Betulinic Acid as a Potential Inhibitor of Ire1/Xbp1/Herp Axis Blocking Both Proliferation and Survival of Glioblastoma Cells

Rationale: Glioblastoma (GBM), a grade IV brain tumor, shows a poor prognosis and is not considered healable even following standard treatment with radiation in combination with temozolomide (TMZ), the first-line chemotherapy, only provides approximately one year survival post diagnosis. Because GBM increases the activation of defensive mechanisms, such as DNA repair enzymes and ER stress sensors, boosting protection against TMZ, to find a novel/second-line agent active against the cellular defenses is critical for treating primary and recurrent GBMs. Preliminary results: Since betulinic acid (BA), a natural product of plant origin, can pass the blood–brain barrier, we investigated the anti-tumor effect of BA using GBM cell lines and their TMZ-resistant cells, and found that BA treatment significantly reduced the proliferation and cell survival in all cell lines via inhibiting the IRE1-XBP1-Herp axis, a signal pathway of ER-stress response, and increasing γH2AX, a marker of DNA damage. These results include the following: (1) BA treatment attenuated cell proliferation and elevated apoptotic cell death in both parental and TMZ-resistant GBM cells; (2) The microarray data showed that ER stress-related genes were up-regulated by BA; (3) The protein levels were further examined, and the results showed that HSPA6/Hsp70, HSB90B1/Hsp90b1, and ERN1/IRE1 indeed increased in a dose-dependent manor following BA treatment, but decreases in XBP1/XBP1 and its downstream target HERPUD1/Herp; (4) BA significantly induced DNA damage markers. Hypothesis: We hypothesize that BA kills malignant GBM cells via targeting the IRE1-XBP1-Herp pathway, which causes the accumulation of ER stress and DNA damage. Specific Aims: Based on this hypothesis, we will investigate the role of IRE1-XBP1-Herp pathway in recurrent or TMZ-resistant GBM cells. First, we will study whether BA attenuates XBP1 expression through inhibiting IRE1 auto-phosphorylation or RNase activity. Second, we will examine whether XBP1 and its downstream targets are key effectors protecting GBM cells against ER stress and chemotherapy. Third, we will evaluate the in vivo anti-cancer effects of BA in orthotopic GBM animal models. Fourth, we will confirm the relationship between the IRE1-XBP1-Herp pathway and GBM recurrence in clinical specimens. Novelty and application: This project will increase understanding of the mechanism of IRE1-XBP1-Herp pathway activity and its protective functions against ER stress in malignant GBMs, and reveal whether BA targeting this pathway is a good therapeutic strategy for overcoming the GBM tumor recurrence after the initial therapy.