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Background and purpose Growing evidence suggested the coexistence of cancer stem cells (CSCs) within solid tumors. We aimed to study radiosensitivity parameters for the CSCs and differentiated tumor cells (TCs) and the correlation of the fractions of CSCs to the overall tumor radioresistance. Material and methods Surviving fractions of breast cancer cell lines were analyzed using a dual-compartment Linear-quadratic model with independent fitting parameters: radiosensitive α TC , β TC , α CSC , β CSC , and fraction of CSCs f . The overall tumor radio-resistance, the biological effective doses and tumor control probability were estimated as a function of CSC fraction for different fractionation regimens. The pooled clinical outcome data were fitted to the single- and dual-compartment linear-quadric models. Results CSCs were more radioresistant characterized by smaller α compared to TCs: α TC = 0.1 ± 0.2, α CSC = 0.04 ± 0.07 for MCF-7 ( f = 0.1%), α TC = 0.08 ± 0.25, α CSC = 0.04 ± 0.18 for SUM159PT ( f = 2.46%). Higher f values were correlated with increasing radioresistance in cell lines. Analysis of clinical outcome data is in accordance of a dual-compartment CSC model prediction. Higher percentage of BCSCs resulted in more overall tumor radioresistance and less biological effectiveness. Conclusions Percentage of CSCs strongly correlated to overall tumor radioresistance. This observation suggested potential individualized radiotherapy to account for heterogeneous population of CSCs and their distinct radiosensitivity for breast cancer. [ABSTRACT FROM AUTHOR]

Background Experimental and clinical data suggest that solid cancers contain treatment-resistant cancer stem cells that will impair treatment efficacy. The objective of this study was to investigate if head and neck squamous cell carcinoma (HNSCC) also contain cancer stem cells that can be identified by low 26S proteasome activity and if their presence correlates to clinical outcome. Methods Human HNSCC cells, engineered to report lack of proteasome activity based on accumulation of a fluorescent fusion protein, were separated based on high (ZsGreen-cODCneg ) or low (ZsGreen-cODCpos) proteasome activity. Self-renewal capacity, tumorigenicity and radioresistance were assessed. Proteasome subunit expression was analyzed in tissue microarrays and correlated to survival and locoregional cancer control of 174 patients with HNSCC. Results HNSCC cells with low proteasome activity showed a significantly higher self-renewal capacity and increased tumorigenicity. Irradiation enriched for ZsGreen-cODC neg cells. The survival probability of 82 patients treated with definitive radio- or chemo-radiotherapy exhibiting weak, intermediate, or strong proteasome subunit expression were 21.2, 28.8 and 43.8 months (p = 0.05), respectively. Locoregional cancer control was comparably affected. Conclusions Subpopulations of HNSCC display stem cell features that affect patients' tumor control and survival. Evaluating cancer tissue for expression of the proteasome subunit PSMD1 may help identify patients at risk for relapse. [ABSTRACT FROM AUTHOR]

Breast cancers are thought to be organized hierarchically with a small number of breast cancer stem cells (BCSCs) able to regrow a tumor while their progeny lack this ability. Recently, several groups reported enrichment for BCSCs when breast cancers were subjected to classic anticancer treatment. However, the underlying mechanisms leading to this enrichment are incompletely understood. Using non-BCSCs sorted from patient samples, we found that ionizing radiation reprogrammed differentiated breast cancer cells into induced BCSCs (iBCSCs). iBCSCs showed increased mammosphere formation, increased tumorigenicity, and expressed the same stemness-related genes as BCSCs from nonirradiated samples. Reprogramming occurred in a polyploid subpopulation of cells, coincided with re-expression of the transcription factors Oct4, sex determining region Y-box 2, Nanog, and Klf4, and could be partially prevented by Notch inhibition. We conclude that radiation may induce a BCSC phenotype in differentiated breast cancer cells and that this mechanism contributes to increased BCSC numbers seen after classic anticancer treatment. S TEM C ELLS 2012;30:833-844 [ABSTRACT FROM AUTHOR]

