RESUMO
BACKGROUND: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression. METHODS: Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease. RESULTS: BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group. CONCLUSION: This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Moduladores de Receptor Estrogênico/uso terapêutico , Terapia de Alvo Molecular/métodos , Quinazolinas/uso terapêutico , RNA não Traduzido/metabolismo , Receptor ErbB-2/metabolismo , Tamoxifeno/uso terapêutico , Adulto , Idoso , Antineoplásicos/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/análise , Neoplasias da Mama/mortalidade , Proliferação de Células , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/farmacologia , RNA Longo não Codificante , RNA Mensageiro/metabolismo , RNA não Traduzido/efeitos dos fármacos , RNA não Traduzido/genética , Receptor ErbB-2/efeitos dos fármacos , Receptor ErbB-2/genética , Receptores de Estrogênio/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Breast cancer anti-oestrogen resistance 4 (BCAR4) was identified in a search for genes involved in anti-oestrogen resistance in breast cancer. We explored whether BCAR4 is predictive for tamoxifen resistance and prognostic for tumour aggressiveness, and studied its function. METHODS: BCAR4 mRNA levels were measured in primary breast tumours, and evaluated for association with progression-free survival (PFS) and clinical benefit in patients with oestrogen receptor (ERα)-positive tumours receiving tamoxifen as first-line monotherapy for advanced disease. In a separate cohort of patients with lymph node-negative, ERα-positive cancer, and not receiving systemic adjuvant therapy, BCAR4 levels were evaluated for association with distant metastasis-free survival (MFS). The function of BCAR4 was studied with immunoblotting and RNA interference in a cell model. RESULTS: Multivariate analyses established high BCAR4 mRNA levels as an independent predictive factor for poor PFS after start of tamoxifen therapy for recurrent disease. High BCAR4 mRNA levels were associated with poor MFS and overall survival, reflecting tumour aggressiveness. In BCAR4-expressing cells, phosphorylation of v-erb-b2 erythroblastic leukaemia viral oncogene homolog (ERBB)2, ERBB3, and their downstream mediators extracellular signal-regulated kinase 1/2 and v-akt murine thymoma viral oncogene homolog (AKT) 1/2, was increased. Selective knockdown of ERBB2 or ERBB3 inhibited proliferation, confirming their role in BCAR4-induced tamoxifen resistance. CONCLUSION: BCAR4 may have clinical relevance for tumour aggressiveness and tamoxifen resistance. Our cell model suggests that BCAR4-positive breast tumours are driven by ERBB2/ERBB3 signalling. Patients with such tumours may benefit from ERBB-targeted therapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Proteína Substrato Associada a Crk/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Tamoxifeno/uso terapêutico , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adulto , Idoso , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Farmacológicos/análise , Biomarcadores Farmacológicos/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Carcinoma/genética , Carcinoma/mortalidade , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA Longo não Codificante , RNA não Traduzido , Estudos Retrospectivos , Análise de SobrevidaRESUMO
BACKGROUND: Endocrine therapies of breast cancer are effective but ultimately fail because of the development of treatment resistance. We have previously revealed several genes leading to tamoxifen resistance in vitro by retroviral insertion mutagenesis. To understand the manner in which these genes yield tamoxifen resistance, their effects on global gene expression were studied and those genes resulting in a distinct gene expression profile were further investigated for their clinical relevance. METHODS: Gene expression profiles of 69 human breast cancer cell lines that were made tamoxifen resistant through retroviral insertion mutagenesis were obtained using oligonucleotide arrays and analysed with bioinformatic tools. mRNA levels of NCOR2 and CITED2 in oestrogen receptor-positive breast tumours were determined by quantitative RT-PCR. mRNA levels were evaluated for association with metastasis-free survival (MFS) in 620 patients with lymph node-negative primary breast cancer who did not receive systemic adjuvant therapy, and with clinical benefit in 296 patients receiving tamoxifen therapy for recurrent breast cancer. RESULTS: mRNA expression profiles of most tamoxifen-resistant cell lines were strikingly similar, except for the subgroups of cell lines in which NCOR2 or CITED2 were targeted by the retrovirus. Both NCOR2 and CITED2 mRNA levels were associated with MFS, that is, tumour aggressiveness, independently of traditional prognostic factors. In addition, high CITED2 mRNA levels were predictive for a clinical benefit from first-line tamoxifen treatment in patients with advanced disease. CONCLUSIONS: Most retrovirally targeted genes yielding tamoxifen resistance in our cell lines do not impose a distinctive expression profile, suggesting that their causative role in cell growth may be accomplished by post-transcriptional processes. The associations of NCOR2 and CITED2 with outcome in oestrogen receptor-positive breast cancer patients underscore the clinical relevance of functional genetic screens to better understand disease progression, which may ultimately lead to the development of improved treatment options.
