Abstract Background Ovarian cancer (OC) remains the most lethal gynecologic malignancy in the United States due to its late diagnosis, aggressive nature, and poor responsiveness to existing therapies. Dissecting the molecular mechanisms and identifying molecular drivers of aggressiveness and therapy resistance is critical for devising new therapies and improving patient outcomes. Methods MYB expression was evaluated in a panel of OC cell lines by immunoblotting. Gain and loss of function studies were performed by developing stable control and forced-MYB-expressing and -silenced cell lines, respectively. Functional assays included growth kinetics, clonogenicity, cell cycle, live-dead cell measurements, and annexin-V staining, followed by flow cytometry, migration and invasion assays, and MTT assays following drug treatment. Gene expression profiling was done using the nanoString PanCancer Progression panel. Chromatin immunoprecipitation (ChIP) was performed to confirm MYB binding to the responsive gene promoter, followed by siRNA-mediated silencing to establish the intermediary role in potentiating the downstream effects. Results Low to high MYB expression was reported in all OC cell lines, with negligible expression reported in normal ovarian surface epithelial cells. MYB expression was significantly higher in aggressive (SKOV3-ip) and chemoresistant (A2780-CP) OC cell lines compared to the parental (SKOV3 and A2780) cells. Functional assays in MYB-overexpressing and -silenced OC cell lines demonstrated a role of MYB overexpression in increased cell proliferation, survival, migration, invasion, EMT, and chemoresistance. nanoString analysis and comparison of transcriptomic data of MYB-silenced SKOV3-ip and MYB-overexpressing SKOV3 cells with their respective control cells identified MYB-dependent genes. Interestingly, these target genes showed a limited overlap between cell lines, suggesting a cell-specific MYB-regulated gene regulation. AKT3 was consistently identified as a common MYB-regulated gene in multiple OC cell lines and confirmed as a direct transcriptional MYB target through confirmation of MYB binding to its promoter. Pathway analysis using the MYB-regulated transcriptomic data also identified PI3K/Akt signaling to be activated in MYB-overexpressing cells. siRNA-mediated silencing of AKT3 confirmed its role in potentiating the oncogenic actions of MYB in OC cells. Conclusion MYB/AKT3 axis drives ovarian cancer growth, aggressiveness, and chemoresistance, highlighting its potential as a therapeutic target in ovarian cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s13048-025-01761-9. Keywords: MYB, AKT3, Ovarian cancer Introduction Ovarian cancer (OC) is a highly aggressive gynecologic malignancy and a leading cause of death among women diagnosed with reproductive system cancers. The American Cancer Society estimates that, about 20,890 new cases of OC will be diagnosed in the United States, with approximately 12,730 deaths attributed to the disease in year 2025 [[38]1]. A major cause of such high mortality is its late diagnosis, often at a stage when it has developed widespread metastases. The clinical management of advanced stage OC primarily involves surgical debulking and platinum-based chemotherapy, but the high rate of recurrence and chemoresistance remains a significant challenge. About 20–30% of patients with high-grade serous OC experience disease recurrence within six months of completing chemotherapy and a median overall survival for these patients is approximately 12–18 months [[39]2]. Furthermore, OC exhibits racial disparities in clinical outcomes, with African American (AA) women showing higher mortality rates than their Caucasian American (CA) counterparts [[40]3]. This aggressive nature of OC, limited response to current therapies, and disparities among racial groups underline the need to better understand the molecular mechanisms driving its progression and develop novel mechanism-based therapies. MYB is a transcription factor, pivotal in regulating cell proliferation, differentiation, and survival. Aberrant MYB expression or genetic rearrangements have been reported in several malignancies, including OC [[41]4–[42]8]. MYB overexpression or activation is frequently associated with increased tumor aggressiveness, metastasis, and poor prognosis [[43]9, [44]10]. We recently reported elevated MYB expression across various histological subtypes of OC, with particularly high levels observed in high-grade serous tumors. Moreover, MYB expression was associated with advanced disease stages and shorter patient survival, especially among AA women [[45]3]. Similarly, MYB overexpression correlates with higher tumor grades and poorer outcomes in prostate cancer, with notable racial disparities in its expression levels [[46]11]. MYB is shown to interact with the androgen receptor (AR) in prostate cancer, sustaining its activity under androgen-deprived conditions, thereby promoting castration resistance [[47]12, [48]13]. MYB overexpression in pancreatic cancer is also associated with enhanced tumor growth and metastasis, and more recently, we have reported its role in supporting hypoxic survival of cancer cells by facilitating metabolic reprogramming [[49]14]. PI3K/AKT signaling pathway is often hyperactivated in cancers due to PIK3CA mutations, PTEN loss, or overexpression of receptor tyrosine kinases (e.g., EGFR, IGF1R), contributing