RESUMO
Interactions between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment significantly influence cancer growth and metastasis. Transforming growth factor-ß (TGF-ß) is known to be a critical mediator of the CAF phenotype, and osteopontin (OPN) expression in tumors is associated with more aggressive phenotypes and poor patient outcomes. The potential link between these two pathways has not been previously addressed. Utilizing in vitro studies using human mesenchymal stem cells (MSCs) and MDA-MB231 (OPN+) and MCF7 (OPN-) human breast cancer cell lines, we demonstrate that OPN induces integrin-dependent MSC expression of TGF-ß1 to mediate adoption of the CAF phenotype. This OPN-TGF-ß1 pathway requires the transcription factor, myeloid zinc finger 1 (MZF1). In vivo studies with xenotransplant models in NOD-scid mice showed that OPN expression increases cancer growth and metastasis by mediating MSC-to-CAF transformation in a process that is MZF1 and TGF-ß1 dependent. We conclude that tumor-derived OPN engenders MSC-to-CAF transformation in the microenvironment to promote tumor growth and metastasis via the OPN-MZF1-TGF-ß1 pathway.
Assuntos
Neoplasias da Mama/patologia , Transformação Celular Neoplásica/metabolismo , Fibroblastos/patologia , Células-Tronco Mesenquimais/patologia , Células-Tronco Neoplásicas/patologia , Osteopontina/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/genética , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Células MCF-7 , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Osteopontina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Células Tumorais CultivadasRESUMO
The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor α (ERα)+ breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase Cα (PKCα) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKCα overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKCα-overexpressing breast cancer cells.Analysis of published microarray data from ERα+ breast carcinomas shows that PKCα expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKCα-overexpressing, TAM-resistant T47D model, PKCα selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKCα-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKCα-expressing T47D cells. In PKCα-overexpressing T47D xenografts, an orally active γ-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKCα overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKCα- and Notch-4-overexpressing, endocrine-resistant breast cancers.
RESUMO
BACKGROUND: We reported that Notch-1, a potent breast oncogene, is activated in response to trastuzumab and contributes to trastuzumab resistance in vitro. We sought to determine the preclinical benefit of combining a Notch inhibitor (γ-secretase inhibitor (GSI)) and trastuzumab in both trastuzumab-sensitive and trastuzumab-resistant, ErbB-2-positive, BT474 breast tumours in vivo. We also studied if the combination therapy of lapatinib plus GSI can induce tumour regression of ErbB-2-positive breast cancer. METHODS: We generated orthotopic breast tumour xenografts from trastuzumab- or lapatinib-sensitive and trastuzumab-resistant BT474 cells. We investigated the antitumour activities of two distinct GSIs, LY 411 575 and MRK-003, in vivo. RESULTS: Our findings showed that combining trastuzumab plus a GSI completely prevented (MRK-003 GSI) or significantly reduced (LY 411 575 GSI) breast tumour recurrence post-trastuzumab treatment in sensitive tumours. Moreover, combining lapatinib plus MRK-003 GSI showed significant reduction of tumour growth. Furthermore, a GSI partially reversed trastuzumab resistance in resistant tumours. CONCLUSION: Our data suggest that a combined inhibition of Notch and ErbB-2 signalling pathways could decrease recurrence rates for ErbB-2-positive breast tumours and may be beneficial in the treatment of recurrent trastuzumab-resistant disease.
