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Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.
Assuntos
Neoplasias da Mama , Receptores de Estrogênio , Tamoxifeno , Humanos , Linhagem Celular Tumoral , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Animais , Camundongos , Modelos Animais de Doenças , Receptores de Estrogênio/genética , Tamoxifeno/farmacologia , Fenótipo , Imuno-Histoquímica , Citometria de Fluxo , Transcriptoma , Camundongos da Linhagem 129 , RNA-Seq , Células Epiteliais , Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genéticaRESUMO
Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes, due to the high propensity to develop distant metastases. Hence, new molecular targets for therapeutic intervention are needed for TNBC. We recently conducted a rigorous phenotypic and genomic characterization of four isogenic populations of MDA-MB-231 human triple-negative breast cancer cells that possess a range of intrinsic spontaneous metastatic capacities in vivo, ranging from nonmetastatic (MDA-MB-231_ATCC) to highly metastatic to lung, liver, spleen and spine (MDA-MB-231_HM). Gene expression profiling of primary tumours by RNA-Seq identified the fibroblast growth factor homologous factor, FGF13, as highly upregulated in aggressively metastatic MDA-MB-231_HM tumours. Clinically, higher FGF13 mRNA expression was associated with significantly worse relapse free survival in both luminal A and basal-like human breast cancers but was not associated with other clinical variables and was not upregulated in primary tumours relative to normal mammary gland. Stable FGF13 depletion restricted in vitro colony forming ability in MDA-MB-231_HM TNBC cells but not in oestrogen receptor (ER)-positive MCF-7 or MDA-MB-361 cells. However, despite augmenting MDA-MB-231_HM cell migration and invasion in vitro, FGF13 suppression almost completely blocked the spontaneous metastasis of MDA-MB-231_HM orthotopic xenografts to both lung and liver while having negligible impact on primary tumour growth. Together, these data indicate that FGF13 may represent a therapeutic target for blocking metastatic outgrowth of certain TNBCs. Further evaluation of the roles of individual FGF13 protein isoforms in progression of the different subtypes of breast cancer is warranted.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/genética , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Células-Tronco Neoplásicas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcriptoma , Neoplasias de Mama Triplo Negativas/genética , Regulação para CimaRESUMO
PURPOSE: We aimed to generate and characterize a novel cell line from a breast cancer bone metastasis to better study the progression of the disease. METHODS: The cell line, P7731, was derived from a metastatic bone lesion of a breast cancer patient and assessed for marker expression. P7731 was analyzed for DNA copy number variation, somatic mutations, and gene expression and was compared with the primary tumor. RESULTS: P7731 cells are negative for estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 (triple-negative); strongly express vimentin (100% of cells positive) and also express cytokeratins 8/18 and 19 but at lower frequencies. Flow cytometry indicates P7731 cells are predominantly CD44+/CD49f+/EpCAM-, consistent with a primitive, mesenchymal-like phenotype. The cell line is tumorigenic in immunocompromised mice. Exome sequencing identified a total of 45 and 76 somatic mutations in the primary tumor and cell line, respectively, of which 32 were identified in both samples and included mutations in known driver genes PIK3CA, TP53, and ARID1A. P7731 retains the DNA copy number alterations present in the matching primary tumor. Homozygous deletions detected in the cell line and in the primary tumor were found in regions containing three known (CDKN2A, CDKN2B, and CDKN1B) and 23 putative tumor suppressor genes. Cell line-specific gene amplification coupled with mRNA expression analysis revealed genes and pathways with potential pro-metastatic functions. CONCLUSION: This novel human breast cancer-bone metastasis cell line will be a useful model to study aspects of breast cancer biology, particularly metastasis-related changes from breast to bone.