to tumorigenesis, progression, and therapeutic resistance [[50]15, [51]16]. AKT has three isoforms, namely AKT1, AKT2, and AKT3, that exhibit tissue/organ-specific expression [[52]17, [53]18]. While AKT1 has been most extensively studied and associated with the pathogenesis of several cancers, the role of AKT3 has only begun to emerge in recent studies [[54]19–[55]21]. AKT3 is upregulated in approximately 20–30% of high-grade serous ovarian carcinoma (HGSOC) cases and has been linked to poor prognosis and advanced disease stage [[56]22]. The mechanisms regulating AKT3 expression and activation remain poorly understood, although there have been some reports of non-coding RNAs, i.e., microRNAs and lncRNAs in AKT3 post-transcriptional regulation [[57]23, [58]24]. In the present study, we investigated the role of MYB in OC growth, aggressiveness, and chemoresistance and delineated the underlying molecular mechanisms. Our study identifies MYB as a regulator of AKT3, where it serves as a critical mediator in promoting its downstream action in OC pathogenesis and chemoresistance. Materials and methods Reagents, gene constructs, and antibodies For RNA interference mediated gene silencing, four independent short hairpin RNA (shRNA) expression constructs for MYB (pGFP-V-RS-shMYB #1, #2, #3 and #4) and non-targeted scrambled control (pGFP-V-RS-NT-Scr) (Origene, Rockville, MD, USA) were used. For Akt3 silencing, Dharmacon™ ON-TARGETplus, SMARTpool Human AKT3 siRNA (cat #L-003002-00-0005) (Lafayette, CO) was used. Primary antibodies used were anti-MYB (cat #12319), AKT3 (cat #14982), pAKT (#4060), E-cadherin (cat #3195), N-Cadherin (cat #13116), anti-GSK3β (cat #9315), -pGSK3β (cat #5558), normal Rabbit IgG (cat #2729), all obtained from (Cell Signaling Technology, Beverly, MA); Rabbit IgG peroxidase-conjugated secondary antibody (cat #HAF008) and Mouse IgG horseradish peroxidase-conjugated antibody (cat # HAF007) (R&D Systems, Inc., Minneapolis, MN, USA). Cell lines Human ovarian cancer cell lines A2780, SKOV3, OVCAR3, CAOV3, TOV112D, ES2, were obtained from ATCC (Manassas, VA USA). SKOV3-ip was obtained from MD Anderson Characterized Cell Line Core Facility (Houston, Texas, USA). Immortalized normal ovarian surface epithelial cell line IOSE-7576 was provided by Dr. S. Singh (Morehouse College of Medicine, Atlanta, GA). A2780-CP70, was generated by us following chronic exposure of A2780 cells with cisplatin. All the ovarian cancer cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA) and Penicillin-Streptomycin solution (Invitrogen, Carlsbad, CA, USA); under humidified atmosphere at 37 °C with 5% CO2. All the cell lines were periodically tested for mycoplasma detection (MP0035, Sigma). Transfection and treatment Cells were transfected with siRNA, plasmids, and vectors using XtremeGENE transfection reagent (Roche, Indianapolis, IN, USA). Transfection was performed in Opti-MEM Reduced-Serum Medium. For stable silencing of MYB expression (in SKOV3-ip and A2780-CP) or to achieve forced overexpression (in SKOV3 and A2780), cells were transfected with pGFP-V-RS-shMYB and pCMV6-MYB plasmid constructs, respectively. In addition, control cell lines were generated by transfection of a non-targeted scrambled sequence vector (pGFP-V-RS-NT-Scr) or an empty vector plasmid (pCMV6-Neo), respectively. Stably transfected cells were selected in puromycin (2 µg/ml; for short hairpin RNA and Scr control) or G418 (200 µg/ml; for forced overexpression and Neo control). After selection, successful MYB overexpression or silencing was confirmed through RT-PCR and immunoblotting. Subsequently, the transfected cells were expanded and routinely monitored for MYB silencing or overexpression via immunoblot and periodic culture in Puromycin or G418-containing media. For transient silencing of AKT3, cells at 50–60% confluence were treated with 50 nM of non-targeting siGENOME™ control pool (NT-Scr), #D‐001206‐13‐05) or SMARTpool HumanAKT3 siRNA (Cat#L-003002-00-0005) (Lafayette, CO) using XtremeGene™ siRNA Transfection Reagent (Roche, Indianapolis, IN) as per manufacturer’s instructions. Cell morphology Actin organization was examined as a measure of EMT in MYB-overexpressing or -silenced ovarian cancer cells. Cells were cultured on FluoroDish™ (World Precision Instruments, LLC), washed once with PBS, then fixed in 4% Paraformaldehyde- PBS. Cells were then washed again and permeabilized in 0.1% Triton X-100 in PBS. Following permeabilization, cells were stained with Alexa Fluor 488-conjugated phalloidin. Post incubation, cells were washed three times with PBS and nuclei stained using NucBlue Fixed DNA stain (2 drops/ mL of PBS). Cells were washed again with PBS, then mounted with Prolong Gold anti-fade mounting medium, covered with a coverslip, and left to set overnight at room temperature. The mounted cells were analyzed and photographed using a fluorescent microscope. Growth kinetics assay MYB overexpressed (SKOV3-MYB & SKOV3-ip-Scr) and MYB silenced (SKOV3-Neo & SKOV3-ip-shMYB) cells were seeded (1 × 10^4 cells/well) in 6-well plates (Corning). The media was changed daily, and cell growth was monitored by counting the number of viable cells every day for 7 days. Every day, cells were trypsinized, washed, and resuspended in media. An equal volume of the Trypan blue solution (Thermo Fisher Scientific) was added to the cell suspension, and cells that did not stain with the dye were counted