# Inhibition of TRAP1 in the C-terminal domain influences mitochondria properties and breast cancer cell metabolism

Clélia Mathieu, Jessica Ristow Branco, Patricia Zancan, Catherine Brenner, Samir Messaoudi, Elias Fattal, Juliette Vergnaud

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► To cite this version:

Clélia Mathieu, Jessica Ristow Branco, Patricia Zancan, Catherine Brenner, Samir Messaoudi, et al. Inhibition of TRAP1 in the C-terminal domain influences mitochondria properties and breast cancer cell metabolism. Biomedicine and Pharmacotherapy, 2026, 196, pp.119060. 10.1016/j.biopha.2026.119060. hal-05524058

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Submitted on 23 Feb 2026

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# Inhibition of TRAP1 in the C-terminal domain influences mitochondria properties and breast cancer cell metabolism

Clélia Mathieu<sup>a</sup>, Jessica Ristow Branco<sup>a,b</sup>, Patricia Zancan<sup>b</sup>, Catherine Brenner<sup>e</sup>, Samir Messaoudi<sup>c,d</sup>, Elias Fattal<sup>a</sup>, Juliette Vergnaud<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

- Chaperone protein

- Mitochondria

- Metabolism

- Breast cancer

## ABSTRACT

Breast cancers are characterized by complex energy metabolisms involving the Warburg effect but also mito-
chondria, although this area is not yet well understood. Tumor cells are particularly flexible by choosing
oxidative phosphorylation (OXPHOS) or glycolysis depending on the needs and aggressiveness. Within the
mitochondria, a HSP90-chaperone protein, TRAP1, exerts regulatory effects on several vital functions such as
OXPHOS, production of reactive oxygen species and apoptosis by interacting with members of the respiratory
chain or the mPTP. However, not all of its roles have yet been elucidated. Here, we propose to modulate TRAP1
functions using a mitochondriotropic molecule (containing triphenylphosphonium) targeting its C-terminal
domain, 6BrCaQ-C10-TPP, in breast tumor cells. Its blocks proliferation with no massive apoptosis, after 24 h of
treatment, and induces dissipation of the mitochondrial membrane potential. 6BrCaQ-C10-TPP also appears to
modulate regulators of epithelial-mesenchymal transition (Snail and ZEB1) without a common response in all cell
lines. Furthermore, the chaperone machinery is affected with a decrease of HSF1 and HSP70, but without
degradation of HSP90 or TRAP1, while decreasing the levels of SDH-A and/or SDH-B, partner of TRAP1. Finally,
short-term treatments (1 and 3 h) with 6BrCaQ-C10-TPP modify energy metabolism by promoting glycolysis. In
conclusion, modulation of TRAP1 on the C-terminal domain by 6BrCaQ-C10-TPP exerts a cell-line dependent anti-
tumor effect by modulating major mitochondrial functions *in vitro*. The differences between cell types need to be
clarified. This study confirms that TRAP1 is a target of interest in breast cancer cells, but some of its functions still
need to be elucidated.

## 1. Introduction

Heat shock proteins (HSP) are chaperone proteins involved in ho-
meostasis in healthy cells but also in all hallmarks of cancer in various
tumor cells [1]. TRAP1 is an isoform of this family, and although it has
been known for several decades, it is less studied than HSP90. However,
through its localization in mitochondria, TRAP1 is involved in cellular
metabolism and apoptosis signaling pathways and thus can be an
exciting target as a new cancer therapeutic approach [2]. Some in-
hibitors have been designed based on N-terminal HSP90 inhibitors, like
Gamitrinibs in which a targeting-mitochondria moiety

(triphenylphosphonium, TPP) has been added thanks to a linker to the
pharmacophore. However, it has been shown that *N*-terminal inhibitors
induced resistance through a heat-shock Response (HSR) based on the
release of the transcription factor HSF-1 after the inhibition of HSP
proteins, leading to upregulation of heat-shock proteins [3,4]. In
contrast to *N*-terminal inhibitors, compounds targeting the *C*-terminal
domain do not imply the HSR and have a different molecular response to
HSP90 inhibition [5,6]. Thus, designing new *C*-terminal TRAP1 in-
hibitors from the already-known Hsp90 inhibitors would be interesting.
In this context, we have previously reported the quinolinone carbox-
amide derivative – 6BrCaQ, as a *C*-terminal HSP90 inhibitor with

<footnote><sup>a</sup> Institut Galien Paris-Saclay UMR CNRS 8612, Univ Paris-Saclay, Orsay 91400, France</footnote>

<footnote><sup>b</sup> The Metabolizsm' Group, Departamento de Biotecnologia Farmacêutica, Faculdade de Farmácia, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil</footnote>

<footnote><sup>c</sup> Laboratoire de Synthèse Organique, Ecole Polytechnique, CNRS, ENSTA, Institut Polytechnique de Paris Palaiseau, France</footnote>

<footnote><sup>d</sup> BioCIS, UMR CNRS 8076, Univ Paris-Saclay, Orsay 91400, France</footnote>

<footnote><sup>e</sup> CNRS, Institut Gustave Roussy, Aspects métaboliques et systémiques de l'oncogenèse pour de nouvelles approches thérapeutiques, Université Paris-Saclay, Villejuif, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: juliette.vergnaud@universite-paris-saclay.fr (J. Vergnaud).</footnote>
promising biological activities against various cancer cell lines [7–9].

In our continued interest to develop new TRAP1 inhibitors based on C-terminal HPS90 inhibition, we reported recently that the conjugation of 6BrCaQ to a triphenylphosphonium anion through a corresponding linker leads to a 6BrCaQ-C₁₀-TPP conjugate [10], with a promising biological activity in colon cancer cell line, HT-29. As for Gamitrinib, 6BrCaQ-C₁₀-TPP is amphiphilic and enters the cells thanks to the positive charge and reaches immediately the mitochondria according to the high mitochondrial membrane potential. We already showed that 6BrCaQ-C₁₀-TPPs derivative induces the depolarization of mitochondria and the degradation of TRAP1 partner proteins such as SDHA-B, but without triggering the HSR response. Furthermore, this led compound displays a sub-micromolar cytotoxicity, with an IC₅₀ of 0.1 µM against several cancer cell lines.

The function of the TRAP1 protein in tumor processes is complex and variable from one tumor type to another. In reality, its overexpression is not always an unfavorable prognostic factor [11]. Depending on the cancer type, TRAP1 may play a tumorigenic or tumor suppressor role [11]. Its function may also depend on the initial metabolism of the cells (glycolytic, aerobic, energetic, or quiescent); thus, an inhibitor of TRAP1 could affect cell metabolism and then tumor progression. It may be interesting to study the new 6BrCaQ-C₁₀-TPP inhibitor on different types of cancer cells, primarily since TRAP1 influences the proliferation of cells and protects them from apoptosis [2]. Breast cancer is the most frequent cancer for women and is highly heterogeneous but TRAP1 is overexpressed in many breast cancer types. In these specific cancer cells, inhibition of TRAP1 increases their sensitivity to anti-cancer treatments and inhibits tumor growth [12]. In addition, overexpression of TRAP1 in breast cancer has been shown to increase cell invasiveness and metastatic abilities [13,14]. Thus, it is essential to understand its role in different cancers and study its inhibition effect.

In this article, a more advanced analysis of the cellular response mechanism involved through the inhibition of TRAP1 was conducted on different cellular models of breast cancer to consider the heterogeneity of these tumors. The molecule's effect has been analyzed on three breast cancer cell lines: MDA-MB-231, MCF-7, and BT474, with distinct

metabolisms and characteristics [15]. MCF-10A has been added to some experiments for comparison as a non-malignant cell line and normal fibroblasts. MDA-MB-231 is a "triple negative breast cancer" (TNBC) cell line that does not express estrogen receptors (ER), progesterone receptors (PR), or human epidermal growth factor receptor 2 (HER-2). They are highly invasive and aggressive epithelial cells, with a low endogenous level of TRAP1. These cells are highly glycolytic and have impaired mitochondrial function. Their duplication time is rather short [16–18]. At the same time, MCF-7 is classified as a luminal A cell line, estrogen-dependent, and positive for ER. These are less invasive and aggressive epithelial cells than MDA-MB-231, with a high endogenous level of TRAP1. These cells are less glycolytic than MDA-MB-231. Their duplication time is also short. At last, BT474 cell lines express ER⁺, PR⁺, and HER2⁺ receptors. These cells do not have a high glycolytic metabolism and thus have a long duplication time [19]. Our strategy to analyze the impact of 6BrCaQ-C₁₀-TPP is illustrated on Fig. 1. First, the global effect of the TRAP1 inhibitor on cells has been studied: inhibition of proliferation, induction of apoptosis and migration. Then the study focused on the mitochondrial targeting. For this purpose, the effect of 6BrCaQ-C₁₀-TPP on the mitochondrial membrane potential, the network and morphology of mitochondria has been investigating. In addition, the regulation of the activity of a TRAP1 partner protein, succinate dehydrogenase (SDH), and the delocalization of specific partner proteins, such as cytochrome c to highlight the effect of inhibition of TRAP1. Finally, the mitochondrial energetic metabolism and the glycolytic flow was studied in real time based on treatment with 6BrCaQ-C₁₀-TPP.

