Targeting the FOXA1/BMI1 Axis to Overcome Chemoresistance and Suppress Tumor Progression in Nasopharyngeal Carcinoma

doi:10.21203/rs.3.rs-5036386/v1

Download PDF

Article

Targeting the FOXA1/BMI1 Axis to Overcome Chemoresistance and Suppress Tumor Progression in Nasopharyngeal Carcinoma

https://doi.org/10.21203/rs.3.rs-5036386/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Nasopharyngeal carcinoma (NPC) is a highly aggressive head and neck cancer characterized by a complex etiology and a propensity for metastasis. The current study explores the intricate relationship between Forkhead Box A1 (FOXA1) and B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) in the cancer progression and chemoresistance of NPC. Our research identified a significant downregulation of FOXA1 in NPC tissues and cell lines, which correlates with advanced clinical stages and poor differentiation, underscoring its potential role as a tumor suppressor. Functional assays demonstrated that the silencing of FOXA1 significantly enhanced the proliferation, migration, and invasive capabilities of NPC cells in vitro. Furthermore, the deficiency of FOXA1 was associated with a diminished sensitivity to cisplatin, as evidenced by increased cell viability, reduced apoptosis, and impaired cell cycle arrest upon drug exposure. Mechanistic studies revealed BMI1 as a critical downstream target of FOXA1. We observed a negative correlation between the expression levels of FOXA1 and BMI1 in NPC tissues. FOXA1 was shown to bind directly to the BMI1 promoter, effectively dampening its transcriptional activity. Rescue experiments indicated that the downregulation of BMI1 could partially reverse the malignant phenotypes induced by FOXA1 silencing, both in vitro and in vivo. Importantly, the knockdown of BMI1 significantly increased the chemosensitivity of FOXA1-depleted NPC cells to cisplatin, effectively counteracting the drug resistance associated with FOXA1 suppression. These findings highlight the pivotal role of FOXA1 in NPC development and progression and suggest that its loss leads to the upregulation of BMI1 and the acquisition of cisplatin resistance. Our study provides novel insights into the molecular mechanisms underlying the malignancy and chemoresistance of NPC and proposes that targeting the FOXA1/BMI1 axis could offer a promising therapeutic strategy for the treatment of this devastating disease.

Biological sciences/Cancer/Head and neck cancer

Health sciences/Diseases/Cancer/Tumour-suppressor proteins

Nasopharyngeal carcinoma

Tumor progression

Chemoresistance

NPC is a unique malignancy of the head and neck, arising from the nasopharyngeal epithelium and exhibiting a significant geographic and ethnic distribution, with a higher incidence noted among populations in Eastern and Southeastern Asia [1]. The disease's elusive nature within the body and its tendency for early lymphatic spread often result in a majority of patients being diagnosed at an advanced stage [2]. The current standard of care involves concurrent chemoradiotherapy (CCRT), primarily with cisplatin, potentially integrated with induction chemotherapy (IC) or adjuvant chemotherapy (AC) [3][4]. Advances in intensity-modulated radiotherapy (IMRT) and chemotherapy have improved local control and reduced mortality [5][6][7][8]; however, the development of cisplatin resistance poses a formidable barrier to treatment efficacy [9][10].

The forkhead protein FOXA1, a key winged-helix transcription factor, plays a pioneering role in gene regulation [11], binding to condensed chromatin to enhance accessibility for other transcription factors, thereby influencing a spectrum of genes tied to cell adhesion, cycle, and differentiation—key players in cancer progression [12][13][14][15]. Meanwhile, FOXA1, closely linked to tumor chemoresistance, enhances EMT, metastasis, and chemoresistance in docetaxel-resistant LAD cells [16], and its upregulation in endocrine therapy-resistant breast cancer promotes drug resistance via transcriptional regulation of downstream genes [17]. Its downregulation in NPC, as observed in studies, suggests a tumor-suppressive function, with restoration of FOXA1 in NPC cells leading to a suppression of proliferation and invasiveness [18][19][20]. Yet, the detailed mechanisms of FOXA1 in NPC chemosensitivity remain to be fully elucidated.

BMI1, a linchpin of the Polycomb Group (PcG) proteins, is an essential component of the Polycomb Repressive Complex 1 (PRC1), exerting a pivotal role in epigenetic regulation [21]. BMI1-mediated ubiquitination of histone H2A at lysine 119 within the PRC1 complex is essential for gene silencing, stem cell preservation, and cell cycle regulation [21][22][23]. BMI1 overexpression in NPC is significantly correlated with heightened malignancy, promoting cancer stem cell self-renewal and tumor invasiveness, thereby adversely impacting patient prognosis [24]. Furthermore, this overexpression is a critical determinant of chemoresistance in NPC, particularly to cisplatin. It diminishes chemosensitivity by inhibiting chemotherapy-induced apoptosis and bolstering cell survival mechanisms [25][26]. Targeting BMI1 offers a strategic oncological intervention, with its inhibition potentially restoring cisplatin sensitivity in NPC cells, thereby enhancing the efficacy of chemotherapy. This targeted strategy not only addresses the chemoresistance challenge in NPC but also heralds a promising avenue for improving patient outcomes, underscoring BMI1 as an innovative therapeutic target in the fight against drug resistance in cancer treatment.

In this study, we delve into the FOXA1/BMI1 axis within NPC, identifying a pivotal link between diminished FOXA1 expression and BMI1's role in promoting malignancy and cisplatin resistance. By targeting this axis, we propose a therapeutic strategy to enhance NPC cells' chemosensitivity to cisplatin and overcome chemoresistance, a major challenge in oncology. Our findings underscore the FOXA1/BMI1 axis as a key target for NPC treatment, offering insights into a potential new approach to improve patient outcomes in this aggressive cancer.

