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1.
Nat Commun ; 10(1): 3840, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477698

RESUMO

Resistant tumours are thought to arise from the action of Darwinian selection on genetically heterogenous cancer cell populations. However, simple clonal selection is inadequate to describe the late relapses often characterising luminal breast cancers treated with endocrine therapy (ET), suggesting a more complex interplay between genetic and non-genetic factors. Here, we dissect the contributions of clonal genetic diversity and transcriptional plasticity during the early and late phases of ET at single-cell resolution. Using single-cell RNA-sequencing and imaging we disentangle the transcriptional variability of plastic cells and define a rare subpopulation of pre-adapted (PA) cells which undergoes further transcriptomic reprogramming and copy number changes to acquire full resistance. We find evidence for sub-clonal expression of a PA signature in primary tumours and for dominant expression in clustered circulating tumour cells. We propose a multi-step model for ET resistance development and advocate the use of stage-specific biomarkers.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Antineoplásicos Hormonais/uso terapêutico , Mama/citologia , Mama/patologia , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Microscopia Intravital , Células MCF-7 , Aprendizado de Máquina , Mutação , Células Neoplásicas Circulantes/efeitos dos fármacos , Análise de Célula Única , Esferoides Celulares
2.
Nat Commun ; 10(1): 3589, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399573

RESUMO

Overcoming cellular growth restriction, including the evasion of cellular senescence, is a hallmark of cancer. We report that PAK4 is overexpressed in all human breast cancer subtypes and associated with poor patient outcome. In mice, MMTV-PAK4 overexpression promotes spontaneous mammary cancer, while PAK4 gene depletion delays MMTV-PyMT driven tumors. Importantly, PAK4 prevents senescence-like growth arrest in breast cancer cells in vitro, in vivo and ex vivo, but is not needed in non-immortalized cells, while PAK4 overexpression in untransformed human mammary epithelial cells abrogates H-RAS-V12-induced senescence. Mechanistically, a PAK4 - RELB - C/EBPß axis controls the senescence-like growth arrest and a PAK4 phosphorylation residue (RELB-Ser151) is critical for RELB-DNA interaction, transcriptional activity and expression of the senescence regulator C/EBPß. These findings establish PAK4 as a promoter of breast cancer that can overcome oncogene-induced senescence and reveal a selective vulnerability of cancer to PAK4 inhibition.


Assuntos
Neoplasias da Mama/patologia , Fator de Transcrição RelB/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Mama/citologia , Mama/patologia , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Senescência Celular/genética , Células Epiteliais , Feminino , Técnicas de Silenciamento de Genes , Humanos , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Cultura Primária de Células , Prognóstico , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/genética
3.
Adv Mater ; 31(40): e1902279, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31414515

RESUMO

Surfactant-stripped micelles are formed from a commercially available cyanine fluoroalkylphosphate (CyFaP) salt dye and used for high contrast photoacoustic imaging (PAI) in the second near-infrared window (NIR-II). The co-loading of Coenzyme Q10 into surfactant-stripped CyFaP (ss-CyFaP) micelles improves yield, storage stability, and results in a peak absorption wavelength in the NIR-II window close to the 1064 nm output of Nd-YAG lasers used for PAI. Aqueous ss-CyFaP dispersions exhibit intense NIR-II optical absorption, calculated to be greater than 500 at 1064 nm. ss-CyFaP is detected through 12 cm of chicken breast tissue with PAI. In preclinical animal models, ss-CyFaP is visualized in draining lymph nodes of rats through 3.1 cm of overlaid chicken breast tissue. Following intravenous administration, ss-CyFaP accumulates in neoplastic tissues of mice and rats bearing orthotopic mammary tumors without observation of acute toxic side effects. ss-CyFaP is imaged through whole compressed human breasts in three female volunteers at depths of 2.6-5.1 cm. Taken together, these data show that ss-CyFaP is an accessible contrast agent for deep tissue PAI in the NIR-II window.