## 2. Materiel and methods

### 2.1. Cell culture

Human breast cancer cells (MDA-MB-231, RRID:CVCL_0062; MCF-7, RRID:CVCL_0031; BT474, RRID:CVCL_0179) were obtained from ATCC® and MCF10A (RRID:CVCL_0598) cell line was purchased from Cell Bank of Rio de Janeiro and (Sigma-Aldrich, France). Cells were regularly tested for the absence of *Mycoplasma*. Fetal bovine serum (FBS)

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Design of our study. A. Theoretical trafficking of 6BrCaQ-C₁₀-TPP to reach mitochondria: 1. Entry of the TRAP1 inhibitor in the cytoplasm, then 2. Targeting of mitochondria by TPP and finally 3. Targeting of TRAP1. B. Goals of the study. To evaluate the effect of 6BrCaQ-C₁₀-TPP on the cellular response: 1. Cell proliferation and cell death. 2. Effect on epithelio-mesenchymal transition (EMT). Then, study of the effect of 6BrCaQ-C₁₀-TPP on TRAP1: 3. Effect on partners proteins. Then, study of the effect of 6BrCaQ-C₁₀-TPP on the mitochondria: 4. To evaluate the reactive oxygen species (ROS) production. 5. Study of the mitochondrial potential. 6. Study of the mitochondria network morphology. Finally, study of the effect on both mitochondria and TRAP1: 7. Study of the cell metabolism.</figcaption></figure>
was purchased from Thermofisher (Gibco™, France). Media were purchased from Sigma-Aldrich® (France). BT474 cells were cultured in DMEM/F-12 medium supplemented with 20% FBS, 7 µg/mL of insulin (Sigma-Aldrich®, France), and 10,000 unit/mL of penicillin and streptomycin (Sigma-Aldrich®, France). MDA-MB-231 cells were cultured in Leibovitz's L-15 medium supplemented with 15% FBS, 2 mM L-glutamine, 20 mM sodium bicarbonate, and 10,000 U/mL of penicillin and streptomycin. MCF-7 cells were cultured in MEM media supplemented with 10% FBS, 2 mM L-glutamine, non-essential amino acids, and 10,000 U/mL of penicillin and streptomycin. MCF10A were cultured in DMEM/F12 medium supplemented with 10% of horse serum (HS; Invitrogen, Sao Paulo, SP, Brazil), 0.02 µg/mL epidermal growth factor (EGF), 5 µg/mL insulin, 1.25 µg/mL hydrocortisone, 0.1 µg/mL cholera toxin (Sigma-Aldrich Co., St. Louis, MO, USA) and 10,000 U/mL of penicillin and streptomycin.

All cell lines were maintained in a humidified incubator supplied with 5 % CO₂ and maintained at 37 °C. The cells were split twice a week with a ratio from 1:6–1:10. For all experiments, cells were seeded between the 2nd and the 20th splits depending on the cell lines when they were in exponential growth in such a way as to controls never reached confluency.

## 2.2. Measurements of cell viability

Formazan powder (M5655, Sigma-Aldrich®, France) and Trypan blue (T8154, Sigma-Aldrich®, France) were purchased from Sigma-Aldrich®. Formazan powder was diluted at 5 mg/mL in water and 0.22 µm filtered. Cell viability was determined by MTT according to the manufacturer's instructions. Briefly, 5000 cells per well were seeded in 96-well plates and treated 24 h after. Each culture condition was analyzed in 4 replicates, and the experiment was repeated at least three times independently. After 72 h of treatment, 20 µL of the formazan solution was added to each well and incubated for 2 h. Then, the medium was removed and replaced by 200 µL of DMSO for each well. Absorbance values (at 570 nm) were corrected by subtracting the average absorbance from blank wells containing DMSO and the products tested without cells. The absorbance recorded (570 nm) with non-treated cells was taken as 100 % of metabolic activity corresponding to the maximum viability.

Cell proliferation was also determined by counting viable and dead cells by trypan blue exclusion. Cells were seeded in 12-well plates (60,000 per well for MDA-MB-231 and MCF-7, 400,000 cells for BT474) and treated 24 h after. Each sample was trypsinized, resuspended into media, and diluted with Trypan Blue in a 1:1 ratio. Cells were then counted by optical microscopy (Kovas slide, Dutscher France). Blue cells were counted as dead. Each culture condition was analysed in 3 wells. The experiment was repeated independently twice.

## 2.3. RNA interference

The siRNA sequences were obtained by Eurogen-tech (Angers, France) and were based on Agorreta et al. [13], with the Forward 5' -CCAGUGGCUUUCAGAUGGU -3' and Reverse 5' -ACCAUCUGAAAGCCACUGG -3' sequence. Lipofectamine® RNAiMAX Reagent (Invitrogen) and different concentrations of siRNA (10 nM, 50 nM and 100 nM) were used for transfection of the siRNA cell lines for 48 h following the protocol provided by the manufacturer N°MAN0007825 Rev 1.0.

## 2.4. q-RT PCR

For RNA extraction, approximately 10⁶ cells were lyzed using 500 µl of Trizol reagent (ThermoFischer, Carlsbad, CA, USA). Samples were vigorously homogenized, and RNA was isolated following the manufacturer's instructions. The High-capacity cDNA Reverse Transcription Kit (ThermoFischer, Carlsbad, CA, USA) was used for cDNA synthesis.

For qPCR, the dye-based GoTaq qPCR Master Mix (Promega, Fitchburg, WI, USA) was used. All reactions were performed according to the Master Mix manufacturers' instructions in a Quant Studio 5 apparatus (ThermoFischer, Carlsbad, CA, USA). The program for dye-based amplifications was 2 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C, and a dissociation curve was performed at the end of the experiment, and dissociation peak was analyzed. The relative mRNA expression was calculated by the 2⁻ΔΔCt method. CCSER2 expression was used as reference gene (housekeeper). The designed primers used were:

CCSER2 forward 5'-TGAGCAGAGGCTCTCCCTAT-3', reverse 5'-GGAAAAAGGGCATCCCTCCA-3'; E-cadherin forward 5'-GACCGAGAGAGTTTCCTACG-3', reverse 5'-TGTGCTTAACCCCTCACCTTG-3'; N-cadherin forward 5'-TCCTGCTTATCCTTGTGCTGAT-3', reverse 5'-AAAAGTTGTTTGGCCTGGCG-3'; Snail forward 5'-TCTAATCCAGAGTTTACCTTCCAGC-3', reverse 5'-GTGGGGTTGAGGATCTCCG-3'; Zeb1 forward 5'-GCCAAATGGAAATCAGGATGAAAG-3', reverse 5'-TTTTGGGCGGTGTAGAATCAG-3'.

## 2.5. Wound healing assay

Cell migration was determined by using 24-well plates (5 × 10⁴ cells/well) treated with siRNA for 48 h, 24 h after the seeding and for 24 h for treatment with 6BrCaQ-C₁₀-TPP, 48 h after the seeding. The scratch was made manually with a 2 µl pipette tip in each well individually the day after the siRNA treatment and at the same time as the 6BrCaQ-C₁₀-TPP treatment. Images were taken after the scratch and 24 h later at specific scratch sites. The images were analyzed using the software BELView (BEL Engineering, It). Each condition was done in 3 replicates.

## 2.6. Measurement of mitochondrial membrane potential

Mitochondrial membrane potential has been measured by the Mitochondrial Membrane Potential Assay Kit (13296) purchased from Cell Signaling® (Ozyme, France) and according to manufacturer's instructions. Briefly, cells were seeded the day before the experiment in a black 96-well flat bottom clear plates (20,000 cells/well for MDA-MB-231; 10,000 cells/well for MCF-7 and BT474) and incubated with 6BrCaQ-C₁₀-TPP at various concentrations for 2, 3, and 6 h. Each condition has been repeated in 3–8 replicates. Some wells have been incubated with carbonylcyanure m-chlorophenylhydrazone (CCCP) at 50 µM for positive control of mitochondrial membrane potential disruption for 20 min. Then, all the wells have been treated with tetramethylrhodamine ethyl ester perchlorate (TMRE) dye at a final concentration of 200 nM for 20 min at 37 °C. The plates were washed 3 times with warm PBS and then read by fluorescence (ex. 530 nm/em. 590 nm) by Envision Xcite plate reader (PerkinElmer®, France). The experiment was repeated three times independently.

Flow cytometry experiments were performed on Accuri C6™ Cytometer (BDbiosciences®, France). Cells were kept on ice until analyzed.

Cells were seeded the day before the experiment in 12-well plates (10⁵ cells per well). They were then incubated with 0.1 and 0.5 µM of 6BrCaQ-C₁₀-TPP for 3 and 6 h. CCCP was incubated for 10 min at 50 µM as a positive control. JC-1 (Mitochondria staining kit, CS0390, Sigma-Aldrich, France®) was solubilized in DMSO at 1 mg/mL. The JC-1 solution was then diluted by 800: 3.12 µL of the stock solution was first diluted in 2 mL of milliQ water until complete dissolution, then 500 µL of JC-1 Staining Buffer (5X) were added. The cells were trypsinized and then rinsed with JC-1 staining buffer (1X), and then incubated with JC-1 solution at 20 min for 37 °C. They were then rinsed 2 times with cold JC-1 staining buffer (1X). They were then kept on ice and read by flow cytometry with FL1 (excitation 488 nm; emission 533/30 nm) and FL2 (excitation 488; emission 585/40 nm) channels.
## 2.7. Cellular proliferation measured with CFSE fluorescence

Carboxyfluorescein succinimidyl ester powder (CellTrace™ CFSE, C34554, Thermofisher®, France) was resuspended in DMSO at 5 mM. Then, the stock solution was diluted in warm PBS at 5 µM the day of the experiment. The cells were resuspended in the solution at 106 cells/mL and incubated at 37 °C for 20 min. To remove the dye excess, 5 volumes of media were added. The cells were then pull down, and resuspended in media, and seeded in 12-well plates at 30,000 cells per well. The cells were treated with 6BrCaQ-C₁₀-TPP (0–0.5 µM) the day after. The cells were detached by trypsin at several time points: 24 h, 48 h and 72 h. 2.5 µL of 7-AAD was added to each sample 15 min before to read the cell fluorescence by flow cytometry. The experiment was repeated thrice independently.

