RESUMEN
Within tumors there appears to be an intricate balance between hyaluronan (HA) synthesis and degradation where the invading edges display increased HA metabolism. The metabolism of HA has not been characterized in breast cancer cell lines; therefore, this study quantitatively identifies and characterizes the enzymes responsible for the synthesis and degradation of HA while correlating gene expression to cancer cell invasiveness and HA receptor status. In ten well-established breast cancer cell lines, the expression of the genes for each hyaluronan synthase (HAS) and hyaluronidase (Hyal) isoform was quantitated using real-time and reverse transcriptase polymerase chain reaction (PCR). The synthesis and degradation rates of hyaluronan were determined by ELISA, while quantitation of HA receptors, CD44 and RHAMM was performed by comparative Western blotting. The molecular weight of HA synthesized by each HAS isoform and the degradation products of each hyaluronidase were characterized by size exclusion chromatography. It was demonstrated that highly invasive cell lines preferentially expressed the HAS2 and Hyal-2 isoforms, while less invasive cells expressed HAS3 and Hyal-3. There was a correlation between elevated levels of HA synthesis, CD44 expression and cancer cell migration thereby highlighting the pivotal role that HA metabolism plays in the aggressive breast cancer phenotype.
Asunto(s)
Neoplasias de la Mama/metabolismo , Moléculas de Adhesión Celular/metabolismo , Glucuronosiltransferasa/metabolismo , Receptores de Hialuranos/metabolismo , Hialuronoglucosaminidasa/metabolismo , Invasividad Neoplásica , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Proteínas Ligadas a GPI , Expresión Génica , Glucuronosiltransferasa/genética , Glicocálix/química , Glicocálix/metabolismo , Humanos , Hialuronano Sintasas , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Peso Molecular , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Regulación hacia ArribaRESUMEN
The progression of several cancers is correlated with the increased synthesis of the glycosaminoglycan, hyaluronan. Hyaluronan is synthesized at the plasma membrane by various isoforms of hyaluronan synthases (HAS). The importance of HAS2 expression in highly invasive breast cancer was characterized by the antisense inhibition of HAS2 (ASHAS2). The effect of HAS2 inhibition on cell proliferation, migration, hyaluronan metabolism, and receptor status was characterized in vitro, whereas the effect on tumorigenicity and metastasis was established in vivo. HAS2 inhibition resulted in a 24-hour lag in proliferation that was concomitant to transient arrest of 79% of the cell population in G0-G1. Inhibition of HAS2 did not alter the expression of the other HAS isoforms, whereas hyaluronidase (HYAL2) and the hyaluronan receptor, CD44, were significantly down-regulated. ASHAS2 cells accumulated greater amounts of high molecular weight hyaluronan (>10,000 kDa) in the culture medium, whereas mock and parental cells liberated less hyaluronan of three distinct molecular weights (100, 400, and 3,000 kDa). The inhibition of HAS2 in the highly invasive MDA-MB-231 breast cancer cell line inhibited the initiation and progression of primary and secondary tumor formation following s.c. and intracardiac inoculation into nude mice, whereas controls readily established both primary and secondary tumors. The lack of primary and secondary tumor formation was manifested by increased survival times where ASHAS2 animals survived 172% longer than the control animals. Collectively, these unique results strongly implicate the central role of HAS2 in the initiation and progression of breast cancer, potentially highlighting the co-dependency between HAS2, CD44, and HYAL2 expression.
Asunto(s)
Adenocarcinoma/enzimología , Neoplasias de la Mama/enzimología , ADN sin Sentido/genética , Glucuronosiltransferasa/antagonistas & inhibidores , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/terapia , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Progresión de la Enfermedad , Femenino , Fase G1/fisiología , Terapia Genética/métodos , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Ácido Hialurónico/metabolismo , Ratones , Ratones Endogámicos CBA , Ratones Desnudos , Fase de Descanso del Ciclo Celular/fisiología , TransfecciónRESUMEN
The concentration and molecular weight of hyaluronan often dictates its physiological function. Consequently full characterisation of the anabolic products and turnover rates of HA could facilitate understanding of the role that HA metabolism plays in disease processes. In order to achieve this it is necessary to interrupt the dynamic balance between concurrent HA synthesis and degradation, achievable through the inhibition of the hyaluronidases, a group of enzymes which degrade HA. The sulphated polysaccharide, dextran sulphate has been demonstrated to competitively inhibit testicular hyaluronidase in a non-biological system, but its application to in vitro biological systems had yet to be developed and evaluated. This study determined the inhibitory concentrations of dextran sulphate against both testicular and Streptomyces hyaluronidase in a cell-free and breast cancer model followed by characterisation of the effect that hyaluronidase inhibition exerted on HA synthesis and degradation. The IC(100) of dextran sulphate for both hyaluronidases in a cell-free and biological system was determined to be >or=400 microg/ml. At concentrations up to 10 mg/ml the dextran sulphate did not effect breast cancer cell proliferation or morphology, while at 400 microg/ml HA degradation was totally inhibited, enabling an accurate quantitation of HA production as well as characterisation of the cell-associated and liberated HA. FACS quantitation of the HA receptor CD44, HA synthase and the hyaluronidases HYAL 1 and HYAL 2 demonstrated that dextran sulphate down-regulated CD44 and HA synthase while upregulating the hyaluronidases. These results suggest dynamic feedback signalling and complex mechanisms occur in the net deposition of HA in vivo.