ABSTRACT
BACKGROUND AND PURPOSE: Radiotherapy (RT) is an integral treatment part for patients with head and neck squamous cell carcinoma (HNSCC), but radioresistance remains a major issue. Here, we use MitoTam, a mitochondrially targeted analogue of tamoxifen, which we aim to stimulate ferroptotic cell death with, and sensitize radioresistant cells to RT. MATERIALS AND METHODS: We assessed viability, reactive oxygen species (ROS) production, disruption of mitochondrial membrane potential, and lipid peroxidation in radiosensitive (UT-SCC-40) and radioresistant (UT-SCC-5) HNSCC cells following MitoTam treatment. To assess ferroptosis specificity, we used the ferroptosis inhibitor ferrostatin-1 (fer-1). Also, total antioxidant capacity and sensitivity to tert-butyl hydroperoxide were evaluated to assess ROS-responses. 53BP1 staining was used to assess radiosensitivity after MitoTam treatment. RESULTS: Our data revealed increased ROS, cell death, disruption of mitochondrial membrane potential, and lipid peroxidation following MitoTam treatment in both cell lines. Adverse effects of MitoTam on cell death, membrane potential and lipid peroxidation were prevented by fer-1, indicating induction of ferroptosis. Radioresistant HNSCC cells were less sensitive to the effects of MitoTam due to intrinsic higher antioxidant capacity. MitoTam treatment prior to RT led to superadditive residual DNA damage expressed by 53BP1 foci compared to RT or MitoTam alone. CONCLUSION: MitoTam induced ferroptosis in HNSCC cells, which could be used to overcome the elevated antioxidant capacity of radioresistant cells and sensitize such cells to RT. Treatment with MitoTam followed by RT could therefore present a promising effective therapy of radioresistant cancers. STATEMENT OF SIGNIFICANCE: Radiotherapy is applied in the treatment of a majority of cancer patients. Radioresistance due to elevated antioxidant levels can be overcome by promoting ferroptotic cell death combining ROS-inducing drug MitoTam with radiotherapy.
ABSTRACT
Background and purpose: Radiotherapy (RT) is a mainstay component of treatment for patients with head and neck squamous cell carcinoma (HNSCC), but responses vary. As RT relies upon oxidative damage, antioxidant expression in response to RT-induced reactive oxygen species (ROS) could compromise treatment response. We aimed to examine local and systemic antioxidant responses to increased RT-induced ROS in relation to treatment success. Materials and methods: Nuclear factor erythroid 2-related factor 2 (NRF2), the main antioxidant transcription factor, was immunofluorescently stained in FaDu cells and in tumor biopsies of patients with oral cavity/oropharynx HNSCC before and after five fractions of RT. Besides, total antioxidant capacity (TAC) was analyzed in HNSCC tumor cells in vitro and in serum of HNSCC patients before, during, and after RT. Results: Data revealed an increase in NRF2 expression and TAC in head and neck cancer cells in vitro over the course of 5 daily fractions of 2 Gy. In accordance, also in patients' tumors NRF2 expression increased, which was associated with increased serum TAC during RT. Increasing serum TAC was related to impaired local tumor control. Conclusion: Radiation induced NRF2 expression and upregulated TAC, which may compromise the effect of RT-induced ROS. Changes in serum TAC during RT could serve as a novel predictor of treatment outcome in HNSCC patients.Medical Ethics Review Committee (CMO) approval - CMO number: 2007/104.
