Role of Notch Signaling Pathway in Breast Cancer Pathogenesis

摘要

Notch proteins are activated upon binding to ligands of the Delta/Serrate family. In previous experiments I had found that activated allele of Notch1 cooperates with low levels of oncogenic Ras expressing HMLE cells(termed HMLER). Since ErbB2 overexpression is one of the leading causes of Ras activation in breast cancer cells, I tested whether ErbB2 overexpression will cooperate with Notch in HMLE cells. While overexpression of activated Notch1 failed to transform wild-type ErbB2-expressing HMLE cells, it caused cell death inconstitutively active ErbB2-expressing cells. These results indicate that Notch-Ras cooperation in transformation is largely dependant on the extent of signal activation. While low levels of Ras signals fails to cooperate withNotch, too much of Ras activation leads to cell death. My previous experiments based on RT-PCR analysis had suggested that the Notch- ligand Jagged1 may be involved in activating Notch signaling in breast cancers. WhileJagged1 expression in HMLER cells led to soft-agar colony formation, it failed to generate tumors in vivo. In order to further demonstrate a role for Notch signaling in breast cancers, I generated siRNA against Jagged1.Screening several of these siRNA led to the selection of siJagB4 that caused greater than 95% suppression ofJagged1 expression in co-transfection experiments undertaken with siJagB4 construct and a myc-tagged Jagged1expresssion construct. Subsequently, I generated lentiviruses encoding this siRNA and introduced this siRNA into several breast cancer cell lines. Upon further characterization, I found that this siRNA had very moderate effects in downmodulating Jagged 1 expression driven by endogenous promoter. The best effect observed was a 25% reduction in MCF-7 cells. Currently, I am making subclones of the MCF7 cells to see if a single clone showing major suppression of Jagged 1 expression could be isolated for further testing their tumorigenic behavior.