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Side Population Cells from Human Melanoma Tumors Reveal Diverse Mechanisms for Chemoresistance Yuchun Luo1, Lixia Z

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Side Population Cells from Human Melanoma Tumors Reveal Diverse Mechanisms for Chemoresistance Yuchun Luo1, Lixia Z ORIGINAL ARTICLE See related commentary on pg 2317 Side Population Cells from Human Melanoma Tumors Reveal Diverse Mechanisms for Chemoresistance Yuchun Luo1, Lixia Z. Ellis1, Katiuscia Dallaglio1,2, Moe Takeda1, William A. Robinson3, Steven E. Robinson3, Weimin Liu1, Karl D. Lewis3, Martin D. McCarter4, Rene Gonzalez3, David A. Norris1,5, Dennis R. Roop1,2, Richard A. Spritz6, Natalie G. Ahn7 and Mayumi Fujita1,2,5 Side population (SP) cells are identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and it represents hematopoietic stem cells and chemoresistant cells from several solid tumors. In this study, we confirmed the presence of SP cells in tumors from melanoma patients. Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5. We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a substrate of ABCB1, both in vitro and in vivo. However, melanoma SP cells were also resistant to temozolomide, which is not a substrate for ABC transporters, through IL-8 upregulation. In addition, gene profiling studies identified three signaling pathways (NF-kB, a6-b4-integrin, and IL-1) as differentially upregulated in melanoma SP cells, and there was a significant increase of PCDHB11 and decrease of FUK and TBX2 in these cells. Therefore, we provide evidence that SP is an enriched source of chemoresistant cells in human melanomas, and suggest that the selected genes and signaling pathways of SP cells may be a potential target for effective melanoma therapies. To our knowledge, this is a previously unreported study to isolate SP cells from melanoma patients and to investigate the gene expression profiling of these cells. Journal of Investigative Dermatology (2012) 132, 2440–2450; doi:10.1038/jid.2012.161; published online 24 May 2012 INTRODUCTION the fluorescent Hoechst 33342 dye has been employed to Malignant melanoma is the fifth most common cancer in the isolate a side population (SP) from hematopoietic cells (Goodell United States (Siegel et al., 2011) and the deadliest form of et al., 1996). This phenotype of low fluorescent staining pattern skin cancer. Despite various types of treatments, advanced results from a family of transporter proteins known as ATP- stages of melanoma cells have proven to be intrinsically binding cassette (ABC) transporters, which are responsible for resistant to many therapies. Multiple mechanisms contribute the exclusion of Hoechst 33342 (Helmbach et al., 2001; to the development of drug resistance in melanoma, Gottesman et al., 2002). These SP cells have stem cell–like including induction of enzyme systems, reduced apoptosis, properties and are capable of self-renewal and differentiation increased DNA repair, and intra- and extra-cellular drug (Dean et al., 2005). These characteristics of SP cells have also transport (Helmbach et al., 2001; La Porta, 2007). been found in various types of tumors (Hirschmann-Jax et al., One of the well-known mechanisms of chemoresistance 2004; Grichnik et al., 2006; Dean, 2009; Dou et al., 2009; in cancers is the enhanced efflux of a broad spectrum of Zhong et al., 2010), and SP cells with a high drug efflux hydrophobic cytotoxic drugs. Recently, the ability to exclude capacity are identified as the potential cause of the chemo- resistant phenotypes of cancer stem cells in human tumors (Hirschmann-Jax et al., 2004; Dean et al., 2005). 1Department of Dermatology, University of Colorado Denver, Aurora, Colorado, USA; 2Charles C Gates Regenerative Medicine and Stem Cell SP cells have previously been isolated from human Biology, University of Colorado Denver, Aurora, Colorado, USA; 3Division of melanoma cell lines (Grichnik et al., 2006), but are yet to Medical Oncology, Department of Medicine, University of Colorado Denver, be isolated directly from patient melanoma tumors. One 4 Aurora, Colorado, USA; Department of Surgery, University of Colorado major challenge of analyzing patient samples is that Denver, Aurora, Colorado, USA; 5Denver Veterans Affairs Medical Center, Denver, Colorado, USA; 6Human Medical Genetics Program, University of substantial quantities of fresh tumor tissues are not available Colorado Denver, Aurora, Colorado, USA and 7Department of Chemistry and for isolation and investigation of SP cells that comprise only Biochemistry, University of Colorado at Boulder, Boulder, Colorado, USA a small percentage of the tumor cells. Another difficulty is Correspondence: Mayumi Fujita, Department of Dermatology, University of that experiments to further characterize biology and function Colorado, Mail Stop 8127, 12801 E. 17th Avenue, Room 4124, Aurora, of these cells are limited by the technical difficulties in Colorado 80045, USA. E-mail: [email protected] preserving and expanding tumor cells that faithfully repre- Abbreviations: ABC, ATP-binding-cassette; qRT-PCR, real-time