ABSTRACT
BACKGROUND: Recently, we reported that tamoxifen-resistant (TAM-R) breast cancer cells are cross-resistant to irradiation. Here, we investigated the mechanisms associated with tamoxifen-induced radioresistance, aiming to prevent or reverse resistance and improve breast cancer treatment. METHODS: Wild-type ERα-positive MCF7 and ERα-negative MDA-MB-231 breast cancer cells and their TAM-R counterparts were analyzed for cellular metabolism using the Seahorse metabolic analyzer. Real-time ROS production, toxicity, and antioxidant capacity in response to H2O2, tamoxifen, and irradiation were determined. Tumor material from 28 breast cancer patients before and after short-term presurgical tamoxifen (ClinicalTrials.gov Identifier: NCT00738777, August 19, 2008) and cellular material was analyzed for NRF2 gene expression and immunohistochemistry. Re-sensitization of TAM-R cells to irradiation was established using pharmacological inhibition. RESULTS: TAM-R cells exhibited decreased oxygen consumption and increased glycolysis, suggesting mitochondrial dysfunction. However, this did not explain radioresistance, as cells without mitochondria (Rho-0) were actually more radiosensitive. Real-time measurement of ROS after tamoxifen and H2O2 exposure indicated lower ROS levels and toxicity in TAM-R cells. Consistently, higher antioxidant levels were found in TAM-R cells, providing protection from irradiation-induced ROS. NRF2, a main activator of the antioxidant response, was increased in TAM-R cells and in tumor tissue of patients treated with short-term presurgical tamoxifen. NRF2 inhibition re-sensitized TAM-R cells to irradiation. CONCLUSION: Mechanisms underlying tamoxifen-induced radioresistance are linked to cellular adaptations to persistently increased ROS levels, leading to cells with chronically upregulated antioxidant capacity and glycolysis. Pharmacological inhibition of antioxidant responses re-sensitizes breast cancer cells to irradiation.
ABSTRACT
Background: Complex clusters of rearrangements are a challenge in interpretation of cancer genomes. Some clusters of rearrangements demarcate clear amplifications of driver oncogenes but others are less well understood. A detailed analysis of rearrangements within these complex clusters could reveal new insights into selection and underlying mutational mechanisms. Patients and methods: Here, we systematically investigate rearrangements that are densely clustered in individual tumours in a cohort of 560 breast cancers. Applying an agnostic approach, we identify 21 hotspots where clustered rearrangements recur across cancers. Results: Some hotspots coincide with known oncogene loci including CCND1, ERBB2, ZNF217, chr8:ZNF703/FGFR1, IGF1R, and MYC. Others contain cancer genes not typically associated with breast cancer: MCL1, PTP4A1, and MYB. Intriguingly, we identify clustered rearrangements that physically connect distant hotspots. In particular, we observe simultaneous amplification of chr8:ZNF703/FGFR1 and chr11:CCND1 where deep analysis reveals that a chr8-chr11 translocation is likely to be an early, critical, initiating event. Conclusions: We present an overview of complex rearrangements in breast cancer, highlighting a potential new way for detecting drivers and revealing novel mechanistic insights into the formation of two common amplicons.
Subject(s)
Breast Neoplasms/genetics , Gene Amplification , Genetic Loci/genetics , Translocation, Genetic , Adult , Age Distribution , Aged , Aged, 80 and over , Algorithms , Breast/pathology , Breast Neoplasms/pathology , Carrier Proteins/genetics , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 8/genetics , Cyclin D1/genetics , Datasets as Topic , Female , Genomics/methods , Humans , Middle Aged , Oncogenes/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Whole Genome SequencingABSTRACT
CONTEXT: Current knowledge on gonadal function in congenital adrenal hyperplasia (CAH) is mostly limited to single-center/country studies enrolling small patient numbers. Overall data indicate that gonadal function can be compromised in men with CAH. OBJECTIVE: To determine gonadal function in men with CAH within the European 'dsd-LIFE' cohort. DESIGN: Cross-sectional clinical outcome study, including retrospective data from medical records. METHODS: Fourteen academic hospitals included 121 men with CAH aged 16-68 years. Main outcome measures were serum hormone concentrations, semen parameters and imaging data of the testes. RESULTS: At the time of assessment, 14/69 patients had a serum testosterone concentration below the reference range; 7 of those were hypogonadotropic, 6 normogonadotropic and 1 hypergonadotropic. In contrast, among the patients with normal serum testosterone (55/69), 4 were hypogonadotropic, 44 normogonadotropic and 7 hypergonadotropic. The association of decreased testosterone with reduced gonadotropin concentrations (odds ratio (OR) = 12.8 (2.9-57.3)) was weaker than the association between serum androstenedione/testosterone ratio ≥1 and reduced gonadotropin concentrations (OR = 39.3 (2.1-732.4)). Evaluation of sperm quality revealed decreased sperm concentrations (15/39), motility (13/37) and abnormal morphology (4/28). Testicular adrenal rest tumor (TART)s were present in 39/80 patients, with a higher prevalence in patients with the most severe genotype (14/18) and in patients with increased current 17-hydroxyprogesterone 20/35) or androstenedione (12/18) serum concentrations. Forty-three children were fathered by 26/113 patients. CONCLUSIONS: Men with CAH have a high risk of developing hypothalamic-pituitary-gonadal disturbances and spermatogenic abnormalities. Regular assessment of endocrine gonadal function and imaging for TART development are recommended, in addition to measures for fertility protection.