For more than a century oxygen has been known to be one of the most powerful radiosensitizers. However, despite decades of preclinical and clinical research aimed at overcoming tumor hypoxia, little clinical progress has been made so far. Ionizing radiation damages DNA through generation of free radicals. In the presence of oxygen these lesions are chemically modified, and thus harder to repair while hypoxia protects cells from radiation (Oxygen enhancement ratio (OER)). Breast cancer stem cells (BSCSs) are protected from radiation by high levels of free radical scavengers even in the presence of oxygen. This led us to hypothesize that BCSCs exhibit an OER of 1. Using four established breast cancer cell lines (MCF-7, T47D, MDA-MB-231, SUM159PT) and primary breast cancer samples, we determined the number of BCSCs using cancer stem cell markers (ALDH1, low proteasome activity), compared radiation clonogenic survival and mammosphere formation under normoxic and hypoxic conditions, and correlated these results to the expression levels of key members of the free radical scavenging systems. The number of BCSCs increased with increased aggressiveness of the cancer. This correlated with increased radioresistance (SF8Gy), and decreasing OERs. When cultured as mammospheres, breast cancer cell lines and primary samples were highly radioresistant and not further protected by hypoxia (OER~1). We conclude that because BCSCs are protected from radiation through high expression levels of free radical scavengers, hypoxia does not lead to additional radioprotection of BCSCs. [ABSTRACT FROM AUTHOR]

Gliomas contain a small number of treatment-resistant glioma stem cells (GSCs), and it is thought that tumor regrowth originates from GSC5, thus rendering GSC5 an attractive target for novel treatment approaches. Cancer cells rely more on glycolysis than on oxidative phosphorylation for glucose metabolism, a phenomenon used in 2-[18F]fluoro-2-deoxy-o-glucose positron emission tomography imaging of solid cancers, and targeting metabolic pathways in cancer cells has become a topic of considerable interest. However, if GSC5 are indeed important for tumor control, knowledge of the metabolic state of GSCs is needed. We hypothesized that the metabolism of GSCs differs from that of their progeny. Using a unique imaging system for GSCs, we assessed the oxygen consumption rate, extracellular acidification rate, intracellular ATP levels, glucose uptake, lactate production, PKM1 and PKM2 expression, radiation sensitivity, and cell cycle duration of GSC5 and their progeny in a panel of glioma cell lines. We found GSC5 and progenitor cells to be less glycolytic than differentiated glioma cells. GSC5 consumed less glucose and produced less lactate while maintaining higher ATP levels than their differentiated progeny. Compared with differentiated cells, GSC5 were radioresistant, and this correlated with a higher mitochondrial reserve capacity. Glioma cells expressed both isoforms of pyruvate kinase, and inhibition of either glycolysis or oxidative phosphorylation had minimal effect on energy production in GSCs and progenitor cells. We conclude that GSCs rely mainly on oxidative phosphorylation. However, if challenged, they can use additional metabolic pathways. Therefore, targeting glycolysis in glioma may spare GSCS. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma is the most common brain tumor in adults. The mechanisms leading to glioblastoma are not well understood but animal studies support that inactivation of tumor suppressor genes in neural stem cells (NSC) is required and sufficient to induce glial cancers. This suggests that the NSC niches in the brain may harbor cancer stem cells (CSCs), Thus providing novel therapy targets. We hypothesize that higher radiation doses to these NSC niches improve patient survival by eradicating CSCs. Methods: 55 adult patients with Grade 3 or Grade 4 glial cancer treated with radiotherapy at UCLA between February of 2003 and May of 2009 were included in this retrospective study. Using radiation planning software and patient radiological records, the SVZ and SGL were reconstructed for each of these patients and dosimetry data for these structures was calculated. Results: Using Kaplan-Meier analysis we show that patients whose bilateral subventricular zone (SVZ) received greater than the median SVZ dose (= 43 Gy) had a significant improvement in progression-free survival if compared to patients who received less than the median dose (15.0 vs 7.2 months PFS; P = 0.028). Furthermore, a mean dose >43 Gy to the bilateral SVZ yielded a hazard ratio of 0.73 (P = 0.019). Importantly, similarly analyzing total prescription dose failed to illustrate a statistically significant impact. Conclusions: Our study leads us to hypothesize that in glioma targeted radiotherapy of the stem cell niches in the adult brain could yield significant benefits over radiotherapy of the primary tumor mass alone and that damage caused by smaller fractions of radiation maybe less efficiently detected by the DNA repair mechanisms in CSCs. [ABSTRACT FROM AUTHOR]

