Epidermal growth factor receptor and integrins meet redox signaling through P66shc and Rac1.

This review examines the concerted role of Epidermal Growth Factor Receptor (EGFR) and integrins in regulating Reactive oxygen species (ROS) production through different signaling pathways. ROS as such are not always deleterious to the cells but they also act as signaling molecules, that regulates numerous indespensible physiological fuctions of life. Many adaptor proteins, particularly Shc and Grb2, are involved in mediating the downstream signaling pathways stimulated by EGFR and integrins. Integrin-induced activation of EGFR and subsequent tyrosine phosphorylation of a class of acceptor sites on EGFR leads to alignment and tyrosine phosphorylation of Shc, PLCγ, the p85 subunit of PI-3 K, and Cbl, followed by activation of the downstream targets Erk and Akt/PKB. Functional interactions between these receptors result in the activation of Rac1 via these adaptor proteins, thereby leading to Reactive Oxygen Species. Both GF and integrin activation can produce oxidants independently, however synergistically there is increased ROS generation, suggesting a mutual cooperation between integrins and GFRs for redox signalling. The ROS produced further promotes feed-forward stimulation of redox signaling events such as MAPK activation and gene expression. This relationship has not been reviewed previously. The literature presented here can have multiple implications, ranging from looking at synergistic effects of integrin and EGFR mediated signaling mechanisms of different proteins to possible therapeutic interventions operated by these two receptors. Furthermore, such mutual redox regulation of crosstalk between EGFR and integrins not only add to the established models of pathological oxidative stress, but also can impart new avenues and opportunities for targeted antioxidant based therapeutics.

ß-Amyloid-evoked apoptotic cell death is mediated through MKK6-p66shc pathway.

We have previously shown the involvement of p66shc in mediating apoptosis. Here, we demonstrate the novel mechanism of ß-Amyloid-induced toxicity in the mammalian cells. ß-Amyloid leads to the phosphorylation of p66shc at the serine 36 residue and activates MKK6, by mediating the phosphorylation at serine 207 residue. Treatment of cells with antioxidants blocks ß-Amyloid-induced serine phosphorylation of MKK6, reactive oxygen species (ROS) generation, and hence protected cells against ß-Amyloid-induced cell death. Our results indicate that serine phosphorylation of p66shc is carried out by active MKK6. MKK6 knock-down resulted in decreased serine 36 phosphorylation of p66shc. Co-immunoprecipitation results demonstrate a direct physical association between p66shc and WT MKK6, but not with its mutants. Increase in ß-Amyloid-induced ROS production was observed in the presence of MKK6 and p66shc, when compared to triple mutant of MKK6 (inactive) and S36 mutant of p66shc. ROS scavengers and knock-down against p66shc, and MKK6 significantly decreased the endogenous level of active p66shc, ROS production, and cell death. Finally, we show that the MKK6-p66shc complex mediates ß-Amyloid-evoked apoptotic cell death.

P66Shc-rac1 pathway-mediated ROS production and cell migration is downregulated by ascorbic acid.

The oxidative role(s) of p66Shc protein has been increasingly expanded over the last decade. However, its relation with the most potent antioxidant molecule, i.e. ascorbic acid has never been studied. We have previously shown that p66Shc mediates rac1 activation, reactive oxygen species (ROS) production and cell death. Here we studied the effect of ascorbic acid on the pathway involving p66Shc and rac1. Our results indicate a decrease in the expression of p66Shc in a dose- and time-dependent manner. We studied the effect of ascorbic acid on rac1 expression and its activity. Ascorbic acid has no effect on total rac1 expression; however, rac1 activation was inhibited in a dose-dependent manner. Results suggest that the decrease in rac1 activity is mediated through ascorbic acid-modulated p66Shc expression. The decrease in rac1 activity was evident in cells transfected with the p66shc mutant (proline motif mutant, at residues P47 to P50). Our studies indicate that p66Shc-mediated ROS upregulation is significantly decreased in the presence of ascorbic acid. Cell migration experiments point towards the inhibition of p66Shc-rac1-mediated migration in the presence of ascorbic acid. Finally, results are suggestive that ascorbic acid-mediated decrease in Shc expression occurs through an increased Shc ubiquitination. Overall, the study brings out the novel role of ascorbic acid in antioxidant signal transduction.