Assuntos
Neoplasias da Mama/metabolismo , Antagonistas de Estrogênios/farmacologia , Correpressor 2 de Receptor Nuclear/metabolismo , Proteínas Repressoras/metabolismo , Tamoxifeno/farmacologia , Transativadores/metabolismo , Adulto , Idoso , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Correpressor 2 de Receptor Nuclear/biossíntese , Correpressor 2 de Receptor Nuclear/genética , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Transativadores/biossíntese , Transativadores/genéticaRESUMO
BACKGROUND: The microRNA-371a-3p (miR-371a-3p) has been reported to be an informative liquid biopsy (serum and plasma) molecular biomarker for both diagnosis and follow-up of patients with a malignant (testicular) germ cell tumor ((T)GCT). It is expressed in all histological cancer elements, with the exception of mature teratoma. However, normal testis, semen, and serum of males with a disrupted testicular integrity without a TGCT may contain miR-371a-3p levels above threshold, of which the cellular origin is unknown. OBJECTIVES: Therefore, a series of relevant tissues (frozen and formalin-fixed paraffin-embedded (FFPE), when available) from the complete male urogenital tract (i.e., kidney to urethra and testis to urethra) and semen was investigated for miR-371a-3p levels using targeted quantitative RT-PCR (qRT-PCR). MATERIALS AND METHODS: In total, semen of males with normospermia (n = 11) and oligospermia (n = 3) was investigated, as well as 88 samples derived from 32 different patients. The samples represented one set of tissues related to the entire male urogenital tract (11 anatomical locations), three sets for 10 locations, and four sets for six locations. RESULTS: All testis parenchyma (n = 17) cases showed low miR-371a-3p levels. Eight out of 14 (57%) semen samples showed detectable miR-371a-3p levels, irrespective of the amount of motile spermatozoa, but related to sperm concentration and matched Johnsen score (Spearman's rho correlation coefficient 0.849 and 0.871, p = 0.000, respectively). In all other tissues investigated, miR-371a-3p could not be detected. DISCUSSION: This study demonstrates that the miR-371a-3p in healthy adult males is solely derived from the germ cell compartment. CONCLUSIONS: The observation is important in the context of applying miR-371a-3p as molecular liquid biopsy biomarker for diagnosis and follow-up of patients with malignant (T)GCT. Moreover, miR-371a-3p might be an informative seminal biomarker for testicular germ cell composition.
