Devora Schiff

Autophagy inhibition by chloroquine sensitizes HT-29 colorectal cancer cells to concurrent chemoradiation.

AIM To investigate whether the inhibition of autophagy by chloroquine (CQ) sensitizes rectal tumors to radiation therapy (RT) or concurrent chemoradiation (chemoRT). METHODS In vitro, HCT-116 and HT-29 colorectal cancer (CRC) cell lines were treated as following: (1) PBS; (2) CQ; (3) 5-fluorouracil (5-FU); (4) RT; (5) CQ and RT; (6) 5-FU and RT; (7) CQ and 5-FU; and (8) 5-FU and CQ and RT. Each group was then exposed to various doses of radiation (0-8 Gy) depending on the experiment. Cell viability and proliferative capacity were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Clonogenic survival curves were constructed and compared across treatment groups. Autophagy status was determined by assessing the LC3-II to LC3-I ratio on western blot analysis, autophagosome formation on electron microscopy and identification of a perinuclear punctate pattern with GFP-labeled LC3 on fluorescence microscopy. Cell cycle arrest and cell death were evaluated by FACS and Annexin V analysis. All experiments were performed in triplicate and statistical analysis was performed by the students t test to compare means between treatment groups. RESULTS RT (2-8 Gy) induced autophagy in HCT-116 and HT-29 CRC cell lines at 4 and 6 h post-radiation, respectively, as measured by increasing LC3-II to LC3-I ratio on western blot. Additionally, electron microscopy demonstrated autophagy induction in HT-29 cells 24 h following irradiation at a dose of 8 Gy. Drug treatment with 5-FU (25 μmol/L) induced autophagy and the combination of 5-FU and RT demonstrated synergism in autophagy induction. CQ (10 μmol/L) alone and in combination with RT effectively inhibited autophagy and sensitized both HCT-116 and HT-29 cells to treatment with radiation (8 Gy; P < 0.001 and 0.00001, respectively). Significant decrease in clonogenic survival was seen only in the HT-29 cell line, when CQ was combined with RT at doses of 2 and 8 Gy (P < 0.5 and P = 0.05, respectively). There were no differences in cell cycle progression or Annexin V staining upon CQ addition to RT. CONCLUSION Autophagy inhibition by CQ increases CRC cell sensitivity to concurrent treatment with 5-FU and RT in vitro, suggesting that addition of CQ to chemoRT improves CRC treatment response.

Riluzole Enhances Ionizing Radiation–Induced Cytotoxicity in Human Melanoma Cells that Ectopically Express Metabotropic Glutamate Receptor 1 In Vitro and In Vivo

Purpose: Brain metastases are a common preterminal event in patients with metastatic melanoma and require radiation therapy. Our group has previously shown that human GRM1 (hGRM1) expressing melanoma cells release excess extracellular glutamate and are growth inhibited by riluzole, an inhibitor of glutamate release. Riluzole-treated cells accumulate in G2/M phase of the cell cycle at 24 hours, and then undergo apoptotic cell death. We evaluated whether riluzole enhanced radiosensitivity in melanoma cells. Experimental Design: Clonogenic assays were performed to evaluate clonogenic survival after treatment in hGRM1 expressing and nonexpressing melanoma cells. Western immunoblots were performed to confirm apoptotic cell death. A xenograft mouse model was used to validate the in vitro experiments. Tumors harvested from the xenografts were fixed and stained for apoptosis and DNA damage markers. Results: In the hGRM1-positive cell lines C8161 and UACC903, riluzole enhanced the lethal effects of ionizing radiation; no difference was seen in the hGRM1-negative UACC930 cell line. C8161 cells treated with riluzole plus irradiation also showed the highest levels of the cleaved forms of PARP and caspase-3; excised C8161 xenografts showed the greatest number of apoptotic cells by immunohistochemistry (P < 0.001). On cell cycle analysis, a sequence-dependent enrichment in the G2/M phase was shown with the combination of riluzole and irradiation. Xenografts treated with riluzole and weekly radiation fractions showed significant growth inhibition and revealed markedly increased DNA damage. Conclusions: We have shown, in vitro and in vivo, that the combination of riluzole and ionizing radiation leads to greater cytotoxicity. These results have clinical implications for patients with brain metastases receiving whole brain radiation therapy. Clin Cancer Res; 17(7); 1807–14. ©2011 AACR.