Assuntos
Mama/citologia , Mama/diagnóstico por imagem , Raios Infravermelhos , Micelas , Imagem Óptica/métodos , Técnicas Fotoacústicas/métodos , Tensoativos/química , Absorção Fisico-Química , Animais , Humanos , Camundongos , Fosfatos/química , Ratos , Ubiquinona/análogos & derivados , Ubiquinona/química
4.
Med Phys ; 46(10): 4405-4416, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31274194

RESUMO

PURPOSE: Segmentation of the chest wall, is an important component of methods for automated analysis of breast magnetic resonance imaging (MRI). Methods reported to date show promising results but have difficulties delineating the muscle border correctly in breasts with a large proportion of fibroglandular tissue (i.e., dense breasts). Knowledge-based methods (KBMs) as well as methods based on deep learning have been proposed, but a systematic comparison of these approaches within one cohort of images is currently lacking. Therefore, we developed a KBM and a deep learning method for segmentation of the chest wall in MRI of dense breasts and compared their performances. METHODS: Two automated methods were developed, an optimized KBM incorporating heuristics aimed at shape, location, and gradient features, and a deep learning-based method (DLM) using a dilated convolution neural network. A data set of 115 T1-weighted MR images was randomly selected from MR images of women with extremely dense breasts (ACR BI-RADS category 4) participating in a screening trial of women (mean age 56.6 yr, range 49.5-75.2 yr) with dense breasts. Manual segmentations of the chest wall, acquired under supervision of an experienced breast radiologist, were available for all data sets. Both methods were optimized using the same randomly selected 36 MRI data sets from a total of 115 data sets. Each MR data set consisted of 179 transversal images with voxel size 0.64 mm3  × 0.64 mm3  × 1.00 mm3 . In the remaining 79 data sets, the results of both segmentation methods were qualitatively evaluated. A radiologist reviewed the segmentation results of both methods in all transversal images (n = 14 141) and determined whether the result would impact the ability to accurately determine the volume of fibroglandular and fatty tissue and whether segmentations masked breast regions that might harbor lesions. When no relevant deviation was detected, the result was considered successful. In addition, all segmentations were quantitatively assessed using the Dice similarity coefficient (DSC) and Hausdorff distance (HD), 95th percentile of the Hausdorff distance (HD95), false positive fraction (FPF), and false negative fraction (FNF) metrics. RESULTS: According to the radiologist's evaluation, the DLM had a significantly higher success rate than the KBM (81.6% vs 78.4%, P < 0.01). The success rate was further improved to 92.1% by combining both methods. Similarly, the DLM had significantly lower values for FNF (0.003 ± 0.003 vs 0.009 ± 0.011, P < 0.01) and HD95 (2.58 ± 1.78 mm vs 3.37 ± 2.11, P < 0.01). However, the KBM resulted in a significantly lower FPF than the DLM (0.018 ± 0.009 vs 0.030 ± 0.009, P < 0.01).There was no significant difference between the KBM and DLM in terms of DSC (0.982 ± 0.006 vs 0.984 ± 0.008, P = 0.08) or HD (24.14 ± 20.69 mm vs 12.81 ± 27.28 mm, P = 0.05). CONCLUSION: Both optimized knowledge-based and DLM showed good results to segment the pectoral muscle in women with dense breasts. Qualitatively assessed, the DLM was the most robust method. A quantitative comparison, however, did not indicate a preference for one method over the other.


Assuntos
Densidade da Mama , Mama/citologia , Mama/diagnóstico por imagem , Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Imagem por Ressonância Magnética , Parede Torácica/diagnóstico por imagem , Idoso , Idoso de 80 Anos ou mais , Automação , Feminino , Humanos , Pessoa de Meia-Idade
5.
Int J Med Sci ; 16(6): 893-901, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31337963

RESUMO

Background: The application of adipose tissue-derived stromal cells (ASCs) in regenerative medicine has become a growing trend due to its abundance and differentiation potentials. However, several breast cancer studies indicated that ASCs promote tumor progression, therefore, the use of ASCs for reconstruction after oncological surgery poses potential risks. In this study, we aimed to examine whether cancerous or non-cancerous breast cells will exhibit different responses to ASC-derived CM. Methods: ASCs were isolated from residuals of subcutaneous adipose tissue obtained from patients undergoing surgery. Cancerous MCF-7, MDA-MB231, and MDA-MB468 cell lines and one non-cancerous M10/H184B5F5 cell line were cultured with variant concentrations of ASC-derived conditioned medium (CM) for analysis. Results: ASC-derived CM significantly reduced cell viability by triggering apoptosis in MCF-7, MDA-MB231, and MDA-MB468 cell lines. ATM-Chk2-dependent DNA damage response was activated early in cancer cells when exposed to ASC-derived CM. By contrast, prompted cell proliferation instead of cell death was detected in M10/H184B5F5 cells under the treatment of lower CM concentration. Even when exposed to the highest concentration of CM, only cell cycle arrest accompanied by a weak DNA damage response were detected in M10/H184B5F5 cells, no cell deaths were observed. Conclusions: Overall, this study demonstrated that cancerous and non-cancerous breast cells respond differently to ASC-derived CM. ASC-derived CM triggered significant cell death in breast cancer cell lines, however non-cancerous breast cells exhibited dissimilar response to ASC-derived CM.


