**2.2 The AIB1**Δ**4 isoform**

*Oncogenes and Carcinogenesis*

**2. Structure of the oncogene**

**2.1 The nuclear coactivator (NCOA) family**

The NCOA/SRC/p160 co-activator family consists of NCOA1 (also called SRC-1), NCOA2 (also called SRC-2, GRIP1, or TIF2), and AIB1 (also called SRC-3, NCOA3, ACTR, TRAM-1, and RAC-3). The three family members share ~55% sequence homology, and common to all members is a bHLH-PAS domain, nuclear receptor interaction domains, and C-terminal activation domains (reviewed in [12]). As a result, the members of the NCOA family interact with many different transcription factors, nuclear receptors, and chromatin modifying enzymes. Apart from mediating transcription factor interactions (detailed in Section 6 of this review), the homologous domains and residues are critical to protein stability and turnover. The bHLH-PAS domain contains residues essential for proteasomal mediated protein turnover (K17 and R18) as well as a nuclear localization signal; site-directed mutagenesis of these residues promote protein hyperstability [13]. Though the bHLH-PAS domain shares homology to DNA recognizing motifs found in both Drosophila and Humans, the NCOA family members do not directly bind the DNA, but rather facilitate transcriptional activity of binding partners [14]. The bHLH-PAS domain mediates the NCOA members' interaction with transcription factors, repressors, and coactivators, but is lost in the clinically relevant Δ4 isoform (see Section 2.2). The middle region of the NCOA family proteins contains three a-helical LXXLL motifs (NR Boxes) that mediate nuclear receptor (NR) recognition and specificity [15, 16]. The affinity of the NCOA is both dependent on the individual nuclear receptors, as well as the specific bound ligands [17]. Interaction with nuclear receptors, including the well understood interaction with the Estrogen Receptor, is detailed in Section 4. The C-terminus contains two activation domains, as well as a glutamine-rich region. These activation domains are critical to the recruitment of chromatin remodeling enzymes CBP/p300, P/CAF, CARM1, and PRMT1 [2, 18]. Similar to the bHLH-PAS domain, the C-terminal activation domains are critical for a variety of transcription factor interactions. Furthermore, SRC-1 and AIB1 both have minimal acyltransferase activity, associated with their C-terminal activation domains, which function as histone acetyltransferases [2, 19]. **Figure 1** illustrates the structure of AIB1 and some of its known binding partners.

*AIB1 interacts with many proteins through its independent domains. Line diagram showing some of the proteins that AIB1 and AIB1Δ4 interact with in a cell. Bolded proteins are described in the text of this chapter. Critical residues for phosphorylation (yellow) and ubiquitination (blue) are shown. bHLH = basic helix-loop-helix* 

*domain; PAS = PER-ARNT-SIM domain. AD = activation domain.*

**50**

**Figure 1.**

We have identified an isoform of the AIB1 oncogene that lacks the N-terminal bHLH-PAS A domain, originally designated Δ3, now called AIB1Δ4. Alternative splicing results in the loss of exon 4, thereby shifting the start codon to exon 7 and forming a N-terminal truncated, yet functional protein, missing 224 amino acids [20, 21]. As the isoform retains its nuclear receptor interacting domain, poly-Q region, and its C-terminal activation domains, the isoform can localize to estrogen response elements in the nucleus and recruits CBP/p300 to activate hormone dependent transcription [22]. Moreover, Δ4 isoform can also utilize its C-terminal activation domains, meaning that it can still co-activate many of hormone receptors and transcription factors on which it usually acts (see Sections 4 and 6). While retaining the ability to perform a significant portion of its normal activity, AIB1Δ4 cannot bind an endogenous repressor, ANCO1, and this may contribute to a significant portion of its hyperactivity [23, 24].

AIB1Δ4 has been shown to be highly responsive to growth factor and estrogen signaling. While much of this chapter will be devoted to the role of AIB1 on promoting transcription, it can also act as a key player in repression complexes. The ANCO1 repressor binds and represses the AIB1-ER complex on an estrogen response element in the Her2 gene body. This mechanism allows for estrogen mediated repression of the Her2 gene, as AIB1 in turn recruits histone deacetylases to the site to suppress transcription. The AIB1Δ4 isoform is not repressed by this mechanism, as it lacks the bHLH-PAS domain that ANCO1 requires as a binding interface, and thus maintains transcription of the Her2 growth factor receptor. As a result, the AIB1Δ4 isoform enhances growth factor signaling input via increasing the mRNA levels of the receptor [24].

Interestingly, by lacking the N-terminal bHLH-PAS domain containing the nuclear localization signal, the Δ4 isoform localizes both to the nucleus and cytoplasm. In the cytoplasm, the isoform interacts with both EGFR and FAK to promote cell migration through promotion of Src phosphorylation. The bHLH-PAS domain generally acts as an inhibitor of this interaction [25]. Knockdown of AIB1 in ovarian cells also results in a mislocalization of FAK, which may be due to loss of the isoform [26].

The role of AIB1Δ4 has been validated in vivo. AIB1Δ4 has been shown to increase mammary cell proliferation in tissue specific genetically engineered mouse models, potentially by increasing levels of IGF-1R levels on the cell surface [27]. Overexpression of the isoform resulted in similar hyperplasia as compared to the overexpression of the full-length protein, however it further presented with an increase in progesterone receptor signaling and cell cycle promoting cyclin levels. Compared to the full-length protein, AIB1Δ4 acted synergistically with ERα to promote mammary gland stromal and epithelial hyperplasia [28]. The isoform is not only seen in estrogen dependent breast cancer, as AIB1Δ4 expression levels are increased in metastatic triple negative (hormone-independent) and pancreatic cancer cell lines, compared to their isogenic parental lines [22]. More data is needed to explain the complete function of AIB1Δ4, which clearly plays a role in normal physiology, and may contribute to disease progression and invasion.