as viable cells under a phase-contrast microscope using a hemocytometer (Hausser Scientific). Colony formation assay Cells were seeded at very low density (1 × 10^3 cells/well) in 6-well plates in complete media for colony formation. Cells were allowed to form colonies for 14 days, after which the colonies were fixed with methanol, stained with crystal violet, photographed and counted using Image analysis software (ImageJ). Drug resistance assay Control or MYB silenced/overexpressed cells (2.5 × 10^3 cells/well) were seeded in 96-well plate, and after 24 h incubation, cells were treated with various concentrations of cisplatin (0–1000 µM) for 72 h. Subsequently, percent viability was measured by WST-1 assay, and IC50 values were obtained in GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA), through the equation: log(inhibitor) vs. normalized response, using a Variable slope model. Cell cycle analysis Cells were seeded in 6-well culture plates at concentration of 1.5 × 10^6 cells/well. After the cells adhered, they were synchronized by growing them in serum-deprived culture media for 72 h and then incubated in regular media for 24 h. Cells were then trypsinized, washed, and fixed with 70% ethanol overnight at 4 °C. Subsequently, cells were processed for staining with propidium iodide using PI/RNase staining buffer for 1 h at 37 °C. The stained cells were analyzed by flow-cytometry on a BD-FACS Canto™ II (Becton–Dickinson, San Jose, CA). Apoptosis and live/dead cell viability assay Apoptosis was measured by using the PE Annexin V apoptosis detection kit (BD Biosciences, San Diego, CA). The cells were grown in serum-depleted conditions for 72 h. The extent of apoptosis was detected by staining the cells with PE Annexin V and 7AAD (7-amino-actinomycin-D) by using the PE Annexin V Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA) and analyzed by flow cytometry. Cell viability was also evaluated using the LIVE/DEAD™ Viability/Cytotoxicity Kit (Thermo Scientific, Logan, UT). Cells (5 × 10^5 cells/well) were plated in six-well plates and allowed to grow for 24 h. Thereafter, cells were washed with PBS and cultured in serum-free media for 72 h. Post incubation, LIVE/DEAD kit reagent mix (4 µM Calcein AM and 2 µM ethidium homodimer (EthD-1) was added and cells incubated for 30 min. Live/dead cells were monitored by fluorescence imaging using a Cytation 7 (BioTek) imaging System. Migration and invasion assay For migration assay, cells (1.5 × 10^5 cells/well) were plated with serum-free media in the top chamber of a noncoated membrane (six-well insert, pore size 8 μm; BD Biosciences, Bedford, MA). For the invasion assay, the cells (1 × 10^5 cells/well) were plated with serum-free media in the top chamber of the transwell with a Matrigel-coated polycarbonate membrane (BD Biosciences). In the bottom chamber, medium with 10% fetal bovine serum was added as a chemoattractant. After 16 h of incubation, cells remaining on the upper surface of the insert membrane were gently removed with a cotton swab. Cells that migrated or invaded through the membrane/Matrigel to the bottom of the insert were fixed and stained using Hema 3™ stat pack (Fisher Scientific) and mounted on a slide. Migrated and invaded cells were counted in 10 random fields of view under a microscope at 10X magnification. Immunoblotting Immunoblotting was performed using standard procedures as described earlier [[59]7]. Briefly, cell lysates were resolved on 10% polyacrylamide gels and transferred to PVDF membranes. Blots were subjected to a standard immunodetection procedure using specific antibodies against various proteins and visualized using SuperSignal West Femto Maximum Sensitivity Substrate Kit with a LAS-3000 image analyzer (Fuji Photo Film Co., Tokyo, Japan). RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA from cells was isolated as described previously [[60]25] using the RNeasy Mini Kit iRNA isolation kit (Qiagen, CA, USA). Briefly, RNA was quantified using Nanodrop 1000 (Thermo Scientific, MA, USA). For mRNA expression analysis, 2 µg total RNA was reverse transcribed using random primers. The quantitative real-time PCR (qRT-PCR) was performed using Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, MA, USA) on a CFX96 real-time PCR system (Bio-Rad, CA, USA). Fold change in miRNA expression was calculated using the ΔΔCt method. Actin Ct was used to normalize the data. Gene expression profiling To examine MYB-associated transcriptomic changes in ovarian cancer SKOV3 and SKOV3-ip, MYB-silenced and -ectopically expressing cells with their respective controls were employed for a targeted transcriptomic approach using a 770 gene PanCancer Progression panel on the NanoString nCounter^® system (NanoString Technologies, Seattle, WA). Total RNA from cells was isolated using the RNeasy Mini Kit RNA isolation kit (Qiagen, CA, USA) according to the manufacturer’s protocol, and RNA concentration was determined using Qubit 4 Fluorometer (Thermo Scientific). Samples were processed for analysis on the NanoString nCounter system according to the manufacturer’s instructions. Using our dataset from the PanCancer Progression Panel, differentially expressed genes (DEGs) were identified using a cutoff value of fold change ≥|±1.5| with adjusted p-value < 0.05. These DEGs were then used to identify altered pathways using the KEGG pathway analysis. Chromatin Immunoprecipitation (ChIP) assay The