FOXA1 is frequently downregulated in NPC tissues and cell lines and its downregulation is association with aggressive clinicopathological characters in NPC

To investigate the influence of FOXA1 in the pathogenesis and tumorigenesis of NPC, we utilized immunohistochemistry to assess FOXA1 protein expression in 175 samples of NPC and 61 samples of non-cancerous nasopharyngeal epithelium (NPE). Uniformly strong nuclear FOXA1 staining was observed in all NPE tissues. Conversely, a significant reduction in FOXA1 protein levels was noted in 58.3% (102/175) of NPC samples (Fig. 1A, B; Table 1). Western blot analysis revealed a significant decrease in FOXA1 expression in most NPC cell lines, including CNE1, CNE2, SUNE1, HONE1, HONE1-EBV, S18, S26, 5-8F, and HK1-EBV, compared to NP69 and SWSX-14890 nasopharyngeal epithelial cell lines (Fig. 1E). The consistent downregulation of FOXA1 in NPC tissues, coupled with its notable deficiency in various NPC cell lines, implicates the diminished expression of FOXA1 as a potentially pivotal molecular alteration in the oncogenesis of NPC. These findings suggest FOXA1 downregulation may be a key molecular change in NPC oncogenesis.

Table 1

Expression of FOXA1 in 61 non-cancerous epithelial tissues and 175 NPC
Variables	n	FOXA1 expression		χ2	P
		Low(n, %)	High(n, %)
non-cancerous epithelial tissues	61	0 (0.0)	61 (100.0)	36.842	<0.001
NPC	175	102(58.3)	73 (41.7)

Our subsequent analysis showed that FOXA1 expression was significantly inversely correlated with WHO classification and clinical TNM staging. Lower FOXA1 expression was primarily linked to UDC and advanced stages, marked by higher T stage (tumor sizes, T3-T4), N stage (lymph node metastasis, N2-N3), M stage (Distant metastasis, M1) and clinical stage (III-IV) (Fig. 1C, D; Table 2). The data above indicate that reduced FOXA1 expression is significantly associated with aggressive NPC traits, suggesting a role in tumor progression.

Table 2

Correlation between the clinicopathological features and expression of FOXA1
Characteristics	Case No.(n)	FOXA1 expression		χ2	P
Characteristics	Case No.(n)	High(n, %)	Low(n, %)	χ2	P
Sex		73	102
Female	45	15(33.3)	30(66.7)	0.078	0.781
Male	130	58(44.6)	72(55.4)	0.078	0.781
Age (years)
<50	81	31(38.3)	50(61.7)	0.735	0.443
≥50	94	42(44.7)	52(55.3)	0.735	0.443
T classification
T1-T2	117	59(50.4)	58(49.6)	11.023	0.001
T3-T4	58	14(24.1)	44(75.9)	11.023	0.001
N classification
N0-N1	114	56(49.1)	58(50.9)	7.383	0.01
N2-N3	61	17(27.9)	44(72.1)	7.383	0.01
M classification
M0	150	68(45.3)	82(54.7)	5.656	0.027
M1	25	5(20.0)	20(80.0)	5.656	0.027
Clinical stage
I - II	58	31(53.4)	27(46.6)	8.831	0.004
III - IV	117	32(29.9)	75(70.1)	8.831	0.004
Histological type
DNKC	27	21(77.8)	6(22.2)	17.078	<0.001
UDC	148	52(35.1)	96(64.9)	17.078	<0.001
Abbreviations: T: tumor size; N: lymph node metastasis; M: distant metastasis; DNKC: differentiated nonkeratinizing carcinoma; UDC: undifferentiated carcinoma.

FOXA1 silencing enhances proliferation, migration and invasion of NPC cells

To investigate the role of FOXA1 in NPC cells, we utilized lentiviruses encoding FOXA1 shRNA and overexpression constructs to modulate FOXA1 levels in CNE1 and CNE2 cell lines. Western blot analysis confirmed the successful knockdown and overexpression of FOXA1 (Fig. 2A; Fig. S2A). Reduction in FOXA1 expression was associated with increased cell proliferation as evidenced by CCK-8 (Fig. 2B) and colony formation assays (Fig. 2C). Additionally, FOXA1 depletion significantly enhanced the migration and invasion of CNE1 and CNE2 cells as shown by wound healing (Fig. 2D) and Transwell assays (Fig. 2E and F). Conversely, overexpression of FOXA1 in CNE1 cells resulted in decreased proliferation, migration, and invasion capabilities (Fig. S2B-E). These findings collectively suggest that FOXA1 acts as a negative regulator of cell proliferation, migration, and invasion in NPC cell lines, highlighting its potential as a tumor suppressor and a promising target for NPC therapeutic intervention.

FOXA1 silencing diminishes Cisplatin chemosensitivity in NPC Cells

To elucidate the impact of FOXA1 suppression on NPC cells' response to cisplatin, we exposed CNE1 and CNE2 cells to varying cisplatin concentrations for 48 hours and determined the IC50 values. NPC cells with diminished FOXA1 expression exhibited an elevated IC50, suggesting a lower sensitivity to cisplatin (Fig. 3A). Further analysis using CCK-8 and colony formation assays showed that downregulation of FOXA1 led to a significant rise in cisplatin resistance, evidenced by enhanced cell viability and colony counts (Fig. 3B-C). Additionally, the wound healing and Transwell assays indicated that FOXA1 depletion reduced the inhibitory impact of cisplatin on cell migration and invasion, pointing to a less effective drug response (Fig. 3D-F). Flow cytometry data confirmed that cisplatin's induction of G2/M cell cycle arrest in CNE1 and CNE2 cells was counteracted by FOXA1 silencing (Fig. 3G). The Annexin V/PI assay also demonstrated that cells with FOXA1 knockdown had a significantly decreased rate of apoptosis in response to cisplatin treatment compared to the control cells (Fig. 3H). These results collectively indicate that diminished FOXA1 expression is associated with a reduced chemotherapeutic response to cisplatin in NPC cells, highlighting the significance of FOXA1 in the modulation of drug sensitivity.

