Therapeutic resistance is a major barrier to improvement of outcomes for patients with glioblastoma. The endoplasmic reticulum stress response (ERSR) has been identified as a contributor to chemoresistance in glioblastoma; however the contributions of the ERSR to radioresistance have not been characterized. In this study we found that radiation can induce ER stress and downstream signaling associated with the ERSR. Induction of ER stress appears to be linked to changes in ROS balance secondary to irradiation. Furthermore, we observed global induction of genes downstream of the ERSR in irradiated glioblastoma. Knockdown of ATF6, a regulator of the ERSR, was sufficient to enhance radiation induced cell death. Also, we found that activation of ATF6 contributes to the radiation-induced upregulation of glucose regulated protein 78 (GRP78) and NOTCH1. Our results reveal ATF6 as a potential therapeutic target to enhance the efficacy of radiation therapy.
Non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM) have poor median survival. NSCLC and GBM overexpress glucose regulated protein 78 (GRP78), which has a role in radioresistance and recurrence. In this study, we determined the effect of anti-GRP78 antibody and the combined effect of the anti-GRP78 antibody with ionizing radiation (XRT) on NSCLC and GBM cell lines both and NSCLC and GBM cancer cell lines were treated with anti-GRP78 antibodies and evaluated for proliferation, colony formation, cell death, and PI3K/Akt/mTOR signaling. The efficacy of anti-GRP78 antibodies on tumor growth in combination with XRT was determined in mouse xenograft models. GBM and NSCLC cells treated with anti-GRP78 antibodies showed attenuated cell proliferation, colony formation, and enhanced apoptosis. GBM and NSCLC cells treated with anti-GRP78 antibodies also showed global suppression of PI3K/Akt/mTOR signaling. Combining antibody with XRT resulted in significant tumor growth delay in both NSCLC and GBM heterotopic tumor models. Antibodies targeting GRP78 exhibited antitumor activity and enhanced the efficacy of radiation in NSCLC and GBM both and GRP78 is a promising novel target, and anti-GRP78 antibodies could be used as an effective cancer therapy alone or in combination with XRT. .
The aggressive nature and inherent therapeutic resistance of glioblastoma multiforme (GBM) has rendered the median survival of afflicted patients to 14 months. Therefore, it is imperative to understand the molecular biology of GBM to provide new treatment options to overcome this disease. It has been demonstrated that the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is an important regulator of the endoplasmic reticulum (ER) stress response. PERK signaling has been observed in other model systems after radiation; however, less is known in the context of GBM, which is frequently treated with radiation-based therapies. To investigate the significance of PERK, we studied activation of the PERK-eIF2α-ATF4 pathway in GBM after ionizing radiation (IR). By inhibiting PERK, it was determined that ionizing radiation (IR)-induced PERK activity led to eIF2α phosphorylation. IR enhanced the prodeath component of PERK signaling in cells treated with Sal003, an inhibitor of phospho-eIF2α phosphatase. Mechanistically, ATF4 mediated the prosurvival activity during the radiation response. The data support the notion that induction of ER stress signaling by radiation contributes to adaptive survival mechanisms during radiotherapy. The data also support a potential role for the PERK/eIF2α/ATF4 axis in modulating cell viability in irradiated GBM. The dual function of PERK as a mediator of survival and death may be exploited to enhance the efficacy of radiation therapy. http://mcr.aacrjournals.org/content/16/10/1447/F1.large.jpg .
Radiation-induced lymphopenia (RIL) is associated with treatment of different tumors (lung, colon, pancreas, breast, sarcomas, and glioblastoma). It is a significant clinical problem affecting the survival of cancer patients. The biologic mechanisms leading to RIL are not clearly understood. In this study, we established a mouse model of RIL representing therapeutic clinical regimen for lung cancer. Flow cytometry was used to analyze circulating levels of T and B cells and bone marrow (BM) stem cells. We found that fractionated radiation to the thorax significantly reduced circulating T and B cells as well as BM stem cells. Ex-vivo irradiation of blood and autologous reinjection to mice also significantly induced lymphopenia. Furthermore, we found that mobilization of stem cells from the BM and autologous stem cell transplant rescued RIL in mice. Overall, our results suggest that RIL has not only direct effect on circulating lymphocytes, but also has indirect effect on circulating lymphocytes as well as stem cells in the nonirradiated BM. These results open a new window for investigating the direct and indirect biologic mechanisms leading to RIL, and provide a preclinical basis to test the effect of stem cell transplantation for treatment of RIL in cancer patients. CancerRes; 75(17); 3442-5. Ó2015 AACR.
