Regulation of IMP3 by EGFR signaling and repression by ERβ: implications for triple-negative breast cancer

Oncogene (2012) 31, 4689 - 4697 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Regulation of IMP3 by EGFR signaling and repression by ERb: implications for triple-negative breast cancer S Samanta1, VM Sharma1, A Khan2 and AM Mercurio1 Insulin-like growth factor II (IGF-II) mRNA-binding protein 3 (IMP3) is emerging as a useful indicator of the progression and outcome of several cancers. IMP3 expression is associated with triple-negative breast carcinomas (TNBCs), which are aggressive tumors associated with poor outcome. In this study, we addressed the hypothesis that signaling pathways, which are characteristic of TNBCs, impact the expression of IMP3 and that IMP3 contributes to the function of TNBCs. The data obtained reveal that IMP3 expression is repressed specifically by estrogen receptor b (ERb) and its ligand 3bA-diol but not by ERa. Epidermal growth factor receptor (EGFR) signaling and consequent activation of the mitogen-activated protein kinase pathway induce IMP3 transcription and expression. Interestingly, we discovered that the EGFR promoter contains an imperfect estrogen response element and that ERb represses EGFR transcription. These data support a mechanism in which ERb inhibits IMP3 expression indirectly by repressing the EGFR. This mechanism relates to the biology of TNBC, which is characterized by diminished ERb and increased EGFR expression. We also demonstrate that IMP3 contributes to the migration and invasion of breast carcinoma cells. Given that IMP3 is an mRNA-binding protein, we determined that it binds several key mRNAs that could contribute to migration and invasion, including CD164 (endolyn) and MMP9. Moreover, expression of these mRNAs is repressed by ERb and enhanced by EGFR signaling, consistent with our proposed mechanism for the regulation of IMP3 expression in breast cancer cells. Our findings show that IMP3 is an effector of EGFR-mediated migration and invasion and they provide the first indication of how this important mRNA-binding protein is regulated in cancer. Oncogene (2012) 31, 4689 -- 4697; doi:10.1038/onc.2011.620; published online 23 January 2012 Keywords: breast cancer; IMP3; ERb; EGFR INTRODUCTION IMP3 is a member of a family of insulin-like growth factor II (IGF-II) mRNA-binding proteins (IMPs), consisting of IMP1, IMP2 and IMP3.1 IMPs have important roles in RNA trafficking, stabilization, localization and cell migration, especially during early stages of both human and mouse embryogenesis.2 IMPs are expressed in developing epithelium, placenta and muscle, but they are undetectable in normal adult tissues.3 Importantly, however, IMP3 is re-expressed in several malignant tissues, including pancreatic, lung, renal cell, ovarian, endometrial and cervical cancers.4 - 9 This phenomenon has been exploited for the prognostic assessment of specific cancers. In particular, IMP3 is an accurate predictor of renal-cell carcinoma metastasis and prognosis,4 and similar trends are emerging for other cancers.10 - 12 We are interested in breast cancer in this context, especially in light of the finding that IMP3 is expressed preferentially in triplenegative breast cancers.10 Triple-negative breast cancer (TNBC) is a molecular subtype of breast cancer characterized by the absence of estrogen receptor-a (ERa), progesterone receptor and human epidermal growth factor receptor-2 amplification. Clinically, TNBCs are usually of high histological grade, poorly differentiated and more aggressive compared with other subtypes of breast cancer.13 In fact, treatment of TNBC patients remains a challenge because of the lack of targeted therapeutic options and the high metastatic potential of TNBC cells. These observations indicate that IMP3 could prove useful for the clinical management of TNBC but much remains to be learned about its regulation and function in these tumors. In this study, we addressed the hypothesis that signaling pathways, which are characteristic of TNBCs, impact the expression of IMP3 and that IMP3 contributes to the function of TNBCs. A salient feature of TNBC is the absence of or diminished ER signaling. More specifically, TNBCs are ERa negative and they express low levels of ERb.14 A reasonable possibility based on these observations is that ER signaling represses IMP3. Interestingly, our data reveal that ERb and its ligand 3bA-diol (5aAndrostane-3b, 17b-diol) repress IMP3 expression but that ERa does not contribute to this repression. Further analysis demonstrated that epidermal growth factor receptor (EGFR) signaling induces IMP3 expression and that ERb represses EGFR transcription, revealing a mechanism in which ERb inhibits IMP3 expression indirectly by repressing the EGFR. This finding is relevant to the biology of TNBC because these tumors are characterized by overexpression of the EGFR and concomitant EGFR signaling.15 Our observation that IMP3 contributes to the migration and invasion of TNBC cells provides a functional role for this IMP in breast cancer, and it suggests that IMP3 can be an effector of EGFR-mediated migration and invasion. RESULTS ERb suppresses IMP3 expression To assess the possible role of ERs in regulating IMP3 expression, we used MDA-MB-231 and MDA-MB-468 cells, which are TNBC cell lines that express IMP3 but lack ERa and express low levels of ERb. Exogenous expression of ERa in MDA-MB-231 cells did not alter 1 Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA and 2Department of Pathology, UMass Memorial Medical Center, Worcester, MA, USA. Correspondence: Dr AM Mercurio, Department of Cancer Biology, University of Massachusetts Medical School, 364 Plantation Street, Lazare Research Building, Worcester, MA 01605, USA. E-mail: Received 25 September 2011; revised 17 November 2011; accepted 1 December 2011; published online 23 January 2012 Regulation of IMP3 in breast cancer S Samanta et al IMP3 expression significantly compared with IgG-treated cells (Figure 2a). This Ab also diminished EGFR phosphorylation and MAP (mitogen-activated protein) kinase activation, which is a bona fide downstream effector of the EGFR signaling pathway18 (Figure 2a). To substantiate the involvement of EGFR signaling pathway in regulating IMP3 expression, we inhibited MEK1/2 (upstream component of MAPK) using two different inhibitors (PD98059 and U0126). As shown in Figure 2b, IMP3 mRNA and protein expression is reduced significantly upon treatment with these inhibitors. Similar results were obtained using MDA-MB-468 cells (Figure 2c). We also assayed the activity of the IMP3 promoter in the presence of the MEK1/2 inhibitors using a reporter construct containing the human IMP3 proximal promoter. As shown in Figure 2d, inhibition of MEK1/2 using either U0126 or PD98059 decreased luciferase activity considerably. Collectively, our data indicate that an EGFR/MEK/MAPK pathway regulates IMP3 expression. IMP3 expression (Figure 1a). A (...truncated)