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BACKGROUND: Squamous cell carcinoma (SCC) is the most common type of tongue and larynx cancer and a common type of lung cancer. In this study, we attempted to specifically evaluate the signaling pathway underlying HGF/Met induced EGFR ligand release in SSCs. The Met proto-oncogene encodes for a tyrosine kinase receptor which is often hyperactivated in human cancers. Met activation correlates with poor patient outcome. Several studies revealed a role of Met in receptor-crosstalk inducing either activation of other receptors, or inducing their resistance to targeted cancer treatments. In an epithelial tumor cell line screen we recently showed that the Met ligand HGF blocks the EGFR tyrosine kinase and at the same time activates transcriptional upregulation and accumulation in the supernatant of the EGFR ligand amphiregulin (Oncogene 32:3846-56, 2013). In the present work we describe the pathway responsible for the amphiregulin induction. FINDINGS: Amphiregulin is transcriptionally upregulated and is released into the supernatant. We show that Erk2 but not Erk1 mediates amphiregulin upregulation upon treatment with monocyte derived HGF. A siRNA knockdown of Erk2 completely abolishes amphiregulin release in squamous cell carcinomas. CONCLUSIONS: These results identify Erk2 as the key downstream signal transducer between Met activation and EGFR ligand upregulation in squamous cell carcinoma cell lines derived from tongue, larynx and lung.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Anfirregulina/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno , Modelos Biológicos , Comunicação Parácrina , Ligação Proteica , Proto-Oncogene Mas , Receptor ErbB-2/metabolismoRESUMO
BACKGROUND: Tumor-associated macrophages (TAM) promote malignant progression, yet the repertoire of oncogenic factors secreted by TAM has not been clearly defined. We sought to analyze which EGFR- and STAT3-activating factors are secreted by monocytes/macrophages exposed to tumor cell-secreted factors. METHODS: Following exposure of primary human monocytes and macrophages to supernatants of a variety of tumor cell lines, we have analyzed transcript and secreted protein levels of EGFR family ligands and of STAT3 activators. To validate our findings, we have analyzed TAM infiltration levels, systemic and local protein levels as well as clinical data of primary breast cancer patients. RESULTS: Primary human monocytes and macrophages respond to tumor cell-derived factors by secreting EGFR- and STAT3-activating ligands, thus inducing two important oncogenic pathways in carcinoma cells. Tumor cell-secreted factors trigger two stereotype secretory profiles in peripheral blood monocytes and differentiated macrophages: monocytes secrete epiregulin (EREG) and oncostatin-M (OSM), while macrophages secrete heparin-binding EGF-like growth factor (HB-EGF) and OSM. HB-EGF and OSM cooperatively induce tumor cell chemotaxis. HB-EGF and OSM are co-expressed by TAM in breast carcinoma patients, and plasma levels of both ligands correlate strongly. Elevated HB-EGF levels accompany TAM infiltration, tumor growth and dissemination in patients with invasive disease. CONCLUSIONS: Our work identifies systemic markers for TAM involvement in cancer progression, with the potential to be developed into molecular targets in cancer therapy.
