Resistance to cytotoxic agents is one of the obstacles in the treatment of AML. We found that combined AurA inhibition and Ara-C treatment enhanced leukemic cell death in Ara-C-resistant leukemia cells by inducing apoptosis and mitotic catastrophe [14
]. Because LSCs are also a major cause of relapse and resistance to treatment, we explored the effects of AurA inhibition in LSCs.
AurA is overexpressed in various cancers, including hematological malignancies [3
], and its overexpression is related to progression or poor prognosis subtypes in some malignancies [4
]. Therefore, inhibition of AurA by selective inhibitors is the subject of several clinical trials. However, AurA expression and the effects of AurA inhibition in cancer stem cells are not yet fully understood. In a few reports on cancer stem cells of epithelial origins, AurA expression was found to be upregulated, and inhibition of AurA led to cell cycle arrest or restricted cell growth [10
Most LSCs are known to exist as a fraction of the CD34+
population and are able to transmit AML to nonobese diabetic/severe combined immunodeficient mice [16
]. Our data show that a CD34+
subpopulation of primary AML cells that contains LSCs exhibited significantly increased expression of AurA when compared to normal HSCs. Similarly, Ye et al. reported increased AurA expression in CD34+
blast cells from patients with AML or myelodysplastic syndrome [17
]. Because the CD34+
population includes non-LSC blasts, to our knowledge, this is the first report on AurA expression in LSCs. As expected, AurA expression was high in non-LSC blast cells, which may be because non-LSC blasts are actively cycling, whereas the majority of LSCs are quiescent. Similar results have been seen in colorectal cancer stem cells (CR-CSC) and ovarian cancer stem cells (EOC stem cells) [10
]. Cammareri et al. [10
] found that the expression of AurA in CR-CSC was lower than that in primary colorectal cancer cells. However, in our findings, LSCs expressed a significantly higher level of AurA than did normal HSCs, and upon AurA inhibition, LSCs showed increased apoptosis while AurA inhibition caused no changes in apoptosis in normal HSCs. We think this difference between normal HSCs and LSCs may be useful for developing treatments that target LSCs.
The role of AurA in LSCs is not yet fully defined. AurA exerted no transforming activity in normal colon cells by itself, but induced centrosomal amplification, which can result in oncogenesis with additional genetic changes [10
]. Although the function of AurA in LSC survival has not been fully analyzed, experiments in both CR-CSC and EOC stem cells showed that silencing or inhibition of AurA was related to a decreased G1 phase population and an increased number of cells in G2/M arrest. Our results in LSCs were similar. Selective AurA inhibition increased the sub-G1 phase fraction in NB4 cells, and in K562, KG1, and U937 cells in another study [14
Our previous report focused on the synergistic effects of Ara-C and the AurA inhibitor in leukemia cells and its effects on related molecular pathways. Here, we examined the effects of selective AurA inhibition on LSC survival. CD34+
populations containing LSCs, obtained from AML patients, showed increased apoptosis after treatment with either Ara-C or the AurA inhibitor, and the effect was significantly enhanced upon co-treatment with Ara-C and the AurA inhibitor. This co-treatment effect was greater in non-LSCs. Following stimulation with G-CSF, AurA expression increased, leading to enhanced apoptosis with Ara-C/AurA co-treatment. Because the main roles of AurA are in spindle formation, centrosome maturation, and duplication [2
], this result may indicate that G-CSF stimulated some LSCs to enter the cell cycle or increased asymmetric cell division.
KG1 cells exhibited lower rates of apoptosis compared to NB4 cells following AurA inhibitor treatment. In both cell lines, Ara-C-induced cell death involved the activation of p38MAPK. In KG1 cells, NF-κB was constitutively activated after Ara-C treatment, perhaps explaining the lower apoptosis rates in these cells. In EOC stem cells [11
], selective AurA inhibition resulted in reduced NF-κ activation. Guzman et al. reported increased NF-κB levels in CD34+
human AML cells [18
], and Colado et al. tested bortezomib as an NF-κB inhibitor to overcome drug resistance in CD34+
immature myeloid leukemia cells [19
]. Reduced apoptosis in KG1 cells induced by AurA inhibition may result from insufficient NF-κB inhibition. Because NF-κB activation levels differ in various cancers, the required dose of AurA inhibitor may vary among cell lines. Nevertheless, taken together, these results suggest that AurA inhibition and potent NF-κB activation can reduce the number of LSCs.
In summary, our study demonstrated increased expression of AurA in LSCs. Furthermore, our findings suggest the possibility that selective AurA inhibitors can be used to reduce the numbers of LSCs, an effect that can be enhanced by stimulation with G-CSF. Further exploration of the relationships between NF-κB and AurA inhibition, and the potential utility of AurA inhibition in leukemia treatment is needed.