BCL(X)L and BCL2 increase mitochondrial dynamics in breast cancer cell: Evidence from functional and genetic studies

BBA - Molecular Cell Research 1868 (2021) 119095 Available online 30 June 2021 0167-4889/© 2021 Elsevier B.V. This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/). BBA research letter BCL(X)L and BCL2 increase mitochondrial dynamics in breast cancer cell: Evidence from functional and genetic studies Federico Lucantoni 1 , Manuela Salvucci , Heiko Dussmann , Jochen H.M. Prehn * Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland Centre for System Medicine, Royal College of Surgeons in Ireland, Dublin 2, Ireland ARTICLE INFO Keywords: Single cell imaging BCL2 proteins Mitochondria Fusion/fission Breast cancer ABSTRACT BCL2 family proteins are important regulators of mitochondrial outer membrane permeabilization (MOMP). In recent years, BCL2 family proteins have also been linked to the regulation of mitochondrial bioenergetics and dynamics. Given their overexpression in breast cancer cells, we sought to explore whether two key members of this family, BCL2 and BCL(X)L impacted on mitochondrial fusion/fission processes. By employing a single cell imaging and RNA sequencing we found that overexpression of BCL2 or BCL(X)L increases mitochondrial dynamics and alters the expression profile of genes involved in this process. Collectively, our data show that overexpression of BCL2 proteins regulates mitochondrial dynamics in breast cancer tumor cells. 1. Introduction Mitochondria are highly plastic organelles, which are susceptible to several morphological changes. The dynamic interplay between the different mitochondrial shapes creates conditions to maintain proper cellular homeostasis. Proteins such as MFN1 and 2 (at the outer mitochondrial membrane - OMM) and OPA1 (at the inner mitochondrial membrane - IMM) mediate the process where two mitochondria undergo fusion [1]. The process of separation of a mitochondrion in two or more organelles is called fission and is mediated by proteins such as DRP1 and FIS1 [1]. The maintenance of the mitochondrial network is part of the delicate integration of energy demands, stress response regulation and clearance of damaged mitochondria. Mitochondria are able to adapt their shape and connections to the metabolic needs of the cells in our body [2]. As an example, alteration of IMM fusion affects cristae shape and respiratory chain supercomplexes [3] and supports increased oxidative phosphorylation (OXPHOS) in proliferating cells [4]. BCL2 proteins, master regulators of intrinsic apoptosis and MOMP [5], are overexpressed in a variety of cancers, including breast cancer [6]. These proteins are fundamental in controlling carcinogenesis and treatment responses. In recent years, BCL2 family members have been found to participate in several cellular processes, not directly related to cell death [7]. We recently observed that overexpression of BCL2 and BCL(X)L at levels comparable to those observed in patients, improves mitochondrial bioenergetics of breast cancer cells, in particular in low energy supply conditions as occurring in the tumor microenvironment, where glucose and oxygen are scarce [8]. Breast cancer cells overexpressing these proteins were able to maintain higher ATP levels in the absence of nutrients [8]. Studies in neurons likewise showed that BCL(X) L increases metabolic efficiency [9]. BCL2 family proteins are also known to control mitochondrial fusion through interaction with the fusion machinery [10]. For example, it has been found that BCL(X)L interacts with DRP1 [11] and MFN1/2 [12,13]. Other studies have shown that BCL(X)L increases the rates of both fusion and fission and increases mitochondrial biomass in neurons [14]. Due to the close link between bioenergetics and mitochondrial networks, we here explored using single cell imaging approaches whether overexpression of BCL2 or BCL(X)L improves mitochondrial dynamics, and explored RNA sequencing data to investigate whether this phenomenon was associated Abbreviations: BCL2, B-cell lymphoma; OXPHOS, oxidative phosphorylation; ΔΨ m , mitochondrial membrane potential; ER + , estrogen receptor positive; TMRM, tetramethylrhodamine methyl ester; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; MOMP, mitochondrial outer membrane permeabilization; ATP, adenosine triphosphate. * Corresponding author at: Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin 2, Ireland. E-mail address: [email protected] (J.H.M. Prehn). 