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Neoplasias da Mama/sangue , Carcinoma/sangue , Quimiotaxia , Fator de Crescimento Epidérmico/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Oncostatina M/metabolismo , Neoplasias da Mama/patologia , Carcinoma/secundário , Linhagem Celular Tumoral , Células Cultivadas , Epirregulina , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Metástase Linfática , Macrófagos/metabolismo , Monócitos/metabolismo , Comunicação Parácrina , Fator de Transcrição STAT3/metabolismoRESUMO
The growing number of recently identified negative feedback regulators of receptor tyrosine kinases (RTKs) highlights the importance of signal attenuation and modulation for correct signaling outcome. Mitogen-inducible gene 6 (Mig6 also known as RALT or Gene 33) is a multiadaptor protein thought to be involved in the regulation of RTK and stress signaling. Here, we show that deletion of the mouse gene encoding Mig6 (designated Errfi1, which stands for ERBB receptor feedback inhibitor 1) causes hyperactivation of endogenous epidermal growth factor receptor (EGFR) and sustained signaling through the mitogen-activated protein kinase (MAPK) pathway, resulting in overproliferation and impaired differentiation of epidermal keratinocytes. Furthermore, Errfi1-/- mice develop spontaneous tumors in various organs and are highly susceptible to chemically induced formation of skin tumors. A tumor-suppressive role for Mig6 is supported by our finding that MIG6 is downregulated in various human cancers. Inhibition of endogenous Egfr signaling with the Egfr inhibitor gefitinib (Iressa) or replacement of wild-type Egfr with the kinase-deficient protein encoded by the hypomorphic Egfr(wa2) allele completely rescued skin defects in Erffi1-/- mice. Carcinogen-induced tumors displayed by Errfi1-/- mice were highly sensitive to gefitinib. These results indicate that Mig6 is a specific negative regulator of Egfr signaling in skin morphogenesis and is a novel tumor suppressor of Egfr-dependent carcinogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Morfogênese , Neoplasias/metabolismo , Pele/crescimento & desenvolvimento , Pele/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proliferação de Células , Gefitinibe , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Quinazolinas/metabolismo , Pele/citologia , Pele/metabolismoRESUMO
A major obstacle in the successful treatment of cancer is the occurrence of chemoresistance. Cancer cells surviving chemotherapy and giving rise to a recurrence of the tumor are termed cancer stem cells and can be identified by elevated levels of certain stem cell markers. Eradication of this cell population is a priority objective in cancer therapy. Here, we report elevated levels of stem cell markers in MCF-7 mammospheres. Likewise, an upregulation of HER2 and its differential expression within individual cells of mammospheres was observed. Sorting for HER2(high) and HER2(low) cells revealed an upregulation of stem cell markers NANOG, OCT4 and SOX2 in the HER2(low) cell fraction. Accordingly, HER2(low) cells also showed reduced proliferation, ductal-like outgrowths and an increased number of colonies in matrigel. Xenografts from subcutaneously injected HER2(low) sorted cells exihibited earlier onset but slower growth of tumors and an increase in stem cell markers compared to tumors developed from the HER2(high) fraction. Treatment of mammospheres with salinomycin reduced the expression of SOX2 indicating a selective targeting of cancer stem cells. Trastuzumab however, did not reduce the expression of SOX2 in mammospheres. Furthermore, a combinatorial treatment of mammospheres with trastuzumab and salinomycin was superior to single treatment with each drug. Thus, targeting HER2 expressing tumors with anti-HER2 therapies will not necessarily eliminate cancer stem cells and may lead to a more aggressive cancer cell phenotype. Our study demonstrates efficient killing of both HER2 positive cells and cancer stem cells, hence opening a possibility for a new combinatorial treatment strategy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sequência de Bases , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Primers do DNA , Feminino , Humanos , Piranos/administração & dosagem , Piranos/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , TrastuzumabRESUMO
[This corrects the article DOI: 10.18632/oncotarget.26817.].
RESUMO