Assuntos
Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proteínas de Neoplasias/genética , Neoplasias de Mama Triplo Negativas/patologia , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Mama/patologia , Variações do Número de Cópias de DNA/genética , Exoma/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Mutação , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Annexin A1 is a multifunctional protein characterised by its actions in modulating the innate and adaptive immune response. Accumulating evidence of altered annexin A1 expression in many human tumours raises interest in its functional role in cancer biology. In breast cancer, altered annexin A1 expression levels suggest a potential influence on tumorigenic and metastatic processes. However, reports of conflicting results reveal a relationship that is much more complex than first conceptualised. In this review, we explore the diverse actions of annexin A1 on breast tumour cells and various host cell types, including stromal immune and structural cells, particularly in the context of cancer immunoediting.
Assuntos
Anexina A1/imunologia , Neoplasias da Mama/patologia , Mama/patologia , Imunidade Adaptativa , Animais , Anexina A1/análise , Anexina A1/genética , Anexina A1/metabolismo , Mama/imunologia , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Celular , Imunidade Inata , Invasividade Neoplásica/genética , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Microambiente TumoralRESUMO
Inflammation has been implicated in tumor initiation, angiogenesis, and metastasis, and linked to the development of more aggressive, therapy-resistant estrogen receptor (ER)-positive breast cancer. Resolvin D2 (RvD2) is a potent anti-inflammatory lipid mediator. As RvD2 may be synthesized within breast tumors by both tumor cells and the surrounding stroma cells and is present in plasma at bioactive concentrations, we sought to characterize the impact of RvD2 on cell processes underlying breast tumor growth and spread. Trypan-blue exclusion, transfection with estrogen response element (ERE) reporter, real-time quantitative polymerase chain reaction, competitive radioligand binding assays, Western blotting, and immunofluorescence were the techniques used. Unexpectedly, whereas RvD2 (10-1000 nM) supported the proliferation of the ER-positive breast tumor (MCF-7) cells, it did not affect the ER-negative MDA-MB-231 cell number. The proliferative effect of RvD2 in MCF-7 cells was attenuated by the ER antagonist ICI 182,780 (7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17ß-diol). Furthermore, RvD2 increased ERE transcriptional activity in a number of ER-positive breast and ovarian tumor cell lines. This activation was also inhibited by ICI 182,780. RvD2 altered the expression of a subset of estrogen-responsive genes. Although binding experiments showed that RvD2 did not directly compete with [(3)H]17ß-estradiol for ER binding, prior exposure of MCF-7 cells to RvD2 resulted in a significant reduction in the apparent cytosolic ER density. Confocal immunocytochemistry and Western blotting studies showed that RvD2 promoted nuclear localization of ERα. These observations indicate that RvD2 displays significant but indirect estrogenic properties and has the potential to play a role in estrogen-dependent breast cancer progression.
Assuntos
Proliferação de Células/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Receptores de Estrogênio/metabolismo , Ácidos Docosa-Hexaenoicos/farmacocinética , Estradiol/análogos & derivados , Estradiol/farmacologia , Fulvestranto , Humanos , Células MCF-7 , Ligação Proteica , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/antagonistas & inibidoresRESUMO
It is now understood that epigenetic alterations occur frequently in sporadic breast carcinogenesis, but little is known about the epigenetic alterations associated with familial breast tumors. We performed genome-wide DNA-methylation profiling on familial breast cancers (n = 33) to identify patterns of methylation specific to the different mutation groups (BRCA1, BRCA2, and BRCAx) or intrinsic subtypes of breast cancer (basal, luminal A, luminal B, HER2-amplified, and normal-like). We used methylated DNA immunoprecipitation (MeDIP) on Affymetrix promoter chips to interrogate methylation profiles across 25,500 distinct transcripts. Using a support vector machine classification algorithm, we demonstrated that genome-wide methylation profiles predicted tumor mutation status with estimated error rates of 19% (BRCA1), 31% (BRCA2), and 36% (BRCAx) but did not accurately predict