FOXA1 transcriptionally inhibits the expression of BMI1 in NPC cells

To investigate FOXA1's role in NPC progression and chemosensitivity, we utilized the hTFtarget database to identify FOXA1-target genes, focusing on BMI1, known for its association with cisplatin resistance in NPC [26]. Bioinformatics analysis implicated BMI1 as a potential transcriptional target of FOXA1 (https://guolab.wchscu.cn/hTFtarget//#!/targets/chipseq_tf?tf=FOXA1). To further delineate the transcriptional regulation of BMI1 by FOXA1, an evaluation of the expression correlation between FOXA1 and BMI1 across NPC tissues and cell lines was conducted. Thereafter, the regulatory influence of FOXA1 on BMI1 transcription was empirically validated through ChIP PCR and a dual-luciferase reporter gene assay.

Immunohistochemical analysis of NPC tissues revealed a significant inverse correlation between FOXA1 and BMI1 expression levels (Fig. 4A). Western blot analysis indicated that upon FOXA1 knockdown, BMI1 protein levels were upregulated in CNE1 and CNE2 cells (Fig. 4B), whereas overexpression of FOXA1 in CNE1 cells resulted in decreased BMI1 levels (Fig. S2A). Furthermore, the epigenetic modification, histone H2A monoubiquitination at lysine 119 (H2AK119ub1), mediated by the BMI1-associated PRC1 complex, mirrored these changes in FOXA1-knockdown CNE1 and CNE2 cells and was downregulated in FOXA1-overexpressing CNE1 cells. This suggests that BMI1-mediated epigenetic effects are congruent with the expression levels of BMI1 in NPC cells.

To reinforce the transcriptional control of BMI1 by FOXA1, a luciferase reporter assay incorporating a FOXA1-responsive element in the BMI1 promoter was conducted. Transfection of CNE1 and CNE2 cells with this reporter alongside LV-FOXA1 or LVcon, followed by incubation, resulted in diminished luciferase activity with LV-FOXA1, thereby validating FOXA1's enhancer function on the BMI1 promoter (Fig. 4C). ChIP assays confirmed specific FOXA1 binding to the BMI1 promoter sequence, in contrast to the non-specific IgG control (Fig. 4D). These findings collectively demonstrate FOXA1's direct interaction with and regulation of the BMI1 promoter in NPC cells.

BMI1 downregulation abrogates the malignant phenotypes conferred by FOXA1 suppression in NPC cells

To clarify the molecular interplay between FOXA1 and BMI1 in NPC, rescue assays were conducted by specifically reducing BMI1 levels in CNE1 and CNE2 cells after the initial knockdown of FOXA1. Western blot analysis confirmed the successful knockdown of BMI1 in FOXA1-silenced NPC cells, accompanied by a reduction in the level of its epigenetic effector, H2AK119ub1, which had been upregulated upon FOXA1 silencing (Fig. 4E). The CCK-8 assay (Fig. 5A) and colony formation assay (Fig. 5B) demonstrated that the enhanced cell viability observed in the FOXA1 knockdown group was significantly reversed by the additional knockdown of BMI1. Meanwhile, the wound healing assay (Fig. 5C) and Transwell assay (Fig. 5D-E) showed that BMI1 knockdown effectively mitigated the enhanced migratory and invasive capacities induced by FOXA1 knockdown in CNE1 and CNE2 cells. Moreover, the xenograft tumor model (Fig. 5F-H) showed that the increased tumor growth rate, consequent to FOXA1 knockdown, was significantly reversed by the knockdown of BMI1.

These findings highlight the critical role of the FOXA1-BMI1 regulatory axis in the progression of NPC, indicating that the inhibition of BMI1 could offer a promising therapeutic approach for treating NPC, particularly in cases with diminished FOXA1 activity.

BMI1 downregulation reverses FOXA1-silencing-induced cisplatin resistance in NPC cells

To elucidate the role of BMI1 in FOXA1-mediated cisplatin sensitivity in NPC, we performed in vitro assays to evaluate cell viability (Fig. 6A-B), migration (Fig. S1A-B), and invasiveness (Fig. S1C) after BMI1 knockdown in cells with silenced FOXA1. These assays revealed that BMI1 knockdown significantly increased cisplatin sensitivity, as evidenced by reduced cell viability, migration, and invasiveness, suggesting an amelioration of chemoresistance associated with FOXA1 deficiency. Concurrently, FOXA1 knockdown was found to decrease cisplatin-induced apoptosis (Fig. 6C) and G2/M cell cycle arrest (Fig. S1D), effects that were markedly reversed by BMI1 knockdown. In direct contrast, overexpression of FOXA1 in CNE1 cells, as measured by the aforementioned assays, inhibited malignant phenotypes and enhanced sensitivity to cisplatin, an effect that was counteracted by the concurrent overexpression of BMI1 (Figs. S3 and S4).

Moreover, Western blotting validated that BMI1 knockdown mitigated the chemoresistance-associated upregulation of MDR1 and MRP1 proteins induced by FOXA1 knockdown (Fig. 6E). Conversely, in CNE1 cells overexpressing FOXA1, the levels of MDR1 and MRP1 proteins were observed to be diminished with BMI1 overexpression (Fig. S4C).

In the xenograft model, the co-knockdown of FOXA1 and BMI1 in CNE1 cells resulted in an increased sensitivity to cisplatin, as indicated by reduced tumor size (Fig. 6F, H) and decelerated growth rates (Fig. 6G), in stark comparison to the FOXA1-knockdown cells that displayed resistance. In xenografted CNE1 cells with combined FOXA1 and BMI1 knockdown, immunohistochemical analysis revealed a reduction in the overexpression of MDR1 and MRP1 proteins, which are associated with FOXA1 knockdown alone (Fig. 6I). The collective findings emphasize the significance of the FOXA1-BMI1 interaction in regulating cisplatin sensitivity in NPC, suggesting that targeting this axis could be key to enhancing chemosensitivity and optimizing treatment efficacy.

