Regulation of cancer stem cell properties by CD9 in human B-acute lymphoblastic leukemia

https://doi.org/10.1016/j.bbrc.2011.04.098Get rights and content

Abstract

Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some of the cases still exhibit therapy-resistant. We have previously reported that CD9 was expressed heterogeneously in B-ALL cell lines and CD9+ cells exhibited an asymmetric cell division with greater tumorigenic potential than CD9 cells. CD9+ cells were also serially transplantable in immunodeficient mice, indicating that CD9+ cell possess self-renewal capacity. In the current study, we performed more detailed analysis of CD9 function for the cancer stem cell (CSC) properties. In patient sample, CD9 was expressed in the most cases of B-ALL cells with significant correlation of CD34-expression. Gene expression analysis revealed that leukemogenic fusion proteins and Src family proteins were significantly regulated in the CD9+ population. Moreover, CD9+ cells exhibited drug-resistance, but proliferation of bulk cells was inhibited by anti-CD9 monoclonal antibody. Knockdown of CD9 remarkably reduced the leukemogenic potential. Furthermore, gene ablation of CD9 affected the expression and tyrosine-phosphorylation of Src family proteins and reduced the expression of histone-deubiquitinase USP22. Taken together, our results suggest that CD9 links to several signaling pathways and epigenetic modification for regulating the CSC properties of B-ALL.

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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