Implication des cellules exprimant le transporteur ABCB5 dans la chimiorésistance du mélanome métastatique

Bien qu'au 8ème rang des cancers les plus fréquents, le mélanome est un problème médical majeur car son incidence double tous les dix ans chez les populations occidentales. Cette pathologie, au stade métastatique, est réfractaire à la chimiothérapie avec moins de 20% de réponses objectives sans amélioration du temps de survie pour le traitement de référence : la dacarbazine. Même si des thérapies ciblées prometteuses sont développées avec l'ipilimumab (inhibiteur de CTL-A4) ou le vemurafenib (inhibiteur de BRAF), la chimiorésistance reste au centre des préoccupations. Récemment, des sous-populations cellulaires présentant des caractéristiques particulières en termes d'agressivité tumorale ont été mises en évidence dans le mélanome. Parmi elles, les cellules exprimant ABCB5 ont été définies comme "Cellules Initiatrices de Mélanome" pour leur capacité à régénérer des tumeurs chez l'animal. ABCB5 appartient à la famille des transporteurs ABC (pour ATP-Binding Cassette) qui permettent l'efflux de nombreux composés de façon ATP-dépendante et qui sont largement impliqués dans la chimiorésistance des cancers. Dans ce contexte, j'ai étudié l'implication d'ABCB5 dans les phénomènes de chimiorésistance du mélanome métastatique. Dans un premier temps, j'ai analysé la résistance globale de plusieurs modèles in vitro de mélanome en relation avec plusieurs transporteurs ABC dont ABCB5. Ces modèles ne permettant pas une modélisation satisfaisante de la chimiorésistance, je me suis intéressée à la résistance de sous-populations cellulaires. J'ai ainsi confirmé que la protéine ABCB5 est exprimée sous forme d'une sous-population minoritaire au sein de plusieurs modèles in vitro de mélanome et j'ai mis en place une technique expérimentale permettant d'évaluer l'impact de substances anti-mélanome sur ces cellules. J'ai montré dans un modèle de xénogreffe WM-266-4 que le traitement par le temozolomide aboutit in vivo à l'enrichissement de la sous-population exprimant ABCB5 et ce, malgré une régression des tumeurs. Par la même approche transposée in vitro, j'ai montré que les cellules exprimant ABCB5 survivent à la dacarbazine, à des doses pharmacologiquement actives. Cette observation a également été étendue à d'autres composés cytotoxiques et notamment aux agents de thérapies ciblées actuellement testés pour le traitement du mélanome. Par ailleurs, mes résultats indiquent que les cellules de mélanome peuvent moduler l'expression de la protéine ABCB5 en réponse à un stress pharmacologique. Cette étude montre l'intérêt de la protéine ABCB5 en tant que marqueur d'une sous-population cellulaire caractérisée par une résistance accrue. Néanmoins, un rôle fonctionnel direct d'ABCB5 dans ce phénotype reste à démontrer. Cette question a été abordée par la mise en place de modèle de surexpression d'ABCB5 au sein de l'équipe ainsi que par des approches d'extinction par siRNA mais les premiers résultats indiquent qu'il est difficile d'étayer l'hypothèse d'un rôle actif pour ABCB5. Il est donc aujourd'hui primordial de statuer sur le rôle de cette protéine et de déterminer son importance dans le processus de chimiorésistance du mélanome. L'ensemble de ce travail ouvre de nouvelles perspectives pour la recherche de cibles pharmacologiques innovantes dans le contexte du mélanome métastatique.Melanoma is one of the most aggressive form of skin cancer and its incidence is increasing worldwide. Metastatic melanoma is highly resistant to conventional chemotherapies and dacarbazine, the reference treatment has a very low response rate (<20%) without impact on overall survival. A number of promising targeted therapies have been developed recently such as ipilimumab (an anti-CTL-A4 antibody) or vemurafenib (a specific inhibitor of BRAF) but resistance invariably develops and still remains an unsolved question. Recently, relevant markers have been identified to select a restricted sub-population of melanoma cells with particular stem cell-like properties. Among them, ABCB5 expressing cells have been defined as "Melanoma initiating cells" since they display an enhanced tumorigenicity in mice. As a member of the ABC (ATP-Binding Cassette) transporters family, ABCB5 is thought to participate to the melanoma chemoresistance through a function of efflux. Here, we have studied the participation of ABCB5 expressing cells to chemoresistance in metastatic melanoma. We first investigated in vitro models of melanoma for their ability to mimic global resistance, in relation with thier ABC transporters expression. We then focused our study on the resistance of minor sub-populations. We confirmed that ABCB5 is expressed at the surface of a sub-population and we developed an experimental procedure to monitor this sub-population upon chemoterapeutic treatment. Using the WM-266-4 melanoma xenograft model, we demonstrated in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment despite a significant tumor regression. We then monitored in vitro the evolution of the ABCB5-expressing cell sub-population upon pharmacologically active doses of dacarbazine. This observation has been extended to various chemotherapeutic drugs including the new targeted therapy developed in melanoma. We also demonstrated that melanoma cells are able to modulate ABCB5 expression upon pharmacological treatment. Taken together, our findings show that ABCB5-expressing cells are more resistant to the anti-melanoma chemotherapeutic drugs than cells of the tumor bulk. Nevertheless, a functional property of ABCB5 protein remains unclear in this phenotype. The question was addressed by using cellular model which stably overexpressed ABCB5 and by performing siRNA extinction experiments. Our data failed to show any functional evidence that ABCB5 can confer a selective advantage to melanoma cell. This remains a determining question we need in order to answer to establish the importance of ABCB5 in melanoma chemoresistance. Our results may be of particular importance in determining the clinical outcome of chemotherapeutic treatments and reinforce the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma

Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells

Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5+ cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma

Implication des cellules exprimant le transporteur ABCB5 dans la chimiorésistance du mélanome métastatique

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains

Microtubules are nucleated from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). Small complexes (γTuSCs) comprise two molecules of γ-tubulin bound to the C-terminal domains of GCP2 and GCP3. γTuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes γTuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called γ-tubulin ring complex (γTuRC). GCPs 2–6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N- and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for γTuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with γ-tubulin. Moreover, we show that binding to γ-tubulin is not essential for integrating into the helical complex

Enrichment in ABCB5-expressing cells is associated with protein neo-synthesis and ABCB5 relocation at the cell-surface.

<p>WM-266-4 cells were treated with dacarbazine, doxorubicin or vehicle (NT) for 72 h. Cycloheximide (<b>A–B</b>) or brefeldin A (<b>C–D</b>) were added respectively 24 h and 4 h before the treatment end-point. Cells were labelled for ABCB5 and analyzed by flow cytometry. The means of fluorescence intensity of the ABCB5⁺ cells from three independent experiments were reported in <b>A</b> and <b>C</b>. Fluorescence intensity histograms of representative experiments are shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036762#pone.0036762.s001" target="_blank">figure S1</a>. The number of ABCB5⁺ cells was reported in <b>B</b> and <b>D</b> as a percentage of the ABCB5⁺ cell number in the vehicle-treated sample.</p

Quantification of ABCB5- and ABCB1-expressing cells after cytotoxic treatments.

<p>WM-266-4 cells were treated for 72 h with the indicated concentrations of doxorubicin, dacarbazine, vemurafenib, gemcitabine or with vehicle (NT) and ABCB5 expression was analyzed by Western blot. Band intensities were quantified and variations are indicated as fold increases in treated versus untreated samples (<b>A</b>). WM-266-4 cells were treated with various drugs at their EC50 for 72 h. The percentages of positive cells among surviving cells were measured by cell surface labelling and flow cytometry analysis for ABCB5 (<b>B</b>) or ABCB1 (<b>C</b>). The relative mRNA expression of ABCB5, ABCB1, ABCC1, ABCG2 and HMBS as the house-keeping gene was measured by Q-PCR (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036762#pone.0036762.s002" target="_blank">table S1</a>) and the amplified products were run on agarose gel after 29 cycles except for ABCB1 (32 cycles) (<b>D</b>).</p