NPC is a highly invasive form of head and neck cancer, with radiotherapy established as an effective treatment for its early stages [1][27][28]. The high invasiveness of nasopharyngeal carcinoma, which frequently results in lymph node infiltration and distant metastasis and is often diagnosed at advanced stages, contributes to a poor prognosis and an elevated recurrence rate [29][30]. The standard treatment for advanced NPC is chemotherapy based on cisplatin [3][4]; however, the development of resistance to cisplatin poses a significant obstacle to successful therapya [10][31]. The propensity for chemoresistance underscores the urgent need for novel therapeutic strategies that can overcome resistance mechanisms and improve patient outcomes.

The transcription factor FOXA1 has been extensively studied for its role in tumorigenesis and cancer progression, exhibiting context-dependent functions that can range from promoting to suppressing tumor growth [32][33]. In various cancer types, FOXA1 has been implicated as an oncogene; its overexpression is significantly associated with the oncogenesis, development, and prognosis of several cancers, including breast, prostate, and lung [34][35][36][37].

In the context of NPC, a highly malignant cancer often diagnosed at advanced stages, FOXA1 appears to play a protective role. Prior studies have reported significant downregulation of FOXA1 in NPC tissues and cell lines, correlating with advanced clinical stages and poor differentiation, which suggests its potential as a tumor suppressor [18][19][20]. Utilizing a larger cohort of 175 NPC and 61 non-cancerous nasopharyngeal epithelia cases, we demonstrated that loss of FOXA1 was significantly associated with larger tumor size, lymphatic metastasis, distant metastasis, advanced clinical stages, and the undifferentiated histological subtype. In NPC cells and corresponding xenografts, assays indicated that FOXA1 functions as a tumor suppressor, with its knockdown promoting malignant behaviors such as increased cell proliferation, migration, and invasion.This research not only supports the notion of FOXA1 as a tumor suppressor, in line with previous studies, but also underscores its critical role in tumor progression.

Furthermore, FOXA1 is reported to play an essential role in chemoresistance, with high levels contributing to chemoresistance in lung and cervical cancers [16][38]. However, in estrogen receptor-positive breast cancer, FOXA1 downregulation enhances sensitivity to doxorubicin and paclitaxel, while its upregulation in basal-like breast cancer cells confers increased drug resistance. These findings highlight FOXA1's pivotal role in chemoresistance, which is cell context-dependent. Moreover, FOXA1 silencing in NPC cells increased cisplatin resistance, characterized by heightened proliferation, migration, and invasion, and attenuated the apoptotic and cell cycle arrest effects typically associated with cisplatin treatment. However, a previous study noted an upregulation of FOXA1 in a cisplatin-resistant NPC cell line (CNE2/DDP), with FOXA1 knockdown enhancing cisplatin sensitivity, suggesting a role for FOXA1 in chemoresistance [39]. These inconsistencies with prior studies highlight that the functional impact of FOXA1 is contingent upon the specific genetic and molecular context of the cell lines involved.

FOXA1, utilizing its intrinsically disordered regions (IDRs) to mediate phase separation via the formation of biomolecular condensates, enables the binding to and unpacking of condensed chromatin [40]. By facilitating the opening of these specific chromatin regions, FOXA1 acts as a "pioneer" transcription factor, collaborating with a variety of transcription factors and co-factors to regulate the expression profile of target genes. In prostate cancer, FOXA1's interaction with the androgen receptor (AR) enhances AR-mediated transcription, potentially driving tumor progression and castration resistance [36]. In breast cancer, the upregulation of FOXA1 leads to the reprogramming of estrogen receptor function, contributing to endocrine resistance and the promotion of metastasis in ER-positive tumors through a High-FOXA1/ER-dependent secretory mechanism [41]. Concurrently, this heightened FOXA1 expression triggers a comprehensive reorganization of the genomic enhancer landscape, activating the HIF-2α transcription factor via superenhancers, which in turn initiates a metastatic transcriptional program [42]. Collectively, FOXA1 can alter the affinity of specific cis-regulatory elements for their associated transcription factors, thereby regulating the expression of target gene profiles, leading to changes in the malignant biological behaviors and therapeutic responses of tumors.

In NPC, the target genes of FOXA1 include oncomiRs such as miR-100-5p and miR-125b-5p [20]. Through transcriptome assays, FOXA1 has been shown to activate the tumor-suppressive transcriptional program induced by TGF-β, instead of tumor-promotive transcriptional program in NPC cells [19]. In our study, by mining the FOXA1 downstream target gene set from the JASPAR database, we identified BMI1 as a potential transcriptional target of FOXA1. Concurrently, a significant inverse correlation between the expression levels of FOXA1 and BMI1 was observed in NPC tissues and in NPC cells. Utilizing ChIP-PCR and luciferase reporter assays, we documented that FOXA1 binds to the promoter region of BMI1 and negatively modulates its transcription.

BMI1 has been reported to promote tumor progression in NPC by regulating cell cycle progression and stemness [24]. It is also implicated in cisplatin resistance through mechanisms including DNA repair enhancement, apoptosis inhibition, and autophagy modulation [23]. Given the pivotal role of BMI1 in NPC malignancy and drug resistance, this study investigates whether BMI1 mediates the oncogenic effects and cisplatin resistance induced by FOXA1 downregulation. To elucidate the molecular interplay between FOXA1 and BMI1, we downregulated BMI1 using shRNA-expressing lentivirus in NPC cells with endogenous FOXA1 silenced. BMI1 downregulation was found to reverse the malignant phenotypes and cisplatin resistance associated with FOXA1 suppression in these cells.