Assuntos
Tecido Adiposo/citologia , Neoplasias da Mama/patologia , Meios de Cultivo Condicionados/farmacologia , Medicina Regenerativa/métodos , Tecido Adiposo/transplante , Apoptose/efeitos dos fármacos , Mama/citologia , Mama/patologia , Mama/cirurgia , Neoplasias da Mama/cirurgia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Células Epiteliais , Feminino , Humanos , Mamoplastia/métodos , Mastectomia/efeitos adversos , Cultura Primária de Células , Células-Tronco/metabolismo , Células Estromais/fisiologia , Células Estromais/transplante , Transplante Autólogo/métodos
6.
Life Sci ; 232: 116610, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31254584

RESUMO

AIMS: The aim of this study was the characterization of the in vitro cytotoxic properties of a recently isolated diterpene compound, 7ß-acetoxy-20-hydroxy-19,20-epoxyroyleanone (compound 1), extracted from Salvia corrugata, versus human cell lines. MAIN METHODS: We used as model study immortalized breast epithelial cells MCF10A and two ERBB2+ breast cancer (BCa) cell lines, SKBR-3 and BT474. Compound 1 was isolated by methanolic extraction from regenerated shoots of Salvia corrugata Vahl, and purified by high pressure liquid chromatography (HPLC). Flow cytometry (FCM) was employed for cell cycle, apoptosis and reactive oxygen species (ROS) analysis. Cell morphology was assessed by immunofluorescence and transmission electron microscopy (TEM). KEY FINDINGS: Compound 1 inhibited cell survival of all breast cell lines. In particular, compound 1 promoted cell cycle arrest in the G0/G1 phase and apoptosis along with impairment of the mitochondrial function, which was reflected in a gross alteration of the mitochondrial network structure. Furthermore, we also detected a potent activation of the ERK1/2 kinase, which suggested the induction of reactive oxygen species (ROS). Partial rescue of survival obtained with n-acetylcysteine (NAC) when coadminstered with compound 1 further supported a contribution of ROS mediated mechanisms to the growth-arrest and proapoptotic activity of compound 1 in both BCa cell lines. ROS production was indeed confirmed in SKBR-3. SIGNIFICANCE: Our findings show that compound 1 has a cytotoxic activity against both human normal and cancer cell lines derived from breast epithelia, which is mediated by ROS generation and mitochondrial damage.


Assuntos
Mama/efeitos dos fármacos , Diterpenos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Células Epiteliais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Mama/citologia , Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Diterpenos/isolamento & purificação , Células Epiteliais/metabolismo , Feminino , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos
7.
BMC Cancer ; 19(1): 565, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185953

RESUMO

BACKGROUND: Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. METHODS: We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. RESULTS: In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. CONCLUSIONS: Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.


Assuntos
Inativação Gênica , Invasividade Neoplásica/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , /genética , Biomarcadores Tumorais/genética , Mama/citologia , Neoplasias da Mama/patologia , Movimento Celular , Ciclina D1/metabolismo , Éxons/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/citologia , Neoplasias Pulmonares/patologia , Células MCF-7 , Fenótipo , RNA Interferente Pequeno/genética , Transfecção
8.
BMC Cancer ; 19(1): 561, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185986

RESUMO

BACKGROUND: Over-expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including induction of stem-like cells (SLC). Combined gene expression and microRNA microarray analyses of empty vector vs COX-2- transfected COX-2 low MCF7 breast cancer cell line identified two COX-2-upregulated microRNAs, miR-526b and miR-655, both found to be oncogenic and SLC-promoting. Cytoplasmic Polyadenylation Element-Binding Protein 2 (CPEB2) was the single common target of both microRNAs, the functions of which remain controversial. CPEB2 has multiple isoforms (A-F), and paradoxically, a high B/A ratio was reported to impart anoikis-resistance and metastatic phenotype in triple- negative breast cancer cells. We tested whether CPEB2 is a tumor suppressor in mammary epithelial cells. METHODS: We knocked-out CPEB2 in the non-tumorigenic mammary epithelial cell line MCF10A by CRISPR/Cas9-double nickase approach, and knocked-down CPEB2 with siRNAs in the poorly malignant MCF7 cell line, both lines being high CPEB2-expressing. The resultant phenotypes for oncogenity were tested in vitro for both lines and in vivo for CPEB2KO cells. Finally, CPEB2 expression was compared between human breast cancer and non-tumor breast tissues. RESULTS: CPEB2 (isoform A) expression was inversely correlated with COX-2 or the above microRNAs in COX-2-divergent breast cancer cell lines. CPEB2KO MCF10A cells exhibited oncogenic properties including increased proliferation, migration, invasion, EMT (decreased E-Cadherin, increased Vimentin, N-Cadherin, SNAI1, and ZEB1) and SLC phenotype (increased tumorsphere formation and SLC marker-expression). Tumor-suppressor p53 protein was shown to be a novel translationally-regulated target of CPEB2, validated with polysome profiling. CPEB2KO, but not wild-type cells produced lung colonies upon intravenous injection and subcutaneous tumors and spontaneous lung metastases upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice, identified with HLA immunostaining. Similarly, siRNA-mediated CPEB2 knockdown in MCF7 cells promoted oncogenic properties in vitro. Human breast cancer tissues (n = 105) revealed a lower mRNA expression for CPEB2 isoform A and also a lower A/B isoform ratio than in non-tumour breast tissues (n = 20), suggesting that CPEB2A accounts for the tumor-suppressor functions of CPEB2. CONCLUSIONS: CPEB2, presumably the isoform A, plays a key role in suppressing tumorigenesis in mammary epithelial cells by repressing EMT, migration, invasion, proliferation and SLC phenotype, via multiple targets, including a newly-identified translational target p53.


