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BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease, and although no effective targeted therapies are available to date, about one-third of patients with TNBC achieve pathologic complete response (pCR) from standard-of-care anthracycline/taxane (ACT) chemotherapy. The heterogeneity of these tumors, however, has hindered the discovery of effective biomarkers to identify such patients. METHODS AND FINDINGS: We performed whole exome sequencing on 29 TNBC cases from the MD Anderson Cancer Center (MDACC) selected because they had either pCR (n = 18) or extensive residual disease (n = 11) after neoadjuvant chemotherapy, with cases from The Cancer Genome Atlas (TCGA; n = 144) and METABRIC (n = 278) cohorts serving as validation cohorts. Our analysis revealed that mutations in the AR- and FOXA1-regulated networks, in which BRCA1 plays a key role, are associated with significantly higher sensitivity to ACT chemotherapy in the MDACC cohort (pCR rate of 94.1% compared to 16.6% in tumors without mutations in AR/FOXA1 pathway, adjusted p = 0.02) and significantly better survival outcome in the TCGA TNBC cohort (log-rank test, p = 0.05). Combined analysis of DNA sequencing, DNA methylation, and RNA sequencing identified tumors of a distinct BRCA-deficient (BRCA-D) TNBC subtype characterized by low levels of wild-type BRCA1/2 expression. Patients with functionally BRCA-D tumors had significantly better survival with standard-of-care chemotherapy than patients whose tumors were not BRCA-D (log-rank test, p = 0.021), and they had significantly higher mutation burden (p < 0.001) and presented clonal neoantigens that were associated with increased immune cell activity. A transcriptional signature of BRCA-D TNBC tumors was independently validated to be significantly associated with improved survival in the METABRIC dataset (log-rank test, p = 0.009). As a retrospective study, limitations include the small size and potential selection bias in the discovery cohort. CONCLUSIONS: The comprehensive molecular analysis presented in this study directly links BRCA deficiency with increased clonal mutation burden and significantly enhanced chemosensitivity in TNBC and suggests that functional RNA-based BRCA deficiency needs to be further examined in TNBC.
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Antraciclinas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Taxoides/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Biomarcadores , Estudos de Coortes , Exoma , Feminino , Genômica , HumanosRESUMO
BACKGROUND: Molecular heterogeneity of tumors suggests the presence of multiple different subclones that may limit response to targeted therapies and contribute to acquisition of drug resistance, but its quantification has remained challenging. RESULTS: We performed simulations to evaluate statistical measures that best capture the molecular diversity within a group of tumors for either continuous (gene expression) or discrete (mutations, copy number alterations) molecular data. Dispersion based metrics in the principal component space best captured the underlying heterogeneity. To demonstrate utility of these measures, we characterized the diversity in transcriptional and genomic profiles of different breast tumor subtypes, and showed that basal-like or triple-negative breast cancers (TNBC) are significantly more heterogeneous molecularly than other subtypes. Our analysis also suggests that transcriptional diversity is a global characteristic of the tumors observed across the majority of molecular pathways. Among basal-like tumors, those that were resistant to multi-agent chemotherapy showed greater transcriptional diversity compared to chemotherapy-sensitive tumors, suggesting that potentially multiple mechanisms may be contributing to chemotherapy resistance. CONCLUSIONS: We proposed and validated measures of transcriptional and genomic diversity that can quantify the molecular diversity of tumors. We applied the new measures to genomic data from breast tumors and demonstrated that basal-like breast cancers are significantly more diverse than other breast cancers. The observation that chemo-resistant tumors are significantly more diverse molecularly than chemosensitive tumors implies that multiple resistance mechanisms may be active, thus limiting the sensitivity and accuracy of predictive markers of chemotherapy response.
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Neoplasias da Mama/genética , Genes Neoplásicos , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Variações do Número de Cópias de DNA , Bases de Dados Genéticas/estatística & dados numéricos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
INTRODUCTION: Human Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer. METHODS: We have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed. RESULTS: Overexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this isoform. CONCLUSIONS: Together, we present isoform-specific roles of ERBB4 during puberty and early pregnancy, and reveal a novel oncogenic property of CYT-1 ERBB4. The results may be exploited to develop better therapeutic strategies in breast cancer.
