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Effects of T-DM1 on 750 cyclin B1 (CCNB1) mRNA expression in T751 DM1 sensitive and resistant breast cancer cells.

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Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance.Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants.Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation.Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006–19. ©2017 AACR.

Representative immunohistochemistry images of control and 765 T-DM1 treated explant obtained from a liver metastasis.

Alzheimer's disease (AD) is tightly linked to oxidative stress since amyloid beta-peptide (Aβ) aggregates generate free radicals. Moreover, the aggregation of Aβ is increased by oxidative stress, and the neurotoxicity induced by the oligomers and fibrils is in part mediated by free radicals. Interestingly, it has been reported that oxidative stress can also induce BACE1 transcription and expression. BACE1 is the key enzyme in the cleavage of the amyloid precursor protein to produce Aβ, and the expression of this enzyme has been previously shown to be enhanced in the brains of Alzheimer's patients. Here, we have found that BACE1 expression is increased in the hippocampi from AD patients at both the early (Braak stage II) and late (Braak stage VI) stages of the disease as studied by immunohistochemistry and western blot. To address the role of Aβ and oxidative stress in the regulation of BACE1 expression, we have analyzed the effect of subtoxic concentrations of Aβ oligomers (0.25 μM) and H2O2 (10 mM) on a human neuroblastoma cell line. Firstly, our results show that Aβ oligomers and H2O2 induce an increase of BACE1 mRNA as we studied by qPCR. Regarding BACE1 translation, it is dependent on the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), since BACE1 mRNA bears a 5′UTR that avoids its translation under basal conditions. BACE1 5′UTR contains four upstream initiating codons (uAUGs), and its translation is activated when eIF2α is phosphorylated. Consistently, we have obtained that Aβ oligomers and H2O2 increase the levels of BACE1 and p-eIF2α assayed by western blot and confocal microscopy. Our results suggest that Aβ oligomers increase BACE1 translation by phosphorylating eIF2α in a process that involves oxidative stress and conforms a pathophysiological loop, where the Aβ once aggregated favors its own production continuously by the increase in BACE1 expression as observed in AD patients., Spanish Ministry of Economy and Business through the grant Plan Estatal SAF2017-83372-R and SAF2014-52228-R (FEDER funds/UE) to FJM and RV, Chilean Government through Fondecyt 11611065 and AFB170005 to AA and REDES 180084 to AA and FJM, and MDM-2014-0370 through the María de Maeztu Programme for Units of Excellence in R&D to Departament de Ciències Experimentals i de la Salut. Silvia Menéndez is supported by the Health Deparment of the Generalitat de Catalunya, Spain

Glioblastoma is the most frequent and aggressive primary tumor of the central nervous system. Prognosis is poor, with a median survival of 15 months after diagnosis. Various tumor biomarkers show prognostic value for glioblastomas, including VEGFR2, which is a receptor of VEGF related to the growth of the blood vessel network. VEGFR2 expression associates with poor prognosis in some tumors. Here we studied the prognostic value of the VEGFR2 immunohistochemical expression in glioblastoma. We used tissue microarrays to analyze 45 surgically excised samples from glioblastomas. Clinical data (age, sex, and Karnofsky Performance Status [KPS]) and morphological data (tumor necrosis, palisading, and vascular thrombosis) were collected. We performed a molecular study of MGMT and IDH1 expression (which are potential prognostic factors for glioblastomas) and an immunohistochemical study of VEGFR2 expression. Our results indicate that age, KPS, tumor necrosis, vascular thrombosis, treatment (STUPP versus other), and VEGFR2 immunoreactivity were related to prognosis (p < .005). In a multivariate analysis, only age > 65 years (Hazard Ratio (HR) (95% CI): 4.9 (2.1¿11.4), p < .01), and VEGFR2 immunoexpression (HR (95% CI): 2.8 (1.3¿6.1), p = .008), were found to have a statistically significant relation to prognosis. We conclude that immunohistochemical evaluation of VEGFR2 provides added prognostic value to the study of glioblastoma., This work was supported by grants from the Fundació La Marató (num.20130332) to FA, and the Spanish Ministerio de Economía y Competitividad/ ISCIIIFEDER (PI14/00125; PI17/00199) and the "Generalitat de Catalunya" (2017/SGR/225) to PN.

[Purpose]: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance. [Experimental Design]: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants. [Results]: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation. [Conclusions]: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer., This work was supported by ISCiii (CIBERONC CB16/12/00481, RD12/0036/0051, RD12/0036/0070, RD12/0036/0003, PIE15/00008, PI13/00864, PI15/00146, PI15/00934, PI15/01617, PT13/0010/0005), Generalitat de Catalunya (2014 SGR 740), and the >Xarxa de Bancs de tumors sponsored by Pla Director d'Oncologia de Catalunya (XBTC). MINECO through BFU2015-71371-R grant supported work in A. Pandiella's laboratory. Our work was supported by the EU through the regional funding development program (FEDER). P. González-Alonso was supported by Fundación Conchita Rábago de Jiménez Díaz grant.