Subject(s)
Adrenal Hyperplasia, Congenital/blood , Androstenedione/blood , Gonadotropins/blood , Hypogonadism/blood , Testosterone/blood , Adolescent , Adrenal Hyperplasia, Congenital/complications , Adrenal Hyperplasia, Congenital/epidemiology , Adrenal Rest Tumor/blood , Adrenal Rest Tumor/epidemiology , Adult , Aged , Cross-Sectional Studies , Europe/epidemiology , Humans , Hydroxyprogesterones/blood , Hypogonadism/complications , Male , Middle Aged , Odds Ratio , Oligospermia/complications , Prevalence , Semen Analysis , Sperm Count , Sperm Motility , Testicular Neoplasms/blood , Testicular Neoplasms/epidemiology , Young AdultABSTRACT
Ideally, each patient with a malignancy who is eligible for radiation therapy should receive the most tumoricidal form of this this treatment with the lowest possible risk of toxicity. To overcome radiotherapy resistance, some patients would benefit from a more aggressive approach. This could be treatment intensification, for example by acceleration of the treatment to prevent the negative effects of accelerated tumor cell proliferation, or by boosting certain areas to specifically address intrinsic radioresistance, or a combination of radiotherapy with, for example, a hypoxic cell sensitizer or chemotherapy to reduce the radiotherapy resistance caused by hypoxia. For some patients, one of these approaches can be beneficial but for others could lead to unacceptable side effects. Therefore, it is highly desirable to make the selection upfront. The use of imageable biomarkers could be the key to a more patient-tailored treatment. Different biomarkers for hypoxia and proliferation that could be valuable for radiotherapy are discussed here, including their mechanism, the imaging procedure, quantification, and the value of the results.
ABSTRACT
The transformation of polarized epithelial cells into cells with mesenchymal characteristics by the morphogenetic process of epithelial-mesenchymal transition (EMT) is a well-characterized process essential for embryonic development and associated with cancer progression. EMT is a program driven by changes in gene expression induced by several EMT-specific transcription factors, which inhibit the expression of cell-cell adhesion proteins and other epithelial markers, causing a characteristic loss of cell-cell adhesion, a switch to mesenchymal cell morphology, and increased migratory capabilities. Recently, it has become apparent that in addition to these transcriptionally regulated changes, EMT may also be regulated posttranscriptionally, that is, by alternative splicing. Specifically, the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) have been described as epithelial-specific splicing master regulators specifically involved in EMT-associated alternative splicing. Here, we discuss the regulation of ESRP activity, as well as the evidence supporting a causal role of ESRPs in EMT.
