Targeting p38δ MAPK In Oesophageal Cancer: A Possible Future Therapeutic Goal?”

C O'Callaghan1, L Fanning0,2, A Houston0,2, O Barry1. 1Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland, 2Department of Medicine, University College Cork, Cork, Ireland

Background : Oesophageal cancer is an aggressive tumour which responds poorly to both chemotherapy and radiation therapy and has a poor prognosis. Approximately half of patients diagnosed with localized oesophageal cancer die of metastatic disease within the first 2 years following tumour resection. Thus, a greater understanding of the biology of oesophageal cancer is needed in order to identify novel therapeutic targets. Among these targets p38 MAPK isoforms are becoming increasingly important for a variety of cellular functions in mammalian cells (New, L. and Han, J.Trends Cardiovascular Medicine,1998, 8:220-229). Their roles, however, are more complex than previously thought with distinct members appearing to have different functions. A better understanding of the role(s) of p38 MAPKs may provide useful therapeutic tools for the management of human cancers. Methods : We analysed p38 MAPK isoform expression in a range of cancer cell lines and corresponding human normal and tumour tissue including oesophageal, liver, lung, prostate, colon, renal and pancreatic and observed that over 75% of both cell lines and human tumour tissue samples fail to express the specific isoform p38δ MAPK at the protein level. To evaluate the role(s) of p38δ and active p-p38δ MAPK in oesophageal cancer progression we developed a series of constructs:pcDNA3-FLAG-p38δ MAPK, pcDNA3-MKK6b(E)-(Gly-Glu)5-FLAG-p38δ andpcDNA3-MKK6b(E)-(Gly-Glu)5-FLAG-p38δDN. Briefly, the active p-p38δ plasmid was generated as follows: the MKK6(E) stop codon in pcDNA3-MKK6b(E) was replaced by Swa1 recognition sequence. The plasmid was linearised by digestion with Swa1 and 5′ phosphate removed with Shrimp Alkaline Phosphatase. Secondly, FLAG-p38δ was amplified from pcDNA3-FLAG-p38δ with a 5′(Gly-Glu)5 linker and 5′ and 3′ Dra1 restriction sites. Following Dra1 digestion (Gly-Glu)5-FLAG-p38δ was gel purified with an Illustra GFX PCR DNA & Gel Band Purification kit. Linear blunt-ended pcDNA3-MKK6b(E) and blunt ended (Gly-Glu)5-FLAG-p38δ were ligated using T4 DNA ligase with an insert:vector ratio of 10:1. The dominant negative p38δ plasmid (p38δDN) was generated from pcDNA3-MKK6b(E)-(Gly-Glu)5-FLAG-p38δ by replacing Thr180 with Ala and Tyr182 with Phe. Stable oesophageal squamous cancer cell lines expressing these plasmids were then generated. Results: Re-introduction of p38δ and p-p38δ proved to be (a) anti-proliferative, (b) anti-migratory and (c) pro-apoptotic. Briefly, we observed a time-dependent decrease in proliferation in oesophageal cancer cell lines when stably transfected with p38δ MAPK. This anti-proliferative effect was exacerbated with cells transfected with p-p38δ MAPK (pcDNA3-MKK6b(E)-(Gly-Glu)5-FLAG-p38δ). Using a Boyden chamber and a wound healing assay we observed a decrease in cellular migration and invasion when p38δ or p-p38δ MAPK was introduced to our cancer cells. Finally using Annexin V-FITC/PI double labeled flow cytometry we observed that p38δ and p-p38δ MAPK confers a greater sensitivity of oesophageal cancer to certain chemotherapeutic drugs but resistance to others. Conclusion: We now provide data for the anti-tumourigenic effect of p38δ MAPK in oesophageal cancer. Our research may provide a new potential target for the treatment of oesophageal cancer and metastases and increase our understanding of the mechanisms of oesophageal cancer progression. Interestingly, our findings are also applicable to other cancer types namely renal, prostate and pancreatic cancer which also lack p38δ MAPK isoform expression.