Targeting hexokinase 2 to induce breast cancer cell senescence

Targeting hexokinase 2 to induce breast cancer cell senescence | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL British Journal of Pharmacology This is a preprint and has not been peer reviewed. Data may be preliminary. 25 February 2025 V1 Latest version Share on Targeting hexokinase 2 to induce breast cancer cell senescence Authors : Helmut Bischof [email protected] , Katarina Vizar-Cisarova , Sandra Burgstaller , Markus Absenger-Novak , Philipp Jost , Roland Malli , Wolfgang Graier 0000-0003-1871-3298 , and Robert Lukowski Authors Info & Affiliations https://doi.org/10.22541/au.174046901.14415288/v1 Published British Journal of Pharmacology Version of record Peer review timeline 545 views 221 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Hexokinase 2 (HK2) is a key enzyme linked to high tumor cell proliferation. Its inhibitors such as 3-bromopyruvic acid (3-BP) induce cancer cell death, highlighting HK2 modulation as potential anti-cancer treatment. However, standard chemotherapies often cause the emergence of cellular senescence, which goes along with cell metabolic reprogramming and treatment failure. This study explores whether targeting HK2 can induce cancer cell senescence and whether metabolic changes in senescent cancer cells are tied to the cellular HK2 status. Experimental Approach: The expression of hexokinase 1 (HK1) and HK2 was assessed using immunoblot and immunofluorescence analysis in cell lines and in primary murine breast cancer (BC) cells. The senescence inducing potential of HK2 inhibition, and the effect of chemotherapy-induced senescence on HK1 and HK2 expression was assessed. Cell-based approaches were complemented by analyzing single-cell RNA sequencing data from BC patients. Key Results: BC cell sensitivity to HK2 inhibition did not correlate with HK2 expression levels. Consistently, senescence was linked to a decrease in HK2, but an increase in HK1 expression. Moreover, genetic knockdown of HK2 induced senescence, indicating that a change in the HK2/HK1 ratio drives, rather than results from, cellular senescence. This shift in HK2/HK1 ratio was confirmed in single-cell RNA sequencing data of BC biopsies. Conclusion and Implications: Expressional shifts in the HK2/HK1 ratio may serve as a novel marker for BC cell senescence. While targeting HK2 shows promise in untreated cancers, senescence-inducing anti-cancer therapies may limit the effectiveness of HK2-targeted treatments in pre-treated cancer patients. Targeting hexokinase 2 to induce breast cancer cell senescence Helmut Bischof 1,2,3,4,* , Katarina Cisarova 2 , Sandra Burgstaller 5 , Markus Absenger-Novak 5 , Philipp J. Jost 2 , Roland Malli 4,5 , Wolfgang F. Graier 1,4 , Robert Lukowski 3 1 Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/IV EAST, 8010 Graz, Austria 2 Division of Oncology, Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria 3 Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany 4 BioTechMed Graz, Mozartgasse 12, 8010 Graz, Austria 5 Center for Medical Research, CF Bioimaging, Medical University of Graz, Neue Stiftingtalstraße 6/VI, 8010 Graz, Austria *Correspondence to [email protected] Word count (Introduction, Results, Discussion and Conclusion): 4791 Acknowledgements This work was funded by the Deutsche Forschungsgemeinschaft (DFG) with individual grants Lu1490/10-1 and Lu1490/12-1 to RL. RL is a member of the GRK2381: ”cGMP: From Bedside to Bench”, DFG grant number 335549539, and he acknowledges additional financial support from the ICEPHA Graduate Program “Membrane-associated Drug Targets in Personalized Cancer Medicine”. HB and WFG are grateful to the Austrian Science Fund (FWF) for the Erwin-Schrödinger-Program, project number J-4457, and for the excellence cluster 10.55776/COE14, respectively. Author contributions HB initiated the study. HB, KC and SB designed experiments. HB, KC and MAN performed experiments and were involved in data curation. HB, KC and SB assisted in formal analysis. HB, SB, RM, WFG and RL contributed materials and protocols. HB and RL acquired financial support. HB and RL performed project administration, supervised the project and the personnel. SB, RM, PJJ, WFG and RL provided resources and infrastructure. HB and RL wrote the original draft. All authors critically reviewed the manuscript and stated comments. Data availability Data generated and analyzed over the course of the current study are included within the main document of the study or in the supplementary material. Raw data for the main figure graphs can be found in source data files. Declaration of interests The authors declare no competing interests. Abstract: Background and Purpose: Hexokinase 2 (HK2) is a key enzyme linked to high tumor cell proliferation. Its inhibitors such as 3-bromopyruvic acid (3-BP) induce cancer cell death, highlighting HK2 modulation as potential anti-cancer treatment. However, standard chemotherapies often cause the emergence of cellular senescence, which goes along with cell metabolic reprogramming and treatment failure. This study explores whether targeting HK2 can induce cancer cell senescence and whether metabolic changes in senescent cancer cells are tied to the cellular HK2 status. Experimental Approach: The expression of hexokinase 1 (HK1) and HK2 was assessed using immunoblot and immunofluorescence analysis in cell lines and in primary murine breast cancer (BC) cells. The senescence inducing potential of HK2 inhibition, and the effect of chemotherapy-induced senescence on HK1 and HK2 expression was assessed. Cell-based approaches were complemented by analyzing single-cell RNA sequencing data from BC patients. Key Results: BC cell sensitivity to HK2 inhibition did not correlate with HK2 expression levels. Consistently, senescence was linked to a decrease in HK2, but an increase in HK1 expression. Moreover, genetic knockdown of HK2 induced senescence, indicating that a change in the HK2/HK1 ratio drives, rather than results from, cellular senescence. This shift in HK2/HK1 ratio was confirmed in single-cell RNA sequencing data of BC biopsies. Conclusion and Implications: E xpressional shifts in the HK2/HK1 ratio may serve as a novel marker for BC cell senescence. While targeting HK2 shows promise in untreated cancers, senescence-inducing anti-cancer therapies may limit the effectiveness of HK2-targeted treatments in pre-treated cancer patients. Keywords: Breast cancer, Hexokinase 1, Hexokinase 2, Metabolism , Senescence What is already known? Chemotherapeutic treatments are associated with the emergence of senescent cancer cells Cancer cell senescence is associated with treatment resistance and cell metabolic alterations What does this study add? Cancer cell senescence is associated with hexokinase 2 (HK2) downregulation and hexokinase 1 (HK1) upregulation Senescent cancer cells are resistant to hexokinase 2 inhibitors What is the clinical significance? The expressional ratio of HK2/HK1 correlates with senescence in breast cancer patients HK2/HK1 ratio may serve as an additional marker of cancer cell senescence and provides an estimate for therapy success Introduction: Cellular senescence is a well-described biological phenomenon characterized by stable cell cycle arrest and typically occurs in aging or consequently to DNA damage or other cellular stress factors (Chen et al., 2007; Di Micco et al., 2021). It is typically characterized by four independent hallmarks: i) durable and generally irreversible cell cycle arrest, ii) evolvement of a senescence-associated secretory phenotype (SASP), iii) macromolecular damage and iv) altered cell metabolic activities (Gorgoulis et al., 2019). In recent years, senescence has gained significant attention not only in ageing, but also in the context of cancer, as senescent cells are frequently observed within tumors (Wyld et al., 2020; Domen et al., 2022). Besides other anti-cancer treatments, chemotherapeutic anti-cancer interventions are