## 2.8. Evaluation of production of reactive oxygen species (ROS)

ROS production was measured through the oxidation of DiHydR-hodamine 123 (DHR, D1054, Sigma-Aldrich®, France). A stock solution for DHR was made in DMSO at 10 mM. Cells were seeded in 12-well plates at 10⁵ cells/well and treated the next day with 6BrCaQ-C₁₀-TPP (0–0.5 µM) for 24, 48, and 72 h. As a positive control of ROS induction, cells were treated with 10 µM of phorbol-12-myristate-13-acetate (PMA) for 15 min. Cells were trypsinized, centrifuged, and resuspended in 200 µL of media supplemented with DHR (5 µM, 2,000-fold diluted). The cells were then analyzed by flow cytometry (FL1, 488/530 nm) after fifteen minutes of incubation in the dark at 37°C. This experiment was repeated twice independently.

## 2.9. Induction of apoptosis by Annexin/7-AAD

Apoptosis/necrosis detections following exposure to 6BrCaQ-C₁₀-TPP were performed using the FITC Annexin V Apoptosis Detection Kit with 7-amino-actinomycin D (7-AAD) (#640922, Biolegend®, France). MDA-MB-231 cells were seeded in 12-well plates (10⁵ cells/wells) and treated the next day with 6BrCaQ-C₁₀-TPP (0–0.5 µM) for 24, 48 and 72 h. After incubation, cells were rinsed with Annexin Binding buffer and then incubated with 200 µL of 2.5 µL of FITC-Annexin V in its binding buffer for 15 min at 37 °C (following provider's instructions). Cells were trypsinized and resuspended in 200 µL of media, supplemented with 2.5 µL of 7-AAD.

Cells were immediately analyzed by flow cytometry using the fluorescent channels FL-1 (excitation 488 nm; emission 533/30 nm) for Annexin V-FITC and FL-3 (excitation 488 nm; emission LP670nm) for 7-AAD. Staurosporine (1 µM, 4 h) was used as a positive control for apoptosis induction. Cells with double negative labeling are considered healthy cells, while annexin V-positive and 7-AAD-negative cells are in early apoptosis, and double-positive cells are in late apoptosis or necrosis.

## 2.10. Staining of mitochondria for confocal microscopy

Cells were seeded the day before the experiment on a glass coverslip in a 12-well plate. They were treated for 30 min and 3 h with 0.1 or 0.5 µM of 6BrCaQ-C₁₀-TPP and then rinsed with warm PBS and fixed with ROTI®Histofix, 4 % (Carl Roth®, Germany), 15 min at RT. Cells were rinsed 3 times with warm PBS, and then permeabilized with Triton X-100, 0.1 %, 10 min at RT. They were then incubated with a primary antibody: anti-TOM20 (612278, BDBiosciences®, France) 1:100 for 1 h at 37 °C in PBS-BSA 5 %. The antibody was then rinsed 3 times 5 min with PBS. Afterward, the cells were incubated with a secondary antibody coupled with a fluorophore Alexa-Fluor594 (A21125, Thermofisher®, France). 1:5000 for 37 °C, in PBS-BSA 5 %. The cells were then rinsed 3 times with PBS for 5 min and then mounted with DAPI (Antifade mounting medium with DAPI, Vectashield®). The slides were sealed by nail varnish the next day and stored at 4 °C.

## 2.11. Western blotting

RIPA buffer (Cat# R0278) and protease inhibitor cocktail (Cat #P8349) were purchased from Sigma-Aldrich (RRID:SCR_008988) (France). Pierce™ BCA protein assay (Cat# 23227) was purchased from Thermo Fisher Scientific (RRID:SCR_008452) (France). Laemmli buffer (Cat# 1610737), β-Mercaptoethanol (Cat# 1610710), 4–15 % Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Cat# 4568084), PVDF-low fluorescence membrane (Cat# 1620260), Tween-20 (Cat# 1662404), Clarity Western ECL Substrate (Cat# 1705060) and all buffers were purchased from Bio-Rad Laboratories (RRID:SCR_008426) (France). The primary antibodies are listed in Table 1. Secondary antibodies were purchased from SouthernBiotech (RRID:SCR_019250) (Clinisciences, France).

Frozen cell pellets (from 24h-treated cells) were lysed in RIPA buffer supplemented with protease inhibitor cocktail (10:100) for 30 min on ice (100 µL for 10⁶ cells). Protein concentrations in lysates were assayed

<table><caption>**Table 1**
Antibodies and dilutions used for western blotting.</caption><thead><tr><th>Target (MW)</th><th>Clone</th><th>Reference & RRIDs</th><th>Species</th><th>Dilution</th></tr></thead><tbody><tr><td>TRAP-1<br>(75 kDa)</td><td>42/Hsp75</td><td>(BD Biosciences Cat#<br>612344, RRID:<br>AB_399710)</td><td>Mouse</td><td>1:1000</td></tr><tr><td>HSF-1<br>(80 kDa)</td><td>10H8</td><td>(Enzo Life Sciences Cat#<br>ADI-SPA-950-D, RRID:<br>AB_2039201)</td><td>Rat</td><td>1:500</td></tr><tr><td>Hsp27<br>(27 kDa)</td><td>C-20</td><td>(Santa Cruz<br>Biotechnology Cat# sc-<br>1048, RRID:AB_631673)</td><td>Goat</td><td>1:500</td></tr><tr><td>SDH-A<br>(70 kDa)</td><td>D-4</td><td>(Santa Cruz<br>Biotechnology Cat# sc-<br>166947, RRID:<br>AB_10610526)</td><td>Mouse</td><td>1:500</td></tr><tr><td>SDH-B<br>(30 kDa)</td><td>Polyclonal</td><td>(GeneTex Cat#<br>GTX113833, RRID:<br>AB_2037951)</td><td>Rabbit</td><td>1:250</td></tr><tr><td>HSP70<br>(70 kDa)</td><td>W27</td><td>(Santa Cruz<br>Biotechnology Cat# sc-<br>24, RRID:AB_627760)</td><td>Mouse</td><td>1:500</td></tr><tr><td>HSP90<br>(90 kDa)</td><td>H-114</td><td>(Santa Cruz<br>Biotechnology Cat# sc-<br>7947, RRID:AB_2121235)</td><td>Rabbit</td><td>1:500</td></tr><tr><td>Cytochrome c<br>(12 kDa)</td><td>136F3</td><td>(Cell Signaling<br>Technology Cat# 4280,<br>RRID:AB_10695410)</td><td>Rabbit</td><td>1:1000</td></tr><tr><td>β-Tubulin<br>(50 kDa)</td><td>3F3-G2</td><td>(Santa Cruz<br>Biotechnology Cat# sc-<br>53140, RRID:AB_793543)</td><td>Mouse</td><td>1:5000</td></tr><tr><td>Tom-20<br>(20 kDa)</td><td>19</td><td>(BD Biosciences Cat#<br>612278, RRID:<br>AB_399595)</td><td>Mouse</td><td>1:10000</td></tr><tr><td>E cadherin<br>(120 kDa)</td><td>36</td><td>(BD Biosciences Cat#<br>610181, RRID:<br>AB_397580)</td><td>Mouse</td><td>1:10000</td></tr><tr><td>Occludin<br>(65 kDa)</td><td>Polyclonal</td><td>(Thermo Fisher Scientific<br>Cat# 71–1500, RRID:<br>AB_2533977)</td><td>Goat</td><td>1:750</td></tr><tr><td>GAPDH<br>(34–36 kDa)</td><td>Polyclonal</td><td>(Sigma-Aldrich Cat#<br>G9545, RRID:<br>AB_796208)</td><td>Rabbit</td><td>1:5000</td></tr><tr><td>Anti-rat (HRP-<br>coupled)</td><td>Polyclonal</td><td>(SouthernBiotech Cat#<br>3030–05, RRID:<br>AB_2716837)</td><td>Goat</td><td>1:4000</td></tr><tr><td>Anti-goat (HRP-<br>coupled)</td><td>Polyclonal</td><td>(SouthernBiotech Cat#<br>6420–05, RRID:<br>AB_2796335)</td><td>Donkey</td><td>1:4000</td></tr><tr><td>Anti-mouse<br>(HRP-<br>coupled)</td><td>Polyclonal</td><td>(SouthernBiotech Cat#<br>1030–05, RRID:<br>AB_2619742)</td><td>Goat</td><td>1:4000</td></tr><tr><td>Anti-rabbit<br>(HRP-<br>coupled)</td><td>Polyclonal</td><td>(SouthernBiotech Cat#<br>4030–05, RRID:<br>AB_2687483)</td><td>Goat</td><td>1:10000</td></tr></tbody></table>

MW: molecular weight of the target protein
using BCA protein assay and 30 µg of proteins were boiled at 95°C for 10 min in presence of 2X Laemmli buffer containing β-Mercaptoethanol (final concentration 355 mM) (v:v). SDS-PAGE was performed using stain-free precast gels 4–15 %, or 4–20 % (150 V for 1 h). Trihalo compounds contained in gels were covalently bound to tryptophan residues under UV light (photoactivation) to detect the separated proteins in the gel (Bio-Rad Stain-Free technology). Proteins were then transferred onto a low fluorescence-PVDF membrane (100 V for 45 min). The transfer quality was then checked thanks to the stain-free imaging, and the fluorescence of the proteins enabled total protein loading visualization and further normalization. For immunodetection, membranes were saturated in 5 % non-fat milk or 3 % Bovine Serum Albumin (BSA) in TBS-Tween 0.05–0.1 % for one hour at room temperature under agitation and were then incubated with primary antibody dilutions overnight at 4 °C. After washing (3 × 10 min), horseradish peroxidase-coupled secondary antibodies were added for 1 h (1:10,000 for anti-rabbit antibody dilution and 1:4 000 anti-mouse, rat or goat antibody dilution) and then washed. Detection was achieved using the Clarity Western ECL Substrate, and chemiluminescent signals were analyzed using the Chemidoc system® (Bio-rad, France) and were normalized to stain-free images or GAPDH signals by the Image Lab Software (RRID:SCR_014210) (Bio-rad, France).

