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• W1995103178 abstract "•Slow-cycling melanoma cells expressing JARID1B show intrinsic multidrug resistance•Slow-cycling melanoma cells show upregulation of OXPHOS•Inhibition of OXPHOS sensitizes slow-cycling cells to therapy•OXPHOS inhibition can overcome drug resistance, irrespective of melanoma genotypes Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1Bhigh subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation. Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1Bhigh subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation. Metastatic melanoma is a heterogeneous tumor of neuroectodermal origin with a median survival less than 9 months. All chemotherapies, immunotherapies, and radiotherapies have failed to persistently increase survival. Despite the encouraging response rates following BRAFV600E targeting, relapses occur after a median duration of ∼5 months, because melanoma cells acquire multiple resistance mechanisms. However, even among functionally and genetically heterogeneous tumors, common and intrinsic mechanisms exist that support the immediate survival of certain cells against the attack of cytotoxic agents. We have identified a subpopulation of slow-cycling, tumor-maintaining melanoma cells with intrinsic phenotypic resistance to various therapies, irrespective of their mode of action. Targeting the slow-cycling subpopulation could increase the efficacy of current treatment regimens and reduce relapses. Malignant melanoma is a heterogeneous tumor of neuroectodermal origin that can be cured if excised at an early stage; however, once disseminated to distant organs, the median survival of patients drops below 9 months. All major cancer therapies have failed to persistently increase melanoma patients’ survival rates. This failure is attributed to different resistance mechanisms, including increased DNA repair and overexpression of antiapoptotic proteins, such as BCL-2, and drug efflux pumps, such as ABCB5 (Chen et al., 2009Chen K.G. Valencia J.C. Gillet J.P. Hearing V.J. Gottesman M.M. Involvement of ABC transporters in melanogenesis and the development of multidrug resistance of melanoma.Pigment Cell Melanoma Res. 2009; 22: 740-749Crossref PubMed Scopus (114) Google Scholar). Despite encouraging response rates seen with the BRAF inhibitor vemurafenib in BRAFV600E-positive individuals, relapses occur after a median duration of ∼5 months following initial tumor shrinkage (Sosman et al., 2012Sosman J.A. Kim K.B. Schuchter L. Gonzalez R. Pavlick A.C. Weber J.S. McArthur G.A. Hutson T.E. Moschos S.J. Flaherty K.T. et al.Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.N. Engl. J. Med. 2012; 366: 707-714Crossref PubMed Scopus (1769) Google Scholar). Several groups have identified various mechanisms of acquired therapy resistance in BRAF-targeted melanomas. Surviving melanomas reactivate pivotal networks, such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways (e.g., through activation of platelet-derived growth factor receptor β, cRAF-1 kinase, and insulin growth factor receptor 1 [Nazarian et al., 2010Nazarian R. Shi H. Wang Q. Kong X. Koya R.C. Lee H. Chen Z. Lee M.K. Attar N. Sazegar H. et al.Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation.Nature. 2010; 468: 973-977Crossref PubMed Scopus (1733) Google Scholar; Villanueva et al., 2010Villanueva J. Vultur A. Lee J.T. Somasundaram R. Fukunaga-Kalabis M. Cipolla A.K. Wubbenhorst B. Xu X. Gimotty P.A. Kee D. et al.Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K.Cancer Cell. 2010; 18: 683-695Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar], dimerization of aberrantly spliced BRAF isoforms [Poulikakos et al., 2011Poulikakos P.I. Persaud Y. Janakiraman M. Kong X. Ng C. Moriceau G. Shi H. Atefi M. Titz B. Gabay M.T. et al.RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E).Nature. 2011; 480: 387-390Crossref PubMed Scopus (1121) Google Scholar], or by secondary genetic events, such as genomic amplification of COT, NRAS mutations, or the MEK1C121S mutation [Johannessen et al., 2010Johannessen C.M. Boehm J.S. Kim S.Y. Thomas S.R. Wardwell L. Johnson L.A. Emery C.M. Stransky N. Cogdill A.P. Barretina J. et al.COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.Nature. 