PURPOSE: Establishment of antiapoptotic signaling pathways in tumor cells is a major cause for the failure of chemotherapy against cancer. To investigate the underlying mechanisms, we developed an experimental approach that is based on the genetic plasticity of cancer cells and the selection for cell survival on treatment with chemotherapeutic agents. EXPERIMENTAL DESIGN: Gene expression changes of surviving cell clones were analyzed by macroarrays. Involvement of fibroblast growth factor receptor 4 (FGFR4) in antiapoptotic pathways was elucidated by apoptosis assays, small interfering RNA experiments, and an antagonistic antibody. RESULTS: We show that FGFR4 gene expression is up-regulated in doxorubicin-treated, apoptosis-resistant cancer cell clones. Ectopic expression of FGFR4 in cancer cells led to reduced apoptosis sensitivity on treatment with doxorubicin or cyclophosphamide, whereas knockdown of endogenous FGFR4 expression in breast cancer cell lines had the opposite effect. FGFR4 overexpression resulted in Bcl-xl up-regulation at both mRNA and protein levels. Knockdown of FGFR4 expression by small interfering RNA caused a decrease in phospho-extracellular signal-regulated kinase 1/2 levels and reduced Bcl-xl expression. Moreover, an antagonistic FGFR4 antibody suppressed the resistance of cancer cells with endogenous FGFR4 expression against apoptosis-inducing chemotherapeutic agents. CONCLUSION: Based on these findings, we propose an antiapoptotic signaling pathway that is initiated by FGFR4 and regulating the expression of Bcl-xl through the mitogen-activated protein kinase cascade. Our findings are exemplary for a novel strategy toward the elucidation of diverse signaling pathways that define antiapoptotic potential in cancer cells. These observations open new avenues toward the diagnosis of chemoresistant tumors and therapies targeting FGFR4-overexpressing cancers.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Sistema de Sinalização das MAP Quinases , RNA Mensageiro/análise , Regulação para Cima , Proteína bcl-X/análiseRESUMO
Exposure to extensive ultraviolet (UV) rays is a major cause of skin cancer, which is thought to be initiated by DNA mutations. Members of the epidermal growth factor receptor (EGFR) family are important in various pathophysiologic processes like cancer and are shown to be phosphorylated upon UV exposure. Here we show that EGFR phosphorylation by modest UV doses is dependent on metalloprotease activity and resultant epidermal growth factor (EGF) family proligand shedding. This proligand cleavage releases the mature ligand, which then binds to and activates EGFR. We show that UV induced EGFR phosphorylation in transformed cell lines of melanocyte and keratinocyte origin, which was reduced upon preincubation with a broad-spectrum metalloprotease inhibitor, BB94. UV also activated EGFR downstream signaling via Erk and Akt pathways in a BB94-sensitive manner. Furthermore, using neutralizing antibodies we found that proligand amphiregulin was required for UV-induced EGFR activation in SCC-9 cells. Using RNAi this EGFR activation was further shown to depend on the metalloproteases ADAM9 and ADAM17 in SCC-9 cells. cDNA array hybridization and RT-PCR analysis showed overexpression of a Disintegrin and a Metalloproteases (ADAMs) and EGF family proligands in melanoma cell lines. Additionally, blocking EGFR signal transactivation by BB94 led to increased apoptosis in UV-irradiated cells. EGFR signal transactivation also led to increased stability of the DNA repair protein, PARP, under UV stress. Thus, both antiapoptotic and DNA repair pathways are activated simultaneously by EGFR signal transactivation. Together, our data provide novel insights into the mechanism of UV-induced EGFR activation, suggesting broad relevance of the UV-ADAM-proligand-EGFR-Erk/Akt pathway and its significance in skin cancer.
Assuntos
Receptores ErbB/genética , Receptores ErbB/efeitos da radiação , Metaloproteases/metabolismo , Neoplasias Cutâneas/metabolismo , Ativação Transcricional/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genéticaRESUMO
PURPOSE: The epidermal growth factor receptor family member HER3 is overexpressed in diverse human cancers and has been associated with poor prognosis in breast, lung, and ovarian cancer. However, the relevance of HER3 with regard to its prognostic significance and function in primary melanoma and metastases remains largely elusive. EXPERIMENTAL DESIGN: HER3 protein expression was analyzed immunohistochemically using tissue microarrays of 130 primary melanoma and 87 metastases relative to established clinical variables. The possibility of an influence of HER3 on melanoma cell proliferation, migration, invasion, and chemotherapy-induced apoptosis was studied in human melanoma cell lines. RESULTS: We show that HER3 is frequently expressed in malignant melanoma and metastases at elevated levels. High HER3 expression may serve as a prognostic marker because it correlates with cell proliferation, tumor progression, and reduced patient survival. Suppression of HER3 expression by RNA interference reduces melanoma cell proliferation, migration, and invasion in vitro. In addition, down-regulation of HER3 synergistically enhances dacarbazine-induced apoptosis. Moreover, monoclonal antibodies specific for the extracellular portion of HER3 efficiently block heregulin-induced proliferation, migration, and invasion of melanoma cell lines. CONCLUSION: Our results provide novel insights into the role of HER3 in melanoma and point out new possibilities for therapeutic intervention.
