Enhanced Radiation Sensitivity Research Articles

Abstract 4904: β-catenin silencing enhances radiation sensitivity through antagonizing effect of AMPK against Ku70/80 in head and neck cancer cells

Abstract The Wnt/β-catenin pathway regulates the cell growth and survival following radiation in various types of cancer cells. Our previous report show that activation of the Wnt/β-catenin signaling pathway is a key radioprotective mechanism in irradiated head and neck cancer (HNC) cells. However, the molecular mechanisms by which β-catenin regulates radiation sensitivity are not clear. Here we attempted to elucidate the mechanism of cell death following radiation by studying how β-catenin silencing controls the radiation sensitivity of radioresistant HNC cells. Of nine cell lines examined, the most radioresistant cell line (AMC-HN-9) were selected for this experiments. β-catenin silencing using small interfering RNA(siRNA) down-regulated β-catenin expression up to 72 h, which was confirmed by western blot analysis. The sensitivity to radiation was anlayzed by clonogenic analysis and MTT assay. As a result, β-catenin silencing remarkably decreased the survival of irradiated AMC-HN-9 cells and the cell viability also significantly reduced more by the combination treatment with β-catenin siRNA and radiation (0.37±0.034 fold) than when treated with β-catenin siRNA or radiation alone (0.68±0.055 fold and 0.90±0.043 fold, respectively). Interestingly, whereas expression of Ku70/80 was up regulated in AMC-HN-9 cells following irradiation (4Gy), in the cells treated with combination of radiation and β-catenin siRNA, Ku70/80 expression was dramatically decreased. In additionally, when exposed to radiation after β-catenin silencing, the up-regulation of irradiation-induced Ku80 completely was prevented by β-catenin silencing-induced AMPK. Taken together, these results suggest that suppression of Ku70/80 expression through β-catenin silencing-induced AMPK is associated with its radio-sentitizing effect in AMC-HN-9 cells, thus supporting a novel radiosensitive mechanism of radioresistant HNC cells. Citation Format: Hyo Won Chang, Hae Yun Nam, Mi Ra Kim, Hyang Ju Lee, Ji Hyun Seo, So Yeon Lee, Seong Who Kim, Sang Yoon Kim. β-catenin silencing enhances radiation sensitivity through antagonizing effect of AMPK against Ku70/80 in head and neck cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2014-4904

Inhibition of N-acetylglucosaminyltransferase V enhances sensitivity of radiotherapy in human prostate cancer

The purpose of this study was to investigate the relationship between N-acetylglucosaminyltransferase V (GnT-V) and radiation sensitivity of prostate cancer (PCa) cells both in vitro and in vivo. Firstly, the GnT-V expression was studied in 84 cases of PCa tissues, in which higher level of GnT-V was detected more frequently in the advanced tumors. Secondly, the GnT-V stably suppressed cell lines PCa/1079 (Lncap/1079 and PC3/1079) were constructed from PCa cell lines (Lncap and PC3) in vitro. Attenuation of GnT-V inhibited cell proliferation, migration and increased apoptosis, which resulted in enhanced radiation sensitivity of PCa cells. The underlying mechanism may be relevant to the increasing ratio of Bax/Bcl-2, the blocking transcription of NF-κB and the reduction of cell cycle G2-M arrest. Finally, in in vivo study, compared with control groups, the irradiated PCa xenograft nude mice of PCa/1079 indicated to reduce tumor-growth rate and enhance survival time. Summary, our studies showed that inhibition of GnT-V probably improved PCa cells’ radiation sensitivity.

MicroRNA-223 Enhances Radiation Sensitivity of U87MG Cells In Vitro and In Vivo by Targeting Ataxia Telangiectasia Mutated

