Abstract
Members of the steroid receptor coactivator (SRC) family are overexpressed in numerous types of cancers. In particular, steroid receptor coactivator 3 (SRC-3) has been recognized as a critical coactivator associated with tumor initiation, progression, recurrence, metastasis, and chemoresistance where it interacts with multiple nuclear receptors and other transcription factors to enhance their transcriptional activities and facilitate cross-talk between pathways that stimulate cancer progression. Because of its central role as an integrator of growth signaling pathways, development of small molecule inhibitors (SMIs) against SRCs have the potential to simultaneously disrupt multiple signal transduction networks and transcription factors involved in tumor progression. Here, high-throughput screening was performed to identify compounds able to inhibit the intrinsic transcriptional activities of the three members of the SRC family. Verrucarin A was identified as a SMI that can selectively promote the degradation of the SRC-3 protein, while affecting SRC-1 and SRC-2 to a lesser extent and having no impact on CARM-1 and p300 protein levels. Verrucarin A was cytotoxic toward multiple types of cancer cells at low nanomolar concentrations, but not toward normal liver cells. Moreover, verrucarin A was able to inhibit expression of the SRC-3 target genes MMP2 and MMP13 and attenuated cancer cell migration. We found that verrucarin A effectively sensitized cancer cells to treatment with other anti-cancer drugs. Binding studies revealed that verrucarin A does not bind directly to SRC-3, suggesting that it inhibits SRC-3 through its interaction with an upstream effector. In conclusion, unlike other SRC SMIs characterized by our laboratory that directly bind to SRCs, verrucarin A is a potent and selective SMI that blocks SRC-3 function through an indirect mechanism.
Highlights
The p160 steroid receptor coactivator (SRC) family contains three members, SRC-1[1], SRC-2/GRIP1/TIF2 [2,3] and SRC3/Amplified in Breast Cancer-1 [4] that interact with multiple nuclear receptors (NRs) and other transcription factors to regulate gene transcription
SRC-1 and steroid receptor coactivator 3 (SRC-3) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and coactivator-associated arginine methyltransferase 1 (CARM1) and SRC-2 antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). b-actin and p300 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA)
As a coactivator for NRs and many other transcription factors, SRC-3 simultaneously drives the activity of multiple cellular signal transduction pathways
Summary
The p160 steroid receptor coactivator (SRC) family contains three members, SRC-1[1], SRC-2/GRIP1/TIF2 [2,3] and SRC3/Amplified in Breast Cancer-1 [4] that interact with multiple nuclear receptors (NRs) and other transcription factors to regulate gene transcription. The C-terminal region of SRCs contains two activation domains (ADs), AD1 and AD2 that interact with other coactivators. SRC-3 overexpression has been found in multiple types of cancers, including breast [21], pancreatic [22], ovarian [23], gastric [24], prostate [25], and colorectal carcinomas [26]. SRC-3 overexpression can promote spontaneous tumor initiation and progression in an animal overexpression model system [36] Together these findings demonstrate that SRC-3 is a key oncoprotein involved in cancer initiation, progression and metastatic growth, pointing to its importance as an important target for therapy [37]. We found that verrucarin A does not detectably bind SRC-3 at its effective concentration in cell culture, implicating an upstream effector of SRC-3 as a likely target of this compound
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