## 2.12. Fractionation of cytosol and mitochondria

According to the manufacturer's instructions for the Biobasics® kit (#BDP051, France), a differential centrifugation method was used to extract cytosol/mitochondria contents with buffers. Cells were previously pelleted down, rinsed with PBS, dried, and stored at -80°C.

All the reagents were cooled down to 4 °C. The mitochondrial buffer was warmed to 37 °C if precipitates were visible. Mitochondrial and cytosolic buffers were supplemented with 0.5 %v of phosphatase inhibitor, 0.1 %v of Dithiothreitol (DTT), and 0.1 %v of protease inhibitor. Then 1 mL per 10 million cells of the modified cytosolic buffer was added to the cell pellet and kept on ice for 15 min. The solution was then stroked 40 times with a homogenizer. The debris of the cytosolic membrane and the nucleus were pulled down first by centrifugation (4 °C, 600 g, 10 min) and again to pellet mitochondria (4 °C, 10,000 g, 30 min). Then cytosolic sample (supernatant) was again centrifuge to clear any residue of mitochondria (4 °C, 18,000 g, 20 min), and protein content was quantified by BCA and stored at -80 °C. The pellet containing the mitochondria was rinsed with 100 µL and pulled down again (4 °C, 10,000 g, 10 min) to clear any cytosolic residue. Then, 100 µL of modified mitochondrial buffer was added for 30 min on ice. The solution was vortexed for 10 s and spinned down (4 °C, 10,000 g, 10 min) to remove the mitochondrial membrane residues. The mitochondrial protein fraction was then stored at -80 C.

## 2.13. Succinate dehydrogenase (SDH) activity measurement

Following exposure to 6BrCaQ-C₁₀-TPP, SDH activity evaluation was performed using the Succinate Dehydrogenase Assay Kit (#MAK197, Sigma Aldrich®, France). Cells were seeded the day before the experiment (10⁶ cells in a 25-cm² flask) and then treated for 30 min at various concentrations of 6BrCaQ-C₁₀-TPP (0–2 µM) and with 1 µM of 17-AAG as control. Cells were then trypsinized, rinsed by cold PBS, and pulled down. They were then homogenized by pipetting in 100 µL of cold SDH assay buffer and kept on ice for 10 min, then pulled down for 10,000 g for 5 min. The supernatant was then transferred to a cold Eppendorf®. 48 µL of the reaction mix (46 µL of SDH assay buffer and 2 µL of SDH probe) was equilibrated at room temperature and added to the 96 wells, with 50 µL of the lysed cells. The whole was equilibrated at 37 °C and shaken. Two µL of the mix probe was then added. The absorbance at 600 nm was followed for 30 min with a measure every 30 s. In parallel, a calibration curve of DCIP was realized. The slope of the linear region was determined (by GraphPad Prism (RRID:SCR_002798)), and the

following formula calculated the activity:

$$ 
\begin{align*}
\text{Succinate Dehydrogenase activity} &= \frac{B}{\Delta T + V} \\
\text{With:} \quad &B = \text{amount of reduced DCIP in the well (nmol).} \\
\text{V} = \text{Volume of sample added in the well (µL).} \\
\Delta T = \text{Time of the reaction (minutes).}
\end{align*}
$$

The experiment was repeated 3 times independently. The data shown are representative of 3 different experiments. However, the enzymatic activity is modified according to the time elapsed between the lysis of the cells and the reading of the activity, so statistics cannot be applied in these experiments. For each experiment, two wells of each condition were considered.

## 2.14. Seahorse experiments

MDA-MB-231 (40,000 cells/well), MCF-7 (20,000 cells/well) or BT474 (10,000–20,000 cells/wells) were seeded in a 96-well Seahorse analyzer plate, the day before the experiment. OCR, ECAR, and PER were measured using the Seahorse XF-96 Analyzer (Seahorse Bioscience, North Billerica, MA, USA), as recommended by the manufacturer. Briefly, the cells were treated 1 h or 3 h with 6-BrCaQ-TPP (0–0.5 µM). Cells were rinsed with Seahorse medium (DMEM, pH 7.4, supplemented with 2 mM glutamine, 1 mM glucose and 1 mM pyruvate, Agilent®, France) one hour before the assay and incubated in an incubator without CO₂. Basal measurements were taken (three readings at 5 min of intervals), the medium was injected with two schemes: Mitostress (103010–100, Seahorse XF Cell Mito Stress Test Kit) or Glycolysis rate (103344–100, Glycolytic Rate Assay Kit) as marked in the graphs. For Mitostress, there were 3 injections: A, oligomycin (1 µM); B, FCCP (0.5–1 µM); C, antimycin A plus rotenone (Rot/AA, 0.5 µM). There were 2 injections for glycolysis rate: A, Rot/AA (0.5 µM); B, 2-Deoxyglucose (2-DG, 50 mM). After each injection, three measures were taken. To measure the impact of glycolytic stress, the PER (Proton Efflux Rate) and glycoPER (Glycolytic Proton Efflux Rate) were used during experiments. PER is the sum of the protons produced by glycolysis, OXPHOS and other non-glycolytic sources (like Krebs cycle). The glycoPER is calculated a *posteriori* using Agilent Seahorse Wave (RRID:SCR_024491), thanks to the value after the inhibition of the glycolysis by 2-DG. The inhibition of mitochondrial respiration by the Rot/AA mixture allows the determination of the compensatory glycolysis, i.e. glycolysis set up in case of increased demand for ATP.

To normalize data, either a BCA (Bicinchoninic acid) protein assay was conducted on the plates, or the wells were pictured. The medium was removed after the assay for the BCA normalization, and the plates were conserved at -80 °C if necessary. Then, 50 µL of lysis buffer RIPA was added to each well for 30 min at 4°C. 30 µL were transferred in a new 96-well plate, and 200 µL of BCA reagent was added. The standard curve was obtained with BSA at several concentrations (0, 125, 250, 500, 750, 1000, 1500 and 2000 µg/mL). Hoechst reagent was added to each well for counted cells at a final concentration of 1–2.5 µg/mL. The plates were incubated for 20 min at 37 °C. Images were taken with a fluorescence microscope. The images were treated with ImageJ (RRID: SCR_003070), and the cells were counted using the AnalyzeParticles plugins. The results were plotted as pmol/min/1000 cells or pmol/min/1000 µg of proteins. Each condition had between 7 and 8 replicates. The data presented for MCF-7 were not normalized, but based on the results that we obtained with other cell lines, we consider that normalization did not influence the trends in the data. Nevertheless, standardization step has reduced variability (error bars). Mortality at the time points and concentrations studied being low, the uncertainties on the number of cells per well are attributable to seeding, the 8 replicates compensating for this variation.
## 2.15. Statistics

All graphics and statistical analyses were performed with the software Prism 8 (GraphPad Prism (RRID:SCR_002798)). All data were expressed as mean ± SD. The obtained data followed a Normal distribution, as evaluated. One-way ANOVA followed by Dunnett's post hoc test or a Mann-Whitney analysis was used to calculate the p-value compared to the control conditions was used to determine the significance of the differences. Statistical significance was reported: ns> 0.5, *< 0.5, **< 0.1 and ***< 0.01 or *P < 0.05 depending on the figure.

The data generated in this study are available upon request from the corresponding author.

## 3. Results

As mentioned in the Fig. 1, we focused our work first on the global cellular response, followed by the evaluation of the impact of 6BrCaQ-C₁₀-TPP on TRAP1/HSP90 machinery and finally on mitochondrial functions related to metabolism regulation.

### 3.1. TRAP1 inhibition affects cell proliferation and cell death

To have a clear view of the sensitivity of these cell lines to 6BrCaQ-C₁₀-TPP, the cellular response was first assessed in all the cell lines (MDA-MB-231, MCF-7, and BT474). Two methods were used to determine the IC₅₀, as 6BrCaQ-C₁₀-TPP can affect mitochondria and cellular metabolism: MTT assay, which measured the mitochondrial metabolic activity and viability by trypan blue staining dead cells. For both methods, 6BrCaQ-C₁₀-TPP has an IC₅₀ of 0.1 µM for the MDA-MB-231 and MCF-7 cell lines (Fig. 2 A, B). BT474 cells have a slower metabolism and are less sensitive than the two other cell lines, with an IC₅₀ of 0.4 µM. Few cells died under the effect of treatment according to the

Trypan Blue count as it is shown on Figure S1 where images of cells treated with 6BrCaQ-C₁₀-TPP indicate low proliferative cells without floating dead cells.

Apoptosis induction has been also assessed even if inhibitors of HSP90 induce mainly an arrest in cell cycle (G2/M) as we showed earlier. A double labeling of cells with a mixture of FITC-Annexin V and 7-amino actinomycin D (7-AAD) was carried out at several time points (24 h, 48 h, and 72 h) and two concentrations (0.1 and 0.5 µM) of 6BrCaQ-C₁₀-TPP. The induction of apoptosis by 6BrCaQ-C₁₀-TPP remains low, even at an IC₅₀ concentration at 72 h (0.1 µM) but increases at 0.2 and 0.5 µM (72 h) (Fig. 2D). To compare, siRNA targeting of TRAP1 displays limited apoptosis induction (data not shown).