Background There is increasing evidence that solid cancers contain cancer-initiating cells (CICs) that are capable of regenerating a tumor that has been surgically removed and/or treated with chemotherapy and/or radiation therapy. Currently, cell surface markers, like CD133 or CD44, are used to identify CICs in vitro; however, these markers cannot be used to identify and track CICs in vivo. The 26S proteasome is the main regulator of many processes within a proliferating cell, and its activity may be altered depending on the phenotype of a cell. Methods Human glioma and breast cancer cells were engineered to stably express ZsGreen fused to the carboxyl-terminal degron of orriithine decarboxylase, resulting in a fluorescent fusion protein that accumulates in cells in the absence of 26S proteasome activity; activities of individual proteases were monitored in a plate reader by detecting the cleavage of fluorogenic peptide substrates. Proteasome subunit expression in cells expressing the fusion protein was assessed by quantitative reverse transcription--polymerase chain reaction, and the stem cell phenotype of CICs was assessed by a sphere formation assay, by immunohistochemical staining for known stem cell markers in vitro, and by analyzing their tumorigenicity in vivo. CICs were tracked by in vivo fluorescence imaging after radiation treatment of tumor-bearing mice and targeted specifically via a thymidine kinase-degron fusion construct. All P values were derived from two-sided tests. Results Cancer cells grown as sphere cultures in conditions, which enrich for cancer stem cells (CSCs), had decreased proteasome activity relative to the respective monolayers (percent decrease in chymotryptic-like activity of sphere cultures relative to monolayers-U87MG: 26.64%, 95% confidence interval [Cl] = 10.19 to 43.10, GL261, 52.91%, 95% Cl = 28.38 to 77.43). The cancer cells with low proteasome activity can thus be monitored in vitro and in vivo by the accumulation of a fluorescent protein (ZsGreen) fused to a degron that targets it for 26S proteasome degradation. In vitro, ZsGreen-positive cells had increased sphere-forming capacity, expressed CSC markers, and lacked differentiation markers compared with ZsGreen-negative cells. In vivo, ZsGreen-positive cells were approximately 100-fold more tumorigenic than ZsGreen-negative cells when injected into nude mice (ZsGreen positive, 30 mice per group; ZsGreen negative, 31 mice per group), and the number of CICs in tumors increased after 72 hours post radiation treatment. CICs were selectively targeted via a proteasome-dependent suicide gene, and their elimination in vivo led to tumor regression. Conclusion Our results demonstrate that reduced 26S proteasome activity is a general feature of CICs that can easily be exploited to identify, track, and target them in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Background: If cancer arises and is maintained by a small population of cancer-initiating cells within every tumor, understanding how these cells react to cancer treatment will facilitate improvement of cancer treatment in the future. Cancer-initiating cells can now be prospectively isolated from breast cancer cell lines and tumor samples and propagated as mammospheres in vitro under serum-free conditions. Methods: CD24-/low/CD44+ cancer-initiating cells were isolated from MCF-7 and MDA-MB-231 breast cancer monolayer cultures and propagated as mammospheres. Their response to radiation was investigated by assaying clonogenic survival and by measuring reactive oxygen species (ROS) levels, phosphorylation of the replacement histone H2AX, CD44 levels, CD24 levels, and Notch-1 activation using flow cytometry. All statistical tests were two-sided. Results: Cancer-initiating cells were more resistant to radiation than cells grown as monolayer cultures (MCF-7: monolayer cultures, mean surviving fraction at 2 Gy ISF2Gy] = 0.2, versus mammospheres, mean SF2Gy = 0.46, difference = 0.26, 95% confidence interval [CI] = 0.05 to 0.47; P = .026; MDA-MB-231: monolayer cultures, mean SF2Gy = 0.5, versus mammospheres, mean SF2Gy = 0.69, difference = 0.19, 95% CI = -0.07 to 0.45; P = .09). Levels of ROS increased in both mammospheres and monolayer cultures after irradiation with a single dose of 10 Gy but were lower in mammospheres than in monolayer cultures (MCF-7 monolayer cultures: 0 Gy, mean = 1.0, versus 10 Gy, mean = 3.32, difference = 2.32, 95% CI = 0.67 to 3.98; P = .026; mammospheres: 0 Gy, mean = 0.58, versus 10 Gy, mean =1.46, difference 0.88, 95% CI = 0.20 to 1.56; P = .031); phosphorylation of H2AX increased in irradiated monolayer cultures, but no change was observed in mammospheres. Fractionated doses of irradiation increased activation of Notch-i (untreated, mean = 10.7, versus treated, mean = 15.1, difference = 4.4, 95% CI = 2.7 to 6.1, P = .002) and the percentage of the cancer stem/initiating cells in the nonadherent cell population of MCF-7 monolayer cultures (untreated, mean = 3.52%, versus treated, mean = 7.5%, difference = 3.98%, 95% CI = 1.67% to 6.25%, P = .009). Conclusions: Breast cancer-initiating cells are a relatively radioresistant subpopulation of breast cancer cells and increase in numbers after short courses of fractionated irradiation. These findings offer a possible mechanism for the accelerated repopulation of tumor cells observed during gaps in radiotherapy. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Transient hypoxia and subsequent reoxygenation are common phenomena in solid tumors that greatly influence the outcome of radiation therapy. This study was designed to determine how varying cycles of hypoxia/reoxygenation affect the response of cervical carcinoma cells irradiated under oxic and hypoxic conditions and whether this could be modulated by proteasome inhibition. MATERIALS AND METHODS: Plateau-phase SiHa cervical carcinoma cells in culture were exposed to varying numbers of 30-minute cycles of hypoxia/reoxygenation directly before irradiation under oxic or hypoxic conditions. 26S Proteasome activity was blocked by addition of MG-132. Clonogenic survival was measured by a colony-forming assay. RESULTS: Under oxic conditions, repeated cycles of hypoxia/reoxygenation decreased the clonogenic survival of SiHa cells. This effect was even more pronounced after the inhibition of 26S proteasome complex. In contrast, under hypoxic conditions, SiHa cells were radioresistant, as expected, but this was increased by proteasome inhibition. CONCLUSIONS: Proteasome inhibition radiosensitizes oxygenated tumor cells but may also protect tumor cells from ionizing radiation under certain hypoxic conditions. [ABSTRACT FROM AUTHOR]

