Back to the early 1970s, it was discovered that non-histone proteins interact with DNA-binding nuclear receptors (NRs) to form complexes and support their functions [1]. These proteins are now known as NR coregulators, including coactivators and corepressors, to modulate gene expression in mammalian cells. The coactivator is a type of transcriptional coregulator that accomplish reactions required for activation of transcription. p160/steroid receptor coactivators (SRC) are about 160 kDa in size and consist of three members including SRC-1 (NCOA1), SRC-2 (NCOA2/TIF-2/GRIP-1) and SRC-3 (NCOA3/AIB1/ACTR/RAC-3/pCIP/TRAM-1) [2]. SRC-1 was the first NR coactivator identified by Bert W. O’Malley’s laboratory in 1995 [3]. A year later, SRC-2 was discovered as the second member of the SRC family [4]. Soon after, SRC-3 was identified on the long arm of chromosome 20 (20q12-13) known to be amplified in breast cancer and termed AIB1 (Amplified in breast cancer 1) [5,6]. SRCs were initially recognized as promoting the transcriptional activity of steroid hormone receptors, and then it was discovered that they can also modulate the activity of multiple other transcription factors.

The SRC-3 gene was first cloned from BT-474, a breast cancer cell line that contains high copy of chromosome 20 [5], five groups subsequently characterized SRC-3 as a coactivator, belonging to SRC family (AIB1, ACTR, RAC-3, pCIP, TRAM-1) [6,7,8,9,10]. In the past two decades, numerous studies have demonstrated that SRC-3 plays crucial roles in its physiology [11]. SRC-3 not only regulates body development but also maintains homeostasis. Deletion of the SRC-3 gene in mice caused growth retardation, dwarfism, abnormal mammary gland development and reproductive function [12]. Loss of SRC-3 also improved mitochondrial function via acetyltransferase GCN5-mediated peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) activity, thereby increasing basal metabolism in BAT and preventing obesity [13]. In addition, SRC-3 plays important roles in innate immunity. SRC-3 null mice exhibited severe endotoxin-induced inflammatory response through enhancing pro-inflammatory cytokine mRNA translation [14], and were more susceptible to bacterial infections [15]. Besides its physiological effects, SRC-3 is best known for its oncogenic functions.

SRC-3 has a similar structure to the other members of SRC family, including three structural and functional regions (Figure 1) [16]. At the N-terminus, the basic–loop–helix (bHLH) and Per/ARNT/Sim (PAS) domain is the well-conserved region through SRCs, which is essential for a member of protein-protein interactions and nuclear localization. The central region is regarded as receptor-interaction domain (RID) and contains three LXXLL (L, Leucine; X, any amino acid) motifs that contribute to interactions with ligand-dependent NRs. At the C-terminus, there are two transcriptional activation domains (AD1 and AD2) as well as a polyglutamate sequence (polyQ) between them. In addition to exhibiting histone acetyltransferase (HAT) activity between the two AD regions, the AD1 and AD2 can interact with HAT (CBP/p300) or histone methyltransferases (CARM1, PRMT1 and NSD2), respectively, and then promote chromatin remodeling. In summary, these structure elements of SRC-3 provide a place where allow transcription factors interact with other coregulators to regulate gene expression.

Thus far, it has been reported that there are two splice variants of SRC-3, such as SRC-3Δ3 and SRC-3Δ4. SRC-3Δ3, an SRC-3 splice isoform with a deletion of exon 3, was determined to lack the N-terminal bHLH and PAS motif [17]. SRC-3Δ3 was more potent than full-length SRC-3 in enhancing estrogen receptor (ER) and progesterone receptor (PR) signaling, and its overexpression was found in breast cancer specimens [17,18]. These findings suggest that this isoform may play a role in pathogenesis of breast cancer. Based on SRC-3Δ3 transgenic mice, overexpression of this isoform promoted mammary epithelial cell growth, and eventually resulted in mammary hyperplasia and adenocarcinoma [19,20]. Similarly, SRC-3Δ4 is another splicing isoform of SRC-3 with a deletion of exon 4, which is predominantly localized in cytoplasm due to lack of nuclear localization sequence (NLS). However, SRC-3Δ4 could enter the nucleus to promote ER signaling by a non-canonical nuclear import pathway [17,21]. SRC-3Δ4 also worked as a signaling adaptor that mediates the interaction with epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK), thus promoting cell migration in an FAK-dependent manner. This was because p21-activted kinase 1 (PAK1) induced phosphorylation of SRC-3Δ4 to promote its membrane localization, thus bridging the interaction [22]. These findings imply that SRC-3 isoforms have more effective and distinct functions to regulate the development of cancer.

