Despite the efficacy of AIs in the treatment of breast cancer, tumors can develop resistance to estrogen deprivation and relapse with recurrent disease. To explore mechanisms involved in the development of AIR
, we recently discovered that expression of a naturally-occurring K303R ERα mutation in MCF-7 breast cancer cells conferred resistance to the non-steroidal AI anastrozole (Barone et al., 2009
). This somatic mutation (A908G) has been identified in about a third of premalignant lesions, and one-half of invasive breast tumors (Fuqua et al., 2000
; Herynk et al., 2007
), although using another detection method the mutation was identified in only 6% of tumors (Conway et al., 2005
). This transition introduces a lysine to arginine substitution at residue 303 (K303R) within the hinge domain. The region between the DNA-binding and the ligand-binding domains, known as the hinge, has long been considered to simply serve as a flexible linker to orient these functional domains. However, it has now shown that this region is a multifunctional domain that binds co-regulatory proteins, participates in DNA binding, and undergoes post-translational modifications (Cui et al., 2004
; Haelens et al., 2007
; Kim et al., 2006
). Naturally-occurring mutations have been also identified in the hinge of the androgen receptor in prostate cancer, and the progesterone receptor in ovarian cancer. These mutations affect acetylation or phosphorylation of the hinge, and result in hyper-active receptors with enhanced hormone sensitivity (Agoulnik et al., 2004
; Deeb et al., 2008
; Romano et al., 2006
). Our studies also suggest a new critical role for the hinge domain of the K303R ERα mutant receptor. We reported that overexpression of K303R mutation in MCF-7 breast cancer cells conferred increased sensitivity to subphysiological levels of estrogen, and decreased sensitivity to Tam when engaged in cross-talk with growth factor receptor signaling (Fuqua et al., 2000
; Giordano et al., 2009
). We also demonstrated that K303R ERα expression conferred resistance to the AI anastrozole in ERα-positive cells also co-expressing human aromatase; resistance occurred via activation of the PI3K/Akt prosurvival signaling pathway to which the mutant cells had become “addicted” for maintenance of growth (Barone et al., 2009
). In this study, we investigated the role of a post-translational modification of the hinge region of the K303R mutant receptor as a determinant of resistance to AI, and as a possible therapeutic target to reverse resistance.
Additional biomarkers to identify patients who are resistant to AIs are lacking in clinical practice. Thus, a better understanding of resistance mechanisms may enable accurate prediction of response, which is relevant since alternative therapeutic strategies are available. To identify genes whose expression were associated with the development of AIR, we performed expression microarray analyses, and found increased expression of several IGF activators, such as IRS-1, and decreased expression of specific IGF inhibitors, such as IGFBP3 in the mutant cells. This altered gene expression resulted in enhanced phosphorylation and activation of IGF signaling molecules, and increased sensitivity to IGF-1 stimulation. We also found an enhanced association between the mutant receptor and IGF-1R, consistent with our earlier observations of enhanced binding of the mutant receptor to other regulatory molecules. This enhanced binding along with the increased ligand-independent transcriptional activity of the mutant we previously reported may help to sustain enhanced bidirectional cross-talk between IGF-1R and K303R ERα, further augmenting signaling between these important growth pathways.
Studies have shown that increased signaling via IGF-1R with consequent activation of IRS-1 and the PI3K/Akt survival pathway leads to resistance to Tam and AI treatments (Campbell et al., 2001
; Santen et al., 2005
; Wiseman et al., 1993
). Indeed, IGF-1R inhibitors have been used to inhibit breast tumor growth and metastasis (Arteaga and Osborne, 1989
; Burtrum et al., 2003
; Sachdev et al., 2004
). We found that an IGF-1R inhibitor, AG1024, completely suppressed the proliferation of mutant-expressing cells, and the combination of an AI with AG resulted in a greater growth inhibition. These results suggest that the persistence of active IGF-1R signaling may be a functional mechanism of resistance to AI therapies in cells expressing the mutant receptor.