frequently associated with the induction of cancer cell senescence, a process known as chemotherapy-induced senescence. Thereby, cancer cells can protect themselves against chemotherapeutic cytotoxicity, driving cancer cell resistance and tumor heterogeneity (Guillon et al., 2019). Despite senescent cells do not actively proliferate, they can contribute to tumor progression by the secretion of pro-inflammatory cytokines, growth factors or proteases (Dong et al., 2024). Alternatively, however, senescence of cancer cells may be a reversible as senescent cancer cells may reenter the cell cycle and subsequently again actively contribute to promoting tumor progression (Saleh et al., 2019). Therefore, understanding how cellular senescence influences cancer therapy and resistance mechanisms has become essential in the development of more effective treatment strategies. Usually, cancer cells show a high metabolic activity and a preference for glucose metabolism towards oxidative phosphorylation. One mediator of this phenomenon, also referred to as the Warburg effect, is hexokinase isoform 2 (HK2), a cancer-associated hexokinase isoform, catalyzing the first and rate-limiting step of glucose phosphorylation in glycolysis (Bischof et al., 2021). Besides HK2, hexokinase isoform 1 (HK1) represents the most abundant cellular hexokinase isoform. Both isoenzymes are associated with the outer mitochondrial membrane, where they interact with the voltage-dependent anion-selective channel 1 (VDAC1), albeit their cytosolic localization or even nuclear translocation has also been observed (Anflous-Pharayra et al., 2007; Thomas et al., 2022). While HK1 is ubiquitously expressed in almost all mammalian tissues, HK2 was described as the “cancer-associated” isoform (Bischof et al., 2021) and has therefore emerged as an interesting target for anti-cancer therapies. One promising compound acting on HK2 represents 3-bromopyruvic acid (3-BP), a HK2 inhibitor with potential for anti-tumor therapies (Pedersen, 2012). Targeting the cancer cell’s metabolism, however, may inadvertently lead to the emergence of senescent cancer cells that exhibit alterations in cellular morphology and impaired organelle functionality (Prasanna et al., 2021). Under these conditions, abnormal metabolic features may emerge, but deeper insights into expressional alterations of single proteins that contribute to the exit, entry or maintenance of senescence remain, however, largely enigmatic (Kim et al., 2024). Within this study, we aimed to explore the relationship between the expression of the key-glycolytic enzymes HK1 and HK2 in commonly used (senescent) breast cancer (BC) cell lines and primary murine BC cells. Furthermore, we evaluated the association of cancer cell senescence with altered expression levels of HK1 and HK2, and whether a combination of established chemotherapeutic agents with the HK2 inhibitor 3-bromopyruvic acid (3-BP) may represent a suitable anti-cancer strategy. Material and Methods Buffers and solutions If not otherwise stated, all chemicals were purchased from Carl Roth GmbH (Karlsruhe, Germany). Buffers and solutions used for Western blotting comprised the following: Cell lysis buffer: 6x Laemmli SDS sample buffer (reducing, Thermo Fisher Scientific, Frankfurt am Main, Germany) diluted to 2x concentration. Separation gel, 12.5%: 4.2 mL Rotiphorese Gel 30, 2.5 mL Tris 1.8 M pH = 8.8, 3.2 mL H 2 O, 100 µL 10% SDS, 20 µL TEMED, 67 µL 30% APS. Stacking gel, 5%: 830 µL Rotiphorese Gel 30, 500 µL Tris 0.6 M pH = 6.8, 3.5 mL H 2 O, 50 µL 10% SDS, 10 µL TEMED, 20 µL 30% APS. Anode 1 buffer: 36.3 g Tris, 200 mL methanol, ad 1 L with H 2 O, pH = 10.4. Anode 2 buffer: 3.6 g Tris, 200 mL methanol, ad 1 L with H 2 O, pH = 10.4. Cathode buffer: 3.0 g Tris, 5.8 g 6-aminocaproic acid, 200 mL methanol, ad 1 L with H 2 O, pH = 7.6. 1x TBS-T: 1.21 g Tris, 8,23 g NaCl, 0.5 mL Tween 20, ad 1 L with H 2 O, pH = 8.0. Blocking and antibody dilution buffer: TBS-T + 5% BSA. Buffers used for live-cell imaging comprised the following: Physiologic buffer: 138 mM NaCl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Glucose, pH = 7.4. 3-BP (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) and Benserazide (Sigma-Aldrich GmbH) were added at a final concentration of 100 µM or 200 µM, respectively. H 2 O 2 was added at a final concentration of 2.0 mM. Docetaxel and Capecitabine were purchased from Bio-Techne Ltd. (Abingdon, UK). Cell treatments 3-BP treatment during live-cell imaging was performed using a 3-BP concentration of 100 µM in physiologic buffer. Benserazide treatment was done at a concentration of 200 µM. For mitoHyPer7 saturation, 2.0 mM H 2 O 2 in physiologic buffer were administered to the cells. IC 50 curves were generated by cell treatment with the respective concentration of 3-BP or Benserazide for 7 days. In case of cotreatment with Docetaxel or Capecitabine, cells were treated for 24 hours using 50 nM of Docetaxel, followed by 3-BP treatments for 7 days, or treated with 50 µM (HEK293) or 100 µM (all others) Capecitabine simultaneously with different 3-BP concentrations for 7 days. Docetaxel, Capecitabine and H 2 O 2 treatment for SA-β-Gal activity staining and Western blot was performed as follows: Docetaxel treatment was performed 24 hours after cell seeding for 24 hours at 50 nM followed by incubation of cells for 10 days. Capecitabine treatment was performed 24 hours after cell seeding for 10 days at 50 µM (HEK293) or 100 µM (all other cell lines). H 2 O 2 treatment was performed 24 hours after cell seeding at a concentration of 1300 µM. 48 hours after treatment, medium was exchanged and H 2 O 2 was renewed, followed by incubation for futher 8 days. SA-β-Gal activity staining following siRNA treatment was performed as follows: Cells were transfected with siRNAs 24 hours after cell seeding, following incubation for 7 days. Cell culture and transfection Human BC cell lines including MCF-7 and MDA-MB-453 cells and HEK293 cells were purchased from ATCC: The Global Bioresource Center. Cells were cultured in Dulbecco’s modified eagle’s medium (DMEM, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS), 100 U mL -1 penicillin and 100 µg mL -1 streptomycin and 1 mM sodium pyruvate (Thermo Fisher Scientific). Mouse mammary tumor virus polyoma middle T antigen (MMTV-PyMT) cells were isolated from tumors of MMTV-PyMT transgenic FVB/N WT mice. Tumor growth in vivo and biopsies were authorized by the local ethics Committee for Animal Research (Regierungspräsidium Tübingen (PZ1/16, PZ2/17, PZ4/20 G), Germany), and were performed in accordance with the German Animal Welfare Act. Animals were kept on a 12 hr light/ dark cycle under temperature- and humidity-controlled conditions with unlimited access to food (Altromin, Lage, Germany) and water. MMTV-PyMT cells used in this study were isolated from three to seven different female breast-cancer bearing WT animals at an age of minced into pieces using atraumatic forceps, lysed by 1 mg mL–1 Collagenase-D (Roche, Basel, Switzerland) for 10 min, and cultured in modified improved minimal essential medium (IMEM) supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate and 100 U mL −1 penicillin and 100 µg mL −1 streptomycin (all purchased from Thermo Fisher Scientific) at 37 °C and 5% CO 2 in a humidified incubator. Fibroblasts were removed by exposure of the cultures to 0.25% trypsin-EDTA in PBS (Thermo Fisher Scientific) and short incubation at 37 °C (~1 min). After gently tapping the plate, trypsin-EDTA with detached fibroblasts was removed and cells were further cultured in supplemented modified IMEM at 37 °C and 5% CO 2 until subculturing. All cell lines were regularly tested negative for mycoplasma contamination using PCR analysis. Transfection of cells was performed either using PolyJet transfection reagent (SignaGen laboratories, Maryland, USA) for plasmid transfection or ScreenFect A-Plus (ScreenFect GmbH, Eggenstein-Leopoldshafen, Germany) for transfection of siRNAs according