Transdifferentiation of MALME-3M and MCF-7 Cells toward Adipocyte-like Cells is Dependent on Clathrin-mediated Endocytosis.pdf
RESEARCH Open Access Transdifferentiation of MALME-3M and MCF-7 Cells toward Adipocyte-like Cells is Dependent on Clathrin-mediated Endocytosis Jaime Carcel-Trullols†, Cristóbal Aguilar-Gallardo†, Fernando Garcia-Alcalde, Miguel Angel Pardo-Cea, Joaquin Dopazo, Ana Conesa and Carlos Simón* Abstract: Enforced cell transdifferentiation of human cancer cells is a promising alternative to conventional chemotherapy. We previously identified albumin-associated lipid- and, more specifically, saturated fatty acid-induced transdifferentiation programs in human cancer cells (HCCLs). In this study, we further characterized the adipocyte-like cells, resulting from the transdifferentiation of human cancer cell lines MCF-7 and MALME-3M, and proposed a common mechanistic approach for these transdifferentiating programs. We showed the loss of pigmentation in MALME-3M cells treated with albumin-associated lipids, based on electron microscopic analysis, and the overexpression of perilipin 2 (PLIN2) by western blotting in MALME-3M and MCF-7 cells treated with unsaturated fatty acids. Comparing the gene expression profiles of naive melanoma MALME-3M cells and albumin-associated lipid-treated cells, based on RNA sequencing, we confirmed the transcriptional upregulation of some key adipogenic gene markers and also an alternative splicing of the adipogenic master regulator PPARG, that is probably related to the reported up regulated expression of the protein. Most importantly, these results also showed the upregulation of genes responsible for Clathrin (CLTC) and other adaptor-related proteins. An increase in CLTC expression in the transdifferentiated cells was confirmed by western blotting. Inactivation of CLTC by chlorpromazine (CHP), an inhibitor of CTLC mediated endocytosis (CME), and gene silencing by siRNAs, partially reversed the accumulation of neutral lipids observed in the transdifferentiated cells. These findings give a deeper insight into the phenotypic changes observed in HCCL to adipocyte-like transdifferentiation and point towards CME as a key pathway in distinct transdifferentiation programs. Disclosures: Simon C and Aguilar-Gallardo C are co-inventors of the International Patent Application No. PCT/EP2011/004941 entitled “Methods for tumor treatment and adipogenesis differentiation”. Keywords: Cell Transdifferentiation, Unsaturated Fatty Acids, PPARG, Perilipin 2, Loss of Pigmentation, Adipogenic Gene Markers, Clathrin, Clathrin-mediated Endocytosis Introduction Transdifferentiation involves reprogramming one type of adult cell into another mature cell type, without having to generate intermediary stem cells. It has received sig- nificant attention because it may have a considerable number of applications in cell and cancer therapy. Successful transdifferentiation from one cell type to another by overexpressing lineage-specific genes in vivo (Takeuchi & Bruneau 2009; Zhou et al. 2008) and in vitro (Graf & Enver 2009; Ieda et al. 2010) has been previously reported. Not only can cell transdifferentiation be enforced through the overexpression of the appropriate set of genes, but also through the application of specific molecules, which is a promising alternative to conven- tional chemotherapy for cancer treatment, for example, the use of all-trans-Retinoid acid (ATRA) for the treat- ment of acute promyelocitic leukemia (APL) (Kakizuka et al. 1991). The fact that there was no treatment based on cell differentiation therapy for solid tumors prompted us to study whether novel transdifferentiating molecules secreted by human embryonic stem cells (hESCs) could prevent cancer progression. Our studies showed that, * Correspondence: carlos.simon@ivi.es †Equal contributors Bioinformatics and Genomics Department, Prince Felipe Research Centre (CIPF), Avda. Autopista del Saler, 16-3 46012, Valencia, Spain a SpringerOpen Journal © 2012 Carcel-Trullols et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Carcel-Trullols et al. SpringerPlus 2012, 1:44 http://www.springerplus.com/content/1/1/44 irrespective of the presence of hESCs, albumin- associated lipids (albuMAXW), and specifically poly- and monounsaturated fatty acids (linoleic, oleic, petroselinic and palmitoleic acids) accounted for the transdifferentia- tion of several distinct human cancer cell lines (HCCLs) into adipocyte-like cells (Ruiz-Vela et al. 2011). Endocytosis regulates the