Proteomic analysis of DNA damage induced stress signaling with focus on p53 : S100A6 regulation, function and potential as biomarker in lung cancer and as a novel therapeutic target

In this thesis powerful proteomics methods were used to reveal novel proteins involved in the cellular response to DNA damaging treatment. The goal was to find proteins with potential as biomarkers for prediction of cancer prognosis and response to cancer therapy, but also to find potential novel targets of cancer therapy. In addition, the impact of the tumor suppressor p53 on the cellular response to DNA damaging treatment was investigated.

When comparing two isogenic colon cancer cell lines (HCT116 p53wt and p53-/-) we were unable to detect p53 dependent differences in sensitivity to ionizing radiation (IR) or DNA damaging drugs. p53 did however alter the localization and reduce the abundance of Rad51, a key protein in homologous recombination repair, in response to IR. Our data thus indicate that p53 is involved in negative regulation of homologous recombination.

The same cell line pair was also used in a proteomics time course study to identify novel proteins involved in the cellular response to DNA damaging agents (IR). In this study we discovered that the small calcium binding protein S100A6 was upregulated in a p53 dependent manner in irradiated cells. In addition to the upregulation of S100A6 post irradiation we also discovered that the post translational modification pattern and the sub cellular localization were altered. The biological functions of S100A6 were not known at the time of this discovery, but several studies had implicated S100A6 in carcinogenesis as it was shown upregulated in several different types of cancer. We therefore decided to pursue this finding and study the biological role of S100A6 in detail.

The expression of S100A6 was investigated in stage I non-small cell lung cancer by immunohistochemistry using tissue microarrays. S100A6 was found overexpressed in cancer cells, and S100A6 expression correlated with wt p53 expression. S100A6 expression also correlated with patient survival in a subset of the cohort.

In order to elucidate the function of S100A6 we set out to identify novel S100A6 interacting proteins. Using immunoprecipitation and proteomics methods we identified Ubiquilin-1 as a novel S100A6 interacting protein. Ubiquilin-1 is involved in regulationof proteasome mediated degradation of ubiquitinated proteins such as p53 and IkappaBa. siRNA mediated silencing of S100A6 resulted in stabilisation of both p53 and IkappaBa. Mass spectrometry based proteomics further revealed that S100A6 silencing reduced proteasomal processing of NFkappaB2 p100. S100A6 has earlier been suggested involved also in degradation of beta-catenin as S100A6 interacts with a component of the complex responsible for ubiquitination of beta-catenin. Using S100A6 silencing we were able to show increased degradation of beta-catenin. S100A6 silencing also increased the cellular sensitivity to ionizing radiation.

In conclusion our data indicates that overexpression of S100A6 in cancer cells would result in a survival benefit through inhibition of apoptosis via increased p53 degradation and stimulation of proliferation via increased NFkappaB and beta-cateninsignaling. Our data also indicates that S100A6 is upregulated at later timepoints post irradiation to inhibit apoptosis and cell cycle arrest, allowing cells with repaired DNA damage to re-enter the cell cycle. These findings suggest the potential of S100A6 as a novel target of cancer therapy.

List of scientific papers

I. Orre LM, Stenerlöw B, Dhar S, Larsson R, Lewensohn R, Lehtiö J (2006). p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line. Biochem Biophys Res Commun. 342(4): 1211-7. Epub 2006 Feb 24
https://pubmed.ncbi.nlm.nih.gov/16516153

II. Orre LM, Pernemalm M, Lengqvist J, Lewensohn R, Lehtiö J (2007). Up-regulation, modification, and translocation of S100A6 induced by exposure to ionizing radiation revealed by proteomics profiling. Mol Cell Proteomics. 6(12): 2122-31. Epub 2007 Sep 4
https://pubmed.ncbi.nlm.nih.gov/17785350

III. De Petris L, Orre LM, Kanter L, Pernemalm M, Koyi H, Lewensohn R, Lehtiö J (2008). Tumor expression of S100A6 correlates with survival of patients with stage I non-small cell lung cancer. [Submitted]

IV. Orre LM, Vernet E, Lengqvist J, Gräslund T, Lewensohn R, Lehtiö J (2008). S100A6 interacts with Ubiquilin-1 and regulates degradation of p53, I(kappa)B(alpha) and NF(kappa)B2. [Submitted]