Assuntos
Neoplasias da Mama/patologia , Mama/citologia , Proteínas de Ligação a RNA/metabolismo , Animais , Sistemas CRISPR-Cas , Movimento Celular , Proliferação de Células , Ciclo-Oxigenase 2/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/metabolismo , Isoformas de Proteínas , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/metabolismo
9.
Int J Mol Sci ; 20(11)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181796

RESUMO

Different phenotypes of normal cells might influence genetic profiles, epigenetic profiles, and tumorigenicities of their transformed derivatives. In this study, we investigate whether the whole mitochondrial genome of immortalized cells can be attributed to the different phenotypes (stem vs. non-stem) of their normal epithelial cell originators. To accurately determine mutations, we employed Duplex Sequencing, which exhibits the lowest error rates among currently-available DNA sequencing methods. Our results indicate that the vast majority of the observed mutations of the whole mitochondrial DNA occur at low-frequency (rare mutations). The most prevalent rare mutation types are C→T/G→A and A→G/T→C transitions. Frequencies and spectra of homoplasmic point mutations are virtually identical between stem cell-derived immortalized (SV1) cells and non-stem cell-derived immortalized (SV22) cells, verifying that both cell types were derived from the same woman. However, frequencies of rare point mutations are significantly lower in SV1 cells (5.79 × 10-5) than in SV22 cells (1.16 × 10-4). The significantly lower frequencies of rare mutations are aligned with a finding of longer average distances to adjacent mutations in SV1 cells than in SV22 cells. Additionally, the predicted pathogenicity for rare mutations in the mitochondrial tRNA genes tends to be lower (by 2.5-fold) in SV1 cells than in SV22 cells. While four known/confirmed pathogenic mt-tRNA mutations (m.5650 G>A, m.5521 G>A, m.5690 A>G, m.1630 A>G) were identified in SV22 cells, no such mutations were observed in SV1 cells. Our findings suggest that the immortalization of normal cells with stem cell features leads to decreased mitochondrial mutagenesis, particularly in RNA gene regions. The mutation spectra and mutations specific to stem cell-derived immortalized cells (vs. non-stem cell derived) have implications in characterizing the heterogeneity of tumors and understanding the role of mitochondrial mutations in the immortalization and transformation of human cells.


Assuntos
Neoplasias da Mama/genética , Células Epiteliais/metabolismo , Genoma Mitocondrial , Taxa de Mutação , Células-Tronco Adultas/metabolismo , Mama/citologia , Linhagem Celular Tumoral , Feminino , Humanos , Mutação Puntual , RNA de Transferência/genética
10.
Breast ; 46: 101-107, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31132475