quantitative reverse transcription–PCR; siRNA, small interfering RNA; SP, side population sent patient tumor biology. To that end, a direct in vivo Received 29 October 2011; revised 9 March 2012; accepted 22 March 2012; xenograft model provides cohorts of tumor-bearing mice published online 24 May 2012 suitable for further biological analyses and drug treatment 2440 Journal of Investigative Dermatology (2012), Volume 132 & 2012 The Society for Investigative Dermatology Y Luo et al. Gene Expression Profiling of SP in Human Melanoma (Rubio-Viqueira et al., 2006). Maintaining tumor xenografts a With verapamil Without verapamil 128 128 exclusively in vivo retains fundamental genotypic features of Non-SP Non-SP the primary tumor of direct relevance to preclinical drug 96 96 testing (Rubio-Viqueira et al., 2006; Daniel et al., 2009). 64 64 Therefore, to isolate and characterize the molecular features 32 SP 32 SP of a small fraction of SP cells in patient melanoma samples, 0.01% 0.34% we generated a direct in vivo xenograft model from human 0 0 0 32 64 96 128 0 32 64 96 128 melanoma tumors. Furthermore, our gene profiling analysis Hoechst blue discovered a series of genes and pathways potentially Hoechst red involved in chemoresistance in melanoma SP cells. b Frequencies Frequencies Name Type Origin of SP (F0) of SP (F1) RESULTS MF347 Primary Skin 0.7% 0.9% Patient melanoma tumors contain SP that excludes Hoechst dye 111308 Primary Skin 0.3% NA The presence of SP cells in patient melanoma tissues was first MB947p Primary Skin 0.43% 0.17% investigated by staining single-cell suspensions with Hoechst MB929 Metastasis Skin 0.27% NA MB947m Metastasis Lymph node 0.29% NA 33342 dye. Flow cytometry plots depict Hoechst excitation MB952 Metastasis Lymph node 0.34% 0.46% at blue and red wavelengths. The cells with the least amount MB1009 Metastasis Lymph node 0.16% 0.15% of Hoechst staining, which disappear in the presence of 82908 Metastasis Adrenal gland 0.13% NA verapamil, an inhibitor of Hoechst 33342 transport, are gated as SP (Figure 1a). All of the patient melanoma tumors, c For SP analysis including three skin primary melanomas and five metastatic (MF347-F0, 111308-F0, MB947p-F0, MB929-F0, F0 Patient tumor MB947m-F0, MB952-F0, MB1009-F0, 82908-F0) melanomas, contained a small but clear SP fraction, ranging For copy number analysis from 0.13 to 0.7% of all gated cells (Figure 1b). We then (MB929-F0, MB947m-F0) generated a direct in vivo xenograft model from human For microarray melanoma specimens to characterize SP cells from patient- (MB952-F1, MB1009-F1) For qRT-PCR derived tumors (Figure 1c). Original patient tumors were (MF347-F1, MB952-F1, MB1009-F1) designated as F0 tumors, whereas those grown from F0 For in vitro drug treatment F1 1st xenograft (MB947m-F1, MB952-F1, MB1009-F1) tumors were designated as F1 tumors. F1 tumors were further For IL-8 ELISA implanted in F2 mice for in vivo drug treatment (Figure 1c). (MB952-F1) For copy number analysis After characterizing SP cells using this model, cells from (MB929-F1, MB947m-F1) HS294T were utilized for further biological and mechanistic studies because the cell line contains a larger SP fraction For in vivo drug treatment (MB952-F2, MB1009-F2) (Supplementary Figure S1 online). F2 2nd xenograft For copy number analysis (MB929-F2, MB947m-F2) Melanoma SP cells are resistant to paclitaxel and temozolomide For copy number analysis in vitro and in vivo Fn nth xenograft (MB929-F4) SP cells expressing ABC transporters have been shown to be Figure 1. Side population (SP) is a rare population in patient melanoma resistant to multiple drugs (Duan et al., 2004; Dean, 2009). tissues. (a) A representative SP flow cytometry profile from a patient tumor Paclitaxel, a substrate of ABCB1 transporter (Duan et al., tissue (MB952-F0). Cells were stained with Hoechst 33342 dye in the 2004), has been widely evaluated and shown to have positive presence (left panel) or absence (right panel) of verapamil. SP cells are shown outcomes in phase II/III clinical trials of malignant melanoma in the lower left gated area. (b) Frequency of SP fractions in eight patient (Bedikian et al., 2004; Hauschild et al., 2009). Therefore, we melanoma specimens (F0 tumors) and selected F1 tumors. (c) Study schema. treated human melanomas with paclitaxel to examine the Clinical melanoma specimens (F0) were used for SP analysis and copy drug sensitivity of SP cells. Temozolomide, an alkylating number analysis. Tumor specimens used in each analysis are listed in parentheses. Tumors were serially passaged into mice (F1, F2,- - -, Fn) for agent for melanoma treatment (Bleehen et al., 1995), is not a further analyses. NA, not analyzed. substrate of ABC transporters and thus was used as a control. As shown in Figure 2a, melanoma cells from MB1009-F1 tumor were sensitive to 20 nM paclitaxel treatment in vitro. in tumor size (79.4±5.9% of original size) was observed This treatment resulted in a 32.5-fold increase in SP cells after 7 days, but the tumors grew thereafter (125.1±13.2% of compared with vehicle control (Figure 2b).
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