Introduction
Ovarian cancer ranks as the eighth most common malignancy among females globally, with 3,24,398 new cases and 2,06,839 new deaths in 2022 [1]. Early diagnosis of ovarian cancer is particularly challenging as the absence of typical early clinical manifestations. Consequently, nearly 70% of individuals are identified at a late stage, leading to a 5-year survival rate below 50% [2]. Notably, cytoreductive surgery followed by platinum-based chemotherapy, such as cisplatin (CDDP), remains the primary therapeutic approach for ovarian cancer [3]. However, a majority of patients eventually suffer from platinum resistance, resulting in recurrence and increased mortality [4]. Thus, it is imperative to further investigate the mechanism behind platinum resistance in OC, aiming to facilitate the development of innovative therapeutic approaches and ultimately enhance patient outcomes.
The KLF family is a class of zinc-finger transcription factors, distinguished by three conserved Cys2/His2 zinc-finger regions located at the C-terminal end [5]. They were first identified as developmental regulators in drosophila embryos and subsequently proved to be highly conserved in mammals, with a total of 18 family members identified in the human genome [6]. The KLF gene participate in regulating cell proliferation, cell differentiation, embryonic stem cell fate and maintenance of tissue and system homeostasis [7]. In this condition, aberrant KLF expression has been proved to be closely associated with tumorigenesis through multiple mechanism, including enhancing cell proliferation, inhibiting apoptosis, and maintaining the stemness of cancer stem cells [8]. KLF4, a member of the KLF gene family, has been associated with the development and progression of several type of cancer, such as non-small cell lung cancer, colorectal cancer and pancreatic cancer [9–13]. However, the association between cancer progression and KLF4 exhibits heterogeneity across diverse cancer types. For example, the cell proliferation and migration of prostate cancer cells were suppressed by KLF4 [14], whereas KLF4 promoted stemness maintenance and tumor metastasis capacity of breast cancer cells [15]. Generally, the dual role of KLF4 in different cancers may depends on tumor types or subtypes, stages of progression, diverse molecular regulatory pathways and cellular context [16]. At present, the important role of KLF4 in OC remains inconclusive. One study compared the DNA methylation and transcriptome changes in patient-derived primary tumor cells, it was observed that KLF4 was up-regulated in platinum-resistant OC cells and may induce the expression of IL-6 and other components in response to cisplatin treatment [17]. Therefore, it is necessary to elucidate the possible involvement of KLF4 in platinum resistance of OC.
The mTORC1 signaling is a highly conserved transduction network that critical to cell metabolism, proliferation and response to environmental stimuli [18]. Dysregulation of this pathway has been recognized as a key driver of tumor progression [19–21]. Notably, hyperactivation of mTORC1 pathway is also strongly linked to chemotherapy resistance and unfavorable prognosis in various tumors, including the ovarian cancer [22–24]. However, it is disappointing that no mTOR pathway inhibitors have been formally approved for clinical use as for their inconsistent efficacy and harmful side effects [25, 26]. Consequently, new predictive molecular markers and new therapeutic target combinations are urgently needed to overcome chemotherapy resistance.
Here, we identified the role of KLF4 in OC platinum resistance. Further investigation revealed that KLF4 promotes platinum resistance in OC, which can be ascribed to the activation of mTORC1 signaling pathway. Therefore, targeting KLF4 could be a promising strategy to overcome platinum resistance in ovarian cancer.
Methods and materials
Clinical samples
From 2017 to 2022, 68 samples of ovarian cancer patients who received cisplatin chemotherapy and provided informed consent at Shanghai Sixth People’s Hospital were collected. Patients with disease progression or tumor recurrence within 6 months after cisplatin chemotherapy were considered cisplatin-resistant group. Patients relapsing for 6 months or more were considered cisplatin-sensitive group. The Research Ethics Committee of Shanghai Sixth People’s Hospital approved this study.
Cell culture and treatment
Human OC cell lines A2780, SKOV3 and the cisplatin-resistant cell lines A2780DDP and SKOV3DDP were conserved in the State Key Laboratory of Oncogenes and related genes, Shanghai Cancer Institute. These cells were cultured in DMEM (C11995500BT, Gibco) supplemented with 10% fetal bovine serum (FBS) and 100 μg/mL penicillin/streptomycin at 37 °C under a 5% CO2 atmosphere. Cisplatin (HY-17394, MCE), MLN0128 (HY-13328, MCE) and rapamycin (AY-22989, MCE) were individually dissolved in sterile ddH2O or DMSO to prepare 10 mM stock solutions. Cisplatin (10 µM for A2780DDP and SKOV3DDP, 5 µM for A2780 and SKOV3) was added to the cell culture medium alone or in combination with either MLN0128 (50 nM) or rapamycin (100 nM) as required for the assay.
Cell transfection
Lentivirus with short hairpin RNAs (shRNA) targeting KLF4 as well as the control virus were constructed by GeneChem (Shanghai, China), and Lentivirus expressing exogenous KLF4 along with the control virus were constructed by GeneChem (Shanghai, China). The viruses were transfected respectively into cells together with 5 μg/mL polybrene. 10 μg/mL of puromycin (A1113803, Thermo Fisher) was then administered to eliminate uninfected cells. The KLF4 stable knockdown cells were transfected with KLF4 overexpression lentivirus to restore KLF4 expression.