Assuntos
Genitália Masculina/metabolismo , MicroRNAs/metabolismo , Sêmen/metabolismo , Sistema Urinário/metabolismo , Humanos , Masculino , Oligospermia/metabolismo , Valores de ReferênciaRESUMO
In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 22 , Leucemia Mieloide/genética , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Quimera , Clonagem Molecular , DNA/metabolismo , Enzimas de Restrição do DNA , Humanos , Hibridização de Ácido NucleicoRESUMO
Epidermal growth factor (EGF) receptor is inversely related to expression of estrogen receptor (ER) and progesterone receptor in primary breast tumors and is a negative predictor for response to endocrine therapy. To investigate a possible causal role of EGF receptor expression in breast cancer progression to hormone independence, we have created an experimental cell system. Epidermal growth factor receptor complementary DNA was introduced in estrogen-dependent ZR-75-1 breast cancer cells, and the resulting ZR/HERc cells exhibited a mitogenic response to epidermal growth factor, thus bypassing estrogen dependence. This EGF-induced proliferation could not be inhibited by antiestrogens. In addition, we noted changes in cell morphology and keratin expression of EGF-stimulated ZR/HERc cells, suggestive of an altered differentiation state. Furthermore, intolerance of functional ER and EGF receptor signal transduction pathways in ZR/HERc cells was observed during simultaneous activation, which possibly explains the inverse relationship of ER and EGF receptor expression in primary tumors. In contrast to the parental cells, ZR/HERc cells rapidly progressed to a stable ER-negative phenotype when cultured in the presence of the antiestrogen hydroxy-tamoxifen. These results suggest a possible role for EGF receptor in progression of breast cancer to hormone independence.
Assuntos
Neoplasias da Mama/patologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Estrogênios/fisiologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Receptores ErbB/genética , Expressão Gênica , Técnicas In Vitro , Receptores de Estradiol/fisiologia , Transdução de Sinais , Tamoxifeno/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
In Acute Nonlymphocytic Leukemia (ANLL) specified by a reciprocal translocation (6;9), defined clinical and morphological features are observed. This suggests that genes located near the breakpoints of the translocation chromosomes are involved in the generation of this subtype of leukemia. The human pim-1 gene has been mapped near the t(6;9) breakpoint on chromosome 6. Using somatic cell hybrids we demonstrated that the pim-1 gene remains on chromosome 6. We investigated whether pim-1 plays a role in t(6;9) ANLL. The expression of pim-1 is elevated in two out of three patients with t(6;9) ANLL. However, the pim-1 gene has a size of only 6 kb and using field inversion gel electrophoresis, no chromosomal breakpoint can be detected within a distance of 165 kb from the pim-1 locus. Therefore it seems more likely that the elevated expression is due to the differentiation state of the cells rather than transcriptional activation by the translocation.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 6 , Cromossomos Humanos Par 9 , Regulação da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/biossíntese , Translocação Genética , Adolescente , Adulto , Idoso , Animais , Southern Blotting , Pré-Escolar , Cricetinae , Eletroforese em Gel de Ágar/métodos , Feminino , Humanos , Células Híbridas , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-pim-1 , Proto-OncogenesRESUMO
We have tested a panel of recombinant hematopoietic growth factors (HGF) including the interleukins (IL) 1, 2, 3, 4, and 6 and the colony stimulating factors GM-CSF, G-CSF, M-CSF for their ability to induce proliferation of precursor B acute lymphoblastic leukemia cells (ALL) from 19 patients. In Ficoll-Isopaque isolated and T cell-depleted ALL bone marrow samples, IL2 (two cases), IL3 (four cases), and GM-CSF (one case) infrequently stimulated DNA synthesis measured by 3H-thymidine (TdR) uptake, and the other recombinant growth factors completely failed to do so. In repeat experiments with ALL blasts purified by fluorescence activated cell sorting (FACS), IL2, IL3, and GM-CSF responses could not be reproduced, suggesting that nonleukemic contaminant cells, and not the ALL blasts, had been stimulated by these factors. Cocktails containing combinations of IL1-IL4 and IL6 also lacked proliferation inducing potency. Depending on the purity of the incubated ALL cell samples, an impure preparation of B cell growth factors that has been reported to contain a highly effective stimulatory activity for precursor B ALL cells induced proliferation of residual normal cells as well as the ALL cells, as was evident from combined analysis of DNA synthesis and karyotyping. Exposure of the ALL blasts to artificial activators of protein kinase C and Ca2+ mobilization resulted in significant rises in 3H-TdR uptake, suggesting that these intracellular compounds are involved in transducing signals that upregulate proliferation. Although it remains possible that some of the human recombinant growth factors promote the growth of precursor B ALL cells in combination with other stimuli, a dominant role in the regulation of proliferation of these cells cannot be attributed to any of these cytokines at the present time.