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1.
J Clin Invest ; 128(4): 1355-1370, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29480816

RESUMO

Metastatic breast cancers are still incurable. Characterizing the evolutionary landscape of these cancers, including the role of metastatic axillary lymph nodes (ALNs) in seeding distant organ metastasis, can provide a rational basis for effective treatments. Here, we have described the genomic analyses of the primary tumors and metastatic lesions from 99 samples obtained from 20 patients with breast cancer. Our evolutionary analyses revealed diverse spreading and seeding patterns that govern tumor progression. Although linear evolution to successive metastatic sites was common, parallel evolution from the primary tumor to multiple distant sites was also evident. Metastatic spreading was frequently coupled with polyclonal seeding, in which multiple metastatic subclones originated from the primary tumor and/or other distant metastases. Synchronous ALN metastasis, a well-established prognosticator of breast cancer, was not involved in seeding the distant metastasis, suggesting a hematogenous route for cancer dissemination. Clonal evolution coincided frequently with emerging driver alterations and evolving mutational processes, notably an increase in apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like-associated (APOBEC-associated) mutagenesis. Our data provide genomic evidence for a role of ALN metastasis in seeding distant organ metastasis and elucidate the evolving mutational landscape during cancer progression.


Assuntos
Neoplasias da Mama/genética , Evolução Molecular , Mutação , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Feminino , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Metástase Neoplásica
2.
BMC Cancer ; 17(1): 802, 2017 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-29187174

RESUMO

BACKGROUND: Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. METHODS: In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. RESULTS: Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. CONCLUSIONS: Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.


Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica/métodos , Heterogeneidade Genética , Biomarcadores Tumorais/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Análise de Sequência de RNA
3.
J Natl Cancer Inst ; 109(3): 1-14, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28376210

RESUMO

Background: Breast cancer cells with tumor-initiating capabilities (BSCs) are considered to maintain tumor growth and govern metastasis. Hence, targeting BSCs will be crucial to achieve successful treatment of breast cancer. Methods: We characterized mammospheres derived from more than 40 cancer patients and two breast cancer cell lines for the expression of estrogen receptors (ERs) and stem cell markers. Mammosphere formation and proliferation assays were performed on cells from 19 cancer patients and five healthy individuals after incubation with ER-subtype selective ligands. Transcriptional analysis was performed to identify pathways activated in ERß-stimulated mammospheres and verified using in vitro experiments. Xenograft models (n = 4 or 5 per group) were used to study the role of ERs during tumorigenesis. Results: We identified an absence of ERα but upregulation of ERß in BSCs associated with phenotypic stem cell markers and responsible for the proliferative role of estrogens. Knockdown of ERß caused a reduction of mammosphere formation in cell lines and in patient-derived cancer cells (40.7%, 26.8%, and 39.1%, respectively). Gene set enrichment analysis identified glycolysis-related pathways (false discovery rate < 0.001) upregulated in ERß-activated mammospheres. We observed that tamoxifen or fulvestrant alone was insufficient to block proliferation of patient-derived BSCs while this could be accomplished by a selective inhibitor of ERß (PHTPP; 53.7% in luminal and 45.5% in triple-negative breast cancers). Furthermore, PHTPP reduced tumor initiation in two patient-derived xenografts (75.9% and 59.1% reduction in tumor volume, respectively) and potentiated tamoxifen-mediated inhibition of tumor growth in MCF7 xenografts. Conclusion: We identify ERß as a mediator of estrogen action in BSCs and a novel target for endocrine therapy.


