There is increasing evidence that tumor cells often reside in a low oxygen tension environment that promotes accumulation of HIF-1α and is involved in tumor progression and aggressiveness. However, the effect of hypoxia on propagation of T-ALL cells and the mechanisms by which hypoxia exerts its effects are still not clear. Notch1 signalling has been shown to play an oncogenic role in the majority of hematological malignancies including T-ALL. Further, Notch1 is related to resistance to chemotherapy, a major cause of treatment failure and poor prognosis in T-ALL. Hypoxia-induced expression of Notch receptors and ligands has been demonstrated in stem cells and malignant tumors [22, 23]. In the present study, we showed that Notch1 signalling was activated by hypoxia and its transducer HIF-1α. Furthermore, we provide the first evidence that hypoxia/HIF-1α promoted the progression of T-ALL through activation of the Notch1 pathway, resulting in altered expression of downsteam genes regulating cellular proliferation, invasion and chemoresistance. HIF-1α-dependent overexpression of Notch1 in T-ALL cells is one of the major mechanisms underlying T-ALL aggressiveness and resistance to chemotherapy.
The role of hypoxia and HIF-1α in cellular proliferation has been investigated in various cell types. It has been revealed that hypoxia and HIF-1α convey stimulating or inhibiting effects on cellular proliferation and viability, depending on cell type. Studies on epithelial ovarian tumors and esophageal cancer showed that HIF-1α overexpression correlated with tumor apoptosis and patient survival [24, 25]. However, in an animal model of chronic myeloid leukemia (CML), inhibition of HIF-1α impaired propagation of CML by impairing cell cycle progression and inducing apoptosis of LSCs, suggesting that HIF-1α plays a crucial role in survival maintenance of LSCs . In the current study, we showed that hypoxia and HIF-1α, by reducing the proportion of cells in the G0/G1 phase of the cell cycle, promote the proliferation of T-ALL cells. Molecular investigation revealed that, under hypoxic conditions, expression of Cyclin D1 and CDK2 was increased whereas p21 expression was decreased. We also found that Notch1 ICN was augmented by hypoxia and this effect was dependent on HIF-1α accumulation, consistent with previous results . Potentiation of Notch1 signalling by hypoxia was further corroborated by elevated expression of the Notch1 downstream gene Hes1 in hypoxic conditions. Inhibition of Notch1 activity using Notch1-targeted siRNA reversed hypoxia-induced changes in expression of cell cycle regulatory proteins (CDK2, CyclinD1 and p21), thus repressed the hypoxia-induced proliferation of T-ALL cells. On the basis of these results, we concluded that hypoxia potentiated Notch1 signalling in T-ALL, leading to altered expression of cell cycle regulatory proteins and increased cell proliferation.
T-ALL is characterized as leukemic cell infiltration of various organs such as lymph nodes, liver, spleen and lungs. MMPs, in particular the gelatinases MMP2 and MMP9, are an important group of zinc- and calcium-dependent proteolytic enzymes responsible for degradation of the vascular basement membrane and the extracellular matrix of lymphoid tissues . A previous study suggested that hypoxia and HIF-1α overexpression contribute to tumor cell invasion and dissemination, probably through activation of MMPs . Considerable evidence has accumulated that MMP9 and MMP2 play an important role in the invasiveness and propagation of several hematological malignancies [29–31]. Our studies indicate that hypoxia may be an initiating event that results in enhanced Notch1 signalling, increased expression of MMP2 and MMP9, and ultimately increased invasiveness of T-ALL. Furthermore, inhibition of Notch1 signalling abrogated hypoxia/HIF-1α-induced cell invasion, probably through down-regulation of MMP2 and MMP9. This indicates that Notch1 may serve as a critical intermediary that transforms the hypoxic response into invasion.
In addition to cellular invasion, as discussed above, chemoresistance has been recognized as another major cause of treatment failure among patients who suffer from T-ALL. Previous studies have shown that hypoxia is closely related to tumor resistance to anticancer therapy in a wide spectrum of neoplastic cells [32, 33]. We hypothesized that chemoresistance to dexamethasone, an anti-leukemia drug commonly used in T-ALL treatment, was acquired by T-ALL cells in response to hypoxia. Under our hypoxic culture conditions (2% oxygen tension), dexamethasone exhibited less effectiveness. Further investigation revealed that hypoxia and HIF-1α increased expression of Bcl-2 and Bcl-xL, leading to a marked decrease in caspase activity.
Several studies have demonstrated the contribution of the Notch pathway to chemoresistance in human malignancies. Previous studies on solid tumors suggested that Notch is involved in the formation of cancer stem cells and the acquisition of the epithelial-mesenchymal transition phenotype, which are both critically associated with chemoresistance [34, 35]. In myeloma and other malignant lymphoid cell lines, studies have demonstrated that Notch1 is closely related to bone marrow stroma–mediated drug resistance, and that inhibition of Notch signalling sensitizes cells to chemotherapy and prevents bone marrow-mediated chemoresistance [36, 37]. Here, we showed that knockdown of Notch1 prevented the protective effect of hypoxia/HIF-1α against dexamethasone-induced apoptosis. This sensitization correlated with loss of the effect of hypoxia/HIF-1α on Bcl-2 and Bcl-xL expression.
Rohwer et al. reported that HIF-1α suppressed gastric cancer chemosensitivity via modulation of p53, indicating that hypoxia-induced chemoresistance was dependent on a functional p53 pathway . Because the leukemic T-cell lines used in our study do not have functional p53, our results provide evidence that activation of the Notch1 pathway may represent an alternative mechanism for hypoxia-induced chemoresistance in mutant p53 cell lines.
HIF-1α knockdown by siRNA or antisense techniques has been shown to suppress cell growth, proliferation and migration in both normal human cells and malignant tumor cells, including umbilical vascular endothelial cells, medulloblastoma, prostate cancer and glioma [39–42]. Silencing HIF-1α also has been shown to reverse chemotherapy resistance in tumor cells . Our finding, that blocking HIF-1α sensitized T-ALL cells to dexamethasone treatment, suggests that HIF-1α may be a potential target for gene therapy in T-ALL cells.