binding of MYB to AKT3 gene promoter was analyzed by performing a ChIP assay as described earlier [[61]23]. Briefly, DNA-protein cross-linking was done with paraformaldehyde (37%) followed by enzymatic DNA shearing using ChIP-IT Express Enzymatic Kit (Active Motif, CA, USA). Sheared DNA was then subjected to immunoprecipitation using anti-MYB or normal rabbit IgG (as control). Subsequently, cross-linking was reversed, proteins were digested with proteinase K, and DNA was isolated. PCR was performed using specific primer sets flanking MYB-binding promoter regions, and amplification products were resolved on a 1.5% agarose gel and visualized using ethidium bromide staining. Input DNA (without immunoprecipitation) and normal IgG-precipitated DNA were used as positive and negative controls, respectively. Statistical analysis All experiments were performed at least three times, and numerical data were expressed as mean ± S.D. Statistical comparisons between two groups were performed by an unpaired Student’s t-test. All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc.). A p-value of less than 0.05 was considered statistically significant. Results MYB promotes ovarian cancer cell growth, clonogenicity, and chemoresistance We evaluated the expression of MYB across a panel of ovarian cancer (OC) cell lines (TOV112D, SKOV3, SKOV3-ip, A2780, A2780-CP, CAOV3, OVCAR3, and ES2) and compared it to that observed in a non-cancerous ovarian epithelial cell line (IOSE 7576). The data revealed that MYB was expressed in all OC cell lines, albeit at varying levels, while being undetectable in the non-cancerous IOSE 7576 cells (Fig. [62]1A). Notably, MYB expression was significantly higher in the aggressive SKOV3-ip and cisplatin-resistant A2780-CP OC cell lines compared to their less aggressive, chemotherapy-sensitive parental counterparts SKOV3 and A2780 (Fig. [63]1A). Therefore, we chose these cell lines as a model system to investigate the role of MYB in OC pathogenesis. MYB expression was silenced in A2780-CP and SKOV3-ip cell lines using stable transfection of lentiviral particles containing MYB-targeting shRNA (pGFP-V-RS-shMYB ). In parallel, we overexpressed MYB in the SKOV3 and A2780 cell lines via stable transfection of a pCMV6-MYB construct. In addition, we generated control cell lines transfected with non-targeted scrambled sequence expressing lentiviral particles (pGFP-V-RS-NT-Scr) or the empty vector (pCMV6-Neo). MYB silencing and overexpression were confirmed by immunoblot analysis (Fig. [64]1B & C). The overexpression of MYB led to a significant increase in the growth of SKOV3-MYB (1.6-fold) and A2780-MYB (1.65-fold) cells compared to their respective controls by day 7 (Fig. [65]1D & E). In contrast, silencing MYB drastically impaired the growth of SKOV3-ip-shMYB (1.93-fold) and A2780-CP-shMYB (2-fold) cells, relative to their control cell lines (Fig. [66]1F & G). Further, MYB-overexpressing cells (SKOV3-MYB and A2780-MYB) exhibited a dramatic increase in colony formation (2.7- and 3.0-fold, respectively) compared to controls (Fig. [67]1H), while MYB-silenced cells (SKOV3-ip-shMYB and A2780-CP-shMYB) showed a significant decrease in clonogenic potential (3.5- and 3.2-fold, respectively) (Fig. [68]1I). Notably, MYB downregulation in the ES2, another OC cell line with high expression of MYB, also impaired both growth and clonogenicity (Supplementary Fig. [69]S1). Given the elevated MYB expression in the chemo-resistant A2780-CP cell line compared to its parental A2780 counterpart, we next explored if MYB overexpression was also associated with chemotherapy resistance. Cells with altered MYB expression were treated with various concentrations of cisplatin (0-1000 µM) for 72 h, and cell viability was assessed by WST-1 assay. Our results showed that MYB-overexpressing OC cells had significantly higher viability following cisplatin treatment than their low MYB-expressing counterparts. The IC[50] values for SKOV3-MYB and A2780-MYB cells increased by approximately 2.8- and 2.4-fold, respectively, compared to controls (Fig. [70]1J & K), whereas MYB silencing in SKOV3-ip-shMYB and A2780-CP-shMYB cells resulted in a substantial reduction in IC[50] (3.3- and 2.1-fold, respectively) (Fig. [71]1L & M). Together, these findings underscore the critical role of MYB in promoting the growth, clonogenicity, and chemotherapy resistance of OC cells. Fig. 1. [72]Fig. 1 [73]Open in a new tab MYB promotes ovarian cancer cell growth, clonogenicity, and chemoresistance. A-C. MYB expression in ovarian cancer cell lines and genetically engineered cells. Total protein was isolated from cells at sub-confluence and analyzed by immunoblotting using MYB-specific antibody. β-actin was used as a loading control. Relative band intensity was calculated using ImageJ software. D-G. Growth kinetics of MYB modulated OC cell lines. 