Assuntos
Secretases da Proteína Precursora do Amiloide/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/metabolismo , Receptores Notch/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Linhagem Celular Tumoral , Óxidos S-Cíclicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Marcação de Genes , Genes erbB , Genes erbB-2 , Humanos , Lapatinib , Camundongos , Camundongos Nus , Transplante de Neoplasias , Quinazolinas/administração & dosagem , Receptores Notch/genética , Recidiva , Tiadiazóis/farmacologia , TrastuzumabRESUMO
Approximately 80% of breast cancers express the estrogen receptor-alpha (ERalpha) and are treated with anti-estrogens. Resistance to these agents is a major cause of mortality. We have shown that estrogen inhibits Notch, whereas anti-estrogens or estrogen withdrawal activate Notch signaling. Combined inhibition of Notch and estrogen signaling has synergistic effects in ERalpha-positive breast cancer models. However, the mechanisms whereby Notch-1 promotes the growth of ERalpha-positive breast cancer cells are unknown. Here, we demonstrate that Notch-1 increases the transcription of ERalpha-responsive genes in the presence or absence of estrogen via a novel chromatin crosstalk mechanism. Our data support a model in which Notch-1 can activate the transcription of ERalpha-target genes via IKKalpha-dependent cooperative chromatin recruitment of Notch-CSL-MAML1 transcriptional complexes (NTC) and ERalpha, which promotes the recruitment of p300. CSL binding elements frequently occur in close proximity to estrogen-responsive elements (EREs) in the human and mouse genomes. Our observations suggest that a hitherto unknown Notch-1/ERalpha chromatin crosstalk mediates Notch signaling effects in ERalpha-positive breast cancer cells and contributes to regulate the transcriptional functions of ERalpha itself.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/fisiologia , Quinase I-kappa B/genética , Receptor Notch1/fisiologia , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Quinase I-kappa B/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligopeptídeos/farmacologia , Regiões Promotoras Genéticas/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Accumulating preclinical and clinical evidence supports a pro-oncogenic function for Notch signaling in several solid tumors, particularly but not exclusively in breast cancer. Notch inhibitory agents, such as gamma-secretase inhibitors, are being investigated as candidate cancer therapeutic agents. Interest in therapeutic modulation of the Notch pathway has been increased by recent reports, indicating that its role is important in controlling the fate of putative 'breast cancer stem cells'. However, as is the case for most targeted therapies, successful targeting of Notch signaling in cancer will require a considerable refinement of our understanding of the regulation of this pathway and its effects in both normal and cancer cells. Notch signaling has bidirectional 'cross talk' interaction with multiple other pathways that include candidate therapeutic targets. Understanding these interactions will greatly increase our ability to design rational combination regimens. To determine which patients are most likely to benefit from treatment with Notch inhibitors, it will be necessary to develop molecular tests to accurately measure pathway activity in specific tumors. Finally, mechanism-based toxicities will have to be addressed by a careful choice of therapeutic agents, combinations and regimens. This article summarizes the current state of the field, and briefly describes opportunities and challenges for Notch-targeted therapies in oncology.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Receptores Notch/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Terapia Combinada , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Neoplasias/fisiopatologia , Neoplasias/terapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/fisiologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Receptores Notch/genética , Receptores Notch/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Proteínas Supressoras de Tumor/fisiologiaRESUMO
ErbB-2 overexpression in breast tumors is associated with poor survival. Expression of Notch-1 and its ligand, Jagged-1, is associated with the poorest survival, including ErbB-2-positive tumors. Trastuzumab plus chemotherapy is the standard of care for ErbB-2-positive breast cancer. A proportion of tumors are initially resistant to trastuzumab and acquired resistance to trastuzumab occurs in metastatic breast cancer and is associated with poor prognosis. Thus, we investigated whether Notch-1 contributes to trastuzumab resistance. ErbB-2-positive cells have low Notch transcriptional activity compared to non-overexpressing cells. Trastuzumab or a dual epidermal growth factor receptor (EGFR)/ErbB-2 tyrosine kinase inhibitor (TKI) increased Notch activity by 2- to 6-fold in SKBr3, BT474 and MCF-7/HER2-18 cells. The increase in activity was abrogated by a Notch inhibitor, gamma-secretase inhibitor (GSI) or Notch-1 small-interfering RNA (siRNA). Trastuzumab decreased Notch-1trade mark precursor, increased amount and nuclear accumulation of active Notch-1(IC) and increased expression of targets, Hey1 and Deltex1 mRNAs, and Hes5, Hey1, Hes1 proteins. Importantly, trastuzumab-resistant BT474 cells treated with trastuzumab for 6 months expressed twofold higher Notch-1, twofold higher Hey1, ninefold higher Deltex1 mRNAs and threefold