the intrinsic subtypes defined by gene expression. Furthermore, using unsupervised hierarchical clustering, we identified a distinct subgroup of BRCAx tumors defined by methylation profiles. We validated these findings in the 33 tumors in the test set, as well as in an independent validation set of 47 formalin-fixed, paraffin-embedded familial breast tumors, by pyrosequencing and Epityper. Finally, gene-expression profiling and SNP CGH array previously performed on the same samples allowed full integration of methylation, gene-expression, and copy-number data sets, revealing frequent hypermethylation of genes that also displayed loss of heterozygosity, as well as of genes that show copy-number gains, providing a potential mechanism for expression dosage compensation. Together, these data show that methylation profiles for familial breast cancers are defined by the mutation status and are distinct from the intrinsic subtypes.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Metilação de DNA/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Mutação , Ilhas de CpG , Feminino , Perfilação da Expressão Gênica , Genes BRCA1 , Genes BRCA2 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
The role of the calcium- and phospholipid-binding protein annexin I (ANXA1) in cell cycle regulation has been investigated in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast tumor cell lines. In MCF-7 cells, ANXA1-targeting small interfering RNA (siRNA) reduced ANXA1 mRNA and protein levels and attenuated cell proliferation induced by FCS, estradiol, or epidermal growth factor. Well-characterized agonists for the known ANXA1 receptor, FPR2, including the ANXA1 N-terminal proteolytic product ANXA1(2-26), lipoxin A(4) (LXA(4)), and the synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), stimulated proliferation of MCF-7 and MDA-MB-231 cells that was attenuated by incubation with FPR2 antagonists WRW(4) (1 µM) or Boc2 (100 nM) or by siRNA against FPR2. FCS-induced mitogenic responses were attenuated by each of the FPR antagonists and by siRNA against FPR2 and, to a lesser extent, FPR1. LXA(4) increased phosphorylation of Akt, p70(S6K) but not ERK1/2. Increases in cyclin D1 protein induced by FCS or LXA(4) were blocked by the PI3 kinase inhibitor, LY294002, and attenuated by FPR2 antagonism using Boc2. In invasive breast cancer, immunohistochemistry revealed the presence of ANXA1 and its receptor, FPR2, in both tumor epithelium and stromal cells. These observations suggest a novel signaling role for ANXA1 in mitogen-activated proliferation of breast tumor epithelial cells that is mediated via activation of FPR1 and FPR2.
Assuntos
Anexina A1/metabolismo , Neoplasias da Mama/metabolismo , Mitógenos/farmacologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Anexina A1/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lipoxinas/metabolismo , Mitógenos/metabolismo , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Interferência de RNA , RNA Interferente Pequeno , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/antagonistas & inibidores , Receptores de Lipoxinas/genética , Transdução de SinaisRESUMO
Inflammatory breast cancer (IBC) describes a highly aggressive form of breast cancer of diverse molecular subtypes and clonal heterogeneity across individual tumors. Accordingly, IBC is recognized by its clinical signs of inflammation, associated with expression of interleukin (IL)-6 and other inflammatory cytokines. Here, we investigate whether sub-clonal differences between expression of components of the IL-6 signaling cascade reveal a novel role for IL-6 to mediate a proliferative response in trans using two prototypical IBC cell lines. We find that SUM149 and SUM 190 cells faithfully replicate differential expression observed in a subset of human IBC specimens between IL-6, the activated form of the key downstream transcription factor STAT3, and of the HER2 receptor. Surprisingly, the high level of IL-6 produced by SUM149 cells activates STAT3 and stimulates proliferation in SUM190 cells, but not in SUM149 cells with low IL-6R expression. Importantly, SUM149 conditioned medium or co-culture with SUM149 cells induced growth of SUM190 cells, and this effect was abrogated by the IL-6R neutralizing antibody Tocilizumab. The results suggest a novel function for inter-clonal IL-6 signaling in IBC, whereby IL-6 promotes in trans proliferation of IL-6R and HER2-expressing responsive sub-clones and, therefore, may provide a vulnerability that can be exploited therapeutically by repurposing of a clinically approved antibody.