Ataxia telangiectasia mutated (ATM) protein is important in the DNA damage response because it repairs radiation-induced damage in cancers. We examined the effect of microRNA-223 (miR-223), a regulator of ATM expression, on radiation sensitivity of cancer cells. Human embryonic kidney 293 T (293T) cells were infected with pLL3.7-miR-223 plasmid to generate the pLL3.7-miR-223 and -empty virus (EV) lentivirus (miR-223 and EV). A dual luciferase assay in which the reporter contained wild-type 3' untranslated region (UTR) of ATM was performed. U87MG cells were infected with miR-223 or EV to establish the overexpressed stable cell lines (U87-223 or U87-EV, respectively). Cells were irradiated in vitro, and dose enhancement ratios at 2 Gy (DER2) were calculated. Hind legs of BALB/c athymic mice were injected with U87-223 or U87-EV cells; after 2 weeks, half of the tumors were irradiated. Tumor volumes were tracked for a total of 5 weeks. The dual luciferase reporter assay showed a significant reduction in luciferase activity of 293T cells cotransfected with miR-223 and the ATM 3'UTR compared to that in EV control. Overexpression of miR-223 in U87MG cells showed that ATM expression was significantly downregulated in the U87-223 cells compared to that in U87-EV (ATM/β-actin mRNA 1.0 vs 1.5, P<.05). U87-223 cells were hypersensitive to radiation compared to U87-EV cells in vitro (DER2 = 1.32, P<.01). Mice injected with miR-223-expressing tumors had almost the same tumors after 3 weeks (1.5 cm(3) vs 1.7 cm(3)). However, irradiation significantly decreased tumor size in miR-223-expressing tumors compared to those in controls (0.033 cm(3) vs 0.829 cm(3)). miR-223 overexpression downregulates ATM expression and sensitizes U87 cells to radiation in vitro and in vivo. MicroRNA-223 may be a novel cancer-targeting therapy, although its cancer- and patient-specific roles are currently undefined.

Suppression of telomere-binding protein TPP1 resulted in telomere dysfunction and enhanced radiation sensitivity in telomerase-negative osteosarcoma cell line

Mammalian telomeres are protected by the shelterin complex that contains the six core proteins POT1, TPP1, TIN2, TRF1, TRF2 and RAP1. TPP1, formerly known as TINT1, PTOP, and PIP1, is a key factor that regulates telomerase recruitment and activity. In addition to this, TPP1 is required to mediate the shelterin assembly and stabilize telomere. Previous work has found that TPP1 expression was elevated in radioresistant cells and that overexpression of TPP1 led to radioresistance and telomere lengthening in telomerase-positive cells. However, the exact effects and mechanism of TPP1 on radiosensitivity are yet to be precisely defined in the ALT cells. Here we report on the phenotypes of the conditional deletion of TPP1 from the human osteosarcoma U2OS cells using ALT pathway to extend the telomeres.TPP1 deletion resulted in telomere shortening, increased apoptosis and radiation sensitivity enhancement. Together, our findings show that TPP1 plays a vital role in telomere maintenance and protection and establish an intimate relationship between TPP1, telomere and cellular response to ionizing radiation, but likely has the specific mechanism yet to be defined.

Radiation Tolerance of Devices and Circuits in a 3D Technology Based on the Vertical Integration of Two 130-nm CMOS Layers

Total ionizing dose effects are studied in 130-nm transistors and pixel sensors in a vertically integrated two-layer CMOS technology, evaluating the possible impact of 3D integration on radiation tolerance and damage mechanisms. Measurements of static characteristics and noise voltage spectra before and after exposure to high total ionizing doses demonstrate that the analog performance of transistors as well as their radiation hardness are not degraded by mechanical and thermal stresses occurring during the fabrication of the 3D chips. The paper also presents irradiation results on 3D CMOS pixel sensors with a sparsified readout architecture. After exposure to ionizing radiation, these devices behave in a very similar way as analogous counterparts in a standard 2D 130-nm process, confirming that performance advantages associated with 3D integration are not impaired by an enhanced radiation sensitivity.

Enhanced Radiographic Response and Retreatment Rates Following Whole Brain Irradiation Among Non-Small Cell Lung Cancer Patients With EGFR-Activating Mutations

Epidermal growth factor receptor (EGFR) activating mutations in non-small cell lung cancer (NSCLC) are correlated with prolonged survival, and several studies suggest enhanced radiation sensitivity in vivo. EGFR mutant NSCLC has a propensity for development of brain metastases. We compared radiographic response, re-irradiation rates, and survival among EGFR mutant and wild type (WT) patients following whole brain radiation therapy (WBRT).