higher Notch-1 and Hes5 proteins, compared to trastuzumab-sensitive BT474 cells. The increase in Hey1 and Deltex1 mRNAs in resistant cells was abrogated by a Notch-1 siRNA. Cell proliferation was inhibited more effectively by trastuzumab or TKI plus a GSI than either agent alone. Decreased Notch-1 by siRNA increased efficacy of trastuzumab in BT474 sensitive cells and restored sensitivity in resistant cells. Trastuzumab plus a GSI increased apoptosis in sensitive cells by 20-30%. A GSI alone was sufficient to increase apoptosis in trastuzumab-resistant BT474 cells by 20%, which increased to 30% with trastuzumab. Notch-1 siRNA alone decreased cell growth by 30% in sensitive and more than 50% in resistant BT474 cells. Furthermore, growth of both trastuzumab sensitive and resistant cells was completely inhibited by combining trastuzumab plus Notch-1 siRNA. More importantly, Notch-1 siRNA or a GSI resensitized trastuzumab-resistant BT474 cells to trastuzumab. These results demonstrate that ErbB-2 overexpression suppresses Notch-1 activity, which can be reversed by trastuzumab or TKI. These results suggest that Notch-1 might play a novel role in resistance to trastuzumab, which could be prevented or reversed by inhibiting Notch-1.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Receptor Notch1/metabolismo , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Interferente Pequeno/farmacologia , Receptor ErbB-2/imunologia , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/fisiologia , Trastuzumab , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Estrogen suppression through the use of an aromatase inhibitor is an effective endocrine treatment option for postmenopausal breast cancer patients with estrogen receptor (ER)-positive disease, however, there are concerns that long-term estrogen deprivation will inevitably lead to resistance. To address the issue of acquired resistance to long-term estrogen deprivation our laboratory has developed an ER+/PR- hormone-independent breast cancer cell line, MCF-7:5C which is a variant clone of wild-type MCF-7 cells. Originally, these cells were cultured in estrogen-free MEM containing 5% charcoal-stripped calf serum and were found to be resistant to both estradiol (E(2)) and antiestrogens. Interestingly, a completely different phenomenon was observed when MCF-7:5C cells were cultured in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped fetal bovine serum (SFS). Using DNA quantitation assays, we examined the effect of E(2) on the growth of MCF-7:5C cells under different media conditions. Our results showed that 10(-9)M E(2) caused a dramatic 90% reduction in the growth of MCF-7:5C cells cultured in RPMI medium containing 10% SFS but did not have any significant inhibitory effects on cells cultured in MEM media. Additional experiments were performed to determine whether the medium or the serum facilitated the inhibitory effects of E(2) and the results indicated that it was the serum. Annexin V and DAPI staining confirmed that the E(2)-induced growth inhibition of MCF-7:5C cells was due to apoptosis. We also examined the tumorigenic potential of MCF-7:5C cells by injecting 1x10(7)cells/site into ovariectomized athymic mice and found that these cells, previously cultured in RPMI media, spontaneously grew into tumors in the absence of E(2). Overall, these results show that low concentrations (>10(-11)M) of E(2) are capable of inducing apoptosis in an aromatase resistant breast cancer cell model and that this effect is highly influenced by the medium in which the cells are grown.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Estrogênios/farmacologia , Anexina A5/análise , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultura , Estradiol/farmacologia , Feminino , Humanos , Cinética , Síndrome de Abstinência a SubstânciasRESUMO
The oestrogen receptor (ER) has proven to be an extraordinarily successful target for breast cancer treatment and prevention. The clinical use of tamoxifen, a nonsteroidal antioestrogen, demonstrated (1) that the strategic use of adjuvant tamoxifen in ER-positive patients could save lives and (2) that a selective ER modulator (SERM) could reduce the incidence of breast cancer in high-risk women. The ER is now the target for new and safer therapies such as the aromatase inhibitors and the pure antioestrogens that either block oestrogen synthesis or destroy the ER. However, the use of raloxifene, a SERM to prevent osteoporosis with the potential to prevent breast cancer has introduced a new dimension into preventive oncology. The widespread use of endocrine modulators (SERMs, aromatase inhibitors, and pure antioestrogens) raised the question of drug resistance. It is now clear that endocrine resistance can evolve through stages. Once a breast tumour becomes resistant to SERMs, the growth is stimulated by either the SERM or oestrogen. This is why an aromatase inhibitor is effective following SERM resistance and withdrawal. However, the extended use of repeated endocrine therapies now supersensitized the cells to oestrogen that causes apoptosis through the ER. We suggest that future clinical treatment strategies incorporate an 'oestrogen purge' to both enhance the actions of chemotherapy or completely reverse endocrine resistance and restore endocrine sensitivity. These new data build on the idea that breast cancer can be controlled as a chronic disease and will permit patients to live long and productive lives during targeted maintenance treatment.