Half maximal inhibitory concentration (IC50) assay
To test the sensitivity of cisplatin, OC cells were cultured in 96-well plates (2 × 103 cells/well) for 24 h. The original culture medium was discarded and incubated with various concentrations of cisplatin alone or combined with mTOR inhibitor for 48 h. The viability of cells was evaluated by cell counting kit-8(CCK-8) (B34304, Bimake), and OD450mm was detected 1 h after addition of 10% CCK-8. Afterward, the IC50 values were calculated with GraphPad Prism 8.0.
Colony formation assay
The different groups cells were cultured into 6-well plates (1 × 103 cells/ well) and treated with cisplatin alone or combined with mTOR inhibitor. After 2 weeks, the clones were treated with 4% paraformaldehyde (PFA) for a period of 15 min followed by staining with 1% crystal violet for 30 min.
Cell apoptosis rate assay
Target cells were treated with cisplatin alone or combined with mTOR inhibitor and then harvested after 48 h. Flow cytometry technology was used to quantitative apoptosis ratio by Annexin V-FITC-PI staining, according to the established protocol.
Protein extraction and western blotting
Cultured Cells were separated by trypsin (25,300,062, Gibco), and lysed with RIPA buffer (G2033, Servicebio) to extract total protein. BCA protein quantification kit (23,227, Thermo Fisher) was employed to measure protein concentration. Equal quantities of protein from each group were separated using SDS-PAGE gel electrophoresis and then transferred to a nitrocellulose membrane (66,485, PALL). After blocking with 5% BSA for 1 h, the nitrocellulose membrane was incubated with primary antibodies against the target protein overnight at 4 °C, followed by incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Antibodies used in this study were as follows: anti-KLF4 (1:1000, ab215036, Abcam), anti-mTOR (1:1000, #2983, CST), anti-Phospho-mTOR Ser2448 (1:1000, #5536, CST), anti-S6K (1:1000, #2708, CST), anti-Phospho-S6K Thr389 (1:1000, #9234, CST), anti-4E-BP1 (1:1000, #9644, CST), anti-Phospho-4E-BP1 Ser65 (1:1000, #9451, CST), anti-β-tubulin (1:1000, 10,094–1-AP, Proteintech), HRP-Goat anti-Ribbit IgG (1:8000, 111–035-003, Jackson). Enhanced chemiluminescence reagent (WBKLS0500, Merck) and Bio-Rad imaging system (Bio-Rad, USA) were used to visual protein bands.
RNA extraction and quantitative real-time PCR (qRT-PCR)
mRNA isolation and qRT-PCR assay were conducted as previously described [27]. All data was normalized to gene 18 s. Primer sequences included the following:
18 s forward: GGCCCTGTAATTGGAATGAGTC.
18 s reverse: CCAAGATCCAACTACGAGCTT.
KLF4 forward: CAGCTTCACCTATCCGATCCG.
KLF4 reverse: GACTCCCTGCCATAGAGGAGG.
Mouse subcutaneous xenograft models
6-to 8-week-old female BALB/c nude mice were selected for in vivo experiments, and all animal studies receiving approval from the Shanghai Sixth People’s Hospital Research Ethics Committee. For subcutaneous xenograft models, 2 × 106 shNC or shKLF4 A2780DDP cells were subcutaneously injected. Once the xenografts became visible, the shNC or shKLF4 group mice were divided randomly into 2 subgroups (n = 5), and either PBS or CDDP solution (5 mg/kg) was administered intraperitoneally in equal volumes twice a week. The xenografts sizes were measured every week with calipers. The xenografts volume was estimated based on the equation: V = 1/2 (width × width × length). The mice were euthanized using CO2 after 4 weeks form injection, and the xenografts were weighed. Afterward, the tumor specimens were treated with 4% paraformaldehyde and prepare for subsequent immunohistochemistry (IHC) analysis.
Immunohistochemistry
Tumor samples from patients and xenografts from mice were embedded in paraffin blocks and sliced into tissue sections for IHC assay. IHC and scoring were performed according to previous research [27], and photographed under an optical microscope. The primary antibodies utilized in this investigation were as follows: anti-KLF4 (1:2000, ab215036, Abcam) and anti-Ki67 (GB111141, Servicebio).
Statistical analyses
GraphPad Prism 8.0 software and IBM SPSS Statistics 22.0 software were employed to statistical analyses in this study. The significance of data comparison was calculated using the unpaired t-test, ANOVA or chi-square test, depending on suitability. For all results, the statistical significance was determined by P-value < 0.05.
Results
High KLF4 expression associated with platinum resistance and unfavorable prognosis in OC patients
In order to investigate the possible target genes affecting the chemotherapy resistance of OC, we initially gathered the clinical data of OC patients from the TCGA database and categorized them into platinum-resistant groups and platinum-sensitive groups based on a 6-month platinum-free interval (Fig. 1A). Compared with the platinum-sensitive group, we identified a cluster of genes with higher expression in the platinum-resistant group. Subsequently, we obtained the InIC50 for cisplatin in 25 OC cell lines from the GDSC database and paired them with mRNA expression profile gained from CCLE database. The 25 OC cell lines were divided into two cohorts according to the median InIC50 value for further investigation of differentiated expressed genes (DEGs) (Fig. 1B). Additional DEGs analysis was performed using two datasets obtained from the GEO database. The GSE58470 dataset comprised the expression profile data of cisplatin sensitive and resistant IGROV-1 cell line. The GSE23553 dataset comprised the expression profile data of cisplatin sensitive and resistant A2780 cell line. Intersecting the four aforementioned gene clusters, we identified KLF4 as the target gene for further investigation (Fig. 1C).
Fig. 1 [Images not available. See PDF.]