RESUMO
MiR-21 was identified as a gene whose expression correlated with the extent of metastasis of murine mammary tumours. Since miR-21 is recognised as being associated with poor prognosis in cancer, we investigated its contribution to mammary tumour growth and metastasis in tumours with capacity for spontaneous metastasis. Unexpectedly, we found that suppression of miR-21 activity in highly metastatic tumours resulted in regression of primary tumour growth in immunocompetent mice but did not impede growth in immunocompromised mice. Analysis of the immune infiltrate of the primary tumours at the time when the tumours started to regress revealed an influx of both CD4+ and CD8+ activated T cells and a reduction in PD-L1+ infiltrating monocytes, providing an explanation for the observed tumour regression. Loss of anti-tumour immune suppression caused by decreased miR-21 activity was confirmed by transcriptomic analysis of primary tumours. This analysis also revealed reduced expression of genes associated with cell cycle progression upon loss of miR-21 activity. A second activity of miR-21 was the promotion of metastasis as shown by the loss of metastatic capacity of miR-21 knockdown tumours established in immunocompromised mice, despite no impact on primary tumour growth. A proteomic analysis of tumour cells with altered miR-21 activity revealed deregulation of proteins known to be associated with tumour progression. The development of therapies targeting miR-21, possibly via targeted delivery to tumour cells, could be an effective therapy to combat primary tumour growth and suppress the development of metastatic disease.
RESUMO
MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. We studied miRNA deregulation in human epithelial ovarian cancer by integrative genomic approach, including miRNA microarray (n = 106), array-based comparative genomic hybridization (n = 109), cDNA microarray (n = 76), and tissue array (n = 504). miRNA expression is markedly down-regulated in malignant transformation and tumor progression. Genomic copy number loss and epigenetic silencing, respectively, may account for the down-regulation of approximately 15% and at least approximately 36% of miRNAs in advanced ovarian tumors and miRNA down-regulation contributes to a genome-wide transcriptional deregulation. Last, eight miRNAs located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumor suppressor genes. Therefore, our results suggest that miRNAs may offer new biomarkers and therapeutic targets in epithelial ovarian cancer.
Assuntos
Epigênese Genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , DNA de Neoplasias , Regulação para Baixo/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease III/genética , Análise de SobrevidaRESUMO
Triple-negative breast cancer (TNBC) has a poor outcome compared to other breast cancer subtypes, and new therapies that target the molecular alterations driving tumor progression are needed. Annexin A1 is an abundant multi-functional Ca2+ binding and membrane-associated protein. Reported roles of Annexin A1 in breast cancer progression and metastasis are contradictory. Here, we sought to clarify the functions of Annexin A1 in the development and progression of TNBC. The association of Annexin A1 expression with patient prognosis in subtypes of TNBC was examined. Annexin A1 was stably knocked down in a panel of human and murine TNBC cell lines with high endogenous Annexin A1 expression that were then evaluated for orthotopic growth and spontaneous metastasis in vivo and for alterations in cell morphology in vitro. The impact of Annexin A1 knockdown on the expression of genes involved in mammary epithelial cell differentia tion and epithelial to mesenchymal transition was also determined. Annexin A1 mRNA levels correlated with poor patient prognosis in basal-like breast tumors and also in the basal-like 2 subset of TNBCs. Unexpectedly, loss of Annexin A1 expression had no effect on either primary tumor growth or spontaneous metastasis of MDA-MB-231_HM xenografts, but abrogated the growth rate of SUM149 orthotopic tumors. In an MMTV-PyMT driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation in both immuno-competent and immuno-deficient mice and induced epithelial to mesenchymal transition and upregulation of basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1- population containing putative tumor initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in a model of human breast cancer and suggest that some basal-like TNBCs may require high endogenous tumor cell Annexin A1 expression for continued growth.