Since little apoptosis induction were observed, proliferation inhibition was evaluated using CarboxyFluorescein Succinimidyl Ester (CFSE) labeling. The number of cell divisions that occurred can be estimated by monitoring the mean fluorescence per cell. The MDA-MB-231 cells were stained with CFSE and then seeded into 12-well plates. The day after seeding, cells were treated and then analyzed by flow cytometry after 24 h, 48 h, and 72 h of treatment. For untreated cells, there is a significant decrease in CFSE fluorescence after 3 days of culture (80 % decrease), confirming cell division – loss of fluorescence at each division (Fig. 2C). Upon treatment by 6BrCaQ-C₁₀-TPP, especially at 0.5 µM, fluorescence intensity of cells is significantly higher than in control condition from the first day (76,000 for 0.5 µM compared to 38,000 for untreated ones). A dose effect is observable. The fluorescence of treated cells decreases less over the days compared to the control cells. The BT474 seems again to be less sensitive than two others. To conclude, 6BrCaQ-C₁₀-TPP inhibits more cell proliferation than cell death especially at short time of treatment (24 h), but its effect on invasion and migration through its impact on epithelial-mesenchymal transition (EMT) has to be explored.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Cellular response of breast cancer cells to 6BrCaQ-C₁₀-TPP. A. Number of alive and dead cells counted by Trypan Blue exclusion on 3 cell lines (MDA-MB-231, MCF-7 and BT474) after 72 h of treatment, n = 2 (2 independent experiments with 3 replicates) and B. Impact of 6BrCaQ-C₁₀-TPP on the metabolic activity at 72 h on 3 cell lines (MDA-MB-231, MCF-7 and BT474), n = 3 (3 independent experiments with 4 replicates). C. MDA-MB-231 CFSE staining for 1, 2 or 3 days of treatment, n = 3 (3 independent experiments with 3 replicates). D. Apoptosis induction was determined by 7-AAD and Annexin V staining on the MDA-MB-231 cell line. Staurosporine was used as positive control of apoptosis induction.</figcaption></figure>
## 3.2. Inhibition of TRAP1 influences the epithelial-mesenchymal transition

Some breast cancers are highly metastatic and the development of metastasis is due to a cell transition from an epithelial to a mesenchymal phenotype accompanied with metabolic adaptations. These properties confer a resistant phenotype to chemotherapy in tumor cells.

As 6BrCaQ-C₁₀-TPP acts more as an anti-proliferative drug than a cell death inducer, we wondered if the resistance to cell death would be due to a secondary mechanism involving epithelial-mesenchymal transition (EMT). In ovarian cancer patients, TRAP1 expression has been shown to decrease in tumor metastasis in correlation with a reduced level of epithelial marker E-Cadherin [20]. In this case, the 6BrCaQ-C₁₀-TPP could modulate the markers of EMT in the two most sensitive cell lines: MDA-MB-231 and MCF-7. Whereas MDA-MB-231 is highly migratory and minimally invasive, MCF-7 is neither migratory nor invasive [21]. As our hypothesis is that C-terminal inhibitor of TRAP1 has an impact on ATPase activity, for comparison, we knocked down TRAP1 after 48 h of treatment with a small interfering RNA (siTRAP1) and observed a substantial reduction of TRAP1 protein without touching HSP90 level (see supplementary data).

In MDA-MB-231 cells, E-cadherin and Occludin expressions (epithelial markers) are decreased by both silencing TRAP1 with siTRAP1 and its inhibition by 6BrCaQ-C₁₀-TPP and only E-cadherin is decreased in MCF-7 with both treatments (Fig. 3A&B). Paradoxically, the Snail transcription factor (mesenchymal marker) is also diminished as ZEB1 in MDA-MB-231 cells but not in MCF-7 (Fig. 3C). Thus, both 6BrCaQ-C₁₀-TPP and siTRAP1 treatment do not affect the tumor cell lines in a same way affecting the more invasive and migratory cell line MDA-MB-231. But the response is partial as E-Cadherin/Occludin proteins are downregulated and Snail/ZEB1 transcripts are downregulated. Moreover, they do not affect the non-malignant breast cell line MCF10A, not even on E-Cadherin protein level (Figure S3A). These results indicate that both siTRAP1 and TRAP1 inhibitors induce partially a negative modulation of EMT, especially in the aggressive lineage. In addition, the effect of inhibition or knockdown of TRAP1 on EMT are comparable.

Cell migration, a part of invasive properties of cancer cells, was also differently affected by siRNA treatment and pharmacological inhibition. In both MDA-MB-231 and MCF-7 cells, the migration was increased by siTRAP1. But 6BrCaQ-C₁₀-TPP has only an effect on MCF-7 cells, in opposition to the knockdown of TRAP1 (Fig. 3D). Migration of MDA-MB-231 is not affected by 6BrCaQ-C₁₀-TPP. In MCF10A, siTRAP1, but not TRAP1 inhibitor, decreased the migratory ability of the cell line (Figure S3C). We did not evaluate the cell invasion capacity after TRAP1 downregulation, but these results suggest that TRAP1 may have a different role in non-malignant cells compared in breast cancer cells and depending on the described aggressiveness of cell lines.

## 3.3. 6BrCaQ-C₁₀-TPP influences the HSP90/TRAP1 machinery and partner proteins

To characterize phenotypically the inhibition of TRAP1, the expression level of TRAP1 partner proteins was analyzed by Western Blot after a 24 h-treatment by 6BrCaQ-C₁₀-TPP, on 3 breast cancer lines (MCF-7, MDA-MB-231 and BT-474) (Fig. 4). SDH-B is showing a sharp decrease for all 3 cell lines (up to 50 %), as well as SDH-A to a lesser extent. Similarly, to explore the Heat Shock Response, Heat-shock proteins and their transcription factor HSF-1 were studied. The level of expression of HSP70 decreases slightly for the 3 cell lines (around 20–30 % of decrease depending on the experiment) while the level of expression of HSP90 is stable. The level of expression of HSP27 remains stable for the MDA-MB-231 cell line but appears to increase slightly for the MCF7 cell line. Concerning HSF-1, its expression is reduced in both MDA-MB-231 and MCF-7 lines (up to 50 %). These results show that, overall, no heat-shock response is induced for these cell lines by 6BrCaQ-C₁₀-TPP whereas partner proteins of TRAP1 are affected by its modulation. TRAP1 expression level is not affected by the TRAP1 inhibitor

treatment whatever the cell line is as well as HSP90 (Supplementary data, Figure S7).

## 3.4. 6BrCaQ-C₁₀-TPP affects reactive oxygen species production capacity and mitochondrial functions

As 6BrCaQ-C₁₀-TPP was designed to target the mitochondria, we focused the next experiments on mitochondrial functions, ROS production capacity, and membrane potential. The network of mitochondria and relocation of proteins were also characterized.

### 3.4.1. ROS production

As TRAP1 is known to protect against reactive oxygen species (ROS), ROS production was also measured in MDA-MB-231 cell line, the most aggressive, through detection of oxidized dihydrorhodamine-123 (DHR) labeled cells. PMA was used as an inducer of ROS production (positive control). ROS production increases over time, and for higher concentrations of 6BrCaQ-C₁₀-TPP (Fig. 5). At 24 h, the maximum of the production of ROS was probably reached for each concentration of inhibitor because fluorescent did not increase with time. Level of ROS are higher than the PMA-induced ROS. Then, TRAP1 inhibition removes the protective effect of the protein against ROS.

### 3.4.2. Mitochondria targeting of 6BrCaQ-C₁₀-TPP and effect on mitochondrial proteins

After assessing the sensitivity of the cell lines to 6BrCaQ-C₁₀-TPP and its antiproliferative effect, the targeting of mitochondria had to be confirmed as well as its effect. Mitochondrial membrane potential is a good parameter to validate mitochondrial targeting. Indeed, it is essential for the process of energy storage and for accumulation of lipophilic cations as 6BrCaQ-C₁₀-TPP, into the mitochondria matrix. Mitochondrial membrane potential is a good indicator for healthy mitochondria and viable cells. Several breast-cancer cell lines (MDA-MB-231, MCF-7 and BT474) were first, seeded in 96-wells plate, treated by 6BrCaQ-C₁₀-TPP and then stained with TMRE. A drop of fluorescence, according to the disturbance of the mitochondria, is happening as soon as 2 h, at 0.1 µM for MCF-7 (Fig. 6A). It is significant for all cell lines at 6 h. The strength of the depolarization depends on the concentration of 6BrCaQ-C₁₀-TPP. These results validated the effect of 6BrCaQ-C₁₀-TPP on the mitochondria on breast cancer cell lines.

However, the TMRE staining does not take into account the possible loss of mitochondrial mass which would also result in a decrease of the fluorescence of TMRE due to cell mortality, for example. An MTT test, conducted in parallel, confirmed that there was no excess mortality for the times and concentrations of 6BrCaQ-C₁₀-TPP used. To complete these results, JC-1 staining was realized in the same cell lines, in flow cytometry, as a marker of mitochondrial membrane potential. The ratio of green/red fluorescence then makes it possible to determine the state of depolarization of mitochondria considering the mitochondrial mass. Mitochondria are depolarized to nearly 50 % from 3 h to 0.1 µM of 6BrCaQ-C₁₀-TPP and this increases with concentration and time for both cell lines (Fig. 6B). It is also noted that regardless of the staining used, mitochondria are more depolarized by the TRAP1 inhibitor (from 0.1 µM, 3 h), than by CCCP treatment (50 µM). These results obtained are consistent with the labeling of mitochondria by TMRE and therefore confirm the disruption of mitochondrial membrane potential induced by 6BrCaQ-C₁₀-TPP. These results are consistent with the results on colorectal cancer cell lines HT-29, previously published [10].

