Abstract
Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP) cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2–4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC) drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assay(EMSA), we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia.
Highlights
Leukemic stem cells (LSCs) are cells that can give rise to leukemia in transplant murine models, and have selfrenewal property
We found that SALL4 could promote the expression of the ATP binding cassette (ABC) transporter genes, such as ABCA3 and ABCG2, and knocking down of SALL4 expression led to a decreased side population (SP)
By quantitative reverse transcription polymerase chain reaction (qRT-PCR), we found that the expression level of ABCA3 in SALL4 overexpressing cells was increased by 33% compared to those of the controls (Figure 5A)
Summary
Leukemic stem cells (LSCs) are cells that can give rise to leukemia in transplant murine models, and have selfrenewal property. Several methods are currently being used to isolate and study hematopoietic stem cells (HSCs) or LSCs, including the use of HSCs or LSCs cell surface markers [5,6,7,8,9,10] and the use of Hoechst 33342 dye efflux to identify side population (SP) [11,12,13,14,15,16]. Isolation of HSCs or LSCs by cell surface markers is better suited for subsequent functional studies such as in vivo transplantation since the Hoechst dye is toxic to the cells and might impair their physiological functions. On the other hand, studying SP cells might be a better approach to identify LSCs with potential resistance to chemotherapeutic agents that may account for leukemic recurrence. In addition to blood cells, the SP approach has been used for enrichment for other cancer stem cells, such as breast cancer stem cells [15]
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