Abstract AS31: MEK inhibition reverses antiestrogen resistance in ovarian cancer (OVCA) via alteration of cell cycle pathways and MAPK/estrogen regulated gene expression
Authors: Hew K. , Miller P. , Sun J. , Wei Z. , Zhang G. , Lu Y. , Mills G. , Slingerland J. , El-Ashry D. , Simpkins F. 
Published: August 2015

BACKGROUND: It is estimated that 67% of epithelial OVCAs are estrogen receptor (ER) positive. However, the response to anti-estrogen therapy in OVCA remains marginal. The Ras/Raf/MEK/MAPK pathway is hyperactivated in 40% of OVCAs. We have previously shown that estrogens further activate kinases such as Src, ER and Src kinase binding in the cytoplasm suggesting a non-genomic role or ER in OVCA. We postulated that estrogens further activate MAPK signaling and combination ER blockade with MEK inhibition would block cross-talk and increase the efficacy of ER blockade.

METHODS: The effects of treatment with MEK inhibitor (AZD6244) and anti-estrogen (Fulvestrant), each alone or together, on cell cycle and cell survival were evaluated in ER+ OVCA lines in vitro. Drug effects on xenograft tumor growth were assayed in vivo in NOD/SCIDs. Reverse phase protein lysate array (RPPA) analysis and gene expression analysis (GEA) were performed to evaluate biomarkers of drug response. Finally, a previously reported MAPK gene signature identified in breast cancer was analyzed in the OVCA lines treated with AZD6244 and combination treatment. And using alteration of gene expression upon MEKi treatment as suggestive of MAPK regulation, we define a MAPK gene signature originating from ovarian cancer cells.

RESULTS: RPPA analysis of high grade serous tumors from the TCGA (n=408) demonstrates that over 70% of tumors have phosphorylated MEK and MAPK, and patients with ER+ cancers and high pMAPK or pMEK (top50%), have a worse overall survival than those with low pMAPK or pMEK. Estrogen (E2) increases phosphorylation of MEK in ER+ OVCA cells. Fulvestrant caused minimal growth arrest after treatment demonstrating intrinsic resistance. AZD6244 caused loss of pMAPK, partial G1 cell cycle arrest and a modest increase in p27 levels in a dose dependent manner after treatment. However, responsiveness of OVCA cells to fulvestrant increased by addition of AZD6244 in vitro, with synergistic cell cycle arrest mediated by p27 binding to Cyclin E/cdk2 and much greater inhibition of MAPK activity. Gene enrichment analysis showed an increase in the ERB4/MAPK gene set with Fulv alone and the addition of AZD6244 showed that the top 20 gene sets downregulated were all related to replication and cell cycle (ie FOXM1, CyclinE). RPPA confirmed that combination was more effective in decreasing cell cycle promoting proteins (ie FOXM1, Cyclin B1) and upregulating p27. AZD6244 treatment of OVCA lines resulted in differential expression of about ¼ of the breast cancer defined MAPK gene expression signature, and of these, fulvestrant addition to MEK inhibition (MEKi) differentially affected 19 genes, reflective of these being E2 regulated genes. Similarly, of the total MEKi affected genes, a subset were differentially regulated by the addition of fulvestrant indicating putative E2 regulation underlying these genes. Xenograft data showed the greatest decrease in tumor volume with the drug combination compared to either drug alone.

CONCLUSION: Given the majority of primary OVCAs express high MEK/MAPK activity may underlie failure of anti-estrogen therapy. MEK inhibition reverses anti-estrogen resistance in our OVCA models. These data support further pre-clinical and clinical evaluation of combined fulvestrant and MEK inhibition in OVCA.

Citation Format: K. Hew, P. Miller, J. Sun, Z. Wei, G. Zhang, Y. Lu, G. Mills, J. Slingerland, MD, PhD, D. El-Ashry, F. Simpkins. MEK inhibition reverses antiestrogen resistance in ovarian cancer (OVCA) via alteration of cell cycle pathways and MAPK/estrogen regulated gene expression [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS31.