Subject(s)
Alternative Splicing/genetics , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition/genetics , RNA-Binding Proteins/metabolism , Animals , Carcinogenesis/genetics , Humans , Models, BiologicalABSTRACT
Patients with congenital adrenal hyperplasia (CAH) are often clinically less severely affected by cortisol deficiency than anticipated from their enzymatic defect. We hypothesize that adrenal steroid hormone precursors that accumulate in untreated or poorly controlled CAH have glucocorticoid activity and partially compensate for cortisol deficiency. We studied the in vitro effects of 17-hydroxyprogesterone (17OHP), progesterone (P), 21-deoxycortisol (21DF), and androstenedione (Δ4) on the human glucocorticoid receptor (hGR). Competitive binding assays were performed in HeLa cells. Nuclear translocation of the hGR was studied by transfection of COS-7 cells with a GFP-tagged hGR and fluorescence microscopy. Transactivation assays were performed in COS-7 cells and in HEK 293 cells after cotransfection with hGR and luciferase reporter vectors using a dual luciferase assay. 17OHP, P, and 21DF are able to bind to the hGR with binding affinities of 24-43% compared with cortisol. Δ4 has a low binding affinity. Incubation with 21DF led to complete nuclear translocation of the hGR, whereas treatment with 17OHP or P resulted in partial nuclear translocation. 21DF transactivated the hGR with an EC50 approximately 6 times the EC50 of cortisol. 17OHP and P transactivated the hGR with EC50s of more than 100 times the EC50 of cortisol. No hGR transactivation was detected after incubation with Δ4. 21DF, 17OHP, and P are able to bind, translocate, and transactivate the hGR in vitro and thus may have glucocorticoid activity. 21DF might have a clinically relevant agonistic effect on the hGR and could potentially partially compensate the cortisol deficiency in CAH patients.
Subject(s)
Adrenal Glands/metabolism , Adrenal Hyperplasia, Congenital/metabolism , Receptors, Glucocorticoid/metabolism , Steroids/metabolism , 17-alpha-Hydroxyprogesterone/chemistry , Active Transport, Cell Nucleus , Androstenedione/chemistry , Animals , Binding, Competitive , COS Cells , Chlorocebus aethiops , Cortodoxone/chemistry , Glucocorticoids/metabolism , Green Fluorescent Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Microscopy, Fluorescence , Progesterone/chemistry , Protein Binding , Transcriptional ActivationABSTRACT
The CC-chemokine receptor CCR5 has been associated with cancer progression and metastasis. CCR5 blockers such as Maraviroc are tested in metastatic cancer patients. A mutant allele of CCR5, CCR5-delta32 (CCR5del32), which encodes for a protein with a trans-dominant negative effect on the wildtype protein, is frequently found in populations of northern European origin. We set out to determine if the CCR5del32 genotype is associated with progression of breast cancer. Here, we genotyped 414 breast cancer patients and investigated whether the CCR5 genotype had an association with the likelihood to metastasize within specific subgroups of this cohort. The findings were subsequently confirmed in an independent cohort of 1,017 breast cancer patients. Specifically within the postmenopausal subgroup of the initial cohort (n = 325) individuals carrying the CCR5del32 genotype exhibited a significantly longer metastasis-free survival (MFS, p = 0.038). In an independent cohort, CCR5del32 genotype was confirmed to be associated with prolonged MFS only in postmenopausal patients (n = 579, hazard ratio [HR] = 0.61, 95% confidence interval [95% CI] = 0.38-0.99, p = 0.044), and not in premenopausal patients (n = 438, HR = 1.01, 95% CI = 0.70-1.48, p = 0.94). Our results indicate that CCR5del32 genotype is associated with good prognosis in postmenopausal breast cancer patients. Considering this result, postmenopausal breast cancer patients who are wildtype for CCR5 genotype might benefit from CCR5 blockers, such as Maraviroc.