High KLF4 expression is associated to platinum resistance and unfavorable prognosis in OC. A. According to 6-month platinum-free interval, OC patients were categorized into resistant group (n = 67) and sensitive group (n = 135), based on TCGA database. B. According to median cisplatin InIC50 values (InIC50 = 3.76), 25 OC cell lines were divided into resistant group (n = 11) and sensitive group (n = 14), based on GDSC database. C. The Venn diagram illustrated the identification of the target gene KLF4 from the overlap of four distinct gene clusters, as follow: 1. Gene clusters upregulated in platinum-resistant versus platinum-sensitive patients in TCGA dataset; 2. Gene cluster with higher expression in cisplatin-resistant OC cell lines compared to cisplatin-sensitive OC cell lines in GDSC database; 3. Gene cluster upregulated in cisplatin-resistant IGROV-1 cells compared to cisplatin-sensitive IGROV-1 cells in GSE58470; 4. Gene cluster upregulated in cisplatin-resistant A2780 cells compared to cisplatin-sensitive A2780 cells in GSE23553. D. Expression analysis of KLF4 in various OC cell lines under conditions with or without cisplatin treatment, based on dataset GSE47856. E. Kaplan–Meier analysis was performed to evaluate the overall survival of OC patients based on KLF4 expression, utilizing dataset GSE26712. F. Detailed expression analysis of KLF4 in the TCGA, GDSC, GSE58470, and GSE23553 databases. The P-value was calculated by an unpaired t-test. G. Representative IHC images and statistical analysis showed the KLF4 expression between cisplatin-sensitive tissues and cisplatin-resistant tissues. Scale bar: 200 μm. The P-value was calculated by chi-square test. *P < 0.05, **P < 0.01
In the previous database analysis, we observed a significant overexpression of KLF4 mRNA in cisplatin-resistant OC cell lines and tissue samples, as compared to cisplatin-sensitive counterparts (Fig. 1F). The finding was validated in an additional OC cells cohort, wherein the mRNA levels of KLF4 was upregulated in response to cisplatin treatment (Fig. 1D). The Kaplan–Meier analysis further indicated a close association between unfavorable prognosis and high KLF4 expression in ovarian cancer (Fig. 1E). Furthermore, we collected the OC tissue specimens from patients and adopted IHC assay to measure KLF4 protein levels. Consistent with the findings from mRNA level analysis, the protein level of KLF4 was significantly upregulated in cisplatin-resistant OC tissues (Fig. 1G). Taken together, our findings suggested that increased expression level of KLF4 is correlated with chemotherapy resistance and unfavorable outcomes in OC patients.
KLF4 enhances cisplatin resistance in ovarian cancer cells
To investigate the functions of KLF4 in chemotherapy resistance of OC, we assessed the KLF4 mRNA expression level in multiple OC cell lines. KLF4 exhibited significant expression levels in A2780DDP and SKOV3DDP cells, the platinum-resistant strain of A2780 and SKOV3 (Fig. 2A). Western blotting experiment also revealed that KLF4 was overexpressed in the resistant strains compared to the parent strains (Fig. 2C). Besides, the cisplatin IC50 was significantly higher in cisplatin-resistant strains compared to the parental strains, as determined by the CCK-8 assay (Fig. 2B). Therefore, A2780DDP and SKOV3DDP were selected for subsequent experiments. Using two independent shRNAs targeting KLF4, we established stable KLF4-knockdown cell lines in both A2780DDP and SKOV3DDP cell types, and validated the interference efficiency by qRT-PCR and western blotting (Fig. 2D). The CCK-8 assay findings showed that A2780DDP and SKOV3DDP cell lines with KLF4 knockdown exhibited a significantly lower calculated IC50 values compared to shNC cells (Fig. 2E). This result revealed that suppression of KLF4 might enhance the cisplatin sensitivity of OC cells. Furthermore, colony formation assay demonstrated that knocking down KLF4 markedly reduced the OC cells proliferation when treated with cisplatin (Fig. 2F). Besides, flow cytometry was adopted to examine cell apoptosis. We observed that knockdown of KLF4 significantly elevated the cell apoptosis rate after administration of cisplatin for 48 h (Fig. 2G). Accordingly, our findings indicated that KLF4 could enhance the cisplatin resistance of OC cells in vitro.
Fig. 2 [Images not available. See PDF.]