1 × 10^4 cells per well were seeded in 6-well plates and counted daily by trypan blue staining over a week period. The linear graph was plotted using the mean values from three separate experiments. H & I. Effect of MYB modulation on anchorage-dependent clonogenic potential of OC cells. MYB-modulated OC cells, along with their respective controls, were seeded at low density (1 × 10^3 cells per well) in six-well plates and grown for 2 weeks. Colonies were stained with crystal violet and visualized and photographed under a microscope. J-M. Effect of MYB modulation on cisplatin sensitivity. MYB-modulated OC cells were seeded in 96-well plates and treated at sub-confluence with various concentrations of cisplatin. After 72 h, cell viability was measured by WST-1 assay and values plotted in a linear graph. IC50 concentrations were obtained in GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA), through the equation: log(inhibitor) vs. normalized response, using a Variable slope model. Bar graphs represent mean ± standard deviation, * p < 0.05 MYB facilitates cell cycle progression and promotes apoptosis resistance Since tumor growth and clonogenic potential are linked to cell-cycle progression and resistance to apoptosis, we investigated the role of MYB in these processes. Cell cycle analysis revealed that MYB overexpression in SKOV3 cells led to their higher proportion in the S-phase compared to the control cells (SKOV3-Neo, 36.65%; SKOV3-MYB, 52.16%). In contrast, MYB silencing in SKOV3-ip cells led to their arrest in the G0/G1 phase (SKOV3-ip-Scr, 18.3%; SKOV3-ip-shMYB, 72.45%) (Fig. [74]2A and B). To evaluate the role of MYB in apoptosis resistance, we performed live-dead cell staining after culturing the cells in serum-free media for 72 h. The results showed a significant reduction in dead cells in MYB-overexpressing SKOV3-MYB (3.2-fold) compared to SKOV3-Neo cells. Conversely, MYB-silenced SKOV3-ip-shMYB exhibited a markedly higher proportion of dead cells (3.3-folds) compared to the control SKOV3-ip-Scr cells. These findings were further confirmed by PE Annexin V and 7AAD dual staining, followed by flow cytometric analysis. A significantly lower percentage of apoptotic cells were observed in SKOV3-MYB (20.2%) compared to SKOV3-Neo (45.2%), while a higher percentage of apoptosis was observed in SKOV3-ip-shMYB cells (84.9%) compared to SKOV3-ip-Scr (18%) (Fig. [75]2E and F). Collectively, these findings indicate that MYB overexpression promotes cell cycle progression while conferring a protective effect against apoptosis in OC cells. Fig. 2. [76]Fig. 2 [77]Open in a new tab MYB facilitates cell cycle progression and promotes apoptosis resistance. A & B. Cell cycle analyses of OC cell lines genetically engineered for altered MYB expression. Synchronized cultures of MYB-modulated OC cells were allowed to enter the cell cycle in serum-containing media for 24 h. Subsequently, the distribution of cells in different phases of the cell cycle was analyzed by propidium iodide (PI) staining followed by flow cytometry. C & D. Live-dead cell assay. MYB-modulated OC cells were cultured in serum-deprived media for 72 h, followed by staining with ethidium homodimer and Calcein-AM. Live (green) and dead (red) cells were visualized under a fluorescence microscope. The bar graph depicts the mean counts of dead cells per well ± standard deviation, *p < 0.05. E & F. Apoptosis assay. MYB-modulated OC cells were cultured in serum-deprived media for 72 h, stained with PE Annexin V/7AAD, and subjected to flow cytometry. The combined population of cells in Q2 (late apoptosis) and Q4 (early apoptosis) was used to determine the total percentage of apoptotic cells. *p < 0.05 MYB enhances migration, invasion, and EMT in ovarian cancer cells To understand the role of MYB in the aggressiveness of OC cells, we investigated if altering its expression affected cell motility and invasiveness. Our data revealed that overexpression of MYB significantly increased the motility of SKOV3-MYB (3.5-fold) and A2780-MYB (3.8-fold) cells when compared to their respective control cell lines (Fig. [78]3A). In contrast, silencing MYB expression resulted in a marked reduction in motility, with SKOV3-ip-shMYB and A2780-CP-shMYB cells showing a 4.2-fold and 3-fold decrease, respectively, in comparison to their control counterparts (Fig. [79]3B.). Our results also indicated that MYB-overexpressing SKOV3-MYB and A2780-MYB cells had a significantly higher invasive capability (5.3- and 4.3-fold, respectively) compared to control cells. On the other hand, MYB-silenced SKOV3-ip-shMYB and A2780-CP-shMYB cells exhibited a substantial reduction in invasiveness (4.4-fold and 3.8-fold, respectively) compared to their respective control lines (Fig. [80]3C and D). Fig. 3. [81]Fig. 3 [82]Open in a new tab MYB enhances migration, invasion, and EMT in ovarian cancer cells. A-D. Effect of MYB modulation on the migration and invasion of OC cells. Cells were plated (1 × 10^5 cells/well) in the non-coated (for migration) or Matrigel-coated (for invasion) transwell chambers and allowed to migrate for 16 h under a chemotactic drive. Cells that had migrated through the membrane were fixed, stained, and counted in 10 random view fields. Bars represent the mean ± standard deviation. *p < 0.05. E. Actin organization visualization. OC cells were seeded and cultured for 48 h. After that, cells were fixed, stained with Alexa Fluor 488-conjugated phalloidin, and then photographed using a fluorescent microscope. Filopodial projections are marked by red arrows. F. Expression of EMT markers was examined by immunoblotting using specific antibodies. β-actin was used as a loading control A hallmark of aggressive cancer cells is the acquisition of mesenchymal characteristics through a process known as epithelial-to-mesenchymal transition (EMT). This process is typically accompanied by changes in the actin cytoskeleton along with altered expression of key biomarker proteins. Therefore, we