Subject(s)
Breast Neoplasms/genetics , Frameshift Mutation , Receptors, CCR5/genetics , Adolescent , Adult , Aged , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Child , Child, Preschool , DNA Mutational Analysis , Disease-Free Survival , Female , Genetic Association Studies , Humans , Infant , Infant, Newborn , Kaplan-Meier Estimate , Middle Aged , Postmenopause , Prognosis , Proportional Hazards Models , Retrospective Studies , Sequence Deletion , Young AdultABSTRACT
The purpose of this study was to assess the effect of bevacizumab on vasculature and hypoxia in a colorectal tumor model. Nude mice with subcutaneous LS174T tumors were treated with bevacizumab or saline. To assess tumor properties, separate groups of mice were imaged using (18) F-Fluoromisonidazole (FMISO) and (18) F-Fluorodeoxyglucose (FDG) positron emission tomography or magnetic resonance imaging before and 2, 6 and 10 days after the start of treatment. Tumors were harvested after imaging to determine hypoxia and vascular density immunohistochemically. The T2 * time increased significantly less in the bevacizumab group. FMISO uptake increased more over time in the control group. Vessel density significantly decreased in the bevacizumab-treated group. The Carbonic anhydrase 9 (CAIX) and glucose uptake transporter 1 (GLUT1) fractions were higher in bevacizumab-treated tumors. However, the hypoxic fraction showed no significant difference. Bevacizumab led to shorter T2 * times and higher GLUT1 and CAIX expression, suggesting an increase in hypoxia and a higher glycolytic rate. This could be a mechanism of resistance to bevacizumab. The increase in hypoxia, however, could not be demonstrated by pimonidazole/FMISO, possibly because distribution of these tracers is hampered by bevacizumab-induced effects on vascular permeability and perfusion.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/pathology , Hypoxia/diagnosis , Neovascularization, Pathologic/diagnosis , Radiation-Sensitizing Agents , Angiogenesis Inhibitors/blood , Animals , Antibodies, Monoclonal, Humanized/blood , Bevacizumab , Colorectal Neoplasms/drug therapy , Enzyme-Linked Immunosorbent Assay , Female , Fluorodeoxyglucose F18 , Hypoxia/drug therapy , Hypoxia/metabolism , Immunoenzyme Techniques , Magnetic Resonance Imaging , Mice , Mice, Inbred BALB C , Mice, Nude , Misonidazole/analogs & derivatives , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Nitroimidazoles , Positron-Emission Tomography , RadiopharmaceuticalsABSTRACT
Epidermal growth factor receptor (EGFR) inhibition using cetuximab improves the efficacy of radiotherapy in only a subgroup of head and neck squamous cell carcinoma (HNSCC) patients. Therefore, to improve patient selection a better understanding of tumor characteristics that affect treatment is necessary. Here, we investigated the effect of cetuximab on repair of radiation-induced DNA damage in a HNSCC xenograft model, which shows a synergistic effect to cetuximab and radiotherapy (SCCNij185) and a HNSCC model, which shows no additive effect of cetuximab to radiotherapy (SCCNij153). In both tumor models, clear increases were seen in the number of 53BP1 and Rad51 foci after irradiation. 53BP1 foci were present at comparable levels in hypoxic and normoxic tumor areas of the tumor xenografts, while the number of Rad51 foci was significantly higher in normoxic areas compared to hypoxic areas (P < 0.05). In both SCCNij185 and SCCNij153 xenografts an increased number of 53BP1 foci was observed in tumors treated with cetuximab and radiotherapy compared to radiotherapy alone. In SCCNij185 this increase was statistically significant in normoxic tumor areas (P = 0.04) and in SCCNij153 in both hypoxic and normoxic areas (P = 0.007 and P = 0.02, respectively). The number of Rad51 foci was not significantly different when cetuximab was added to radiotherapy compared to radiotherapy alone. Levels of pEGFR and pERK1/2 were decreased when cetuximab was added to radiotherapy in SCCNij185, but not in SCCNij153. Apoptosis was also only increased in SCCNij185 tumors at 4 days after cetuximab and radiotherapy treatment (P < 0.01). In conclusion, cetuximab inhibited DNA repair in both HNSCC models, but this effect was not predictive for the radiosensitizing effect of cetuximab in vivo. This lack of correlation may be related to differential effects of cetuximab and radiotherapy on ERK1/2 signaling and a decreased induction of apoptosis by cetuximab and radiotherapy in the resistant model.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Apoptosis/drug effects , Apoptosis/radiation effects , DNA Repair/drug effects , DNA Repair/radiation effects , ErbB Receptors/antagonists & inhibitors , Head and Neck Neoplasms/pathology , Animals , Antibodies, Monoclonal, Humanized/therapeutic use , Cell Hypoxia/drug effects , Cell Hypoxia/radiation effects , Cell Line, Tumor , Cetuximab , Combined Modality Therapy , DNA Damage , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/radiation effects , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/radiotherapy , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphoproteins/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Tumor Suppressor p53-Binding Protein 1 , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: To assess the value of breast MRI in size