The function and level of SRC-3 are regulated by diverse PTMs including phosphorylation, methylation, acetylation, SUMOylation, ubiquitination and many other modifications [23]. Studying the PTM of SRC-3 is essential for understanding how to regulate the activity and level of SRC-3. Previous reviews summarized the modified residues, the responsible modifying proteins and the relative functions of SRC-3’s PTM [24,25]. Here, we update some new findings related to phosphorylation of SRC-3. SRC-3 has multiple phosphorylation sites including seven Serine/Threonine (T24, S505, S543, S601, S857, S860 and S867) phosphorylation sites [26] and one Tyrosine site (Y1357) [27], which control its transcriptional activity, protein stability and subcellular localization. Human epidermal growth factor receptor 2 (HER-2) is amplified in about 20% of all breast cancers and is associated with endocrine resistance and poor prognosis. A recent study indicated that decreased HER-2 signaling inhibited SRC-3 phosphorylation at T24, S543, S857 and S860, and the enhanced HER-2 signaling cooperated with phosphorylated SRC-3 to promote breast cancer cell proliferation [28]. According to reports, the most common phosphorylation site of SRC-3 is S857 [29]. Recently, two signaling axes were found to regulate phosphorylation of SRC-3 at this site [30,31]. Dasgupta et al. performed a kinome-wide RNA interference-based screening to identify fuctose-2,6-bisphosphatase 4 (PFKFB4) that phosphorylates SRC-3 at S857 and promotes the intrinsic SRC-3 transcriptional activity [30]. PFKFB4 is an isoform of phosphofructokinase 2 that synthesizes fructose 2,6-bisphosphate (F2,6-BP), an important glycolysis-inducing metabolite. The phosphorylation of SRC3 in cells was induced by glucose in a PFKFBP4-dependent manner, leading to transcriptional reprogramming and aggressive metastatic tumor. This study provides evidence for insight into the metabolism of cancer cells and their preference for glycolysis [32]. Based on in vitro phosphorylation assay, Shrestha et al. indicated that MK2 was responsible for phosphorylation of SRC-3 at S857 and p38 is the upstream of MK2. Activation of the p38-MK2 signaling leaded to the nuclear translocation of SRC-3, which contributed to NF-kB activation and downstream target expression [31]. In conclusion, PTMs are critical molecular events, which change SRC-3 conformation and generate diverse SRC-3 properties by regulating its stability, localization and interacting partners, consequently exerting pivotal roles in regulating the functions of SRC-3. Unraveling the specific role of SRC-3’s PTM and the responsible proteins in carcinogenesis is potentially useful for anti-cancer therapy.

Since it was discovered in 1997 that SRC-3 is frequently amplified in breast cancer [6], extensive research has been conducted on the oncogenic role of SRC-3. SRC-3 amplification and overexpression have been found to be associated with tumor aggressiveness or poor prognosis in a number of clinical studies (Table 1), and SRC-3 promotes tumorigenesis and malignancy through multiple pathways (Figure 2). Based on previous reviews [24,33], we will summarize and update the important progress of SRC-3 relevant to cancers, including hormone-sensitive and non-hormone targeted cancers.