1 Present address: Departamento de Farmacología, Universitat de Val` encia; FISABIO (Fundaci´ on para el Fomento de la Investigaci´ on Sanitaria y Biom´ edica de la Comunidad Valenciana), Valencia, Spain. Contents lists available at ScienceDirect BBA - Molecular Cell Research journal homepage: www.elsevier.com/locate/bbamcr https://doi.org/10.1016/j.bbamcr.2021.119095 Received 19 April 2021; Received in revised form 23 June 2021; Accepted 25 June 2021 BBA - Molecular Cell Research 1868 (2021) 119095 2 with alterations in the transcriptome of breast cancer cells. 2. Results Materials and Methods: To examine whether BCL2 or BCL(X)L have an effect on fusion dynamics, we employed previously characterised MCF7 cells overexpressing either BCL2 (MCF7-BCL2) or BCL(X)L (MCF7-BCL(X)L) and compared their responses to empty vector transfected cells (MCF7-pSFFV) [8]. Cells were transfected with the photoconvertible mitochondrial-targeted probe mitoKaede [15]. This fluorescent protein is red-shifted following a brief irradiation in the area of interest and allows the detection of the dynamics of a mitochondrial subpopulation over time (Supplementary Fig. 1). A decrease in signal intensity of photo-converted Kaede represents mitochondrial dynamics events as the red signal is diluted from surrounding non photo-converted mitochondria. Following transfection, cells were placed in Krebs buffer (KB), in the presence of 2 mM pyruvate, which we previously demonstrated is capable of activating mitochondrial respiration in these cells [8]. After photo-conversion of a subset of mitochondria in mitoKaedetransfected cells, fluorescence intensity was followed over a time period of 30 min. We monitored fusion/fission processes by observing the decrease in signal intensity from photo-converted Kaede protein. Interestingly, time lapse experiments revealed that both MCF7-BCL2 and BCL(X)L cell lines possessed increased rates of mitochondrial fusion when fuelled with pyruvate compared to MCF7-pSFFVcells (Fig. 1A and B). Moreover, we observed that both overexpressing cell lines had faster fusion kinetics compared to control cells (Fig. 1C). To confirm that the results observed were dependent on BCL2 or BCL(X)L, we employed a selective BCL2 and BCL(X)L inhibitor, ABT199 and WEHI-539, respectively. These are specific drugs that target the BH3 domain of the above-mentioned BCL2 family members [16]. The addition of 1 μ M ABT199 to BCL2or 1 μ M WEHI-539 to BCL(X)L-overexpressing cells, inhibited fusion dynamics (Fig. 1B and C). Concentrations of ABT199 and WEHI-539 were chosen based on previous study where we found that these inhibitors specifically inhibited BCL2 or BCL(X)L-facilitated cell survival [17]. We also explored the effects of the inhibitors on mitochondrial bioenergetics, and measured ΔΨ m (mitochondrial membrane potential) using time lapse imaging of TMRM fluorescence changes. Again, cells were placed in KB with 2 mM pyruvate and a baseline TMRM fluorescence was established for both MCF7-BCL2 and BCL(X)L cells. After 20 min, 1 μ M ABT-199 or 1 μ M WEHI-539 were added to the medium, [17] and TMRM fluorescence intensity recorded for 1 h. Interestingly, we found that TMRM fluorescence intensity increased in MCF7-BCL2 when treated with ABT199, and in MCF7-BCL(X)L when exposed to WEHI-539 (Fig. 1D, E and F). In order to establish a link between mitochondrial dynamics and gene expression changes induced by BCL2 and BCL(X)L, we next conducted RNA-sequencing experiments in the three MCF7 cell lines. Additionally, we compared these data with publicly available gene expression profiles from breast cancer cell lines and patient samples. A comparison between expressions of the genes from the fusion/fission mitochondrial processes, grouped by process, is shown in Supplementary Fig. 2. Overall, we observed different expression patterns regarding genes for the regulation of mitochondrial dynamics in both overexpressing cell lines, with BCL(X)L showing more pronounced differences in gene expression (Supplementary Fig. 2). Interestingly, OPA1, FIS1, and UBA52 expression was higher in MCF7 pSFFV and BCL2overexpressing clones and reduced in BCL(X)L overexpressing cells. VPS35 recorded similar expression levels in both overexpressing clones, while PPARGC1A was increased in MCF7-BCL(X)L. In the TCGA and METABRIC data sets we found that OPA1 and VPS35 genes expression was negatively correlated with BCL(X)L (small blue dots), consistent with our in house data. Similar correlations were also