| Abstract |
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
[1-4 mug/mL], 3 days on/3 days 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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