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Interleucinas/farmacologia , Leucemia Linfoide/patologia , Células-Tronco Neoplásicas/patologia , Linfócitos B , Calcimicina/farmacologia , Divisão Celular/efeitos dos fármacos , Humanos , Interleucina-4 , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Antagonists of steroid hormones are clinically important in the management of breast cancer. However, the duration of response is limited due to the development of hormone-independent tumors in virtually all cases. In an attempt to obtain insight into the mechanisms underlying antiestrogen resistance, the consequences of epigenetic changes in gene expression were studied in vitro. Estrogen-dependent ZR-75-1 human breast cancer cells were treated with 5-azacytidine, an inhibitor of DNA methylation, and cultured in the absence of estradiol or in the presence of antiestrogens. Estrogen-independent cell colonies developed within 3 weeks at high frequency in 5-azacytidine-treated cultures (0.7 x 10(-3), in contrast to control cultures (< or = 10(-8). The derived cells (ZR/AZA) were resistant to 4-hydroxytamoxifen and ICI 164,384, independent of the selection protocol, but had lost the ability to grow anchorage-independent. Whereas expression of estrogen receptor, progesterone receptor, and pS2 were down-regulated, expression of epidermal growth factor (EGF) receptor and HER2/neu were increased in ZR/AZA cells. In contrast to the stable altered expression patterns of estrogen receptor and EGF receptor, transient keratin 7 expression was observed. Transforming growth factor-alpha mRNA was identified in ZR-75-1 cells and ZR/AZA cells and EGF-like peptides were secreted in the culture medium. Proliferation of ZR/AZA cells could be partially inhibited with an EGF receptor-blocking antibody. Presence of both growth factor receptors and possible ligands suggests the development of an autocrine growth mechanism. Our data show that epigenetic alterations of gene expression result in rapid progression of breast cancer cells to hormone independence.
Assuntos
Neoplasias da Mama/secundário , Estrogênios/fisiologia , Neoplasias Hormônio-Dependentes/genética , Proteínas , Receptores de Estradiol/metabolismo , Azacitidina/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Regulação para Baixo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinas/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Alcamidas Poli-Insaturadas , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Fator Trefoil-1 , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
Duration of response to antiestrogen therapy in metastatic breast cancer is limited due to the development of antiestrogen-resistant tumors. The mechanisms involved are not understood but could originate from (epi)genetic alterations within the tumor cells. We have applied in vitro random insertional mutagenesis with replication defective retroviruses to identify those genes playing a key role in development of antiestrogen resistance in human breast cancer cells. Eighty antiestrogen-resistant cell clones were isolated from 7 x 10(8) estrogen-dependent ZR-75-1 cells, mass-infected with defective retroviruses and subjected to 4-OH-tamoxifen selection. Integration site-specific DNA probes were made by inverse polymerase chain reaction techniques and used to search for common integration sites. Six cell clones were identified with retroviral genome integrations in the same orientation in a single locus, designated breast cancer antiestrogen resistance locus-1 (bcar-1). These bcar-1 cell clones had lost estrogen receptor expression and had become estrogen independent. Our results strongly suggest that alteration of the bcar-1 locus is responsible for development of antiestrogen resistance in human breast cancer cells in vitro. In addition, we have shown that in vitro insertional mutagenesis using defective retroviruses can be applied for gene tagging in human cells.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Elementos de DNA Transponíveis/genética , DNA de Neoplasias/genética , DNA Viral/genética , Antagonistas de Estrogênios/farmacologia , Retroviridae/genética , Integração Viral , Autorradiografia , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Sondas de DNA , Resistência a Medicamentos , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , Reação em Cadeia da Polimerase , Tamoxifeno/farmacologia , Células Tumorais Cultivadas , Integração Viral/genéticaRESUMO