to manufacturer’s instructions. Plasmids, siRNAs and oligos Plasmids encoding genetically-encoded sensors used for live-cell imaging included the following: mtAT1.03 for measurements of [ATP] mito was a gift from Hiromi Imamura, HyPer7.2DAAO-mito was a gift from Thomas Michel (Addgene plasmid # 168304 ; http://n2t.net/addgene:168304 ; RRID:Addgene_168304). Plasmid preparation was performed using the HiSpeed plasmid midi kit (Qiagen GmbH, Hilden, Germany). siRNAs were purchased from Microsynth AG (Balgach, Switzerland) and showed the following sequences: siScrbl: 5’-UUCUCCGAACGUGUCACGUdTdT-3’, siHK1: 5’-GGAAGAUCAAGGACAAGAAdTdT-3’, siHK2 human: 5’-AGACAUCUCAGACAUUGAAdTdT-3’, siHK2 mouse: 5’-AGAUGUCUCGGAUAUUGAAdTdT-3’. Primers used for qPCR were purchased from Microsynth AG had the following sequences: Tub for: 5’-CCATGGTAAATACATGGCTTG-3’, Tub rev: 5’- CAACCTTGAAGCCAGTGG-3’, HK1 for: 5’-TGGTGGCTGTGGTCAACGACAC-3’, HK1 rev: 5’-TTCATCTCCTCCATGTAGCAGGC-3’, HK2m for: 5’- GATTGTGCGCAACATCCTGATCGA-3’, HK2m rev: 5’- TGTCTTGAGGCGCTCTGAGAT-3’, HK2h for: 5’-GATTGTCCGTAACATTCTCATCGA-3’, HK2h rev: 5’- TGTCTTGAGCCGCTCTGAGAT-3’. qPCR was performed using the GoTaq qPCR System (Promega GmbH, Walldorf, Germany) according to manufacturer’s instructions on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Western blotting For Western blot analysis, cells showing 100% confluency in a 10 cm dish were lysed using 6x Laemmli buffer (Thermo Fisher Scientific) diluted to a 2x concentration with H 2 O and heated to 95°C, followed by incubation at 95°C for 10 minutes. Subsequently, lysates were sonicated for 5 seconds for sample homogenization (Model FB120, Fisher Scientific). Protein concentration determination was performed using trichloroacetic acid (Carl Roth GmbH). Therefore, samples were diluted 1:7, and 10 µL of the samples were mixed with 90 µL of a 28 % trichloroacetic acid solution. A BSA standard curve served for calibration. Typically, 30 µg of protein were used per slot of the SDS-PAGE gel, which was assembled of a 12.5 % separation and 5 % stacking gel. Gel was transferred to an Immobilon-FL PVDF membrane (Merck KGaA, Darmstadt, Germany) using semi-dry transfer method. 5 % BSA in TBS-T was used for blocking. Primary antibodies included anti-Hexokinase 1 rabbit mAb (C35C4, 1:750), anti-Hexokinase 2 rabbit mAb (C64G5, 1:750) and anti-β-Tubulin rabbit mAb (9F3, 1:1.000), all purchased from Cell Signaling Technology, Leiden, Netherlands. Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, 1:2.500) served as a secondary antibody for visualization, which was performed using an Amersham Imager 600 (GE, Massachusetts, USA). Immunofluorescence imaging For immunofluorescence experiments, cells were seeded in Ibidi µ-Slide 8 Well high slides (Ibidi GmbH, Gräfelfing, Germany). Mitochondria were stained using MitoTracker Red CMXRos at a dilution of 1:10.000 in DMEM for 45 minutes. Subsequently, cells were fixed using 4% PFA in PBS at room temperature for 15 minutes. Cells were washed twice with PBS followed by incubation in 5% BSA and 0.3% Triton X-100 (both Carl Roth GmbH) in PBS for 1 hours. Anti-Hexokinase 1 rabbit mAb (C35C4, 1:750) and anti-Hexokinase 2 rabbit mAb (C64G5, 1:750) served for HK visualization. Cells were incubated over-night at 4°C with the primary antibody. The next day, cells were washed thrice with 5% BSA in PBS solution, followed by addition of the secondary antibody (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, 1:2.500) for 1 hour. Ultimately, cells were washed thrice in PBS, with DAPI (1 µM) added during the third washing step and mounted using 90 % glycerol and 10 % PBS, pH = 9.0. Cells were imaged on a Nikon Eclipse Ti2 microscope. The microscope was equipped with a 100×/1.45 NA oil objective (CFI Apochromat, Nikon) and two Kinetix Scientific CMOS cameras (Photometrics). Excitation of dyes was achieved with 405 nm, 476 nm and 568 nm laser light (Celesta, Light Engine). Live-cell imaging of [ATP] mito and [H 2 O 2 ] mito Live-cell imaging of [ATP] mito and [H 2 O 2 ] mito was performed using a Zeiss AXIO Observer Z1 microscope (Carl Zeiss AG, Oberkochen, Germany). The microscope was connected to a LEDHub high-power LED light engine (OMICRON Laserage, Rodgau-Dudenhofen, Germany) and equipped with a EC Plan-Neofluar 40x/1.3 Oil DIC M27 objective (Carl Zeiss AG), an Optosplit II emission image splitter (Cairn Research Ltd, Faversham, UK), and a pco.panda 4.2 bi sCMOS camera (Excelitas PCO GmbH, Kelheim, Germany). The microscope possessed a BioPrecision2 automatic XY-Table (Ludl Electronic Products, Ltd., New York, USA). Optical filters included a 459/526/596 dichroic mirror and a 475/543/702 emission filter for FRET-based measurements, and an additional 409/493/573/652 dichroic mirror combined with a 514/605/730 emission filter for mitoHyPer7-based measurements (all purchased from AHF Analysentechnik, Tuebingen, Germany), which was excited at 427 nm and 473 nm. The Optosplit II emission image splitter was equipped with a T505lpxr long-pass filter (AHF Analysentechnik). The LEDHub high-power LED light engine was equipped with a 340 nm, 385 nm, 455 nm, 470 nm and 505-600 nm LED, followed by the following emission filters, respectively: 340x, 380x, 427/10, 473/10 and 510/10 or 575/15 (AHF Analysentechnik). Cells were transfected with the plasmids encoding the respective biosensor 24 hours prior to transfection. Viability assays To assess cell viability, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT, Thermo Fisher Scientific) assays were performed according to manufacturer’s instructions. For IC 50 determination of 3-BP and Benserazide, cells were seeded in 96-well plates (2.500 HEK293 and MMTV-PyMT, 5.000 MCF-7 and 7.500 MDA-MB-453). The next day, cells were treated with 3-BP or Benserazide at the indicated concentrations and cells were cultivated for 7 days, followed by performing MTT-assay. Synergistic effects of Docetaxel or Capecitabine co-treatment with 3-BP treatment was assessed as follows: Cells were seeded in 96-well plates (2.500 HEK293 and MMTV-PyMT, 5.000 MCF-7 and 7.500 MDA-MB-453). Docetaxel treatment was performed for 24 hours before 3-BP treatment. Capecitabine treatment was performed for the full duration of the experiment (7 days) using 100 µM (MCF-7, MDA-MB-453 or MMTV-PyMT) or 50 µM Capecitabine (HEK293) in the presence of different concentrations of 3-BP as indicated in the panels. For experiments using proliferating or senescent cells, cells were treated with 50 nM of Docetaxel for 24 hours, followed by cultivation for 7 days. After 7 days, untreated control cells or senescent cells were treated with the indicated concentration of 3-BP (0 µM, 50 µM, 100 µM or 300 µM) for 7 further days, prior to conducting the MTT assay or using an image-based read-out following DAPI staining (1 µM in PBS) on a Zeiss Cell Discoverer 7 (Carl Zeiss AG). Senescence associated β-galactosidase activity staining SA-β-Gal staining was performed either using senescence β-galactosidase staining kit (Cell Signaling Technology) or CellEvent senescence green detection kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Colorimetric assay was performed in 12 well plates. Images were acquired on a Zeiss Axiovert 200m microscope equipped with a 5MP USB3.0 Digital Camera for Microscopes (AMScope, London, UK). Fluorigenic assay was performed in 96 well plates. Staining was performed according to manufacturer’s instructions with additional DAPI staining (1 µM in PBS). Detection was performed on a Zeiss Cell Discoverer 7 (Carl Zeiss AG). Analysis of single cell RNA-sequencing data Single-cell RNA-sequencing (scRNA-seq) data from Wu et al. (2021) [GEO: GSE176078], comprising primary tumor samples from 26 breast cancer patients across ER+, HER2+, and TNBC subtypes, was analyzed (Wu et al., 2021). Cell-type annotations at three resolution levels, as provided by the authors, were included in the analysis. Data preprocessing and analysis was conducted in R 4.1 using Seurat 5.2.0. A Seurat