Abstract
BackgroundThe ErbB family consists of four proteins including (EGFR)/ErbB1, ErbB2, ErbB3, and ErbB4, and plays a crucial role in the promotion of multiple tumorigenic processes. In addition to the traditional pathways of EGFR signaling, EGFR translocates to the nucleus and acts as a transcription factor in the proliferation of cancer cells. Heregulin is known as both an ErbB3 and an ErbB4 ligand. This study aimed to investigate the expression of heregulin and its relevant EGFR family members as well as their phosphorylated forms in human colorectal cancer (CRC) tissues and to determine the relationship between their expression and clinicopathological factors including patient prognosis.MethodsWe analyzed the effects of exogenous heregulin on ErbB2, ErbB3 and ErbB4 phosphorylation in Caco-2, DLD-1, and HCT 116 colon cancer cell lines by western blot analysis. We examined 155 surgical resections from colorectomy patients. Cellular localization of ErbB1-4, their phosphorylated forms and heregulin protein was analyzed in CRC surgical resections by immunohistochemical analysis. Immunohistochemical results were compared with clinicopathological factors and patient prognosis.ResultsPhosphorylated ErbB2 (pErbB2) and phosphorylated ErbB3 (pErbB3) were detected in both nuclear and cytosolic fractions of Caco-2 and DLD-1 cells stimulated by exogenous heregulin. Whereas, phosphorylated ErbB4 (pErbB4) was detected only in cytosolic fractions of HCT 116 cells stimulated by exogenous heregulin. Phosphorylated EGFR (pEGFR) immunoreactivity was observed in the cytoplasm and nuclei of cancer cells, whereas the pattern of EGFR staining was membranous and cytoplasmic. Subcellular localization of pErbB2, cytoplasmic, membranous, or nuclear, varied among cases. pErbB3 immunoreactivity was exclusively observed in the nuclei of cancer cells. pErbB4 immunoreactivity was observed in the cell membrane of cancer cells. Statistically, heregulin immunoreactivity correlated with pErbB2 and pErbB4 expression. In multivariate analysis for disease free survival, lymph node status, pErbB3 and pErbB4 expression retained independent prognostic significance. In multivariate analysis for overall survival, lymph node status, pEGFR and pErbB4 retained independent prognostic significance.ConclusionsErbB2 and ErbB3 phosphorylated by heregulin localized in the nucleus of CRC cells. Phosphorylated ErbB1-4 and heregulin contribute to poorer patient prognosis in CRC. This heregulin-ErbB family member autocrine loop may be a candidate for targeted treatment of CRC.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-863) contains supplementary material, which is available to authorized users.
Highlights
The ErbB family consists of four proteins including (EGFR)/ErbB1, ErbB2, ErbB3, and ErbB4, and plays a crucial role in the promotion of multiple tumorigenic processes
In this study we examined the effects of Recombinant human heregulin-β1 EGF domain (rHRG) on tyrosine phosphorylation of the ErbB2, ErbB3, and ErbB4 proteins in nuclear fractions of colon cancer cell lines
PEGFR, pErB2 and phosphorylated ErbB3 (pErbB3) localized in the nuclei of colorectal cancer (CRC) cells; and we found for the first time that Phosphorylated EGFR (pEGFR), Phosphorylated ErbB2 (pErbB2), pErbB3 and phosphorylated ErbB4 (pErbB4) correlated with poorer patient prognosis
Summary
The ErbB family consists of four proteins including (EGFR)/ErbB1, ErbB2, ErbB3, and ErbB4, and plays a crucial role in the promotion of multiple tumorigenic processes. This study aimed to investigate the expression of heregulin and its relevant EGFR family members as well as their phosphorylated forms in human colorectal cancer (CRC) tissues and to determine the relationship between their expression and clinicopathological factors including patient prognosis. The EGFR and all other family members contain an extracellular ligand-binding domain (except in the case of ErbB2), a transmembrane region and a cytoplasmic domain with kinase activity [1,2]. Activation of EGFR receptors by the binding of the cognate ligands induces formation of homo- and hetero-dimers between different members of the EGFR family, and subsequent phosphorylation of specific sites in the cytoplasmic tail and recruitment of protein adaptors results in activation of multiple downstream signaling pathways. EGFR and ErbB2, two of four members of the EGFR tyrosine kinase family, have been widely studied on CRC [4]. The relationship between the expression of these proteins and clinicopathological factors, as evaluated by immunohistochemical techniques, has proven to be controversial in those studies [4]
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