entry of nutrients, hormones and signaling factors into the cell, and serves to regulate the internalization of transmembrane receptors. During endocytosis the plasma membrane invaginates to form a new intracellular vesicle containing various cargo mole- cules and material to be internalized from the external environment (Conner & Schmid 2003). Clathrin (CLTC)- mediated endocytosis (CME) is the major endocytic route and is assumed to regulate ligand mediated signal transduction by disrupting ligand-receptor interactions after their uptake into endosomal compartments (Andersson 2012). It can sequester the receptors in intra- cellular compartments, target ligand-receptor complexes to lysosomes for degradation, or recycle them by sending them back to the cell surface for re-use (Maxfield & McGraw 2004). Several in vitro studies have reported evidence for a relationship between the endocytosis of ligands such as Bone morphogenetic protein 2 (BMP2) and oxidized Low-density lipoprotein (ox-LDL), bound to their respective receptors, and their key roles in dif- ferent transdifferentiation models (Rauch et al. 2002; Yu et al. 2010). Others have shown that the incorporation of insulin regulated glucose transporter 4 (GLUT4) into the cell plasma membrane, a process required for adi- pogenesis, is dependent on clathrin coated vesicles (Huang et al. 2007). However, the existence of a direct correlation between endocytic ligand-receptor complex internalization and the transcription of genes controlling critical physiological events, such as cell transdifferentia- tion or adipogenesis, remains largely uninvestigated. Our previous report identified similar transdifferentia- tion programs in HCCLS of completely different origins such as in cells from ovarian carcinoma, hepatocarci- noma, breast adenocarcinoma and melanoma (Ruiz-Vela et al. 2011). In this study we aimed to characterize the adipocyte-like cells resulting from melanoma MALME-3M and breast carcinoma MCF-7 transdifferentiation pro- grams and to set the key pathways that were concomi- tantly affected in these programs. In order to document induced-transdifferentiation we report the loss of pig- mentation in MALME-3M cells, the upregulation of PLIN2 protein expression in the studied HCCLs and the upregulation of gene expression of PLIN2 and other commonly considered key adipogenic markers such as lipoprotein lipase (LPL) and peroxisome proliferator- activated receptor alpha (PPARA) in MALME-3M cells. These results also revealed an alternative gene splicing of the adipogenic master regulator PPARG that was likely to be related to the upregulation of the protein found in transdifferentiated MALME-3M cells, previously reported by our group (Ruiz-Vela et al. 2011). Most importantly, our results also showed an increase in Clathrin (CLTC) expression and we provided evidence that Clathrin- mediated Endocytosis (CME) was essential for the trans- differentiation programs of the breast adenocarcinoma and melanoma HCCLs we investigated into adipocyte-like cells. Materials and methods Cell culture and transfection MALME-3M melanoma (ATTC# HTB-64) and MCF-7 breast adenocarcinoma (ATTC#HTB-22) cells were cultured in growth media containing RPMI-1640, 10% fetal bovine serum (FBS) and 2 mM glutamine, follow- ing a standard 3 T3 protocol (Todaro & Green 1963). Albumin-associated lipids were obtained by adding GIBCOTM AlbuMAX II from Invitrogen at a concentra- tion of 1.6% w/v in RPMI media containing 10% FBS. To induce cell transdifferentiation, cells were cultured with albumin-associated lipids, whereas mock-treated cells were cultured in RPMI containing 10% FBS. After 24 hours cells were trypsinized, collected in plastic tubes and centrifuged at 5000 rpm. CLTC-targeted siRNA and scrambled siRNA (control siRNA) were obtained from Dharmacon (Dharmacon RNA i Technologies). Sequences from CLTC-targeted siRNA and control siRNA were commercially provided. Cells were seeded according to the manufacturer’s protocol in serum-containing media without antibiotics and transfected with either 50 nM siRNA or 50 nM control siRNA with DharmaFECT (Dharmacon RNA i Technolo- gies) in antibiotic-free media for 72 h. Gene expression analysis Whole genome transcriptional profiles of both control MALME-3M cells and transdifferentiated cells, after 24 hours incubation with albumin-associated lipids, were obtained by RNA-seq. Briefly, total RNA was purified from cell cultures using Quick-RNA MiniPrepTM (Zymo Research) protocol and strand-specific 