In this study, we present the first report of IRS-2 expression in normal human breast and breast tumors. In the normal breast, IRS-2 is expressed strongly in the myoepithelial cell layer, with a lower level of diffuse cytoplasmic staining in the luminal epithelial cells. This expression pattern persists in benign breast disease. In invasive breast tumors, IRS-2 is localized in one of three staining patterns: diffusely cytoplasmic, punctate in the cytoplasm, and at the plasma membrane. IRS-2 is absent from the nucleus in both normal and tumor tissue. With regard to clinical relevance, IRS-2 membrane staining is associated with decreased OS of breast cancer patients. In addition, IRS-2 membrane staining identifies a sub-population of patients with PR− tumors that have significantly worse OS outcomes. Taken together, our results demonstrate that IRS-1 and -2 have distinct intracellular localization patterns in human breast tumors, and they reveal a potential role for IRS-2 in the aggressive biology of PR− breast tumors.
Our observation that IRS-1 and -2 are expressed in distinct intracellular compartments reveals a potential mechanism for their divergent roles in breast cancer [12
]. The targeting of IRS-1 and -2 to unique intracellular compartments would localize the signals that are generated and determine access of these adaptor proteins to distinct subsets of downstream effectors. As a result, different functional outcomes would occur. In the nucleus, IRS-1 interacts with ER-α
and regulates its transcriptional activity [29
]. IRS-1 can also interact with β
-catenin, the androgen receptor, and upstream binding factor-1 to positively regulate target gene expression [35
]. Regulation of genes such as Cyclin D and c-Myc is likely to contribute to the IRS-1-dependent stimulation of proliferation [35
]. In contrast, IRS-2 is excluded from the nucleus and instead can be found in the cytoplasm or at the cell membrane in many invasive breast carcinomas. The localization of IRS-2 at or near the cell membrane would provide access to downstream effectors that are involved in regulating dynamic adhesive and cytoskeletal rearrangements that are required for cell movement [38
]. Membrane recruitment of IRS-2 would also localize its signaling to regulate the surface expression of glucose transporter 1 (GLUT1) to promote aerobic glycolysis [17
Our finding that membrane localization of IRS-2 is associated with poor prognosis supports the hypothesis that IRS-2-mediated signaling promotes tumor progression and metastasis [10
]. Upon ligand stimulation, the IRS proteins are recruited to upstream receptors where they are phosphorylated on tyrosine residues and initiate signaling [8
]. We hypothesize that IRS-2 at the cell membrane is more likely to be tyrosine phosphorylated and actively signaling than the population of IRS-2 that is diffusely expressed in the cytoplasm because the upstream activating receptors are present at the cell membrane. In support of this hypothesis, diffuse localization of IRS-2 was associated with improved survival outcomes in patients with PR− tumors in our study. The population of punctate IRS-2 may result from internalization of the adaptor proteins with surface receptors [39
]. However, the question of whether receptor internalization would enhance or attenuate signaling remains to be determined. Generation of phosphospecific antibodies that can distinguish “active” from “inactive” IRS-2 will be necessary for future studies to confirm the functional status of the membrane, diffuse cytoplasmic, and punctate populations of IRS-2. It will also be important to investigate the expression and activity of upstream regulatory receptors, such as the IGF-1R or insulin receptor, to establish their connection with IRS-2 localization.
For tumors exhibiting membrane staining, those that are PR− demonstrate the worst OS outcomes. Loss of PR expression during disease progression, especially following endocrine therapy, is associated with decreased survival [41
]. In general, loss of PR expression is indicative of a more aggressive tumor behavior. To date, a biological explanation for the increased aggressiveness associated with PR− tumors has not been fully elucidated. The loss of PR expression is thought to represent a down-regulation of ER signaling, a pathway that positively regulates IRS-1 [45
]. Previous studies in our lab have shown that loss of IRS-1 results in upregulation of IRS-2 expression and function and promotes tumor progression [12
]. In PR− tumors, downregulation of IRS-1 function due to absent ER activity would enhance IRS-2 signaling, leading to increased metastatic potential and risk of death. The fact that we did not observe significant correlations between ER expression, IRS-2 membrane staining, and survival may be due to the fact that many of the ER+ tumors in our dataset were PR−, indicating that the ER pathway was not active [46
]. Alternatively, loss of PR expression may be the result of enhanced IGF-1R/IRS-2 signaling through PI3K/Akt/mTOR, which can downregulate PR expression independent of ER activity [47
]. Further studies are warranted to determine if specific signaling pathways, particularly PI3K/Akt/mTOR, are preferentially activated in PR− tumors with IRS-2 membrane localization. Interestingly, patients with PR+ tumors that express IRS-2 at the membrane did not exhibit significantly diminished survival outcomes in our combined dataset. This finding supports the hypothesis that ER signaling, and potentially IRS-1, may be dominant with regard to suppressing the impact of IRS-2-mediated signaling. Additional studies to address this question will be important to understand fully the cross talk between these hormone and growth factor signaling pathways.
Our study is the first report on the expression of IRS-2 in human breast cancer. The fact that the correlations of IRS-2 membrane localization and poor outcomes in PR− patients were observed in tumors from two separate institutions and in both whole tumor sections and a tissue array support the validity of these observations. However, to confirm IRS-2 localization as a predictive biomarker in breast cancer, these results need to be carefully validated through evaluation of a much larger cohort of patients with longer follow-up times to increase the statistical power of these findings. Ideally, a prospective study in which tumor biopsies could be taken before and after adjuvant treatment would better control for effects of therapy on IRS-2 localization. We established rules to classify the IRS-2 staining patterns in our study to address the subjectivity of determining IRS-2 localization, and two independent investigators were in greater than 90% concordance with identifying the localization pattern. Analysis of additional cohorts of patients by independent investigators will be important to confirm that the identification of IRS-2 staining patterns is not investigator dependent. The molecular mechanisms underlying the present findings also require further investigation. The membrane IRS-2 staining pattern could represent activation of specific pathways, which promote aggressive tumor behavior. Receptor activation upstream as well as activation of downstream signaling pathways need to be evaluated and correlated to IRS-2 expression patterns. In vitro studies in cell lines are also needed to further explore the role of the localization of IRS-2 in breast carcinoma cell motility, invasion, and metabolism.
In summary, we have identified an IRS-2 staining pattern that has prognostic significance for the OS of breast cancer patients. This association was found to be even stronger for patients with PR− tumors. Evaluation of IRS-2 staining patterns could potentially be used to identify patients with PR− tumors who would most benefit from aggressive treatment.