Original ArticlesAnchorage-dependent multicellular aggregate formation induces CD44 high cancer stem cell-like ATL cells in an NF-κB- and vimentin-dependent manner
Introduction
Adult T-cell leukemia/lymphoma (ATL) is an intractable and fatal T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) [1]. A striking feature of ATL is a massive invasion of lymphoma cells into various tissues such as the gastrointestinal tract, lung, skin, and central nervous system [2], [3]. Leukemia/lymphoma cells that have invaded the tissues become more resistant to chemotherapy, presenting a major obstacle to the treatment of ATL patients [4], [5], [6]. However, the mechanisms underlying the acquisition of intractable phenotypes by tissue-invading ATL cells are largely unknown.
Cancer stem cells (CSCs) are defined as a small fraction of tumor cells that display properties of self-renewal, high tumorigenicity, and drug resistance, resulting in cancer metastasis and recurrence [7], [8]. Of many CSC markers identified thus far, CD44 is of particular biologic importance [9]. Recent work has revealed that nuclear CD44 directly reprograms stem cell properties in colon cancer cells [10]. Also, expression of CD44 splice variants containing exons 8–10 (CD44v8–10) contributes to reactive oxygen species (ROS) defense by up-regulating the synthesis of a major antioxidant, glutathione [11]. In ATL patients, CD44 is expressed on skin-infiltrating tumor cells, with the plasma levels of soluble CD44 showing correlation with disease severity [12], suggesting the existence of CD44+ ATL stem cells. Cancer cells with constitutively activated NF-κB tend to become resistant to various anticancer drugs and radiotherapy [13], [14], suggesting a close link between CSC phenotypes and NF-κB activation [15], [16]. Notably, constitutive activation of NF-κB is observed in ATL cells, despite the fact that they do not express detectable levels of viral gene products such as Tax with potent NF-κB-activating activities [17], [18].
CSC properties are thought to be regulated and maintained by the surrounding microenvironment (niche) composed of cellular structures such as epithelium, vasculature, mesenchymal cells and extracellular matrix [19], [20], [21]. However, the mechanisms of how CSCs emerge and are maintained in the niche are poorly understood mainly because of the difficulty in expanding CSCs in vitro. Feeder cells have been used to support the growth and stemness potential of stem cells in vitro [22], [23], [24], [25]. Mesenchymal cells such as mouse embryonic fibroblasts and human mesenchymal stem cells have been successfully employed as feeder cells to support the growth of human embryonic and induced pluripotent stem cells [23], [24], [25]. Although human embryonic cells can be maintained under feeder-free conditions, the absence of feeder cells often results in karyotype abnormalities due to chromosomal instability [26], [27]. Given the basic similarities of CSCs and normal stem cells, it is reasonable to assume that feeder cells play a similarly important role in the emergence of CSCs and the formation of CSC niches.
We previously showed that ATL cells undergo apoptosis when cultured in the presence of histone deacetylase (HDAC) inhibitors; however, when ATL cells were directly cocultured with HEK293T cells, a normal tissue-derived epithelial-like cell line, they acquired resistance to HDAC-inhibitor-induced apoptosis [28]. Interestingly, the majority of ATL cells adhering to HEK293T cells became quiescent and slowed down cell cycle progression, and some ATL cells acquired CD44 high phenotypes. The present study was undertaken to investigate the nature of CD44 high ATL cells induced by simple coculture with HEK293T cells and to study the mechanism by which coculture induces such ATL cells.
Section snippets
Cells
ATL-CR and ATL-TH, both IL-2-independent ATL cell lines in which HTLV-1 gene expression was silenced [28], were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. HEK293T cells, human embryo kidney-derived epithelial-like cells used as normal tissue-derived feeder cells, were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% FBS and penicillin/streptomycin. 1C3IKEI, a primary normal human embryonic
Anchorage-dependent multicellular aggregate (Ad-MCA) formation induces a population of CD44 high ATL cells
To induce mass formation by ATL cells, we cocultured ATL cells directly with HEK293T cells (Fig. 1A). When ATL cells were cocultured overnight on a monolayer of HEK293T cells, the majority of ATL cells adhered to and formed masses on the monolayer, but coculture with normal-tissue-derived fibroblasts failed to induce such mass formation by ATL cells [28]. To determine whether mass formation by ATL cells was induced by clonal proliferation or cellular aggregation of ATL cells, we cocultured a
Discussion
The present study has demonstrated that ATL cells can acquire intractable CSC-like phenotypes when they form Ad-MCAs on a layer of epithelial-like HEK293 feeder cells (Fig. 1). Inhibition of NF-κB signaling suppressed Ad-MCA formation and subsequent emergence of CD44 high CSC-like ATL cells (Fig. 4), demonstrating the critical role of the NF-κB pathway in the induction of CSC-like ATL cells.
ATL cells adhering to a layer of HEK293T feeder cells showed enhanced expression of vimentin, a
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number 26460464 and the Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Ireland. We thank Drs. Shuichi Ota and Takanori Teshima for supplying us with samples of blood from an ATL patient.
References (47)
- et al.
The epithelial-mesenchymal transition generates cells with properties of stem cells
Cell
(2008) - et al.
CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth
Cancer Cell
(2011) - et al.
Involvement of osteopontin and its signaling molecule CD44 in clinicopathological features of adult T cell leukemia
Leuk. Res
(2011) - et al.
Hyaluronan-CD44 interaction activates stem cell marker Nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells
J. Biol. Chem
(2008) - et al.
NF-kappaB pathway inhibitors preferentially inhibit breast cancer stem-like cells
Breast Cancer Res. Treat
(2008) - et al.
