Herceptin-Refractory Ovarian Carcinoma Cells Differentially Express Genes Involved in Angiogenesis, Invasion, and Metastasis

Leopoldo L. Luistro, James A. Rosinski, Hongjin Bian, Nicholas M. Ponzio, and Steve R. Ritland.
Departments of Oncology, Bioinformatics/Genetics/Genomics, Genomics, and Pharmaceutical Development Early Strategic Planning, Hoffmann-La Roche Inc., Nutley, NJ, and Department of Pathology and Laboratory Medicine, UMDNJ, New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, NJ.
 

We have previously reported the generation of a preclinical model of ovarian carcinoma with diminished response to Herceptin (Trastuzumab). SKOV-3 HR (Herceptin-refractory) subcutaneous xenograft tumors, unlike parental SKOV-3 xenografts, were not growth inhibited by Herceptin over a 4-week treatment period when implanted in athymic nu/nu mice. Herceptin has a 3-fold lower binding affinity on HER2 (erbB2) cell surface receptors of SKOV-3 HR cells, and was unable to inhibit the proliferation of these cells. To understand the cause of the experimentally-induced lack of response to Herceptin, microarray expression profiling (MEP) was performed on both the parental/Herceptin-susceptible and Herceptin-refractory cell lines. SKOV-3 and SKOV-3 HR cells were incubated with or without Herceptin up to 72 hours, cells harvested, RNA extracted, and cDNA scanned onto Affymetrix (U133A, U133B) gene chips. MEP results revealed an increased expression of angiogenic, metastatic, and invasive genes such as Angiopoietin 2, ICAM2, nestin, fibulin, MMP7, ceruloplasmin, CD24, FGFR3, and Vav3 oncogene in SKOV-3 HR cells. Alternatively, keratin 19, integrin alpha 3 and beta 4, uPAR and thrombomodulin were downregulated potentiating an epithelial to mesenchymal phenotypic transition. Interestingly, Mucin1 (MUC1) gene expression was also upregulated (4-5 fold) in basal and Herceptin-treated SKOV-3 HR cells. Glycosylated MUC1 is aberrantly overexpressed in breast and ovarian carcinomas. HER2 has been shown to regulate MUC1 expression in transformed mammary epithelial cells. MUC1 transmembrane overexpression on SKOV-3 HR cells could possibly affect Herceptin binding by direct steric hindrance onto HER2 receptors. Additionally, the MUC1 cytoplasmic tail has been reported to bind all four members of the HER/erbB family of tyrosine kinase receptors. Increased phosphorylation of MUC1 in SKOV-3 HR cells could potentiate downstream signaling pathways such as Ras/Raf/Erk (MAPK) leading to increased proliferation. Through the use of MEP, we hypothesize that increased expression of MUC1 and genes involved in angiogenesis, invasion, and metastasis may play a role in lack of Herceptin-response which drives an aggressive HER2-positive primary tumor with greater metastatic potential, thus leading to poor prognosis and shortened survival. It remains to be determined if similar changes occur in Herceptin-treated patients whose tumors progress.