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Extensive expression profiling studies have shown that sporadic breast cancer is composed of five clinically relevant molecular subtypes. However, although BRCA1-related tumours are known to be predominantly basal-like, there are few published data on other classes of familial breast tumours. We analysed a cohort of 75 BRCA1, BRCA2 and non-BRCA1/2 breast tumours by gene expression profiling and found that 74% BRCA1 tumours were basal-like, 73% of BRCA2 tumours were luminal A or B, and 52% non-BRCA1/2 tumours were luminal A. Thirty-four tumours were also analysed by single nucleotide polymorphism-comparative genomic hybridization (SNP-CGH) arrays. Copy number data could predict whether a tumour was basal-like or luminal with high accuracy, but could not predict its mutation class. Basal-like BRCA1 and basal-like non-BRCA1 tumours were very similar, and contained the highest number of chromosome aberrations. We identified regions of frequent gain containing potential driver genes in the basal (8q and 12p) and luminal A tumours (1q and 17q). Regions of homozygous loss associated with decreased expression of potential tumour suppressor genes were also detected, including in basal tumours (5q and 9p), and basal and luminal tumours (10q). This study highlights the heterogeneity of familial tumours and the clinical consequences for treatment and prognosis.
Assuntos
Neoplasias da Mama/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Aberrações Cromossômicas , Análise por Conglomerados , Estudos de Coortes , Hibridização Genômica Comparativa , Metilação de DNA , Feminino , Perfilação da Expressão Gênica/métodos , Hereditariedade , Humanos , Perda de Heterozigosidade , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Polimorfismo de Nucleotídeo Único , PrognósticoRESUMO
The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.
Assuntos
Caderinas/metabolismo , Movimento Celular , Proteína Oncogênica p21(ras)/metabolismo , Ductos Pancreáticos/citologia , Ductos Pancreáticos/patologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Junções Aderentes/patologia , Animais , Carcinoma Ductal Pancreático/patologia , Técnicas de Cultura de Células , Transformação Celular Neoplásica , Cistos/patologia , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Camundongos , Mutação/genética , Invasividade Neoplásica/patologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteína Oncogênica p21(ras)/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.
Assuntos
Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Priônicas/metabolismo , Adulto , Antraciclinas/uso terapêutico , Antibióticos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Epirubicina/farmacologia , Epirubicina/uso terapêutico , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Seleção de Pacientes , Proteínas Priônicas/sangue , Proteínas Priônicas/genética , Prognóstico , Ligação Proteica , Isoformas de Proteínas/sangue , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes. Hence, new molecular targets for therapeutic intervention are necessary. Analyses of panels of human or mouse cancer lines derived from the same individual that differ in their cellular phenotypes but not in genetic background have been instrumental in defining the molecular players that drive the various hallmarks of cancer. To determine the molecular regulators of metastasis in TNBC, we completed a rigorous in vitro and in vivo characterisation of four populations of the MDA-MB-231 human breast cancer line ranging in aggressiveness from non-metastatic to spontaneously metastatic to lung, liver, spleen and lymph node. Single nucleotide polymorphism (SNP) array analyses and genome-wide mRNA expression profiles of tumour cells isolated from orthotopic mammary xenografts were compared between the four lines to define both cell autonomous pathways and genes associated with metastatic proclivity. Gene set enrichment analysis (GSEA) demonstrated an unexpected association between both ribosome biogenesis and mRNA metabolism and metastatic capacity. Differentially expressed genes or families of related genes were allocated to one of four categories, associated with either metastatic initiation (e.g. CTSC, ENG, BMP2), metastatic virulence (e.g. ADAMTS1, TIE1), metastatic suppression (e.g. CST1, CST2, CST4, CST6, SCNNA1, BMP4) or metastatic avirulence (e.g. CD74). Collectively, this model system based on MDA-MB-231 cells should be useful for the assessment of gene function in the metastatic cascade and also for the testing of novel experimental therapeutics for the treatment of TNBC.This article has an associated First Person interview with the first author of the paper.