assessment of breast cancers in high risk patients, including those with a BRCA 1 or 2 mutation. Guidelines recommend invariably breast MRI screening for these patients and therapy is thus based on these findings. However, the accuracy of breast MRI for staging purposes is only tested in sporadic cancers. METHODS: We assessed concordance of radiologic staging using MRI with histopathology in 49 tumors in 46 high risk patients (23 BRCA1, 12 BRCA2 and 11 Non-BRCA patients). The size of the total tumor area (TTA) was compared to pathology. In invasive carcinomas (n=45) the size of the largest focus (LF) was also addressed. RESULTS: Correlation of MRI measurements with pathology was 0.862 for TTA and 0.793 for LF. TTA was underestimated in 8(16%), overestimated in 5(10%), and correctly measured in 36(73%) cases. LF was underestimated in 4(9%), overestimated in 5(11%), and correctly measured in 36(80%) cases. Impact of BRCA 1 or 2 mutations on the quality of size estimation was not observed. CONCLUSIONS: Tumor size estimation using breast MRI in high risk patients is comparable to its performance in sporadic cancers. Therefore, breast MRI can safely be used for treatment planning.
Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Genetic Predisposition to Disease/genetics , Magnetic Resonance Imaging/methods , Tumor Burden , Adult , Female , Heterozygote , Humans , Middle Aged , Mutation/genetics , Neoplasm Staging , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Clinical studies have shown that bevacizumab beyond progression to first line therapy is beneficial for overall survival in advanced stage colorectal cancer. We studied the utility of several functional imaging modalities to assess the efficacy of bevacizumab beyond progression (BBP). All BALB/c mice with s.c. LS174T xenografts were treated with capecitabine, oxaliplatin and bevacizumab combination therapy. Tumor volume was assessed using caliper measurements. Increase of 1.5 times the initial volume on two subsequent measurements, was considered progression. In half of the mice bevacizumab treatment was continued (n = 13) after progressive disease was established, while the others received saline injections (n = 12). Within 3 days after progression, multi-modal imaging was performed using FDG-PET, diffusion weighted imaging, T2* and dynamic contrast enhanced MRI. Measurements were repeated 7 and 10 days after the first measurements. Afterwards, tumors were analyzed for expression of carbonic anhydrase IX, glucose transporter 1, 9 F1 to stain the vasculature and Ki67 to assess proliferation. In the BBP group tumor growth after progression was reduced compared to the control group (p < 0.01). FDG-PET showed a trend towards lower FDG uptake in the BBP group (p = 0.08). DWI, T2* and DCE-MRI parameters were not significantly different between both groups. The immunohistochemical analyses showed higher CAIX-positive fraction (p < 0.01) and lower Ki67 expression (p = 0.06) in the BBP group. The relative vascular area was significantly lower in the BBP group (p = 0.03). GLUT-1 expression and vascular density did not significantly differ between both groups. Bevacizumab after progression resulted in significant changes in the tumor proliferation and microenvironment compared to discontinuation of bevacizumab. FDG-PET may be sensitive to BBP-induced effects.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Colorectal Neoplasms/drug therapy , Disease Progression , Magnetic Resonance Imaging , Positron-Emission Tomography , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Bevacizumab , Colorectal Neoplasms/diagnostic imaging , Colorectal Neoplasms/pathology , Disease Models, Animal , Female , Fluorodeoxyglucose F18 , Ki-67 Antigen/metabolism , Mice , Mice, Inbred BALB C , Tumor Burden/drug effectsABSTRACT
BACKGROUND: Previously we demonstrated that an mRNA signature reflecting cellular proliferation had strong prognostic value. As clinical applicability of signatures can be controversial, we sought to improve our marker's clinical utility by validating its biological relevance, reproducibility in independent data sets and applicability using an independent technique. METHODS: To facilitate signature evaluation with quantitative PCR (qPCR) a novel computational procedure was used to reduce the number of signature genes without significant information loss. These genes were validated in different human cancer cell lines upon serum starvation and in a 168 xenografts panel. Analyses were then extended to breast cancer and non-small-cell lung cancer (NSCLC) patient cohorts. RESULTS: Expression of the qPCR-based signature was dramatically decreased under starvation conditions and inversely correlated with tumour volume doubling time in xenografts. The signature validated in breast cancer (hazard ratio (HR)=1.63, P<0.001, n=1820) and NSCLC adenocarcinoma (HR=1.64, P<0.001, n=639) microarray data sets. Lastly, qPCR in a node-negative, non-adjuvantly treated breast cancer cohort (n=129) showed that patients assigned to the high-proliferation group had worse disease-free survival (HR=2.25, P<0.05). CONCLUSION: We have developed and validated a qPCR-based proliferation signature. This test might be used in the clinic to select (early-stage) patients for specific treatments that target proliferation.