### 3.4.3. Impact of 6BrCaQ-C₁₀-TPP on mitochondrial network morphology

Mitochondria are organized in a network that can be filamentous, fragmented, or intermediate. Indeed, this mitochondrial network is dynamic and governed by fusion/fission equilibrium. To complete our study of the impact of 6BrCaQ-C₁₀-TPP on mitochondria, the morphology of the mitochondrial network was observed by confocal microscopy on MCF-7 and MDA-MB-231 breast cancer cells. The TOM-
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Epithelium-Mesenchymal transition markers and migration ability upon 48 h of siTRAP1 treatment and 24 h of 6BrCaQ-C₁₀-TPP. A and B, Western blotting of E-Cadherin and Occludin in MDA-MB-231 and MCF-7 cells, left and right panel respectively (For molecular weight, see Table 1). C. mRNA expression of the transcription factors Snail and ZEB1 (RT-qPCR) in tumor cells (MCF-7 and MDA-MB-231) treated with siRNA or 6BrCaQ-C₁₀-TPP. D. Wound healing assay for MDA-MB-231 and MCF-7 cells treated with anti-TRAP1 siRNA or 6BrCaQ-C₁₀-TPP. For Western Blot and qRT-PCR, each point was obtained in duplicates, and the wound healing assay was in triplicates.</figcaption></figure>
A. BT474

<figure><img src="image_6.png" /></figure>

B. MDA-MB-231

<figure><img src="image_7.png" /></figure>

MDA-MB-231

<figure><img src="image_8.png" /></figure>

C. MCF7

<figure><img src="image_9.png" /></figure>

MCF7

<figure><img src="image_10.png" /></figure>

**Fig. 4.** Influence of 6BrCaQ-C₁₀-TPP on HSP90 machinery (partners proteins and Heat-Shock proteins) analysed by Western Blot) after 24 h treatment (0.01–1 µM). n = 2–3 (depending on the cells). A. BT474, B. MDA-MB-231, C. MCF-7. Expression levels were normalized with the “Stain free” signal, representing accurately the protein loading (For molecular weight of proteins, see Table 1).
<figure><img src="image_11.png" /><figcaption>**Fig. 5.** ROS production induced by 6BrCaQ-C₁₀-TPP in MDA-MB-231 cells. Oxidized Rhodamine 123 was analyzed by flow cytometry after 24, 48 and 72 h for 0.1–0.5 µM. n = 2 (2 independent experiments). A, representative cytograms of flow cytometry; B, Comparison of mean fluorescence intensity according to time and dose of treatment.</figcaption></figure>

20 protein (Translocase of Outer Membrane) of the mitochondrial outer membrane was detected on fixed and permeabilized cells and the nuclei were labeled with DAPI. In these images (Fig. 6C and supplementary data Figure S4), there is a change in the morphology of the mitochondrial network under the effect of 6BrCaQ-C₁₀-TPP. Mitochondria take the form of hollow disks, which can be called "donuts" (see on Figure S4). These new forms appear from 3 h (0.1 µM), but are more numerous and more marked at 6 h (0.5 µM). Untreated cells have a filamentous network, as expected for healthy MDA-MB-231 cells. The appearance of "donuts" has also been confirmed on the MCF-7 cell line (data not shown).

### 3.4.4. Transfer of proteins between cytosol and mitochondria

Inhibition of TRAP1 may be accompanied by a change in the distribution of certain proteins between cytosol and mitochondria (Fig. 6D). Indeed, TRAP1 has been shown to control the apoptotic mechanisms through the regulation of the mitochondrial transition permeability pore (mPTP) [22]. Cell fractionation was carried out to observe these possible relocations. The cytosolic fraction was separated from the mitochondrial one by a series of differential centrifugations, and each fraction was then analyzed by Western blot. Tubulin and TOM20 were used as markers of each compartment to validate the fractionation quality, as both proteins are not known to be partners of TRAP1. As cytochrome c and mPTP are both protein partners of TRAP1, the repartition of cytochrome c between cytosol and mitochondria was blotted. The immunoblotting results obtained on the MDA-MB-231 cell line (Fig. 6E) show a decrease in cytosolic and mitochondrial HSP70 (25 %), a decrease in cytosolic HSP90, in mitochondrial TRAP1, and appearance of TRAP1 in cytosolic sample. Finally, a minor cytochrome c release into the cytosol is observed. Then,

treatment with 6BrCaQ-C₁₀-TPP might induce a relocation of proteins from mitochondria to cytosol, confirming mitochondria disturbance.

### 3.5. Modulation of TRAP1 by 6BrCaQ-C₁₀-TPP has an impact on cell metabolism

To go further, we performed metabolism-based experiments to evaluate the role of inhibition of TRAP1 in tumor metabolism. Succinate dehydrogenase (SDH) activity, Oxygen consumption rate (OCR), and glycolytic activity have been characterized at short term (30 min or 1 h and 3 h) of treatment to avoid modulation of protein expression levels at longer treatment times.

#### 3.5.1. SDH activity upon 6BrCaQ-C₁₀-TPP treatment is affected at short time

TRAP1 plays a crucial role in the activity of SDH by stabilizing the levels of the proteins SDH-A and B (subunits of SDH) and by interacting with and inhibiting activity SDH-A. The effect of TRAP1 inhibition was observed after treating MDA-MB-231 cells for a short time (30 min) to avoid the degradation of SDH proteins. For this purpose, the reduction of DiChlorophenolIndoPhenol (DCIP) by FADH2 during the oxidation of succinate to fumarate was monitored by absorbance, as the oxidized form of DCIP is blue, with maximum absorption at 600 nm, while its reduced form is colorless. The activity of SDH is expressed in units of enzymatic activity (U) normalized by the reaction volume, i.e., in the number of µmol of DCIP reduced per minute per microliter. 17-AAG serves as a positive control of the inhibitory action of TRAP1 on SDH. Indeed Xie et al. [23] showed that 17-AAG was partly localized in the
<figure><img src="image_12.png" /><figcaption>**Fig. 6.** Mitochondrial membrane potential dissipation and relocation of proteins from mitochondria to cytosol. A. The Mean fluorescence intensity of TMRE staining on the 3 cell lines (MDA-MB-231, MCF-7 and BT474) treated by 6BrCaQ-C₁₀-TPP, n = 3. B. Ratio of the both fluorescence peaks of JC-1 staining on MCF-7 and MDA-MB-231, n = 3. C. Morphology of the mitochondrial network of MDA-MB-231 cells analyzed by STED confocal microscopy. n = 2. D. Principle of the theoretical molecular mechanism of protein transfer from mitochondria to cytosol applied to cytochrome c. E. Immunoblots of HSP70, HSP90, TOM20, Tubulin and</figcaption></figure>
Cytochrome c from cytosolic and mitochondrial samples of MDA-MB-231. n = 2. F. Normalization of blot results. Panels are representative of two independent experiments.

mitochondria by complexing with theVDAC protein, thanks to its ben-
zoquinone function. The effect of TRAP1 inhibition by 6BrCaQ-C₁₀-TPP
on SDH activity shown in Fig. 7. This activity tends to increase with the
concentration of 6BrCaQ-C₁₀-TPP up to 1 µM, then stabilizes at 2 µM
(the control is at about 15 µU/µL, and rises to 25 µU/µL for 1 and 2 µM,
an increase of a factor of 1.7). The loss of inhibition of SDH activity by
TRAP1 is close to the one induced by treatment with 17-AAG (2 µM).

3.5.2. *TRAP1 modulation alters energetic metabolism of breast cancer cells*

Since -i) TRAP1 is known to control several mitochondrial metabolic
processes like OXPHOS (through interaction with SDH, complex IV) and
-ii) cancer cells present metabolic alterations compared to normal cells,
the respiratory and mitochondrial capacities of cells treated with
6BrCaQ-C₁₀-TPP were further analyzed using Agilent's Seahorse® XFe96
Analyzer, which measures in a real-time manner both the extracellular
pH and the oxygen consumption and offers several assays for live-cell
assessment of bioenergetic parameters such as glycolysis and mito-
chondrial stress. The mitochondrial stress® assay includes three in-
hibitors of the respiratory chain (Oligomycin/FCCP/Rot-AA) and was
used for the 3 breast cancer cell lines (MDA-MB-231, MCF-7 and BT474)
with different metabolic properties, for 5 different concentrations
(0.005 µM; 0.01 µM; 0.05 µM; 0.1 µM and 0.5 µM) of 6BrCaQ-C₁₀-TPP
and 2 incubation times (1 h and 3 h). Short times of treatment are
important to eliminate the potential consequence of apoptosis induction
which can occur at 24 h on the respiratory capacity, as shown earlier.
The Seahorse® medium contains specific substrates essential for the
study of cell metabolism to estimate the maximum respiratory response:
pyruvate (1 mM), glutamine (2 mM), and glucose (10 mM) (Figure S5).
All experiments were standardized by protein titration (BCA, Bicincho-
ninic acid assay) or automated cell counting (Hoechst labeling). Ac-
cording to these normalizations, no cytotoxicity was observed after
6BrCaQ-C₁₀-TPP treatment at these concentrations and times (1 and
3 h).

First, we chose 3 cell lines with different basal levels of OCR, BT-474
having the highest basal respiration. Accordingly, the production of ATP
in the absence of any treatment was higher for BT474 than for MDA-MB-
231 (respectively 69 and 29 pmol/min/1000 cells), as shown in Fig. 8A-
C. MCF-7 has an intermediate OCR. The 6BrCaQ-C₁₀-TPP induces a
decrease in basal respiration of MDA-MB-231 and MCF-7 cells, which
depends on treatment duration and concentration (decrease between
30 % and 50 %), while there is no significant variation in basal respi-
ration for BT474 induced by the treatment. The spare respiratory ca-
pacity (supplementary data, Figure S6), tends to decrease upon
treatment with 6BrCaQ-C₁₀-TPP for the two cell lines MDA-MB-231 and
BT474 (not statistically significant) and is between 20 % and 30 % at
0.5 µM and 3 h of treatment (effect depending on time and concentra-
tion). The results on MCF-7 were not statistically significant. In addition,
ATP production is strongly reduced in MDA-MB-231 and MCF-7 when

cells are treated with 6BrCaQ-C₁₀-TPP (80–85 % decrease for 3 h of
treatment at 0.5 µM). This decrease is lower for BT474 (30 % after 3 h).
Moreover, reduction of ATP production was dependent on time and drug
concentrations in all cases. Our results indicates that the cell line that is
the less influenced is the most respiratory, BT474. Finally, the proton
leak is increased for the three cell lines (supplementary data, Figure S6
about 150–300 %). This parameter is related to the proton gradient on
either side of the mitochondrial inner membrane; it is independent of
ATP synthase activity but depends on membrane potential, modified in
the presence of 6BrCaQ-C₁₀-TPP.