Assuntos
Biomarcadores Tumorais/análise , Melanoma/metabolismo , Melanoma/patologia , Receptor ErbB-3/biossíntese , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Melanoma/mortalidade , Pessoa de Meia-Idade , Prognóstico , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/mortalidade , Análise Serial de TecidosRESUMO
PURPOSE: The receptor tyrosine kinase Axl has recently been identified as a critical element in the invasive properties of glioma cell lines. However, the effect of Axl and its ligand growth arrest--specific gene 6 (Gas6) in human gliomas is still unknown. EXPERIMENTAL DESIGN: Axl and Gas6 expression was studied in 42 fresh-frozen and 79 paraffin-embedded glioma specimens by means of reverse transcription-PCR and immunohistochemistry. The prognostic value of Axl and Gas6 expression was evaluated using a population-based tissue microarray derived from a cohort of 55 glioblastoma multiforme (GBM) patients. RESULTS: Axl and Gas6 were detectable in gliomas of malignancy grades WHO 2 to 4. Moderate to high Axl mRNA expression was found in 61%, Axl protein in 55%, Gas6 mRNA in 81%, and Gas6 protein in 74% of GBM samples, respectively. GBM patients with high Axl expression and Axl/Gas6 coexpression showed a significantly shorter time to tumor progression and an association with poorer overall survival. Comparative immunohistochemical studies showed that Axl staining was most pronounced in glioma cells of pseudopalisades and reactive astrocytes. Additionally, Axl/Gas6 coexpression was observed in glioma cells and tumor vessels. In contrast, Axl staining was not detectable in nonneoplastic brain tissue and Gas6 was strongly expressed in neurons. CONCLUSIONS: In human gliomas, Axl and Gas6 are frequently overexpressed in both glioma and vascular cells and predict poor prognosis in GBM patients. Our results indicate that specific targeting of the Axl/Gas6 signaling pathway may represent a potential new approach for glioma treatment.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Intervalo Livre de Doença , Feminino , Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Análise Serial de Tecidos , Receptor Tirosina Quinase AxlRESUMO
The lung cancer stem cell (LuCSC) model comprises an attractive framework to explore acquired drug resistance in non-small cell lung cancer (NSCLC) treatment. Here, we used NSCLC cell line model to translate cellular heterogeneity into tractable populations to understand the origin of lung cancers and drug resistance. The epithelial LuCSCs, presumably arising from alveolar bipotent stem/progenitor cells, were lineage naïve, noninvasive, and prone to creating aggressive progeny expressing AT2/AT1 markers. LuCSC-holoclones were able to initiate rimmed niches, where their specialization created pseudo-alveoli structures. Mechanistically, LuCSC transitioning from self-renewal (ß-catenin and Nanog signaling) to malignant lineage differentiation is regulated by EGFR activation and the inverse inhibition of tumor suppressor MIG6. We further identified the functional roles of endogenous EGFR signaling in mediating progeny invasiveness and their ligands in LuCSC differentiation. Importantly, drug screening demonstrated that EGFR driving progeny were strongly responsive to TKIs; however, the LuCSCs were exclusively resistant but sensitive to AMPK agonist Metformin, antibiotic Salinomycin and to a lesser degree Carboplatin. Our data reveals previously an unknown mechanism of NSCLC resistance to EGFR-TKIs, which is associated with LuCSCs bearing a silenced EGFR and inversely expressed MIG6 suppressor gene. Taken altogether, successful NSCLC treatment requires development of a novel combination of drugs, efficiently targeting both LuCSCs and heterogeneous progeny.
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Clinical investigations of an FGFR4 germline polymorphism, resulting in substitution of glycine by arginine at codon 388 (G388 to R388), have shown a correlation between FGFR4 R388 and aggressive disease progression in cancer patients. Here, we studied the differential effects of the two FGFR4 isotypes on cellular signalling and motility in the MDA-MB-231 human breast cancer cell model. cDNA array analysis showed the ability of FGFR4 G388 to suppress expression of specific genes involved in invasiveness and motility. Further investigations concentrating on cell signalling and motility revealed an abrogation of phosphatidylinositol-3-kinase-dependent LPA-induced Akt activation and cell migration due to downregulation of the LPA receptor Edg-2 in FGFR4 G388-expressing MDA-MB-231 cells. Moreover, FGFR4 G388 expression attenuated the invasivity of the breast cancer cell line and decreased small Rho GTPase activity. We conclude that FGFR4 G388 suppresses cell motility of invasive breast cancer cells by altering signalling pathways and the expression of genes that are required for metastasis. Therefore, the positive effect of FGFR4 R388 on disease progression appears to result from a loss of the tumour suppressor activity displayed by FGFR4 G388 rather than the acquisition or enhancement of oncogenic potential.