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Carcinogênese/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Neoplasias Mamárias Experimentais/genética , Gravidez/genética , Isoformas de Proteínas/genética , Receptor ErbB-4/genética , Processamento Alternativo , Animais , Carcinogênese/metabolismo , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Vírus do Tumor Mamário do Camundongo , Camundongos , Camundongos Transgênicos , Gravidez/metabolismo , Isoformas de Proteínas/metabolismo , Receptor ErbB-4/metabolismoRESUMO
The effect of scheduling of targeted therapy combinations on drug resistance is underexplored in triple-negative breast cancer (TNBC). TNBC constitutes heterogeneous cancer cell populations the composition of which can change dynamically during treatment resulting in the selection of resistant clones with a fitness advantage. We evaluated crizotinib (ALK/MET inhibitor) and navitoclax (ABT-263; Bcl-2/Bcl-xL inhibitor) combinations in a large design consisting of 696 two-cycle sequential and concomitant treatment regimens with varying treatment dose, duration, and drug holiday length over a 26-day period in MDA-MB-231 TNBC cells and found that patterns of resistance depend on the schedule and sequence in which the drugs are given. Further, we tracked the clonal dynamics and mechanisms of resistance using DNA-integrated barcodes and single-cell RNA sequencing. Our study suggests that longer formats of treatment schedules in vitro screening assays are required to understand the effects of resistance and guide more realistically in vivo and clinical studies.
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BACKGROUND: Inflammatory breast cancer (IBC) has a highly invasive and metastatic phenotype. However, little is known about its genetic drivers. To address this, we report the largest cohort of whole-genome sequencing (WGS) of IBC cases. METHODS: We performed WGS of 20 IBC samples and paired normal blood DNA to identify genomic alterations. For comparison, we used 23 matched non-IBC samples from the Cancer Genome Atlas Program (TCGA). We also validated our findings using WGS data from the International Cancer Genome Consortium (ICGC) and the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We examined a wide selection of genomic features to search for differences between IBC and conventional breast cancer. These include (i) somatic and germline single-nucleotide variants (SNVs), in both coding and non-coding regions; (ii) the mutational signature and the clonal architecture derived from these SNVs; (iii) copy number and structural variants (CNVs and SVs); and (iv) non-human sequence in the tumors (i.e., exogenous sequences of bacterial origin). RESULTS: Overall, IBC has similar genomic characteristics to non-IBC, including specific alterations, overall mutational load and signature, and tumor heterogeneity. In particular, we observed similar mutation frequencies between IBC and non-IBC, for each gene and most cancer-related pathways. Moreover, we found no exogenous sequences of infectious agents specific to IBC samples. Even though we could not find any strongly statistically distinguishing genomic features between the two groups, we did find some suggestive differences in IBC: (i) The MAST2 gene was more frequently mutated (20% IBC vs. 0% non-IBC). (ii) The TGF ß pathway was more frequently disrupted by germline SNVs (50% vs. 13%). (iii) Different copy number profiles were observed in several genomic regions harboring cancer genes. (iv) Complex SVs were more frequent. (v) The clonal architecture was simpler, suggesting more homogenous tumor-evolutionary lineages. CONCLUSIONS: Whole-genome sequencing of IBC manifests a similar genomic architecture to non-IBC. We found no unique genomic alterations shared in just IBCs; however, subtle genomic differences were observed including germline alterations in TGFß pathway genes and somatic mutations in the MAST2 kinase that could represent potential therapeutic targets.
