Notch signaling is a highly conserved pathway which plays a vital role in normal embryonic development and morphogenesis as well as in stem cell maintenance (Yu et al., 2008
). However, aberrant expression of Notch signaling is also frequently observed in many solid tumors and leukemia, suggesting a critical role of this pathway in tumor pathology and cancer progression (Ellisen et al., 1991
; Koch & Radtke, 2007
). The results of our immunohistochemical analysis also indicate that Notch signaling is strongly activated in normal glands of breast tissue and generally down-regulated in DCIS and advanced tumor cells; however, it is significantly up-regulated at the invasive front. These results illustrate an intricate role of Notch signaling in tumor progression. A hallmark of Notch signaling is the requirement of the ligand-receptor interaction through direct cell-cell contact, which may occur between tumor cells or tumor cell-stroma interactions (Felli et al., 1999
). Therefore, dysregulation of Notch signaling in cancer cells is likely caused by either alteration of the receptor expression in cancer cell and/or abnormal expression of ligand in tumor-surrounding cells. In mammalian cells, there are four Notch receptors, Notch 1 to 4, and they are cleaved by γ-secretase upon ligand binding to become the activated form, NICD (Artavanis-Tsakonas et al., 1999
). On the other hand, less is known about the expression status of Notch ligand in tumor and tumor-associated stroma. The results of our existing microarray data analysis indicate that Jagged2 is the ligand, and in some cases the only ligand, whose expression is significantly correlated with metastasis-free survival in many patient cohorts. It should be noted that Reedijk et al (Reedijk et al., 2005
) and Dickson et al (Dickson et al., 2007
) previously reported that mRNA expression of Jagged1 had significant correlation to patient survival. The reason for this apparent contradiction is not clear; however, there are several differences between these analyses and our approach. First, their analysis is based on in situ
hybridization, while our analysis is based on existing data set of microarray analysis which used gene chip arrays. Although in situ
hybridization is more accurate for localization, it is less quantitative. Secondly, the design and location of probes for Jagged1 gene in these analyses are different. The gene chip generally uses multiple probes. Thirdly, the numbers of patients are different. We analyzed all together more than 600 patients in three different cohorts while they examined about 100 patients in each of their experiments. These differences may have contributed to the different outcome of the results of mRNA expression of Jagged1 in breast cancer patients.
We also demonstrated that high expression of Jagged2 and concomitant activation of Notch signaling often coincides with the hypoxic regions of invasive front in breast cancer and that hypoxic condition indeed significantly up-regulates the Jagged2 expression in both tumor and stromal cells in vitro
. Although there is a general concern whether the cell lines that have been adapted for years to grow in 21% oxygen reflects in vivo
hypoxic condition, which may be indeed a limitation of the in vitro
experiment, our results using multiple cell lines clearly indicate that Jagged2 specifically responds to hypoxia which is also consistent with the result of immunohistochemical analysis for clinical sample using hypoxic markers. Hypoxia is a hallmark of tumor, which contributes to tumor cell survival, angiogenesis and chemo-resistance (Maynard & Ohh, 2007
). The central area of tumor mass is often hypoxic due to necrotic cell death; however, it is well established that invasive front of tumor is also strongly hypoxic because of the rapid rate of proliferation of tumor cells (Horree et al., 2007
). It has been reported that hypoxia increases the transcriptional activity of NICD by inhibiting its degradation in neuronal stem cells and myogenic cells (Gustafsson et al., 2005
). Similar results were also observed in NSCL (Chen et al., 2007
; Eliasz et al., 2010
). It was also found that HIF1 alpha can directly bind to NICD and stabilize this protein and activity (Cejudo-Martin & Johnson, 2005
). This protein stabilization is certainly considered as one of the mechanisms which contribute to the Notch activation at the invasive front; however, our results indicate that the major factor in the Notch activation is the hypoxia-induced expression of Jagged2 through cell-cell interaction of tumor cells. In fact, we have shown that hypoxia can significantly activate Jagged2 expression as well as the Notch signaling in breast tumor cell lines, but this activation is strongly cell-density dependent and the clustered cells showed much higher sensitivity to Notch activation by hypoxia than a single cell in the culture (). How hypoxia promotes invasiveness of tumor cells through activation of Notch signaling is an intriguing question. Our results indicate that inhibition of hypoxia-induced Notch signaling significantly decreased cell survival and invasiveness by blocking Akt pathway and also by suppressing EMT. It should be noted that Akt was previously found to be activated by Notch signaling in melanoma and lung cancers (Zhao et al., 2010
; Bedogni et al., 2008
). In addition, Notch signaling has been known to be associated with chemo-resistance and cell survival (Wang et al., 2010
; Eliasz et al., 2010
). Therefore, hypoxia-induced Notch activation may render tumor cells to become more resistant to cell death through activation of Akt. Taken together, the aggressive nature of tumor cells at the invasive front appears to be mediated by Akt/EMT induction through up-regulation of Jagged2 followed by the activation of Notch signaling.
Bone is one of the most common sites of breast cancer metastasis (Kominsky & Davidson, 2006
). It is becoming clear that there are many regions that are hypoxic in the bone, and these areas play an important role in the self-renewal ability of hematopoietic stem cells by providing stem cell niche (Yin & Li, 2006
). Because metastatic cells must have stem-like characteristics, it is highly plausible that cancer stem-like cells take advantage of these niches once they reach the bone. In fact, our results indicate that bone marrow stromal cells significantly over-expressed Jagged2 under hypoxic condition and that co-culturing the stromal cells with the cancer stem-like cells significantly promoted proliferation of cancer stem-like cells by activating Notch signaling. Therefore, Jagged2 expression in bone marrow stromal cells at the hypoxic regions in the bone is considered to provide appropriate niche for metastatic cancer stem-like cells and promote their self-renewal.
Collectively, our results indicate that the expression of Jagged2 is significantly augmented by hypoxia at the invasive front and the interaction between breast cancer cells and the Jagged2-expressing cells significantly enhanced the invasiveness and survival of cancer cells through the activation of Akt/EMT pathways. Furthermore, hypoxic regions in the bone marrow provide the niche for cancer stem-like cells by expressing the hypoxia-induced Jagged2. Therefore, Jagged2 is considered to be a valuable marker to predict the outcome in patients with metastatic disease, and more importantly, intervention in Jagged2 expression and cell-cell interaction may serve as a novel therapeutic target for metastatic breast cancer.