Slug expression inhibits calcitriol‐mediated sensitivity to radiation in colorectal cancer

Recently, a reciprocal relationship between calcitriol and epithelial-to-mesenchymal transition has been described. Therefore, we hypothesized that calcitriol (1α,25-dihydroxyvitamin D₃) would enhance radiation sensitivity in colorectal cancer regulated by epithelial mesenchymal transition. Vitamin-D receptor, E-cadherin and vimentin protein as well as E-cadherin, Snail and Slug mRNA levels were assessed in a panel of human colorectal cancer cell lines at baseline and in response calcitriol. We defined cell lines as calcitriol sensitive based on demonstrating an enhanced epithelial phenotype with increased E-cadherin, reduced vimentin and decreased expression of Snail and Slug as well as decreased cellular migration in response to calcitriol. In calcitriol sensitive cells, including DLD-1 and HCT116, 24 h calcitriol pre-treatment enhanced the radiation sensitivity by 2.3- and 2.6-fold, respectively, at 4 Gy (P < 0.05). In contrast, SW620 cells with high baseline mesenchymal features including high Slug and vimentin expression with low E-cadherin expression demonstrated no significant radiation sensitizing response to calcitriol treatment. Similarly, transfection of Slug in the calcitriol sensitive colon cancer cell lines, DLD-1 and HCT 116, completely inhibited the radiation sensitizing effect of calcitriol. Collectively, we demonstrate that calcitriol can enhance the therapeutic effects of radiation in colon cancer cells and Slug expression mitigates this observed effect potentially representing an effective biomarker for calcitriol therapy.

Update on hepatocellular carcinoma breakthroughs: Poly(ADP-ribose) polymerase inhibitors as a promising therapeutic strategy

Hepatocellular carcinoma is the most common form of primary liver cancer which is the fifth most common cancer in men and the seventh in women and the third most common cause of cancer-related death worldwide. Only 10-20% of patients are eligible for curative treatments that result in a 5-year survival rate of 40% to 70%. Therefore, the development of novel treatment options is necessary for the majority of patients and remains a considerable challenge. Conformal radiotherapy is used in certain circumstances and preliminary data obtained from phase 1/2 trials are showing promising curative effects. There is thus an interest in identifying drugs that can be exploited to enhance radiation sensitivity that could be used in therapy and might improve clinical outcome. Small molecules inhibitors of poly(ADP-ribose) polymerases (PARP) are an example of a radio- and chemo-sensitizing drug, as well as being an efficient single agent treatment in certain genetic backgrounds. In this review, we discuss the role of PARP-1 in hepatocellular carcinoma and present the results of preclinical studies that have assessed the potential of PARP inhibition as a single treatment or combined with chemotherapy or radiotherapy for the treatment of hepatocellular carcinoma.

Enhanced Radiation Sensitivity in HPV-Positive Head and Neck Cancer

Patients with human papillomavirus (HPV+)-associated head and neck cancer (HNC) show significantly improved survival outcome compared with those with HPV-negative (HPV-) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV- HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV-: 0.59 vs. HPV+: 0.22; P < 0.0001), corresponding with a prolonged G2-M cell-cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%; P = 0.002). A genome-wide microarray was used to compare gene expression 24 hours following radiation between HPV+ and HPV- cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6-fold increase in TP53 (P < 0.0001). Using immortalized human tonsillar epithelial (HTE) cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV- HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.

Inhibition of β2-Microglobulin/Hemochromatosis Enhances Radiation Sensitivity by Induction of Iron Overload in Prostate Cancer Cells

BackgroundBone metastasis is the most lethal form of several cancers. The β2-microglobulin (β2-M)/hemochromatosis (HFE) complex plays an important role in cancer development and bone metastasis. We demonstrated previously that overexpression of β2-M in prostate, breast, lung and renal cancer leads to increased bone metastasis in mouse models. Therefore, we hypothesized that β2-M is a rational target to treat prostate cancer bone metastasis.ResultsIn this study, we demonstrate the role of β2-M and its binding partner, HFE, in modulating radiation sensitivity and chemo-sensitivity of prostate cancer. By genetic deletion of β2-M or HFE or using an anti-β2-M antibody (Ab), we demonstrate that prostate cancer cells are sensitive to radiation in vitro and in vivo. Inhibition of β2-M or HFE sensitized prostate cancer cells to radiation by increasing iron and reactive oxygen species and decreasing DNA repair and stress response proteins. Using xenograft mouse model, we demonstrate that anti-β2-M Ab sensitizes prostate cancer cells to radiation treatment. Additionally, anti-β2-M Ab was able to prevent tumor growth in an immunocompetent spontaneous prostate cancer mouse model. Since bone metastasis is lethal, we used a bone xenograft model to test the ability of anti-β2-M Ab and radiation to block tumor growth in the bone. Combination treatment significantly prevented tumor growth in the bone xenograft model by inhibiting β2-M and inducing iron overload. In addition to radiation sensitive effects, inhibition of β2-M sensitized prostate cancer cells to chemotherapeutic agents.ConclusionSince prostate cancer bone metastatic patients have high β2-M in the tumor tissue and in the secreted form, targeting β2-M with anti-β2-M Ab is a promising therapeutic agent. Additionally, inhibition of β2-M sensitizes cancer cells to clinically used therapies such as radiation by inducing iron overload and decreasing DNA repair enzymes.