ERα is known to be a target of several post-translational modifications (Faus and Haendler, 2006
). ERα phosphorylation results from the activation of various cellular kinases and substantially alters its function. For example, ERα can be differentially phosphorylated by Akt and extracellular regulated kinase (Erk
1/2) MAPK resulting in diverse responses to ligand (Likhite et al., 2006
). Recently, we and others have shown that PKA and PAK1-mediated phosphorylation of ERα S305 impacted both estrogen hypersensitivity and Tam response (Cui et al., 2004
; Michalides et al., 2004
; Rayala and Kumar, 2007a
). Our search for kinase consensus sequences identified the S305 site as a novel Akt phosphorylation site in the mutant receptor. Although the target site sequence around S305 is not a perfect match to the consensus target site for Akt (R-x-R-x-x-S/T) (Manning and Cantley, 2002
), it is known for other protein kinases that the inclusion of additional amino acids of the same charge in this target site sequence enhanced substrate activity (Brenet et al., 2009
). In addition, Akt may phosphorylate substrates efficiently which conform less rigorously to the requirement for arginine at positions n
-3 and n
-5 (Ksiezak-Reding et al., 2003
). In vitro
Akt kinase assays demonstrated that the K303R ERα mutant was more efficiently phosphorylated compared to WT ERα, and phosphorylation of the K303R mutant receptor at S305 was comparable with S167 phosphorylation. Both mutation of S305 to A to remove the phosphorylation site, or the use of a selective blocking peptide mimicking the sequence of ERα surrounding S305, prevented in vitro
phosphorylation by Akt. We previously showed that the mutation leads to enhanced constitutive phosphorylation of Akt, and subsequent phosphorylation of the Akt S167 site within ER (Barone et al., 2009
). Here we show that activated Akt can also elicit effects on phosphorylation of S305 in the mutant receptor, and that this phosphorylation increased its ligand-independent activity. To study the involvement of S305 phosphorylation in the AIR
phenotype associated with the K303R mutation, we generated stable transfectants expressing exogenous K303R/S305A receptor. Double mutant-expressing cells exhibited soft agar growth similar to WT cells, showing a significant reduction of the colony numbers compared to K303R-expressing cells, no increase in anchorage-independent growth under low doses of IGF-1, and a marked sensitivity to anastrozole. These results suggested a prominent role for the S305 residue in mediating the mutant-resistant phenotype.
Others have synthesized small antagonistic peptides capable of interfering with signaling pathway activation (Fontenot et al., 2007
; Rayala et al., 2006
; Varricchio et al., 2007
) Nakajima et al. demonstrated that a five amino acid peptide inhibited ERBB2 signaling, and proliferation equivalent to treatment with the ERBB2-antagonist trastuzumab (Nakajima et al., 2008
). The S305 peptide used in this study blocked heregulin-induced S305 ERα phosphorylation, and prevented downstream phosphorylation events in K303R-expressing cells (Giordano et al., 2009
). Thus, the contribution of the S305 site to the AIR
phenotype associated with the K303R mutation was also evaluated using the S305 blocking peptide. We found specific inhibition of IGF-1R/IRS-1/Akt phosphorylation, and a significant reduction in basal and IGF-1-stimulated growth in the presence of the S305 peptide in mutant-expressing cells. Importantly, inhibition of IGF-1R and the mutant receptor cross-talk by blocking S305 phosphorylation reversed resistance in the mutant cells. Our studies suggest that the K303R/S305 ERα residues play a crucial role in ERα action in breast cancer by enhancing cross-talk between IGF-1R and the mutant receptor which leads to AIR
. We propose a model in which the mutation enhances the ability of the receptor to bind to IGF-1R and activates IRS-1/Akt downstream signaling pathway. Activated Akt in turn phosphorylates ERα at S305, and increases the transcription of IGF signaling molecules, thus establishing a positive feedback loop between growth factor signaling and ERα to bypass normal endocrine controls and responsiveness ().
Figure 7 Model depicting potential cross-talk between the ERα and IGF-signaling components in breast cancer cells expressing the mutation. K303R ERα mutant receptor binds to IGF-1R and activates the IGF-1R signaling cascades, including IRS-1 and (more ...)
The role of S305 phosphorylation in AIR represents a novel mechanism. We speculate that blockade of this phosphorylation site may be a new therapeutic strategy to block ERα hyperactivation and overcome resistance in mutation-positive patients that are resistant to aromatase inhibitor therapy.