microRNAs (miRs) are short non-coding RNA molecules (≈21 nucleotides) involved in regulation of translation. As such they are crucial for normal cell development and differentiation as well as cellular maintenance. Dysregulation of miRs has been reported in various diseases, including cancer. Interestingly, miRs can be informative as tumor classifiers and disease biomarkers. Recent studies demonstrated the presence of miRs in body fluids like serum, thus providing a putative non-invasive tool to study and monitor disease state. Earlier targeted studies by several independent groups identified specific embryonic miRs as characteristic for germ cell tumors (GCT) (miR-371-2-3 & miR-302/367 clusters). This study reports a high-throughput miR profiling (≈750 miRs) approach on serum from testicular germ cell tumor patients (14 seminoma and 10 non-seminoma) and controls (n = 11), aiming at independent identification of miRs as candidate biomarkers for testicular GCT. A magnetic bead capture system was used to isolate miRs from serum. Subsequently, the TaqMan Array Card 3.0 platform was used for profiling. The previously identified miRs 371 and 372 were confirmed to be specifically elevated in serum from germ cell tumor patients. In addition, several novels miRs were identified that were discriminative between germ cell cancer and controls: miR-511, -26b, -769, -23a, -106b, -365, -598, -340, and let-7a. In conclusion, this study validates the power of the embryonic miRs 371 and 372 in detecting malignant GCT (SE and NS) based on serum miR levels and identifies several potential novel miR targets.
Assuntos
Biomarcadores Tumorais/sangue , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/sangue , Neoplasias Embrionárias de Células Germinativas/sangue , Seminoma/sangue , Neoplasias Testiculares/sangue , Sequência de Bases , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Análise Discriminante , Humanos , Masculino , MicroRNAs/genética , Dados de Sequência Molecular , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Valor Preditivo dos Testes , Seminoma/genética , Seminoma/patologia , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologiaRESUMO
Tamoxifen has been used for the systemic treatment of patients with breast cancer for nearly three decades. Treatment success is primarily dependent on the presence of the estrogen receptor (ER) in the breast carcinoma. While about half of patients with advanced ER-positive disease immediately fail to respond to tamoxifen, in the responding patients the disease ultimately progresses to a resistant phenotype. The possible causes for intrinsic and acquired resistance have been attributed to the pharmacology of tamoxifen, alterations in the structure and function of the ER, the interactions with the tumour environment and genetic alterations in the tumour cells. So far no prominent mechanism leading to resistance has been identified. The recent results of a functional screen for breast cancer antiestrogen resis- tance (BCAR) genes responsible for development of tamoxifen resistance in human breast cancer cells are reviewed. Individual BCAR genes can transform estrogen-dependent breast cancer cells into estrogen-independent and tamoxifen-resistant cells in vitro. Furthermore, high levels of BCAR1/pl30Cas protein in ER-positive primary breast tumours are associated with intrinsic resistance to tamoxifen treatment. These results indicate a prominent role for alternative growth control pathways independent of ER signalling in intrinsic tamoxifen resistance of ER-positive breast carcinomas. Deciphering the differentiation characteristics of normal and malignant breast epithelial cells with respect to proliferation control and regulation of cell death (apoptosis) is essential for understanding therapy response and development of resistance of breast carcinoma.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Tamoxifeno/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Antagonistas de Estrogênios/farmacologia , Feminino , Humanos , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/genética , Receptores de Estrogênio/fisiologiaRESUMO
Recent studies have suggested that interleukin-2 (IL-2), tumor necrosis factor (TNF) and a crude preparation of B-cell growth factors (BCGF) have a regulatory role in the proliferation of B-CLL cells. However, interpretation of the experimental data has been complicated by potential methodological pitfalls. To establish the role of these factors in B-CLL growth, we investigated their stimulatory effects under accurately defined in vitro conditions. Purified B-CLL cells were obtained by fluorescence activated cell sorting on the basis of coexpression of CD5 and CD19/CD20/CD24 surface antigens in order to avoid interference of normal cell contamination. Subsequently, the cells were cultured free of serum. TNFs alpha and beta and BCGF induced DNA synthesis in the purified B-CLL cells in five out of six cases, as assessed