analyzed actin organization in control and MYB-modulated cells by staining the filamentous actin with FITC-conjugated phalloidin. We observed that MYB-overexpressing cells (SKOV3-MYB and SKOV3-ip-Scr) exhibited numerous filopodial protrusions, characteristic of mesenchymal cells, while low MYB-expressing cells (SKOV3-Neo and SKOV3-ip-shMYB) had fewer or no filopodial structures (Fig. [83]3E). Additionally, our data revealed a significant decrease in the expression of the epithelial marker E-cadherin in MYB-overexpressing cells (SKOV-MYB & SKOV3-ip-Scr) along with an upregulation of mesenchymal markers, Vimentin and N-cadherin, compared to the respective control cells (SKOV3-Neo & SKOV3-ip-shMYB) (Fig. [84]3F). Taken together, these data underscore the critical role of MYB in promoting the malignant phenotype of OC cells by driving the acquisition of mesenchymal traits through EMT. MYB-induced gene expression alterations vary among ovarian cancer cell types Since MYB is a transcription factor, we wanted to identify MYB-regulated genes to gain insights into its role in influencing OC cell phenotypes. For this, we subjected RNA isolated from MYB-modulated (SKOV3-ip-shMYB and SKOV3-MYB) and their respective control cells (SKOV3-ip-Scr and SKOV3-Neo) to gene expression profiling using nCounter^® PanCancer Progression Panel on NanoString nCounter^® system (Fig. [85]4A). A total of 87 differentially expressed genes (DEGs) were identified, of which 75 genes were upregulated, and 12 genes were downregulated in the MYB-overexpressing SKOV3-MYB cells (Fig. [86]4B and C). Similarly, we observed a total of 164 DEGs between control and knockdown SKOV3-ip cells, of which 149 genes were downregulated, and 15 genes were upregulated in MYB-silenced SKOV3-ip-shMYB cells (Fig. [87]4D and E). Interestingly, when we compared the DEGs between the MYB-modulated SKOV3 (MYB overexpression vs. Neo) and SKOV3-ip (MYB silencing vs. Scr) cells, we found that only 12 genes were common between these two model cell lines (Fig. [88]4F). This finding suggests that MYB exerts its regulatory effects on gene expression in a cell-specific manner, with distinct gene sets being regulated depending on the cellular context. The data highlights that besides its overexpression, MYB-mediated gene regulation is dependent on other intrinsic properties of the cell, possibly its interaction with other gene regulatory factors exhibiting altered expression. Fig. 4. [89]Fig. 4 [90]Open in a new tab Gene expression profiling in ovarian cancer cells having altered MYB expression. A. Total RNA was isolated from OC cells engineered for either forced overexpression or silenced expression of MYB and subjected to gene expression analysis using the NanoString PanCancer Progression Panel. B-E. Volcano plots and heatmaps were developed depicting the differentially expressed genes (DEGs) and fold-changes in the expression levels. F. Venn diagram showing the comparison of the DEGs from SKOV3 MYB gain of function and SKOV3-ip MYB loss of function models AKT3 is a direct transcriptional target of MYB in ovarian cancer cells Upon observing that 12 genes were commonly altered in two model cell lines upon MYB modulation, we validated the expression of these genes in a panel of other OC cell lines after genetically manipulating MYB expression (Supplementary Fig. [91]2). Among the genes tested, AKT3 emerged as the most consistently altered across all MYB-modulated cell lines, suggesting it as a direct downstream target of MYB (Fig. [92]5A). To confirm this, we conducted an in-silico analysis of the AKT3 promoter using the ALGGEN-PROMO tool. A total of seven putative MYB binding regions (denoted as P1-P7) were identified in AKT3 promoter (Fig. [93]5B). Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) analysis in SKOV3 and SKOV3-ip cells confirmed that MYB directly bound to the P4 region of the AKT3 promoter (Fig. [94]5C). An enhanced pull-down of the P4 MYB-binding sequence in the AKT3 promoter was observed in MYB-overexpressing SKOV3 cells while it was reduced in MYB silenced SKOV3-ip cells compared to their respective control cells (Fig. [95]5D). To ascertain whether MYB-induced transcriptional upregulation of AKT3 is reflected at the protein level, we assessed protein levels of AKT3. MYB-overexpressing OC cells had an elevated expression of AKT3 relative to control cells, which was also accompanied by elevated levels of phosphorylated AKT (p-AKT) and phosphorylated form of its downstream target, GSK3-β (Fig. [96]6A and B). Moreover, KEGG pathway enrichment analysis of DEGs in MYB-altered OC cell lines demonstrated PI3K-Akt signaling pathway as the most upregulated pathway (Fig. [97]6C). Collectively, these data suggest that AKT3 is a common MYB target genes in OC, associated with the activation of AKT signaling. Fig. 5. [98]Fig. 5 [99]Open in a new tab AKT3 is a direct transcriptional target of MYB in ovarian cancer cells. A. AKT3 mRNA expression was analyzed in a panel of MYB-modulated OC cell lines by qPCR. Actin Ct was used to normalize the data. B. In silico analysis of AKT3 promoter was performed using PROMO- ALGGEN to identify putative MYB binding sites (MBS). Seven putative MBS (P1-P7) were identified. C. Chromatin immunoprecipitation was performed using anti-MYB antibody or IgG control, followed by qPCR analysis using site-specific primers in SKOV3 and SKOV3-ip cells. The highest binding was recorded at the