Abstract
Abstract ErbB-2 (HER2) is over expressed in 15 - 25% of breast cancer. First line therapy for HER2+ breast cancer is a humanized, monoclonal antibody, trastuzumab. Resistance is a major clinical problem. Previous work has suggested that Notch-1 is critical for resistance. PTEN, a breast tumor suppressor and potent inhibitor of the PI3-K/AKT is decreased in HER2+ breast cancer and loss of PTEN predicts for worst outcome. Notch-1 downregulates PTEN expression and we sought to determine the mechanism by which Notch-1 attenuates PTEN and whether this decrease is necessary for trastuzumab resistance. Our hypothesis is that Notch-1 decreases PTEN to maintain trastuzumab resistance. Human HER2+ breast cancer cell lines: sensitive (BT474HS) or resistant (BT474HR and HCC1954) to trastuzumab were used to measure Notch-1 modulation of PTEN expression when treated with trastuzumab. Protein and transcript expression of PTEN were measured by Western blot and RT-PCR, respectively. ChIP assays were preformed to identify whether Notch-1 regulated PTEN expression was mediated by direct recruitment of Notch-1 to the PTEN promoter. Growth and mammosphere forming assays were performed to determine if trastuzumab resistance could be rescued in trastuzumab treated cells with simultaneous Notch-1 and PTEN knockdown, or increased downstream PI3K pathway activity using a constitutive active form of AKT1 (Myr-AKT1). Western blot and RT-PCR results showed that Notch-1 protein levels are increased while PTEN mRNA and protein levels are decreased in trastuzumab resistant cells (BT-474HR) compared to sensitive (BT-474HS) cells. Results revealed that Notch-1 knockdown increased PTEN transcript and protein expression. Notch-1 ChIP assays demonstrated that Notch-1 was enriched on the PTEN promoter and a Notch target gene promoter, HEY1 in resistant cells. Notch1 knockdown significantly decreased proliferation and mammosphere forming efficiency (MFE) of resistant cells regardless of trastuzumab treatment. PTEN knockdown rescued the decrease in proliferation and MFE mediated by Notch-1 knockdown. As PTEN is an attenuator of PI3-K/AKT signaling, we asked whether constitutive activation of AKT1 was sufficient to rescue proliferation upon Notch-1 knockdown. The results showed that myr-AKT1 increased AKT1 signaling and rescued the decrease in proliferation mediated by Notch-1 knockdown. These results demonstrate, to our knowledge, for the first time that Notch-1 is a direct transcriptional repressor of PTEN in HER2+ breast cancer cells. Increased activation of the PI3K pathway through Notch-1 mediated inhibition of PTEN is necessary to maintain proliferation and possibly survival of the cancer stem cell population of trastuzumab resistant cells. Inhibiting the expression and/or activity of Notch-1 in HER2+ breast cancer could prove to be an effective therapy to prevent expansion of cancer stem cells and/or re-sensitize HER2+ breast cancer to ant-HER2 therapy. Citation Format: Andrew T. Baker, Kinnari Pandya, Clodia Osipo. Notch-1 mediated down regulation of PTEN in trastuzumab resistant HER2+ breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2124.
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