Knockdown of KLF4 promotes the cisplatin sensitivity of OC cells in vitro. A. KLF4 mRNA expression level between different OC cell lines. B. The relative cell viability of A2780, A2780DDP, SKOV3 and SKOV3DDP cells following exposure to varying different concentrations of cisplatin. IC50 value as follow: IC50 A2780 = 14.81 µM, IC50 A2780DDP = 36.98 µM, IC50 SKOV3 = 15.82 µM, IC50 SKOV3DDP = 42.98 µM. C. Protein expression of KLF4 in A2780DDP and SKOV3DDP cells and their parental strains were assessed using western blotting. D. Interference efficiency of KLF4 in OC cells was assessed using qRT-PCR and western blotting analysis. E. The sensitivity of shNC, shKLF4-1 and shKLF4-2 OC cells to cisplatin were evaluated by CCK-8 assays. IC50 value of A2780DDP cells: IC50 shNC = 29.75 µM, IC50 shKLF4-1 = 22.10 µM, and IC50 shKLF4-2 = 23.46 µM; IC50 value of SKOV3DDP cells: IC50 shNC = 34.02 µM, IC50 shKLF4-1 = 14.77 µM, and IC50 shKLF4-2 = 14.03 µM. F. The colony formation assay was conducted in shNC, shKLF4-1 and shKLF4-2 OC cells after treatment with cisplatin, the representative images and statistical analysis were shown. G. The apoptotic rate of shNC, shKLF4-1 and shKLF4-2 OC cells treated with cisplatin were detected by Flow cytometry assay, the representative images and statistical analysis were shown. The P-value was calculated by unpaired t-test. *P < 0.05, **P < 0.01
KLF4 knockdown sensitized OC cells to cisplatin in vivo
Our above research showed that interfering KLF4 expression effectively reduces cisplatin resistance in OC cells in vitro. Subsequently, subcutaneous xenograft model was utilized to explore the biological functions of KLF4 in vivo. Nude mice were inoculated separately with shNC or shKLF4 A2780DDP cells, and then treated with cisplatin or PBS twice a week. The results indicated that knockdown of KLF4 led to a reduction in both tumor volume and weight, while significantly increased the tumors responsiveness to cisplatin in comparison to the control group (Fig. 3A–C). Further IHC analysis showed that positive rate of the proliferation marker Ki67 decreased after KLF4 knockdown, which was more significant after cisplatin treatment (Fig. 3D). The results demonstrated that silencing KLF4 can enhance responsiveness of cisplatin-resistant cells to the drug and impeded tumor proliferation in vivo, suggesting that KLF4 could be a prospective target for overcoming cisplatin resistance in OC.
Fig. 3 [Images not available. See PDF.]
Knockdown of KLF4 suppresses the cisplatin resistance of OC cells in vivo. A. A2780DDP cells transfected with shKLF4 or shNC lentivirus were subcutaneously injected into BALB/c nude mice. Each group was randomly divided into two subgroups and treatment with CDDP (5 mg/kg) or an equivalent PBS control. After 4 weeks, xenograft tumors from each group were excised and exhibited. B. Growth curves of xenografts from each group. C. The quantitative analysis of xenografts weight from each group. D. Representative IHC images and statistical analysis for Ki67 in xenografts from each group. Scale bar: 200 μm. Two-way ANOVA was selected to compare growth curves, unpaired t-test was selected to compare the data between two groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Silencing KLF4 inhibits the activation of mTORC1 signaling
Given the crucial role of KLF4 in facilitating chemoresistance in OC, we intended to further elucidate the potential mechanisms by which KLF4 functions. We isolated RNA from KLF4-knockdown and control A2780DDP cells respectively, and compared the gene expression profiles of two groups via high-throughput transcriptome sequencing analysis (RNA-seq) (Fig. 4A–B). Gene set enrichment analysis (GSEA) using Hallmarks gene sets showed that dysregulated expression of KLF4 profoundly enriched in the pathways associated with tumor growth, cell survival, and cellular metabolism, including mTORC1 signaling, PI3K/AKT/mTOR signaling, cholesterol homeostasis, P53 pathway, xenobiotic metabolism (Fig. 4D). Among these results, mTORC1 signaling is the most affected target pathways related to KLF4 with a normalized enrichment score (NES) of 2.590 (Fig. 4C). Previous research have demonstrated that irregular activation of mTORC1 pathway is crucial in tumor progression and drug resistance [25]. In our study, we conducted single-sample gene set enrichment analysis (ssGSEA) on the OC patients from several GEO dataset based on mTORC1 signaling genes and calculated the mTORC1 activity score for each patient. The results showed that patient with higher mTORC1 scores had a shorter overall survival time (Supplementary Fig. 1). These finding provides strong evidence supporting the critical role of mTORC1 activation in ovarian cancer. Therefore, we further investigated whether KLF4 mediates the activation of mTORC1 signaling. Through analysis of the TCGA database, we observed a positive correlation between the expression of KLF4 and mTORC1-signaling signatures genes (Fig. 4E). Further GSEA derived from TCGA database revealed a significant enrichment of genes involved in the mTOCR1 pathway within the KLF4-overexpression group (Fig. 4F). To further elucidate the role of KLF4 in mTORC1 activation, we knockdown KLF4 and devised corresponding experiments for validation. Western blotting assay was employed to assess the activation of mTOR and its downstream targets in shKLF4 OC cell lines. Consistent with RNA-seq results, knocking down KLF4 led to a decreased in the phosphorylation levels of mTOR, S6K, as well as 4E-BP1, without affecting the total protein abundance (Fig. 4G). More significantly, after reintroducing KLF4 into KLF4-knockdown cells, we observed that downregulation of KLF4 led to a decrease in IC50, colony formation ability and apoptosis resistance upon cisplatin treatment (Supplementary Fig. 2A–C); however, these effects were restored following the reintroduction of KLF4. Moreover, the re-expression of KLF4 restored the activation levels of mTOR and its downstream molecules (Supplementary Fig. 2D). The findings confirmed that the effects observed in KLF4-knockdown cells were specific due to the down-regulation of KLF4.Taken together, our results suggested that KLF4 may promote OC cisplatin resistance by activating the mTORC1 signaling pathway.
Fig. 4 [Images not available. See PDF.]