BUB1 and BUBR1 inhibition decreases proliferation and colony formation, and enhances radiation sensitivity in pediatric glioblastoma cells

Glioblastoma (GBM) is a very aggressive and lethal brain tumor with poor prognosis. Despite new treatment strategies, patients' median survival is still lower than 1 year in most cases. The expression of the BUB gene family has demonstrated to be altered in a variety of solid tumors, pointing to a role as putative therapeutic target. The purpose of this study was to determine BUB1, BUB3, and BUBR1 gene expression profiles in glioblastoma and to analyze the effects of BUB1 and BUBR1 inhibition combined or not with Temozolomide and radiation in the pediatric SF188 GBM cell line. For gene expression analysis, 8 cell lines and 18 tumor samples were used. The effect of BUB1 and BUBR1 inhibition was evaluated using siRNA. Apoptosis, cell proliferation, cell cycle kinetics, micronuclei formation, and clonogenic capacity were analyzed after BUB1 and BUBR1 inhibition. Additionally, combinatorial effects of gene inhibition and radiation or Temozolomide (TMZ) treatment were evaluated through proliferation and clonogenic capacity assays. We report the upregulation of BUB1 and BUBR1 expression and the downregulation of BUB3 in GBM samples and cell lines when compared to white matter samples (p < 0.05). Decreased cell proliferation and colony formation after BUB1 and BUBR1 inhibition were observed, along with increased micronuclei formation. Combinations with TMZ also caused cell cycle arrest and increased apoptosis. Moreover, our results demonstrate that BUB1 and BUBR1 inhibition sensitized SF188 cells to γ-irradiation as shown by decreased growth and abrogation of colony formation capacity. BUB1 and BUBR1 inhibition decreases proliferation and shows radiosensitizing effects on pediatric GBM cells, which could improve treatment strategies for this devastating tumor. Collectively, these findings highlight the potentials of BUB1 and BUBR1 as putative therapeutic targets for glioblastoma treatment.

A potential role for resveratrol as a radiation sensitizer for melanoma treatment

BackgroundRadiotherapy (XRT) is used to improve local control of melanoma and for palliation of metastatic disease. Clinical use of XRT for melanoma is often limited by extent of disease and the relative radioresistance of melanoma may limit the effectiveness of XRT. Our group and others have previously shown that resveratrol (RSV) enhances radiation sensitivity in radioresistant prostate cancer cell lines. Material and MethodsIn this study, the effects of XRT in combination with RSV on radioresistant melanoma lines, SK-Mel-5 and HTB-65, were evaluated by assessment of proliferation and apoptosis. Clonogenic assay, comparison of proliferating cell nuclear antigen staining, Quick Cell Proliferation assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and caspase-3 activity assay were used to assess proliferation and apoptosis, as appropriate. ResultsWe found that the percentage of colonies, proliferating cell nuclear antigen + cells and the optical density value of melanoma cells were decreased after addition of RSV to XRT (XRT/RSV). TUNEL + cells and the relative caspase-3 activity in melanoma cells were increased after addition of RSV to XRT (XRT/RSV). We investigated the possible molecular mechanisms of decreased proliferation and increased apoptosis by using reverse transcriptase-polymerase chain reaction and immunohistochemical staining. The anti-proliferative effect of XRT/RSV correlated with decreased expression of pro-proliferative molecule cyclin B, cyclin D, cdk2 and cdk4. The pro-apoptotic effect of XRT/RSV correlated with decreased expression of the anti-apoptotic molecule FLIP, Bcl-2, and survivin. ConclusionThese data suggest that RSV enhances radiation sensitivity of melanoma cells by inhibiting proliferation and promoting apoptosis. Resveratrol may have a potential role as a radiation sensitizer for melanoma treatment.