38 nt pair-end Solexa libraries were prepared following the dUTP method (Parkhomchuk et al. 2009; Levin et al. 2010). Sequencing reads were mapped to the human genome (GRCh37/hg19 assembly) using Tophat 1.1.4 with stand- ard parameters and indicating the corresponding mate inner distance (46 bp for 0 h and 44 bp for 24 h) (Trapnell et al. 2009). Gene counts were estimated using htseq- count (http://www-huber.embl.de/users/anders/HTSeq) with standard parameters, using the human annotations obtained from Ensembl 60. Differentially expressed genes between MALME-3M and adipose cell types were estimated by means of the NOISeq non parametrical Carcel-Trullols et al. SpringerPlus 2012, 1:44 Page 2 of 12 http://www.springerplus.com/content/1/1/44 Izpisua Belmonte 2008) is far more complex than previ- ously anticipated. In addition to maintaining self- renewal and pluripotency in hESCs (Garcia-Gonzalo & Izpisua Belmonte 2008), a role for albumin (Kallee 1996) and its associated lipids (Davis & Dubos 1947; Thomas et al. 1995; Lafond et al. 1994) has been discovered in adipogenesis in other cell types (Schopfer et al. 2005). Our results also indicate that certain albumin complex associated poly- and monounsaturated fatty acids induce terminal differentiation and arrest cancer progression (Ruiz-Vela et al. 2011). Concomitanlty to our studies, Khan et al. have also described the specific growth inhib- ition of esters of oleic acid and ricinoleic acid against the human skin malignant melanoma cell line (SK-MEL-1) (Khan et al. 2012). Despite the important findings that we describe here, many key questions remain un- answered in the identification of novel genes and pro- teins that mediate the terminal transdifferentiation of human cancer cells. To get a better understanding of the mechanisms that lead to transdifferentiation we concen- trated our efforts on the role of those genes that are dif- ferentially regulated during the process. EM revealed a marked loss of pigmentation in the melanoma MALME-3M cells treated with albumin- associated lipids, in accordance with the downregulation of MLANA gene expression. Melan-A is known to form a complex with Pmel17 which affects its expression, sta- bility, trafficking, and the processing which is required for melanosome maturation. Its expression is indispens- able for Pmel17 function and the formation of cell pig- mentation (Hoashi et al. 2005). Quantification of gene expression by RNA-seq led to the characterization of the MALME-3M cells treated with albumin-associated lipids. Several adipocytic mar- kers such as PLIN2, LPL and PPARA were signifi- cantly upregulated. PPARA could be upregulated merely as a consequence of fatty acid accumulation as it has been shown to be involved in the regulation of obesity in rodents by increasing hepatic fatty acid oxi- dation (Kersten et al. 1999). LPL is a well describeda- dipocyte marker as it regulates the hydrolysis of triglycerides in the adipose tissue (Mead et al. 2002). Interestingly, PPARG1, an adipogenesis marker shown to be upregulated upon HCCL to adipocyte transdifferen- tiation (Ruiz-Vela et al. 2011), showed a differential map- ping pattern between the albumin-associated lipid-treated and non-treated cells. The PPARG gene has eight exons which are translated and spliced into different isoforms (http://www.ensembl.org/Homo_sapiens/Gene/Summary? g=ENSG00000132170;r=3:12328867–12475855), with the PPARG1 isoform being the primary transcript expressed in adipocytes. In treated cells, PPARG showed whole tran- script expression, while in mock-treated cells no expres- sion of exons 4 and 5 was evident (Additional file 3: Supporting information B). These two exons encode the Zinc finger binding site domain of the PPARG1 transcrip- tion factor (Finn et al. 2010). The lack or downregulation of these functional domains in the non-treated cells might be indicative of the differential processing of this gene in the MALME-3M cell lines and of differential expression of the protein (Ruiz-Vela et al. 2011). The characterization of the adipocyte-like cells showed that the expression of PLIN2 was increased in MALME- 3M, and in MCF-7 cells treated with albumin-associated lipids and with petroselinic acid. PLIN2 is a principal adi- pocytic marker, which coats lipid droplets in adipocytes (Brasaemle et al. 1997; Heid et al. 1998) and its expres- sion has been linked to PPARG1. We previously reported that PPARG1 expression was increased in MALME-3M cells treated with albumin-associated