Background: By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition. Methods: Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNAPK activity were monitored. Radiosensitivity was assessed using a clonogenic assay. Results: Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity. Conclusion: We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132. [ABSTRACT FROM AUTHOR]

Anemia in cancer patients is associated with reduced quality of life and local failure after radiation treatment. However, the use of erythropoietin to correct cancer anemia and to improve radiation efficacy was disappointing. Erythropoietin-receptor signaling mainly acts via activation of STAT 5, but also crossactivates the antiapoptotic transcription factor NF-?B. This causes neuroprotection against oxidative stress and implies radioprotection. In order to investigate possible radioprotective effects of erythropoietin-receptor signaling, we used an in vitro model system employing HeLa TetOff cells, stably transfected with an expression vector for the erythropoietin-receptor gene. Using electrophoretic mobility shift assays, we could demonstrate strong activation of NF-?B by erythropoietin-receptor signaling in HeLa cells. Activation of NF-?B did not require degradation of I?Baand was not prevented by proteasome inhibition. Furthermore, stimulation with erythropoietin resulted in a 50%increased clonogenicity of erythropoietin-receptor-expressing cells but did not alter radiation sensitivity itself. As most human tumors express erythropoietin receptor, we advocate a restricted use erythropoietin to patients suffering from erythropoietin-receptor-expressing cancers.Oncogene (2004) 23, 8987-8991. doi:10.1038/sj.onc.1208140 Published online 11 October 2004 [ABSTRACT FROM AUTHOR]

Presents information on a study that looked at the impact of transcription factor NF-kB on the intrinsic of human cancer cells. Response of mammalian cells to ionizing radiation; Methodology; Results and discussion.

