Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen receptor, progesterone receptor, and HER2 overexpression. Patients with TNBC have a generally poor prognosis due to metastasis, high rates of recurrence, and lack of FDA-approved targeted therapies. We previously showed using functional proteomics that patients with high-ERK2-expressing TNBC tumors had a higher risk of death than those with low-ERK2-expressing tumors. Moreover, ERK2 but not ERK1 plays an important role in epithelial-mesenchymal transition (EMT) and is required for acquisition of stem cell-like characteristics. Compared to other breast cancer subtypes, TNBC has a higher proportion of cancer stem cells (CSCs) and is linked to EMT, two critical features associated with breast cancer progression, metastasis, and recurrence in patients. The MAPK signaling pathway is activated in TNBC, but the roles of ERK isoforms in tumor progression and metastasis are not well defined. We hypothesized that ERK2 but not ERK1 promotes EMT, the CSC phenotype, and metastasis in TNBC.
Methods and Results: Knockdown of ERK2 in SUM149 and BT549 TNBC cells significantly inhibited anchorage-independent colony formation (p<0.0001), inhibited formation of mammospheres (p = 0.003), and reduced the CSC population (CD44+/CD24-) (p = 0.002) in vitro. This effect correlated with a reduction in migration (p = 0.0004) and invasion (p<0.0001). SCID-beige mice injected via the tail vein with SUM149 shERK2 cells had a significantly lower lung metastatic burden than control mice (p = 0.0034) or mice injected with shERK1 cells (p = 0.0012), suggesting that ERK2 is a mediator of metastatic burden. To determine the mechanism by which ERK2 mediates this phenotype, we performed an Affymetrix Human Genome U133 Plus 2.0 array and compared the gene expression levels between SUM149 cells with ERK2 or ERK1 knockdown or transfection with control shRNA. Analysis of microarray data revealed that global gene expression changes associated with ERK2 knockdown predominantly altered regulation of the EMT pathway. Among the genes with ERK2-knockdown-associated expression change was EGR1, an immediate early response transcription factor whose downstream targets affect cell growth and differentiation. EGR1 is down-regulated 6-fold (p = 0.00013) compared to control and shERK1 cells. Knockdown of ERK2, but not ERK1, resulted in significantly lower EGR1 at both the mRNA and protein levels, validating our microarray data.
Conclusions and Future Directions: Our findings support our hypothesis, indicating that ERK2 promotes EMT and the CSC phenotype through EGR1 and mediates metastasis in TNBC. Future studies will determine ERK activity and pathway engagement using a novel peptide sensor based on the Sox fluorophore. We will pursue a therapeutic approach using siRNA against ERK2 incorporated in a DOTAP:cholesterol liposome.
Citation Format: Mary Kathryn Pitner, Hitomi Saso, Richard Larson, Rachel M. Sammons, Huiqin Chen, Caimiao Wei, Gaurav Chauhan, Kimie Kondo, Naoto T. Ueno, Kevin Dalby, Bisrat G. Debeb, Chandra Bartholomeusz. Silencing of ERK2 reverses EMT and suppresses the CSC phenotype, inhibiting lung metastasis in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1624.