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Genoma Humano , Neoplasias Inflamatórias Mamárias/genética , Mutação/genética , Sequenciamento Completo do Genoma , Células Clonais , Variações do Número de Cópias de DNA/genética , Evolução Molecular , Humanos , Neoplasias Inflamatórias Mamárias/microbiologia , Neoplasias Inflamatórias Mamárias/patologia , Anotação de Sequência Molecular , Fenótipo , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Aberrant ErbB receptor signaling is associated with various types of malignancies. gamma-Tocotrienol is a member of the vitamin E family of compounds that displays potent anticancer activity that is associated with suppression in ErbB receptor phosphorylation and mitogenic signaling. Erlotinib and gefitinib are tyrosine kinase inhibitors that block ErbB1 receptor activation, whereas trastuzumab is a monoclonal antibody that has been designed to specifically inhibit ErbB2 receptor activation. However, the clinical effectiveness of these agents have been disappointing because of cooperation between different ErbB family members that can rescue cancer cells from agents directed against a single ErbB receptor subtype. It was hypothesized that targeting multiple ErbB receptor subtypes with combined treatment of gamma-tocotrienol and ErbB receptor inhibitors would provide greater anticancer effects than monotherapy targeting only a single ErbB receptor subtype. METHODS: Highly malignant mouse +SA mammary epithelial cells were maintained in culture on serum-free defined media containing 10 ng/ml EGF as a mitogen. Cell viability wase determined by MTT assay, whereas Western blot and immunofluorescent staining was used to determine treatment effects on ErbB receptor subtype level and activation. Treatment-induced apoptosis was determined using annexin V staining and Western blot analysis of cleaved caspase-3 and PARP levels. RESULTS: Treatment with 3.5 microM gamma-tocotrienol, 0.5 microM erlotinib or 1.0 microM gefitinib alone, significantly inhibited +SA tumor cell growth. Combined treatment with subeffective doses of erlotinib (0.25 microM) or gefitinib (0.5 microM) with subeffective doses of gamma-tocotrienol (0.5-3.0 microM) significantly inhibited the growth and induced apoptosis in a dose-responsive manner. Trastuzumab treatment alone or in combination had no effect on +SA cell growth and viability. Combined treatment of gamma-tocotrienol with erlotinib or gefitinib also cause a large decrease in ErbB3, ErbB4, and to a lesser extent ErbB2 receptor levels, and EGF-dependent ErbB2-4 tyrosine phosphorylation (activation), but had no effect on ErbB1 receptor levels or activation. CONCLUSION: Combination treatment of gamma-tocotrienol with specific ErbB receptor inhibitors is more effective in reducing mammary tumor cell growth and viability than high dose monotherapy, suggesting that targeting multiple ErbB receptors with combination therapy may significantly improve the therapeutic response in breast cancer patients.
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Apoptose , Cromanos/administração & dosagem , Neoplasias Mamárias Animais/tratamento farmacológico , Quinazolinas/administração & dosagem , Vitamina E/análogos & derivados , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Meios de Cultura Livres de Soro/farmacologia , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Gefitinibe , Camundongos , Transdução de Sinais , Trastuzumab , Vitamina E/administração & dosagemRESUMO
Vitamin E (VE) is a generic term that represents a family of compounds composed of various tocopherol and tocotrienol isoforms. Tocotrienols display potent anti-angiogenic and antiproliferative activities. Redox-silent tocotrienol analogues also display potent anticancer activity. The ultimate objective of this study was to develop semisynthetically C-6-modified redox-silent tocotrienol analogues with enhanced antiproliferative and anti-invasive activities as compared to their parent compound. Examples of these are carbamate and ether analogues of alpha-, gamma-, and delta-tocotrienols (1-3). Various aliphatic, olefinic, and aromatic substituents were used. Steric limitation, electrostatic, hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) properties were varied at this position and the biological activities of these derivatives were tested. Three-dimensional quantitative structure-activity relationship (3D QSAR) studies were performed using Comparative Molecular Field (CoMFA) and Comparative Molecular Similarity Indices Analyses (CoMSIA) to better understand the structural basis for biological activity and guide the future design of more potent VE analogues.
