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Uncivering mechanisms of acquired resistance to trastuzumab-emtansine (T-DM1) in HER2 positive breast cancer

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Universitat de Barcelona. Facultat de Biologia

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[eng] Trastuzumab-emtansine (T-DM1) is an antibody-cytotoxic agent (DM1) conjugated drug. DM1 delivery by trastuzumab inside the HER2 positive cells affects microtubule polymerization, cell cycle arrest and finally cell death. Although T-DM1 is approved for the treatment of HER2 positive metastatic breast cancer patients, primary and acquired resistance towards this drug is still a main challenge. Looking for the mechanisms of resistance is necessary to improve patient selection and to develop novel treatment strategies. Here, we focused on finding mechanisms of acquired resistance to T-DM1 in a panel of HER2 positive breast cancer cell lines (HCC1954, HCC1419 and SKBR3 parental vs. resistant cells) generated by an established protocol of T-DM1 exposure, increasing the concentration of T-DM1[1-4µg/mL], 3days on/3days off, for 54 days overall. We generated acquired resistant cells with different level of resistance to T-DM1 evaluated by 3, 7 and 10 days proliferation assay, using automated cell counting in SKBR3, HCC1419 and HCC1954 parental and the acquired resistant cells. Analysis of T-DM1 effects on cell cycle showed a significant induction of G2/M arrest in the parental cells, while this effect was not observed in the resistant cells. Expression/activity analysis of cyclin B1/CDK1 complex, the main apparatus involve in G2/M cell cycle arrest induction, showed a remarkable decrease in the basal level of cyclin B1 in the resistant cells. Cyclin B1 accumulation induced by T-DM1 in the parental cells was not observed in the resistant cells. CDK1 activity assay was also correlated with cyclin B1 expression, increasing following T-DM1 treatment in the parental cells, but not in the resistant cells. Functional analysis revealed that cyclin B1 knock down in the parental cells induced a significant T-DM1 resistance. Furthermore, the silencing of cdc20, a protein mainly involved in APC complex related cyclin B1 degradation, could sensitize the resistant cells to T-DM1. Finally, cyclin B1 induction by T-DM1 was confirmed in in vivo and ex vivo xenograft animal model and patients’ explants, respectively. By cyclin B1 induction pattern, we could categorize T-DM1 responsive/non-responsive in fresh breast cancer explants from HER2 positive breast cancer patients. Our results showed that T-DM1 induced G2/M cell cycle arrest in a cyclin B1/CDK1 dependent-manner. Lack of these effects appeared in acquired T-DM1 resistant cells. Besides, similar pattern in G2/M and cyclin B1 was verified in vivo and in patients explants. These data strongly suggest that induction of cyclin B1 is necessary for T-DM1 antitumor effects and emerges as a potential pharmacodynamic marker. Our finding also raises the question on what are the mechanisms leading to cyclin B1 dysregulation in resistant cells.

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Uncivering mechanisms of acquired resistance to trastuzumab-emtansine (T-DM1) in HER2 positive breast cancer