Ligand-activated androgen receptor (AR) drives prostate cancer (CaP) growth. Heterogeneity in AR action exists and AR often remains active when therapies targeting its ligand-activation have failed. Understanding the mechanisms by which AR mediates CaP progression may lead to new treatments. Previously, we identified a mechanism in which AR imparts androgen-responsiveness to the transcription factor Serum Response Factor (SRF). SRF-dependent AR action is enriched in CaP, correlates with CaP recurrence and is maintained in late-stage CaP. SRF binds constitutively at its target genes and becomes transcriptionally active via upstream signaling cascades or SRF cofactors. Our prior work showed that RhoA signaling transduces androgen regulation to a significant fraction, but not all, SRF target genes. Here, we isolate the homeodomain protein Nkx2-5 as a novel regulator of androgen-responsive SRF action. Nkx2-5 co-operates with SRF in transcriptional control of heart development. In CaP, Nkx2-5 promoter hypermethylation is a biomarker that distinguishes CaP from benign prostate, yet consequences of differential Nkx2-5 expression on CaP are unknown. In the CaP cell models LNCaP, C4-2 and VCaP, siRNA-mediated loss of Nkx2-5 increased basal and androgen-dependent expression of exogenously and endogenously expressed SRF target genes, and these effects were reversed after silencing of SRF. Conversely, overexpression of Nkx2-5 decreased androgen-responsiveness of SRF target genes. Co-IP and ChIP assays indicated androgen-stimulated SRF-Nkx2-5 interaction in CaP cell nuclei. Loss of Nkx2-5 resulted in distinct morphological changes in CaP cells, which depended entirely on SRF. In rhodamine phalloidin staining assays, loss of Nkx2-5 increased significantly (p&lt;0.01) cell length, width and circumference, and the number of cell protrusions in cultures with and without androgen stimulation. Changes in cell shape were accompanied by modest decreases in CaP cell proliferation and migration, but significant (p&lt;0.05) decreases in CaP cell-matrix adhesion. Nkx2-5 silencing increased the size of the cell nucleus and led to multilobed and grooved nuclei and enlarged nucleoli. These nuclear shape changes were not associated with alterations in DNA ploidy, but were associated with changed heterochromatin organization as reflected in a reduction in heterochromatin protein 1 immunostaining and nuclear content. Such Nkx2-5-dependent changes in nuclear shape and size and heterochromatin organization occurred also in non-malignant prostate epithelial RWPE2 cells. This work uncovered a novel role for Nkx2-5 in the transition from benign prostate to CaP, showing that Nkx2-5 regulates SRF transcriptional activity in CaP cells and demonstrating that differential Nkx2-5 expression alters cell-matrix adhesion and nuclear morphology, both processes that are associated with aggressive CaP progression.

Citation Format: Salma Ben-Salem, Dhirodatta Senapati, Giridhar Mudduluru, Yixue Su, Varadha Balaji Venkadakrishnan, Hannelore V. Heemers. The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in 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 LB-211.