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Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Desenho de Fármacos , Tocotrienóis/síntese química , Tocotrienóis/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Modelos Moleculares , Estrutura Molecular , Invasividade Neoplásica , Oxirredução , Relação Quantitativa Estrutura-Atividade , Estereoisomerismo , Tocotrienóis/químicaRESUMO
Tocotrienols are vitamin E members with potent antiproliferative activity against preneoplastic and neoplastic mammary epithelial cells with little or no effect on normal cell growth or functions. However, physicochemical and pharmacokinetic properties greatly limit their use as therapeutic agents. Tocotrienols' chemical instability, poor water solubility, NPC1L1-mediated transport, and rapid metabolism are examples of such obstacles which hinder the therapeutic use of these valuable natural products. Vitamin E esters like α-tocopheryl succinate were prepared to significantly improve chemical and metabolic stability, water solubility, and potency. Thus, 12 semisynthetic tocotrienol ester analogues 4-15 were prepared by direct esterification of natural tocotrienol isomers with various acid anhydrides or chlorides. Esters 4-15 were evaluated for their ability to inhibit the proliferation and migration of the mammary tumor cells +SA and MDA-MB-231, respectively. Esters 5, 9, and 11 effectively inhibited the proliferation of the highly metastatic +SA rodent mammary epithelial cells with IC(50) values of 0.62, 0.51, and 0.86µM, respectively, at doses that had no effect on immortalized normal mouse CL-S1 mammary epithelial cells. Esters 4, 6, 8-10, and 13 inhibited 50% of the migration of the human metastatic MDA-MB-231 breast cancer cells at a single 5µM dose in wound-healing assay. The most active ester 9 was 1000-fold more water-soluble and chemically stable versus its parent α-tocotrienol (1). These findings strongly suggest that redox-silent tocotrienol esters may provide superior therapeutic forms of tocotrienols for the control of metastatic breast cancer.
Assuntos
Antineoplásicos/química , Neoplasias da Mama/metabolismo , Tocotrienóis/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Ésteres , Feminino , Humanos , Glândulas Mamárias Animais/citologia , Camundongos , Oxirredução , Ratos , Solubilidade , Tocotrienóis/síntese química , Tocotrienóis/farmacologiaRESUMO
PURPOSE: We performed whole-exome sequencing (WES) of pre- and posttreatment cancer tissues to assess the somatic mutation landscape of tumors before and after neoadjuvant taxane and anthracycline chemotherapy with or without bevacizumab. EXPERIMENTAL DESIGN: Twenty-nine pretreatment biopsies from the SWOG S0800 trial were subjected to WES to identify mutational patterns associated with response to neoadjuvant chemotherapy. Nine matching samples with residual cancer after therapy were also analyzed to assess changes in mutational patterns in response to therapy. RESULTS: In pretreatment samples, a higher proportion of mutation signature 3, a BRCA-mediated DNA repair deficiency mutational signature, was associated with higher rate of pathologic complete response (pCR; median signature weight 24%, range 0%-38% in pCR vs. median weight 0%, range 0%-19% in residual disease, Wilcoxon rank sum, Bonferroni P = 0.007). We found no biological pathway level mutations associated with pCR or enriched in posttreatment samples. We observed statistically significant enrichment of high functional impact mutations in the "E2F targets" and "G2-M checkpoint" pathways in residual cancer samples implicating these pathways in resistance to therapy and a significant depletion of mutations in the "myogenesis pathway" suggesting the cells harboring these variants were effectively eradicated by therapy. CONCLUSIONS: These results suggest that genomic disturbances in BRCA-related DNA repair mechanisms, reflected by a dominant mutational signature 3, confer increased chemotherapy sensitivity. Cancers that survive neoadjuvant chemotherapy frequently have alterations in cell-cycle-regulating genes but different genes of the same pathways are affected in different patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Genômica/métodos , Mutação , Terapia Neoadjuvante/métodos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Distúrbios no Reparo do DNA/genética , Feminino , Humanos , Prognóstico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Cancer cells employ various defense mechanisms against drug-induced cell death. Investigating multi-omics landscapes of cancer cells before and after treatment can reveal resistance mechanisms and inform new therapeutic strategies. We assessed the effects of navitoclax, a BCL2 family inhibitor, on the transcriptome, methylome, chromatin structure, and copy number variations of MDA-MB-231 triple-negative breast cancer (TNBC) cells. Cells were sampled before treatment, at 72 h of exposure, and after 10-day drug-free recovery from treatment. We observed transient alterations in the expression of stress response genes that were accompanied by corresponding changes in chromatin accessibility. Most of these changes returned to baseline after the recovery period. We also detected lasting alterations in methylation states and genome structure that suggest permanent changes in cell population composition. Using single-cell analyses, we identified 2350 genes significantly upregulated in navitoclax-resistant cells and derived an 18-gene navitoclax resistance signature. We assessed the navitoclax-response-predictive function of this signature in four additional TNBC cell lines in vitro and in silico in 619 cell lines treated with 251 different drugs. We observed a drug-specific predictive value in both experiments, suggesting that this signature could help guiding clinical biomarker studies involving navitoclax.