Assuntos
Genômica , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Bovinos , Ciclo Celular/genética , Linhagem Celular Tumoral , Cistatina M/genética , Cistatina M/metabolismo , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Metástase Neoplásica , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
Insulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP-3 in esophageal tumor biology remains elusive. To delineate the functional consequences of IGFBP-3 overexpression, we stably transduced Ha-Ras(V12)-transformed human esophageal cells with either wild-type or mutant IGFBP-3, the latter incapable of binding Insulin-like growth factor (IGFs) as a result of substitution of amino-terminal Ile56, Leu80, and Leu81 residues with Glycine residues. Wild-type, but not mutant, IGFBP-3 prevented IGF-1 from activating the IGF-1 receptor and AKT, and suppressed anchorage-independent cell growth. When xenografted in nude mice, in vivo bioluminescence imaging demonstrated that wild-type, but not mutant IGFBP-3, abrogated tumor formation by the Ras-transformed cells with concurrent induction of apoptosis, implying a prosurvival effect of IGF in cancer cell adaptation to the microenvironment. Moreover, there was more aggressive tumor growth by mutant IGFBP-3 overexpressing cells than control cell tumors, without detectable caspase-3 cleavage in tumor tissues, indicating an IGF-independent growth stimulatory effect of mutant IGFBP-3. In aggregate, these data suggest that IGFBP-3 contributes to esophageal tumor development and progression through IGF-dependent and independent mechanisms.
Assuntos
Neoplasias Esofágicas/patologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Neoplasias Esofágicas/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/metabolismoRESUMO
Glucocorticoids are commonly used to prevent chemotherapy-induced nausea and vomiting despite a lack of understanding of their direct effect on cancer progression. Recent studies suggest that glucocorticoids inhibit cancer cell migration. However, this action has not been investigated in estrogen receptor (ER)-negative breast tumour cells, although activation of the glucocorticoid receptor (GR) is associated with a worse prognosis in ER-negative breast cancers. In this study we have explored the effect of glucocorticoids on the migration of the ER-negative MDA-MB-231 human breast tumour cell line and the highly metastatic MDA-MB-231-HM.LNm5 cell line that was generated through in vivo cycling. We show for the first time that glucocorticoids inhibit 2- and 3-dimensional migration of MDA-MB-231 cells. Selection of cells for high metastatic potential resulted in a less migratory cell phenotype that was resistant to regulation by glucocorticoids and showed decreased GR receptor expression. The emergence of glucocorticoid resistance during metastatic selection may partly explain the apparent disparity between the clinical and in vitro evidence regarding the actions of glucocorticoids in cancer. These findings highlight the highly plastic nature of tumour cells, and underscore the need to more fully understand the direct effect of glucocorticoid treatment on different stages of metastatic progression.
Assuntos
Movimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Invasividade Neoplásica , Receptores de Estrogênio/metabolismoRESUMO
We analysed breast tumors and breast cancer cell lines for the expression of beta-parvin (ParvB), an adaptor protein that binds to the integrin-linked kinase (ILK). Quantitative RT-PCR indicated that ParvB mRNA was downregulated, by at least 60%, in four of nine breast tumors, relative to patient-matched normal mammary gland tissue. We also found that ParvB protein levels were reduced by > or =90% in five of seven advanced tumors, relative to matched normal breast tissue. Conversely, ILK protein and kinase activity levels were elevated in these tumors, suggesting that downregulation of ParvB stimulates ILK signaling. Western blot analyses indicated very low levels of ParvB protein in MDA-MB-231 and MCF7 breast cancer cells, facilitating functional studies of the effects of ParvB on ILK signaling. Expression of ParvB in MDA-MB-231 and MCF7 cells increased cell adhesion to collagen. ParvB inhibited ILK kinase activity, anchorage-independent cell growth and in vitro matrigel invasion by MDA-MB-231 cells. EGF-induced phosphorylation of two ILK targets, PKB (Ser473) and glycogen synthase kinase 3beta (Ser9), was also inhibited by ParvB. These results indicated that ParvB inhibits ILK signaling downstream of receptor tyrosine kinases. Our results suggest that loss of ParvB expression is a novel mechanism for upregulating ILK activity in tumors.
