Overexpression 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 signaling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feed-back 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 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 membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation.

Localization on membrane and interaction of PC-PLC with HER2, EGFR and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, 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 membrane, and on the levels of overall HER2, HER2-HER3 and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, following 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.

PC-PLC selectively accumulates on plasma membrane of HER2-overexpressing cells, where it co-localizes and associates with HER2 in raft domains. PC-PLC inhibition resulted into enhanced HER2 internalization and lysosomal degradation, inducing downmodulation of HER2 expression on the membrane. Moreover, PC-PLC inhibition resulted in strong retardation of HER2 re-expression on membrane and decrease in the overall cellular contents of HER2, HER2-HER3 and HER2-EGFR heterodimers. The PC-PLC inhibitor also induced anti-proliferative effects, especially in trastuzumab-resistant cells.

The 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.

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