KLF4 regulates the activation of mTORC1 signaling in OC. A. Gene expression heatmap of A2780DDP cells after KLF4 knockdown. B. Volcano plotting of A2780DDP cells after KLF4 knockdown. C–D. GSEA using the hallmark gene set was performed to identified the pathway affected by KLF4 knockdown based on RNA-seq result. The bubble chart visually presented the significant enrichment pathways (D), GSEA result showing that mTOR signaling was enriched in A2780DDP cells with high KLF4 expression (C). E. The correlation analysis between KLF4 expression and mTORC1 activity based on TCGA database. F. GSEA using the hallmark gene set showed mTORC1 signaling was enriched in KLF4 high expression group. G. Western blotting result of KLF4 and total and phosphorylated mTOR, S6K, 4E-BP1 in shNC and shKLF4 OC cell lines
KLF4 promotes OC cisplatin resistance via mTORC1 signaling cascades
To further validate that KLF4 activates mTORC1 pathway, we overexpressed KLF4 in the parental A2780 and SKOV3 cell lines, considering their lower expression level of KLF4 and increased sensitivity to cisplatin, and subsequently conducted experiments to investigate the alterations in cisplatin sensitivity and activation of the mTORC1 pathway. Rapamycin (Rapa), a mTOR phosphorylation inhibitor, was employed to modify the activation status of the mTORC1 signaling in A2780 and SKOV3 cell lines. Western blotting analysis showed that KLF4 overexpression enhanced the phosphorylation levels of mTOR, S6K, 4EBP1. However, treated with rapamycin depressed the KLF4-induced mTOR, S6K and 4EBP1 phosphorylation (Fig. 5D). By examining the cisplatin IC50 of each group, we found that KLF4 overexpression improved the platinum resistance in OC cells, while treatment with rapamycin restored cellular sensitivity to cisplatin (Fig. 5A). Similarly, rapamycin reduced the promotive effect of KLF4 overexpression in colony formation under cisplatin exposure (Fig. 5B). Apoptosis assays indicated that KLF4 overexpression significantly decreased the apoptosis in response to cisplatin. However, rapamycin treatment could abolish the tolerance caused by KLF4 overexpression (Fig. 5C). The above experiments were repeated when employing MLN0128, a pan-mTOR inhibitor, as the therapeutic intervention. In KLF4-overexpressing cells, the cisplatin resistance assessed by CCK8 assay, colony formation assay as well as apoptotic assay (Supplementary Fig. 3A–C) and the mTORC1 signaling activation (Supplementary Fig. 3D) were also attenuated following MLN0128 treated. The results further verify the effect of mTORC1 signaling inhibition in reversing KLF4-mediated cisplatin resistance.
Fig. 5 [Images not available. See PDF.]
KLF4 promotes OC cisplatin resistance through mTORC1 pathway. A. CCK-8 assay to detect the cisplatin IC50 of Vector + DMSO, KLF4 + DMSO, Vector + Rapa, KLF4 + Rapa cell groups. The IC50 values for A2780 cells in turn: 21.59, 27.89, 13.99, 18.65. The IC50 values for SKOV3 cells in turn: 15.32, 23.70, 8.51, 13.66. B. The colony formation assay of Vector + DMSO, KLF4 + DMSO, Vector + Rapa, KLF4 + Rapa cell groups treated with cisplatin, the representative images and statistical analysis are shown. C. Apoptotic assay and statistical analysis of Vector + DMSO, KLF4 + DMSO, Vector + Rapa, KLF4 + Rapa cell groups treated with cisplatin. D. Western blotting result in Vector + DMSO, KLF4 + DMSO, Vector + Rapa, KLF4 + Rapa cell groups. The P-value was calculated by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Taken together, these findings suggested that overexpressed KLF4 enhanced the cisplatin resistance in OC cells, conversely, inhibition of mTORC1 activity abrogated such effect. It supports our hypothesis that KLF4 promotes OC cisplatin resistance via the activation of mTORC1 signaling cascades.
Discussion
Currently, platinum-based chemotherapy remains the cornerstone of primary therapeutic intervention in ovarian cancer [28]. Despite 80% of OC patients initially respond well to platinum drugs, almost all patients eventually develop chemotherapy resistance due to various mechanisms, such as DNA repair abnormalities, drug efflux pumps, and suppression of apoptosis signaling pathways [29]. Therefore, searching for a novel target to overcome chemotherapy resistance may significantly improve the prognosis of OC patients. Here, we have described the crucial role of KLF4 in platinum resistance in ovarian cancer for the first time. Our findings highlight that KLF4 could work as a potential therapeutic target gene for reversing platinum resistance. We propose that the KLF4’ role in activating mTORC1 pathway is key to facilitate cisplatin resistance. These results contribute to further uncover the cisplatin resistance mechanism in ovarian cancer and provide novel insight into targeted therapy for cisplatin-resistant ovarian cancer.
Based on current research, aberrant expression of KLF4 has been firmly associated with tumor progression [16, 30]. However, the role of KLF4 in tumors is intricate and heterogeneous. The context-dependent function of KLF4 relies on diverse environmental stimuli, types of cancer, and molecular regulation [16, 31–33]. Through bioinformatics analysis on the TCGA, GDSC, CCLE, and GEO databases, along with IHC examination of clinical tissue samples, we demonstrated a significant upregulation of KLF4 in platinum-resistant OC patients, which was found to be associated with an unfavorable overall survival. Consistent with the findings of Lund et al. [33] we also found an overexpression of KLF4 in cisplatin-resistant OC cells. Moreover, by employing both in vitro and in vivo experiments, we revealed the role of KLF4 in facilitating cisplatin resistance in OC. Excitingly, Tuo et al. [34] had demonstrated that suppressing KLF4 could promote cisplatin-induced apoptosis and enhance drug sensitivity in lung cancer cells, which further supports our standpoint. Conversely, another study showed that KLF4 could inhibit the migration and proliferation of OC cells by inhibiting TGF-β-induced EMT pathway [35]. This disparity might be attributed to the variations of cell lines subtypes employed in the experiments, as well as the difference in specific stimulus. Thus, further investigation is still needed to distinguish the correlation between subtypes of OC, cancer staging, isomers, and subcellular localization with the function of KLF4.