Inhibition of COX-2 expression by topical diclofenac enhanced radiation sensitivity via enhancement of TRAIL in human prostate adenocarcinoma xenograft model

BackgroundCOX-2 inhibitors have an antitumor potential and have been verified by many researchers. Treatment of cancer cells with external stressors such as irradiation can stimulate the over-expression of COX-2 and possibly confer radiation resistance. In this study, we tested if topical diclofenac, which inhibits both COX-1 and COX-2, administration rendered prostate tumor cells sensitize to the effects of radiation.MethodsLNCaP-COX-2 and LNCaP-Neo cells were treated with 0 to 1000 μM diclofenac. Next, a clonogenic assay was performed in which cells were subjected to irradiation (0 to 4 Gy) with or without diclofenac. COX-2 expression and other relevant molecules were measured by real-time PCR and immunohistochemistry after irradiation and diclofenac treatment. In addition, we assessed the tumor volumes of xenograft LNCaP-COX-2 cells treated with topical diclofenac with or without radiation therapy (RT).ResultsLNCaP-COX-2 and LNCaP-Neo cell lines experienced cytotoxic effects of diclofenac in a dose related manner. Clonogenic assays demonstrated that LNCaP-COX-2 cells were significantly more resistant to RT than LNCaP-Neo cells. Furthermore, the addition of diclofenac sensitized LNCaP-COX-2 not but LNCaP-Neo cells to the cytocidal effects of radiation. In LNCaP-COX-2 cells, diclofenac enhanced radiation-induced apoptosis compared with RT alone. This phenomenon might be attributed to enhancement of RT-induced TRAIL expression as demonstrated by real-time PCR analysis. Lastly, tumor volumes of LNCaP-COX-2 cells xenograft treated with diclofenac or RT alone was >4-fold higher than in mice treated with combined diclofenac and radiation (p<0.05).ConclusionsThese in vitro and in vivo findings suggest that conventional COX inhibitor, diclofenac enhances the effect of RT on prostate cancer cells that express COX-2. Thus, diclofenac may have potential as radiosensitizer for treatment of prostate cancer.

Effect of PF-02341066 and radiation on non-small cell lung cancer cells

Recently, a fusion protein of echinoderm microtubule associated protein like-4 (EML4) and anaplastic lymphoma kinase (ALK) has been found in non-small cell lung cancer (NSCLC) patients. In addition, endogenous expression of phosphorylated c-Met was found to be increased in many invasive NSCLC cases. PF-02341066 (crizotinib) is a novel dual c-Met and EML4-ALK inhibitor, and preclinical studies have shown that treatment with ALK inhibitors leads to drastic tumor regression in xenograft models. A phase I trial of PF-02341066 yielded a 53% response rate and a disease control rate of 79%. We evaluated crizotinib as a potential radiation-sensitizing agent in multiple established NSCLC cell lines with varying expression levels of c-Met and EML4-ALK. The combined effect of ionizing radiation (IR) and PF-02341066 was determined by the surviving cell fraction, cell cycle distribution, apoptosis, DNA double-strand break repair in 5 NSCLC cell lines (A549, H460, H3122, H2228 and H1993) and in in vivo xenograft studies. Treatment of NSCLC cells with either PF-02341066 alone or PF-02341066 + IR did not significantly alter cellular radiosensitivity, DNA repair kinetics and cell cycle distribution; no significant enhancement of tumor growth delay was noted in response to the combined treatment of PF-02341066 + IR. EML4-ALK and c-Met inhibition leads to activation of parallel pathways that converge on Akt signaling which abrogates any radiation-sensitizing effect. Although PF-02341066 is an effective therapy able to suppress tumor growth in tumors that exhibit positivity for either EML4-ALK or c-Met, it did not affect the intrinsic radiation response of tumor cell lines. In the present study, we demonstrated that PF-02341066 did not enhance radiation sensitivity in a panel of NSCLC cell lines.

Abstract P5-03-02: Targeting mRNA Translation to Enhance the Radiosensitivity of Inflammatory Breast Cancer Stem Cells