observed for CLPB and UBA52 as found in TCGA and METABRIC datasets, respectively. Additionally, we observed a negative correlation of BCL2 with TFPT, VPS35 and PPARGC1A and a positive correlation with MIEF2 in both TCGA and METABRIC datasets, again consistent with our in house data. 3. Discussion In this paper we provide evidence that BCL2 and BCL(X)L are implicated in the regulation of mitochondrial dynamics in breast cancer. As highlighted above, mitochondrial bioenergetics and dynamics are tightly interconnected. Because we showed previously that BCL2 and BCL(X)L increase mitochondrial bioenergetics in breast cancer cells [8] it was crucial to measure fusion/fission events in this system. Interestingly, we observed that BCL2 and BCL(X)L overexpressing cells possessed a higher rate of mitochondrial fusion. This is in line with previous literature data, in which it has been observed that BCL(X)L overexpression is able to increase fusion/fission and mitochondrial biomass in neurons [14]. Strikingly, treatment with BCL2and BCL(X)Lselective inhibitors led to complete inhibition of mitochondrial fusion in overexpressing cells. In the case of BCL(X)L, this effect could be due to direct interaction between fission regulator Drp1 and BCL(X)L [18]. The increase in TMRM fluorescence we observed upon exposure to the selective BCL2 and BCL(X)L inhibitors could be explained by several possible mechanisms: inhibition of BCL2 and BCL(X)L could lead to a decrease in mitochondrial ATP synthase activity causing a buildup of a higher H + gradient across the mitochondrial inner membrane. This is supported by studies showing that BCL(X)L increases mitochondrial ATP synthase activity [9]. The inhibition of BCL(X)L by WEHI-539 could therefore affect ATP synthase activity. It is also possible that inhibition of BCL2 and BCL(X)L leads to an increase in mitochondrial citric acid cycle activity, producing more mitochondrial NADH and therefore generating a higher H + gradient with no concurrent increase in ATP synthase activity, however there are to our knowledge no studies supporting this hypothesis. In contrast, it has been found that ABT199 inhibited mitochondrial function independent of its target BCL2 by blocking TCA cycle with the concurrent increase in reductive carboxylation [19]. Alternatively, BCL2 and BCL(X)L inhibition may, through their inhibition of mitochondrial fusion, increase the process of mitophagy [13], thereby replacing non-functional mitochondria with a new population of mitochondria that have a higher membrane potential. However this process is likely occurring too slow to explain the rapid increase in TMRM fluorescence seen in our study. These results are also in line with previous data, where both inhibitors were used on MCF7 control cells and increased the levels of TMRM significantly, while decreasing fusion. With our current data we cannot rule the specificity of these inhibitors in altering the effect observed. Our previous study found that, at the cell death level, these inhibitors at the concentration chosen are however specific. At a more general level the same study highlighted that ABT199 or BCL(X)L were more efficient in decreasing mitochondrial activity in BCL2 or BCL(X)L cells, respectively [17]. Nonetheless, increasing the concentration of the inhibitors at a higher level to the one used in this study, will most likely inhibit other BCL2 proteins or other metabolic processes as discussed above [19]. We also found that BCL2 and BCL(X)L-overexpressing cells possessed distinct mRNA expression profiles for genes involved in mitochondrial dynamics, pointing at a regulatory effect that the overexpression of these proteins might have on fusion/fission components. BCL(X)L cells had decreased levels of the fission regulators FIS1 and MIEF2. Both BCL2 and BCL(X)L cells recorded decreased levels of VPS35 mRNA encoding a protein involved in multiple processes such as membrane stability, fusion and fission [20]. This was observed both in our in house data and in patient datasets. These alterations may clinically also be highly relevant since mitochondrial dynamics regulate cancer progression and metastasis, as seen in triple negative breast cancer [21]. Nonetheless, these are preliminary and descriptive data that need to be further explored. Collectively, we demonstrate that overexpression of BCL2 and BCL (X)L in breast cancer cells contributes to the regulation of mitochondrial dynamics, and that BCL2 antagonists decrease mitochondrial