In addition, while monitoring OCR in cells, it was possible to simultaneously observe extracellular acidification levels (ECAR *Extra-Cellular Acidity Rate*) as an indirect indicator of glycolysis. Indeed, this value is correlated with lactate efflux, and the concentration of lactate increases with the implementation of anaerobic glycolysis, often present in cancer cells (Warburg effect), especially under relative hypoxia conditions. The ratio between OCR and ECAR makes determining the metabolic profile of cells makes possible and allows the detection of metabolic reprogramming induced by the treatment. Cells with high OCR and ECAR produce a lot of ATP and use both OXPHOS and glycolysis pathways. Conversely, cells are quiescent with a slow metabolism if both parameters are low. When OCR is high but ECAR is low, cells mainly use the mitochondrial respiratory chain, while when OCR is low, but ECAR is high, they mainly use glycolysis (and lactate production). Thus, Fig. 8D shows OCR as a function of ECAR for cells treated or not treated with 6BrCaQ-C₁₀-TPP. The results indicate that while MDA-MB-231 and MCF-7 cells are shifting from aerobic to rather more glycolytic metabolism, BT474 cells are less metabolically affected by TRAP1 inhibition becoming more energetic. It also confirmed the difference between the basal metabolism of the cell lines.

The results obtained on the three cell lines are shown in Fig. 9.
Without any treatment, the 3 cell lines exhibit different metabolic be-
haviors. BT474 cells would rely more on the Krebs cycle than the other 2
cell lines: the %PER induced by glycolysis is only 31 ± 20 % for BT474
while it is 86 ± 5 % and 81 ± 10 % for MDA-MB-231 and MCF-7,
respectively.

When cells are treated, basal glycolysis increases for MDA-MB-231 and BT474 lines as a function of increasing 6BrCaQ-C₁₀-TPP concentrations. The variations in basal glycolysis for MCF-7 are not significant for all concentrations; however, there is a clear increase for the 0.5 µM concentration regardless of the treatment time (1 h or 3 h) (supplementary data Figure S6). Regardless of the lineage, variations in compensatory glycolysis are not significant. In addition, the treatment induces an increase in the %PER from glycolysis in BT474 cells (from 32 % to 48 %), and in MDA cells (from 86 % to 96 %). The variations for MCF-7 do not show any significant trends.

In conclusion, inhibition of TRAP1 by 6BrCaQ-C₁₀-TPP has a meta-
bolic impact depending on breast cancer cell type based on the type of

<figure><img src="image_13.png" /><figcaption>**Fig. 7.** Succinate dehydrogenase (SDH) activity in MDA-MB-231. A. Interaction of TRAP1 with the subunit A of SDH. B. SDH activity after 30 min of treatment by 6BrCaQ-C₁₀-TPP (0.1 µM; 0.5 µM; 1 µM; 2 µM) and 17-AAG as control (2 µM).</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Fig. 8.** Evaluation of the mitochondrial stress by Seahorse® technology and switch of metabolism, n = 9. A, B, C, Left panel: OCR according to time, Right panel: characteristics values of mitochondrial respiration, Basal respiration and ATP-linked respiration. A. MDA-MB-231. B. MCF-7. C. BT-474. D. Metabolic phenotype determination of non-treated cells, and treated cells (0.5 µM at 1 h and 3 h). Left: MDA-MB-231, middle: BT474, right: MCF-7.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Fig. 9.** Evaluation of glycolytic rate by Seahorse® Technology, n = 9. A. Map of the experiment. B, C, D. Proton Efflux Rate (PER) according to time. B. MCF-7. C. BT474. D. MDA-MB-231. GlycoPER was calculated by subtraction.</figcaption></figure>

basal metabolism more glycolytic or aerobic, as shown in Fig. 9. This correlates with Matassa *et al.*, who showed different roles of TRAP1 according to their metabolism (Glycolytic or aerobic) [11].

## 4. Discussion

Based on our first results obtained for the HSP90 C-terminal domain inhibitor, 6BrCaQ – which has an anti-tumor activity [7,8], chemical modulation of 6BrCaQ was focused on mitochondrial targeting to explore the metabolic field of the breast cancer biology and the role of modulation of HSP90 chaperone family on it [10]. This work led to the synthesis of 6BrCaQ-C₁₀-TPP, a amphiphilic compound (logP 8 and displaying self-assembly properties) whose inhibitory capacity and anti-tumor activity have already been validated on HT-29 colon cancer cell line [10]. The docking of 6BrCaQ-C₁₀-TPP has been modelized by Kang et al. in 2022 based on our work but we failed to show the interaction with TRAP1. Indeed, as 6BrCaQ-C₁₀-TPP is not water soluble, it makes self-assembly in aqueous solution that prevent the analysis of protein-inhibitor interaction at high concentrations of drug that is needed to obtain the sufficient signal [10,24]. However, for *in vitro* studies, the molecule is diluted from a stock solution in DMSO into the cell culture medium containing protein-rich serum to obtain final concentration. The entry of 6BrCaQ-C₁₀-TPP into the cells is at that moment facilitated by the auto-organization of the molecule that interact with plasma membrane lipids. In the cytosol, 6BrCaQ-C₁₀-TPP is highly diluted in the complex biological environment, which facilitates its solubilization and therefore its access to its target. Once in the cytosol and after crossing freely the outer mitochondrial membrane (exclusion limit about 6 kDa) the positive charge allows the passage of inner mitochondrial membrane, according to its potential ΔΨm.

This new study focused on the effect of 6BrCaQ-C₁₀-TPP on breast cancer lines, more specifically on their proliferation and metabolic phenotype. Three cell lines was chosen to represent different tumoral

properties, BT474, MDA-MB-231 and MCF-7. First, the cytotoxicity was assessed on those cancer cell lines to confirm rationale of our study. The sensitivity of the breast cancer cells is the same as that of the colorectal cancer cell line HT-29, shown in our previous work [10]. However, as shown by Trypan blue counting or 7-AAD/Annexin staining, the cytotoxic mechanism of action of the compound does not rely on the induction of apoptosis but rather on the inhibition of cell proliferation, as confirmed by CFSE staining. We confirmed the preferential effect of 6BrCaQ-C₁₀-TPP on proliferation using several techniques with different measurement principles. In addition, this blockade in cell cycle induced by HSP90 inhibitors is well established by us and in the literature. Furthermore, as we explored the impact of the TRAP1 inhibitor on markers of the EMT to predict its impact on migration and invasion, we observed that epithelial markers (at the protein level) are reduced (E-Cadherin and Occludin). In contrast, EMT-related transcription factors expressions (at the gene level) are affected especially in MDA-MB-231 cells. On MCF-7, 6BrCaQ-C₁₀-TPP decreased the cell migration, as observed with 6BrCaQ (encapsulated in liposomes) in prostate cancer PC-3 cells [9]. We assume that 6BrCaQ-C₁₀-TPP induces a partial effect on pro-invasive properties of tumor cells at the time and dose points that we chose. One of our hypotheses is that the treatment times are too short to visualize the impact of EMT at the protein level. The increase in these proteins may be a response to mitochondrial stress or compensatory pathways (e.g., Wnt/beta catenin or NFkappa B) [25]. The comparison with knockdown of TRAP1 by siRNA indicates that the impact is quite the same. Inhibition of TRAP1 by 6BrCaQ-C₁₀-TPP in its C-terminal domain may stabilize the protein avoiding correct chaperoning activity and inducing degradation of partners. Those results confirmed that the role of TRAP1 is complex in cancer cells and the complete understanding must be based on a knock-out model to eliminate TRAP1.

In order to confirm the mitochondrial targeting of 6BrCaQ-C₁₀-TPP, the study focused next on the evaluation of the mitochondrial network.
Mitochondrial depolarization induced by 6BrCaQ-C₁₀-TPP and, therefore, by mitochondrial targeting was confirmed to occur after 3 h and identified by two markers with distinct mechanisms (TMRE and JC-1). JC-1 confirmed that depolarization was not due to mitochondrial mass loss. After 3 h of treatments, some mitochondria network in the form of "donuts" appeared for the two cell lines MDA-MB-231 and MCF-7. These characteristic shapes of mitochondria have already been observed during treatment with mitochondrial uncouplers (FCCP, CCCP) [26]. The formation of "donuts" can be triggered either by the depolarization of mitochondria or by the opening of the mPTP pore. But this hypothesis is not completely supported since our molecule does not induce an increase in OCR equivalent to that observed with FCCP in Seahorse® experiments. TRAP1 can also influence the morphology of the mitochondrial network (tubular, fragmented, or intermediate) by regulating the expression and activity of Drp1 (dynamin-related protein 1) and Mff (mitochondrial fission factor), mitochondrial fission proteins. TRAP1 overexpression or silencing has different effects depending on its endogenous level – low for MDA-MB-231 and high for MCF-7 [12]. In both cases, a low level of TRAP1 correlated with a rod-shaped mitochondrial network, whereas a high level of TRAP1 correlated with a tubular-shaped mitochondrial network. The effect of 6BrCaQ-C₁₀-TPP treatment can be the result of two mechanisms, the first related to the TPP moiety which can disturb mitochondria trough the positive charge and the second based on the direct modulation of TRAP1 functions which are related directly to mechanisms in charge of the maintenance of membrane potential. However, the TPP moiety could have another effect, as proposed in two recent literature reviews that suggests a mechanism of inhibition of TRAP1 involving both the interaction of the 6BrCaQ moiety with the C-terminal domain and the interaction of the TPP moiety with the middle domain of TRAP1 [27,28].