by 3H-thymidine (TdR) uptake on days 3 and 6 of culture. The stimulating activity of BCGF could be suppressed by the addition of specific anti-TNF monoclonal antibodies, indicating that the BCGF activity had been, to a major extent, due to the presence of TNFs in this impure preparation. IL-2, when added as a single stimulus, induced DNA synthesis in the B-CLL cells in three out of six cases on day 3, but not on day 6 of culture. In some patients, cooperative effects of IL-2 and TNF were observed. Cytogenetic analysis was applied to confirm the CLL origin of the proliferating cells. The phorbol ester TPA or anti-IgM were not required for induction of DNA synthesis in the CLL cells, although TPA potentiated 3H-TdR uptake in three out of six cases, and anti-IgM in one case. Our data demonstrate that IL-2 and TNFs alpha and beta can act as growth factors for B-CLL cells under fully defined in vitro conditions, essentially without the need of TPA or anti-IgM as primary activation signals.
Assuntos
Linfócitos B/patologia , Interleucina-2/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfotoxina-alfa/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Antígenos CD/análise , Antígenos CD19 , Antígenos de Diferenciação/análise , Antígenos de Diferenciação de Linfócitos B/análise , Antígenos CD5 , Divisão Celular/efeitos dos fármacos , Humanos , Técnicas Imunológicas , Interleucina-4/farmacologia , Cariotipagem , Receptores de Antígenos de Linfócitos B/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Endocrine therapy is effective in the treatment of breast cancer. Adjuvant treatment with tamoxifen reduces tumor recurrence and achieves increased survival. In metastatic disease, tamoxifen treatment accomplishes objective responses in +/- 50% of the patients with estrogen receptor-positive primary tumors. However, the response duration is limited due to the inevitable development of metastases resistant to tamoxifen. The mechanisms leading to tamoxifen resistance are largely unknown. We have set out to identify genetic pathways in the tumor cells causing failure of tamoxifen therapy. We selected an estrogen-dependent human breast cancer cell line (ZR-75-1) and demonstrated that genetic and epigenetic alterations can change the hormone-response phenotype of these cells. Subsequently, we applied insertional mutagenesis with defective retroviruses to these ZR-75-1 breast cancer cells. Integration of a retrovirus in the cellular DNA alters the genome structure and may modify the expression of genes in its surroundings. As a result of the altered gene expression, the biological phenotype of the infected cell may be changed. The infected ZR-75-1 cells were subjected to tamoxifen selection and a panel of tamoxifen-resistant cell lines has been established. Screening for a common integration site for the retrovirus has provided, so far, compelling evidence for the involvement of at least one genetic locus (BCAR 1) in breast cancer antiestrogen resistance in vitro.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Estrogênios/fisiologia , Feminino , HumanosAssuntos
Interleucina-1/farmacologia , Leucemia Mieloide Aguda/patologia , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Substâncias de Crescimento/biossíntese , Humanos , Leucemia Mieloide Aguda/metabolismo , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaAssuntos
Neoplasias da Mama/genética , Estrogênios/fisiologia , Regulação Neoplásica da Expressão Gênica , Azacitidina/farmacologia , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular/genética , DNA/efeitos dos fármacos , DNA/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Humanos , Metilação , Receptores de Estrogênio/metabolismo , Células Tumorais CultivadasRESUMO
The antiestrogen tamoxifen is important in the treatment of hormone-dependent breast cancer, although development of resistance is inevitable. To unravel the molecular mechanisms of antiestrogen resistance, a search for involved genes was initiated. Retrovirus-mediated insertional mutagenesis was applied to human ZR-75-1 breast cancer cells. Infected cells were subjected to tamoxifen selection and a panel of resistant cell clones was established. Screening for a common integration site resulted in the identification of a novel gene designated BCAR3. Transfer of this locus by cell fusion or transfection of the BCAR3 cDNA to ZR75-1 and MCF-7 cells induces antiestrogen resistance. BCAR3 represents a putative SH2 domain-containing protein and is partly homologous to the cell division cycle protein CDC48.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Antagonistas de Estrogênios/farmacologia , Receptores de AMP Cíclico/genética , Tamoxifeno/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Fusão Celular , Clonagem Molecular , DNA Complementar , DNA de Neoplasias , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica , Humanos , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.