2010; 468: 968-972Crossref PubMed Scopus (1179) Google Scholar; Wagle et al., 2011Wagle N. Emery C. Berger M.F. Davis M.J. Sawyer A. Pochanard P. Kehoe S.M. Johannessen C.M. Macconaill L.E. Hahn W.C. et al.Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling.J. Clin. Oncol. 2011; 29: 3085-3096Crossref PubMed Scopus (780) Google Scholar]). However, even among functionally and genetically heterogeneous tumors, common and intrinsic survival mechanisms exist. Cytotoxic agents, including conventional chemotherapy, targeted therapy, or radiation, most effectively eliminate rapidly dividing cells (Blagosklonny, 2005Blagosklonny M.V. Why therapeutic response may not prolong the life of a cancer patient: selection for oncogenic resistance.Cell Cycle. 2005; 4: 1693-1698Crossref PubMed Scopus (96) Google Scholar). This concept is gaining prominence in the field of cancer research, with several studies reporting the enrichment of quiescent cancer stem cells after chemotherapy. For example, slow-cycling glioblastoma cells survive temozolomide treatment (Chen et al., 2012Chen J. Li Y. Yu T.S. McKay R.M. Burns D.K. Kernie S.G. Parada L.F. A restricted cell population propagates glioblastoma growth after chemotherapy.Nature. 2012; 488: 522-526Crossref PubMed Scopus (1575) Google Scholar). Accordingly, if cell subpopulations can act as major drivers for tumor maintenance and mediate universal therapeutic resistance, approaches that target this phenotype could increase the efficacy of treatment regimens and reduce the risk of melanoma relapses. We have recently demonstrated that, even within highly proliferative melanomas, there is a slow-cycling cell subpopulation that is identifiable by the expression of the histone 3 K4 demethylase JARID1B (Roesch et al., 2005Roesch A. Becker B. Meyer S. Wild P. Hafner C. Landthaler M. Vogt T. Retinoblastoma-binding protein 2-homolog 1: a retinoblastoma-binding protein downregulated in malignant melanomas.Mod. Pathol. 2005; 18: 1249-1257Crossref PubMed Scopus (72) Google Scholar, Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). JARID1B (KDM5B/PLU-1/RBP2-H1) is a member of the highly conserved family of jumonji/ARID1 H3K4 demethylases, which are involved in tissue development, cancer, and stem cell biology (Christensen et al., 2007Christensen J. Agger K. Cloos P.A. Pasini D. Rose S. Sennels L. Rappsilber J. Hansen K.H. Salcini A.E. Helin K. RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3.Cell. 2007; 128: 1063-1076Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar; Dey et al., 2008Dey B.K. Stalker L. Schnerch A. Bhatia M. Taylor-Papidimitriou J. Wynder C. The histone demethylase KDM5b/JARID1b plays a role in cell fate decisions by blocking terminal differentiation.Mol. Cell. Biol. 2008; 28: 5312-5327Crossref PubMed Scopus (103) Google Scholar; Yamane et al., 2007Yamane K. Tateishi K. Klose R.J. Fang J. Fabrizio L.A. Erdjument-Bromage H. Taylor-Papadimitriou J. Tempst P. Zhang Y. PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation.Mol. Cell. 2007; 25: 801-812Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). The JARID1Bhigh slow-cycling subpopulation is required for the continuous tumor growth of melanoma; however, it does not follow a unidirectional cancer stem cell hierarchy. The JARID1Bhigh phenotype is temporarily distinct, dynamic, and can be acquired depending on the microenvironmental context (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). In this respect, another study indicates that other types of cancers, such as breast or lung cancer, also harbor populations of quiescent cells that are drug resistant and whose phenotypes can switch dynamically (Sharma et al., 2010Sharma S.V. Lee D.Y. Li B. Quinlan M.P. Takahashi F. Maheswaran S. McDermott U. Azizian N. Zou L. Fischbach M.A. et al.A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.Cell. 2010; 141: 69-80Abstract Full Text Full Text PDF PubMed Scopus (1703) Google Scholar). Interestingly, in the latter study, slow-cycling cells were