object was created with curated count matrices and associated metadata. The data were normalized using the SCTransform method, variable features were identified with FindVariableFeatures and scaled with ScaleData. Quality control filtering retained cells with 500–7500 detected features, 1000–100,000 total counts. Notably, mitochondrial content was not used as a filtering criterion, as mitochondria may play a key role in cellular senescence, but the impact on mitochondrial content in scRNA-seq studies remains unclear. Cells showing a CDKN1A, CDKN2A, TP53 and GLB1 expression = 0 were excluded from their respective analysis due to our interest in senescent cancer cells. Gene expression levels were analyzed in ”cancer epithelial cells” identified through annotation. Data representation and statistical analysis Data were visualized and statistically analyzed using GraphPad Prism 8 software (GraphPad Software, Boston, USA). Data was tested for normal distribution using D’Agostino and Pearson omnibus normality test. A Brown-Forsythe and Welch ANOVA test followed by Holm-Sidak’s MC test was used for normally distributed data showing significantly different SDs. A Kruskal-Wallis test followed by Dunn’s MC test was performed if data were not normally distributed. A One sample Wilcoxon Signed Rank test was used for the analysis of normalized data (Western blot) showing no normal distribution A Multiple t-test without assumption of equal SDs was used for the comparison of multiple pairwise data (Figure 4l and 4m). The statistical tests used are indicated in the figure legends. p-values of ≤0.05 were considered as significant, where *p≤0.05, **p≤0.01 and ***p≤0.001. No priory sample size estimation was performed. HK1 and HK2 are located on the outer mitochondrial membrane of murine and human breast cancer cells HK1 and HK2 represent key enzymes for cell metabolic activities, especially for glycolysis, which subsequently delivers intermediates for further metabolic pathways (Bischof et al., 2021). We first used Western blot experiments to evaluate HK1 and HK2 expression levels in all cell models. Therefore, the commonly used BC cell lines MCF-7 and MDA-MB-453, and primary BC cells isolated from a mouse model of BC based on the mammary tumor virus (MMTV) polyoma middle T-antigen (PyMT) model, MMTV-PyMT cells, were used (Bischof et al., 2024). Furthermore, the human embryonic kidney cell line HEK293 served as a surrogate model, to investigate whether our findings are relevant in other cell models. We detected HK1 and HK2 in all of the tested cell lines, despite pronounced expressional differences using β-Tubulin as independent reference for protein loading ( Figure 1a-c and Figure S1a ). The highest expression of HK1 was observed in MCF-7 cells (relative expression/β-Tubulin 5.14 ± 3.07) and MDA-MB-453 cells (2.61 ± 1.18). HEK293 and MMTV-PyMT cells possessed similar expression levels of HK1 (0.53 ± 0.20 and 0.49 ± 0.23), and showed considerably lower protein levels of HK1 compared to MCF-7 and MDA-MB-453 cells ( Figure 1a and b ). Quantification unveiled highest HK2 protein levels in MDA-MB-453 cells (1.11 ± 0.25), followed by HEK293 (0.52 ± 0.09), MMTV-PyMT (0.40 ± 0.18) and MCF-7 cells (0.38 ± 0.18) ( Figure 1a and c ). To simplify the representation of these analyses, we subsequently calculated the HK2/HK1 expression ratio as a measure of cellular HK2 dependency for maintaining their glycolytic activity ( Figure 1d ). HK2/HK1 ratios in HEK293 and the primary murine MMTV-PyMT BCCs were high (1.09 ± 0.44 and 1.06 ± 0.76), whereas the ratio was significantly lower in MDA-MB-453 cells (0.52 ± 0.31) and lowest in MCF-7 cells (0.09 ± 0.05) ( Figure 1d ). These findings suggest that the relevance of HK2 for maintaining the glycolytic activity may strongly differ within the cell lines used. As HK1 and HK2 sustain their enzymatic activity by associating with the outer mitochondrial membrane (Bischof et al., 2021), we next performed immunofluorescence analysis to i) investigate the subcellular localizations of HK1 and HK2 and ii) validate the immunoblot analysis. Utilizing established isoenzyme-specific antibodies, cells were stained for HK1 or HK2, respectively, and nuclei and mitochondria were visualized using 4′,6-Diamidin-2-phenylindol (DAPI) and MitoTracker CMXRos staining. Subsequently, high-resolution confocal imaging was performed. In all the cell lines tested, protein expression of HK1 and HK2 was confirmed and in line with Western blot data ( Figure 1a – c ). Importantly, we observed a tight mitochondrial association of both isoenzymes, as the signals observed for HK1 ( Figure 1e ) and HK2 ( Figure 1f ) surrounded the fluorescence signals received from MitoTracker Red CMXRos, a dye that accumulates in the mitochondrial matrix (Pendergrass et al., 2004). From these experiments we concluded that HK1 and HK2, are tightly mitochondria-associated proteins in all analyzed BC cell lines, albeit their individual expression levels as well as the HK2/HK1 expression ratio differ greatly from each other. HK2 is of functional relevance for maintaining the [ATP] mito - and H 2 O 2mito - homeostasis and thereby the cell viability Cancer cell mitochondria utilize glycolytically derived adenosine-5’-triphosphate (ATP) to maintain the mitochondrial ATP (ATP mito ) homeostasis (Bischof et al., 2021). To test the implications of HK2 inhibition on cellular energy homeostasis, we investigated the ATP mito concentration ([ATP] mito ) using a well-established, genetically-encoded, Förster resonance energy transfer (FRET)-based ATP indicator, mtAT1.03 (Imamura et al., 2009). We utilized 3-BP, a HK2 inhibitor (Pedersen, 2012), to study whether HK2 activity is linked to ATP mito homeostasis in BC cells. In all BC cell lines, [ATP] mito was differentially affected upon 3-BP application ( Figure 2a and b ). While MMTV-PyMT cells showed the strongest and a very acute response to 3-BP treatment (∆Ratio 0.49 ± 0.08), the [ATP] mito decreased gradually in HEK293 cells (∆Ratio 0.23 ± 0.09). Contrary, in MCF-7 cells, [ATP] mito was only slightly affected (∆Ratio 0.07 ± 0.07) and it remained virtually unaffected in MDA-MB-453 cells (∆Ratio 0.05 ± 0.04). Interestingly, the drop in [ATP] mito upon 3-BP treatment correlated positively and best with the HK2/HK1 ratio (R 2 = 0.587), negatively with HK1 expression (R 2 = 0.543) and negatively with the expression of HK2 (R 2 = 0.270) ( Figure 1b – 1d , Figure 2a and b and Figure S2a ). These results indicate that the 3-BP sensitivity of the cells is dependent on both, the HK1 and HK2 expression levels and especially on their ratio, suggesting that HK1 may compensate for inhibited HK2 activity. As disturbances in cellular ATP homeostasis are tightly associated with the generation of reactive oxygen species (ROS) (Kim et al., 2016), we visualized mitochondrial concentrations of hydrogen peroxide ([H 2 O 2 ] mito ) as a surrogate for cellular ROS formation using mitoHyPer7 (Pak et al., 2020). To calibrate [H 2 O 2 ] mito , we administered 2.0 mM H 2 O 2 to the cells at the end of the measurement to yield saturating mitoHyPer7 signals. In line with the [ATP] mito measurements ( Figure 2a and b ) the highest [H 2 O 2 ] mito production in response to 3-BP treatment was evoked in MMTV-PyMT cells (94.71 % of maximal mitoHyPer7 signal), followed by HEK293 cells (83.64 %), MCF-7 (16.50 %) and MDA-MB-453 cells (15.20 %), highlighting ROS, i.e. H 2 O 2 , production as a consequence of 3-BP treatment ( Figure 2c and d ). We observed correlations between the HK1 and HK2 expression status and [H 2 O 2 ] mito signals comparable to the correlations observed upon analysis of [ATP] mito . In detail, R 2 values were as follows: 0.753, 0.264 and 0.837 for [H 2 O 2 ] mito with HK1, HK2 and HK2/HK1 expression ratio, respectively ( Figure 1b – 1d , Figure 2c and d and Figure S2b ). Based on the observation that 3-BP induces alterations in [ATP] mito and an increase in [H 2 O 2 ] mito production, we aimed to