To further explore the underlying mechanism that KLF4 promotes drug resistance, we performed RNA-seq on cisplatin-resistant OC cells in both shKLF4 group and control group. Subsequent GSEA analysis revealed that decreased expression of KLF4 mainly affected pathways related to cell survival and metabolism, including mTORC1 signaling, cholesterol homeostasis, p53 pathway, and so on. Interestingly, we noted a robust correlation between the mTORC1 pathway and KLF4. It’s well-established that mTORC1 pathway is essential in regulating cell growth, cellular metabolism as well as tumor progression [36–38]. Indeed, aberrant activation of mTORC1 pathway has been observed to participate in the progression and drug resistance in OC [25]. For instance, increased levels of p-S6K was observed in the ascites samples obtained from chemotherapy non-responsive patients [39]. IHC analysis revealed an increased expression level of p-4EBP1 and p-S6K in OC tissues, and the former exhibited a significant correlation with an unfavorable prognosis [40]. Here, we showed a negative correlation between mTORC1 activity score and OS time in ovarian cancer patients, consistently. We also observed that silencing KLF4 can effectively attenuates the activation of mTORC1 signaling. Through further functional rescue experiments, we confirmed the crucial role of mTORC1 pathway activation in KLF4-mediated OC drug resistance. However, one limitation of this study as the underlying molecular mechanisms by which KLF4 activates mTORC1 signaling remains unclear. Further investigation is warranted to elucidate this aspect.
In summary, our study demonstrates that KLF4 is upregulated in platinum-resistant OC and associated with platinum resistance. The aberrant expression of KLF4 enhances the chemoresistance of OC via activating the mTORC1 pathway. Our findings suggest that targeting KLF4 may improve the response rate to platinum drugs in chemotherapy-resistant OC patients. These findings will offer novel strategies for overcoming cisplatin resistance in OC.
Author contributions
Conceptualization: W.Z. and H.H. Funding acquisition: Y.T. Methodology: W.Z. and H.H. Statistical analysis: W.Z. and H.H. Project administration: Y.H. and X.L. Supervision: Y.H. and X.L. Writing-original draft: W.Z. and R.H. Writing review and editing: R.H. and X.L. All authors read and approved the final manuscript.
Funding
The research was supported by National Natural Science Foundation of China (82172934).
Data availability
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Declarations
Conflict of interest
The authors declare no competing interests.
Ethical approval
All patients gave written informed consent for using clinical data in the study in accordance with the Declaration of Helsinki. This study was approved by the Research Ethics Committee of Shanghai Sixth People’s Hospital, affiliated with Shanghai Jiao Tong University School of Medicine (2021-YS-075). All methods were performed in accordance with the guiding principles of the Research Ethics Committee of Shanghai Sixth People’s Hospital. All animal procedures were conducted in accordance with the National Laboratory Animal Guideline for Ethical Review of Animal Welfare and the ARRIVE guidelines. The tumor burden of the experimental animals involved in this study did not exceed the maximum tumor burden allowed by the guidelines.
Abbreviations
Eukaryotic translation initiation factor 4E-binding protein 1
Analysis of variance
Cancer cell line encyclopedia
Cis-Diaminodichloroplatinum
Dulbecco’s modified eagle medium
Dimethyl sulfoxide
Epithelial-mesenchymal transition
Fetal bovine serum
Fluorescein isothiocyanate
Genomics of drug sensitivity in cancer
Gene expression omnibus
Gene set enrichment analysis
Immunohistochemistry
Kyoto Encyclopedia of Genes and Genomes
KLF transcription factor 4
Negative control
Normalized enrichment score
Ovarian cancer
Optical density at 450 nm
Tumor protein p53
Paraformaldehyde
Phosphatidylinositol 3-kinase
Rapamycin
Ribosomal protein S6 kinase
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
The cancer genome atlas
Transforming growth factor-β
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
1. Bray, F; Laversanne, M; Sung, H; Ferlay, J; Siegel, RL; Soerjomataram, I; Jemal, A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin; 2024; 74,
2. Siegel, RL; Miller, KD; Jemal, A. Cancer statistics, 2019. CA Cancer J Clin; 2019; 69,
3. Lheureux, S; Gourley, C; Vergote, I; Oza, AM. Epithelial ovarian cancer. Lancet; 2019; 393,
4. Song, M; Cui, M; Liu, K. Therapeutic strategies to overcome cisplatin resistance in ovarian cancer. Eur J Med Chem; 2022; 232, 114205.[COI: 1:CAS:528:DC%2BB38XkvFSksrY%3D] [DOI: https://dx.doi.org/10.1016/j.ejmech.2022.114205] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35217497]
5. Ghaleb, AM; Nandan, MO; Chanchevalap, S; Dalton, WB; Hisamuddin, IM; Yang, VW. Krüppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation. Cell Res; 2005; 15,
