RESUMEN
Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1 was bioactive as shown by its proper inhibitory effect on MMP-2 activity, and its stimulatory effect on cell growth when added to the growth medium of four different breast cancer cell lines. This study provides an easy set-up for large scale production and purification of bioactive, tagged recombinant human TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.
Asunto(s)
Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Secuencia de Aminoácidos , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Femenino , Expresión Génica , Células HEK293 , Humanos , Células MCF-7 , Inhibidores de la Metaloproteinasa de la Matriz , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Inhibidor Tisular de Metaloproteinasa-1/biosíntesisRESUMEN
BACKGROUND: Worldwide more than one million women are annually diagnosed with breast cancer. A considerable fraction of these women receive systemic adjuvant therapy; however, some are cured by primary surgery and radiotherapy alone. Prognostic biomarkers guide stratification of patients into different risk groups and hence improve management of breast cancer patients. Plasma levels of Matrix Metalloproteinase-9 (MMP-9) and its natural inhibitor Tissue inhibitor of metalloproteinase-1 (TIMP-1) have previously been associated with poor patient outcome and resistance to certain forms of chemotherapy. To pursue additional prognostic information from MMP-9 and TIMP-1, the level of the MMP-9 and TIMP-1 complex (MMP-9:TIMP-1) was investigated in plasma from breast cancer patients. METHODS: Detection of protein:protein complexes in plasma was performed using a commercially available ELISA kit and, for the first time, the highly sensitive in-solution proximity ligation assay (PLA). We screened plasma from 465 patients with primary breast cancer for prognostic value of the MMP-9:TIMP-1 complex. Both assays were validated and applied for quantification of MMP-9:TIMP-1 concentration. In this retrospective study, we analyzed the association between the concentration of the MMP-9:TIMP-1 complex and clinicopathological data and disease free survival (DFS) in univariate and multivariate survival analyses. RESULTS: Following successful validation both assays were applied for MMP-9:TIMP-1 measurements. Of the clinicopathological parameters, only menopausal status demonstrated significant association with the MMP-9:TIMP-1 complex; P = 0.03 and P = 0.028 for the ELISA and PLA measurements, respectively. We found no correlation between the MMP-9:TIMP-1 protein complex and DFS neither in univariate nor in multivariate survival analyses. CONCLUSIONS: Despite earlier reports linking MMP-9 and TIMP-1 with prognosis in breast cancer patients, we here demonstrate that plasma levels of the MMP-9:TIMP-1 protein complex hold no prognostic information in primary breast cancer as a stand-alone marker. We demonstrate that the highly sensitive in-solution PLA can be employed for measurements of protein:protein complexes in plasma.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Carcinoma Ductal de Mama/sangre , Metaloproteinasa 9 de la Matriz/sangre , Inhibidor Tisular de Metaloproteinasa-1/sangre , Anciano , Análisis Químico de la Sangre/normas , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Ensayo de Inmunoadsorción Enzimática/normas , Femenino , Humanos , Límite de Detección , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Modelos de Riesgos Proporcionales , Estándares de Referencia , Estudios RetrospectivosRESUMEN
High levels of Tissue Inhibitor of Metalloproteinases-1 (TIMP1) are associated with poor prognosis, reduced response to chemotherapy, and, potentially, also poor response to endocrine therapy in breast cancer patients. Our objective was to further investigate the hypothesis that TIMP1 is associated with endocrine sensitivity. We established a panel of 11 MCF-7 subclones with a wide range of TIMP1 mRNA and protein expression levels. Cells with high expression of TIMP1 versus low TIMP1 displayed significantly reduced sensitivity to the antiestrogen fulvestrant (ICI 182,780, Faslodex®), while TIMP1 levels did not influence the sensitivity to 4-hydroxytamoxifen. An inverse correlation between expression of the progesterone receptor and TIMP1 was found, but TIMP1 levels did not correlate with estrogen receptor levels or growth-promoting effects of estrogen (estradiol, E2). Additionally, the effects of fulvestrant, 4-hydroxytamoxifen, or estrogen on estrogen receptor expression were not associated with TIMP1 levels. Gene expression analyses revealed associations between expression of TIMP1 and genes involved in metabolic pathways, epidermal growth factor receptor 1/cancer signaling pathways, and cell cycle. Gene and protein expression analyses showed no general defects in estrogen receptor signaling except from lack of progesterone receptor expression and estrogen inducibility in clones with high TIMP1. The present study suggests a relation between high expression level of TIMP1 and loss of progesterone receptor expression combined with fulvestrant resistance. Our findings in vitro may have clinical implications as the data suggest that high tumor levels of TIMP1 may be a predictive biomarker for reduced response to fulvestrant.
Asunto(s)
Resistencia a Antineoplásicos/genética , Estradiol/análogos & derivados , Regulación Neoplásica de la Expresión Génica , Receptores de Progesterona/genética , Inhibidor Tisular de Metaloproteinasa-1/genética , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Células Clonales/metabolismo , Análisis por Conglomerados , Regulación hacia Abajo , Estradiol/farmacología , Femenino , Fulvestrant , Humanos , Células MCF-7 , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Progesterona/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence 418KRPR421, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin beta/alpha1,3,7 whereas hMSH2 specifically recognizes importin beta/alpha3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.