As a member of the Polycomb group (PcG) proteins, BMI1 plays a multifaceted role in epigenetic regulation [21]. Being a core component of the Polycomb repressive complex 1 (PRC1), BMI1 is instrumental in the ubiquitination of histone H2A (H2AK119ub), which is a process that leads to chromatin compacting [43][44]. Through PRC1-mediated chromatin remodeling in gene silencing, BMI1 involved in a panel of physiological and patholphysiological processes [22]. BMI1 has been reported to possess potent oncogenic and therapeutic resistance properties through the regulation of a specific gene spectrum [21]. In our study, the expression of H2AK119ub is consistent with changes in the expression level of BMI1, suggesting that chromatin remodeling has occurred, which could be a potential mechanism by which BMI1 intervenes in the function of FOXA1. Additionally, we found that the expression of multidrug resistance genes in nasopharyngeal carcinoma cells was also affected by the intervention of BMI1 expression. However, the specific gene spectrum regulated by BMI1 needs further clarification.

Our findings validate BMI1’s role as a transcriptional target and downstream effector of FOXA1, participating in the progression of nasopharyngeal carcinoma and cisplatin resistance. This research not only consolidates the role of FOXA1 as a transcription factor but also uncovers its capacity to engage in chromatin remodeling via its target genes, thereby expanding our comprehension of the intricate functions of FOXA1. We advocate for the targeting of the FOXA1/BMI1 axis to potentiate the chemosensitivity of NPC cells to cisplatin, offering a novel strategy to counteract tumor progression and chemoresistance.

Patient specimens

Formalin-fixed, paraffin-embedded non-cancerous nasopharyngeal tissues (61 cases) and NPC tissues (175 cases), along with their corresponding clinical and pathological data, were collected from the Second Affiliated Hospital of Guilin Medical University (Guilin, China) between 2015 and 2022. The study was approved by the Research Ethics Committee and conducted with patient consent (Approval No. EFY-GZR2022007).

Hematoxylin-Eosin (H&E) Staining and Immunohistochemistry (IHC)

Hematoxylin-eosin (H&E) staining [45] and immunohistochemical assays [46][47] were performed as described previously. Immunohistochemical scores were categorized as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong), based on staining intensity. Scores of 0–1 were considered low expression, while 2–3 indicated high expression. The antibodies utilized for IHC are detailed in Table S1.

Cell lines and culture

Human NPC cell lines, including CNE1, CNE2, SUNE-1, HONE1, HONE1-EBV, S18, S26, 5-8F, and HK1-EBV, as well as immortalized nasopharyngeal epithelial cell lines NP69 and SXSW-1489, were generously provided by Prof. Dong Xiao of the Institute of Cancer Research, Southern Medical University, Guangzhou, China. The cells were routinely cultured in RPMI-1640 medium (Gibco, California, USA) supplemented with 10% fetal bovine serum (Excellbio, Shanghai, China) and 1% penicillin/streptomycin (Biosharp, Beijing, China). Cultures were maintained in a humidified incubator at 37°C with an atmosphere of 5% CO₂.

Stable cell line generation via lentiviral transduction

Lentiviral particles, including shRNA-expressing constructs targeting FOXA1 (sc-37930-V) and BMI1 (sc-29814-V), as well as overexpression constructs for FOXA1 (sc-400743-LAC) and BMI1 (sc-417606-LAC), were procured from Santa Cruz Biotechnology (Shanghai, China). Control lentiviral particles comprised shRNA (sc-108080) and Activation Particles (sc-437282). Transfection was performed using the manufacturer's recommended protocol, followed by puromycin selection.

Western blotting

Western blot analysis was performed according to established protocols [10][48]. Primary antibodies are detailed in Table S1. Protein expression was quantified using ImageJ software.

Cisplatin treatment

To determine the IC50 values of cisplatin in CNE1 and CNE2 cells and select an appropriate concentration for further chemosensitivity assays, NPC cells were treated with varying concentrations of cisplatin (0.0–4 µg/mL; QiLu Pharmaceutical, Jinan, China, Cat. No. H20023461) for 48 h, followed by CCK-8 assay as described [10].

CCK-8 and Plate colony‑forming assays

The CCK-8 assay and the plate colony-forming assay were performed as previously described [10][48].

Wound healing

The wound healing assay was conducted as reported previously [45].

Transwell migration and invasion assays

NPC cells (1 × 10⁵ or 2 × 10⁵) in serum-free medium were seeded into the upper 8-µm-pore transwell chambers (Corning, NY, USA) with or without Matrigel (BD Biosciences, Bedford, MA, USA). The lower chambers received 500 µL medium with 10% FBS. After 48-h incubation, migrated or invaded cells on the membranes were fixed, stained, and observed under a microscope [45][48].

Flow cytometry

Flow cytometry procedures were previously detailed [10][48]. For cell cycle analysis, cells exposed to cisplatin for 48 h were fixed in 75% ethanol at 4°C overnight. Post-centrifugation, they were stained with 500 µL propidium iodide (PI) staining buffer (Life-iLab Biotech, Shanghai, China) for 30 min at room temperature in darkness. The cell cycle distribution was determined using a flow cytometer (BD Biosciences, San Jose, CA, USA). For the apoptosis assay, cells treated with cisplatin for 48 h were stained with Annexin V-647 and PI for 15 min in the dark prior to analysis on a FACS Calibur cytometer (BD Biosciences, San Jose, CA, USA).

Chromatin immunoprecipitation (ChIP) and PCR

The SimpleChIP™ Enzymatic Chromatin IP Kit (Cell Signaling Technology, Cat. No. 9003S) was employed for ChIP analysis, following the manufacturer's protocol. CNE1 cells were cross-linked with 1% formaldehyde and the reaction was quenched with Glycine. DNA fragments were extracted after digestion with micrococcal nuclease and subsequent ultrasonication. The lysate was immunoprecipitated using anti-FOXA1, normal IgG, or anti-Histone H3 antibodies conjugated to magnetic beads (the information of antibodies listed in Table S1). Enrichment of the BMI1 promoter in the precipitates was assessed by PCR with the following primers: BMI1-forward, 5’-TCTACAGGAGAGCGTCACAT-3’ and BMI1-reverse, 5’-ACTTAGCCCGAAACCGTCAG-3’.