Abstract Purpose/Objective(s): Inflammatory breast cancer (IBC) is a highly aggressive and radiation resistant malignancy with a dismal prognosis despite multimodality therapy, including ionizing radiation. We have previously shown that the unique pathogenic properties of IBC result in part from over-expression of translation initiation factor eIF4G1, which is part of the eIF4F translation initiation complex, along with eIF4E and eIF4A. eIF4F is regulated by mTOR, providing a promising target for anti-cancer therapeutics. We demonstrated that protein synthesis is highly regulated during IR by the DNA-damage response (DDR) pathway through mTOR signaling. Many key proteins required for the DDR pathway are encoded by mRNAs that require high levels of the eIF4F complex and mTOR activity for their efficient translation. We hypothesized that upregulation of eIF4F in IBC plays a crucial role in the radio-resistance of disease. Materials/Methods: Experiments were conducted in IBC SUM149 cells. eIF4G1, eIF4E and eIF4A were silenced through the generation of stable cell lines that express tetracycline-inducible shRNAs. eIF4A was also inhibited using the pharmacologic investigational inhibitor DAMD-PatA. Radiation sensitivity in vitro was determined by cell survival assay. Tumor xenografts were generated by the injection of stable shRNA inducible cell lines into nude mice. IBC SUM149 cancer stem cells (CSC) from both in vitro and in vivo experiments were analyzed by a combination of cell surface marker analysis, mammosphere formation and Aldefluor assays. Results: We show that moderate inhibition by silencing of individual components (or by pharmacologic inhibition of eIF4A) of the eIF4F complex prevents IBC xenograft tumor growth and strongly enhances radiosensitivity. In contrast to results obtained for non-transformed breast epithelial cells, reducing the high levels of eIF4G1 in epithelial IBC cells in 2D cultures provides no enhancement in radiation sensitivity. Rather, SUM149 IBC cells harbor a substantial population of CSCs, which are the cells that are strongly dependent on high levels of eIF4G1, and which are selectively radio-sensitized as a result of eIF4G1-silencing. CSCs also require eIF4E and eIF4A activity in order to survive radiation treatment. We also demonstrate that silencing of eIF4G1 radio-sensitizes the stem cell population within IBC tumor xenografts. Radio-resistance of IBC cells is likely mediated by differential responses to the DDR in the stem-cell populations and by selective mRNA translation of proteins involved in the DDR pathway. Conclusions: Our results demonstrate that regulation of mRNA translation plays an important role in conferring radio-resistance to advanced breast cancers, particularly by allowing the survival of the CSC compartment. While inhibition of eIF4F enhances radiation sensitivity in non-transformed cells, this process is abrogated in IBC due to enrichment of a radiation resistant CSC population, demonstrating translational control of the breast cancer stem cell population, and providing a novel understanding of the role of the regulation of mRNA translation in radiation resistance of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-02.

DAB2IP Regulates Autophagy in Prostate Cancer in Response to Combined Treatment of Radiation and a DNA-PKcs Inhibitor

Radiation therapy (RT) is an effective strategy for the treatment of localized prostate cancer (PCa) as well as local invasion. However, some locally advanced cancers develop radiation resistance and recur after therapy; therefore, the development of radiation-sensitizing compounds is essential for treatment of these tumors. DOC-2/DAB2 interactive protein (DAB2IP), which is a novel member of the Ras-GTPase activating protein family and a regulator of phosphatidylinositol 3-kinase-Akt activity, is often downregulated in aggressive PCa. Our previous studies have shown that loss of DAB2IP results in radioresistance in PCa cells primarily because of accelerated DNA double-strand break (DSB) repair kinetics, robust G(2)/M checkpoint control, and evasion of apoptosis. A novel DNA-PKcs inhibitor NU7441 can significantly enhance the effect of radiation in DAB2IP-deficient PCa cells. This enhanced radiation sensitivity after NU7441 treatment is primarily due to delayed DNA DSB repair. More significantly, we found that DAB2IP-deficient PCa cells show dramatic induction of autophagy after treatment with radiation and NU7441. However, restoring DAB2IP expression in PCa cells resulted in decreased autophagy-associated proteins, such as LC3B and Beclin 1, as well as decreased phosphorylation of S6K and mammalian target of rapamycin (mTOR). Furthermore, the presence of DAB2IP in PCa cells can lead to more apoptosis in response to combined treatment of NU7441 and ionizing radiation. Taken together, NU7441 is a potent radiosensitizer in aggressive PCa cells and DAB2IP plays a critical role in enhancing PCa cell death after combined treatment with NU7441 and radiation.