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gamma-Tocotrienol, a member of the vitamin E family of compounds, induces apoptosis in a variety of cancer cell types. However, previous studies have clearly demonstrated that gamma-tocotrienol-induced apoptosis in neoplastic mouse +SA mammary epithelial cells is not mediated through mitochondrial stress or death receptor apoptotic signaling. Therefore, studies were conducted to determine the role of endoplasmic reticulum (ER) stress in mediating gamma-tocotrienol-induced apoptosis in +SA mammary tumor cells. Treatment with 15-40 microM gamma-tocotrienol induced +SA cell death in a dose-responsive manner, and these effects were associated with a corresponding increase in poly (ADP-ribose) polymerase (PARP)-cleavage and activation of protein kinase-like endoplasmic reticulum kinase/eukaryotic translational initiation factor/activating transcription factor 4 (PERK/eIF2alpha/ATF-4) pathway, a marker of ER stress response. These treatments also caused a large increase in C/EBP homologous protein (CHOP) levels, a key component of ER stress mediated apoptosis that increases expression of tribbles 3 (TRB3). Knockdown of CHOP by specific siRNAs attenuated gamma-tocotrienol-induced PARP-cleavage, CHOP and TRB3 expression. gamma-Tocotrienol treatment also reduced full-length caspase-12 levels, an indication of caspase-12 cleavage and activation. Intracellular levels of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase, an ER-transmembrane enzyme catalyzing the synthesis of mevalonate, decreased following gamma-tocotrienol treatment, but combined treatment with mevalonate did not reverse gamma-tocotrienol-induced apoptosis, suggesting that a decrease in HMGCoA reductase activity is not required for gamma-tocotrienol induced apoptosis. These results demonstrate that ER stress apoptotic signaling is associated with gamma-tocotrienol-induced apoptosis in +SA mammary tumor cells.
Assuntos
Apoptose/efeitos dos fármacos , Cromanos/farmacologia , Retículo Endoplasmático/patologia , Neoplasias Mamárias Animais/patologia , Vitamina E/análogos & derivados , Animais , Caspase 12/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hidroximetilglutaril-CoA Redutases/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Vitamina E/farmacologiaRESUMO
Statins and gamma-tocotrienol (a rare isoform of vitamin E) both inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase activity and display anticancer activity. However, clinical application of statins has been limited by high dose toxicity. Previous studies showed that combined statin and gamma-tocotrienol treatment synergistically inhibits growth of highly malignant +SA mammary epithelial cells in culture. To investigate the mechanism mediating this growth inhibition, studies were conducted to determine the effect of combination low dose gamma-tocotrienol and statin treatment on +SA mammary tumor cell cycle progression. Treatment with 0.25 microM simvastatin, lovastatin, mevastatin, 10 microM pravastatin or 2.0 microM gamma-tocotrienol alone had no effect, while combined treatment of individual statins with gamma-tocotrienol significantly inhibited +SA cell proliferation during the 4-day culture period. Flow cytometric analysis demonstrated that combined treatment induced cell cycle arrest in G1. Additional studies showed that treatment with 0.25 microM simvastatin or 2 microM gamma-tocotrienol alone had no effect on the relative intracellular levels of cyclin D1, CDK2, CDK4 and CDK6, but combined treatment caused a large reduction in cyclin D1 and CDK2 levels. Combined treatments also caused a relatively large increase in p27, but had no effect on p21 and p15 levels, and resulted in a large reduction in retinoblastoma (Rb) protein phosphorylation at ser780 and ser807/811. Similar effects were observed following combined treatment of gamma-tocotrienol with low doses of lovastatin, mevastatin and pravastatin. These findings demonstrate that combination low dose statin and gamma-tocotrienol treatment induced mammary tumor cell cycle arrest at G1, resulting from an increase in p27 expression, and a corresponding decrease in cyclin D1, CDK2, and hypophosphorylation of Rb protein. These findings suggest that combined treatment of statins with gamma-tocotrienol may provide significant health benefits in the treatment of breast cancer in women, while avoiding myotoxicity associated with high dose statin monotherapy.