6. Kaczynski, J; Cook, T; Urrutia, R. Sp1- and Krüppel-like transcription factors. Genome Biol; 2003; 4,
7. Zeng, L; Zhu, Y; Moreno, CS; Wan, Y. New insights into KLFs and SOXs in cancer pathogenesis, stemness, and therapy. Semin Cancer Biol; 2023; 90, pp. 29-44.[COI: 1:CAS:528:DC%2BB3sXjsFCrsrc%3D] [DOI: https://dx.doi.org/10.1016/j.semcancer.2023.02.003] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36806560][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10023514]
8. Yuce, K; Ozkan, AI. The kruppel-like factor (KLF) family, diseases, and physiological events. Gene; 2024; 895, 148027.[COI: 1:CAS:528:DC%2BB3sXisVyqtbbO] [DOI: https://dx.doi.org/10.1016/j.gene.2023.148027] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38000704]
9. Yuan, L; Meng, Y; Xiang, J. KLF4 induces colorectal cancer by promoting EMT via STAT3 activation. Dig Dis Sci; 2024; [DOI: https://dx.doi.org/10.1007/s10620-024-08473-y] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/39446200][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11026227]
10. Zuo, X; Wang, L; Liu, Y; Wang, H; Hafley, M; Gagea, M; Chen, R; Xiong, Y; Pan, S; Shureiqi, I et al. Dysregulated KLF4 expression plays a pivotal role in the pathogenesis of pancreatic intraductal papillary mucinous neoplasms. Gut; 2024; [DOI: https://dx.doi.org/10.1136/gutjnl-2024-332255] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/39562050]
11. Bai, J; Chen, Y; Sun, Y; Wang, X; Wang, Y; Guo, S; Shang, Z; Shao, Z. EphA2 promotes the transcription of KLF4 to facilitate stemness in oral squamous cell carcinoma. Cell Mol Life Sci; 2024; 81,
12. Shi, Y; Ou, L; Han, S; Li, M; Pena, MM; Pena, EA; Liu, C; Nagarkatti, M; Fan, D; Ai, W. Deficiency of Kruppel-like factor KLF4 in myeloid-derived suppressor cells inhibits tumor pulmonary metastasis in mice accompanied by decreased fibrocytes. Oncogenesis; 2014; 3,
13. Liu, M; Li, X; Peng, KZ; Gao, T; Cui, Y; Ma, N; Zhou, Y; Hou, G. Subcellular localization of Klf4 in non-small cell lung cancer and its clinical significance. Biomed Pharmacother; 2018; 99, pp. 480-485.[COI: 1:CAS:528:DC%2BC1cXivVSgsrc%3D] [DOI: https://dx.doi.org/10.1016/j.biopha.2018.01.090] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29665649]
14. Wang, X; Xia, S; Li, H; Wang, X; Li, C; Chao, Y; Zhang, L; Han, C. The deubiquitinase USP10 regulates KLF4 stability and suppresses lung tumorigenesis. Cell Death Differ; 2020; 27,
15. Wang, R; Yu, W; Zhu, T; Lin, F; Hua, C; Ru, L; Guo, P; Wan, X; Xue, G; Guo, Z et al. MED27 plays a tumor-promoting role in breast cancer progression by targeting KLF4. Cancer Sci; 2023; 114,
16. He, Z; He, J; Xie, K. KLF4 transcription factor in tumorigenesis. Cell Death Discov; 2023; 9,
17. Lund, RJ; Huhtinen, K; Salmi, J; Rantala, J; Nguyen, EV; Moulder, R; Goodlett, DR; Lahesmaa, R; Carpén, O. DNA methylation and transcriptome changes associated with cisplatin resistance in ovarian cancer. Sci Rep; 2017; 7,
18. Liu, GY; Sabatini, DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol; 2020; 21,
19. Mao, X; Wang, L; Chen, Z; Huang, H; Chen, J; Su, J; Li, Z; Shen, G; Ren, Y; Li, Z et al. SCD1 promotes the stemness of gastric cancer stem cells by inhibiting ferroptosis through the SQLE/cholesterol/mTOR signalling pathway. Int J Biol Macromol; 2024; 275,
20. Shen, WJ; Zhang, Y. RPN1 promotes the proliferation and invasion of breast cancer cells by activating the PI3K/AKT/mTOR signaling pathway. Discov Oncol; 2024; 15,
21. Lin, L; Li, X; Wu, AJ; Xiu, JB; Gan, YZ; Yang, XM; Ai, ZH. TRPV4 enhances the synthesis of fatty acids to drive the progression of ovarian cancer through the calcium-mTORC1/SREBP1 signaling pathway. iScience; 2023; 26,
22. Chang, HC; Yang, CC; Loi, LK; Hung, CH; Wu, CH; Lin, YC. Interplay of p62-mTORC1 and EGFR signaling promotes cisplatin resistance in oral cancer. Heliyon; 2024; 10,
23. Zhu, J; Tong, H; Sun, Y; Li, T; Yang, G; He, W. YTHDF1 promotes bladder cancer cell proliferation via the METTL3/YTHDF1-RPN2-PI3K/AKT/mTOR Axis. Int J Mol Sci; 2023; [DOI: https://dx.doi.org/10.3390/ijms24086905] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38203566][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10778951]
24. Zhang, M; Wang, J; Guo, Y; Yue, H; Zhang, L. Activation of PI3K/AKT/mTOR signaling axis by UBE2S inhibits autophagy leading to cisplatin resistance in ovarian cancer. J Ovarian Res; 2023; 16,
25. Rinne, N; Christie, EL; Ardasheva, A; Kwok, CH; Demchenko, N; Low, C; Tralau-Stewart, C; Fotopoulou, C; Cunnea, P. Targeting the PI3K/AKT/mTOR pathway in epithelial ovarian cancer, therapeutic treatment options for platinum-resistant ovarian cancer. Cancer Drug Resist; 2021; 4,
26. Glaviano, A; Foo, ASC; Lam, HY; Yap, KCH; Jacot, W; Jones, RH; Eng, H; Nair, MG; Makvandi, P; Geoerger, B et al. PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer. Mol Cancer; 2023; 22,