Inhibition of cyclin-dependent kinase 6 suppresses cell proliferation and enhances radiation sensitivity in medulloblastoma cells

Medulloblastoma accounts for 20% of all primary pediatric intracranial tumors. Current treatment cures 50-80% of patients but is associated with significant long-term morbidity and thus new therapeutic targets are needed. One such target is cyclin-dependent kinase6 (CDK6), a serine/threonine kinase that plays a vital role in cell cycle progression and differentiation. CDK6 is overexpressed in medulloblastoma patients and is associated with an adverse prognosis. To investigate the role of CDK6 in medulloblastoma, we assayed the effect of CDK6 inhibition on proliferation by depleting expression with RNA interference (RNAi) or by inhibiting kinase function with a small molecule inhibitor, PD0332991. Cell proliferation was assessed by colony focus assay or by the xCELLigence system. We then investigated the impact of CDK6 inhibition on differentiation of murine neural stem cells by immunofluorescence of relevant markers. Finally we evaluated the effects of PD0332991 treatment on medulloblastoma cell cycle and radiosensitivity using colony focus assays. Gene expression analysis revealed that CDK6 mRNA expression is higher than normal cerebellum in fifteen out of sixteen medulloblastoma patient samples. Inhibition of CDK6 by RNAi significantly decreased medulloblastoma cell proliferation and colony forming potential. Interestingly, CDK6 inhibition by RNAi increased differentiation in murine neural stem cells. PD0332991 treatment significantly decreased medulloblastoma cell proliferation and led to a G0/G1 cell cycle arrest. Furthermore, PD0332991 pretreatment sensitized medulloblastoma cells to ionizing radiation. Our findings suggest that targeting CDK6 with small molecule inhibitors may prove beneficial in the treatment of medulloblastoma, especially when combined with radiation.

Increased aldehyde reductase expression mediates acquired radioresistance of laryngeal cancer cells via modulating p53

The main obstacle to cure tumors by radiotherapy has been ascribed to tumor radioresistance. To determine the mechanisms underlying resistance to irradiation, it is essential to compare proteins differentially expressed from radiotherapy-sensitive and -resistant cancer cells. Aldehyde reductase (AKR1A1) was recently identified as increased in radioresistant laryngeal cancer cells by comparative proteomics approach. Here, we provide the mechanism of AKR1A1-mediated radioresistance via p53 regulation in laryngeal cancer cells. AKR1A1 induction was correlated with the radioresistant phenotype of laryngeal cancer HEp-2 cells. AKR1A1 depletion with siRNA significantly enhanced radiation sensitivity of radioresistant HEp-2 cells by promoting radiation-induced cell death and accelerated radiation-mediated inhibition of cell proliferation, without affecting either the PI3K-Akt or MAPK-ERK pathways. Intriguingly, AKR1A1 depletion induced phosphorylation of p53 at serine 15 and G2/M transition in response to irradiation. We further found that AKR1A1 interacted with p53 and this interaction was dramatically increased in the irradiated radioresistant cells compared with the control cells. AKR1A1 expression also regulated p53 stability in response to irradiation. Furthermore, AKR1A1 depletion only sensitized HCT116 cells expressing p53 to irradiation and not p53-deficient cells. Therefore, our data suggest that radiation-inducible AKR1A1 contributes to acquired radioresistance of laryngeal cancer cells by suppressing p53 activation through inhibitory interaction.

Preliminary Data from a Multicenter, Phase II, Randomized, Non-Comparative Clinical Trial of Radiation and Temozolomide with or without Vandetanib in Newly-Diagnosed Glioblastoma (GBM) (S45.006)

