Resistance of Glioblastoma-Initiating Cells to Radiation Mediated by the Tumor Microenvironment Can Be Abolished by Inhibiting Transforming Growth Factor-β

Abstract

The poor prognosis of glioblastoma (GBM) routinely treated with ionizing radiation (IR) has been attributed to the relative radioresistance of glioma initiating cells (GIC). Other studies suggest that GIC are sensitive but the response is mediated by undefined factors in the microenvironment. GBM produce abundant transforming growth factor-β (TGFβ), a pleotropic cytokine that promotes effective DNA damage response. Consistent with this, radiation sensitivity, as measured by clonogenic assay, of cultured murine (GL261) and human (U251, U87MG) glioma cell lines, increased approximately 25% when treated with LY364947, a small molecule inhibitor of TGFβ type I receptor kinase, prior to irradiation. Mice bearing GL261 flank tumors treated with 1D11, a pan-isoform TGFβ neutralizing antibody, exhibited significantly increased tumor growth delay following IR. GL261 neurosphere cultures were used to evaluate GIC. LY364947 had no effect on primary or secondary neurosphere-forming capacity. IR decreased primary neurosphere formation by 28%, but did not reduce secondary neurosphere formation. In contrast, LY364947 prior to IR decreased primary neurosphere formation by 75% and secondary neurosphere formation by 68%. Notably, GL261 neurospheres produced 3.7-fold more TGFβ per cell compared to traditional culture, suggesting that TGFβ production by GIC promotes the DNA damage response and self-renewal and creates microenvironment mediated resistance. Consistent with this, LY364947 treatment in irradiated GL261 neurosphere-derived cells decreased DNA damage responses, H2AX and p53 phosphorylation, and induction of self-renewal signals, Notch1 and CXCR4. These data motivate the use of TGFβ inhibitors with radiation to improve therapeutic response in GBM patients.

TGFβ1 Protects Cells from γ-IR by Enhancing the Activity of the NHEJ Repair Pathway

“…This delayed effect of TGFb1 raises a possibility that the decrease in foci represent the accelerated NHEJ process in the presence of TGFb1, as there is a close correlation between the rate of foci loss and DSB repair (27). Similarly, high levels of TGFb1 and reduced frequency of g-H2AX foci in glioma-initiating cells correlates with better clonogenic survival (20).…”

“…TGFb1 inhibition before g-irradiation attenuated the DNA damage response, increased cell death and delayed tumor growth (4,5), and TGFb1 pretreatment protected Mv1Lu epithelial cells from g-irradiation, an effect that was dependent on de novo protein synthesis (19). Moreover, high levels of TGFb production in gliomainitiating cells in oncosphere culture provide a clonogenic benefit to this population following radiation exposure (20). In addition, as HaCaT and A431 are known to be defective in p53 pathway (21,22), the effect of TGFb1 is independent of p53.…”

“…TGFb1 treatment facilitated the DNA repair process and subsequently enhanced long-term survival upon g-irradiation in cells. In clinical applications using genotoxic damage, TGFb1 at cancer site can sustain cancer cell survival after cancer therapy (4,20); Blockade of TGFb signaling by the TGFbR-I kinase inhibitor LY2109761 enhances radiation response and prolongs survival in glioblastoma (5). This idea is currently being tested in clinical trials for glioblastoma.…”

Tumor microenvironment as a target of malignant gliomas treatment

“…Блокирование TGF-с помощью специфических моноклональных антител [61,62], ингибиторов рецептора TGF-(TGF-R1) [63] или антисмысловых олигонуклеоти-дов представляется заманчивой целью терапии и может, в частности, усиливать эффективность вакцинирования ввиду более выраженного иммунного ответа. Фресолимумаб (Fresolimumab), моноклональное антите-ло к TGF 1, находится в I фазе клинических исследований при меланоме и почечно-клеточном раке [64], а кроме это-го ожидаются результаты исследования NCT 01472731, где оценивалась возможность его применения при ЗГ.…”

Tumor microenvironment as a target of malignant gliomas treatment

“…Фресолимумаб (Fresolimumab), моноклональное антите-ло к TGF 1, находится в I фазе клинических исследований при меланоме и почечно-клеточном раке [64], а кроме это-го ожидаются результаты исследования NCT 01472731, где оценивалась возможность его применения при ЗГ. Пан-TGF -антитело 1D11, нейтрализующее все изоформы TGF-, изучалось на животных моделях злокачественных глиом, где показало значимую противоопухолевую активность [61,65].…”