Inhibition of phosphatidylcholine-specific phospholipase C downregulates HER2 overexpression on plasma membrane of breast cancer cells

Abstract

IntroductionOverexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop. We recently showed that inactivation of phosphatidylcholine-specific phospholipase C (PC-PLC) may exert a pivotal role in selectively modulating the expression on the membrane of specific receptors or proteins relevant to cell function. In the present study, we investigated the capability of PC-PLC inhibition to target the molecular mechanisms controlling HER2 overexpression on the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation.MethodsLocalization on the membrane and interaction of PC-PLC with HER2, EGFR, and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, by using confocal laser scanning microscopy, flow cytometry, cell-surface biotinylation, isolation of lipid rafts, and immunoprecipitation experiments. The effects of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) on HER2 expression on the membrane and on the levels of overall HER2, HER2-HER3, and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, after either transient or continuous receptor engagement with anti-HER2 monoclonal antibodies, including trastuzumab. Changes of HER2 expression and cell proliferation were examined in SKBr3, BT-474, and MDA-MB-453 cells continuously exposed to D609 alone or combined with trastuzumab.ResultsPC-PLC selectively accumulates on the plasma membrane of HER2-overexpressing cells, where it colocalizes and associates with HER2 in raft domains. PC-PLC inhibition resulted in enhanced HER2 internalization and lysosomal degradation, inducing downmodulation of HER2 expression on the membrane. Moreover, PC-PLC inhibition resulted in strong retardation of HER2 reexpression on the membrane and a decrease in the overall cellular contents of HER2, HER2-HER3, and HER2-EGFR heterodimers. The PC-PLC inhibitor also induced antiproliferative effects, especially in trastuzumab-resistant cells.ConclusionsThe results pointed to PC-PLC inhibition as a potential means to counteract the tumorigenic effects of HER2 amplification and complement the effectiveness of current HER2-targeting therapies.

Choline metabolism in malignant transformation

“…PC-PLC inhibition reduced HER2 expression on the plasma membrane, thereby inducing antiproliferative effects, which suggests that it may provide a valuable strategy to counteract the tumorigenic effects of HER2 and thus complement HER2-targeting therapies 81 . The antiviral, potentially antitumoural xanthate D609 was shown to be a potent inhibitor of PC-PLC 167 , and significantly reduced PCho levels along with cell proliferation in ovarian cancer cells 59 .…”

“…In ovarian cancer cells, the elevated PCho levels, which are linked to ovarian carcinogenesis, are partially caused by PC-PLC activation 80 . In breast cancer cells, PC-PLC accumulates on the plasma membrane of HER2 (also known as ERBB2)-overexpressing cells and associates with HER2 in lipid raft domains 81 .…”

Development and Analytical Validation of a BT-474 Anti-Proliferation Assay Targeting HER2

“…When searching papers or information regarding the anti-proliferation bioactivity study targeting HER2, in more than 50%, the cell-based assay would be developed under the breast cancer cell line Sk-Br-3 (Lee et al 2009;Cuello et al 2001;Paris et al 2010), which may be useful in scientific study when the purpose is focused on the drug candidate's anti-proliferation ratio. However, when applying the assay in the industry for bioactivity evaluation, for example drug candidates screening, or especially drug substance and drug product quality control, it would not be preferable.…”

Lipid Rafts as Master Regulators of Breast Cancer Cell Function

“…HER2 is rapidly recycled back to the cell membrane if endocytosed (Worthylake et al, 1999), which maintains its overexpression at the cell membrane of breast cancer cells. Modulation of lipid metabolism may control HER2 overexpression by increasing its endocytosis and preventing redistribution back to the cell membrane (Paris et al, 2010). For instance, phospholipase C (PLC) has been shown to co-localise with HER2 in lipid raft domains of HER2-overexpressing breast cancer cell lines, and PLC antagonism enhances HER2 internalisation and delays its recycling to the cell membrane, reducing breast cancer cell proliferation (Paris et al, 2010).…”

“…Modulation of lipid metabolism may control HER2 overexpression by increasing its endocytosis and preventing redistribution back to the cell membrane (Paris et al, 2010). For instance, phospholipase C (PLC) has been shown to co-localise with HER2 in lipid raft domains of HER2-overexpressing breast cancer cell lines, and PLC antagonism enhances HER2 internalisation and delays its recycling to the cell membrane, reducing breast cancer cell proliferation (Paris et al, 2010). This implies that lipid rafts play a key role in transduction of this oncogenic signal.…”