Assuntos
Neoplasias da Mama/fisiopatologia , Movimento Celular/genética , Polimorfismo de Nucleotídeo Único , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Alelos , Substituição de Aminoácidos , Arginina/genética , Neoplasias da Mama/genética , Linhagem Celular , Células Cultivadas , Progressão da Doença , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Glicina/genética , Humanos , Isoformas de Proteínas , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/fisiologia , Transdução de SinaisRESUMO
The biologic plausibility of an association between type 2 diabetes mellitus (T2D) and lung cancer has received increasing attention, but the results of investigations remain largely inconclusive. In the present study we investigated the influence of the anti-diabetic drug metformin on the cytotoxic effects of EGFR targeted therapy and chemotherapy in 7 non-small cell lung cancer (NSCLC) cell lines and a cohort of lung cancer patients with/without T2D. In vitro cell viability assays indicated that metformin didn't potentiate the growth inhibitory effects of erlotinib at different doses in cell lines that are of distinct genetic background. EGFR downstream signaling evaluation further demonstrated that metformin, at its IC50 value, modified apoptosis caused in erlotinib or chemotherapeutic agent-treated cells via AKT activation and the inhibition of caspase 3 and PARP cleavages. These regulations were driven independently from EGFR, LKB1, KRAS, PTEN and p53 status. Metformin triggered autophagy (LC3B expression) was identified to interplay with apoptosis to attenuate the drug effect and postpone cancer cell death. In the retrospective study of 8 NSCLC patients, the administration of metformin did not induce statistically significant changes as assessed by immunohistochemical staining of pERK, pAKT and cleaved PARP. Consequently, the application of metformin for T2D NSCLC patients receiving chemo or EGFR targeted therapy should be considered with caution.
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Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Receptores ErbB/genética , Metformina/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/complicações , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/administração & dosagem , Humanos , Camundongos , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Signal transduction via tyrosine phosphorylation, normally fine-tuned by the concerted action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is a key mechanism in tumorigenesis. PTP-PEST, a ubiquitously expressed cytoplasmic tyrosine phosphatase, is thought to play an important role in cell adhesion and motility, and may be involved in metastasis. A search for sequence variations within the gene PTPN12 (alias PTP-PEST) was performed in breast cancer cell lines, leading to the identification of three amino acid substitutions at positions 322, 573, and 709. These alterations were also found in squamous cell carcinoma cell lines and could be verified in primary human breast and kidney tumor samples. Analysis of peripheral blood samples confirmed the germline origin of these alterations. Furthermore, functional characterization of the Ile322 and Ala573 PTP-PEST mutants revealed an enhancement of in vitro phosphatase activity, whereas the Lys709 variant showed reduced catalytic activity. These data demonstrate the existence of PTP-PEST variants that might be meaningful for human cancer and underscore the need for further characterizing PTP-PEST and its signaling pathways in context of this disease.
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Neoplasias da Mama/enzimologia , Variação Genética , Proteínas Tirosina Fosfatases/genética , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 12RESUMO
Luminal A breast cancer is the most common breast cancer subtype which is usually treated with selective estrogen receptor modulators (SERMS) like tamoxifen. Nevertheless, one third of estrogen receptor positive breast cancer patients initially do not respond to endocrine therapy and about 40% of luminal A breast tumors recur in five years. In this study, we investigated an alternative treatment approach by combining tamoxifen and salinomycin in luminal A breast cancer cell lines. We have found that salinomycin induces an additional cytotoxic effect by inhibiting the ligand independent activation of ERα. Thereby salinomycin increases the intracellular calcium level. This leads to a premature fusion of endosomes with lysosomes and thus to the degradation of Egfr family members. Since this process is essential for luminal A breast cancer cells to circumvent tamoxifen treatment, the combination of both drugs induces cytotoxicity in tamoxifen sensitive as well as resistant luminal A breast cancer cell lines.