Low p53 Binding Protein 1 (53BP1) Expression Is Associated With Increased Local Recurrence in Breast Cancer Patients Treated With Breast-Conserving Surgery and Radiotherapy

PURPOSE To investigate whether the expression of p53 binding protein 1 (53BP1) has prognostic significance in a cohort of early-stage breast cancer patients treated with breast-conserving surgery and radiotherapy (BCS+RT). METHODS AND MATERIALS A tissue microarray of early-stage breast cancer treated with BCS+RT from a cohort of 514 women was assayed for 53BP1, estrogen receptor, progesterone receptor, and HER2 expression by immunohistochemistry. Through log-rank tests and univariate and multivariate models, the staining profile of each tumor was correlated with clinical endpoints, including ipsilateral breast recurrence-free survival (IBRFS), distant metastasis-free survival (DMFS), cause-specific survival (CSS), recurrence-free survival (RFS), and overall survival (OS). RESULTS Of the 477 (93%) evaluable tumors, 63 (13%) were scored as low. Low expression of 53BP1 was associated with worse outcomes for all endpoints studied, including 10-year IBRFS (76.8% vs. 90.5%; P=.01), OS (66.4% vs. 81.7%; P=.02), CSS (66.0% vs. 87.4%; P<.01), DMFS (55.9% vs. 87.0%; P<.01), and RFS (45.2% vs. 80.6%; P<.01). Multivariate analysis incorporating various clinico-pathologic markers and 53BP1 expression found that 53BP1 expression was again an independent predictor of all endpoints (IBRFS: P=.0254; OS: P=.0094; CSS: P=.0033; DMFS: P=.0006; RFS: P=.0002). Low 53BP1 expression was also found to correlate with triple-negative (TN) phenotype (P<.01). Furthermore, in subset analysis of all TN breast cancer, negative 53BP1 expression trended for lower IBRFS (72.3% vs. 93.9%; P=.0361) and was significant for worse DMFS (48.2% vs. 86.8%; P=.0035) and RFS (37.8% vs. 83.7%; P=.0014). CONCLUSION Our data indicate that low 53BP1 expression is an independent prognostic indicator for local relapse among other endpoints in early-stage breast cancer and TN breast cancer patients treated with BCS+RT. These results should be verified in larger cohorts of patients to validate their clinical significance.

Clinicopathologic Significance of Excision Repair Cross-Complementation 1 Expression in Patients Treated With Breast-Conserving Surgery and Radiation Therapy

PURPOSE The excision repair cross-complementation 1 (ERCC1) enzyme plays a rate-limiting role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in cancers of the head and neck and the lung. The purpose of this study was to evaluate the clinicopathologic and prognostic significance of ERCC1 expression in a cohort of early-stage breast cancer patients treated with breast conservation therapy. METHODS AND MATERIALS Paraffin specimens from 504 women with early-stage breast cancer treated with breast conservation therapy were constructed into tissue microarrays. The array was stained for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) and ERCC1. This was then correlated with clinicopathologic factors and outcomes data. RESULTS ERCC-1 expression was evaluable in 366 cases (72%). In this group, 32% and 38% of patients received adjuvant chemotherapy and hormonal therapy, respectively. Increased ERCC-1 expression was found to be correlated with ER positivity (p < 0.005), lower T stage (p < 0.017), nodal negativity (p < 0.013), age >50 (p < 0.006), reduced use of adjuvant chemotherapy (p < 0.02), and increased use of adjuvant hormonal therapy (p < 0.004). ERCC1 expression did not correlate with locoregional recurrence-free survival, distant metastasis-free survival, cause-specific survival, or overall survival. In patients who were both ERCC1-negative and -positive, the use of chemotherapy predicted for worse distant metastasis-free survival (p = 0.05 and p = 0.07, respectively) but not cause-specific survival or overall survival. CONCLUSIONS Although ERCC1 expression did not predict for outcome measures in this dataset, overexpression correlated with favorable prognostic factors such as ER positivity, lower T stage, nodal negativity, and age >50. To our knowledge, this is the first study investigating ERCC1 expression in patients receiving adjuvant radiation therapy for breast cancer.