Subject(s)
Neoplasms/genetics , Neoplasms/pathology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Growth Processes/genetics , Cell Line, Tumor , Cohort Studies , Disease-Free Survival , Female , Gene Expression Profiling/methods , HCT116 Cells , HT29 Cells , HeLa Cells , Hep G2 Cells , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Prognosis , Real-Time Polymerase Chain Reaction/methodsABSTRACT
Paragangliomas (PGLs) have widely different metastastic potentials. Two different types of PGLs can be defined by expression profiling. Cluster 1 PGLs exhibit VHL and/or succinate dehydrogenase (SDH) mutations and a pseudohypoxic phenotype. RET and neurofibromatosis type 1 (NF1) mutations occur in cluster 2 tumors characterized by deregulation of the RAS/RAF/MAP kinase signaling cascade. Sporadic PGLs can exhibit either profile. During sustained hypoxia, a natural antisense transcript of hypoxia-inducible factor 1 (aHIF) is expressed. The role of aHIF in the metastatic potential of PGL has not yet been investigated. The aim was to test the hypothesis that genotype-specific overexpression of aHIF is associated with an increased metastatic potential. Tumor samples were collected from 87 patients with PGL. Quantitative PCR was performed for aHIF, vascular endothelial growth factor (VEGF), aquaporin 3, cytochrome b561, p57Kip2, slit homolog 3, and SDHC. Expression was related to mutation status, benign versus malignant tumors, and metastasis-free survival. We found that both aHIF and VEGF were overexpressed in cluster 1 PGLs and in metastatic tumors. In contrast, slit homolog 3, p57Kip2, cytochrome b561, and SDHC showed overexpression in non-metastatic tumors, whereas no such difference was observed for aquaporin 3. Patients with higher expression levels of aHIF and VEGF had a significantly decreased metastasis-free survival. Higher expression levels of SDHC are correlated with an increased metastasis-free survival. In conclusion, we not only demonstrate a higher expression of VEGF in cluster 1 PGL, fitting a profile of pseudohypoxia and angiogenesis, but also of aHIF. Moreover, overexpression of aHIF and VEGF marks a higher metastatic potential in PGL.
Subject(s)
Adrenal Gland Neoplasms/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Pheochromocytoma/genetics , RNA, Messenger/biosynthesis , Adolescent , Adrenal Gland Neoplasms/metabolism , Adult , Aged , Aquaporin 3/biosynthesis , Aquaporin 3/genetics , Child , Cyclin-Dependent Kinase Inhibitor p57/biosynthesis , Cyclin-Dependent Kinase Inhibitor p57/genetics , Cytochrome b Group/biosynthesis , Cytochrome b Group/genetics , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Middle Aged , Pheochromocytoma/metabolism , RNA, Messenger/genetics , RNA, Neoplasm/chemistry , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/genetics , Young AdultSubject(s)
Hypoxia/genetics , Neoplasms/genetics , RNA, Ribosomal, 18S/genetics , Cell Line , Humans , ResearchABSTRACT
Nuclear receptors (NR) are intracellular receptors that execute a transcriptional program upon binding to hormones, vitamins and metabolic products. They are key regulators of distinct physiological processes, including growth and differentiation, metabolism, and immunity. The impact of NR activation on a given cell can differ from proliferation induction to programmed cell death. NR malfunction is associated with different diseases, such as diabetes, chronic inflammatory diseases and cancer. Much progress has been made towards understanding the transcriptional regulation by individual NR at the molecular level. However, essentially every cell expresses multiple NR and will encounter complex mixtures of NR ligands during its life cycle. In this review, we will focus on novel insights in balancing NR activity via NR crosstalk and DC-SCRIPT/ZNF366, a bi-functional NR coregulator. The impact on breast cancer development and prognosis will be discussed.
