Regulation of cancer stem cell properties by CD9 in human B-acute lymphoblastic leukemia
Highlights
► We performed more detailed analysis of CD9 function for CSC properties in B-ALL. ► Leukemogenic fusion/Src family proteins were markedly regulated in the CD9+ cells. ► Proliferation of B-ALL cells was inhibited by anti-CD9 monoclonal antibody. ► Knockdown of CD9 by RNAi remarkably reduced the leukemogenic potential. ► CD9-knockdown affected the expression and phosphorylation of Src family and USP22.
Introduction
CD9 is a 24–27kD cell-surface glycoprotein which belongs to a tetraspanin super family, expressed in a variety of normal tissues, and reported to involve in cell adhesion, motility, and many signaling events [1]. In blood cells, although hematopoietic stem cells do not express CD9, pre-B lymphocytes express CD9 [2]. The importance of CD9 for cancer progression has often been indicated in recent years. CD9 was reported to be involved in invasion of cancer cells, and associated with cancer progression, metastasis, recurrence, and clinical outcome [3].
Although the prognosis of pediatric acute lymphoblastic leukemia (ALL) has improved considerably in recent years, a significant number of cases exhibit therapy-resistant. It has been postulated that the relapse may be due to cancer stem cell (CSC) [4]. CSC has self-renewal capacity and multipotency similar with normal stem cells, and proliferates by asymmetric cell division, generating mature leukemia cells. Therefore, CSC is believed to be a potential reason for chemoresistance, metastasis, and recurrence [5].
Previous studies using primary samples have shown that B-ALL cells capable of long-term proliferation in vivo were the CD34+/CD10−/CD19− fraction [6]. More recent study has also reported that CD133 was expressed on leukemia-initiating cells of childhood ALL [7]. This fraction was more resistant to anti-cancer drugs than the bulk leukemic cells.
To understand therapy-resistance in lymphoid leukemia, we have explored CSC markers using cell lines. In T-lineage malignancy [8], adult T-cell leukemia/lymphoma often contained SP cells [9]. We also found that CD90 and CD110 correlated with stem cell properties in T-ALL [10]. In B-ALL, we have previously reported that CD9 was expressed heterogeneously and only CD9+ cells proliferated by asymmetric cell division-like manner in vitro [11]. CD9+ cells also exhibited greater tumorigenic potential in immunodeficient mice than CD9− cells. Moreover, these CD9+ cells were serially transplantable in mice and reconstituted the original pattern of CD9 expression, indicating that CD9+ cell possesses the self-renewal potential.
In this report, we conducted more detailed analysis of the CSC properties in CD9-expressing cells of B-ALL cell lines and patient samples. We found that leukemia-related genes and Src family genes were markedly regulated between CD9+ and CD9− populations. We also found that CD9 is a promising target for CSC-oriented therapy by monoclonal antibody treatment and RNAi-knockdown. Moreover, ablation of CD9 significantly affected the expression and tyrosine-phosphorylation of Src family and histone ubiquitination through novel CSC marker ubiquitin-specific protease 22 (USP22) [12].
Section snippets
Cell lines, culture, flow cytometry, and cell sorting
The protocols were described in our previous paper [11].
Patient specimens and antibodies
Diagnostic bone marrow or peripheral blood samples were obtained from pediatric patients with precursor B-ALL as described in Supplementary Table 1 (Approval number: 20-32-1006). For phenotypic analysis and transplantation, mononuclear cells were first gated by PI and lineage-markers mixture (APC-conjugated anti-CD3, CD33, CD56, and Immunoglobulin light chain κ/λ), then analyzed using anti-CD9-PE and CD34-FITC antibodies. For RT-PCR
Correlation between CD9 and CD34-expressions in pediatric B-ALL clinical samples
In our previous report, we used three B-ALL cell lines to analyze stem cell properties of B-ALL both in vitro and in vivo assays [11]. We conducted further analysis using several primary samples of childhood precursor B-ALL patients to examine the expression of CD9 and CD34. We obtained eight primary samples (peripheral blood and bone marrow), whose characteristics are summarized in Supplementary Table 1. As shown in Fig. 1, both CD9 and CD34 were heterogeneously expressed in all cases.
Discussion
In our previous report, CD9+ cells were suggested to be the stem cell-like subpopulation of B-ALL having the leukemogenic and self-renewal potentials [11]. In this report, we showed that CD9 was expressed with highly correlation of CD34 and correlated with the engraftment potential in pediatric patient samples. Regulation of cancer-related genes was observed in the CD9+ cells, and the CD9+ cells exhibited the drug-resistance. Moreover, CD9-knockdown reduced the leukemogenic potential in the
Acknowledgments
This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan, and by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (C. Morimoto). CWX was supported by a grant from the National Institutes of Health (CA108795).
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