unravel whether the 3-BP treatment affects cell viability in a dose- and cell-line specific manner. Therefore, cells were treated for 7 days with increasing concentrations of 3-BP, followed by assessment of cell viability using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. These analyses revealed pronounced differences, with HEK293 cells reacted most sensitively to 3-BP (IC 50 = 21.8 (20.5 – 23.3) µM), followed by MMTV-PyMT (26.9 (25.5 – 28.2) µM), MCF-7 (64.6 (61.0 – 68.2) µM) and MDA-MB-453 cells (68.2 (61.7 – 75.2) µM) ( Figure 2e and f ). This set of data demonstrates that 3-BP leads to a disruption of [ATP] mito homeostasis ( Figure 2a ) and to [H 2 O 2 ] mito formation ( Figure 2b ), thereby affecting cell viability ( Figure 2c ). Interestingly, these events well correlated with the HK2/HK1 ratio and the HK1 expression status of the cells ( Figure S2a and S2b ). Nevertheless, we observed a very different sensitivity of the BC cell lines exposed to the potential antitumor agent 3-BP. To support our data on the physiological relevance of HK2 in the utilized cell lines, we furthermore tested Benserazide (Bens), a dopa decarboxylase- and HK2 inhibitor (Li et al., 2017). We started investigating the [ATP] mito over-time using mtAT1.03 (Imamura et al., 2009). 200 µM of Benserazide were administered to the cells for 15 minutes after 5 minutes of baseline recording. The highest sensitivity of [ATP] mito towards HK2 inhibition was observed in MMTV-PyMT (∆Ratio 0.35 ± 0.07), followed by HEK293 cells (∆Ratio 0.27 ± 0.08) ( Figure 2g and h ). Contrary to the measurements using 3-BP, however, Benserazide induced significant changes in [ATP] mito in MDA-MB-453 cells (∆Ratio 0.26 ± 0.15), and in MCF-7 cells (∆Ratio 0.11 ± 0.01) ( Figure 2g and h ). Considering the selectivity of Benserazide for HK2, we do not know whether these results point to a different inhibitory efficiency or mode of action of HK2 inhibition by 3-BP compared to Benserazide. We further performed analysis of the correlation status between HK1 and HK2 expression, and their interdependence (HK2/HK1 expression ratio). Unexpectedly, this analysis unveiled the highest correlation of [ATP] mito sensitivity for Benserazide treatment with HK1 expression (R 2 = 0.863), followed by the HK2/HK1 ratio (R 2 = 0.795), and little or no correlation with HK2 expression (R 2 = 0.013) ( Figure 1b – 1d , Figure 2g and h and Figure S2c ). Next, we aimed to investigate changes in the [H 2 O 2 ] mito levels as a measure of HK2 inhibition. These experiments were inconclusive, however, as Benserazide itself seemed to liberate H 2 O 2 as mitoHyPer7 signals immediately increased with the administration of the buffer. This would align with a previous report (Kelts et al., 2015), hampering quantification of Benserazide-mediated H 2 O 2 production. To investigate the consequences of Benserazide treatment on cell viability, we again conducted MTT-based experiments. Cells were cultured for 7 days in the presence of increasing concentrations of Benserazide. Comparable to results obtained from 3-BP treatment ( Figure 3e and f ), cell viability was affected in a concentration-dependent manner in all cell lines tested. The highest sensitivity for Benserazide treatment was thereby again observed in HEK293 cells (IC 50 = 58.0 (51.3 – 66.5) µM), followed by MMTV-PyMT (146.6 (130.6 – 164.9) µM), MCF-7 (164.1 (152.5 – 177.2) µM) and MDA-MB-453 cell (200.2 (191.2 – 210.2) µM) ( Figure 2i and j ). Altogether, these results point to a strong linkage between the HK2 inhibitors, 3-BP and Benserazide, the cellular [ATP] mito , and H 2 O 2 homeostasis (endogenously triggered or exogenously generated) and cell viability, thereby confirming earlier previously recognized anti-cancer properties of HK2 inhibitors. Inhibition of HK2 induces senescence and alters expression levels of mitochondria-associated HK isoforms Perturbances of cellular metabolism frequently induce cellular senescence (Liu et al., 2023). To elucidate potential effects of 3-BP and Benserazide treatment on the induction of senescence, we visualized the activity of senescence-associated β-galactosidase (SA-β-Gal) following cell treatment with 3-BP and Benserazide at the determined IC 50 values for 10 days for each cell line (21.8 µM, 64.6 µM, 68.4 µM and 26.9 µM of 3-BP and 58.0 µM, 164.1 µM, 200.2 µM and 146.6 µM of Benserazide for HEK293, MCF-7, MDA-MB-453 and MMTV-PyMT, respectively) ( Figure 3a ). These experiments unveiled that HK2 inhibition by 3-BP and Benserazide at half-maximal inhibitory concentration indeed induced senescence in the utilized cell lines ( Figure 3a ). We next utilized Western blot analysis to study the effects of HK2 inhibition on the expression levels of the pace-maker enzymes of glycolysis, HK1 and HK2. Throughout all cell lines tested, we observed increasing HK1 and decreasing HK2 levels compared to untreated control cells upon cell treatment with 3-BP or Benserazide ( Figure 3b, d, f and h and Figure S3a – h ). In detail, normalized to the expression of β-tubulin as an internal reference protein, HK1 expression levels increased to 1.19 ± 0.13 and 1.13 ± 0.07 in HEK293, 1.65 ± 0.31 and 1.63 ± 0.62 in MCF-7, 1.42 ± 0.36 and 1.81 ± 0.66 in MDA-MB-453 and 1.40 ± 0.29 and 1.60 ± 0.70 in MMTV-PyMT cells for 3-BP and Benserazide treatment, respectively ( Figure S3a – d ). In contrast, the expression level of HK2 decreased to 0.36 ± 0.16 and 0.27 ± 0.19 in HEK293, 0.49 ± 0.11 and 0.55 ± 0.15 in MCF-7, 0.77 ± 0.20 and 0.60 ± 0.16 in MDA-MB-453 and 0.74 ± 0.19 and 0.73 ± 0.20 in MMTV-PyMT cells upon treatment with 3-BP and Benserazide, respectively ( Figure S3a – d ). To simplify the representation of these data, we calculated the HK2/HK1 ratio under these conditions, respectively, as a measure of HK2/HK1 dependency of the cells. This analysis confirmed the switch from HK2- to HK1-dependent glycolysis, as the HK2/HK1 ratios significantly decreased in a senescence-associated manner in all cell lines ( Figure 3a, c, e, g, i ) (3-BP / Benserazide: HEK293: 0.30 ± 0.14 / 0.24 ± 0.17; MCF-7: 0.30 ± 0.08 / 0.37 ± 0.15; MDA-MB-453: 0.55 ± 0.14 / 0.37 ± 0.18; MMTV-PyMT: 0.54 ± 0.17 / 0.50 ± 0.19). Together, these experiments point to a switch in the expression levels of glycolytic key-proteins when cells underwent HK2-inhibition-induced senescence. Alteration of HK expression levels is a senescence-associated phenomenon and is associated with increased resistance to HK2 inhibitors Chemotherapy treatments of cancers frequently induce senescence (Guillon et al., 2019). Thus, we aimed to elucidate whether the altered expression levels of HK1 and HK2, as observed in 3-BP- and Benserazide-induced senescence, are a common senescence-associated phenomenon. Therefore, senescence in HEK293, MCF-7, MDA-MB-453 and MMTV-PyMT cells was differentially induced applying docetaxel and capecitabine, two frequently used chemotherapeutic agents for anti-BC therapy (O’Shaughnessy et al., 2002), or H 2 O 2 , a frequently used inductor of cellular senescence (Duan et al., 2005). SA-β-Gal staining was conducted to investigate senescent cell status after 10 days of treatment, which confirmed senescence induction in all cell lines under all conditions tested ( Figure 4a ). Next, Western blot analysis was performed to determine the HK1 and HK2 expression in the total protein lysates from these cells. Throughout the cells investigated, treatment with docetaxel, capecitabine or H 2 O 2 yielded significant decreases of the HK2 expression level to 0.37 ± 0.28, 0.39 ± 0.23 and 0.47 ± 0.18 in HEK293, 0.33 ± 0.23, 0.58 ± 0.32 and 0.58 ± 0.36 in MCF-7, 0.47 ± 0.17, 0.85 ± 0.13 and 0.60 ± 0.20 in MDA-MB-453 and 0.65 ± 0.18, 0.75 ± 0.30 and 0.73 ± 0.20 in MMTV-PyMT cells in response to docetaxel, capecitabine and H 2 O 2 treatment, respectively ( Figure 4b – e and Figure S4a – h ). The expression levels of HK1 were affected differentially, despite it tendentially increased under most of the tested conditions to (Docetaxel / Capecitabine / H 2 O 2 ) 0.95 ± 0.37, 1.20 ± 0.4 and 1.64 ± 0.69 in HEK293, 1.27 ± 1.06, 2.12 ± 0.65 and 1.68 ± 0.43 in MCF-7, 0.84 ± 0.63, 2.08 ± 1.03 and 1.56 ± 0.51 in MDA-MB-453 and 1.45 ± 0.60, 1.27 ± 0.55 and 1.73 ± 0.81 in MMTV-PyMT cells ( Figure 4b – e , and Figure S4a – h ). Again, to simplify the data representation, we calculated the HK2/HK1 ratio, which consistently decreased under all treatments compared to control conditions in all cell lines tested, including HEK293, MCF-7, MDA-MB-453, and the primary murine BCCs derived from the MMTV-PyMT BC model, mostly below HK2/HK1 ratios of 0.5 ( Figure 4 f – i ). In detail, the HK2/HK1 ratios changed to the following values upon treatment (Docetaxel / Capecitabine / H 2 O 2 ): 0.35 ± 0.17 / 0.33 ± 0.16 / 0.33 ± 0.16 in HEK293, 0.37 ± 0.25 / 0.31 ± 0.22 / 0.36 ± 0.24 in MCF-7, 0.58 ± 0.27 / 0.46 ± 0.14 / 0.40 ± 0.11 in MDA-MB-453 and 0.49 ± 0.17 / 0.63 ± 0.16 and 0.51 ± 0.27 in MMTV-PyMT cells. To investigate whether BC cell treatment with the established chemotherapeutic agents in combination with the proposed anti-cancer agent 3-BP may have synergistic effects, we again performed concentration-response studies by co-treating cells with increasing concentrations of 3-BP and docetaxel or capecitabine ( Figure 4j ). Unexpectedly, under these conditions, we could not observe synergistic effects between the treatments, as the IC 50 values for HEK293 were 21.8 (20.5 – 23.3) µM, 22.0 (20.3 – 23.8) µM and 28.1 (25.9 – 32.1) µM, for MCF-7 were 64.6 (61.0 – 68.2) µM, 78.4 (76.5 – 80.4) µM and 78.6 (75.7 – 81.6) µM, and MMTV-PyMT were 26.9 (25.5 – 28.2) µM, 23.7 (15.5 – 54.5) µM and 37.5 (31.5 – 45.1) µM of 3-BP for control conditions, cotreatment with docetaxel, and cotreatment with capecitabine, respectively ( Figure 4j and k ). Rather, the cotreatment of 3-BP – especially with capecitabine – the 3-BP potency as IC 50 values increased significantly in HEK293 and MMTV-PyMT cells. This was especially pronounced in MDA-MB-453 cells, where the IC 50 increased from 68.2 µM (61.7 µM – 75.2 µM) under control conditions to 191.6 µM (160.4 µM – 218.2 µM) and 177.7 µM (165.2 µM – 190.5 µM) upon cotreatment with docetaxel and capecitabine, respectively ( Figure 4j and k ). Based on these results, we hypothesized that the switch in protein expression from HK2 to HK1 in senescent cancer cells desensitizes these cells to HK2 inhibition. To test whether senescent cells show lower sensitivity for HK2 inhibition, we induced senescence in HEK293, MCF-7, MDA-MB-453 and MMTV-PyMT cells by treatment with docetaxel ( Figure 4a ). After 1 week of docetaxel treatment, we treated the cells for 7 days with different concentrations of 3-BP (50 and 100 µM 3-BP for HEK293, MCF-7 and MMTV-PyMT, and 100 and 300 µM of 3-BP for MDA-MB-453 cells) ( Figure 2e and f and Figure 4j ). Subsequently, we analyzed cell viability using MTT assay and aimed to validate these findings using image-based analysis of DAPI-, i.e. nuclei. stained cells. Interestingly, both, the MTT-based ( Figure 4l ) and the image-based read-out ( Figure 4m and Figure S4i and j ) unveiled increased resistance to 3-BP treatment if BCCs were treated with docetaxel, i.e. senescent, compared to untreated control, i.e. proliferating, BCCs. This effect was not visible in HEK293 cells. This may indicate that the increased resistance of senescent cells to 3-BP might be specific for BC cells. Genetic ablation of HK2-, but not HK1 expression induces senescence To complement our pharmacological approaches, we tested whether a genetic downregulation of HK2 by siRNA induces senescence in the utilized cell lines. Therefore, cells were transiently transfected with siRNAs targeted against HK1 or HK2, or a scrambled siRNA (siScrbl) as a control. mRNA expression analysis of HK1 and HK2 by qPCR confirmed a pronounced knockdown of HK1 ( Figure S5a ) and HK2 ( Figure S5b ) under these conditions in all cell lines to 51.68 % ± 15.71 %, 46.35 % ± 14.84 %, 57.56 % ± 18.47 %, 54.58 % ± 13.69 % for HK1 and 39.75 % ± 7.87 %, 14.07 % ± 8.32 %, 44.60 % ± 10.32 %, 53.32 % ± 21.64 %, for HK2 in HEK293, MCF-7, MDA-MB-453 and MMTV-PyMT cells, respectively. Subsequently, we assessed SA-β-Gal activity using senescence green staining under these conditions in combination with DAPI staining to visualize cell nuclei. These experiments showed that neither HK1 silencing nor cell treatment with scrambled siRNA induced senescence in HEK293 ( Figure 5a ), MCF-7 ( Figure 5b ), MDA-MB-453 ( Figure 5c ) or MMTV-PyMT cells ( Figure 5d ). Contrary, when cells were treated with siRNAs against HK2, we observed increased senescence green staining in HEK293 cells ( Figure 5a ), MCF-7 cells ( Figure 5b ) and MMTV-PyMT cells ( Figure 5d ). In MDA-MB-453 cells a strong trend towards an increasing senescence green staining was observed that did not reach significance, however ( Figure 5c ). These results highlight that a genetic downregulation of HK2, but not HK1, mediates cellular senescence induction as measured by assessing SA-β-Gal activity. HK2/HK1 expression negatively correlates with senescent cell status in primary breast cancer biopsies To address the potential clinical relevance of our in vitro studies, we assessed whether the observed alteration of the HK2/HK1 expression ratio can also be found in human patient samples. Therefore, we analyzed single-cell RNA (scRNA) sequencing data of 26 BC patients using a dataset which was available from Wu et al., 2021 (Wu et al., 2021), and determined the HK1 and HK2 transcript levels and the expression of frequently used senescence markers including the levels for mRNAs encoding for the proteins p21, p16, p53 and β-galactosidase, respectively (Yan et al., 2024) ( Figure 5e and 5f and Figure S5c – h ). These analyses revealed no correlation of HK1 with p21 (Slope = 0.023, R 2 = 0.0002, p = 0.2958) ( Figure 5e , left panel). The mRNA expression level of HK2, however, showed a negative and highly significant linear correlation with p21, as HK2 levels tended to decrease with increasing p21 levels (Slope = - 0.192, R 2 = 0.0050, p ≤ 0.001) ( Figure 5e, right panel). To improve data interpretation and representation, we calculated the HK2/HK1 ratio as a read-out of hexokinase isoform-dependency and correlated this value with p21 levels. As a result of this transformation, we found the highest correlation of our analyses, as HK2/HK1 ratio negatively and highly significantly correlated with p21 expression levels (Slope = -0.331, R 2 = 0.02718, p ≤ 0.001) ( Figure 5f ). The same tests we conducted for p14 ( Figure S5c and d ), p53 ( Figure S5e and f ) and β-galactosidase ( Figure S5g and h ), where we observed decreases in HK2/HK1 expression ratios with increasing expression of p16 ( Figure S5c and d ), p53 ( Figure S5e and f ) and β-galactosidase ( Figure S5g and h ). These data support our previous findings on the association of altered HK1 and HK2 expression with cancer cell senescence. Within this set of data, however, both, HK1 and HK2 correlated negatively with p16 ( Figure S5c ), p53 ( Figure S5e ) and β-galactosidase ( Figure S5g ) expression. In summary, we conclude that a switch from HK2- to HK1-dependent cell metabolism affects cellular senescence and may therefore be a clinically relevant factor determining the efficacy of chemotherapy in BC patients. Discussion and Conclusion Within this study, we describe a novel role of HK2 and HK1 as key-proteins determining cellular senescence. We show, that i) the direct pharmacologic inhibition of HK2 by either 3-BP or Benserazide and ii) the treatment of cells with sublethal concentrations of docetaxel, capecitabine or H 2 O 2 induces cellular senescence, which is associated with an upregulation of HK1 and a downregulation of HK2 expression levels. Furthermore, iii) a direct siRNA-mediated knockdown of HK2 induced cellular senescence in the utilized BC cell lines and HEK293 cells (Figure 5g ). These findings suggest that an analysis of the HK2/HK1 expression ratio