lipids (Ruiz-Vela et al. 2011). It has been recently reported that pretreat- ment of murine 3T3-L1 preadipocytes with Rosiglita- zone, a potent PPARG1 agonist, decreased lipolysis and increased PLIN2 expression (Kim et al. 2007). Quantification of gene expression by RNA-seq also gave us some clues about the possible mechanistic path- ways involved in transdifferentiation. Among the genes that were identified as differentially expressed between the treated and the untreated conditions, many of them were related to endocytic functions. Albumin-associated lipid induced transdifferentiation was accompanied by the upregulation of CLTC and other important adaptor- related complexes such as AP1B1, AP1G1, AP1S3, AP2A1, Synergin and AP3M1. Adaptor-related proteins (See figure on previous page.) Figure 5 Inhibition of CLTC levels by SiRNA affects LD biogenesis. After silencing CLTC expression, biogenesis was examined by Nile Red (C, D) quantification and Oil Red O semi-quantitative determination (E, F). Experiments were performed in MALME-3M cells (C and E) and in MCF-7 cells (D and F). Cells were transiently transfected with CONT (Control) SiRNA or with CLTC SiRNA for 72 hours prior to the addition of the vehicle 24 hours prior to cell lysation. Cell lysates were then separated in a 4-12% Bis-Tris gel and probed by western blotting with antibodies against CLTC and Actin. The experiment was repeated three times and a representative western blot is shown for MALME-3M cells (A) and for MCF-7 cells (B). For the neutral lipid determinations, after transfection, cells were treated with either the vehicle or petroselinic acid (100 μg/ml) for 24 hours prior to the testing. Nile Red (C, D) and Oil Red O and hematoxylin (E, F) staining were performed as described in Materials and Methods. Quantification was assessed using the FlowJo software. Fold induction was estimated by calculating the ratio between treated conditions (Petroselinic acid) and the untreated condition (vehicle). The average values from three independent analyses are shown. *P <0.05 compared to each control in C and D. Representative photos are shown in E and F. The length of the shown size bars is 20 μm. Carcel-Trullols et al. SpringerPlus 2012, 1:44 Page 10 of 12 http://www.springerplus.com/content/1/1/44 are key components of clathrin coated vesicles that can bind directly to both the clathrin lattice and to the lipid and protein components of membranes (Pearse et al. 2000). AP1 and AP3 are found at the coated vesicles located at the Golgi complex and it has been suggested that both associate with GLUT4 transporting vesicles and mediate distinct intracellular sorting events at the level of the TGN and endosomes in rat adipocytes (Gillingham et al. 1999). The observed upregulation of proteins involved in GLUT4 trafficking could be related to the acquisition of the adipocytic phenotype. Encouraged by the western blot results that showed a clear overexpression of CLTC protein in the transdif- ferentiated MCF-7 and MALME-3M cells, we decided to determine the role of CME in the adipogenic trans- differentiation process in MALME-3M and in MCF-7 cells. Transient CLTC silencing accomplished by siRNA, caused a significant reduction in LD accumulation induced by petroselinic acid, and further microscopic analysis of Oil Red O and hematoxylin stained cells sug- gested that not only LD accumulation but also neutral lipid composition in LDs was affected by CLTC silencing. So far no study has reported the induction of CLTC expression or CME stimulation by monounsaturated fatty acids, although polyunsaturated fatty acids have been found to play a role in the formation of synaptic vesicles and in promoting vesicle budding and mem- brane trafficking (Darios & Davletov 2006; Chernomor- dik et al. 1997; Chernomordik et al. 1999). It was recently reported that α-Synuclein expression, coupled with exposure to physiological levels of polyunsaturated fatty acids, enhanced CLTC mediated endocytosis in neuronal and non-neuronal cultured cells (Ben Gedalya et al. 2009). Transferrin receptor (TfR), whose gene expression was upregulated by a fold change of 3.33 in the albumin- associated lipid-treated MALME-3M cells (Table 1A), is internalized from the cell plasma membrane and recycled back to the cell plasma membrane specifically via CME (Hanover et al. 1984). El-Jack et al. reported that murine 3T3-L1 preadipocytes, in a differentiation media previ- ously described (Stephens et al. 1997), showed a gradual increase in whole cell