RESUMEN
INTRODUCTION: To investigate the expression of EPLIN-alpha, epithelial protein lost in neoplasm, in human breast cancer tissues/cells and investigate the cellular impact of EPLIN-alpha on breast cancer cells. EXPERIMENTAL DESIGN: EPLIN-alpha was determined in tumour (n = 120) and normal mammary tissues (n = 32), and cancer cell lines (n = 16). Cell invasion, in vitro and in vivo growth of cells transfected with EPLIN-alpha were evaluated using in vitro invasion assay, in vitro and in vivo tumour model. Cellular migration was analysed using Electric Cell Impedance Sensing assays. RESULTS: Low level of EPLIN-alpha was seen in tumour tissues. Grade-2/3 tumours had significantly lower levels of EPLIN-alpha compared with grade-1 (p = 0.047 and p = 0.046 vs grade-1, respectively). Patients with poor prognosis had a significantly lower levels of EPLIN-alpha compared with those with good prognosis (p = 0.0081). Patients who developed recurrence and died of breast cancer had significantly lower levels of EPLIN-alpha compared with those who remained disease free (p = 0.0003 and p = 0.0008, respectively) (median follow-up 10 years). Patients with high levels of EPLIN-alpha transcript had a longer survival than those with low levels. Over-expression of EPLIN-alpha in breast cancer cells by way of transfection rendered cells less invasive, less motile and growing at a slower pace in vitro and in vivo. An ERK inhibitor was shown to be able to abolish the effect of EPLIN expression. CONCLUSION: It is concluded that expression of EPLIN-alpha in breast cancer is down-regulated in breast cancer cells and tissues, a change linked to the prognosis. EPLIN-alpha acts as a potential tumour suppressor by inhibition of growth and migration of cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas del Citoesqueleto/genética , Regulación Neoplásica de la Expresión Génica , Animales , Movimiento Celular , Estudios de Cohortes , Proteínas del Citoesqueleto/metabolismo , Regulación hacia Abajo , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Invasividad NeoplásicaRESUMEN
Both HGF and BMP-7 have been implicated in the prostate cancer, particularly in bone metastasis. We recently demonstrated an up-regulation of BMP receptors by HGF in prostate cancer cells. Whether HGF has an effect on BMP-7 is still unknown. In the current study, we investigated the effects of HGF on the expression of BMP-7 in prostate cancer cells. Human prostate cancer cells, PC-3 and DU-145, were exposed to HGF at different concentrations for up to 24 hours. The mRNA levels of BMP7 were detected using RT-PCR and Quantitative PCR, and protein levels using Western blotting and immunocytochemistry. The levels of the BMP-7 transcripts in both PC-3 and DU-145 cells were up-regulated by the treatment with HGF in a concentration dependent and time related manner. The rise in BMP-7 mRNA was accompanied by an increase in protein levels, an effect completely blocked by the HGF antagonist, NK4. We further assessed this effect in an in vivo murine tumor model and showed that HGF up-regulated BMP-7 in prostate tumors. The antagonist of HGF, NK4 similarly blocked the induction of BMP-7 by HGF under the in vivo conditions. Taken together, HGF/SF can regulate BMP-7 expression in prostate cancer cells, both in vitro and in vivo. It may have a significant influence on the progression of prostate cancer and/or the development of bone metastasis.
Asunto(s)
Proteínas Morfogenéticas Óseas/biosíntesis , Factor de Crecimiento de Hepatocito/fisiología , Neoplasias de la Próstata/patología , Factor de Crecimiento Transformador beta/biosíntesis , Regulación hacia Arriba , Proteína Morfogenética Ósea 7 , Humanos , Masculino , Células Tumorales CultivadasRESUMEN
PURPOSE: We investigated the effect of manipulating endogenous BMP-7 expression on the invasion and motility of prostate cancer cells and the resulting effect on its antagonists using a ribozyme transgene. MATERIALS AND METHODS: A hammerhead ribozyme transgene was synthesized and cloned into a mammalian expression vector (pcDNA3.1/nt-GFP-TOPO). PC-3 cells (American Type Culture Collection, Manassas, Virginia) were transfected with the ribozyme transgene (PC-3(DeltaBMP7)) or with an empty plasmid (PC-3(pcDNA/GFP)) by electroporation. Invasion and motility were accessed by in vitro invasion and motility assays. RESULTS: The ribozyme decreased BMP-7 expression at the mRNA and protein levels in PC-3 cells. Invasive potential was significantly increased following the loss of BMP-7 expression. The mean +/- SD invading cell number for PC-3(DeltaBMP7) was 231.3 +/- 28.6 vs 7.1 +/- 4.4 for the WT cell line PC-3(WT) and 2.7 +/- 2 for PC-3(pcDNA/GFP) (each p <0.001). BMP-7 knockdown in PC-3 cells significantly increased motility with a migrating cell number for PC-3(DeltaBMP7) of 24 +/- 7.5 compared with 10.2 +/- 4.5 for PC-3(WT) and 11.3 +/- 7.5 for PC-3(pcDNA/GFP) (p <0.01 and 0.011, respectively). The change in motility was seen together with changes in the cellular location of paxillin and focal adhesion kinase (p125(FAK)). Interestingly the loss of BMP-7 resulted in decreased noggin and follistatin expression. CONCLUSIONS: The loss of endogenous BMP-7 from prostate cancer cells is associated with increased invasiveness and motility, which appears to be facilitated by changes in the level of the BMP antagonists noggin and follistatin. Endogenous BMP-7 has an important role in controlling noggin and follistatin expression.
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Movimiento Celular , Neoplasias de la Próstata/fisiopatología , Factor de Crecimiento Transformador beta/fisiología , Animales , Proteína Morfogenética Ósea 7 , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/fisiología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Adhesión Celular/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Folistatina/metabolismo , Folistatina/farmacología , Humanos , Masculino , Invasividad Neoplásica/fisiopatología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Catalítico/genética , Transfección , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Hepatocyte growth factor (HGF) plays multiple roles in cancer, by acting as a motility, invasion and angiogenesis stimulating factor, which promotes metastasis and tumour growth. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily. The effects of BMPs are mediated by two subgroups of receptors, type I and type II. Recent studies have shown that some BMPs, via their signaling pathways, affect the growth of prostate cancer cells. BMPR-IB and BMPR-II have been reported to be expressed at low levels in prostate cancer. However, little is known about the crosstalk between HGF and BMP pathways. In this study, prostate cancer cells (PC-3 and DU-145) were exposed to HGF at different concentrations (1-75 ng/ml) for 18 h, or were treated with HGF at 40 ng/ml over various time periods (up to 24 h). The effect of HGF on BMP receptor expression was further investigated in a nude mouse PC-3 xenograft model. Mice were treated with either HGF, the HGF antagonist NK4, or a combination of both. The expression of BMPR-IB and BMPR-II mRNA was up-regulated by HGF, as shown by both conventional PCR and quantitative PCR. An elevation of BMPR-IB and BMPR-II at the protein level was confirmed by both Western blot analysis and immunocytochemical staining. In a murine prostate tumour model, infusion of recombinant HGF resulted in an increase in the levels of both BMPR-IB and BMPR-II transcript in prostate tumours. Concomitant delivery of NK4, an HGF antagonist, prevented this effect. In conclusion, HGF up-regulates the expression of the bone morphogenetic protein receptors, BMPR-IB and BMPR-II, in prostate cancer cells, both in vitro and in vivo. This may have important implications in the development of bone metastasis in prostate cancer.