ABCB5-expressing cells are enriched in the residual tumors after an anti-melanoma treatment <i>in vivo.</i>

<p>WM-266-4 cells (5×10⁶ cells) were injected subcutaneously in Swiss nude mice. Fourteen days later, mice were treated by repeated i.p. injections of either temozolomide (80 mg/kg) or vehicle following the schedule indicated by the black arrows. The tumoral volumes were monitored and the means of measured volumes respectively for temozolomide-treated and vehicle treated tumors are as follows: 180 mm³ and 175 mm³ at day 14; 264 mm³ and 295 mm³ at day 16; 178 mm³ and 444 mm³ at day 18; 84 mm³ and 699 mm³ at day 21. (<b>A</b>). 24 h after the injections at days 16 and 21 (d17 and d22), tumors were recovered, dissociated and the cell suspensions were searched for the presence of human ABCB5⁺ cells by flow cytometry (<b>B</b>) (medians are represented as black lines). Tumors recovered at day 17 were analyzed by immunohistochemistry for their ABCB5 expression (<b>C</b>).</p

ABCB5-expressing cells survive upon dacarbazine treatment.

<p>WM-266-4 (<b>A,D,G</b>), G-361 (<b>B,E,H</b>) and SK-MEL-28 cells (<b>C,F,I</b>) were treated at the indicated concentrations of dacarbazine (<b>A–C</b>), vemurafenib (<b>D–F</b>) and doxorubicin (<b>G–I</b>). After 72 h, the total viable cells were numbered using an automated cell viability analyzer. The percentages of viable ABCB5-expressing cells (among viable cells gated on DAPI-negative cells) were analyzed by flow cytometry. The numbers of total cells (white symbols) are reported as percentages of the number of cells in the untreated control sample. The numbers of viable ABCB5⁺ cells (black symbols) were calculated from the total cell numbers and ABCB5⁺ cells ratio, and reported as percentages of the viable ABCB5⁺ cells number in the control sample. ABCB5⁺ cells represent respectively 3%, 3.5% and 5% of the total cells in the WM-266-4, G-361 and SK-MEL-28 cell lines.</p

ABCB5 is expressed on the surface of a subpopulation of melanoma cells.

<p>WM-266-4 cells were surface-labelled with the ABCB5-Ab^Rock antibody and analyzed by flow cytometry. ABCB5⁺ cells (right contour plot) were gated on viable cells (DAPI-negative) according to the isotype control (left contour plot). Inserts (dot plots) display the gating of the positive cells (<b>A</b>). WM-266-4 cells were treated with a siRNA designed to target ABCB5 (si-ABCB5). After 72 h, the cells were analyzed for their ABCB5 mRNA content and ABCB5 surface expression. The left and right histograms show respectively the relative expression of ABCB5 mRNA normalized to the ABCB5 mRNA in cells treated with a control siRNA (si-ctrl), and the percentage of ABCB5⁺ cells among total cells (n = 3). The corresponding contour plots are shown (<b>B</b>). Different melanoma cell lines were analyzed for their ABCB5 surface expression (<b>C</b>) or their ABCB5 mRNA content (<b>D</b>) (n = 3).</p

ABCB5 expression is increased in melanoma tumors obtained from treated patients.

<p>Skin metastases specimens from respectively 8 untreated and 7 treated patients were analyzed by immunohistochemistry for their ABCB5 protein expression. The ABCB5 staining intensity was ranked in four arbitrary classes according to the intensity and the extent of the labelling. Representative staining of two levels of intensity (left panel: isotypic control) (A). Repartition of the specimens in the different classes (B). The two groups of specimens (untreated versus treated) have been compared with the non parametric Kruskall Wallis test (p<0.30).</p