Functional characterization of HJURP (Holliday junction recognizing protein) in glioblastoma multiforme cells

Abstract

Diffuse astrocytomas are the most common type of primary brain cancer in adults. They present a wide variation in differentiation and aggressiveness, being classified into three different grades: low-grade diffuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (grade IV), the most frequent and the major lethal type. The GBMs are highly invasive and extremely resistant to radio and chemotherapy, and due to these characteristics the majority of GBM patients survive for approximately one year after diagnosis. Recent studies have highlighted the molecular heterogeneity of astrocytomas and demonstrated that large scale analysis of gene expression can help in their classification and treatment. In this context, we previously demonstrated that HJURP (Holliday Junction Recognizing Protein), a novel protein involved in DNA repair and genomic stability, is highly overexpressed in glioblastoma. Recent data of our group have shown that HJURP silencing promotes apoptosis by different mechanisms in two GBM cell lines, while non-tumoral cells are not significantly affected. Thus, we propose to further characterize HJURP functions in GBM cells as follows: i) to evaluate the requirement of HJURP for the viability of other cell lines (tumoral and non-tumoral); ii) to investigate the possible association between HJURP expression levels and the resistance to ionizing radiation; and iii) to characterize the molecular mechanisms of HJURP action in GBM cells. To achieve these goals, we intend to perform functional assays of HJURP over-expression and silencing followed by analysis of proliferation, viability and DNA damage. We also intend to investigate the mechanisms that regulate HJURP activity through the characterization of ATM quinase and p53 roles in HJURP stability and function, once the literature indicates that these proteins can crosstalk with HJURP pathways. Additionally, we will perform immunoprecipitation followed by mass spectrometry experiments to identify proteins that interact with HJURP in different GBM cell lines. Thus, we intend to contribute for the understanding of HJURP biological functions and evaluated its involvement in the maintenance of the genomic stability in astrocytoma cells. These data should provide evidence regarding the potential of HJURP as a novel therapeutic target. (AU)