Assuntos
Actinina/metabolismo , Neoplasias da Mama/metabolismo , Regulação para Baixo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Adenoviridae/genética , Sequência de Aminoácidos , Anticorpos/química , Western Blotting , Neoplasias da Mama/patologia , Adesão Celular , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Colágeno/química , Colágeno/metabolismo , Colágeno/farmacologia , Corantes/farmacologia , DNA Complementar/metabolismo , Combinação de Medicamentos , Fator de Crescimento Epidérmico/metabolismo , Genes Reporter , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Laminina/química , Laminina/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , Fosforilação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteoglicanas/química , Proteoglicanas/farmacologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Regulação para CimaRESUMO
Oncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignant transformation. In mouse, the ARF-p53 pathway mediates Ha-Ras(G12V)-induced senescence, and p19(ARF-/-) and p53(-/-) cells undergo transformation upon Ras activation. In addition, mouse cells, unlike human cells, express constitutively active telomerase and have long telomeres. However, it is unclear how Ras activation affects human cells of epithelial origin with p53 mutation and/or telomerase activation. In order to address this question, Ha-Ras(G12V) was expressed ectopically in primary as well as hTERT-immortalized human esophageal keratinocytes stably expressing dominant-negative p53 mutants. In human esophageal keratinocytes, we found that Ha-Ras(G12V) induced senescence regardless of p53 status and telomerase activation. Ras activation resulted in changes of cellular morphology, activation of senescence-associated beta-galactosidase, and suppression of cell proliferation, all coupled with reduction in the hyperphosphorylated form of the retinoblastoma protein (pRb). Furthermore, Ha-Ras(G12V) upregulated p16(INK4a) and downregulated cyclin-dependent kinase Cdk4 in human esophageal keratinocytes. Thus, Ras-mediated senescence may involve distinct mechanisms between human and mouse cells. Inactivation of the pRb pathway may be necessary for Ras to overcome senescence and transform human esophageal epithelial cells.
Assuntos
Senescência Celular/fisiologia , Genes ras/genética , Queratinócitos/fisiologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética , Substituição de Aminoácidos , Linhagem Celular , Senescência Celular/genética , Proteínas de Ligação a DNA , Esôfago , Vetores Genéticos , Humanos , Retroviridae/genética , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Interleukin (IL)-11 is a member of the IL-6 family of cytokines that is defined by the shared use of the GP130 signal transducing receptor subunit. In addition of its long recognized activities as a hemopoietic growth factor, IL-11 has an emerging role in epithelial cancer biology. Through the activation of the GP130-Janus kinase signaling cascade and associated transcription factor STAT3, IL-11 can confer many of the tumor intrinsic 'hallmark' capabilities to neoplastic cells, if they express the ligand-specific IL-11Rα receptor subunit. Accordingly, IL-11 signaling has recently been identified as a rate-limiting step for the growth tumors arising from the mucosa of the gastrointestinal tract. However, there is less appreciation for a potential role of IL-11 to support breast cancer progression, apart from its well documented capacity to facilitate bone metastasis. Here we review evidence that IL-11 expression in breast cancer correlates with poor disease outcome and discuss some of the molecular mechanisms that are likely to underpin these observations. These include the capacity of IL-11 to stimulate survival and proliferation of cancer cells alongside angiogenesis of the primary tumor and of metastatic progenies at distant organs. We review current strategies to interfere with IL-11 signaling and advocate that inhibition of IL-11 signaling may represent an emerging therapeutic opportunity for numerous cancers.