RESUMO

BACKGROUND: To evaluate whether uPA/PAI-1 protein in hormone receptor-positive (HR+) breast tumor can predict prognosis in early breast cancer (BC). METHODS: 606 women with HR + BC who had ≥5 years of endocrine therapy and in whom tumor tissue was available were included in this analysis. Stromal uPA/PAI-1 protein expression was evaluated by immunohistochemistry and correlated with distant recurrence-free survival (DRFS) and overall survival (OS). RESULTS: Stromal uPA was detected in 292/538 tumors (54.3%) while 269/505 samples (53.3%) exhibited stromal PAI-1. Co-expression of both proteins was found in 163/437 (37.3%) samples. Stromal uPA/PAI-1 co-expression was not associated with tumor size, age, nodal status, grading, or receptor status. Tumor stroma with both uPA and PAI-1 protein expression were more likely to have a shorter DRFS (HR: 1.87; 95%CI 1.18-2.96; p = 0.007) and OS (HR: 1.29; 95%CI 0.93-1.80; p = 0.129) than women without uPA/PAI-1 co-expression. After a median follow-up of 10 years, women with uPA/PAI-1-positive tumors experienced a significantly shorter DRFS (86.5% vs 72.4%; p < 0.001) and OS (70.4% vs 58.9%; p = 0.020) compared to women with uPA/PAI-1 negative tumors. CONCLUSION: Stromal co-expression of uPA and PAI-1 in breast cancer predicts poor DRFS and OS in postmenopausal women with HR + early-stage BC who receive endocrine therapy.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Mama/citologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Idoso , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Intervalo Livre de Doença , Receptores ErbB/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Pós-Menopausa , Prognóstico , Fatores de Risco , Células Estromais/metabolismo , Resultado do Tratamento
11.
Zhonghua Zhong Liu Za Zhi ; 41(5): 331-337, 2019 May 23.
Artigo em Chinês | MEDLINE | ID: mdl-31137165

RESUMO

Objective: To investigate the differential expression profiles of circular RNA (circRNA) in human epidermal growth factor receptor 2 (HER-2) positive breast cancer cells and normal mammary epithelial cells, and to develop novel diagnostic and therapeutic markers for HER-2 positive breast cancer. Methods: Total RNA were extracted from HER-2 positive breast cancer cell SK-BR-3 and normal mammary epithelial cell MCF10A. RNA quality was detected using NanoDrop ND-1000. Rnase R was applied to remove linear RNA and enrich circRNAs. After amplification and reverse transcription into fluorescent complementary RNA (cRNA) using random primer, the labeled cRNAs were hybridized onto the Arraystar Human circRNA Arrays. The raw data were extracted and the acquired array images were subjected to quantile normalization followed by heat map and volcano plot analysis. The expression of circRNAs with large fold change was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed in the differentially expressed circRNAs and circRNA-microRNA (miRNA) network was constructed. Results: The total RNA extracted from SK-BR-3 and MCF10A had high integrity and quality. The expression profiles of circRNA in SK-BR-3 and MCF10A cells were significantly different shown by fluorescent expression signals. Compared with MCF10A cells, there were 6 584 up-regulated circRNAs and 6254 down-regulated circRNAs in SK-BR-3 cells. There were 348 circRNAs with |log(2)FC|≥2, of which 153 were up-regulated and 195 were down-regulated. Moreover, 8 circRNAs with |log(2)FC|>5. Among them, 5 were up-regulated in SK-BR-3 cells, including hsa_circRNA_074595 (|log(2)FC|=7.84), hsa_circRNA_074598 (|log(2)FC|=6.50), hsa_circRNA_085362 (|log(2)FC|=5.86), hsa_circRNA_101379 (|log(2)FC|=5.71) and hsa_circRNA_406683 (|log(2)FC|=5.34); as well as 3 were down-regulated, including hsa_circRNA_021714 (|log(2)FC|=5.46), hsa_circRNA_100777 (|log(2)FC|=5.40), and hsa_circRNA_100796 (|log(2)FC|=5.03). The expression levels of hsa_circRNA_074595, hsa_circRNA_074598 and hsa_circRNA_100777 were further validated by RT-qPCR in consistent with the results of microarray. GO analysis showed that differentially expressed circRNAs were significantly enriched in the biological process of heart development (P<0.001), cellular component in the cell adhesion-related components (P<0.001), molecular function in protein serine/threonine kinase activity (P<0.001). KEGG analysis revealed that differentially expressed circRNAs were significantly enriched in the PI3K-Akt signaling pathway. Conclusions: The expression profile of circRNA in HER-2 positive breast cancer cells is significantly different from that in normal mammary epithelial cells. The differentially expressed circRNAs may be served as potential diagnostic or therapeutic targets for HER-2 positive breast cancer.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Mama/metabolismo , Células Epiteliais/metabolismo , RNA não Traduzido/biossíntese , Receptor ErbB-2/metabolismo , Mama/citologia , Células Epiteliais/citologia , Feminino , Perfilação da Expressão Gênica , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , RNA não Traduzido/genética , Receptor ErbB-2/genética
12.
Toxicol In Vitro ; 59: 55-63, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30974152

