Glioblastoma Multiforme (GBM) is the most common and deadly primary malignant tumour of the central nervous system. Despite considerable research to find novel targets for therapy, standard of care continues to comprise surgery followed by concomitant radiotherapy and chemotherapy with the alkylating agent Temozolomide (TMZ). Resistance to treatment inevitably develops, and life expectancy upon diagnosis remains to this day amongst the shortest of all cancer types. Finding genetic markers of resistance to treatment would allow for individual care to be tailored to the biological profile of each patient, hopefully sparing avoidable side effects, and paves the way for future targeted molecular therapies.Currently, the only clinically relevant marker of that nature is O6-methylguanine–DNA methyltransferase (MGMT), whose methylation status dictates whether it is able or not to repair the cytotoxic O6-methylguanine lesions induced by TMZ. However, TMZ causes numerous other DNA damages includingon the Base Excision Repair (BER) pathway, initiated by N-methylpurine DNA Glycosylase (MPG), for repair. Presence of MPG in glioblastoma cells has been associated with worse
patient outcomes, but precise mechanisms of MPG-regulation remain unknown.
HIV-1 Tat Interactive Protein 2 (HTATIP2) is a known tumour suppressor in certain tumours, although its function in GBM is currently unknown. We found HTATIP2 to be epigenetically downregulated through promoter methylation in multiple GBM datasets. Moreover, HTATIP2 has been shown to participate in the regulation of nuclear transport through its binding of karyopherins. Here we propose a model according to which HTATIP2’s presence in GBM cells interferes with MPG’s translocation to the nucleus, thereby preventing it from exerting its DNA repair function and contributing to development of resistance towards alkylating agent treatments such as TMZ.
Expressing HTATIP2 through a Tet-On system in epigenetically downregulated GBM cell lines (LN-229, BS-153), we studied the effects of HTATIP2 on the subcellular localization of MPG and on the BER DNA repair pathway. First we show that inducing HTATIP2’s expression with doxycycline in BS-153 GBM cells leads to a transition from a nuclear pattern of MPG expression to a cytoplasmic retention in treated cells. Next, we demonstrate a striking similarity in MPG sequestration in the cytoplasm of GBM cells expressing HTATIP2 and cells treated with Inhibitor of Nuclear Import-43 (INI-43) and Importazole (IPZ), inhibitors of Importin-b1 and the Importin-Ran-GTP interaction, respectively. Immunofluorescence staining with Calreticuline (CalR) and Protein Disulfide-Isomerase (PDI) displayed close colocalization of HTATIP2 and the endoplasmic reticulum within GBM cells, indicating a possible lead on HTATIP2’s biological functions. Finally, high content Operetta CLS analysis reveals an association between HTATIP2 expression and double strand breaks (DSB) under alkylating agent treatment, indicating a possible imbalance between MPG and downstream BER enzymes.
Collectively, our data supports HTATIP2 as a key determinant in glioblastoma’s sensitivity to treatment through regulation of the subcellular localization of the DNA repair protein MPG.