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Neoplasias da Mama/tratamento farmacológico , Lisossomos/efeitos dos fármacos , Piranos/administração & dosagem , Receptores Proteína Tirosina Quinases/metabolismo , Tamoxifeno/administração & dosagem , Transporte Biológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Endossomos/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Concentração Inibidora 50 , Ligantes , Lisossomos/metabolismo , Células MCF-7 , Fenobarbital , Moduladores Seletivos de Receptor Estrogênico/farmacologiaRESUMO
The presence of cancer stem cells (CSCs) is linked to preexisting or acquired drug resistance and tumor relapse. Therefore, targeting both differentiated tumor cells and CSCs was suggested as an effective approach for non-small cell lung cancer (NSCLC) treatment. After screening of chemotherapeutic agents, tyrosine kinase inhibitors (TKIs) or monoclonal antibody in combination with the putative stem cell killer Salinomycin (SAL), we found Metformin (METF), which modestly exerted a growth inhibitory effect on monolayer cells and alveospheres/CSCs of 5 NSCLC cell lines regardless of their EGFR, KRAS, EML4/ALK and LKB1 status, interacted synergistically with SAL to effectively promote cell death. Inhibition of EGFR (AKT, ERK1/2) and mTOR (p70 s6k) signaling with the combination of METF and SAL can be augmented beyond that achieved using each agent individually. Phospho-kinase assay further suggested the multiple roles of this combination in reducing oncogenic effects of modules, such as ß-catenin, Src family kinases (Src, Lyn, Yes), Chk-2 and FAK. Remarkably, significant reduction of sphere formation was seen under combinatorial treatment in all investigated NSCLC cell lines. In conclusion, METF in combination with SAL could be a promising treatment option for patients with advanced NSCLC irrespective of their EGFR, KRAS, EML4/ALK and LKB1 status.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Quinases Proteína-Quinases Ativadas por AMP , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metformina/administração & dosagem , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Piranos/administração & dosagem , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Protein Tyrosin Kinase 7 (PTK7) is upregulated in several human cancers; however, its clinical implication in breast cancer (BC) and lymph node (LN) is still unclear. In order to investigate the function of PTK7 in mediating BC cell motility and invasivity, PTK7 expression in BC cell lines was determined. PTK7 signaling in highly invasive breast cancer cells was inhibited by a dominant-negative PTK7 mutant, an antibody against the extracellular domain of PTK7, and siRNA knockdown of PTK7. This resulted in decreased motility and invasivity of BC cells. We further examined PTK7 expression in BC and LN tissue of 128 BC patients by RT-PCR and its correlation with BC related genes like HER2, HER3, PAI1, MMP1, K19, and CD44. Expression profiling in BC cell lines and primary tumors showed association of PTK7 with ER/PR/HER2-negative (TNBC-triple negative BC) cancer. Oncomine data analysis confirmed this observation and classified PTK7 in a cluster with genes associated with agressive behavior of primary BC. Furthermore PTK7 expression was significantly different with respect to tumor size (ANOVA, pâ=â0.033) in BC and nodal involvement (ANOVA, pâ=â0.007) in LN. PTK7 expression in metastatic LN was related to shorter DFS (Cox Regression, pâ=â0.041). Our observations confirmed the transforming potential of PTK7, as well as its involvement in motility and invasivity of BC cells. PTK7 is highly expressed in TNBC cell lines. It represents a novel prognostic marker for BC patients and has potential therapeutic significance.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/genética , Transformação Celular Neoplásica/genética , Receptores Proteína Tirosina Quinases/genética , Adulto , Idoso , Biomarcadores Tumorais , Neoplasias da Mama/mortalidade , Neoplasias da Mama Masculina/genética , Neoplasias da Mama Masculina/mortalidade , Neoplasias da Mama Masculina/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Análise por Conglomerados , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , PrognósticoRESUMO
Biomarkers predicting resistance to particular chemotherapy regimens could play a key role in optimally individualized treatment concepts. PTK7 (protein tyrosine kinase 7) belongs to the receptor tyrosine kinase family involved in several physiological, but also malignant, cell behaviors. Recent studies in acute myeloid leukemia have associated PTK7 expression with resistance to anthracycline therapy. PTK7 mRNA expression in primary tumor tissue (PTT) and corresponding lymph node tissue (LNT) were retrospectively measured in 117 patients with early breast cancer; PTK7 expression was available in 103 PTT and 108 LNT samples. Median age was 60 years (range, 27-87 years). At a median follow-up of 28.5 months, 6 deaths and 16 recurrences had occurred. PTK7 expression correlations with clinicopathological features were computed and PTK7 expression effects on patient outcome were analyzed in three cohorts defined by adjuvant treatment: anthracycline-based treatment, other chemotherapy regimens (including taxane or other substances), or no chemotherapy. Association of PTK7 expression with clinicopathological features was seen only for age in PTT and nodal stage in LNT. High LN PTK7 was associated with poorer disease-free survival (DFS) in the total population (3-year DFS: low [81.7%] versus high [70.4%]; P=0.016) and in patients without adjuvant chemotherapy (3-year DFS: low [91.7%] versus high [22.3%]; P<0.001), but not in patients receiving adjuvant chemotherapy (P=0.552). DFS stratified by PTK7 expression was compared in treatment cohorts: In patients with low LN PTK7 expression, neither chemotherapy cohort showed significantly better survival than the no-chemotherapy cohort. In patients with high LN PTK7 expression, those receiving chemotherapy, including substances other than anthracyclines, but not those receiving only anthracycline-based chemotherapy, showed significantly better DFS than those receiving no chemotherapy (P=0.001). Our results support earlier findings that PTK7 may be a prognostic and predictive marker associated with resistance to anthracycline-based chemotherapy. Further investigations are needed to validate these findings in breast cancer.