RESUMO

The human breast epithelial cell lines MCF-10A and MCF-12A form well-differentiated acinus-like structures when grown in three-dimensional matrigel culture over a period of 20 days. In the present study, both cell lines were tested for their suitability to serve as an effect-based in vitro test system for non-genotoxic carcinogens. A software solution for automated Acinus Detection And Morphological Evaluation (ADAME) was developed to automatically acquire acinus images and to determine morphological parameters such as acinus size, lumen size, and acinus roundness. A number of test compounds were tested for their capacity to affect acinus formation and cellular differentiation. Human epidermal growth factor stimulated acinus growth for both cell lines whereas all-trans retinoic acid inhibited acinus growth. The strong estrogen 17ß-estradiol had no effect on acinus formation of estrogen receptor (ER)-negative MCF-10A cells, but yielded larger MCF-12A (ER-positive) acini. Thus, the parallel use of both cell lines allows the identification of estrogenic properties of a given test compound.


Assuntos
Mama/citologia , Carcinógenos/farmacologia , Técnicas de Cultura de Células , Células Epiteliais/efeitos dos fármacos , Estrogênios/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Estradiol/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Receptores Estrogênicos , Software , Testes de Toxicidade/métodos , Tretinoína/farmacologia
13.
ACS Appl Mater Interfaces ; 11(18): 16347-16356, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31032616

RESUMO

We report new surface coatings that adhesively distinguish three breast epithelial cell lines (MCF-10A, MCF-7, and TMX2-28) when cell suspensions in buffer or breast milk are flowed over the coatings. We also report the selective capture of epithelial cells and rejection of Jurkat lymphocytes, with average selectivities exceeding 60 and captured cell purities often exceeding >99%. The surfaces achieve the dual goals of selective cell capture and resistance to fouling by proteins and other components of breast milk. The coatings do not rely on antibody targeting of cell surface markers but instead contain polycation chains embedded within a layer of end-tethered poly(ethylene glycol) (PEG) chains. The PEG, somewhat shielding the polycations, prevents surface fouling by proteins, nondesired cells, and other milk components, while the polycations produce electrostatic attractions that are heterogeneous on nanoscopic length scales. These electrostatic heterogeneities on the engineered coating, shown to produce curvature-selective particle capture in other studies, produce cell selectivity here. The ability of the engineered surfaces to discriminate these cell lines via an electrostatic driving force is remarkable, as the cells are of very similar surface charge as evidenced by their nearly identical ζ-potentials. The current surfaces, which likely distinguish cells based on their electrostatic surface landscape combined with other factors, adhesively distinguish cell lines that may differ only slightly in their expression of a surface marker, or cancer cells that minimally express EpCAM but which have different distributions of electrostatic charge on their surfaces. These surfaces are among the first to be documented for the compatibility of a polymer brush with human breast milk and may find use in technologies that capture cells from human breast milk or other complex fluids for cancer risk assessment.


Assuntos
Adesivos/farmacologia , Mama/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Leite Humano/química , Adesivos/química , Mama/citologia , Tampões (Química) , Molécula de Adesão da Célula Epitelial/genética , Células Epiteliais/química , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Células MCF-7 , Polietilenoglicóis/química , Polímeros/química , Polímeros/farmacologia , Propriedades de Superfície
14.
Breast Cancer Res ; 21(1): 31, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30813947

RESUMO

INTRODUCTION: In a cancer-free environment in the adult, the skeleton continuously undergoes remodeling. Bone-resorbing osteoclasts excavate erosion cavities, and bone-depositing osteoblasts synthesize osteoid matrix that forms new bone, with no net bone gain or loss. When metastatic breast cancer cells invade the bone, this balance is disrupted. Patients with bone metastatic breast cancer frequently suffer from osteolytic bone lesions that elicit severe bone pain and fractures. Bisphosphonate treatments are not curative. Under ideal circumstances, osteoblasts would synthesize new matrix to fill in erosion cavities caused by osteoclasts, but this is not what occurs. Our prior evidence demonstrated that osteoblasts are diverted from laying down bone matrix to producing cytokines that facilitate breast cancer cell maintenance in late-stage disease. Here, we have new evidence to suggest that there are subpopulations of osteoblasts in the tumor niche as evidenced by their protein marker expression that have distinct roles in tumor progression in the bone. METHODS: Tumor-bearing tibia of mice was interrogated by immunofluorescent staining for the presence of osteoblasts and alterations in niche protein expression. De-identified tissue from patients with bone metastatic breast cancer was analyzed for osteoblast subpopulations via multi-plex immunofluorescent staining. Effects of breast cancer cells on osteoblasts were recapitulated in vitro by osteoblast exposure to breast cancer-conditioned medium. Triple-negative and estrogen receptor-positive breast cancer proliferation, cell cycle, and p21 expression were assessed upon contact with "educated" osteoblasts. RESULTS: A subpopulation of osteoblasts was identified in the bone tumor microenvironment in vivo of both humans and mice with bone metastatic breast cancer that express RUNX2/OCN/OPN but is negative for IL-6 and alpha-smooth muscle actin. These tumor "educated" osteoblasts (EOs) have altered properties compared to "uneducated" osteoblasts and suppress both triple-negative and estrogen receptor-positive breast cancer cell proliferation and increase cancer cell p21 expression. EO effects on breast cancer proliferation were mediated by NOV and decorin. Importantly, the presence of EO cells in the tibia of mice bearing tumors led to increased amounts of alkaline phosphatase and suppressed the expression of inflammatory cytokines in vivo. CONCLUSIONS: Our work reveals that there is a subpopulation of osteoblasts in the bone tumor microenvironment that demonstrate a functional role in retarding breast cancer cell growth.


