We found that cytoplasmic expression of IGF1R in the epithelial cells of normal breast TDLUs in benign breast biopsies was positively associated with subsequent risk of breast cancer. Women whose TDLUs were IGF1R – cytoplasm positive/IGF1R-membrane negative were at a more than a 15- fold increased risk of developing breast cancer subsequent to their biopsy (median time=8 years later).
IGF1R is a tyrosine kinase growth factor receptor that when activated plays an important role in signal transduction pathways regulating cell proliferation, survival migration, and differentiation. Upon ligand binding, IGF1R stimulates downstream pathways responsible for these processes via phosphorylation of substrates including receptor substrate 1 (IRS1), Shc, and phosphatidyl inositol-3 kinase (PI3K).
The differential staining patterns for IGF1R observed in our studies could result from various possible mechanisms; one intriguing possibility is the differential activation status of IGF1R. In several reports (28
) and in our own preliminary studies (unpublished data), cells cultured in the absence of serum or IGF ligands display a predominantly membranous staining pattern for IGF1R; this localized membrane staining pattern is less prominent following ligand stimulation.
Ligand stimulation induces internalization of IGF1R and other receptor tyrosine kinases like Met and EGFR(30
). In the case of IGF1R, the ligand stimulated receptor undergoes endocytosis via both caveolin and clathrin-mediated pathways (32
). Interestingly, recent studies have also reported nuclear localization of intact IGF1R that was dependent on both ligand stimulation and intact IGF1R kinase activity (28
). Furthermore, nuclear localization of IGF1R was also blocked by inhibition of endocytosis, suggesting that IGF1 receptors in the nucleus translocated from the cell membrane following ligand stimulation. Thus, the redistribution of IGF1R from the membrane to various intracellular locations could reflect changes in the activation status of IGF1R.
Although initially considered to be a mechanism by which receptor-mediated signaling was down-regulated, internalization of receptors into various intracellular compartments may actually be required for activation of specific signaling pathways (32
). For example, IGF1R internalization following IGF1 stimulation of oligodendrocyte precursor cells was required for phosphorylation and sustained activation of Akt (32
). Studies of EGFR also suggest that clathrin-mediated receptor internalization is required for sustained activation of specific signaling molecules required for EGF-dependent biological processes including DNA replication and proliferation (35
). The signaling capabilities of internalized receptor tyrosine kinases may also be responsible for the adverse outcomes reported for cytoplasmic Met localization in colon cancer (36
In the current study, women whose benign breast biopsies showed little or no IGF1R expression on the membranes of normal TDLU epithelial cells but high levels internally within the cytoplasm were at the highest risk of subsequent breast cancer. These results raise the possibility that in a population of cells in this subset of women, the decrease in membrane staining ref in the early development of breast cancer which may provide new opportunities for breast cancer chemoprevention. Currently, there are anti-cancer agents in development targeting the IGF pathway either through receptor blockade or kinase inhibition (37
The major limitation of the current study is the small sample size. Given the small sample size, these results should be interpreted cautiously, as chance may be a possible explanation for the observed results. However, the prospective nature of the study with benign tissue obtained several years prior to breast cancer diagnosis makes this study uniquely able to examine the association between IGF1R expression in normal breast epithelium and subsequent risk of breast cancer. In addition, all benign biopsies underwent centralized pathology review and IGF1R scoring with the same expert breast pathologists. The pathologists were blinded to the subsequent case-control status of the participants throughout the study; therefore it is unlikely that there is any differential misclassification of IGF1R scoring. We were unable to obtain tissue samples from all eligible participants. The primary reason for this is due to the long time between BBD diagnosis and collection in our study. Many hospitals are not required to maintain formalin fixed paraffin embedded tissue samples beyond 5 to 10 years. Thus many of these samples had been destroyed by the time we attempted to collect them. There was no significant difference in our ability to obtain BBD tissue samples from cases versus controls. Interestingly, the 15-fold increase in breast cancer risk observed with the IGF1R – cytoplasm positive/IGF1R-membrane negative staining pattern was independent of the category of the benign breast disease category. Women with nonproliferative benign lesions, thought to be at low or no increased risk of breast cancer, were also at high risk of breast cancer if they had this high risk IGF1R staining pattern. It is important that these results be confirmed by other studies. Identification of tissue markers in normal breast tissue that are predictive of subsequent risk has important implication for risk stratification as well as preventive strategies.