Dual luciferase reporter assay

A potential binding site for the BMI1 promoter with FOXA1 binding sequence was obtained from Jaspar (http://jaspar.genereg.net/), and the binding region was PCR amplified and cloned into the pGL3 vector (Promega, Madison, WI, USA) to obtain a Promoter luciferase reporter vector. The above reporter vectors were transfected into LVcon or LV-FOXA1 infected CNE1 and CNE2 cells, using Lipofectamine™ 3000 (Invitrogen, Carlsbad, California, USA) as per the manufacturer’s protocol. After 24 hours, cells were lysed and luciferase and Renilla signals were quantified using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) following the manufacturer's protocol.

Generation of the subcutaneous xenograft model in mice

Male BALB/c nude mice (6–8 weeks old, 18–22 g; Hunan Silaikejingda, Changsha, China) were maintained under specific-pathogen-free (SPF) conditions. Subcutaneous injections of 5 × 10⁶ CNE1 cells expressing shSCR, shFOXA1, or shFOXA1-shBMI1 were administered in the right axillary region. Tumor dimensions were measured every three days from day ten post-inoculation using a vernier caliper, with tumor volume calculated as 1/2 × width² × length. When tumors reached approximately 100 mm³, mice were treated with cisplatin at 4 mg/kg, with intraperitoneal injections given every three days for a total of three treatments. Euthanasia via cervical dislocation was performed three days after the final treatment, after which the primary tumors were excised and weighed. This study was conducted in compliance with the animal welfare guidelines of the Institutional Animal Care and Use Committee (IACUC) of Guilin Medical University (Guilin, China) and was approved under protocol number GLMC-202003113.

Statistical analysis

Quantitative data are presented as mean ± standard deviation (SD). The association of clinicopathological features with FOXA1 expression, as well as the correlation between FOXA1 and BMI1 expression levels, was examined using the chi-square (χ2) test. Statistical significance was determined using a one-way analysis of variance (ANOVA) test. Significance was denoted as not significant (NS) for P > 0.05, * for P < 0.05, ** for P < 0.01, # for P < 0.001.

Acknowledgements

Gratitude is extended to Prof. Dong Xiao, Lab Animal Center, Southern Medical University, for providing crucial NPC and immortalized nasopharyngeal epithelial cell lines, which significantly advanced our study.

Author contributions

Conceptualization of the manuscript was performed by Shengjun Xiao and Xiaoling Zhang; methodology by Shengjun Xiao, Xiaoling Zhang and Yaping Qin; investigation by Yaping Qin, Mingqing Yang, Yunzhu Cao and Yue Fu; resources by Shengjun Xiao and Xiaoling Zhang; writing of the original draft by Yaping Qin, Mingqing Yang, Yunzhu Cao and Yue Fu, Fan Yang; writing, review, and editing of the manuscript by Shengjun Xiao, Xiaoling Zhang and Yaping Qin; visualization by Yaping Qin; supervision by Shengjun Xiao and Xiaoling Zhang; and funding acquisition by Shengjun Xiao. All authors read and approved the final manuscript.

Funding

The project received supporing from the National Science Foundation of China (No. 82260476; No. 82060500; The Basic Scientific Research Ability Improvement Project for Young and Middle-aged Teachers of Guangxi Colleges and Universities (No. 2023KY0507); Guangxi Key Research and Development Plan (GuiKe AB24010096); Guangxi Science and Technology Base and Talent Special Project (GuiKe AD24999032).

Data availability

The datasets pertaining to the binding of FOXA1 to the promoter region of the BMI1 gene are available in the JASPAR database (http://jaspar.genereg.net/). Additional datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. The original full-length Western blots are included in the supplemental material.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

Clinical NPC samples were obtained from the Department of Pathology at the Second Affiliated Hospital of Guilin Medical University, Guilin, China. The use of these clinical samples was reviewed and approved by the Biomedical Ethics Committee of the Second Affiliated Hospital of Guilin Medical College (Approval No. EFY-GZR2022007). Informed consent was obtained from all patients prior to the use of their tissue samples. All animal studies were following was performed in accordance with the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals (NIH publication nos. 80-23, revised 1996) and were approved by Animal Research Committee of Guilin Medical College (GLMC-202003113).

Footnotes

These authors contributed equally: Yaping Qin, Mingqing Yang, Yunzhu Cao, Yue Fu.

Contributor Information

Xiaoling Zhang, Email: [email protected].

Shengjun Xiao, Email: [email protected].

