Abstract 710: Gene expression profile of the Wnt signaling pathway in trastuzumab resistant breast cancer cells
Authors: Wu Yanyuan , Mosher Nicole , Kim Juri , Chung Seyung , Ginther Charles , Slamon Dennis , Vadgama Jaydutt V. 
Published: June 2012

Introduction: We are interested in understanding mechanisms that lead to Trastuzumab (Herceptin) resistance in HER2 overexpressing breast tumors. In this study, we examined the role of the Wnt/β-catenin signaling pathway in Trastuzumab resistance. In addition to the Wnt pathway genes, we examined gene expression profiles using 44K gene chip array and EMT associated genes in Trastuzumab resistant cells.

Methods: Wnt pathway gene profiles were examined by Agilent chip (4×44K) and Wnt/β-catenin specific array. HER2 overexpressing breast cancer cells, SKBR3 and trastuzumab resistant clone, SKBR3 100-8 cells were used as cell models. The EMT (Epithelial to Mesenchymal Transition) markers Snail, Slug, Twist and E-Cadherin were examined by both real-time PCR and western blot analysis. Cell invasiveness was determined by invasive assay. HER2-HER3 dimerization was examined using immunoprecipitation and immunoblotting procedures.

Results: We established a Trastuzumab resistant cell line from the HER2 overexpressing parental SKBR3 cell line. The resistant clone called SKBR3/100-8 is insensitive to 100µg/ml of Trastuzumab. It has a mesenchymal or more basal-like morphology. It overexpresses HER2 protein and mRNA more than the parental line SKBR3. This resistant clone shows increased dimerization of HER2 with HER3. The SKBR3 100-8 cells were found to overexpress EGFR1. It is more invasive compared to wt SKBR3 and shows a significant increase in EMT marker, SLUG, but a decrease in Snail and E-cadherin compared to the wtSKBR3 cells. The resistant clone shows increase in phospho-Akt, pGSK3β and a decrease in β-Catenin. Agilent chip (4×44K) array (containing 84 Wnt pathway genes) identified the following Wnt pathway genes to be upregulated by 2 or more fold in the resistant clone: AXIN1, CSNK1A1, CSNK1D, CTBP2, DAAM1, c-JUN, TLE1, WNT3 and WNT6. Conversely, the following genes were downregulated by 2 or more fold in the resistant clone: AES, CCND3, CTNNB1, FZD1, FZD4, FZD5, PITX2, PPP2CA, PPP2R1A, WIF1, WNT11, and WNT4. The resistant clone also increased Nanog and VEGF significantly. TGFβ treatment in wtSKBR3 increased SLUG, Snail, Twist, and decreased E-Cadherin. The cells showed more mesenchymal morphology than wtSKBR without TGFβ treatment. TGFβ treatment in wtSKBR3 upregulated: c-Jun, C-Myc, CCND1, FosL1, LEF1, and TCF-1. TGFβ also induced Met, MMP1, MMP7, MMP9, MMP26, Nanog, NOS2, POU5F1, PPARd, Survivin, uPAR and VEGF. In contrast, TGFβ upregulated only c-Jun, Met, Nanog, and VEGF in SKBR3 100-8 cells.

Conclusion: Wnt/β-catenin signaling is highly involved in the trastuzumab resistant breast cancer cells. The resistant signature involves c-Jun, Met, Nanog and VEGF.

These results suggest a very specific signature that needs further studies for target therapy for the Trastuzumab resistant tumors and implies the use of anti VEGF therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 710. doi:10.1158/1538-7445.AM2011-710