Abstract 4527: The HER3-EREG axis and its role in colorectal cancer aggression

Abstract

Abstract Yearly, 50,000 Americans die of colorectal cancer metastases to liver and lung. EGFR-targeted agents improve survival in a subset of metastatic colorectal cancer (mCRC) cases, and overexpression of the HER-family ligands amphiregulin (AREG) and epiregulin (EREG) correlates with EGFR-targeted agent susceptibility. Here we present evidence of association between EREG overexpression and disease aggression by showing that knockdown of EREG or HER3 impede anchorage independent growth in the HCT 116 CRC cell line. We also find that a HER3-EREG axis gene signature groups mCRC tumors into prognostically distinct groups. Snap-frozen CRC hepatic metastases (65) with patient matched normal liver, as well as 71 primary CRC tumors with patient matched normal colonic mucosa were analyzed via qRT-PCR for receptors (HER1-4) and ligands (AREG, and EREG). Among the mCRC cases HER2, HER3, AREG, and EREG were expressed at higher levels than unmatched normal mucosa (p<0.005). HER2, AREG, and EREG were also overexpressed in mCRC compared to unmatched primary CRC tissue (p<0.005). Expression of AREG and EREG were highly correlated (Rs=0.90), and higher EREG mRNA expression was associated with markedly decreased post-hepatectomy overall survival (OS) (p=0.018). In a panel of 9 CRC cell lines HCT 116 had the highest EREG expression. Using the pLKO.1 viral vector, shRNAs targeting EREG, and HER3 were introduced to HCT 116 cells. Empty vector was used as a control. mRNA levels for HER3 and EREG were knocked down by 87% and 73% respectively and protein downregulation was confirmed by western blot. Knockdown and control cells were grown in 0.35% soft agar at a density of 5000 cells per well in 6 well plates. Knockdowns impeded anchorage independent growth with a mean colony count (>50 um diameter) of 170 for HER3, and 285 for EREG as compared to 530 for the empty vector (p<0.005). RNA from 3 distinct sets of HER3, EREG, and empty vector infections was assayed on Agilent Human GE 4x44K v2 microarrays. Unsupervised Principle Component Analysis (PCA) suggested broad concordance in the impact of EREG or HER3 knockdown upon the expression profile. Statistical Analysis of Microarrays (SAM) comparing HER3, and EREG knockdowns to empty vector control revealed 205, and 432 differentially expressed genes with at least a 1.5 fold-change and a false discovery rate of 0%. When HER3 and EREG differential gene lists were compared, 95 overlapping genes with 100% directional agreement were observed. For 51 mCRC cases RNA was available for microarray analysis. A predictor of HER3-EREG activity was constructed from 250 genes that discriminate HER3/EREG knockdown and empty vector control cells and are generally variable in tumors. The predictor was used to assign a HER3-EREG activity score to each mCRC case. This score is significantly associated with OS (p=0.005) with the top 50% of patients by HER3-EREG score demonstrating 5 year OS of 23% as compared to 49% for cases in the lower half of HER3-EREG activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4527. doi:1538-7445.AM2012-4527