Evaluation of Single Nucleotide Polymorphisms (SNPs) in the p53 Binding Protein 1 (TP53BP1) Gene in Breast Cancer Patients Treated With Breast-Conserving Surgery and Whole-Breast Irradiation (BCS + RT)

PURPOSE TP53BP1 is a key component of radiation-induced deoxyribonucleic acid damage repair. The purpose of this study was to evaluate the significance of a known common single nucleotide polymorphism in this gene (rs560191) in patients treated with breast-conserving surgery and whole-breast irradiation (BCS + RT). METHODS AND MATERIALS The population consisted of 176 premenopausal women treated with BCS + RT (median follow-up, 12 years). Genomic deoxyribonucleic acid was processed by use of TaqMan assays. Each allele for rs560191 was either C or G, so each patient was therefore classified as CC, CG, or GG. Patients were grouped as GG if they were homozygous for the variant G allele or CC-CG if they carried at least one copy of the common C allele (CC or CG). RESULTS Of the 176 women, 124 (71%) were CC-CG and 52 (29%) were GG. The mean age was 44 years for GG vs. 38 years for CC-CG (p < 0.001). GG was more common in African-American women than white women (69% vs. 13%, p < 0.001) and more commonly estrogen receptor negative (70% vs. 49%, p = 0.02). There were no significant correlations of rs560191 with other critical variables. Despite the fact that GG patients were older, the 10-year rate of local relapses was higher (22% for GG vs. 12% for CC-CG, p = 0.04). CONCLUSIONS This novel avenue of investigation of polymorphisms in radiation repair/response genes in patients treated with BCS + RT suggests a correlation to local relapse. Additional evaluation is needed to assess the biological and functional significance of these single nucleotide polymorphisms, and larger confirmatory validation studies will be required to determine the clinical implications.

Evaluation of Diabetic Patients with Breast Cancer Treated with Metformin during Adjuvant Radiotherapy

Purpose. The purpose of this study was to evaluate acute locoregional toxicity in patients with breast cancer receiving concurrent metformin plus radiation therapy. Methods and Materials. Diabetic breast cancer patients receiving concurrent metformin and radiation therapy were matched with nondiabetic patients and diabetic patients using an alternative diabetes medication. Primary endpoints included the presence of a treatment break and development of dry or moist desquamation. Results. There was a statistically significant increase in treatment breaks for diabetic patients receiving concurrent metformin when compared to the nondiabetic patients (P value = 0.02) and a trend toward significance when compared to diabetic patients receiving an alternate diabetes medication (P value = 0.08). Multiple logistic regression analysis demonstrated concurrent metformin use as being associated with a trend toward the predictive value of determining the incidence of developing desquamation in diabetic patients receiving radiation therapy compared to diabetic patients receiving an alternate diabetes medication (P value = 0.06). Conclusions. Diabetic patients treated with concurrent metformin and radiation therapy developed increased acute locoregional toxicity in comparison with diabetic patients receiving an alternate diabetes medication and nondiabetic patients. Further clinical investigation should be conducted to determine the therapeutic ratio of metformin in combination with radiation therapy.

Disruption of GRM1-mediated signalling using riluzole results in DNA damage in melanoma cells.

Gain of function of the neuronal receptor, metabotropic glutamate receptor 1 (Grm1), was sufficient to induce melanocytic transformation in vitro and spontaneous melanoma development in vivo when ectopically expressed in melanocytes. The human form of this receptor, GRM1, has been shown to be ectopically expressed in a subset of human melanomas but not benign nevi or normal melanocytes, suggesting that misregulation of GRM1 is involved in the pathogenesis of certain human melanomas. Sustained stimulation of Grm1 by the ligand, glutamate, is required for the maintenance of transformed phenotypes in vitro and tumorigenicity in vivo. In this study, we investigate the mechanism of an inhibitor of glutamate release, riluzole, on human melanoma cells that express metabotropic glutamate receptor 1 (GRM1). Various in vitro assays conducted show that inhibition of glutamate release in several human melanoma cell lines resulted in an increase of oxidative stress and DNA damage response markers.

Ionizing Radiation Sensitizes Breast Cancer Cells to Bcl-2 Inhibitor, ABT-737, through Regulating Mcl-1

