Detectable neonatal Nav1.5 (nNav1.5) expression in tumour breast tissue positive for lymph node metastasis and triple-negative subtype serves as a valid tumour-associated antigen to target and prevent breast cancer invasion and metastasis. Therapeutic antibodies against tumour antigens have become the predominant class of new drugs in cancer therapy because of their fewer adverse effects and high specificity.This study was designed to investigate the therapeutic and anti-metastatic potential of the two newly obtained anti-nNav1.5 antibodies, polyclonal anti-nNav1.5 (pAb-nNav1.5) and monoclonal anti-nNav1.5 (mAb-nNav1.5), on breast cancer invasion and metastasis.MDA-MB-231 and 4T1 cells were used as in vitro models to study the effect of pAb-nNav1.5 (59.2 µg/ml) and mAb-nNav1.5 (10 µg/ml) (24 hours treatment) on cell invasion. 4T1-induced mammary tumours in BALB/c female mice were used as an in vivo model to study the effect of a single dose of intravenous pAb-nNav1.5 (1 mg/ml) and mAb-nNav1.5 (1 mg/ml) on the occurrence of metastasis. Real-time PCR and immunofluorescence staining were conducted to assess the effect of antibody treatment on nNav1.5 mRNA and protein expression, respectively. The animals' body weight, organs, lesions, and tumour mass were also measured and compared.pAb-nNav1.5 and mAb-nNav1.5 treatments effectively suppressed the invasion of MDA-MB-231 and 4T1 cells in the 3D spheroid invasion assay. Both antibodies significantly reduced nNav1.5 gene and protein expression in these cell lines. Treatment with pAb-nNav1.5 and mAb-nNav1.5 successfully reduced mammary tumour tissue size and mass and prevented lesions in vital organs of the mammary tumour animal model whilst maintaining the animal's healthy weight. mRNA expression of nNav1.5 in mammary tumour tissues was only reduced by mAb-nNav1.5.Overall, this work verifies the uniqueness of targeting nNav1.5 in breast cancer invasion and metastasis prevention, but more importantly, humanised versions of mAb-nNav1.5 may be valuable passive immunotherapeutic agents to target nNav1.5 in breast cancer.
Epigenetic alterations associated with cancer have been shown to facilitate tumorigenesis and promote metastasis. In the study of cancer metastasis, epigenetics has been revealed to play a crucial role in supporting tumour immune evasion. As a result, epigenetic drugs have been identified as potential agents to activate anti-tumour immune responses and reverse tumour immunologically tolerant states. Mounting evidence is showing aberrant expression of MHC class I antigen processing molecules in cancers and their upregulation as a potential indicator for anti-tumour immunity. In this study, we demonstrate that the epigenetic drug Trichostatin A (TSA), a histone deacetylase inhibitor, can restore MHC I antigen presentation machinery (MHC I APM) genes in human breast cancer cells (MCF-7). Treatment with TSA resulted in the upregulation of MHC I, B2M, and PSMB9 in MCF-7 monolayer cells, and MHC I, B2M, PSMB9, PSMB8, TAP1, and TAP2 in MCF-7 spheroid cells. Interestingly, treatment with TSA also increased CD274 expression in these cells and enhanced the invasion ability of the MCF-7 spheroid. This aggressive behaviour was confirmed by increased expression of metastatic-related genes, SCN5A (nNav1.5 protein) and MMP1. In summary, although the restoration of MHC I APM expression was achieved by TSA, the upregulation of metastatic genes and CD274 also enhanced the invasion ability of breast cancer cells. These findings suggest the need for careful consideration when utilizing epigenetic drugs for breast cancer therapy.
Glutamate and voltage-gated sodium channels, both have been the target of intense investigation for its involvement in carcinogenesis and progression of malignant disease. Breast cancer with increased level of glutamate often metastasize to other organs (especially bone), whilst re-expression of 'neonatal' Nav1.5, nNav1.5 in breast cancer is known to promote cell invasion in vitro, metastasis in vivo and positive lymph node metastasis in patients.In this study, the role of nNav1.5 in regulating glutamate level in human breast cancer cells was examined using pharmacological approach (VGSCs specific blocker, TTX, glutamate release inhibitor, riluzole and siRNA-nNav1.5). Effect of these agents were evaluated based on endogenous and exogenous glutamate concentration using glutamate fluorometric assay, mRNA expression of nNav1.5 using qPCR and finally, invasion using 3D culture assay.Endogenous and exogenous glutamate levels were significantly higher in aggressive human breast cancer cells, MDA-MB-231 cells compared to less aggressive human breast cancer cells, MCF-7 and non-cancerous human breast epithelial cells, MCF-10A. Treatment with TTX to MDA-MB-231 cells resulted in significant reduction of endogenous and exogenous glutamate levels corresponded with significant suppression of cell invasion. Subsequently, downregulation of nNav1.5 gene was observed in TTX-treated cells.An interesting link between nNav1.5 expression and glutamate level in aggressive breast cancer cells was detected and requires further investigation.
'/%3e%3cuse xlink:href='%23a' transform='translate(0 -400)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -600)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -800)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -1000)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -1200)'/%3e%3cpath d='M0 0h1400v800H0z' style='fill:%23010066'/%3e%3cpath d='M576 100c-166.146 0-301 134.406-301 300s134.854 300 301 300c60.027 0 115.955-17.564 162.927-47.783-27.353 9.44-56.71 14.602-87.271 14.602-147.327 0-266.897-119.172-266.897-266.01 0-146.837 119.57-266.01 266.897-266.01 32.558 0 63.746 5.815 92.602 16.468C696.217 118.91 638.305 100 576 100z' style='fill:%23fc0'/%3e%3cpath d='m914.286 471.429-99.538-53.25 29.43 108.982-66.575-91.165-20.77 110.96-20.428-111.024-66.857 90.96L699.314 418l-99.701 52.943 74.065-85.192-112.8 4.441 103.694-44.62-103.555-44.94L673.8 305.42 600 220l99.538 53.25-29.43-108.982 66.575 91.165 20.77-110.96 20.428 111.023 66.857-90.959L814.97 273.43l99.702-52.944-74.065 85.193 112.8-4.441-103.694 44.62 103.555 44.94-112.785-4.79z' style='fill:%23fc0' transform='matrix(1.2738 0 0 1.2423 -89.443 -29.478)'/%3e%3c/svg%3e)
'/%3e%3cuse xlink:href='%23a' transform='translate(288.889 -214.211)'/%3e%3cuse xlink:href='%23a' transform='translate(108 342.749)'/%3e%3cuse xlink:href='%23a' transform='translate(469.189 342.749)'/%3e%3c/svg%3e)