dynamics. F. Lucantoni et al. BBA - Molecular Cell Research 1868 (2021) 119095 3 Photo-converted MitoKaede C BA 0 min 10 min 20 min 30 min MCF7-pSFFV MCF7-BCL2 MCF7-BCL(X)L BCL2 + ABT199 BCL(X)L + WEHI539 0102030 60 70 80 90 100 110 120 Time [min] * Slope (Average intensity fluorescence/time) Photo-converted MitoKaede Average Intensity (normalised to baseline) MCF7-pSFFV MCF7-BCL2 MCF7-BCL(X)L MCF7-BCL2 + ABT199 MCF7-BCL(X)L + WEHI539 *** *** 16-bit color scale −3 −2 −1 0 1 2 * MCF7-pSFFV MCF7-BCL2 MCF7-BCL(X)L MCF7-BCL2 + ABT199 MCF7-BCL(X)L + WEHI539 ** **** **** **** MCF7-BCL2 MCF7-BCL(X)L % TMRM fluorescence (normalised to baseline) Time [min] 04020 60 80 0 100 50 150 200 250 % TMRM fluorescence (normalised to baseline) 0 100 50 150 200 250 MCF7-BCL2 MCF7-BCL(X)L 2 mM Pyruvate Inhibitor 1 µM ABT199 Baseline 1 µM WEHI539 Baseline MCF7-BCL2 MCF7-BCL(X)L 16-bit color scale TMRM Time 0 min 20 min 40 min 60 min 80 min D EF **** 10 μm 10 μm (caption on next page) F. Lucantoni et al. BBA - Molecular Cell Research 1868 (2021) 119095 4 Our data might explain the effect seen in a previous study, where we described that overexpression of BCL2 and BCL(X)L increase mitochondrial bioenergetics efficiency, as increased mitochondrial dynamics translates in increased coupling efficiency [8]. Furthermore, our data indicate that expression levels of core genes regulating mitochondrial dynamics may also be regulated by BCL2 and BCL(X)L. Supplementary data to this article can be found online at https://doi. org/10.1016/j.bbamcr.2021.119095. CRediT authorship contribution statement Federico Lucantoni: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft. Manuela Salvucci: Data curation, Formal analysis. Heiko Dussmann: Conceptualization, Investigation, Methodology, Writing – review & editing. Jochen H.M. Prehn: Conceptualization, Supervision, Funding acquisition, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank Dr. Claus Reimertz for generating the BCL2 and BCL(X)L overexpressing clones, and Dr. Bram Boeckx and Prof Diether Lambrechts for RNA sequencing. This research was funded by grants from the Irish Cancer Society Collaborative Cancer Research Centre BREASTPREDICT (CCRC13GAL) and Science Foundation Ireland and the Health Research Board (16/US/3301) to JHMP. We kindly thank Dr. Geoffrey Owens for providing the mito-Kaede plasmid (Addgene plasmid # 28133). References [1] L. Pernas, L. Scorrano, Mito-morphosis: mitochondrial fusion, fission, and cristae remodeling as key mediators of cellular function, Annu. Rev. Physiol. 78 (2016) 505–531, https://doi.org/10.1146/annurev-physiol-021115-105011. [2] B. 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Cells were placed in krebs buffer (KB) with 2 mM pyruvate. Fluorescent signal was monitored every 10 s for 30 min; a decrease in the fluorescence intensity is accountable for mitochondrial dynamics events. For BCL2 inhibitors, cell was again equilibrated in KB buffer with 2 mM pyruvate and 1 μ M ABT199 or WEHI-539 added for 1 h. Following treatments, cells were selected and photo conversion of mitoKaede performed as before. (B) Representative traces for time-lapse fluorescence intensity in KB with 2 mM pyruvate. The average signal values from each photo-converted area were normalised to the baseline. Data represent mean ±SD from n =3 independent experiments (C) Slope values were assessed using a nonlinear fit –straight line function in GraphPad Prism and represented in a violin plot. Data represent median and interquartile range from n =3 independent experiments and were analysed by two-way ANOVA with Tukey post-test (* indicates a pvalue <0.05, ** indicates a p-value <0.01 and *** indicates a p-value <0.001). (D) Representative images of mitochondrial membrane potential in MCF7-BCL2 and MCF7-BCL(X)L treated with ABT199 or WEHI-539, respectively. (E) Kinetics of TMRM fluorescence were monitored over time in BCL2and BCL(X)L MCF7 clones. Baseline was recorded for 20 min, after which ABT199 or WEHI-539 (1 μ M) were added to the medium and signals recorded for 1 h. All data represent mean ±SD from n =3 independent experiments and both signals are normalised to the baseline levels. (F) TMRM intensity values, normalised to the baseline levels, were analysed by taking into account the maximal value reached during ABT199 or WEHI-539 treatments. Values were evaluated by two-way ANOVA with Tukey post-test for multiple comparison (* indicates a p-value <0.05 and *** indicates a p-value <0.001). F. Lucantoni et al.