Supplementary information

Supplementary information is available at Cell death Discovery’s website

Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394:64–80. 10.1016/S0140-6736(19)30956-0.
Lin M, Zhang XL, You R, Yang Q, Zou X, Yu K, et al. Neoantigen landscape in metastatic nasopharyngeal carcinoma. Theranostics. 2021;11:6427–6444. 10.7150/thno.53229.
Caudell JJ, Gillison ML, Maghami E, Spencer S, Pfister DG, Adkins D, et al. NCCN Guidelines® Insights: Head and Neck Cancers, Version 1.2022. J Natl Compr Canc Netw. 2022;20:224–234. 10.6004/jnccn.2022.0016.
Chen YP, Ismaila N, Chua MLK, Colevas AD, Haddad R, Huang SH, et al. Chemotherapy in Combination With Radiotherapy for Definitive-Intent Treatment of Stage II-IVA Nasopharyngeal Carcinoma: CSCO and ASCO Guideline. J Clin Oncol. 2021;39:840–859. 10.1200/JCO.20.03237.
You R, Liu YP, Xie YL, Lin C, Duan CY, Chen DP, et al. Hyperfractionation compared with standard fractionation in intensity-modulated radiotherapy for patients with locally advanced recurrent nasopharyngeal carcinoma: a multicentre, randomised, open-label, phase 3 trial. Lancet. 2023;401:917-927.10.1016/S0140-6736(23)00269-6.
Sidaway P. Chemoradiotherapy improves NPC outcomes. Nat Rev Clin Oncol. 2020;17:592. 10.1038/s41571-020-0424-9.
Tang LQ, Chen DP, Guo L, Mo HY, Huang Y, Guo SS, et al. Concurrent chemoradiotherapy with nedaplatin versus cisplatin in stage II-IVB nasopharyngeal carcinoma: an open-label, non-inferiority, randomised phase 3 trial. Lancet Oncol. 2018;19:461–473. 10.1016/S1470-2045(18)30104-9.
Liu LT, Liu H, Huang Y, Yang JH, Xie SY, Li YY, et al. Concurrent chemoradiotherapy followed by adjuvant cisplatin-gemcitabine versus cisplatin-fluorouracil chemotherapy for N2-3 nasopharyngeal carcinoma: a multicentre, open-label, randomised, controlled, phase 3 trial. Lancet Oncol. 2023;24:798–810. 10.1016/S1470-2045(23)00232-2.
Zhang B, Li J, Wang Y, Liu X, Yang X, Liao Z, et al. Deubiquitinase USP7 stabilizes KDM5B and promotes tumor progression and cisplatin resistance in nasopharyngeal carcinoma through the ZBTB16/TOP2A axis. Cell Death Differ. 2024;31:309–321. 10.1038/s41418-024-01257-x.
Zhou C, Shen G, Yang F, Duan J, Wu Z, Yang M, et al. Loss of AKR1C1 is a good prognostic factor in advanced NPC cases and increases chemosensitivity to cisplatin in NPC cells. J Cell Mol Med. 2020;24:6438–6447. 10.1111/jcmm.15291.
Teng M, Zhou S, Cai C, Lupien M, He HH. Pioneer of prostate cancer: past, present and the future of FOXA1. Protein Cell. 2021;12:29–38. 10.1007/s13238-020-00786-8.
Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, Zaret KS. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell. 2002;9:279–89. 10.1016/s1097-2765(02)00459-8.
Jin Y, Liang Z, Lou H. The Emerging Roles of Fox Family Transcription Factors in Chromosome Replication, Organization, and Genome Stability. Cells. 2020;9:258. 10.3390/cells9010258.
Zhu J, Wei Y, Deng F, Zhou Y, Yang Z, Ma Y. The Role of FOXA1 in Human Normal Development and Its Functions in Sex Hormone-Related Cancers. Front Biosci (Landmark Ed). 2024;29:225. 10.31083/j.fbl2906225.
Castaneda M, Hollander PD, Mani SA. Forkhead Box Transcription Factors: Double-Edged Swords in Cancer. Cancer Res. 2022;82:2057–2065. 10.1158/0008-5472.CAN-21-3371.
Chen D, Wang R, Yu C, Cao F, Zhang X, Yan F, et al. FOX-A1contributes to acquisition of chemoresistance in human lung adenocarcinoma via transactivation of SOX5. EBioMedicine. 2019;44:150–161. 10.1016/j.ebiom.2019.05.046.
Fu X, Jeselsohn R, Pereira R, Hollingsworth EF, Creighton CJ, LiF, et al. FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer. Proc Natl Acad Sci U S A. 2016;113:E6600-E6609. 10.1073/pnas.1612835113.
Ammous-Boukhris N, Ayadi W, Derbel M, Allaya-Jaafar N, CharfiS, Daoud J, et al. FOXA1 Expression in Nasopharyngeal Carcinoma: Association with Clinicopathological Characteristics and EMT Markers. Biomed Res Int. 2020;2020:4234632. 10.1155/2020/4234632.
Li J, Wang W, Chen S, Cai J, Ban Y, Peng Q, et al. FOXA1 reprograms the TGF-β-stimulated transcriptional program from a metastasis promoter to a tumor suppressor in nasopharyngeal carcinoma. Cancer Lett. 2019;442:1–14. 10.1016/j.canlet.2018.10.036.
Peng Q, Zhang L, Li J, Wang W, Cai J, Ban Y, et al. FOXA1 Suppresses the Growth, Migration, and Invasion of Nasopharyngeal Carcinoma Cells through Repressing miR-100-5p and miR-125b-5p. J Cancer. 2020;11:2485–2495. 10.7150/jca.40709.
Piunti A, Shilatifard A. The roles of Polycomb repressive complexes in mammalian development and cancer. Nat Rev Mol Cell Biol. 2021;22:326–345. 10.1038/s41580-021-00341-1.
Ge W, Yu C, Li J, Yu Z, Li X, Zhang Y, et al. Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase. Nature. 2023;616:176–182. 10.1038/s41586-023-05841-y.
Xu J, Li L, Shi P, Cui H, Yang L. The Crucial Roles of Bmi-1 in Cancer: Implications in Pathogenesis, Metastasis, Drug Resistance, and Targeted Therapies. Int J Mol Sci. 2022;23:8231. 10.3390/ijms23158231.
Lei Y, Shen HF, Li QW, Yang S, Xie HT, Li XF, et al. Hairy gene homolog increases nasopharyngeal carcinoma cell stemness by upregulating Bmi-1. Aging (Albany NY). 2023;15:4391–4410. 10.18632/aging.204742.