Assuntos
Carcinoma/genética , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Esferoides Celulares/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Proliferação de Células/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Fulvestranto , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Esferoides Celulares/efeitos dos fármacos , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral , Regulação para Cima
4.
Cancer Lett ; 367(1): 76-87, 2015 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-26208432

RESUMO

Breast cancer cells with stem cell characteristics (CSC) are a distinct cell population with phenotypic similarities to mammary stem cells. CSCs are important drivers of tumorigenesis and the metastatic process. Tamoxifen is the most widely used hormonal therapy for estrogen receptor (ER) positive cancers. In our study, tamoxifen was effective in reducing proliferation of ER + adherent cancer cells, but not their CSC population. We isolated, expanded and incubated CSC from seven breast cancers with or without tamoxifen. By genome-wide transcriptional analysis we identified tamoxifen-induced transcriptional pathways associated with ribosomal biogenesis and mRNA translation, both regulated by the mTOR-pathway. We observed induction of the key mTOR downstream targets S6K1, S6RP and 4E-BP1 in-patient derived CSCs by tamoxifen on protein level. Using the mTOR inhibitors rapamycin, everolimus and PF-04691502 (a dual PI3K/mTOR inhibitor) and in combination with tamoxifen, significant reduction in mammosphere formation was observed. Hence, we suggest that the CSC population play a significant role during endocrine resistance through activity of the mTOR pathway. In addition, tamoxifen further stimulates the mTOR-pathway but can be antagonized using mTOR-inhibitors.


Assuntos
Antineoplásicos Hormonais/toxicidade , Neoplasias da Mama/enzimologia , Antagonistas de Estrogênios/toxicidade , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tamoxifeno/toxicidade , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Separação Celular/métodos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática , Everolimo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Piridonas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Esferoides Celulares , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica , Ativação Transcricional , Células Tumorais Cultivadas
5.
Lab Invest ; 94(7): 796-805, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24776644

RESUMO

Breast cancer is a unique tumor disease in terms of the stringent requirement of predictive biomarker assessments. As recommended by current international guidelines, the established markers consist of estrogen receptor (ER), progesterone receptor, human epidermal growth factor and Ki67, and are primarily analyzed by immunohistochemistry. However, new diagnostic methods based on microarray or next-generation sequencing on DNA and mRNA level are gaining ground. These analyses require fresh-frozen tumor tissue that is generally not available from tumors <10 mm in diameter, comprising almost 25% of all resected breast cancer at our department. We here present a simple and standardized method to generate material from small tumors without risking the histopathological examination. Furthermore, we show that the quality of this material is sufficient for subsequent analysis on mRNA, DNA, and epigenetic level. We were also able to use this method for isolation and expansion of cancer stem cells from the majority of tumors. Consequently, researches can be provided with clinically relevant material for translational studies. In conclusion, this method opens up a new possibility for usage of valuable fresh tumor material for research purposes, biobanking, and next-generation sequencing.


Assuntos
Bancos de Espécimes Biológicos/normas , Pesquisa Biomédica/métodos , Neoplasias da Mama/patologia , Manejo de Espécimes/métodos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Metilação de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Feminino , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Patologia Molecular/métodos , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Reprodutibilidade dos Testes , Análise de Sequência de DNA
6.
PLoS One ; 5(12): e15064, 2010 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-21179214

RESUMO

BACKGROUND: Recognizing specific protein changes in response to drug administration in humans has the potential for the development of personalized medicine. Such changes can be identified by pharmacoproteomics approach based on proteomic technologies. It can also be helpful in matching a particular target-based therapy to a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism. Warfarin is a commonly prescribed oral anticoagulant in patients with prosthetic valve disease, venous thromboembolism and stroke. METHODS AND FINDING: We used a combined pharmacogenetics and iTRAQ-coupled LC-MS/MS pharmacoproteomics approach to analyze plasma protein profiles of 53 patients, and identified significantly upregulated level of transthyretin precursor in patients receiving low dose of warfarin but not in those on high dose of warfarin. In addition, real-time RT-PCR, western blotting, human IL-6 ELISA assay were done for the results validation. CONCLUSION: This combined pharmacogenomics and pharmacoproteomics approach may be applied for other target-based therapies, in matching a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism.


Assuntos
Anticoagulantes/farmacologia , Biomarcadores/metabolismo , Oxigenases de Função Mista/genética , Farmacogenética/métodos , Pré-Albumina/biossíntese , Proteômica/métodos , Varfarina/farmacologia , Linhagem Celular Tumoral , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Interleucina-6/metabolismo , Fígado/metabolismo , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem/métodos , Vitamina K Epóxido Redutases
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