Objective: We conducted a randomized phase II study of vandetanib in patients with newly-diagnosed GBM in combination with radiation (RT) and temozolomide (TMZ). Background EGFR and VEGFR signaling have been implicated in the development and growth of GBM. In addition, angiogenesis inhibitors may enhance radiation sensitivity. Vandetanib is an inhibitor of VEGFR-2 and EGFR tyrosine kinase activities. Design/Methods: We planned to randomize a total of 114 newly-diagnosed GBM patients in a ratio of 1:2 to Arm A: RT/TMZ or Arm B: vandetanib 100 mg daily + RT/TMZ. All pts received RT (60 Gy) and concurrent TMZ 75 mg/m 2 daily, followed by adjuvant TMZ for up to 12 cycles (150-200 mg/m 2 on days 1-5 of each 28 day cycle). Pts with age ≥ 18, Karnofsky performance status (KPS) ≥ 60, and not on enzyme-inducing antiepileptics were eligible. Primary endpoint was overall survival (OS) from the date of randomization. Secondary endpoints included median progression-free survival (PFS) and safety. Results: We enrolled 106 pts (36 in Arm A, 70 in Arm B) with median age 59 yrs (range: 23-83) and median KPS 90 (range: 60-100). Most frequent Gr ≥ 3 AEs in the vandetanib arm were lymphopenia (20%), thrombosis (12.5%), transaminitis (8%) and leukopenia (5%). Preliminary median OS is 487 days with 95% CI (350, NR) in Arm A and 503 days with 95% CI (451, 634) in Arm B. Median PFS is 141 days with 95% CI (104. 252) in Arm A and 127 days with 95% CI (93. 165) in Arm B. Due to an unplanned interim analysis demonstrating no survival difference, enrollment was terminated early and the study was closed. Conclusions: Although reasonably well-tolerated, the addition of vandetanib to standard chemoradiation in newly-diagnosed GBM may not significantly prolong OS compared to the parallel control arm. Updated efficacy and toxicity data will be presented. Supported by: AstraZeneca. Disclosure: Dr. Lee has received personal compensation for activities with Norvartis and Genentech as an advisory board member.Dr. Lee has received personal compensation in an editorial capacity for UpToDate, Inc. Dr. Batchelor has received personal compensation for activities with Roche Diagnostics Corporation, Genentech, Inc., Merck & Co. Inc., and Millennium. Dr. Batchelor has received personal compensation in an editorial capacity for UpToDate Inc. Dr. Batchelor has received research support from Pfizer Inc, AstraZeneca Pharmaceuticals and Millennium. Dr. Kaley has nothing to disclose. Dr. Schiff has received personal compensation for activities with Novocure and Genentech. Dr. Schiff has received research support from Exelixis. Dr. Lassman has received personal compensation for activities with Schering-Plough Corp., ImClone Systems, Genentech, Inc., Eisai Inc., Cephalon, Inc., Physicians Education Resource, The Robert Michael Educational Institute, and Medical Communications Media.Dr. Lassman has received research support from Keryx Biopharmaceuticals, Genentech, and Schering Plough Corp. Dr. Wong has received research support from AstraZeneca Pharmaceuticals, Exelixis, Novartis and NovoCure as a researcher. Dr. Mikkelsen has received personal compensation for activities with Merck and Roche. Dr. Purow has nothing to disclose. Dr. Drappatz has nothing to disclose. Dr. Norden has nothing to disclose. Dr. Beroukhim has received personal compensation for activities with Novartis as a consultant. Dr. Beroukhim holds stock and/or stock options in AstraZeneca, which sponsored research in which Dr. Beroukhim was involved as an investigator. Dr. Beroukhim holds stock and/or stock options in Baxter, CareFusion, Edwards Lifesciences, and Cardinal Health. Dr. Beroukhim has received research support from Novartis. Dr. Weiss has received personal compensation for activities with a consulting firm as RT expert. Dr. Weiss has received research support from Aposence. Dr. Alexander has nothing to disclose. Dr. McCluskey has nothing to disclose. Dr. Gerard has nothing to disclose. Dr. Teulings has nothing to disclose. Dr. Smith has nothing to disclose. Dr. Muzikansky has nothing to disclose. Dr. Wen has received personal compensation for activities with Merck & Co., and Novartis as consutant and/or speaker. Dr. Wen has received personal compensation in an editorial capacity for UptoDate. Dr. Wen has received research support from Amgen Inc, AstraZeneca Pharmaceuticals, Eisai Inc, Exelixis, Genentech, Inc., Medimmune, Merck & Co., Novartis, Sanofi-Aventis Pharmaceuticals and Vascular Biogenics.

Resveratrol enhances radiation sensitivity in prostate cancer by inhibiting cell proliferation and promoting cell senescence and apoptosis

Radiation therapy (XRT) for treatment of localized prostate cancer (PCA) has outcomes similar to surgery and medical therapy. Toxicities of XRT and the relative radioresistance of PCA limit the effectiveness of this treatment method. Safe and effective radiosensitizing agents are lacking to enhance the effectiveness for XRT for PCA. In this study, the effect of XRT in combination with the radiosensitizing agent resveratrol (RSV) was investigated in a radioresistant PCA cell line, PC-3. Our results show the addition of RSV to XRT (XRT/RSV) synergistically enhanced XRT-induced apoptosis and inhibition of PC-3 proliferation. The antiproliferative effect of XRT/RSV treatment correlated with increased expression of p15, p21, and mutant p53 and decreased expression of cyclin B, cyclin D, and cdk2. Increased apoptosis correlated with increased expression of Fas and TRAILR1. Furthermore, XRT/RSV had little effect on the expression of p-AKT, whereas it increased the expression level of p-H2A.X, a marker for senescence. These data highlight the potential of RSV as a radiation sensitizer for PCA treatment and warrant further investigation.