HER-2 receptor expression, localization, and activation in colorectal cancer cell lines and human tumors

Abstract

The HER-2/neu oncogene encodes a 185 kD protein that is phosphorylated upon ligand binding to other HER/erbB members and regulates cell growth and differentiation. Given that HER-2 receptor blockade can inhibit the growth of colon cancer cell lines and tumor xenografts, we investigated the frequency, localization and phosphorylation status of HER-2 in colon cancer cell lines and in human tumors. Protein expression was analyzed in relation to mRNA levels, HER-2 amplification, and clinicopathological variables. Colon cancer cell lines constitutively expressed HER-2 proteins and none showed HER-2 amplification by fluorescence in situ hybridization. Cell fractionation and immunoblotting showed HER-2 in both the membrane and cytosolic compartments. Primary colorectal carcinomas (n = 96) and their metastases (n = 25) were examined by immunohistochemistry. Strong membrane HER-2 staining was detected in 5 (5%) of primaries and in 3 (12%) metastases (p = 0.36). Membrane but not cytoplasmic localization was strongly associated with HER-2 gene amplification (p = 0.007). Cytoplasmic HER-2 staining was found in 61 (63.5%) of primary tumors and localization was confirmed by immunoelectron microscopy that also showed plasma membrane HER-2. Using real-time quantitative RT-PCR, HER-2 mRNA was increased in tumors with membrane compared to cytoplasmic staining (r = 0.66, p = 0.001). Cytoplasmic HER-2 was associated with tumor differentiation (p = 0.018), but not other clinicopathological variables. By immunoblotting, heterogeneity was seen in HER-2 levels with downregulation in 4 of 7 tumors relative to normal epithelia that uniformly expressed HER-2. Phosphorylated HER-2 was detected in approximately 50% of tumors and in normal mucosa. In conclusion, HER-2 is expressed constitutively in colon cancer cell lines and demonstrates relatively distinct localization patterns in human tumors. Strong membrane immunoreactivity is associated with high levels of HER-2 mRNA and gene amplification whereas cytoplasmic HER-2 is detected frequently and seems to be a marker of tumor differentiation.