Overexpression of miR 375 partially restores trastuzumab sensitivity in vivo To investigate whether miR 375 can reverse the resistance of HER2 positive breast cancers to trastuzumab in vivo, xenograft models were generated using trastuzumab resistant SKBr 3 cells modified to overexpress pre miR 375 or control pre miRNA. These cells were injected into the mammary AG-014699 fat pads of athymic nude mice, and then the mice were intravenously injected with 10 mg/kg trastuzumab twice a week. Compared with mice bearing tumors derived from SKBr 3 cells expressing a control pre miRNA, mice inoculated with pre miR 375 modified SKBr 3 cells displayed significantly suppressed tumor development and growth. A Kaplan Meier survival analysis showed pro longed survival of mice challenged with SKBr 3 cells expressing pre miR 375, compared with those inoculated with the control cells after treatment with trastuzumab.

These data suggest that the overex pression of miR 375 Inhibitors,Modulators,Libraries may sensitize trastuzumab resistant breast cancers to trastuzumab in vivo. Epigenetic mechanisms and PI3K/Akt pathway are involved in miR 375/IGF1R mediated trastuzumab resistance We next probed the mechanisms underlying the sup pressed expression of miR 375 in trastuzumab resistant breast cancer cells. However, the luciferase expression under the control of a miR 375 promoter in an artificial construct were comparable in parental and trastuzumab resistant SKBr 3 cells, suggestive of the involvement of either chromosomal modification or mechanisms other than transcriptional activation in miR 375 suppression in trastuzumab resistant SKBr 3 cells.

To test whether miR 375 expression was regulated by epigenetic mechanisms, trastuzumab resistant Inhibitors,Modulators,Libraries cells were treated with the DNA methyltransferase inhibitor, Inhibitors,Modulators,Libraries 5 Aza 2 deoxycyti dine, and the histone deacetylatase Inhibitors,Modulators,Libraries inhibitor, Trichostatin Inhibitors,Modulators,Libraries A. As a result, blockade of DNA methylation and/or histone deacetylation caused signifi cant upregulation of miR 375 in trastuzumab resistant SKBr 3 cells. Chromosomal immunopre cipitation detected an increased histone H3K9 acetylation in miR 375 promoter after treatment with TSA, and methylation specific PCR vali dated the much higher level of DNA methylation in miR 375 promoter of trastuzumab resistant compared with the parental SKBr 3 cells, suggesting the involvement of these epigenetic modifications in the downregulation of miR 375 in trastuzumab resistant breast cancer cells. Trastuzumab exerts its anti tumor effect http://www.selleckchem.com/products/Lenalidomide.html by inhibiting AKT phosphorylation in HER2 positive breast cancer cells. Consistent with previous reports that AKT is activated downstream of IGF1R signaling, expression of pre miR 375 in trastuzumab resistant cells reduced the levels of IGFR1 and phosphorylated AKT proteins.