BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is associated with decreased adhesion and acquisition of metastatic potential of breast cancer cells. Epithelial-to-mesenchymal transition is mediated, in part, by two transcription repressors, Snail and Slug, that are known to be targets of the Notch signaling pathway, and JAGGED1-induced Notch activation increases EMT. However, the events that lead to increased Notch activity during EMT of breast cancer cells are unknown. METHODS: The accumulation of hypoxia inducible factors (HIFs) under hypoxia was detected by western blot analysis, and their effects on Notch signaling were measured by an in vitro Notch reporter assay. The expression of Notch target genes under hypoxia was tested by real-time PCR. The knockdown of HIF-1a was mediated by retroviral delivery of shRNA. The expression of Slug and Snail under hypoxia was measured by real-time PCR. Breast cancer cell migration and invasion under hypoxia were tested with cell migration and invasion kits. RESULTS: Hypoxia increased the expression of Notch target genes such as HES1 and HEY1 in breast cancer cells, as was expression of Notch receptors and ligands. The mechanism is likely to involve the accumulation of HIF-1a and HIF-2a in these cells by hypoxia, which synergised with the Notch co-activator MAML1 in potentiating Notch activity. Hypoxia inducible factor-1a was found to bind to HES1 promoter under hypoxia. Knockdown of HIF-1a with shRNA inhibited both HES1 and HEY1 expression under hypoxia. Hypoxia increased the expression of Slug and Snail, and decreased the expression of E-cadherin, hallmarks of EMT. Notch pathway inhibition abrogated the hypoxia-mediated increase in Slug and Snail expression, as well as decreased breast cancer cell migration and invasion. CONCLUSION: Hypoxia-mediated Notch signaling may have an important role in the initiation of EMT and subsequent potential for breast cancer metastasis.

Activating mutations in the NOTCH1 gene have been found in about 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL). In order to study the molecular mechanisms by which altered Notch signaling induces leukemia, a zebrafish model of human NOTCH1-induced T-cell leukemia was generated. Seven of sixteen mosaic fish developed a T-cell lymphoproliferative disease at about 5 months. These neoplastic cells extensively invaded tissues throughout the fish and caused an aggressive and lethal leukemia when transplanted into irradiated recipient fish. However, stable transgenic fish exhibited a longer latency for leukemia onset. When the stable transgenic line was crossed with another line overexpressing the zebrafish bcl2 gene, the leukemia onset was dramatically accelerated, indicating synergy between the Notch pathway and the bcl2-mediated antiapoptotic pathway. Reverse transcription-polymerase chain reaction analysis showed that Notch target genes such as her6 and her9 were highly expressed in NOTCH1-induced leukemias. The ability of this model to detect a strong interaction between NOTCH1 and bcl2 suggests that genetic modifier screens have a high likelihood of revealing other genes that can cooperate with NOTCH1 to induce T-ALL.

SummaryInterleukin-2 (IL-2)-deficient (IL-2 -/-) mice develop anemia and colonic inflammatory bowel disease. To elucidate the mechanism of this disease, we have bred IL-2 -/-mice to two strains of immunodeficient mice, RAG-2-deficient (RAG-2 -/-, lacking B and T cells) andJH-deficient mice (JH -/-, lacking B cells). IL-2 -/-, RAG-2 -/-double-mutant mice are disease free, while IL-2-/-,JH -/-double-mutant mice succumb to bowel disease at the same rate as ILo2 -/-littermates. IL-2 -/-, JH -/-mice do not, however, succumb to anemia. Thus, spontaneous intestinal inflammation in IL-2 -/-mice requires mature T cells, not B cells, while anemia is dependent on B cells.