Assuntos
Cromossomos Humanos Par 9 , Leucemia Mieloide/genética , Cromossomo Filadélfia , Translocação Genética , Adolescente , Sequência de Bases , Deleção Cromossômica , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Masculino , Sequências Repetitivas de Ácido NucleicoRESUMO
To create experimental systems that facilitate studies aimed at the responsiveness of hematopoietic progenitors to interleukin (IL)-6 in combination with IL-3, we introduced the human IL-6 receptor (hIL-6R) into the IL-3-dependent cell line 32D. For this purpose, a retroviral vector containing the hIL-6R cDNA was constructed. 32D parental cells did not respond to IL-6, neither alone nor in combination with increasing concentrations of IL-3, and did not express detectable numbers of IL-6R as determined by 125I-IL-6 binding. 32D/hIL-6R cells expressed high-affinity IL-6 binding and responded synergistically to IL-6 in combination with suboptimal amounts of IL-3 in DNA synthesis assays. In addition, IL-6 promoted the short-term survival of IL-3-responsive clonogenic 32D/hIL-6R cells. On the other hand, although introduction of hIL-6R resulted in the formation of high-affinity IL-6 receptor structures in the IL-2-dependent thymocyte cell line CTLL, CTLL/hIL-6R cells did not respond to IL-6 in synergy with IL-2. We conclude that 32D cells possess the intracellular machinery permissive for IL-6 signal transduction. Murine IL-3-dependent cell lines with ectopic IL-6 receptors can serve as models for dissecting the molecular basis of IL-6 responses in primitive hematopoietic cells.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Receptores Imunológicos/fisiologia , Animais , Divisão Celular , Linhagem Celular , Sobrevivência Celular , DNA/biossíntese , DNA/genética , Sinergismo Farmacológico , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Receptores Imunológicos/genética , Receptores de Interleucina-6 , TransfecçãoRESUMO
The prognostic value of epidermal growth factor receptor (EGFR) expression and its biological role in estrogen receptor-positive (ER+) and ER-negative (ER-) primary breast cancer is controversial. In this study, distributions of ER, progesterone receptor and EGFR have been established using immunohistochemistry in both primary breast tumors and their matched axillary lymph node metastases of 26 patients or their matched distant metastases of 2 patients. In addition, 5 patients with bilateral breast cancer were studied. ER+ tumor cells were detected in 22 (69%) and EGFR+ tumor cells were detected in 11 (34%) primary breast carcinomas. Expression of ER and EGFR was inverse regarding the individual tumor cells in both primary tumors and metastases. Relationship of EGFR expression with poorly differentiated and large breast tumors was observed. Furthermore, primary tumors with a predominant lobular component were ER+ and, with one exception, EGFR-. Invasive ductal carcinomas were more frequently EGFR+. No apparent differences in receptor expression were observed between primary tumors and lymph node metastases or chronously or metachronously occurring bilateral breast cancers. Only one ER+ primary tumor showed a switch to EGFR expression in the involved lymph node. Our study shows that a shift in receptor phenotype between primary tumors and lymph node metastases is a rare event and, thus, additional analyses of involved lymph nodes will not likely serve as a better predictor for response to anti-estrogen therapy. We conclude that expression of EGFR is not a prerequisite for development of metastases.