may serve as a novel marker for BC cell senescence, representing a potentially important factor determining the efficacy of anti-BC and other cancer entity therapies. This assumption got strengthened by our observations of a correlation between p21 as a senescence marker and the HK2/HK1 expression ratio in scRNA sequencing data from BC patients (Wu et al., 2021). We first analyzed the basal expression levels of HK1 and HK2 in the BCC lines MCF-7, MDA-MB-453 and MMTV-PyMT. Additionally, HEK293 cells were included as a surrogate model. Our analyses revealed highly differential expression levels of HK1 and HK2 within these cells, although levels of HK1 showed a higher variation between the different cell lines compared to HK2. The highest HK1 protein levels were thereby observed in MCF-7 cells, while HK2 levels in MDA-MB-453 cells were higher than in all other cell lines examined. These findings are well in line with an earlier study (Ma et al., 2023). Interestingly, in MMTV-PyMT cells we observed two bands for the HK2 protein throughout all experiments. We could not distinguish whether these were splice variants of HK2 in the murine BC model or a consequence of unspecific antibody binding. Hence, both bands were quantified for this cell line. Further studies are required to elucidate reasons for the double protein band of HK2 in murine BC cells, despite the effect in our experiments was not observed in lysates derived from human cells. Interestingly, Quach et al. also found that HK2 appeared as two bands in mitochondrial samples derived from T47D cells, a human breast cancer cell line, whereas a single band was observed using the cytosolic fraction (Quach et al., 2016). These dual bands may be a result of post-translational modification, an aspect that needs attention in future, however. Consistently, we found that all the cell lines tested showed sensitivity to HK2 inhibition. Unexpectedly, this sensitivity, especially in the functional read-out upon measuring [ATP] mito and [H 2 O 2 ] mito , correlated worst with the expression level of HK2. Much better correlations were observed with the expression level of HK1 or the expressional ratio of HK2/HK1. These data either indicate that 3-BP and Benserazide show a low selectivity towards HK2 as they also act on HK1, or that HK1 expression and activity are upregulated to compensate for HK2 inhibition, another point which requires further attention. Nevertheless, we highlight that an isolated analysis of HK2 expression cannot reliably predict the sensitivity of cancer cells for HK2 inhibitors. Rather, the expression levels of other HK isoforms, i.e. HK1, must be analyzed in parallel. These results are well in line with a recent study by Xu et al., showing that a knockdown of HK2 only modulates cell proliferation in vitro and xenograft tumor progression in vivo in the absence of HK1, highlighting the compensatory ability of HK1 if HK2 is inhibited (Xu et al., 2019). To investigate whether an inhibition of HK2 at sub-lethal concentrations induces senescence, we treated cells with 3-BP and Benserazide using the respective IC 50 of the compounds. Furthermore, low to moderate treatment concentrations of Docetaxel or Capecitabine were chosen to induce cancer cell senescence, which was also observed by other studies focusing e.g. on lung cancer (Zhao et al., 2023). Throughout all cell lines tested, this treatment regimens induced senescence as measured by the SA-β-Gal activity assay. Whether the accumulation of such senescent cancer cells is desirable or detrimental in anti-cancer therapies remains still a matter of debate (Liu et al., 2018), although senescent cells remain in a viable state which is certainly disadvantageous for the patient. Therefore, anti-cancer treatment dosages must be tightly adjusted to achieve the anticipated effect. Furthermore, we found that the cotreatment of our utilized cell models with chemotherapeutic agents and HK2 inhibitors did not show the expected synergistic effects. This might be caused by a fast adaption of cells to these treatments and expressional alterations of the HK2/HK1 ratio, causing desensitization towards pharmacologic HK2 inhibition. This effect was most drastic in MDA-MB-453 cells, where the effect was even detrimental in terms of cell sensitivity for HK2 inhibition if cells were treated with both, Docetaxel or Capecitabine additionally. Interestingly, if the cancer cells were senescent a priory, a drastic desensitization towards HK2 inhibitors was observed, highlighting that the usage of 3-BP or Benserazide, clinically approved for the treatment of Parkinson’s disease, might only be feasible as first-line treatment in formerly untreated cancers where chemotherapy-induced senescence has not been induced. Moreover, a cautious modification of the HK2/HK1 expression ratio could lead to better and more successful treatment strategies for individual patients. Ultimately, our data highlight a potential clinical relevance, as we found a correlation between high expression levels of CDKN1A mRNA (p21 protein), CDKN2A mRNA (p16 protein), TP53 (p53 protein) and GLB1 mRNA (β-galactosidase protein) as a marker of senescence with low HK2/HK1 expression ratios in primary biopsies from 26 BC patients assessed by single cell RNA sequencing data (Wu et al., 2021). 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IL-1β is involved in docetaxel chemoresistance by regulating the formation of polyploid giant cancer cells in non-small cell lung cancer. Sci Rep 13 : 12763. Main figure legends: Figure 1: Analysis of HK1 and HK2 expression and localization in different cell lines. (a) Representative Western blot showing HK1 (top), HK2 (middle) and β-Tubulin (bottom) expression in HEK293 (first lane), MCF-7 (second lane), MDA-MB-453 (third lane) and MMTV-PyMT cells (fourth lane). ( b, c ) Relative expression levels of HK1 ( b ) and HK2 ( c ) normalized to β-Tubulin of HEK293, MCF-7, MDA-MB-453 and MMTV-PyMT cells as indicated, assessed by Western blot analysis. N (independent experiments) = 7 for all, *p ≤ 0.05, **p ≤ 0.01, Kruskal-Wallis test followed by Dunn’s Multiple Comparison test. ( d ) HK2/HK1 expression calculated based on the data shown in ( b ) and ( c ). **p ≤ 0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s multiple comparison test. ( e, f ) Representative immunofluorescence images of HEK293 (first and second lane), MCF-7 (third and fourth lane), MDA-MB-453 (fifth and sixth lane) and MMTV-PyMT cells (seventh and eight lane) either stained for HK1 ( e ) or HK2 ( f ). Cells were stained with DAPI (blue), MitoTracker (orange), or for HK1 or HK2 (green). A merge of all channels is shown (right columns). The upper lanes of each cell line show an overview image, lower lines show zooms, as indicated. Scale bars in the overview images represent 50 µm, scale bars in the zooms represent 2 µm. N = 5. The contrast of the zoom images was adjusted to the signal present in the image, whereas the contrast in the overview images represents the real and comparable signal between the different cell lines. Figure 2: Pharmacologic inhibition of HK2 affects [ATP] mito , [H 2 O 2 ] mito and cell viability. ( a, b, g, h ) FRET-ratio signal, i.e. [ATP] mito , over-time ( a, g ) and corresponding statistics ( b, h ) of HEK293 (first), MCF-7 (second), MDA-MB-453 (third) and MMTV-PyMT cells (fourth) expressing mtAT1.03, a FRET-based, genetically-encoded ATP sensor targeted to the mitochondrial matrix. At time points indicated in the panels, 100 µM of 3-BP ( a ) or 200 µM of Benserazide ( g ) were administered to the cells. Data show average (black bold curve and bar) and every single cell response (grey curves and circles), SDs are indicated ( b, h ). N (independent experiments) / n (cells analyzed) = 6 / 65, 9 / 48, 8 / 34 and 6 / 148 for 3-BP ( a ) and 6 / 91, 8 / 53, 7 / 53 and 8 / 88 for Benserazide ( g ) using HEK293, MCF-7, MDA-MB-453 and MMTV-PyMT cells, respectively. ***p≤0.001 using Kruskal Wallis test followed by Dunn’s