Since SRC-3 plays an important role in a variety of cancers, recent studies have explored how to target SRC-3 and reported that some drugs targeting SRC-3 are used in cancer treatment. Gambogic acid (GA), a main active ingredient in gamboge, exhibits effective anti-cancer activity in multiple cancer cells. Studies have reported that GA could reduce the expression of SRC-3 and inhibit the proliferation of cancer cells [155,156]. Cullin3, an E3 ligase involved in SRC-3 ubiquitination [90], was up-regulated under GA treatment, which may contribute to SRC-3 degradation [155]. These findings suggest that GA could be used as a potential inhibitor of SRC-3 for cancer therapy. Salinomycin, an antibiotic, was found to down-regulate the transcription of SRC-3 and increase the sensitivity of breast cancer cells to tamoxifen treatment, which indicates salinomycin may be developed as SRC-3 inhibitor for endocrine-resistant breast cancer [157]. However, it has not been examined whether GA and salinomycin have an effect on the expression of SRC-1 and SRC-2. Recently, it was discovered that thevebioside (THB), an active component from traditional Chinese medicine, specifically suppressed the expression of SRC-3 via ubiquitin–proteasome-mediated degradation, but not SRC-1 and SRC-2, thus inhibiting the proliferation of NSCLC cells [158].

To repurpose the chemicals for targeting SRC-3, a high throughput screening assay was performed according to PubChem bioassay bioinformatic resource. 2,2-bis-(Formyl-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene (gossypol) was identified as directly binding to SRC-3 via its RID domain, and down-regulating the protein level of SRC-3 in multiple cancer cell lines through a proteasome-independent pathway [159]. In addition, the cardiac glycoside bufalin was found be as a potent small-molecule inhibitor for SRC-3 through protein degradation [160]. Bufalin not only inhibited tumor growth via p53-mediated senescence [117,160,161], but also improved the sensitivity of cell cells to HDAC inhibitors [162]. However, gossypol and bufalin also affected the expression of SRC-1 [159,160], indicating they are not specific inhibitors of SRC-3. In addition, the cardiotoxicity of bufalin limits its clinical application. Then, Verrucarin A has been determined to selectively promote the degradation of the SRC-3 protein to inhibit proliferation and migration of cancer cells but has little effect on SRC-1 and SRC-2 [163]. Since Verrucarin A has no direct interaction with SRC-3, it may affect the upstream pathway to regulate SRC-3 degradation [163], which cannot rule out the impact on other proteins. Recently, SI-2 was developed as a new and effective small-molecule inhibitor for SRC-3 [164]. SI-2 can reduce the protein level of SRC-3 as well as directly interact with SRC-3 to inhibit its transcriptional activities. Based on toxicity assay, no acute or chronic toxicity of SI-2 was observed in vivo. Therefore, SI-2 is considered as a highly promising inhibitor for SRC-3 and high potential candidate for developing an anti-cancer drug [52,118,153,164,165,166,167,168]. Besides identifying chemicals used to block the activity of SRC-3, recent studies have also developed new methods for targeting SRC-3. Since cancer cells heavily depend on SRCs to maintain homeostasis, excessive SRCs activation may be able to disrupt the critical homeostasis, leading to stress accumulation and cell death [169]. MCB-613 was discovered as an effective SRC stimulator. It caused hyper-activation of SRCs including SRC-3 and resulted in cancer cell death through uncontrolled ER stress and excessive ROS [169]. Therefore, super-activation of SRC-3 is a potential alternative strategy for cancer treatment. Aptamers are single-stranded oligonucleotides that bind to a specific target molecule. They are widely used for targeting molecules due to many advantages, such as high affinity and specificity, low toxicity and immunogenesis, rapid and reproducible synthesis, etc. [170]. Based on SELEX (Systematic evolution of Ligands by Exponential Enrichment), it was found that a DNA aptamer AY-3 interacted with SRC-3 and impaired the interaction between SRC-3 and p300 [171]. It suggests that AY-3 may inhibit the activity of SRC-3 and become a potential candidate for developing an SRC-3 inhibitor. In addition, to silence SRC-3 expression by RNAi technology in vivo, nanoparticles have been developed as carriers to deliver siRNAs against SRC-3 [172,173]. The findings present that nanoparticles are a promising system for effective delivery of siRNA to down-regulate SRC-3 expression and inhibit tumor growth. In short, as technology advances, more drugs and methods will be developed to inhibit SRC-3 activity and treat cancers.