P4 site. The direct MYB binding in the P4 region of the AKT3 promoter increased in MYB-overexpressing cells and decreased in MYB-silenced OC cells. D. MYB-modulated cells were also subjected to ChIP-qPCR analysis, and enhanced pull-down of the P4 MYB-binding sequence in the AKT3 promoter was observed in MYB-overexpressing SKOV3 cells, while it was reduced in MYB-silenced SKOV3-ip cells compared to their respective control cells Fig. 6. [100]Fig. 6 [101]Open in a new tab PI3K/AKT signaling is induced in MYB-overexpressing ovarian cancer cells. A & B. MYB-regulated AKT3 expression was confirmed in OC cell lines with modulated MYB expression by immunoblotting, which correlated with relative increase or decrease in the phosphorylation of AKT and its downstream target, GSK3β. β-actin was used as a loading control. C. KEGG pathway analysis using the DEGs identified PI3K/AKT as the most activated pathway in MYB-overexpressing cells. The x-axis represents the significance of fold enrichment while the y-axis shows the pathway categories AKT3 mediates the actions of MYB on ovarian cancer cell growth, migration, invasion, survival and chemoresistance To further elucidate the functional contribution of AKT3 to the MYB-driven oncogenic phenotype, we silenced its expression in MYB-overexpressing OC cell lines. Efficient knockdown of AKT3 was confirmed by immunoblotting, which also led to a substantial reduction in phospho-AKT and phospho-GSK3-β (Fig. [102]7A). We next investigated the consequences of AKT3 knockdown on OC growth by WST1-assay and observed that the silencing of AKT3 in MYB-overexpressing cells significantly reduced the cell growth, relative to the not-targeted scrambled sequence (NT-Scr)-transfected cells (Fig. [103]7B & C). Additionally, live-dead staining following 72 h of serum starving revealed a notable increase in cell death in AKT3-silenced cells (Fig. [104]7D & E). We also analyzed the sensitivity of MYB-overexpressing cells to cisplatin upon AKT3 silencing. We observed that silencing of AKT3 significantly enhanced the sensitivity of both MYB-overexpressing cell lines to cisplatin relative to respective control transfected OC cells. The IC[50] value for cisplatin was reduced in SKOV3-MYB cells from 22.51 µM to 7.8 µM following AKT3 knockdown (Fig. [105]7F), whereas in SKOV3-ip cells, it decreased from 32.4 µM in control cells to 14.18 µM in AKT3-silenced cells (Fig. [106]7G). We also wanted to assess whether AKT3 silencing had any effect on the MYB mediated increase in cell motility and invasiveness of OC cells. We observed that when AKT3 was silenced, either in SKOV3-MYB or SKOV3-ip, there was a significant reduction in the migration and invasive potential of the cells (Fig. [107]7H-I). These results indicate that AKT3 plays a crucial role in mediating the oncogenic action of MYB in OC cells as well as in promotion of cisplatin resistances, suggesting a significant contribution of the MYB–AKT3 signaling axis in OC pathobiology. Fig. 7. [108]Fig. 7 [109]Open in a new tab AKT3 mediates the actions of MYB on ovarian cancer cell growth, survival, and chemoresistance. A. OC cells with forced (SKOV3-MYB) or endogenously high expression of MYB (SKOV3-ip) were treated with 50 nM of AKT3 siRNA for 48 h as described in the materials and methods. Subsequently, cell lysates were prepared and analyzed by immunoblotting. B & C. OC cell lines were seeded in 96-well culture plates and treated with 50 nM of AKT3 siRNA. After 72 h, cells were subjected to WST assay for assessment of cell growth. Bars represent the relative differences in the growth, *p < 0.05. D & E. SKOV3 and SKOV3-ip cells were transfected with either AKT3 siRNA (50nM) or scrambled siRNA (NT-Scr) and incubated for 24 h. After 24 h, the cells were washed and cultured for 72 h in serum-deprived media. Following this, LIVE/DEAD kit reagents (Ethidium homodimer and Calcein-AM) were added to the culture, which were then incubated for 20 min, rinsed 3 times with PBS. Live cells display green fluorescence while dead cells fluoresce Red/orange. The graph depicts the count of dead cells per well. (*p < 0.05). F & G. OC cells seeded in 96-well plates and transfected with 50 nM of AKT3 or scrambled siRNA were incubated for 24 h. Following that, these cells were treated with different concentrations of cisplatin for 72 h. After 72 h, percent viability was measured by WST-1 assay, and IC50 values were obtained in GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA), through the equation: log(inhibitor) vs. normalized response, using a Variable slope model. Effect of MYB modulation on the migration and invasion of OC cells. H & I. SKOV3-MYB and SKOV3-ip cells were transfected with 50 nM of either non-targeting scrambled siRNA (NT-Scr) or AKT3 siRNA for 48 h and subsequently were plated in the non-coated (for migration) or Matrigel-coated (for invasion) transwell chambers and allowed to migrate for 16 h under a chemotactic drive. Cells that had migrated through the membrane were fixed, stained, and counted in 10 random view fields. Bars represent the mean ± standard deviation. *p < 0.05 Discussion Our findings highlight the crucial role of MYB in OC pathogenesis and chemoresistance, revealing its multifaceted contribution in enhanced cell proliferation, clonogenicity, survival, migratory and invasive behavior, and EMT. While MYB has long been implicated in hematological malignancies and select solid