27. Xu, Q; Zhou, W; Zhou, Y; Zhang, X; Jiang, R; Ai, Z; Chen, J; Ma, L. IRX2 regulates endometrial carcinoma oncogenesis by transcriptional repressing RUVBL1. Exp Cell Res; 2024; 434,
28. Konstantinopoulos, PA; Matulonis, UA. Clinical and translational advances in ovarian cancer therapy. Nat Cancer; 2023; 4,
29. Yang, L; Xie, HJ; Li, YY; Wang, X; Liu, XX; Mai, J. Molecular mechanisms of platinum-based chemotherapy resistance in ovarian cancer (Review). Oncol Rep; 2022; [DOI: https://dx.doi.org/10.3892/or.2022.8293] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36562383][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9827260]
30. Yang, L; Shi, P; Zhao, G; Xu, J; Peng, W; Zhang, J; Zhang, G; Wang, X; Dong, Z; Chen, F et al. Targeting cancer stem cell pathways for cancer therapy. Signal Transduct Target Ther; 2020; 5,
31. Zhou, H; Guan, Q; Hou, X; Liu, L; Zhou, L; Li, W; Liu, H. Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells. Int J Biol Sci; 2022; 18,
32. Pandya, AY; Talley, LI; Frost, AR; Fitzgerald, TJ; Trivedi, V; Chakravarthy, M; Chhieng, DC; Grizzle, WE; Engler, JA; Krontiras, H et al. Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer. Clin Cancer Res; 2004; 10,
33. Wei, D; Wang, L; Kanai, M; Jia, Z; Le, X; Li, Q; Wang, H; Xie, K. KLF4α up-regulation promotes cell cycle progression and reduces survival time of patients with pancreatic cancer. Gastroenterology; 2010; 139,
34. Tuo, Z; Liang, L; Zhou, R. LINC00852 is associated with poor prognosis in non-small cell lung cancer patients and its inhibition suppresses cancer cell proliferation and chemoresistance via the hsa-miR-145-5p/KLF4 axis. J Gene Med; 2021; 23,
35. Chen, Z; Wang, Y; Liu, W; Zhao, G; Lee, S; Balogh, A; Zou, Y; Guo, Y; Zhang, Z; Gu, W et al. Doxycycline inducible Krüppel-like factor 4 lentiviral vector mediates mesenchymal to epithelial transition in ovarian cancer cells. PLoS ONE; 2014; 9,
36. Szwed, A; Kim, E; Jacinto, E. Regulation and metabolic functions of mTORC1 and mTORC2. Physiol Rev; 2021; 101,
37. Saxton, RA; Sabatini, DM. mTOR signaling in growth, metabolism, and disease. Cell; 2017; 169,
38. Popova, NV; Jücker, M. The Role of mTOR Signaling as a Therapeutic Target in Cancer. Int J Mol Sci; 2021; [DOI: https://dx.doi.org/10.3390/ijms22041743] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34948251][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8708083]
39. Carden, CP; Stewart, A; Thavasu, P; Kipps, E; Pope, L; Crespo, M; Miranda, S; Attard, G; Garrett, MD; Clarke, PA et al. The association of PI3 kinase signaling and chemoresistance in advanced ovarian cancer. Mol Cancer Ther; 2012; 11,
40. Castellvi, J; Garcia, A; Rojo, F; Ruiz-Marcellan, C; Gil, A; Baselga, J; Ramon y Cajal, S. Phosphorylated 4E binding protein 1: a hallmark of cell signaling that correlates with survival in ovarian cancer. Cancer; 2006; 107,
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Abstract
Ovarian cancer (OC) is a highly fatal gynecological malignancy worldwide, and cisplatin (CDDP) is commonly used as an initial chemotherapy treatment for OC. Nonetheless, most patients ultimately face recurrence because of resistance to cisplatin. Therefore, it is imperative to investigate the underlying mechanisms of drug resistance in OC. By analyzing differential gene expression using TCGA, GDSC, and GEO public databases, we discovered that increased KLF4 expression is strongly linked to chemotherapy resistance and unfavorable outcomes in OC. Subsequent validation through immunohistochemistry and western blotting confirmed the upregulated KLF4 expression in cisplatin-resistance OC cells lines and tissues. To investigate the function of KLF4, functional experiments were performed both in vitro and in vivo. We observed that knocking down KLF4 impaired cisplatin-resistance of OC. Further mechanism research based on RNA-seq and gene enrichment analysis revealed that interfering KLF4 suppressed the activation of mTORC1 pathway. Finally, rescue experiment demonstrated that using mTORC1 pathway inhibitor could attenuate the cisplatin resistance induced by the overexpression of KLF4. In conclusion, our research indicates that KLF4 promotes cisplatin resistance through the activation of mTORC1 signaling, and proposes that inhibiting KLF4 might serve as a viable therapeutic approach to overcoming drug resistance in ovarian cancer.
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Details
1 Shanghai Sixth People’s Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Department of Gynecology and Obstetrics, Shanghai, People’s Republic of China (GRID:grid.412528.8) (ISNI:0000 0004 1798 5117)
2 Shanghai Jiao Tong University, Shanghai, People’s Republic of China (GRID:grid.16821.3c) (ISNI:0000 0004 0368 8293)
3 Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China (GRID:grid.16821.3c) (ISNI:0000 0004 0368 8293)