Assuntos
Neoplasias Ósseas/patologia , Neoplasias da Mama/patologia , Comunicação Celular , Osteoblastos/patologia , Microambiente Tumoral , Animais , Matriz Óssea/citologia , Matriz Óssea/diagnóstico por imagem , Matriz Óssea/patologia , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/secundário , Mama/citologia , Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultivo Condicionados , Feminino , Humanos , Microscopia Intravital , Camundongos , Camundongos Nus , Células NIH 3T3 , Cultura Primária de Células , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Breast Cancer Res ; 21(1): 17, 2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700319

RESUMO

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


Assuntos
Anemia/sangue , Antineoplásicos/efeitos adversos , Neoplasias da Mama/sangue , Eritropoetina/sangue , Células-Tronco Neoplásicas/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Aldeído Oxirredutases/metabolismo , Anemia/tratamento farmacológico , Anemia/etiologia , Animais , Mama/citologia , Mama/patologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Eritropoetina/administração & dosagem , Eritropoetina/metabolismo , Feminino , Hemoglobinas/análise , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Estudos Prospectivos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Breast Cancer Res ; 21(1): 7, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30654824

RESUMO

INTRODUCTION: Clinical studies suggest that obesity, in addition to promoting breast cancer aggressiveness, is associated with a decrease in chemotherapy efficacy, although the mechanisms involved remain elusive. As chemotherapy is one of the main treatments for aggressive or metastatic breast cancer, we investigated whether adipocytes can mediate resistance to doxorubicin (DOX), one of the main drugs used to treat breast cancer, and the mechanisms associated. METHODS: We used a coculture system to grow breast cancer cells with in vitro differentiated adipocytes as well as primary mammary adipocytes isolated from lean and obese patients. Drug cellular accumulation, distribution, and efflux were studied by immunofluorescence, flow cytometry, and analysis of extracellular vesicles. Results were validated by immunohistochemistry in a series of lean and obese patients with cancer. RESULTS: Adipocytes differentiated in vitro promote DOX resistance (with cross-resistance to paclitaxel and 5-fluorouracil) in a large panel of human and murine breast cancer cell lines independently of their subtype. Subcellular distribution of DOX was altered in cocultivated cells with decreased nuclear accumulation of the drug associated with a localized accumulation in cytoplasmic vesicles, which then are expelled into the extracellular medium. The transport-associated major vault protein (MVP), whose expression was upregulated by adipocytes, mediated both processes. Coculture with human mammary adipocytes also induced chemoresistance in breast cancer cells (as well as the related MVP-induced DOX efflux) and their effect was amplified by obesity. Finally, in a series of human breast tumors, we observed a gradient of MVP expression, which was higher at the invasive front, where tumor cells are at close proximity to adipocytes, than in the tumor center, highlighting the clinical relevance of our results. High expression of MVP in these tumor cells is of particular interest since they are more likely to disseminate to give rise to chemoresistant metastases. CONCLUSIONS: Collectively, our study shows that adipocytes induce an MVP-related multidrug-resistant phenotype in breast cancer cells, which could contribute to obesity-related chemoresistance.