tumors [[110]23, [111]26], our data extends these observations to OC, where MYB appears to serve not only as a biomarker of aggressiveness but as a potential driver of therapeutic resistance. The finding that MYB is more abundant in aggressive and cisplatin-resistant OC cell lines than in their sensitive counterparts provides strong rationale for further investigation in clinical settings, particularly in light of the limited treatment options for recurrent OC [[112]27]. Through a combination of gain- and loss-of-function studies, we demonstrated that MYB expression modulated OC cell growth and clonogenicity, reinforcing previous findings in pancreatic, prostate, breast, and colorectal cancers [[113]7, [114]10, [115]11, [116]28–[117]32]. Our studies revealed that MYB facilitates S-phase entry and protects cells from apoptosis under stress conditions, aligning with its previously described role in maintaining cell cycle progression and survival [[118]33, [119]34]. Notably, MYB knockdown resulted in G0/G1 arrest and a marked increase in apoptosis, underscoring its role in cell fate decisions. Importantly, MYB overexpression was associated with increased resistance to cisplatin, whereas silencing MYB restored sensitivity. This suggests that MYB may be part of a broader regulatory framework influencing DNA repair, drug efflux, and apoptotic thresholds, mechanisms commonly implicated in platinum resistance [[120]35]. However, it yet remains to be explored in more focused and in-depth studies if MYB directly or indirectly regulates the expression or functions of the key resistance mediators such as ERCC1 or ABC transporters. Furthermore, evasion of apoptosis supports both primary and acquired chemoresistance [[121]36]. In that context, our findings demonstrate a role of MYB in integrating growth signals and survival under stress are hold significance from the mechanistic standpoint. Transcriptomic profiling showed that MYB regulates distinct gene sets in different OC cell line models. The limited overlap in differentially expressed genes between overexpression and knockdown models suggests that the transcriptional output of MYB is shaped by additional cell-intrinsic factors such as chromatin architecture or co-regulator availability [[122]37]. Indeed, our recently published reports and several other findings demonstrate that MYB interacts with other transcription factors and likely engages in cooperative gene regulation [[123]12–[124]14, [125]38, [126]39]. This context specificity is particularly pertinent in OC, a molecularly heterogeneous disease comprising multiple histotypes with divergent biology [[127]40]. Thus, our findings point toward the molecular complexity of OC and need for a better understanding of cross-talking gene networks that can be used for better disease stratification and treatment planning. One of the most compelling findings was the identification of AKT3 as a direct MYB transcriptional target. Our data confirmed MYB occupancy at the AKT3 promoter, and functional assays established an essential role of AKT3 in maintaining the MYB-driven malignant phenotypes. While AKT3 has previously been implicated in cancer progression and survival [[128]19, [129]41], our data place it within a novel MYB-regulated axis in OC where it acts as a key driver of oncogenesis. Given that AKT signaling is frequently upregulated in OC through mutations or amplification [[130]42, [131]43], the MYB–AKT3 connection offers a potential new explanation and enforces future investigations into the sustained pathway activation, especially in MYB-high tumors. Regardless, our transcriptomic analyses implicate PI3K-AKT signaling as a dominant effector of MYB, suggesting synergy between transcriptional and signaling networks in promoting ovarian malignancy. Although targeting transcription factors remains a formidable challenge, our results provide an indirect and alternate way of targeting MYB induced oncogenic effects via disruption of key effectors like AKT3. Recent advances in Proteolysis Targeting Chimeras (PROTACs) and selective inhibitors of co-regulators (e.g., CBP/p300, BET proteins) may offer avenues to destabilize MYB-driven transcriptional programs [[132]44]. Furthermore, AKT3, as a kinase, presents a more conventional therapeutic target. Given the distinct roles of AKT isoforms, developing AKT3-specific inhibitors could yield more precise interventions with reduced toxicity compared to pan-AKT blockade [[133]45]. Clinical translation will require in vivo validation, including studies in patient-derived xenografts or organoids, and correlation of MYB-AKT3 expression with treatment outcomes. In summary, our study highlights multifactorial roles of MYB in OC progression, from promoting growth and survival to fostering chemoresistance and EMT. Through its transcriptional control of AKT3, MYB orchestrates a signaling axis that not only promotes malignant progression but also likely underpin the treatment failure. While there are still inherent questions remaining regarding the upstream regulation of MYB and its broader involvement in transcriptional programming under different cellular and environmental contexts, our findings provide a framework for exploiting MYB as a potential therapeutic vulnerability in OC. Supplementary Information Below is the link to the electronic supplementary material. [134]Supplementary Material 1^ (274.8KB, docx) Acknowledgements