Snail is a zinc finger transcriptional repressor that induces epithelial-mesenchymal transitions (EMT) and is expressed at high levels in tumors. Snail contributes to chemotherapy resistance in ovarian, liver, colorectal, and breast carcinomas by activating the MAPK/ERK1/2 (ERK1/2) pathway .

ERK1/2 controls cell proliferation, survival, migration, and adhesion.

However, a role for MAPK in

Snail-mediated EMT in breast cancer is unclear. We hypothesized that Snail promotes EMT by decreasing cell adhesion and increasing cell migration through the MAPK pathway, resulting inresistance to Tamoxifen chemotherapy. Preliminary results suggest that breast cancer cells express variable levels of Snail that is higher than in normal MCF10A cells.

We utilized MCF-7 breast adenocarcinoma cells transfected stably with empty

Neo vector control (MCF-7 Neo) or constitutively active Snail cDNA (MCF-7

Snail). MCF-7 Neo and MCF-7 Snail have previously represented a breast cancer

EMT model; we used this model to examine cell adhesion and cell migration. We injected these cells into female nude mice to examine tumor size. We also examined activation of MAPK pathway using phospo-ERK (p-ERK) antibodies in response to Snail overexpression and the effect of inhibiting this pathway with

UO126 (MEK inhibitor, 20uM). Finally, we tested how Snail overexpression affects response to UO126 and/or Tamoxifen treatments using MTS cell proliferation assay. Our results showed that Snail overexpression led to decreased cell adhesion and increased cell migration on collagen and fibronectin in vitro, and increased tumorigenicity in vivo. Interestingly, immunofluorescent analysis revealed that activated ERK1/2 (p-ERK) had cytoplasmic localization in MCF-7 Neo, and exclusive nuclear localization in

MCF-7 Snail. Inhibition of activated ERK1/2 with UO126 antagonized cell adhesion primarily in MCF-7 Neo cells, while it decreased cell migration primarily in MCF-7 Snail cells. This would suggest that cytoplasmic p-ERK may be important for cell adhesion while nuclear p-ERK may be important for cell migration. MCF-7 Neo proliferation was consistently higher than MCF-7 Snail indicating that Snail may not play a significant role in cell proliferation, but other EMT functions like migration and adhesion. Tamoxifen treatment did not affect MCF-7 Snail cells morphologically, but led to sickly-appearing MCF-7

Neo cells. Additionally, MCF-7 Snail cells treated with Tamoxifen proliferated at similar rates to control untreated cells but co-treatment with UO126 greatly inhibited cell proliferation. This suggests that antagonizing nuclear p-ERK may sensitize breast cancer cells to Tamoxifen therapy. In conclusion, our study shows that Snail activation of MAPK pathway specifically within the nucleus promotes resistance to Tamoxifen and future ERK1/2 inhibition within the nucleus may sensitize cells to chemotherapy.

Citation Format: Bethany N. Smith, Peri Nagappan, Mahandranauth Chetram, LaTonia Taliaferro-Smith, Clayton Yates, Cimona Hinton, Valerie Odero-Marah. Snail activation of MAPK pathway may contributes to tumor insensitivity to tamoxifen chemotherapy in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1485. doi:10.1158/1538-7445.AM2013-1485