Constitutive activation of NF-kappaB in primary adult T-cell leukemia cells
Blood
(1999) - et al.
Tax-independent constitutive IkappaB kinase activation in adult T-cell leukemia cells
Neoplasia
(2004) - et al.
Induction of pluripotent stem cells from adult human fibroblasts by defined factors
Cell
(2007) - et al.
Protective roles of epithelial cells in the survival of adult T-cell leukemia/lymphoma cells
Am. J. Pathol
(2013) - et al.
Role of neuronal interferon-gamma in the development of myelopathy in rats infected with human T-cell leukemia virus type 1
Am. J. Pathol
(2006)
Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells
Cell Stem Cell
Molecular insight in the multifunctional activities of withaferin A
Biochem. Pharmacol
The tumor inhibitor and antiangiogenic agent withaferin A targets the intermediate filament protein vimentin
Chem. Biol
Phase 2 trial of romidepsin in patients with peripheral T-cell lymphoma
Blood
Cell growth in aggregates determines gene expression, proliferation, survival, chemoresistance, and sensitivity to immune effectors in follicular lymphoma
Am. J. Pathol
Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma
Proc. Natl. Acad. Sci. U.S.A.
Cutaneous manifestations associated with HTLV-1 infection
Int. J. Dermatol
Pathological features of diseases associated with human T-cell leukemia virus type I
Cancer Sci
Controversies in targeted therapy of adult T cell leukemia/lymphoma: ON target or OFF target effects?
Viruses
Definition, prognostic factors, treatment, and response criteria of adult T-cell leukemia-lymphoma: a proposal from an international consensus meeting
J. Clin. Oncol
No effect of humanized CCR monoclonal antibody (mogamulizumab) on treatment-resistant adult T cell leukemia with meningeal infiltration
Leuk. Lymphoma
Leukaemia stem cells and the evolution of cancer-stem-cell research
Nat. Rev. Cancer
CD44: can a cancer-initiating cell profit from an abundantly expressed molecule?
Nat. Rev. Cancer
Cited by (17)
Proximity-constructed bifunctional DNA probes for identification of stem-like biomarker in breast cancer
2021, Sensors and Actuators, B: ChemicalCitation Excerpt :By referring to the regression equation, CD44 expressions were calculated to be about 49.3 ng/mL for BCSCs lysates, 46.4 ng/mL for MDA-MB-231 cell lysates, and 0.11 ng/mL for BT-474 cell lysates, which were in concordance with those of flow cytometry analysis. Moreover, Jurkat, a T-cell lymphoma cell line that was short in CD44 expression [28,29], was used as a normal cell control, which only resulted in a very small peak current that approached the background signaling. All the results confirmed the probability of our method in selective identification of surface-expressed CD44.
Adult T-cell Leukemia: Molecular basis for clonal expansion and transformation of HTLV-1-infected T cells
2017, BloodCitation Excerpt :In addition, “microenvironment” of the ATL cells is supposed to be involved in ATL cell survival and resistance to chemotherapy. Several reports suggested importance of interaction with normal epithelial cells and mesenchymal stem cells.121-123 Furthermore, involvement of angiogenesis and fibroblast-derived osteopontin has been suggested in proliferation and survival, as well as possible roles of exosomes produced by ATL cells.124-126
Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles
2016, VirologyCitation Excerpt :We firstly analysed the expression levels of NF90 and NF110 in two HTLV-1 chronically infected cell lines (MT2 and C91), a HTLV-2 chronically infected cell line (Mo) and two ATL cell lines (ATL-CR and ATL-TH) compared to control uninfected Jurkat cells (Fig. 1B). The ATL-CR and ATL-TH cell lines were established from patients with ATL and viral gene expression is silenced in these cells (Miyatake et al., 2013, 2015). The status of HBZ expression in the ATL-CR and ATL-TH cell lines was not investigated.
An alginate-based platform for cancer stem cell research
2016, Acta BiomaterialiaCitation Excerpt :Although there was no significant difference between the control group and the 1.5% LMW-HA group in terms of tumor spheroid size (131.3 ± 8.5 μm), the 1.5% LMW-HA group could lead to a higher spheroid colony number (47 ± 1). Furthermore, the 1.5% LMW-HA group can significantly up-regulate the expression of MDR1, which has been proven as CSCs marker by that MDR1 high expression helps the survival of CD44 high CSC [61]. The objective our study was to identify a suitable material to support CSC proliferation by developing an easily tunable hydrogel system.
The Use of Withaferin A to Study Intermediate Filaments
2016, Methods in EnzymologyCitation Excerpt :Thus, vimentin represents a class of target that has remained undruggable for several decades, despite the numerous studies illuminating the disease-related roles of IFs (Eriksson et al., 2009; Kidd, Shumaker, & Ridge, 2014). Our discovery has provided an unprecedented opportunity to exploit WFA as a pharmacological probe of type III IFs in cell biology and disease (Bargagna-Mohan et al., 2007, 2015, 2010, 2012; Gladilin, Gonzalez, & Eils, 2014; Grin et al., 2012; Lahat et al., 2010; Lynch, Lazar, Iskratsch, Zhang, & Sheetz, 2013; Menko et al., 2014; Miyatake, Sheehy, Ikeshita, Hall, & Kasahara, 2015; Thaiparambil et al., 2011). In this chapter, we will describe some of the salient findings we have made with the use of WFA to investigate cell behavior that are tied to critical IF functions (see also Table 1 for summary of WFA activity on IFs and non-IFs).