Our study showed that both HIF-1α
accumulated in breast cancer cells with hypoxia and they potentiated Notch signaling. Notch target gene expression was induced in these cells when they were cultured at low oxygen conditions. shRNA-mediated knockdown of HIF-1α
abrogated hypoxia-induced HES1
expression, indicating the effect of hypoxia on Notch activation is via HIF factors. Interestingly, HIF-1α
synergised with MAML1 in the activation of Notch pathway in the in vitro
Notch reporter assay and dominant negative MAML1 can block HIF-induced Notch signaling, indicating MAML1 as a key co-activator among the Notch activation complex. MAML1 has been detected earlier as part of a Notch/HIF/MAML1 immuno-complex (Sahlgren et al, 2008
). We did not detect a direct interaction between HIF factors and full length MAML1 or RBP-Jκ
(J Chen and J Griffin, unpublished data). Therefore, MAML1 and HIF factors may form a complex with Notch intracellular domain and function as scaffold proteins to recruit CBP/p300 and other Notch co-activators to the whole complex.
SK-BR-3 cells have a higher basal level expression of both HIF-1α and HIF-2α compared with other breast cancer cells. Under hypoxia, the expression of HIF-1α in these cells was not increased while the expression of HIF-2α was slightly elevated. This might be the reason why there was only two-fold increase of HEY1 expression and no elevated HES1 expression in these cells with hypoxia.
To date, cell autonomous gain-of-function mutations in Notch receptors have not been reported in human breast cancers and other solid tumours, suggesting that ligand-mediated Notch activation predominates in these contexts. We detected the expression of Notch ligands JAGGED1, JAGGED2 and DELTA1 in these breast cancer cells. They might have a role in the stimulation of Notch pathway in these cells. Elevated NOTCH3 expression was detected in MCF7, MDA-468 and T47D cells after hypoxia, and the expression of JAGGED1, JAGGED2 and DELTA1 ligands was also increased in these cells after they were cultured at 1% O2 condition. This indicated that increased ligand levels and stabilisation of Notch receptors are likely to act together to increase Notch signaling under hypoxia. Furthermore, our studies suggested that hypoxia not only potentiate the pre-existing Notch signaling in breast cancer cells, it also induce Notch signaling by increasing the expression of both Notch receptors and ligands.
Our study found that the expression of Notch target genes HES1
was increased in most breast cancer cells with hypoxia. However, the increased HEY1
expression by hypoxia was more dramatic compared with HES1
expression, indicating HEY1
might be a better marker of Notch activation in breast cancer cells. Previous studies also showed that HEY
genes are the most sensitive Notch target genes for Notch pathway inhibition in breast cancer (Leong et al, 2007
). Positive expression correlations between JAGGED1
genes have also been identified in primary human breast cancer (Leong et al, 2007
Epithelial-to-mesenchymal transition is characterised by loss of cell adhesion, repression of E-cadherin expression and increased cell mobility. Induction of EMT in immortalised human mammary epithelial cells results in the expression of stem cell markers (Mani et al, 2008
). Several oncogenic pathways such as TGF-β
, receptor tyrosine kinases (RTKs), integrin, endothelin A receptor, Wnt/beta-catenin, hypoxia, matrix metalloproteinase (MMPs) and Notch induce EMT (Polyak and Weinberg, 2009
). Downregulation of E-cadherin is one of the best markers of EMT in breast cancers. Zinc-finger proteins Snail and Slug are two transcriptional repressors of E-cadherin and their expression induces EMT and promotes malignant cell invasion and dissemination. Other E-cadherin transcriptional repressors include SIP1, TWIST1, FOXC2, FOXC1 and ZEB1 (Polyak and Weinberg, 2009
). Some non-coding microRNAs have also been implicated as regulators of EMT and tumour metastasis (Ma et al, 2007
; Gregory et al, 2008
; Park et al, 2008
; Tavazoie et al, 2008
Previous studies have suggested that Notch signaling induces a particular type of EMT during normal heart development and that Notch increases Snail expression in endothelial cells to promote mesenchymal transformation (Noseda et al, 2004
; Timmerman et al, 2004
). Recent studies showed that JAGGED1-induced Notch signaling induces EMT through Slug-mediated repression of E-cadherin (Leong et al, 2007
). Over-expression of NOTCH1 intracellular domain, but not HES1
, inhibits E-cadherin expression and induces EMT in ovarian cancer cells, probably through the induction of Snail expression (Sahlgren et al, 2008
). Our studies indicate that hypoxia may be an initiative event resulting in enhanced Notch signaling, increasing the expression of Slug and Snail in breast cancer cells, which in turn inhibited E-cadherin expression. Inhibition of Notch signaling by dominant negative MAML1 abrogated the downregulation of E-cadherin observed during hypoxia. Culture of breast cancer cells under hypoxia increased their migration and invasion, which is abolished by Notch pathway inhibition with either GSI treatment or DNMAML1. These studies suggested that Notch signaling is not only required for the proliferation of breast cancer cells under regular culture conditions, it is also required, under hypoxia, to further activate Notch signaling, to initiate EMT, and thereby increasing the likelihood of increased breast cancer cell migration, invasion and metastasis. Over the short term, pharmacological inhibitors of ligand-induced Notch signaling might be able to block EMT and tumour metastasis in the treatment of human breast cancer.