New potential therapeutic approach for HER2-positive breastcancer discovered
EDI3 protein expression in breast cancer subtypes using IHC on a TMA ; high expression was defined as strong positivity in > 50% of tumour cells, intermediate as weak in >50% or strong in ≤ 50% of tumour cells, and low as negative or weak in ≤ 50% of tumour cells.qPCR analysis of EDI3 mRNA expression in a panel of breast cancer cell lines of different molecular subtypes normalized to β-Actin.
]. All cell lines were authenticated by DSMZ according to the ANSI/ATCC ASN-0002–2011 guidelines, and were regularly tested for mycoplasma using the Venor® GeM Classic kit . Culture conditions for all lines are provided in the Supplementary Methods.Total RNA was isolated using the RNeasy Mini Kit , according to the manufacturer’s instructions, and quantified using NanoDrop 2000.
EDI3 activity was measured employing a modified version of the Amplex® Red Phospholipase D Assay Kit , as previously described [Intracellular concentrations of GPC, choline, and PCho were measured using nuclear magnetic resonance spectroscopy at 14.1 T, as described in the Supplementary Methods.
For the EDI3 expression studies in endogenous HER2 expressing cell lines, SKBR3 and HCC1954 cells were seeded onto a 6-well plate and allowed to attach overnight. On the next day, cells were treated with different concentrations of lapatinib, everolimus, CHIR-99021, compound 3i , C188-9, KC7F2, SC75741, 10074-G5 or SR18662 . After one, two and three days , cells were harvested at approximately 80% confluency for RNA and protein expression analyses as described above.
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