yr61 full-length protein into MCF-7 which also resulted in significant increase in proliferation and invasion as compared with WT. Previous studies by Xie et al, and Tsai et al confirm that increased Cyr61 in breast cancer cells result in aggressive properties. Together, our studies indicate that Cyr61 is inducible both by estrogen as well as IGF-1, rendering this molecule highly sensitive to growth factor induction. Physiologically, patients with high IGF-1 circulatory levels have been shown to have reduced survival from breast cancer; hence, the upregulation of Cyr61 which mediates cell migration may result in increased metastasis observed. 8 IGF-1 Regulates Cyr61 in Breast Cancer The mechanism whereby IGF-1 stimulates Cyr61 was also investigated in the present study. The PI3K/Akt pathway was 
assessed given that it is one of the central downstream pathways from IGF-1 and previous studies from our group and others identified a central role of PI3K/Akt in breast cancer outcomes. Transfection of constitutively active Akt resulted in significant increase in Cyr61 expression, whereas inactive Akt transfection resulted in a decrease. This was further confirmed by inducing the Akt pathway with IGF-1 and blocking the Akt pathway with LY294002. The cell proliferative activity and invasion mirrored Cyr61 transcriptional levels. However, the IGF-1 induced transcription of Cyr61 was not completely abolished. Therefore, MAPK the other key downstream pathway from IGF-1 was assessed. Inhibition of MAPK resulted in a significant decrease in Cyr61 1022150-57-7 web expression in the WT cells which was not restored by IGF-1 induction, unlike the response seen by blocking the PI3K/Akt pathway. However, similarly to the LY294002 response, blocking MAPK with PD98059 reduced proliferation and invasion. Together these data suggest that IGF-1 mediated Cyr61 expression, proliferation, and invasion are intrinsically but not exclusively mediated through MAPK and PI3K/Akt signaling with high Cyr61 expressing cells less sensitive to MAPK inhibition than PI3K/Akt inhibition in presence of IGF1. Furthermore, we posit it is the activation of Akt rather than the expression of Akt that is the driver of proliferation and invasion in our model. When the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19659572 PI3K inhibitor is applied, we see loss of the growth and proliferation because phosphorylation of Akt is inhibited, rather than the actual expression. In preliminary evaluation of the cell lines, we identified that baseline levels of pAkt were higher in MCF-7 CYA cells than MCF-7 WT cells. These data are consistent with our previous findings indicating that aggressive breast cancer phenotypes do not necessarily have increased levels of Akt, but do have increased levels of active Akt . This finding was further confirmed in breast cancer patient clinical samples; patient tissues with high pAkt levels had reduced disease free survival. Previous studies have highlighted the role of estrogen-induced Cyr61 expression in breast cancer. However, the presence of the estrogen receptor is implied to be present in these cell lines. Paradoxically, Cyr61 is most highly expressed in ER- cells lines such as MDA-MB231 as reported previously and confirmed in this present study. The finding from our study implicating IGF-1 in Cyr61 expression and subsequent proliferation and invasion can shed light on this paradox as well as present several translational implications. As indicated earlier, IGF-1 levels have been associated with more aggressive breast cancer