Breast-conserving surgery followed by radiation therapy has become the standard of care for early stage breast cancer. However, there are some patients that develop a local failure. We have previously shown that Bcl-2 overexpression was associated with an increased risk of local recurrence in patients with early stage breast cancer. The purpose of this study was to explore an approach to overcome radiation resistance by targeting pro-survival Bcl-2 family proteins in breast cancer cells. The breast cancer cell lines MCF-7, ZR-75-1 and MDA-MB231 were used in this study. siRNAs were employed to silence myeloid cell leukemia 1 (Mcl-1). A small molecule inhibitor of Bcl-2, ABT-737, was used to target anti-apoptotic Bcl-2 family proteins. Apoptosis was identified by FITC Annexin V, PI staining and Western blot analysis. The sensitivity to ionizing radiation and ABT-737 were measured by clonogenic assays. The effect of radiation and ABT-737 was also tested in a MCF-7 xenograft mouse model. Our data demonstrate that the combination of ABT-737 and radiation-induced apoptosis had an inhibitory effect on breast cancer cell proliferation. However, treatment with ABT-737 resulted in elevated Mcl-1 in breast cancer cell lines. Targeting Mcl-1 by siRNA sensitized MCF-7 cells to ABT-737. We revealed that radiation blunted Mcl-1 elevation induced by ABT-737, and that radiation downregulated Mcl-1 by promoting its degradation. Our results indicate that radiation and ABT-737 exert a synergistic effect on breast cancer cell lines through downregulating Mcl-1 and activating the bak-apoptotic pathway. These results support the combination of radiation and pro-survival Bcl-2 family inhibitor as a potential novel therapeutic strategy in the local-regional management of breast cancer.

Is Ki-67 Expression Prognostic for Local Relapse in Early-Stage Breast Cancer Patients Treated With Breast Conservation Therapy (BCT)?

PURPOSE Ki-67 is a human nuclear protein whose expression is strongly up-regulated in proliferating cells and can be used to determine the growth fraction in clonal cell populations. Although there are some data to suggest that Ki-67 overexpression may be prognostic for endpoints such as survival or postmastectomy recurrence, further elucidation of its prognostic significance is warranted. Specifically after breast conservation therapy (BCT) (defined in this setting as breast-conserving surgery and adjuvant radiation therapy), whether Ki-67 predicts for locoregional recurrence has not been investigated. The purpose of this study was to assess Ki-67 expression in a cohort of early-stage breast cancer patients to determine whether a significant independent association between Ki-67 and locoregional relapse exists. METHODS AND MATERIALS Ki-67 staining was conducted on a tissue microarray of 438 patients previously treated with BCT, and expression was analyzed with clinicopathologic features and outcomes from our database. RESULTS Ki-67 expression was more prevalent in black patients (37% of black patients vs 17% of white patients, P<.01), younger patients (27% of patients aged ≤50 years vs 15% of patients aged >50 years, P<.01), estrogen receptor (ER)-negative tumors (25% of ER-negative tumors vs 17% of ER-positive tumors, P=.04), human epidermal growth factor receptor 2 (HER2)/neu-positive tumors (35% of HER2-positive tumors vs 18% of HER2-negative tumors, P=.01), and larger tumors (26% of T2 tumors vs 16% of T1 tumors, P=.03). On univariate/multivariate analysis, Ki-67 did not predict for overall survival (74.4% vs 72.6%), cause-specific survival (82.9% vs 82.1%), local relapse-free survival (83.6% vs 88.5%), distant metastasis-free survival (76.1% vs 81.4%), recurrence-free survival (65.5% vs 74.6%), and locoregional recurrence-free survival (81.6% vs 84.7%): P>.05 for all. CONCLUSIONS Ki-67 appears to be a surrogate marker for aggressive disease and significantly correlates with known prognostic features such as age, race, hormone receptor status, and HER2 status but independently does not predict for locoregional outcomes after BCT when these other prognostic clinicopathologic features are taken into consideration. The independent associations of Ki-67 with race and age appear to be novel to our study.

VX-984 is a selective inhibitor of non-homologous end joining, with possible preferential activity in transformed cells

Purpose DNA double-strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). We demonstrate the selectivity of VX-984, a DNA-PK inhibitor, using assays not previously reported. Experimental Design The class switch recombination assay (CSR) in primary B cells was used to measure efficiency of NHEJ. A cellular reporter assay (U2OS EJ-DR) was used to assess the efficiency of HR and NHEJ in cells treated with VX-984. Immunofluorescence assays (IF) evaluated γ-H2AX foci for DSB repair kinetics in human astrocytes and T98G glioma cells. Western blotting was used to evaluate phosphorylation of DNA-PKcs substrates. Results We found a dose-dependent reduction in CSR efficiency with VX-984, and through the EJ-DR assay, dramatic dose-dependent increases in HR and mNHEJ. Immunofluorescence assays showed an inability of malignant cells to resolve γ-H2AX foci in the presence of VX-984. Radiation-induced phosphorylation of DNA-PK substrates was further reduced by treatment with VX-984. Conclusions VX-984 efficiently inhibits NHEJ, resulting in compensatory increases in alternative repair pathways, increases DSBs, and appears to affect transformed cells preferentially.