Subject(s)
Carrier Proteins/physiology , Receptor Cross-Talk/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Breast Neoplasms/etiology , Female , Humans , Prognosis , Receptors, Estrogen/physiology , Receptors, Retinoic Acid/physiology , Tumor Suppressor Proteins/physiologySubject(s)
Breast Neoplasms/drug therapy , Drug Screening Assays, Antitumor , Gene Expression Profiling , RNA, Messenger/metabolism , Receptors, Estrogen/metabolism , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Humans , Immunohistochemistry , Receptors, Estrogen/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Survival Analysis , Tamoxifen/pharmacologyABSTRACT
BACKGROUND: Nuclear receptors, including estrogen receptor (ER), progesterone receptor (PR)-B, peroxisome proliferator-activated receptor gamma, and retinoic acid receptor alpha, have been implicated in breast cancer etiology and progression. We investigated the role of dendritic cell-specific transcript (DC-SCRIPT) as coregulator of these nuclear receptors and as a prognostic factor in breast cancer. METHODS: The effect of DC-SCRIPT on the transcriptional activity of nuclear receptors was assessed by luciferase reporter assays. DC-SCRIPT expression in normal and tumor tissue from breast cancer patients was analyzed by polymerase chain reaction and immunohistochemistry. The prognostic value of tumor DC-SCRIPT mRNA expression was assessed in three independent cohorts of breast cancer patients: a discovery group (n = 47) and a validation group (n = 97) (neither of which had received systemic adjuvant therapy) and in a tamoxifen-treated validation group (n = 68) by using a DC-SCRIPT to porphobilinogen deaminase transcript ratio cutoff of 0.15 determined in the discovery group. Univariate and multivariable Cox proportional hazards model analyses were performed. All statistical tests were two-sided. RESULTS: DC-SCRIPT suppressed ER- and PR-mediated transcription in a ligand-dependent fashion, whereas it enhanced the retinoic acid receptor alpha- and peroxisome proliferator-activated receptor gamma-mediated transcription. In breast tissue samples from nine patients, DC-SCRIPT mRNA was expressed at lower levels in the tumor than in the corresponding normal tissue (P = .010). Patients in the discovery group with high tumor DC-SCRIPT mRNA levels (66%) had a longer disease-free interval than those with a low DC-SCRIPT mRNA level (34%) (hazard ratio [HR] of recurrence for high vs low DC-SCRIPT level = 0.23, 95% confidence interval [CI] = 0.06 to 0.93, P = .039), which was confirmed in the validation group (HR of recurrence = 0.50, 95% CI = 0.26 to 0.95, P = .034). This prognostic value was confined to patients with ER- and/or PR-positive tumors (discovery group: HR of recurrence = 0.16, 95% CI = 0.03 to 0.89, P = .030; validation group: HR of recurrence = 0.42, 95% CI = 0.19 to 0.91, P = .028) and was also observed in the second validation group (HR = 0.46, 95% CI = 0.22 to 0.97, P = .040). DC-SCRIPT was an independent prognostic factor after correction for tumor size, lymph node status, and adjuvant therapy (n = 145; HR = 0.50, 95% CI = 0.29 to 0.85, P = .010). CONCLUSION: DC-SCRIPT is a key regulator of nuclear receptor activity that has prognostic value in breast cancer.