characterized by the expression of the chromatin-remodeling factor JARID1A, a homolog of JARID1B. Considering JARID1B’s role in continuous tumor maintenance (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar), we asked (1) whether the subpopulation of JARID1Bhigh slow-cycling melanoma cells displays lower drug susceptibility compared to the bulk of tumor cells and (2) whether this resistant phenotype can be pharmacologically eradicated. To assess whether slow-cycling melanoma cells show differences in the therapeutic response compared to the rapidly proliferating bulk, we treated melanoma cultures with various anticancer drugs. To discriminate between slowly and rapidly cycling subpopulations, we used a JARID1B- promoter-EGFP- reporter construct, as previously described (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). This fluorescence-based model allows both the detection of slow-cycling melanoma cells based on their expression of JARID1B and monitoring of the dynamic nature of the JARID1B phenotype while cells are alive and interacting with their microenvironment. Prior in vitro and in vivo expression studies confirmed the correlation between the endogenous expression of JARID1B and the JARID1B promoter-induced expression of EGFP (J/EGFP) using an analytical threshold set to the maximum 5% of the fluorescence signal (Figure S1A available online; Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). Cells with an EGFP signal above this threshold show the highest endogenous JARID1B levels and, thus, can be reliably selected and analyzed. Two genetically different melanoma cell lines, WM3734 and 1205Lu, were stably transduced with this reporter construct and named WM3734JARID1Bprom-EGFP and 1205Lu JARID1Bprom-EGFP. When the cell lines were treated with cisplatin at a highly toxic concentration (20 μM) for 24, 48, and 72 hr, a gradual separation of the total population emerged into live, 7-aminoactinomycin (7AAD)− and dead, 7AAD+ subpopulations (Figure 1A). With increasing incubation time, 7AAD− cells were incrementally enriched for J/EGFPhigh cells, whereas 7AAD+ cells were relatively J/EGFPlow. Control cells stably expressing a cytomegalovirus (CMV)-promoter-EGFP-reporter construct failed to show such a separation/enrichment pattern. While the total number of cells was constantly decreasing with rising cisplatin doses, the relative number of J/EGFPhigh cells was significantly increasing in the drug-resistant population (Figures 1B and S1B). For example, up to 30.2% J/EGFPhigh cells were seen following 20 μM of cisplatin addition, compared to the 5% seen in vehicle controls. Immunoblotting confirmed an ∼3-fold enrichment for endogenous JARID1B protein after densitometric normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Figure 1C). Vemurafenib also failed to eradicate all melanoma cells and enriched for J/EGFPhigh cells, however, to a lesser extent than seen for cisplatin (Figure 1D). Interestingly, even when high concentrations of vemurafenib were applied, a distinct subpopulation of cells remained alive (7AAD−) and displayed an ∼3-fold higher fluorescence intensity for J/EGFP (GMean = 25.55) than the population of vemurafenib-sensitive (7AAD+) cells (GMean = 8.54; Figure 1D). The surviving melanoma cells did not change in their mutational status of BRAF, as confirmed by DNA sequencing before and after treatment (data not shown). In a previous publication, we excluded the acquisition of secondary mutations for a large panel of diverse melanoma cell lines rendered resistant to BRAF inhibitors. We have tested 451Lu, Mel1617, WM983B, WM902B, and SKMel28 (parental and resistant pairs; several passages) for mutations in BRAF, NRAS, TP53, PTEN, KIT, MEK1, MEK2, AKT1, CDKN2A, and CDK4 (Villanueva et al., 2010Villanueva J. Vultur A. Lee J.T. Somasundaram R. Fukunaga-Kalabis M. Cipolla A.K. Wubbenhorst B. Xu X. Gimotty P.A. Kee D. et al.Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K.Cancer Cell. 2010; 18: 683-695Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar). Interestingly, similar results were observed for other anticancer drugs, such as bortezomib and temozolomide (Figure 2A). We also note that a number of cells with relatively low J/EGFP expression can survive various drug treatments, indicating other potential operating mechanisms of phenotypic resistance. In contrast to its proposed effect on cancer stem cells (Gupta et al., 2009Gupta P.B. Onder T.T. Jiang G. Tao K. Kuperwasser C. Weinberg R.A. Lander E.S. Identification of selective inhibitors of cancer stem cells by high-throughput screening.Cell. 2009; 138: 645-659Abstract Full Text Full Text PDF PubMed Scopus (1954) Google Scholar), the potassium ionophore salinomycin also showed a strong enrichment of J/EGFPhigh cells. To rule out an unspecific bias in our model, we tested vitamin D3 as a compound with a different anticancer mechanism (prodifferentiation; Figure 2B). Here, no significant change in J/EGFPhigh cells was seen. Next, we tested compounds that are expected to diminish the slow-cycling phenotype, such as the histone deacetylase inhibitor trichostatin A (Sharma et al., 2010Sharma S.V. Lee D.Y. Li B. Quinlan M.P. Takahashi F. Maheswaran S. McDermott U. Azizian N. Zou L. Fischbach M.A. et al.A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.Cell. 2010; 141: 69-80Abstract Full Text Full Text PDF PubMed Scopus (1703) Google Scholar) or the Sp1 inhibitor mithramycin A (Sp1-binding sites within the JARID1B promoter are indicative of a possible regulatory potential); however, both compounds failed to reduce the J/EGFPhigh subpopulation (Figure 2B). Similarly, leflunomide, which was suggested to interfere with developmental processes in melanocytes and melanoma growth (White et al., 2011White R.M. Cech J. Ratanasirintrawoot S. Lin C.Y. Rahl P.B. Burke C.J. Langdon E. Tomlinson M.L. Mosher J. Kaufman C. et al.DHODH modulates transcriptional elongation in the neural crest and melanoma.Nature. 2011; 471: 518-522Crossref PubMed Scopus (334) Google Scholar), did not reduce the J/EGFPhigh subpopulation in our model (Figure S2). The in vivo relevance of our observations was confirmed in WM3734 cells xenografted to NOD/LtSscidIL2Rγnull (NSG) mice and treated with vemurafenib (Figure 3A). While tumors decreased in size due to treatment, the number and staining intensity of resistant JARID1Bhigh cells significantly increased. Importantly, a clear increase in JARID1B-expressing cells could be observed in three out of four matched pairs of patients’ melanomas that relapsed under vemurafenib (Figure 3B). Together, these data indicate a universal resistance of the slow-cycling JARID1Bhigh melanoma subpopulation against various anticancer agents. Previous studies suggested that the abrogation of the slow-cycling phenotype by JARID1B-specific knockdown leads in vitro and in vivo to increased cell proliferation followed by melanoma exhaustion (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). To quantify the shifts in cell-cycle progression, depending on the presence or absence of JARID1B, we performed cell-cycle experiments in WM35 melanoma cells stably knocked down for JARID1B (Figures 3C and S3). WM35 was chosen because changes in cell cycle of this low tumorigenic cell line could be better assessed compared to lines with high doubling rate. Influences of cell densities and growth factors were considered. Cells were seeded at different densities and serum-starved at 0% fetal calf serum (FCS) for 4 days to synchronize the cell cycles. Under these conditions, knockdown of JARID1B led to an increase in S phase cells when compared to the control. Following stimulation with 2%, 5%, and 10% FCS over 4, 8, 16, and 24 hr, JARID1B knockdown cells shifted to G2/M. The most consistent changes in the cell cycle were seen after 8 hr of 10% FCS stimulation. Across five independently performed experiments, we saw an average decrease in G1 phase cells in JARID1B knocked down cells by ∼20% (ranging from ∼5% to 40%; the latter experiment is depicted in Figure 3C), while S phase cells increased by ∼6% (∼2%–12%), and G2/M phase cells by ∼14% (∼1%–31%). As predicted by previous studies showing JARID1B/retinoblastoma protein (pRB)-dependent cell cycle control (Roesch et al., 2006Roesch A. Becker B. Schneider-Brachert W. Hagen I. Landthaler M. Vogt T. Re-expression of the retinoblastoma-binding protein 2-homolog 1 reveals tumor-suppressive functions in highly metastatic melanoma cells.J. Invest. Dermatol. 