ErbB-2 inhibition activates Notch-1 and sensitizes breast cancer cells to a gamma-secretase inhibitor.

ErbB-2 overexpression in breast tumors is associated with poor survival. Expression of Notch-1 and its ligand, Jagged-1, is associated with the poorest survival, including ErbB-2-positive tumors. Trastuzumab plus chemotherapy is the standard of care for ErbB-2-positive breast cancer. A proportion of tumors are initially resistant to trastuzumab and acquired resistance to trastuzumab occurs in metastatic breast cancer and is associated with poor prognosis. Thus, we investigated whether Notch-1 contributes to trastuzumab resistance. ErbB-2-positive cells have low Notch transcriptional activity compared to non-overexpressing cells. Trastuzumab or a dual epidermal growth factor receptor (EGFR)/ErbB-2 tyrosine kinase inhibitor (TKI) increased Notch activity by 2- to 6-fold in SKBr3, BT474 and MCF-7/HER2-18 cells. The increase in activity was abrogated by a Notch inhibitor, gamma-secretase inhibitor (GSI) or Notch-1 small-interfering RNA (siRNA). Trastuzumab decreased Notch-1trade mark precursor, increased amount and nuclear accumulation of active Notch-1(IC) and increased expression of targets, Hey1 and Deltex1 mRNAs, and Hes5, Hey1, Hes1 proteins. Importantly, trastuzumab-resistant BT474 cells treated with trastuzumab for 6 months expressed twofold higher Notch-1, twofold higher Hey1, ninefold higher Deltex1 mRNAs and threefold higher Notch-1 and Hes5 proteins, compared to trastuzumab-sensitive BT474 cells. The increase in Hey1 and Deltex1 mRNAs in resistant cells was abrogated by a Notch-1 siRNA. Cell proliferation was inhibited more effectively by trastuzumab or TKI plus a GSI than either agent alone. Decreased Notch-1 by siRNA increased efficacy of trastuzumab in BT474 sensitive cells and restored sensitivity in resistant cells. Trastuzumab plus a GSI increased apoptosis in sensitive cells by 20-30%. A GSI alone was sufficient to increase apoptosis in trastuzumab-resistant BT474 cells by 20%, which increased to 30% with trastuzumab. Notch-1 siRNA alone decreased cell growth by 30% in sensitive and more than 50% in resistant BT474 cells. Furthermore, growth of both trastuzumab sensitive and resistant cells was completely inhibited by combining trastuzumab plus Notch-1 siRNA. More importantly, Notch-1 siRNA or a GSI resensitized trastuzumab-resistant BT474 cells to trastuzumab. These results demonstrate that ErbB-2 overexpression suppresses Notch-1 activity, which can be reversed by trastuzumab or TKI. These results suggest that Notch-1 might play a novel role in resistance to trastuzumab, which could be prevented or reversed by inhibiting Notch-1.

High throughput screens for the identification of compounds that alter the accumulation of the Alzheimer's amyloid beta peptide (Abeta).

Evidence gathered over the last two decades suggests that beta amyloid (Abeta), the predominant proteinaceous component of senile plaques, plays an early and critical role in the etiology and pathogenesis of Alzheimer's disease (AD). Thus, it is reasonable to hypothesize that compounds capable of reducing the accumulation of Abeta may be of value therapeutically. Additionally, compounds that influence Abeta accumulation may be useful as tools to further dissect the cellular pathways that regulate Abeta production and accumulation. To screen for compounds that affect Abeta levels, we have established high throughput, cell-based assays capable of the sensitive and selective detection of Abeta40 in parallel with the more amyloidogenic form of the peptide, Abeta42. To validate the approach, we examined the effects of several compounds previously identified to influence Abeta accumulation. Analysis of peptide accumulation following treatment with these compounds showed results similar to those previously published. Currently, we are using this assay to screen drugs that have already received FDA approval for the treatment of other diseases and over-the-counter natural product extracts. If compounds such as these can be identified that lower Abeta in the brain, they may represent one of the fastest and most cost effective methods to therapy.