The incidence of gastrointestinal adenocarcinoma has increased six-fold since 1970. As shown previously, ERBB2 is overexpressed in 15-25% of tumors. In breast cancers with ERBB2 amplification, ERBB2-targeted therapies have improved overall survival and disease-free rates of survival. In this study, we use a transient siRNA model and a stable lentiviral shRNA model to demonstrate that knockdown of ERBB2 in esophageal and gastric cancer cell lines with known ERBB2 amplification effectively decreases ERBB2 protein levels and decreases cell viability mainly via apoptotic pathways. METHODS:An esophageal adenocarcinoma cell line no ERBB2 amplification (Seg-1) and two adenocarcinoma lines with ERBB2 amplification (MKN45 and OE19) were treated with ERBB2 siRNA or control siRNA for 6 hours. Protein lysates were collected on Day 3 and ERBB2 protein levels were analyzed via western blots. Cell viability was measured by MTS assay while apoptosis and cell cycle analysis were assessed by flow cytometry on Day 3 with flow cytometry. Once knockdown was shown in siRNA model, lentiviral ERBB2 shRNA vectors were generated. Cell lines were treated with one of three separate lentiviral GFP-labeled ERBB2 shRNA vectors or a control shRNA vector for 6 hours. Protein lysates were collected on Day 6, and cell viability assay was performed from Day 3 to Day 8. Statistical analysis was performed by Student’s T-test. RESULTS:siRNA transfection efficiency was measured at approximately 75%. ERBB2 protein levels decreased by 80% with siRNA treatment. While ERBB2 knockdown significantly decreased cell viability and increased apoptosis in lines with high ERBB2 levels, there was no difference seen in the cell line with baseline ERBB2(Table). In all cell lines, there was minimal change seen in cell cycle. Stable transfection using lentiviral ERBB2-shRNA vectors decreased ERBB2 protein levels significantly and cell viability decreased by 60% on Day 6. CONCLUSION:ERBB2 suppression based on siRNA transfection and shRNA lentiviral vector effectively decrease cell viability mainly via apoptosis in cell lines with amplification of ERBB2 as compared to cell lines without overexpression. ERBB2-directed therapy may be of benefit in the subset of patients with gastrointestinal adenocarcinomas exhibiting overamplification of ERBB2.
Effect of shRNA ERBB2 treatment on Cell Viability

	Control Lentiviral shRNA	Lentiviral ERBB2 shRNA #1	Lentiviral ERBB2 shRNA #2	Lentiviral ERBB2 shRNA #3
Seg-1	99.20+/-9.93	98.59+/-2.47	9.876+/-7.12	90.02+/-10.01
OE19	91.5+/-5.57	29.09+/-8.01**	31.010+/-6.06**	21.80+/-4.00**
MKN45	88.17+/-13.76	52.83+/-3.36**	45.19+/-4.48**	52.40+/-7.87**

n=5. **p<0.001 when compared to Control Lentiviral shRNA treatments. Data expressed as Mean of % Cell viability +/- SEM.