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Cromanos/farmacologia , Fase G1/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Mamárias Animais/tratamento farmacológico , Vitamina E/análogos & derivados , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/dietoterapia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromanos/agonistas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/agonistas , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Proteínas de Neoplasias/metabolismo , Vitamina E/agonistas , Vitamina E/farmacologiaRESUMO
The goal of this study was to identify a novel target for antibody-drug conjugate (ADC) development in triple negative breast cancer (TNBC), which has limited treatment options, using gene expression datasets and in vitro siRNA/CRISPR and in vivo functional assays. We analyzed 4467 breast cancers and identified GABRP as top expressed gene in TNBC with low expression in most normal tissues. GABRP protein was localized to cell membrane with broad range of receptors/cell (815-53,714) and expressed by nearly half of breast cancers tissues. GABRP gene knockdown inhibited TNBC cell growth and colony formation in vitro and growth of MDA-MB-468 xenografts in nude mice. Commercially available anti-GABRP antibody (5-100 µg/ml) or de novo generated Fabs (20 µg/ml) inhibited TNBC cell growth in vitro. The same antibody conjugated to mertansine (DM1) also showed significant anticancer activity at nanomolar concentrations. Our results indicate that GABRP is a potential novel therapeutic target for ADC development.
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Antineoplásicos/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Imunoconjugados/farmacologia , Receptores de GABA-A/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Conjuntos de Dados como Assunto , Desenvolvimento de Medicamentos , Feminino , Antagonistas de Receptores de GABA-A/uso terapêutico , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imunoconjugados/uso terapêutico , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Maitansina/farmacologia , Maitansina/uso terapêutico , Camundongos , Terapia de Alvo Molecular/métodos , Receptores de GABA-A/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: How the immune microenvironment changes during neoadjuvant chemotherapy of primary breast cancer is not well understood. METHODS: We analyzed pre- and post-treatment samples from 60 patients using the NanoString PanCancer IO360™ assay to measure the expression of 750 immune-related genes corresponding to 14 immune cell types and various immune functions, and assessed TIL counts and PD-L1 protein expression by immunohistochemistry. Treatment associated changes in gene expression levels were compared using t-test with Bonferroni correction. TIL count, PD-L1 protein and immune metagenes were compared using Wilcoxon test. Baseline immune markers were correlated with pathologic complete response (pCR) using estrogen receptor and treatment arm adjusted logistic regression. RESULTS: At baseline, high TIL counts and high expression of chemoattractant cytokines (CCL21, CCL19) and cytotoxic T cell markers were associated with higher pCR rate. High expression of stromal genes (VEGFB, TGFB3, PDGFB, FGFR1, IGFR1), mast and myeloid inflammatory cell metagenes, stem cell related genes (CD90, WNT11, CTNNB1) and CX3CR1, and IL11RA were associated with residual disease (RD). After treatment, in cases with pCR, TIL counts and most immune genes decreased significantly. Among RD cases, TIL counts and PD-L1 expression did not change but cellular stress and hypoxia associated genes (DUSP1, EGR1), and IL6, CD36, CXCL2, CD69 and the IL8/VEGF metagene increased. CONCLUSIONS: Activated T cells in the tumor microenvironment are associated with pCR whereas stromal functions are associated with residual disease. Most immune functions decrease during neoadjuvant chemotherapy but several immunotherapy targets (PD-L1, IL6, IL8) remain expressed in RD suggesting potential therapeutic strategies.
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Antineoplásicos Imunológicos/uso terapêutico , Bevacizumab/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Perfilação da Expressão Gênica/métodos , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/metabolismo , Bevacizumab/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Terapia Neoadjuvante , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacosRESUMO
Two new diterpenes, sinulodurin A (1) and sinulodurin B (2), along with two known sterols, 24 S-methyl cholesterol and 24-methylene cholesterol, were isolated from the Palau soft coral Sinularia dura. The structures of the new metabolites were determined on the basis of spectroscopic methods and by comparison of NMR data with those of related metabolites. Sinulodurin A (1) and sinulodurin B (2) showed antiproliferative activity against highly malignant +SA mammary epithelial cells with an IC 50 range of 20-30 microM. They also displayed anti-invasive activity against human highly metastatic prostate cancer PC-3M-CT+ cells in the spheroid disaggregation assay. Furthermore, the antimicrobial activities of the isolates were tested.
