Abnormal expression and signaling of ErbB receptors has been implicated in multiple epithelial malignancies, including pancreatic cancer. Erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has been recently approved for pancreatic cancer treatment, but there are no reliable predictors of patient response. Expression of additional ErbB receptors seems to influence tumor response to EGFR-targeted therapy. We analyzed the influence of ErbB3 expression on pancreatic cancer cell response to erlotinib treatment. Proliferation assays of five human pancreatic cancer cell lines were performed following treatment with erlotinib. Expression and phosphorylation profiles of ErbB receptors and downstream adaptor protein (Akt, ERK1/2, STAT3, mTOR) were evaluated following stimulation with EGF or neuregulin-beta. The formation of EGFR homodimers and EGFR-ErbB3 heterodimers, necessary to enable ErbB3 downstream signaling, was demonstrated by chemical cross-linking assays. The effects of RNA inhibition of ErbB3 on sensitivity to erlotinib treatment were evaluated in AsPC-1 pancreatic cancer cells. Erlotinib inhibited Akt phosphorylation and proliferation of all the ErbB3-expressing cell lines but did not affect mTOR activation. Cross-linking studies confirmed the presence of EGFR-ErbB3 heterodimers in pancreatic cancer cells. Only the ErbB3-deficient MIA PaCa-2 cells displayed persistent Akt activation and ongoing proliferation in spite of erlotinib treatment. siRNA-mediated inhibition of ErbB3 expression in AsPC-1 cells resulted in acquired resistance to erlotinib treatment. Pancreatic cancer cells which lack ErbB3 do not display activation of the ErbB3-PI3K-Akt cascade induced by EGFR/ErbB3 heterodimers and become less critically dependent on EGFR signaling and therefore resistant to erlotinib. Pancreatic cancer expression of ErbB3 may be useful for EGFR-targeted therapy patient selection.

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Historically, ErbB3 has been overlooked within the ErbB receptor family due to its perceived lack of tyrosine kinase activity. We have previously demonstrated that in pancreatic cancer ErbB3 is the preferred dimerization partner of EGFR, ErbB3 protein expression level directly correlates with the anti-proliferative effect of erlotinib (an EGFR-specific tyrosine kinase inhibitor), and transient knockdown of ErbB3 expression results in acquired resistance to EGFR-targeted therapy. In this study, we develop a stable isogenic model of ErbB3 expression in an attempt to decipher ErbB3's true contribution to pancreatic cancer tumorigenesis and to examine how this receptor affects cellular sensitivity to EGFR-targeted therapy. Analysis of the EGFR-ErbB3 heterodimer demonstrates that ligand-induced PI3K-AKT signaling is limited to ErbB3-expressing cells and that this signaling cascade can be partially abrogated by inhibiting EGFR function with erlotinib. Using our model of exogenous ErbB3 expression we showed a direct relationship between ErbB3 protein levels and increased pancreatic cancer cell proliferation in vitro. In vivo, ErbB3(+)PANC-1 xenografts had a significantly larger tumor volume than PANC-1 control xenografts (ErbB3-PANC-1) and displayed increased sensitivity to EGFR-targeted therapy. In pancreatic cancer, ErbB3 appears to be critically involved in EGFR signaling as evidenced by its profound effect on cellular proliferation and its ability to influence response to EGFR-targeted therapy.

MED1 is a base excision repair enzyme that interacts with the mismatch repair protein MLH1 and maintains genomic integrity by binding methylated DNA and repairing spontaneous deamination events. MED1 mutations have been associated with microsatellite instability and accelerated colorectal cancer (CRC) tumorigenesis. We propose that promoter methylation may serve as an alternative epigenetic mechanism for MED1 gene suppression during sporadic CRC tumorigenesis. Methylation status of the MED1 promoter was investigated in a panel of ovarian and colorectal cancer cell lines. The MED1 promoter region was sequenced following bisulfite treatment and sequence analysis identified a CpG island within the MED1 promoter which is frequently and preferentially methylated (≥ 50%) in ovarian and colorectal cancer cell lines with low/reduced MED1 expression. In vitro reversal of methylation restored MED1 expression. In colorectal cancer patients, when MED1 methylation was present, both tumor and matched mucosa were affected equally (mean frequency of methylation 24%) and there was no correlation between methylation and tumor stage. Patients without history of CRC showed significantly lower frequency of methylation (mean 14%, p<0.05). Decreased MED1 transcript levels were observed in matched normal mucosa when compared to controls (median fold difference 8.0). Additional decreased expression was seen between mucosa and matched tumor (median fold decrease 4.4). Thus, MED1 promoter methylation and gene silencing occur in sporadic CRC patients and represent an early event in CRC tumorigenesis. Detection of MED1 methylation and gene suppression in normal colon mucosa may contribute to identifying patients at higher risk of developing CRC during screening procedures.

Shorter EGFR intron 1 CA repeat length is associated with worse pancreatic cancer clinical prognosis and in vitro response to erlotinib. EGFR intron 1 length can be reliably measured in peripheral blood and may translate into a quantitative predictive marker of both pancreatic cancer aggressiveness and erlotinib sensitivity.