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


METHODS
Localization 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.


RESULTS
PC-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.


CONCLUSIONS
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.
Paris
Luisa
L
Department of Cell Biology and Neurosciences, Section of Molecular and Cellular Imaging, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy. luisa.paris@iss.it
Cecchetti
Serena
S
Spadaro
Francesca
F
Abalsamo
Laura
L
Lugini
Luana
L
Pisanu
Maria Elena
ME
Iorio
Egidio
E
Natali
Pier Giorgio
PG
Ramoni
Carlo
C
Podo
Franca
F
D015153:Blotting, Western; D001940:Breast / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002462:Cell Membrane / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D020893:Receptor, ErbB-3 / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D066246:ErbB Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D010738:Type C Phospholipases; C512478:EGFR protein, human; C105598:phosphatidylcholine-specific phospholipase C
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