Radioresistance of Brain Tumors

Radiation therapy (RT) is frequently used as part of the standard of care treatment of the majority of brain tumors. The efficacy of RT is limited by radioresistance and by normal tissue radiation tolerance. This is highlighted in pediatric brain tumors where the use of radiation is limited by the excessive toxicity to the developing brain. For these reasons, radiosensitization of tumor cells would be beneficial. In this review, we focus on radioresistance mechanisms intrinsic to tumor cells. We also evaluate existing approaches to induce radiosensitization and explore future avenues of investigation

Repurposing Mebendazole as a Replacement for Vincristine for the Treatment of Brain Tumors

Abstract The microtubule inhibitor vincristine is currently used to treat a variety of brain tumors, including low-grade glioma and anaplastic oligodendroglioma. Vincristine, however, does not penetrate well into brain tumor tissue, and moreover, it displays dose-limiting toxicities, including peripheral neuropathy. Mebendazole, a Food and Drug Administration-approved anthelmintic drug with a favorable safety profile, has recently been shown to display strong therapeutic efficacy in animal models of both glioma and medulloblastoma. Importantly, appropriate formulations of mebendazole yield therapeutically effective concentrations in the brain. Mebendazole has been shown to inhibit microtubule formation, but it is not known whether its potency against tumor cells is mediated by this inhibitory effect. To investigate this, we examined the effects of mebendazole on GL261 glioblastoma cell viability, microtubule polymerization and metaphase arrest, and found that the effective concentrations to inhibit these functions are very similar. In addition, using mebendazole as a seed for the National Cancer Institute (NCI) COMPARE program revealed that the top-scoring drugs were highly enriched in microtubule-targeting drugs. Taken together, these results indicate that the cell toxicity of mebendazole is indeed caused by inhibiting microtubule formation. We also compared the therapeutic efficacy of mebendazole and vincristine against GL261 orthotopic tumors. We found that mebendazole showed a significant increase in animal survival time, whereas vincristine, even at a dose close to its maximum tolerated dose, failed to show any efficacy. In conclusion, our results strongly support the clinical use of mebendazole as a replacement for vincristine for the treatment of brain tumors

PDZ-RhoGEF Is a Signaling Effector for TROY-Induced Glioblastoma Cell Invasion and Survival

Glioblastoma multiforme (GBM) is the most common type of malignant brain tumors in adults and has a dismal prognosis. The highly aggressive invasion of malignant cells into the normal brain parenchyma renders complete surgical resection of GBM tumors impossible, increases resistance to therapeutic treatment, and leads to near-universal tumor recurrence. We have previously demonstrated that TROY (TNFRSF19) plays an important role in glioblastoma cell invasion and therapeutic resistance. However, the potential downstream effectors of TROY signaling have not been fully characterized. Here, we identified PDZ-RhoGEF as a binding partner for TROY that potentiated TROY-induced nuclear factor kappa B activation which is necessary for both cell invasion and survival. In addition, PDZ-RhoGEF also interacts with Pyk2, indicating that PDZ-RhoGEF is a component of a signalsome that includes TROY and Pyk2. PDZ-RhoGEF is overexpressed in glioblastoma tumors and stimulates glioma cell invasion via Rho activation. Increased PDZ-RhoGEF expression enhanced TROY-induced glioma cell migration. Conversely, silencing PDZ-RhoGEF expression inhibited TROY-induced glioma cell migration, increased sensitivity to temozolomide treatment, and extended survival of orthotopic xenograft mice. Furthermore, depletion of RhoC or RhoA inhibited TROY- and PDZ-RhoGEF–induced cell migration. Mechanistically, increased TROY expression stimulated Rho activation, and depletion of PDZ-RhoGEF expression reduced this activation. Taken together, these data suggest that PDZ-RhoGEF plays an important role in TROY signaling and provides insights into a potential node of vulnerability to limit GBM cell invasion and decrease therapeutic resistance

Semapimod inhibits tumor growth <i>in vivo</i>.

<p>GL261 cells were implanted and mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095885#pone-0095885-g004" target="_blank">Fig. 4</a>. (A) Quantification of tumor volume. Data shown represent the average +/− SEM of 5 different tumors. (B) Quantification of tumor cell density. Data shown represent the average +/− SEM of 5 different tumors. (C) Quantification of total tumor cell number. Data shown represent the average +/− SEM of 5 different tumors. *: p<0.05 student's 2 tailed t-test.</p

Semapimod increases apoptosis induced by radiation <i>in vivo</i>.

<p>GL261 tumors were generated in mice and treatments were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095885#pone-0095885-g006" target="_blank">Fig. 6</a>, except that the total radiation was 8 Gy. The mice were euthanized the day after the last radiation treatment. Frozen brain sections were analyzed for apoptosis using TUNEL staining as described in Materials and Methods. (A) Micrographs of tumor sections illustrating TUNEL staining. (B) Quantification of TUNEL staining. The percentage of apoptotic cells was calculated using 20–60 micrographs per tumor section. Data shown represent the average +/− SEM of 3–4 different tumors. *: p<0.05 student's 2 tailed t-test.</p

Semapimod removes microglia-induced radioprotection on GL261 <i>in vitro</i>.

<p>GL261 cells were cultured in the presence or absence of microglia and in the presence or absence of 200 nM of semapimod, followed by a colony formation assay. (A) Colony formation assay of GL261 after treatment with microglia and semapimod. (B) Determination of survival of GL261 after treatment with 3 Gy irradiation, microglia and semapimod. Data shown represent the average +/− SEM of 3 independent experiments. *: p<0.05 student's 2 tailed t-test.</p

Semapimod does not affect microglia-stimulated growth of glioblastoma cells <i>in vitro</i>.

<p>GL261 cells were cultured in the presence or absence of microglia at the indicated ratio's, in the presence and absence of 200 nM semapimod. Cell growth over a period of 3 days was determined using the SRB method. Data shown represent the average +/− SEM of 3 independent experiments. *: p<0.05, **: p<0.01 student's 2 tailed t-test.</p

Semapimod inhibits glioblastoma cell invasion <i>in vivo</i>.

<p>GL261 cells were orthotopically implanted into C57Bl/6 mice. Starting 7 days after cell inoculation, the mice were treated intracranially for 1 week with semapimod, delivered via an osmotic pump. (A) Micrographs of tumor sections illustrating inhibition of GL261 cell invasion by semapimod. GL261 cells were visualized using Ki67 staining. The tumor borders are outlined. Scale bar represents 200 µm. (B) Quantification of the number of invaded GL261 cells normalized to the length of the tumor border (expressed in mm). Data shown represent the average +/− SEM of 5 different tumors. **: p<0.01 student's 2 tailed t-test. (C) Micrographs illustrating infiltration of microglia into GL261 tumors. Activated microglia were visualized using Iba1 staining. (D) Quantification of Iba1⁺ microglia infiltrated into the tumor. Scale bar represents 100 µm. Data shown represent the average +/− SEM of 5 different tumors.</p