Assuntos
Proliferação de Células/efeitos dos fármacos , Diterpenos/isolamento & purificação , Invasividade Neoplásica/prevenção & controle , Animais , Antozoários , Diterpenos/química , Diterpenos/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Infravermelho , Espectrofotometria UltravioletaRESUMO
Multi-region sequencing is used to detect intratumor genetic heterogeneity (ITGH) in tumors. To assess whether genuine ITGH can be distinguished from sequencing artifacts, we performed whole-exome sequencing (WES) on three anatomically distinct regions of the same tumor with technical replicates to estimate technical noise. Somatic variants were detected with three different WES pipelines and subsequently validated by high-depth amplicon sequencing. The cancer-only pipeline was unreliable, with about 69% of the identified somatic variants being false positive. Even with matched normal DNA for which 82% of the somatic variants were detected reliably, only 36%-78% were found consistently in technical replicate pairs. Overall, 34%-80% of the discordant somatic variants, which could be interpreted as ITGH, were found to constitute technical noise. Excluding mutations affecting low-mappability regions or occurring in certain mutational contexts was found to reduce artifacts, yet detection of subclonal mutations by WES in the absence of orthogonal validation remains unreliable.
Assuntos
Sequenciamento do Exoma , Heterogeneidade Genética , Neoplasias/genética , Feminino , Humanos , Mutação INDEL/genética , Mutação/genética , Ploidias , Polimorfismo de Nucleotídeo Único/genética , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Age is one of the most important risk factors for developing breast cancer. However, age-related changes in normal breast tissue that potentially lead to breast cancer are incompletely understood. Quantifying tissue-level DNA methylation can contribute to understanding these processes. We hypothesized that occurrence of breast cancer should be associated with an acceleration of epigenetic aging in normal breast tissue. RESULTS: Ninety-six normal breast tissue samples were obtained from 88 subjects (breast cancer = 35 subjects/40 samples, unaffected = 53 subjects/53 samples). Normal tissue samples from breast cancer patients were obtained from distant non-tumor sites of primary mastectomy specimens, while samples from unaffected women were obtained from the Komen Tissue Bank (n = 25) and from non-cancer-related breast surgery specimens (n = 28). Patients were further stratified into four cohorts: age < 50 years with and without breast cancer and age ≥ 50 with and without breast cancer. The Illumina HumanMethylation450k BeadChip microarray was used to generate methylation profiles from extracted DNA samples. Data was analyzed using the "Epigenetic Clock," a published biomarker of aging based on a defined set of 353 CpGs in the human genome. The resulting age estimate, DNA methylation age, was related to chronological age and to breast cancer status. The DNAmAge of normal breast tissue was strongly correlated with chronological age (r = 0.712, p < 0.001). Compared to unaffected peers, breast cancer patients exhibited significant age acceleration in their normal breast tissue (p = 0.002). Multivariate analysis revealed that epigenetic age acceleration in the normal breast tissue of subjects with cancer remained significant after adjusting for clinical and demographic variables. Additionally, smoking was found to be positively correlated with epigenetic aging in normal breast tissue (p = 0.012). CONCLUSIONS: Women with luminal breast cancer exhibit significant epigenetic age acceleration in normal adjacent breast tissue, which is consistent with an analogous finding in malignant breast tissue. Smoking is also associated with epigenetic age acceleration in normal breast tissue. Further studies are needed to determine whether epigenetic age acceleration in normal breast tissue is predictive of incident breast cancer and whether this mediates the risk of chronological age on breast cancer risk.