Cancer-specific targeting sparing normal tissues would significantly enhance cancer therapy outcomes and reduce cancer-related mortality. One approach is to target receptors or molecules that are specifically expressed on cancer cells. Peptides as cancer-specific targeting agents offer advantages such as ease of synthesis, low antigenicity, and enhanced diffusion into tissues. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum stress chaperone that regulates the unfolded protein response and is overexpressed in various cancers. In this study, we evaluated GIRLRG peptide that specifically targets GRP78 for cancer-specific binding (in vitro) and noninvasive tumor imaging (in vivo). Methods: GIRLRG peptide was modeled into the GRP78 ATPase domain using computational modeling. Surface plasmon resonance studies were performed to determine the affinity of GIRLRG peptide to GRP78 protein. GIRLRG was conjugated with PEG to prolong its circulation in mice. Tumor binding efficacy of PEG-GIRLRG peptide was evaluated in nude mice bearing heterotopic cervical (HT3), esophageal (OE33), pancreatic (BXPC3), lung (A549), and glioma (D54) tumors. Nano-SPECT/CT imaging of the mice was performed 48 and 72 h after injection with 111 In-labeled PEG-GIRLRG or PEG-control peptide. Post-SPECT biodistribution studies were performed 96 h after injection of the radiolabeled peptides. Results: Using molecular modeling and surface plasmon resonance, we identified that GIRLRG was binding with an affinity constant of 2.16 · 10 −3 M in the ATPase domain of GRP78. GIRLRG peptide specifically bound to cervical, lung, esophageal, and glioma cells. SPECT imaging revealed that 111 In-PEG-GIRLRG specifically bound to cervical, esophageal, pancreatic, lung, and brain tumors. Post-SPECT biodistribution data also validated the SPECT imaging results. Conclusion: GIRLRG peptide specifically binds to the ATPase domain of GRP78. Radiolabeled PEG-GIRLRG could be used to target various cancers. Further studies would be required to translate PEG-GIRLRG peptide into the clinic.
There are multiple intravenous (IV) iron formulations available, of which several may be administered as single-dose infusions such as low-molecular weight iron dextran (LMWID), ferumoxytol, ferric carboxymaltose, and ferric derisomaltose. However, administration of ferumoxytol as a single-dose infusion is off-label as it is approved as a two-dose series. Previous studies of ferumoxytol alone support the effectiveness and safety of the single-dose regimen, but there is a paucity of data directly comparing single-dose ferumoxytol to other single-dose IV iron formulations. This multicenter cohort study sought to affirm the safety and effectiveness of single-dose ferumoxytol compared to single-dose LMWID. Overall, 906 patients who received single-dose LMWID (n = 439) or ferumoxytol (n = 467) were identified, of whom 351 met criteria for the primary effectiveness endpoint defined as median change in hemoglobin (Hb), hematocrit (Hct), and ferritin 8 to 12 weeks from baseline. All 906 patients were included for the secondary analysis evaluating the incidence of adverse events (AE) and requirement of additional IV iron infusions. Median change in Hb (LMWID 0.5 g/dL; ferumoxytol 0.8 g/dL; P = .24), Hct (LMWID 1.1%; ferumoxytol 1.25%; P = .89), and ferritin (LMWID 87 ng/dL; ferumoxytol 71 ng/dL; P = .47) was not significantly different between groups. Both groups experienced similar rates of AEs (LMWID 2.3%; ferumoxytol 2.8%; P = .63). The LMWID patients more frequently required additional IV iron infusions (LMWID 28.5%; ferumoxtyol 16.1%; P < .001). These findings support that single-dose ferumoxytol is effective and safe, and that patients may require fewer additional infusions compared to patients who received LMWID.
Glioblastoma is the most common primary malignant brain tumor in adults in the United States. While the standard of care for afflicted patients has evolved to include surgery, ionizing radiation (IR) and temozolomide chemotherapy, the prognoses of these patients remain dismal. With the median survival of glioblastoma patients remaining in the range of 12-15 months, there is an unmet need for approaches that can overcome the inherent therapeutic resistance of these tumors. In recent years, several studies have demonstrated that IR can activate pro-survival signaling that may facilitate adaptation to therapy and the development of radio-resistance. The endoplasmic reticulum stress response (ERSR) is a conserved program known to be deregulated in cancer and can mediate pro-survival signaling in the context of therapy. The ATF6, IRE1 and PERK pathways of the ERSR are known to be important mediators in the initiation of this signaling. In this research, we show for the first time that IR, a key component in glioblastoma therapy, can induce genes downstream of ATF6, IRE1 and PERK in glioblastoma cell lines. Furthermore, we identify the ATF6 and PERK-eIF2a-ATF4 pathways as important contributors to the radiation-response in glioblastoma. Our study began with the observation of protein and mRNA induction of targets associated with the ERSR in irradiated glioblastoma cell lines. In addition to ATF6 target genes, we observed robust induction of PERK target genes 48h after 6 Gy IR. This induction of PERK target genes was accompanied with increased phosphorylation of eIF2a in a time and dose-dependent manner. To evaluate the requirement of PERK for radiation-induced eIF2a phosphorylation, we used a specific inhibitor of PERK - GSK2606414, and found that PERK inhibition was sufficient to prevent radiation-induced eIF2a phosphorylation. We also analyzed expression of several genes downstream of PERK after treatment with GSK2606414, and found that PERK inhibition lead to 70-80% reduction in radiation-induction of downstream genes. Since ATF4 is downstream of PERK-eIF2a, we examined ATF4 expression in irradiated glioblastoma and found that it was also induced by radiation. Using RNA-interference, we found that knockdown of ATF4 reduced proliferation and clonogenic survival by 50%, and resulted in 38% increase in PARP cleavage in irradiated glioblastoma. We observed similar trends when we knocked down ATF6, suggesting that multiple aspects of the ERSR can contribute to the radiation response in glioblastoma. These results indicate that IR-induced activation of ER-stress signaling through PERK and ATF6 contributes to adaptive survival mechanisms in glioblastoma. Further characterization of the mechanism by which ATF6 and ATF4 mediate cell survival after irradiation may reveal novel targets for the enhancement of radiotherapy for glioblastoma. Citation Format: David Dadey, Vaishali Kapoor, Arpine Khudanyan, Dinesh Thotala, Dennis Hallahan. Radiation-induced ER stress contributes to survival in glioblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2015-3323
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