Assuntos
Adipócitos/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Obesidade/complicações , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Células 3T3 , Tecido Adiposo/citologia , Adulto , Idoso , Animais , Antineoplásicos/uso terapêutico , Mama/citologia , Mama/patologia , Mama/cirurgia , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Linhagem Celular Tumoral , Técnicas de Cocultura , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Mastectomia , Camundongos , Pessoa de Meia-Idade , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética
17.
Phys Med Biol ; 64(5): 055006, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30650384

RESUMO

Millimeter (mm)-wave imaging has been recently proposed as a new technique for breast cancer detection, based on the significant dielectric contrast between healthy and tumor tissues. Here we propose a procedure to fabricate, electromagnetically characterize and preserve realistic breast tissue-mimicking phantoms for testing mm-wave imaging prototypes. Low-cost, non-toxic and easy-to-produce mixtures made of sunflower oil, water and gelatin were prepared and their dielectric properties were for the first time measured in the (0.5-50) GHz frequency range using a coaxial probe kit. Different oil and gelatin percentages were tested. An alternative recipe based on a waste-oil hardener was also proposed. Finally, water and sunflower oil were investigated as preservation media. The mixtures electromagnetic properties were in good agreement with those of human breast ex vivo samples. By changing the ingredient concentrations or using different solidifying agents it was possible to mimic different tissue types. Besides, we show that sunflower oil represents an effective preservation medium for the developed materials. The first breast phantom mimicking a tumor mass into healthy tissues up to 50 GHz was also successfully fabricated. Results demonstrated the potential of the designed recipes to mimic breast tissues with different biological characteristics, preserving dielectric properties over time. Thus, this study represents a fundamental step towards the development of heterogeneous breast phantoms able to mimic the electromagnetic behavior of healthy and tumor tissues for mm-wave imaging applications.


Assuntos
Mama/citologia , Mama/diagnóstico por imagem , Micro-Ondas , Imagem Molecular/instrumentação , Imagens de Fantasmas , Mama/patologia , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Gelatina , Humanos , Óleo de Girassol , Água
18.
Curr Protein Pept Sci ; 20(7): 696-704, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30678617

RESUMO

Numerous experimental studies have demonstrated that a series of remodeling processes occurred in the adipose tissue during the weaning, such as differentiation. Fibroblasts in the breast at weaning stage could re-differentiate into mature adipocytes. Many transcriptional factors were involved in these processes, especially the PPARγ, C/EBP, and SREBP1. There is cell apoptosis participating in the breast tissue degeneration and secretory epithelial cells loss during weaning. In addition, hormones, especially the estrogen and pituitary hormone, play a vital role in the whole reproductive processes. In this review, we mainly focus on the underlying regulated mechanisms of differentiation of adipose tissue and apoptosis of breast cell to provide a specific insight into the physiological changes during weaning.


Assuntos
Adipócitos/citologia , Apoptose , Mama/citologia , Diferenciação Celular , Desmame , Animais , Mama/metabolismo , Humanos , Fatores de Transcrição/metabolismo
19.
Mol Cancer Res ; 17(4): 937-948, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30651374

RESUMO

Radiation is used in multiple procedures as a therapeutic and diagnostic tool. However, ionizing radiation can induce mutations in the DNA of irradiated cells, which can promote tumorigenesis. As malignant transformation is a process that takes many years, there are intermediate stages of cells that have initiated the process but have not yet evolved into cancer. The study here aimed to investigate the effect of ionizing radiation on normal and partially transformed human mammary epithelial cells. Breast primary epithelial cells were derived from normal breast tissue from two different donors and modified by transduction with the SV40 small and large T antigen and hTERT genes to obtain partially transformed cells and also with HRAS to completely and experimentally transform them. After exposure to different doses of ionizing radiation, oncogenic features were analyzed by means of an anchorage-independent growth assay and 3D cell culture. The addition of radiation exposure resulted in an increase in the number and size of colonies formed in each of the conditions analyzed and in the reduction of the capacity of partially transformed cells to form properly polarized 3D structures. Moreover, partially transformed cells require lower doses of radiation than healthy cells to enhance anchorage-independent growth capacity. Although cells from different donors have a different degree of sensitivity in the response to radiation, a higher sensitivity to the radiation-induced cell transformation process was observed in those cells that had already initiated the oncogenic process, which require higher doses of radiation to complete the transformation process. IMPLICATIONS: Individuals carrying accumulation of genetic alterations may have an increased susceptibility to radiation-induced neoplastic transformation.


Assuntos
Neoplasias da Mama/patologia , Mama/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Lesões Pré-Cancerosas/patologia , Mama/citologia , Mama/patologia , Neoplasias da Mama/etiologia , Células Epiteliais/citologia , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Feminino , Humanos , Neoplasias Induzidas por Radiação/patologia
20.
Oncogene ; 38(17): 3151-3169, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30622340

RESUMO

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Mama/citologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular , Proliferação de Células , Células Epiteliais/citologia , Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Tamoxifeno/farmacologia , Regulação para Cima
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