Trastuzumab stimulation of ribosomal protein S6 kinase 1 (S6K1) predicts de novo trastuzumab resistance.

Breast cancer is a complex disease with at least five different molecular subtypes identified. The breast tumor molecular subtypes guide stratification of patients for specific targeted therapy regimens and each subtype is associated with significantly different patient outcomes. For example, patients with the HER2 positive molecular subtype benefit from the HER2 targeted therapy trastuzumab. Unfortunately, women with the HER2 positive molecular subtype have the worst overall prognosis and nearly 70% of women with HER2 positive breast cancer exhibit de novo or acquired resistance to trastuzumab. Identification of tumor markers predicting trastuzumab response can be used to further stratify patients for life-saving personalized therapeutic options. The aim of this study was to identify clinically useful tumor markers predicting de novo tumor cell resistance to trastuzumab treatment. To identify oncogenic signaling pathways activated in response to trastuzumab treatment, we performed a Human Phospho-Kinase Proteome Profiler Array analysis comparing trastuzumab sensitive MCF-7/HER2.2 and trastuzumab resistant MCF-7/HER2Δ16H cells following acute treatment with 20 µg/ml of trastuzumab for 2 h. We found that of the 43 phosphorylation activated human kinases represented on the array, S6K1 was the only kinase altered greater than 1.5-fold in response to trastuzumab treatment of the trastuzumab resistant MCF-7/HER2Δ16H cells. Trastuzumab activation of S6K1 was confirmed in the two trastuzumab resistant SUM190 and SUM225 cell lines. Significantly, trastuzumab failed to stimulate S6K1 activation in the trastuzumab sensitive MCF-7/HER2.2, BT474, and SKBR3 cell lines suggesting that trastuzumab activation of S6K1 is a tumor cell marker for trastuzumab resistance. Consistent with a role for mTORC1/S6K1 signaling promoting trastuzumab resistance, all cell lines were sensitive to S6K1 inactivation with significant growth inhibition following treatment with the mTORC1 inhibitor rapamycin. In conclusion, characterizing rapid trastuzumab induced molecular alterations resulted in the identification of activated S6K1 as an early breast tumor cell marker for trastuzumab resistance. Our results further suggest that trastuzumab resistant breast tumor cells are addicted to mTORC1/S6K1 oncogenic signaling and targeting mTORC1 with rapamycin reverses trastuzumab resistance.

MicroRNA-7 inhibits multiple oncogenic pathways to suppress HER2Δ16 mediated breast tumorigenesis and reverse trastuzumab resistance.

The oncogenic isoform of HER2, HER2Δ16, is expressed with HER2 in nearly 50% of HER2 positive breast tumors where HER2Δ16 drives metastasis and resistance to multiple therapeutic interventions including tamoxifen and trastuzumab. In recent years microRNAs have been shown to influence multiple aspects of tumorigenesis and tumor cell response to therapy. Accordingly, the HER2Δ16 oncogene alters microRNA expression to promote endocrine resistance. With the goal of identifying microRNA suppressors of HER2Δ16 oncogenic activity we investigated the contribution of altered microRNA expression to HER2Δ16 mediated tumorigenesis and trastuzumab resistance. Using a gene array strategy comparing microRNA expression profiles of MCF-7 to MCF-7/HER2Δ16 cells, we found that expression of HER2Δ16 significantly altered expression of 16 microRNAs by 2-fold or more including a 4.8 fold suppression of the miR-7 tumor suppressor. Reestablished expression of miR-7 in the MCF-7/HER2Δ16 cell line caused a G1 cell cycle arrest and reduced both colony formation and cell migration activity to levels of parental MCF-7 cells. Suppression of miR-7 in the MCF-7 cell line resulted in enhanced colony formation activity but not cell migration, indicating that miR-7 suppression is sufficient to drive tumor cell proliferation but not migration. MiR-7 inhibited MCF-7/HER2Δ16 cell migration through a mechanism involving suppression of the miR-7 target gene EGFR. In contrast, miR-7 inhibition of MCF-7/HER2Δ16 cell proliferation involved a pathway where miR-7 expression resulted in the inactivation of Src kinase independent of suppressed EGFR expression. Also independent of EGFR suppression, reestablished miR-7 expression sensitized refractory MCF-7/HER2Δ16 cells to trastuzumab. Our results demonstrate that reestablished miR-7 expression abolishes HER2Δ16 induced cell proliferation and migration while sensitizing HER2Δ16 expressing cells to trastuzumab therapy. We propose that miR-7 regulated pathways, including EGFR and Src kinase, represent targets for the therapeutic intervention of refractory and metastatic HER2Δ16 driven breast cancer.

Rescue of Mtp siRNA-induced hepatic steatosis by DGAT2 siRNA silencing.

Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response.

Jumonji/ARID1 B (JARID1B) protein promotes breast tumor cell cycle progression through epigenetic repression of microRNA let-7e.

MicroRNAs (miRs) function as tumor suppressors or oncogenes in multiple tumor types. Although miR expression is tightly regulated, the molecular basis of miR regulation is poorly understood. Here, we investigated the influence of the histone demethylase Jumonji/ARID1 B (JARID1B) on miR regulation in breast tumor cells. In MCF-7 cells with stable RNAi-mediated suppression of JARID1B expression we identified altered regulation of multiple miRs including let-7e, a member of the let-7 family of tumor suppressor miRs. Chromatin immunoprecipitation analysis demonstrated JARID1B binding to the let-7e promoter region as well as removal of the of H3K4me3 histone mark associated with active gene expression. These results suggest that JARID1B epigenetically represses let-7e expression. JARID1B stimulates tumor cell proliferation by promoting the G(1) to S transition. As predicted, suppression of JARID1B resulted in an accumulation of MCF-7 cells in G(1). We confirmed that cyclin D1, which also promotes G(1) progression, is a direct target of let-7e, and we show that cyclin D1 expression is suppressed in JARID1B knockdown cells. Cyclin D1 expression and cell cycle progression were restored following inhibition of let-7e, suggesting that JARID1B repression of let-7e contributes to cyclin D1 expression and JARID1B-mediated cell cycle progression. Our results indicate that the JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR.