Abstract 1131: Snail- and ERK2-dependent signaling enhances breast cancer cell resistance to hydroxytamoxifen

• Published in: Cancer research (Chicago, Ill.) Ill.), 2014-10, Vol.74 (19_Supplement), p.1131-1131
• Main Authors: Smith, Bethany N., Nagappan, Peri, Taliaferro-Smith, Latonia, Mezencev, Roman, Yates, Clayton, Hinton, Cimona, Odero-Marah, Valerie
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2014-1131

Snail transcription factor and MAPK/ERK signaling regulate EMT and chemotherapy resistance in various tumor models by binding to target promoters (i.e., E-cadherin, maspin, ER-α). ERK1 is expressed during embryogenesis and in non-metastatic cells; ERK2 is implicated during vasculogenesis and promotes stem cell phenotype in triple negative breast cancer. Nuclear-localized ERK is associated with more active and potentially metastatic breast and ovarian carcinoma cells; cytoplasmic-localized ERK is a good prognostic factor. The role that Snail plays during the transition from cytoplasmic ERK1 to nuclear ERK2 has not been investigated. We hypothesized that both nuclear Snail and ERK2 are required for breast cancer cells to become resistant to 4-hydroxytamoxifen (4-OHT). We utilized MCF-7 breast adenocarcinoma cells, MCF-7 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 analyze ERK signaling and therapy response. Preliminary data suggest that MCF-7 Neo express higher levels of p-ERK, ER-α, and E-cadherin and no Snail. MCF-7 Snail expressed higher levels of Snail and vimentin, low levels of p-ERK, and low ER-α and no E-cadherin. MCF-7 Neo expressed higher ERK1 and MCF-7 Snail expressed higher ERK2. Immunocytochemistry indicated that p-ERK was nuclear and cytoplasmic in MCF-7 Neo, but mostly nuclear in MCF-7 Snail. We treated MCF-7 Neo and MCF-7 Snail with control siRNA, ERK1 siRNA, or ERK2 siRNA, then determined the expression of Snail, p-ERK1/2, ERK1/2, E-cadherin, and ER-α. We found that ERK1 siRNA decreased E-cadherin, while ERK2 siRNA increased E-cadherin in MCF-7 Neo. ERK2 siRNA decreased Snail, but ERK1 siRNA had no effect on Snail. ERK1 siRNA increased active ERK2 and ERK2 siRNA increased ERK1 in MCF-7 Neo and MCF-7 Snail. To determine the role that Snail plays in resistance, we treated MCF-7 Neo and MCF-7 Snail with 4-OHT and/or UO126 then mitochondrial permeability and caspase-3/7 activity assays. MCF-7 Neo had higher mitochondrial permeability than MCF-7 Snail, indicative of the dimmer staining using TMRM stain, and MCF-7 Neo had higher caspase-3/7 activity after treatments. Overall, cell death in MCF-7 Snail was caspase-independent. These data imply that Snail and ERK2 collaborate through an unknown mechanism to promote EMT and therapy resistance in breast cancer cells. Targeting Snail a