2006; 126: 1850-1859Crossref PubMed Scopus (41) Google Scholar), JARID1B knockdown was accompanied by an increase in pRB phosphorylation at the JARID1B-specific phosphorylation site Ser795 (Figure 3C). Consequently, we asked if the inhibition of JARID1B could affect the therapeutic responsiveness of melanoma cells to anticancer drugs while releasing them from their slow-cycling phenotype. Indeed, stable knockdown of JARID1B in WM3734 melanoma cells xenografted to NSG mice led to increased cell proliferation accompanied by a significant sensitization to different treatments (Figure 3D). JARID1B knockdown plus bortezomib showed an additive effect, improving the moderate antimelanoma effect of bortezomib to an almost complete stasis of tumor growth. When we combined JARID1B knockdown with vemurafenib, which by itself dramatically prevents melanoma growth, we still observed a decrease in the average tumor volume below that of the treatment-starting volume. Because of the biological variability of tumor sizes, this effect was not significant in the multivariance analysis of variance (MANOVA), but an analysis of the change in the logarithm of volume per unit time by t test had significantly different rates. In sum, these results support the hypothesis that targeting the slow-cycling cell phenotype can sensitize melanoma to anticancer approaches, including chemotherapeutics and molecular-targeted drugs. According to our previous observations, the progeny of both J/EGFPhigh and J/EGFPlow cells can dynamically interconvert into the opposing phenotype, indicating a flexible system that is dependent on the microenvironmental context (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). Thus, we asked (1) if the newly developing progenies of initially sorted J/EGFPhigh or J/EGFPlow cells show a differential drug response and (2) if the observed enrichment of J/EGFPhigh cells after anticancer treatment results from selection of J/EGFPhigh cells or induction of J/EGFPhigh expression. In regular culture, spontaneous phenotype interconversion usually becomes visible within a few days after fluorescence-activated cell sorting (FACS) of the two populations. For example, 69.9% of FACS-sorted J/EGFPhigh cells turned negative 72 hr after separation (Figure 4Ac, upper left plus lower left quadrant), while in total, 3.5% of the progeny of sorted J/EGFPlow cells became J/EGFPhigh (Figure 4Ae). When sorted cells were treated with 20 μM cisplatin for 72 hr, we saw no substantial enrichment for J/EGFPhigh cells in the progeny of initially J/EGFPhigh cells, indicating that no additional JARID1B-expression cells were induced (summarized upper and lower right quadrants; Figure 4Ac versus Figure 4Ad). Interestingly, most newly developed J/EGFPlow cells became 7AAD+ under cisplatin treatment (Figure 4Ac versus Figure 4Ad, upper and lower left quadrants). The apparent preference for JARID1B induction in the progeny of J/EGFPlow cells (Figure 4Ae versus Figure 4Af) was counteracted by an increased rate of cisplatin-induced cell death in cells derived from the J/EGFPlow population. Manual counts revealed a significantly lower number of surviving cells in the progeny of sorted J/EGFPlow cells compared to the progeny of J/EGFPhigh cells (p < 0.05 for 5 μM and 10 μM cisplatin; data not shown). Together with the results from consecutive measurements of JARID1B messenger RNA (mRNA) under cisplatin treatment that did not show transcriptional upregulation (Figure 4A), we concluded that the major mechanism for the enrichment of J/EGFPhigh cells following therapy is a selection of pre-existent JARID1Bhigh slow-cycling cells. However, to a lesser extent, JARID1B induction may also occur (e.g., shifting cells with intermediate JARID1B expression to higher levels). When cells were long-term challenged with cisplatin, the J/EGFPhigh subpopulation remained constantly elevated over several weeks in a dose-dependent manner. The cisplatin-induced elevation of