TfR levels but a decrease in cell surface TfR levels (El-Jack et al. 1999). The results obtained in this study indicated that the differentiation process might account for the observed alterations in internalization and/or TfR recycling. These results may be useful in understanding why CME is of critical im- portance in HCCL to adipocyte transdifferentiation. The clarification of the roles played by the differen- tially expressed genes and proteins in the process of adi- pogenic transdifferentiation in HCCL cultures should provide the basic foundation to develop novel molecules for cancer therapy. Additional files Additional file 1: Supporting information A. Gene expression in the endocytic pathway and at the PPARG locus after treatment with albumin- associated lipids. (A) Downregulated and upregulated genes after treatment with albumin-associated lipids are colored in blue and red, respectively. Intensity is proportional to the log2 of the ratio between the two conditions. Significantly differentially expressed genes are indicated by bold box lines. Additional file 2: Supporting information B. Gene expression in the endocytic pathway and at the PPARG locus after treatment with albumin- associated lipids. (B) Mapped reads of gene expression at the PPARG locus are shown piled up on mapping positions. Expression at exons 4 and 5 in the PPARG1 transcript was observed only after albumin incubation. Additional file 3: Supporting information C. Electron Microscopy at high magnification of melanosome pigmentation in MALME-3M cells mock-treated or treated with Albumin-associated lipids. MALME-3M cells were mock-treated or treated with albumin-associated lipids for 24 hours. The arrows indicate the dark vesicles that are hallmarks of melanosomes and disappear upon albumin-associated lipid treatment. Competing interests Simon C and Aguilar-Gallardo C are co-inventors of the International Patent Application No. PCT/EP2011/004941 entitled “Methods for tumor treatment and adipogenesis differentiation”. Authors’ contributions C-T J and A-G C carried out the cell biology and molecular biology experiments, G-A F, P-C MA and C A participated in the deep sequencing experiments and S C and C-T J drafted the manuscript. All authors read and approved the final manuscript. 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Title: Transdifferentiation of MALME-3M and MCF-7 Cells toward Adipocyte-like Cells is Dependent on Clathrin-mediated Endocytosis ​
Authors: Jaime Carcel-Trullols†, Cristóbal Aguilar-Gallardo†, Fernando Garcia-Alcalde, Miguel Angel Pardo-Cea, Joaquin Dopazo, Ana Conesa, Carlos Simón* ​
Publisher: SpringerPlus
Date: October 30, 2012 ​
Abstract: Enforced cell transdifferentiation of human cancer cells is a promising alternative to conventional chemotherapy. ​ We previously identified albumin-associated lipid-and, more specifically, saturated fatty acid-induced transdifferentiation programs in human cancer cells (HCCLs). ​ In this study, we further characterized the adipocyte-like cells, resulting from the transdifferentiation of human cancer cell lines MCF-7 and MALME-3M, and proposed a common mechanistic approach for these transdifferentiating programs. ​ We showed the loss of pigmentation in MALME-3M cells treated with albumin-associated lipids, based on electron microscopic analysis, and the overexpression of perilipin 2 (PLIN2) by western blotting in MALME-3M and MCF-7 cells treated with unsaturated fatty acids. ​ Comparing the gene expression profiles of naive melanoma MALME-3M cells and albumin-associated lipid-treated cells, based on RNA sequencing, we confirmed the transcriptional upregulation of some key adipogenic gene markers and also an alternative splicing of the adipogenic master regulator PPARG, that is probably related to the reported up regulated expression of the protein. ​ Most importantly, these results also showed the upregulation of genes responsible for Clathrin (CLTC) and other adaptor-related proteins. ​ An increase in CLTC expression in the transdifferentiated cells was confirmed by western blotting. ​ Inactivation of CLTC by chlorpromazine (CHP), an inhibitor of CTLC mediated endocytosis (CME), and gene silencing by siRNAs, partially reversed the accumulation of neutral lipids observed in the transdifferentiated cells. ​ These findings give a deeper insight into the phenotypic changes observed in HCCL to adipocyte-like transdifferentiation and point towards CME as a key pathway in distinct transdifferentiation programs. ​  