Assuntos
Neoplasias da Mama/fisiopatologia , Receptores de Estrogênio/fisiologia , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/prevenção & controle , Resistência a Medicamentos , Feminino , Humanos , Fosforilação , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêuticoRESUMO
OBJECTIVE: Tamoxifen has mixed agonist/antagonist activities, leading to tissue-specific estrogen-like actions and endometrial cancer. The purpose of this study was to evaluate the effects of antiestrogens on the growth of estrogen receptor (ER)-positive ECC-1 endometrial cancer cells in vitro and in vivo. METHODS: We performed growth studies and luciferase assays using ERE-tK and AP-1 reporters. ERalpha protein expression was measured by Western blot after antiestrogen treatments. We investigated the actions of antiestrogens on the transcription of the pS2 gene in situ measured by Northern blot and the actions of antiestrogens on the VEGF protein secreted by ELISA. ERalpha, ERbeta, EGFR, and HER2/neu mRNAs were determined by RT-PCR. Last, ECC-1 tumors were developed by inoculation of cells into ovariectomized athymic mice and treated with estradiol (E2), tamoxifen, raloxifene, and a combination. RESULTS: E2 induced cell proliferation while antiestrogens did not. E2 and raloxifene down regulated ERalpha protein; in contrast, 4OHT did not. ICI182,780 completely degraded the receptor. ECC-1 cells express ERbeta at insignificant levels. Luciferase assays did not show any induction in ERE- nor AP-1-mediated transcription by antiestrogens. E2 caused a concentration-dependent increase in pS2 mRNA but antiestrogens did not. E2 increased VEGF expression in a dose-dependent manner and antiestrogens blocked E2 action. E2 down regulated HER2/neu while 4OHT and raloxifene did not change HER2/neu levels compared to control. In addition, EGFR mRNA was down regulated by E2 but raloxifene did not change it. Tamoxifen and raloxifene did not promote tumor growth in vivo. However, raloxifene (1.5 mg daily) only partially blocked E2-stimulated growth. CONCLUSION: Tamoxifen and raloxifene are antiproliferative agents and antiestrogens in ECC-1 endometrial cells in vitro and in vivo. The observation that selective estrogen-receptor modulators do not down regulate EGFR and HER2/neu mRNA may provide a potential role for these oncogenes in the development of raloxifene- or tamoxifen-stimulated endometrial cancer. The ECC-1 cell line could provide important new clues about the evolution of drug resistance to tamoxifen and raloxifene.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Adenocarcinoma/metabolismo , Animais , Northern Blotting , Divisão Celular/efeitos dos fármacos , Neoplasias do Endométrio/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Receptores ErbB/biossíntese , Receptores ErbB/genética , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Luciferases/biossíntese , Luciferases/genética , Luciferases/metabolismo , Linfocinas/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cloridrato de Raloxifeno/farmacologia , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator Trefoil-1 , Proteínas Supressoras de Tumor , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The insulin-like growth factor-II receptor (IGF-IIR) is frequently mutated or deleted in some malignant human tumors, suggesting that the IGF-IIR is a tumor suppressor. However, the exact mechanism by which IGF-IIR suppresses growth in tumors has not been definitively established. We demonstrate that IGF-IIR-deficient murine L cells (D9) have higher growth rates than IGF-IIR-positive L cells (Cc2) in response to IGF-II. IGF-II levels are higher in growth-conditioned medium from D9 versus Cc2 cells. Receptor neutralization studies and measurements of insulin receptor substrate 1 phosphorylation confirm that the enhanced growth of D9 cells is due to increased stimulation of the IGF-I and insulin receptors by IGF-II. In contrast, the levels of secreted latent and active transforming growth factor beta (TGF-beta) are similar for both D9 and Cc2 cells, indicating that the slower growth of Cc2 cells is not due to activation of latent TGF-beta by IGF-IIR and growth inhibition. The results directly demonstrate that down regulation of the IGF-IIR promotes the growth of transformed D9 cells by sustaining IGF-II, which binds to and activates IGF-IR and insulin receptor to increase intracellular growth signals.