We have previously identified a patient-derived microRNA signature indicative of hyperactive MAPK (hMAPK) signaling in breast cancer. This signature associates with reduced estrogen receptor (ER) expression, and altered protein expression of a multitude of genes associated with breast cancer aggressiveness and progression. Additionally, this signature predicts poor clinical outcome with reduced disease free survival and overall survival in all comers, and especially among patients with ER+ tumors. Many of the proteins whose expression is altered in tumors with this hMAPK-miRNA signature are predicted or validated targets of miRNAs within the signature. We hypothesize that alterations in expression of hMAPK-miRNAs directly affect post-transcriptional regulation of a subset of these proteins. Using a hMAPK cell line model of breast cancer generated from an MCF-7 background, we investigated the involvement of hMAPK-miRNAs in post-transcriptional regulation and resulting dynamics of expression of several proteins identified as being differentially expressed between hMAPK-tumors and not-hMAPK-tumors.
Here we have investigated the hMAPK regulation of one miRNA family that is upregulated and one miRNA family that is downregulated in the hMAPK signature. Each of these families has key validated targets important in breast cancer, and many other putative gene targets. The miR-221/222 family is upregulated in the hMAPK-miRNA signature, and is a negative regulator of ER and the cell cycle regulator p27. In our breast cancer cell line model, we have previously demonstrated that miR-221/222 are upregulated and ER and p27 are downregulated in hMAPK cells vs control transfected MCF-7 cells. We now show that inhibiting MAPK signaling with pharmacological inhibitors reduces the expression of miR-221/222, and results in enhanced expression of ER and p27; we confirm that this regulation occurs at the post-transcriptional level using a dual-luciferase reporter construct containing the ER 3′ UTR. Additionally, stimulation of MAPK signaling dynamically increases the expression of miR-221/222 and decreases ER and p27 expression. These results indicate that hMAPK signaling directly alters expression of ER and p27 by altering miRNA expression. Members of the miR-30 family are downregulated in the hMAPK-miRNA signature, and several proteins that upregulated in hMAPK-miRNA tumors vs not-hMAPK-miRNA tumors, including SNAIL, NOTCH1, and FOXO3, are all targets of the miR-30 family. Using our cell line model, we have shown that members of the miR-30 family are downregulated by hMAPK signaling, that SNAIL is upregulated, and that abrogation of hMAPK signaling reverses these changes.
It is known that SNAIL and ER exhibit inverse expression in breast cancer cells, and these data suggest that this is due to hMAPK mediated miRNA activity. Thus, hMAPK signaling may contribute to increased EMT, ER-negativity, and poor clinical outcome observed in breast tumors with this hMAPK-miRNA signature via the direct action of these hMAPK-regulated miRNAs. These results suggest novel mechanisms by which hMAPK signaling may contribute to breast cancer disease aggressiveness and progression.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-08.