RESUMO
Acquiring therapy resistance is one of the major obstacles in the treatment of patients with cancer. The discovery of the cancer stem cell (CSC)-specific drug salinomycin raised hope for improved treatment options by targeting therapy-refractory CSCs and mesenchymal cancer cells. However, the occurrence of an acquired salinomycin resistance in tumor cells remains elusive. To study the formation of salinomycin resistance, mesenchymal breast cancer cells were sequentially treated with salinomycin in an in vitro cell culture assay, and the resulting differences in gene expression and salinomycin susceptibility were analyzed. We demonstrated that long-term salinomycin treatment of mesenchymal cancer cells resulted in salinomycin-resistant cells with elevated levels of epithelial markers, such as E-cadherin and miR-200c, a decreased migratory capability, and a higher susceptibility to the classic chemotherapeutic drug doxorubicin. The formation of salinomycin resistance through the acquisition of epithelial traits was further validated by inducing mesenchymal-epithelial transition through an overexpression of miR-200c. The transition from a mesenchymal to a more epithelial-like phenotype of salinomycin-treated tumor cells was moreover confirmed in vivo, using syngeneic and, for the first time, transgenic mouse tumor models. These results suggest that the acquisition of salinomycin resistance through the clonal selection of epithelial-like cancer cells could become exploited for improved cancer therapies by antagonizing the tumor-progressive effects of epithelial-mesenchymal transition.
RESUMO
BACKGROUND: Protein tyrosine kinase-7 (PTK7) plays an important role in cancer. Our aim was to evaluate PTK7 in triple-negative breast cancer (TNBC). MATERIALS AND METHODS: PTK7 Expression was assessed by immunohistochemistry (IHC) in 133 patients with TNBC. Expression levels were correlated with clinicopathological features and survival, taking chemotherapy into account. RESULTS: Positive PTK7 expression was detected in 28.6% of tumors. In the total population, no significant difference was detected in disease-free survival (DFS) or overall survival (OS) related to PTK7 status. However, in patients treated by chemotherapy, patients with PTK7-negative tumors seemed to have better DFS than those with PTK7-positive tumors, particularly for patients treated with only anthracycline-therapy drugs. In patients receiving other chemotherapy regimens, PTK7 status had no significant impact on DFS or OS. CONCLUSION: Our results support earlier findings that PTK7 may be associated with resistance to anthracycline-based chemotherapy.
Assuntos
Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Pessoa de Meia-Idade , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Estudos RetrospectivosRESUMO
Protein tyrosine kinases (PTKs) play a critical role in the manifestation of cancer cell properties, and respective signaling mechanisms have been studied extensively on immortalized tumor cells. To characterize and analyze commonly used cancer cell lines with regard to variations in the primary structure of all expressed PTKs, we conducted a cDNA-based sequence analysis of the entire tyrosine kinase transcriptome of 254 established tumor cell lines. The profiles of cell line intrinsic PTK transcript alterations and the evaluation of 155 identified polymorphisms and 234 somatic mutations are made available in a database designated "Tykiva" (tyrosine kinome variant). Tissue distribution analysis and/or the localization within defined protein domains indicate functional relevance of several genetic alterations. The cysteine replacement of the highly conserved Y367 residue in fibroblast growth factor receptor 4 or the Q26X nonsense mutation in the tumor-suppressor kinase CSK are examples, and may contribute to cell line-specific signaling characteristics and tumor progression. Moreover, known variants, such as epidermal growth factor receptor G719S, that were shown to mediate anticancer drug sensitivity could be detected in other than the previously reported tumor types. Our data therefore provide extensive system information for the design and interpretation of cell line-based cancer research, and may stimulate further investigations into broader clinical applications of current cancer therapeutics.