multiple comparison test ( b ) or Brown-Forsythe and Welch ANOVA test followed by Holm-Sidak’s multiple comparison test ( h ). ( c, d ) [H 2 O 2 ] mito over-time ( c ) and corresponding statistics ( d ) of HEK293 (first), MCF-7 (second), MDA-MB-453 (third) and MMTV-PyMT cells (fourth) upon treatment with 100 µM 3-BP at time-points indicated in the panel, measured using mitoHyPer7. After 15 minutes of cell treatment with 3-BP, 2.0 mM H 2 O 2 were administered to the cells for sensor saturation resulting in maximal responses. Graphs display average (black bold curve and bar) and every single cell response ( c, d, grey curves and circles), SDs are indicated ( d ). N / n = 5 / 42, 5 / 26, 5 / 23 and 5 / 36 for HEK293, MCF-7, MDA-MB-453 and MMTV-PyMT, respectively. ***p≤0.001, Kruskal-Wallis test followed by Dunn’s multiple comparison test. ( e, f, i, j ) Cell viability of HEK293 (first), MCF-7 (second), MDA-MB-453 (third) and MMTV-PyMT cells (fourth) measured using MTT-assay in response to cell cultivation in the presence of increasing concentrations of 3-BP ( e ) or Benserazide ( i ) for IC 50 determination. IC 50 was calculated using sigmoidal dose-response fitting and is indicated in the panel. Data show average ± SEM ( e, i ) or average ± range ( f, j ). N = 5 for all except for MMTV-PyMT, N = 3. Figure 3: Pharmacologic inhibition of HK2 induces senescence and alters expression levels of HK1 and HK2. ( a ) Representative images of senescence associated β-galactosidase activity-stained HEK293 (first line), MCF-7 (second line), MDA-MB-453 (third line) and MMTV-PyMT cells (fourth line). Cells were either untreated (ctrl, first column) or treated with 3-BP (+3-BP, middle column) or Benserazide (+Bens, third column). Scale bar in the lower left image represents 100 µm. ( b, d, f, h ) Representative Western blots of HEK293 ( b ), MCF-7 ( d ), MDA-MB-453 ( f ) and MMTV-PyMT ( h ) cells analyzed for the expression of HK1 (top panels), HK2 (middle panels) and β-Tubulin (bottom panels) as a housekeeper. Cells were treated as in ( a ), including no treatment (ctrl), treatment with 3-BP (+3-BP) or treatment with Benserazide (+Bens). ( c, e, g, i ) Quantification of HK2/HK1 protein expression ratio corresponding to Western blots shown in ( b, d, f, h ). Expression under untreated conditions was normalized to 1. Average (bar) and each replicate (circles) are shown. SDs are indicated. N = 6 for all. *p≤0.05, One sample Wilcoxon Signed Rank test. Figure 4: Alteration of HK expression levels is a senescence-associated phenomenon and mediates increased resistance to HK2 inhibition. ( a ) Representative images of senescence associated β-galactosidase activity-stained HEK293 (first line), MCF-7 (second line), MDA-MB-453 (third line) and MMTV-PyMT cells (fourth line). Cells were either untreated (ctrl, first column) or treated with Docetaxel (+Doc, middle column), Capecitabine (+Cap, third column) or with H 2 O 2 (+H 2 O 2 , fourth column). Scale bar in the lower right image represents 100 µm. ( b, d, f, h ) Representative Western blots of HEK293 ( b ), MCF-7 ( d ), MDA-MB-453 ( f ) and MMTV-PyMT ( h ) cells analyzed for the expression of HK1 (top panels), HK2 (middle panels) and β-Tubulin (bottom panels) as a housekeeper. Cells were treated as in ( a ), including no treatment (ctrl), treatment with Docetaxel (+Doc), treatment with Capecitabine (+Cap) or treatment with H 2 O 2 (+H 2 O 2 ). ( f, g, h, i ) Quantification of HK2/HK1 protein expression ratio corresponding to Western blots shown in ( b, c, d, e ). Expression under untreated conditions was normalized to 1. Average (bar) and each replicate (circles) are shown. SDs are indicated. N = 6 for HEK293, MCF-7 and MDA-MB-453, N = 8 for MMTV-PyMT. *p≤0.05, **p≤0.01, One sample Wilcoxon Signed Rank test. ( j ) Cell viability of HEK293 (first), MCF-7 (second), MDA-MB-453 (third) and MMTV-PyMT cells (fourth) measured using MTT-assay in response to cell cultivation in the presence of increasing concentrations of 3-BP alone (ctrl, grey circles and dashed lines), or in combination with cell treatment using Docetaxel (+Doc, black squares and lines) or Capecitabine (+Cap, dark grey triangles and lines). IC 50 was calculated using sigmoidal dose-response fitting. Data show average ± SEM. N = 5 for all cells and conditions. ( k ) Representation of IC 50 values as determined by experiments shown in ( j ) under control conditions (ctrl, light grey bars), or upon cotreatment of cells with Docetaxel (black bars) or Capecitabine (dark grey bars). Average ± range are indicated. N = 5 for all except for MMTV-PyMT, N = 3. ( l, m ) Cell viability (%) either determined using MTT-assay ( l ) or a microscopy-based read-out of DAPI-stained cell nuclei ( m ) of HEK293 (first panels), MCF-7 (second panels), MDA-MB-453 (third panels) or MMTV-PyMT cells (fourth panels). Cells were treated with different concentrations of 3-BP (0, 50, 100 or 300 µM, as indicated) either under control conditions (Proliferating, white bars) or following Docetaxel treatment and cultivation for 7 days (Senescent, grey bars). Data represents average (bars) and each replicate (circles). SDs are indicated. N = 6 – 7 for all. *p≤0.05, ***p≤0.001, Multiple t-test. Figure 5: Genetic silencing of HK2 induces senescence and the expressional alteration of HK1/HK2 is of clinical relevance in BC patients. ( a – d ) Representative images (left panels) and corresponding quantifications (right panels) of HEK293 ( a ), MCF-7 ( b ), MDA-MB-453 ( c ) and MMTV-PyMT cells ( d ) stained with senescence green for quantification of senescence-associated β-galactosidase activity (green, Senesc. green, top). Cells were treated with a scrambled siRNA as control (siScrbl) or siRNAs directed against HK1 (siHK1) or HK2 (siHK2) and additionally stained with DAPI for nuclei visualization (blue, bottom). Data on the right display average ± SDs and each replicate. Scale bars in the lower right images show 500 µm. *p≤0.05, Kruskal-Wallis test followed by Dunn’s Multiple Comparison test. ( e, f ) Single cell RNA sequencing analysis-based expression of CDKN1A and HK1 ( e , left panel), HK2 ( e , right panel) or HK2/HK1 ratio ( f ), and their correlation (black dashed line) from 26 BC biopsies. Slopes, R 2 and p values are indicated. ( g ) Schematic summary of the proposed mechanisms. Proliferating cells (top) become senescent (bottom) by various tested interventions including HK2 inhibition (left), chemotherapy treatment (middle) or direct genetic downregulation of HK2 (right). These interventions mediate a shift in HK2/HK1 expression ratio as they are associated with upregulation of HK1 and downregulation of HK2 (HK2 inhibition and chemotherapy treatment) or HK2 downregulation only (HK2 silencing). Information & Authors Information Version history V1 Version 1 25 February 2025 Peer review timeline Published British Journal of Pharmacology Version of Record 30 Nov 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection British Journal of Pharmacology Keywords biochemical pharmacology cancer and carcinogenesis cancer pharmacology imaging metabolism Authors Affiliations Helmut Bischof [email protected] Medical University of Graz View all articles by this author Katarina Vizar-Cisarova Medical University of Graz View all articles by this author Sandra Burgstaller Medical University of Graz View all articles by this author Markus Absenger-Novak Medical University of Graz View all articles by this author Philipp Jost Medical University of Graz View all articles by this author Roland Malli Medical University of Graz View all articles by this author Wolfgang Graier 0000-0003-1871-3298 Medical University of Graz View all articles by this author Robert Lukowski University of Tübingen View all articles by this author Metrics & Citations Metrics Article Usage 545 views 221 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Helmut Bischof, Katarina Vizar-Cisarova, Sandra Burgstaller, et al. 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