Background: Cancer is frequently associated with tumor-related anemia, and many chemotherapeutic agents impair hematopoiesis, leading to impaired quality of life for affected patients. The use of erythropoiesis-stimulating agents has come under scrutiny after prospective clinical trials using recombinant erythropoietin to correct anemia reported increased incidence of thromboembolic events and cancer-related deaths. Furthermore, previous preclinical reports indicated expansion of the pool of breast cancer-initiating cells when erythropoietin was combined with ionizing radiation.Methods: Using four established breast cancer cell lines, we test the effects of recombinant human erythropoietin and the number of breast cancer-initiating cells in vitro and in vivo and study if recombinant human erythropoietin promotes the phenotype conversion of non-tumorigenic breast cancer cells into breast cancer-initiating cells. In a prospective study, we evaluate whether elevated endogenous serum erythropoietin levels correlate with increased numbers of tumor-initiating cells in a cohort of breast cancer patients who were scheduled to undergo radiation treatment.Results: Our results indicate that recombinant erythropoietin increased the number of tumor-initiating cells in established breast cancer lines in vitro. Irradiation of breast cancer xenografts caused a phenotype conversion of non-stem breast cancer cells into induced breast cancer-initiating cells. This effect coincided with re-expression of the pluripotency factors c-Myc, Sox2, and Oct4 and was enhanced by recombinant erythropoietin. Hemoglobin levels were inversely correlated with serum erythropoietin levels, and the latter were correlated with disease stage. However, tumor sections revealed a negative correlation between serum erythropoietin levels and the number of ALDH1A3-positive cells, a marker for breast cancer-initiating cells.Conclusions: We conclude that physiologically slow-rising serum erythropoietin levels in response to tumor-related or chemotherapy-induced anemia, as opposed to large doses of recombinant erythropoietin, do not increase the pool of breast cancer-initiating cells. [ABSTRACT FROM AUTHOR]

Purpose: The lack of a molecular target in triple-negative breast cancer (TNBC) makes it one of the most challenging breast cancers to treat. Radiation therapy (RT) is an important treatment modality for managing breast cancer; however, we previously showed that RT can also reprogram a fraction of the surviving breast cancer cells into breast cancer-initiating cells (BCICs), which are thought to contribute to disease recurrence. In this study, we characterize mebendazole (MBZ) as a drug with potential to prevent the occurrence of radiation-induced reprogramming and improve the effect of RT in patients with TNBC.Methods and Materials: A high-throughput screen was used to identify drugs that prevented radiation-induced conversion of TNBC cells into cells with a cancer-initiating phenotype and exhibited significant toxicity toward TNBC cells. MBZ was one of the drug hits that fulfilled these criteria. In additional studies, we used BCIC markers and mammosphere-forming assays to investigate the effect of MBZ on the BCIC population. Staining with propidium iodide, annexin-V, and γ-H2AX was used to determine the effect of MBZ on cell cycle, apoptosis, and double-strand breaks. Finally, the potential for MBZ to enhance the effect of RT in TNBC was evaluated in vitro and in vivo.Results: MBZ efficiently depletes the BCIC pool and prevents the ionizing radiation-induced conversion of breast cancer cells into therapy-resistant BCICs. In addition, MBZ arrests cells in the G2/M phase of the cell cycle and causes double-strand breaks and apoptosis. MBZ sensitizes TNBC cells to ionizing radiation in vitro and in vivo, resulting in improved tumor control in a human xenograft model of TNBC.Conclusions: The data presented in this study support the repurposing of MBZ as a combination treatment with RT in patients with TNBC. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cancer anemia causes fatigue and correlates with poor treatment outcome. Erythropoietin has been introduced in an attempt to correct these defects. However, five recent clinical trials reported a negative impact of erythropoietin on survival and/or tumor control, indicating that experimental evaluation of a possible direct effect of erythropoietin on cancer cells is required. Cancer recurrence is thought to rely on the proliferation of cancer initiating cells (CICs). In breast cancer, CICs can be identified by phenotypic markers and their fate is controlled by the Notch pathway. METHODS: In this study, we investigated the effect of erythropoietin on CICs in breast cancer cell lines. Levels of erythropoietin receptor (EpoR), CD24, CD44, Jagged-1 expression, and activation of Notch-1 were assessed by flow cytometry. Self-renewing capacity of CICs was investigated in sphere formation assays. RESULTS: EpoR expression was found on the surface of CICs. Recombinant human Epo (rhEpo) increased the numbers of CICs and self-renewing capacity in a Notch-dependent fashion by induction of Jagged-1. Inhibitors of the Notch pathway and PI3-kinase blocked both effects. CONCLUSIONS: Erythropoietin functionally affects CICs directly. Our observation may explain the negative impact of recombinant Epo on local control and survival of cancer patients with EpoR-positive tumors. [ABSTRACT FROM AUTHOR]