Assuntos
Neoplasias da Mama/genética , Mama/química , Ilhas de CpG , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adulto , Fatores Etários , Estudos de Casos e Controles , Epigênese Genética , Feminino , Humanos , Pessoa de Meia-Idade , Análise de Sequência de DNA , Bancos de TecidosRESUMO
Statins are potent inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase and display anticancer activity, but their clinical use is limited by their high-dose toxicity. Similarly, gamma-tocotrienol, an isoform of vitamin E, also reduces HMGCoA reductase activity and displays potent anticancer activity. Studies were conducted to determine if combined low dose treatment of gamma-tocotrienol with individual statins resulted in a synergistic antiproliferative effect on neoplastic mouse +SA mammary epithelial cells. Treatment with 3-4 microM gamma-tocotrienol or 2-8 microM simvastatin, lovastatin or mevastatin alone resulted in a significant decrease, whereas treatment with 10-100 microM pravastatin had no effect on +SA cell growth. However, combined treatment of subeffective doses (0.25 or 10 microM) of individual statins with 0.25-2.0 microM gamma-tocotrienol resulted in a dose-responsive synergistic inhibition in +SA cell proliferation. Additional studies showed that treatment with subeffective doses of individual statins or gamma-tocotrienol alone had no effect, whereas combined treatment of these compounds resulted in a relatively large decrease in intracellular levels of phosphorylated (activated) MAPK, JNK, p38, and Akt. These findings strongly suggest that combined low dose treatment of gamma-tocotrienol with individual statins may have potential value in the treatment of breast cancer without causing myotoxicity that is associated with high dose statin treatment.
Assuntos
Proliferação de Células/efeitos dos fármacos , Cromanos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Vitamina E/análogos & derivados , Animais , Western Blotting , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Lovastatina/análogos & derivados , Lovastatina/farmacologia , MAP Quinase Quinase 4/metabolismo , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Pravastatina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sinvastatina/farmacologia , Vitamina E/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Triple-negative breast cancer (TNBC) remains an aggressive disease without effective targeted therapies. In this study, we addressed this challenge by testing 128 FDA-approved or investigational drugs as either single agents or in 768 pairwise drug combinations in TNBC cell lines to identify synergistic combinations tractable to clinical translation. Medium-throughput results were scrutinized and extensively analyzed for sensitivity patterns, synergy, anticancer activity, and were validated in low-throughput experiments. Principal component analysis revealed that a fraction of all upregulated or downregulated genes of a particular targeted pathway could partly explain cell sensitivity toward agents targeting that pathway. Combination therapies deemed immediately tractable to translation included ABT-263/crizotinib, ABT-263/paclitaxel, paclitaxel/JQ1, ABT-263/XL-184, and paclitaxel/nutlin-3, all of which exhibited synergistic antiproliferative and apoptotic activity in multiple TNBC backgrounds. Mechanistic investigations of the ABT-263/crizotinib combination offering a potentially rapid path to clinic demonstrated RTK blockade, inhibition of mitogenic signaling, and proapoptotic signal induction in basal and mesenchymal stem-like TNBC. Our findings provide preclinical proof of concept for several combination treatments of TNBC, which offer near-term prospects for clinical translation. Cancer Res; 77(2); 566-78. ©2016 AACR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias de Mama Triplo Negativas , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Imunoprecipitação , Análise de Componente PrincipalRESUMO
Purpose: The 3-biomarker homologous recombination deficiency (HRD) assay measures the number of telomeric allelic imbalances, loss of heterozygosity, and large-scale state transitions in tumor DNA and combines these metrics into a single score that reflects DNA repair deficiency. The goal of this study is to assess the consistency of these HRD measures in different biopsies from distinct areas of the same cancer.Experimental Design: HRD scores, BRCA mutation status, and BRCA1 promoter methylation were assessed in 99 samples from 33 surgically resected, stage I-III breast cancers; each cancer was biopsied in three distinct areas. Homologous recombination repair (HR) deficiency was defined as either high HRD score (≥42) or tumor BRCA mutation.Results: Eighty-one biopsies from 32 cancers were analyzed. Tumor BRCA status was available for all samples, HRD scores for 70, and BRCA1 methylation values for 76 samples. The BRCA1/2 mutation and promoter methylation status and HR category showed perfect concordance across all biopsies from the same cancer. All tumors with BRCA1/2 mutations or promoter methylation had high HRD scores, as did 17% (4/24) of the BRCA1/2 wild-type and nonmethylated tumors. The HRD scores were also highly consistent between different biopsies from the same tumor with an intraclass correlation coefficient of 0.977, indicating that only 2.3% of the variance is attributed to within-tumor biopsy-to-biopsy variation.Conclusions: These results indicate that within-tumor spatial heterogeneity for HRD metrics and the technical noise in the assay are small and do not influence HRD scores and HR status. Clin Cancer Res; 23(5); 1193-9. ©2016 AACR.