J/EGFPhigh cells was fully reversible following drug withdrawal (Figure 4B). In line with the concept of dynamic phenotype switching (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar), drug-resistant cells that are enriched for JARID1B can regain the original cell distribution and J/EGFP ratios after drug removal. Moreover, cisplatin-surviving JARID1B-enriched cells gave rise to fewer but significantly larger (i.e., rapidly growing) three-dimensional (3D) colonies in soft agar compared to untreated cells (Figure 4C). This observation is reminiscent of earlier clonogenicity and single-cell dilution assays showing that untreated FACS-isolated J/EGFPhigh cells give rise to a rapidly proliferating progeny with elevated repopulation capacity when compared to JARID1Blow cells (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). We next designed experiments to unravel druggable targets that are specifically activated in slow-cycling melanoma cells. Since global gene expression screening with complementary DNA microarrays did not provide significant hit candidates in FACS-sorted J/EGFPhigh versus J/EGFPlow or JARID1B knockdown versus control cells (data not shown), we performed quantitative proteome profiling. Following our established protocols for isolation of slow-cycling melanoma cells (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar), we costained WM3734JARID1Bprom-EGFP cells with the PKH26 dye and then FACS-isolated the label-retaining and J/EGFPhigh subpopulation versus nonlabel-retaining J/EGFPlow cells after a period of 4 weeks of cell divisions. Prior to sorting, the cells were grown as melanospheres to attain a more stringent J/EGFP phenotype, as previously reported (Roesch et al., 2010Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). Tryptic digests of the cell lysates were analyzed by liquid chromatography-mass spectrometry/mass spectrometry. Label-free computational quantitation identified 423 proteins with significant expression changes between label-retaining J/EGFPhigh cells and nonlabel-retaining J/EGFPlow cells with a p value < 0.001. Unexpectedly, neither common cancer target nor drug resistance-related protein could be detected by this approach. Instead, we found a differential regulation of a number of mitochondrial proteins with particular functions in bioenergetic metabolism (Table S1). Many of the upregulated proteins play a major role in cell respiratory electron transport (oxidative phosphorylation [OXPHOS]), such as nicotinamide adenine dinucleotide (NADH) dehydrogenase (complex I of the mitochondrial electron transport chain), ubiquinol cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and ATP synthase. In contrast, glycolytic hexokinase I and II were downregulated. Immunoblotting of lysates from J/EGFP-sorted cells confirmed upregulation of ATP synthase, NADH dehydrogenase, ubiquinol-cyctochrome C reductase-binding protein, and cytochrome c oxidase in J/EGFPhigh cells (Figure 5A). WM3734 cells transiently overexpressing JARID1B had a higher mitochondrial energy production compared to the mock control (Figure 5B). Accordingly, JARID1B-overexpressing WM3734 cells consumed ∼50% more oxygen than control cells (Figure 5C). Treatment with different concentrations of oligomycin and rotenone severely reduced the oxygen consumption by up to 94%, indicating a major role for mitochondria in this process (data not shown). Because high mitochondrial electron transfer activity can lead to increase in superoxide and hydrogen peroxide (H2O2) production, we analyzed the H2O2 level. JARID1B-overexpressing cells generated 76% more H2O2 than control cells 60–72 hr after transfection (Figure 5D). The H2O2 increase was completely abolished by treatment with oligomycin or rotenone. These results" @default.
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• W1995103178 date "2013-06-01" @default.
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• W1995103178 title "Overcoming Intrinsic Multidrug Resistance in Melanoma by Blocking the Mitochondrial Respiratory Chain of Slow-Cycling JARID1Bhigh Cells" @default.
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