TRAP1 binds both complex II and complex IV of the mitochondrial respiratory chain. It binds to SDH-A for complex II, cytochrome c for complex IV, and to the mPTP pore, allowing cytochrome c to be released from mitochondria to cytosol [29]. In our hands, a decrease in the expression levels of the subunits of succinate dehydrogenase, SDH-A, and SDH-B, is observed during treatment with 6BrCaQ-C₁₀-TPP on MCF-7, MDA-MB-231 and BT474 cells. Furthermore, treatment does not induce a Heat Shock Response in these cell lines, consistent with inhibition of the C-terminal domain and the effect on 6BrCaQ-C₁₀-TPP on HT-29 cell line (colorectal cancer) [10]. Subcellular cell fractionation of breast cancer cells highlighted that inhibition of TRAP1 by 6BrCaQ-C₁₀-TPP induced a cytochrome c discharge from the mitochondrial membrane to the cytosol. Indeed, the mechanism of cytochrome c release involves two client proteins of TRAP1: cytochrome c itself and the membrane permeability transition pore (mPTP). Pridgeon et al. showed that PINK1 induced the phosphorylation of TRAP1, preventing cytochrome c release [30]. TRAP1, therefore, protects cells from apoptosis by blocking this signaling pathway. As stated above, the inhibition of TRAP1 at its C-terminal domain and further release of cytochrome c, follow the opening of the mPTP pore. The transient opening of mPTP is consistent with the "donuts" form of mitochondria [31,32]. However, only slight apoptosis has been observed in cells, contrary to what should be observed with mPTP opening. However, Gamitrinib, an N-terminal domain inhibitor targeting the mitochondria, also induce a discharge of cytochrome c into the cytosol but not 17-AAG (without TPP moiety) despite its mitochondrial location. Kang et al. made the hypothesis that mitochondrial depolarization is sufficient to induce the release of cytochrome c, which our results can confirm [33,34]. In addition, the limited release of cytochrome c that we observed could be correlated to the limited induction of apoptosis after 24 h of treatment. Combined with the ROS production which appears to reach a maximum at 24 h, we can propose that the morphology of the mitochondria ("donuts") is not too much deleterious to induce cell death but sufficient to disturb cell metabolism in cell with low aerobic metabolism. Our hypothesis is supported by the computational study published in 2013 that demonstrate that the donut morphology may be an early marker of

cell stress that would be reversible and not correlated to cytotoxicity [35].

The 6BrCaQ-C₁₀-TPP treatment leads to an increase in SDH activity at early time point (30 min). This increase is close to the one for the NTD inhibitor, 17-AAG. Sanchez et al. also measured the influence of several TRAP1 allosteric inhibitors on SDH activity [28]. These inhibitors are able at 100 µM to have the same SDH activity as when TRAP1 is knocked out by single guide RNA with CRISPR/Cas9 technology. 17-AAG induces also an increase in SDH activity for a lower concentration (10 µM). Although concentrations of 6BrCaQ-C₁₀-TPP used in our study are much lower, the increase in SDH activity is only found up to 1 µM but with a similar efficiency as 17-AAG at the same concentration.

The involvement of TRAP1 in metabolism control, mainly via the Krebs cycle, suggests that 6BrCaQ-C₁₀-TPP could impact this metabolism in breast cancer cells. As TRAP1 contributes to the switch toward aerobic glycolysis, combining decreased of OXPHOS activity and enhanced glucose utilization [36] we investigated glycolytic rates and mitochondrial respiration levels through a real-time metabolism measurement technique (Seahorse®). The basal respiration of each cell line (MDA-MB-231, MCF-7, and BT474), as well as the percentage of PER from glycolysis, is consistent with the Agilent® database: 86 ± 5 %, 81 ± 10 %, 31 ± 20 % in our data respectively for MDA-MB-231, MCF-7 and BT474, nearly 85, 76 and 46 % in the Agilent® database. In addition, Winnike et al. have shown that ATP production due to the TCA cycle is significant for BT474 cells [33]. They explored the metabolism of different cell lines according to their tissue type (TBNC, Luminal ...), including MDA-MB-231, MCF-7, and BT474. They compared the concentrations of different substrates of glycolysis and the TCA (Krebs) cycle by NMR of carbon 13. Thus, BT474 has a high production of alanine and a low production of lactate, which indicates a high use of the Krebs cycle. Conversely, the MCF-7 line uses the most glucose and produces the most lactate, which is consistent with the Warburg effect. Curiously, the metabolism of MDA-MB-231 cells is close to BT474 with a strong catabolism of glutamine. Their data are consistent with our results for MCF-7 and BT474 lines, although according to our data MDA-MB-231 cells also rely heavily on glycolysis. To go further, the basal proton production of BT474 was shown to be greater than for MDA-MB-231 cells. This phenomenon correlates with a greater activity of the TCA cycle. It should be noted that the comparison of metabolism studies should also consider the culture media in which the cells are grown. For example, MDA-MB-231 cells are grown in our laboratory in L15 medium containing galactose while very often they are grown in glucose-rich DMEM.

Curiously, treatment with 6BrCaQ-C₁₀-TPP induced a change in metabolic phenotype for MCF-7 and MDA-MB-231. However, this may indicate that cells would become more sensitive to chemotherapy. Indeed, one study showed that cell lines from ovarian cancers highly resistant to chemotherapy (C200 and PEO4) had a very active metabolism (both glycolytic and OXPHOS), while their chemotherapy-sensitive counterparts had only glycolytic metabolism [37]. Furthermore, on cells that combined both metabolisms, 6BrCaQ-C₁₀-TPP induces a shift to glycolytic pathway can be an advantage because some mitochondrial metabolic pathways are impeded like fatty acid oxidation, aminoacid catabolism hampering the replenishment of the stock of metabolic intermediates for cancer cell growth.

Treatment with 6BrCaQ-C₁₀-TPP also increased proton leakage for the 3 cell lines, consistent with the mitochondrial potential that decreases shown by labeling with TMRE and JC-1 at short time of treatment (3 h and 6 h).

Pfleger et al. have shown that the isoforms of pyruvate dehydrogenase kinase PDK regulate reserve respiratory capacity. During hypoxia, hypoxia-inducible factor 1-alpha (HIF-1 α) induces an increase in PDK1, inhibiting glucose oxidation. SDH is the only enzyme in the Krebs cycle that is also a component of Complex II of the electron transport chain, so it is well-placed as a checkpoint of reserve respiratory capacity. The authors were therefore interested in the role of complex II by studying its
gene inhibition and extinction. Inhibition of complex II by 3-Nitropro-
pionic acid (3-NP) completely abolished the reserve respiratory capac-
ity of cells, in addition to a slight decrease in basal OCR (10–18 %).
Reserve respiratory capacity has been shown to correlate with cell sur-
vival in various cell types. Thus, the decrease in basal respiration and
reserve respiratory capacity is compatible with the degradation of SDH,
and, therefore, with the inhibition of TRAP1 (as our results), or by the
extinction of the TRAP1 gene. Indeed, Zhang et al. have shown that
TRAP1 silencing leads to decreased respiration in basal OCR and reserve
respiratory capacity. The effect of Gamitrinibs on basal respiration is not
significant, however treatment with Gamitrinibs decreases ATP pro-
duction and alternative respiratory capacity (PC3-cells) [38].

To conclude, the metabolism of breast cancer cells is modified by 6BrCaQ-C₁₀-TPP treatment depending on cell phenotype. Overall, glycolysis is increased while OXPHOS decreases. Reserve capacities to produce ATP are diminished. This is consistent with less aggressive cell types and is sensitive to chemotherapy [37]. So, our treatment could reduce the invasiveness of the cells and sensitize them to chemotherapy in relation to the observed proliferation inhibition.

Our study gives some new information about the impact of inhibition of TRAP1 in the C-terminal domain on mitochondrial functions in breast cancer cells. Cell proliferation and mitochondrial function are affected, but without inducing apoptosis. We hypothesize that 6BrCaQ-C₁₀-TPP could have a partial effect at the time and concentrations tested. In addition, TRAP1 may have complex role in cancer cells depending on the partner protein it takes charge (oncogene or tumor suppressor). In some cancers, TRAP1 has been shown to be protective for tumor cells inducing chemoresistance, whereas in some others, it is sensitizing [11].

Finally, as the 6BrCaQ-C₁₀-TPP, as a majority of inhibitors is not selective of tumor cells and is not water-soluble, we are currently developing a liposomal formulation of our molecule to solubilize the molecule and accumulate in tumors (on-going) as we already showed with a former derivative for HSP90 inhibition [8]. The PEGylation of the liposomes will increase the circulation time of the molecule after intravenous administration by preventing the hepatic first passage.

5. Conclusion

The TRAP1 inhibitor, 6BrCaQ-C₁₀-TPP was evaluated on three breast cancer cell lines (Luminal and TBNC) displaying three different basal metabolisms. The study of the cellular mechanisms induced by the inhibitor has shown changes in the mitochondrial network, the rate of cell proliferation, and their metabolism. Despite their initial differences in metabolism, all three types of cells react *in vitro* similarly to treatment with 6BrCaQ-C₁₀-TPP. However, the results could be different in *in vivo* studies. We have shown that treatment can reduce reserve respiratory capacity and move from high metabolism to glycolytic metabolism. This could be a sign of possible sensitization of cells to chemotherapy. This metabolism is consistent with the inhibition of proliferation observed in this study.

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.

Acknowledgments

The authors acknowledge support for this project by CNRS, Univer-
sity Paris-Saclay, Ecole Normale Supérieure de Lyon (CM, CDSN) and
Ligue contre le cancer-Comité des Hauts de Seine (2019, 2020) Campus
France (JB, Eiffel scholarship). Authors would like to thank MIPSIT (Dr.
Valérie Nicolas), Frédéric Eghiaian from Abberior Instruments for
confocal images; Delphine Courilleau for her help to acquire fluorescent
data and Stéphanie Denis for her help with cell culture.

Statement of significance

This study helps to understand the role of modulation of TRAP1 in
breast cancer and validate its interest as a therapeutic target through
inhibition of its C-terminal domain.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2026.119060.

Data availability

Data will be made available on request.

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