E3B1/ABI-1 isoforms are down-regulated in cancers of human gastrointestinal tract.

The expression of E3B1/ABI-1 protein and its role in cancer progression and prognosis are largely unknown in the majority of solid tumors. In this study, we examined the expression pattern of E3B1/ABI-1 protein in histologically confirmed cases of esophageal (squamous cell carcinoma and adenocarcinoma), gastro-esophageal junction, colorectal cancers and corresponding normal tissues freshly resected from a cohort of 135 patients, by Western Blotting and Immunofluorescence Staining. The protein is present in its phosphorylated form in cells and tissues. Depending on the extent of phosphorylation it is either present in hyper-phosphorylated (M. Wt. 72 kDa) form or in hypo-phosphorylated form (M. Wt. 68 kDa and 65 kDa). A thorough analysis revealed that expression of E3B1/ABI-1 protein is significantly decreased in esophageal, gastro-esophageal junction and colorectal carcinomas irrespective of age, gender, dietary and smoking habits of the patients. The decrease in expression of E3B1/ABI-1 was consistently observed for all the three isoforms. However, the decrease in the expression of isoforms varied with different forms of cancers. Down-regulation of E3B1/ABI-1 expression in human carcinomas may play a critical role in tumor progression and in determining disease prognosis.

Alpha-1-syntrophin protein is differentially expressed in human cancers.

We studied the expression of α1-syntrophin (SNTA1) protein in histologically confirmed esophageal, stomach, lung, colon, rectal and breast cancerous tissue samples. Our results suggest a significant decrease in the expression level of SNTA1 protein in both esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) compared with their respective controls while a significant increase in expression of SNTA1 protein compared with the normal tissue was observed in breast carcinoma samples. No significant difference in expression of SNTA1 protein was observed in stomach, lung, colon and rectal cancers. Our results suggest that SNTA1 has a role in carcinogenesis and could possibly be used as a novel diagnostic or prognostic marker in esophageal and breast cancers.

P66shc and its downstream Eps8 and Rac1 proteins are upregulated in esophageal cancers.

Members of Shc (src homology and collagen homology) family, p46shc, p52shc, p66shc have known to be related to cell proliferation and carcinogenesis. Whereas p46shc and p52shc drive the reaction forward, the role of p66shc in cancers remains to be understood clearly. Hence, their expression in cancers needs to be evaluated carefully so that Shc analysis may provide prognostic information in the development of carcinogenesis. In the present study, the expression of p66shc and its associate targets namely Eps8 (epidermal pathway substrate 8), Rac1 (ras-related C3 botulinum toxin substrate1) and Grb2 (growth factor receptor bound protein 2) were examined in fresh tissue specimens from patients with esophageal squamous cell carcinoma and esophageal adenocarcinoma using western blot analysis. A thorough analysis of both esophageal squamous cell carcinoma and adenocarcinoma showed p66shc expression to be significantly higher in both types of carcinomas as compared to the controls. The controls of adenocarcinoma show a higher basal expression level of p66shc as compared to the controls of squamous cell carcinoma. The expression level of downstream targets of p66shc i.e., eps8 and rac1 was also found to be consistently higher in human esophageal carcinomas, and hence correlated positively with p66shc expression. However the expression of grb2 was found to be equal in both esophageal squamous cell carcinoma and adenocarcinoma. The above results suggest that the pathway operated by p66shc in cancers does not involve the participation of Ras and Grb2 as downstream targets instead it operates the pathway involving Eps8 and Rac1 proteins. From the results it is also suggestive that p66shc may have a role in the regulation of esophageal carcinomas and represents a possible mechanism of signaling for the development of squamous cell carcinoma and adenocarcinoma of esophagus.