Qin L, Zhang X, Zhang L, Feng Y, Weng GX, Li MZ, et al. Downregulation of BMI-1 enhances 5fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells. Biochem Biophys Res Commun. 2008;371:531–5. 10.1016/j.bbrc.2008.04.117.
Xu XH, Liu Y, Li DJ, Hu J, Su J, Huang Q, et al. Effect of shRNA-Mediated Gene Silencing of Bmi-1 Expression on Chemosensitivity of CD44 + Nasopharyngeal Carcinoma Cancer StemLike Cells. Technol Cancer Res Treat. 2016;15:NP27–39. 10.1177/1533034615599461.
Chua MLK, Wee JTS, Hui EP, Chan ATC. Nasopharyngeal carcinoma. Lancet. 2016;387:1012–1024. 10.1016/S0140-6736(15)00055 – 0.
Wong KCW, Hui EP, Lo KW, Lam WKJ, Johnson D, Li L, et al. Nasopharyngeal carcinoma: an evolving paradigm. Nat Rev Clin Oncol. 2021;18:679–695. 10.1038/s41571-021-00524-x.
Wu LR, Zhang XM, Xie XD, Lu Y, Wu JF, He X. Validation of the 8th edition of AJCC/UICC staging system for nasopharyngeal carcinoma: Results from a non-endemic cohort with 10-year follow-up. Oral Oncol. 2019;98:141–146. 10.1016/j.oraloncology.2019.09.029.
Xu AA, Miao JJ, Wang L, Li AC, Han F, Shao XF, et al. Efficacy of concurrent chemoradiotherapy alone for loco-regionally advanced nasopharyngeal carcinoma: long-term follow-up analysis. Radiat Oncol. 2023;18:63. 10.1186/s13014-023-02247-y.
Hong X, Li Q, Li J, Chen K, He Q, Zhao Y, et al. CircIPO7 Promotes Nasopharyngeal Carcinoma Metastasis and Cisplatin Chemoresistance by Facilitating YBX1 Nuclear Localization. Clin Cancer Res. 2022;28:4521–4535. 10.1158/1078 – 0432.CCR-22-0991.
Xu AA, Miao JJ, Wang L, Li AC, Han F, Shao XF, et al. Efficacy of concurrent chemoradiotherapy alone for loco-regionally advanced nasopharyngeal carcinoma: long-term follow-up analysis. Radiat Oncol. 2023;18:63. 10.1186/s13014-023-02247-y.
Zhang G, Zhao Y, Liu Y, Kao LP, Wang X, Skerry B, et al. FOXA1 defines cancer cell specificity. Sci Adv. 2016;2:e1501473. 10.1126/sciadv.1501473.
Zhang Y, Huang YX, Wang DL, Yang B, Yan HY, Lin LH, et al. LncRNA DSCAM-AS1 interacts with YBX1 to promote cancer progression by forming a positive feedback loop that activates FOXA1 transcription network. Theranostics. 2020;10:10823–10837. 10.7150/thno.47830.
Xie H, Xiao R, He Y, He L, Xie C, Chen J, et al. MicroRNA-100 inhibits breast cancer cell proliferation, invasion and migration by targeting FOXA1. Oncol Lett. 2021;22:816. 10.3892/ol.2021.13077.
Gerhardt J, Montani M, Wild P, Beer M, Huber F, Hermanns T, et al. FOXA1 promotes tumor progression in prostate cancer and represents a novel hallmark of castration-resistant prostate cancer. Am J Pathol. 2012;180:848–61. 10.1016/j.ajpath.2011.10.021.
Hight SK, Mootz A, Kollipara RK, McMillan E, Yenerall P, Otaki Y, et al. An in vivo functional genomics screen of nuclear receptors and their co-regulators identifies FOXA1 as an essential gene in lung tumorigenesis. Neoplasia. 2020;22:294–310. 10.1016/j.neo.2020.04.005.
Wang Z, Sun BS, Chen ZS, Zhao KK, Wang YL, Meng FX, et al. FOXA1 Leads to Aberrant Expression of SIX4 Affecting Cervical Cancer Cell Growth and Chemoresistance. Anal Cell Pathol (Amst). 2022;2022:9675466. 10.1155/2022/9675466.
Li YL, Zhao YG, Chen B, Li XF. MicroRNA-132 sensitizes nasopharyngeal carcinoma cells to cisplatin through regulation of forkhead box A1 protein. Pharmazie. 2016;71:715–718. 10.1691/ph.2016.6764.
Ji D, Shao C, Yu J, Hou Y, Gao X, Wu Y, et al. FOXA1 forms biomolecular condensates that unpack condensed chromatin to function as a pioneer factor. Mol Cell. 2024;84:244–260.e7. 10.1016/j.molcel.2023.11.020.
Fu X, Pereira R, Liu CC, De Angelis C, Shea MJ, Nanda S, et al. High FOXA1 levels induce ER transcriptional reprogramming, a pro-metastatic secretome, and metastasis in endocrine-resistant breast cancer. Cell Rep. 2023;42:112821. 10.1016/j.celrep.2023.112821.
Fu X, Pereira R, De Angelis C, Veeraraghavan J, Nanda S, Qin L, et al. FOXA1 upregulation promotes enhancer and transcriptional reprogramming in endocrine-resistant breast cancer. Proc Natl Acad Sci U S A. 2019;116:26823–26834. 10.1073/pnas.1911584116.
Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, Jones RS, et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature. 2004;431:873-8.2004. 10.1038/nature02985.
Cao R, Tsukada Y, Zhang Y. Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing. Mol Cell. 2005;20:845–54. 10.1016/j.molcel.2005.12.002.
Yang F, Yang M, Liu Y, Zhou C, Chen Y, Wu J, et al. PDLIM7 Promotes Tumor Metastasis in Papillary Thyroid Carcinoma via Stabilizing Focal Adhesion Kinase Protein. Thyroid. 2024;34:598–610. 10.1089/thy.2023.0497.
Yang Y, Wang X, Yang J, Duan J, Wu Z, Yang F, et al. Loss of ARID1A promotes proliferation, migration and invasion via the Akt signaling pathway in NPC. Cancer Manag Res. 2019;11:4931–4946. 10.2147/CMAR.S207329.
Zhang X, Wang X, Jia L, Yang Y, Yang F, Xiao S. CtBP1 Mediates Hypoxia-Induced Sarcomatoid Transformation in Hepatocellular Carcinoma. J Hepatocell Carcinoma. 2022;9:57–67. 10.2147/JHC.S340471.
Chen S, Guan X, Xie L, Liu C, Li C, He M, et al. Aloe-emodin targets multiple signaling pathways by blocking ubiquitin-mediated degradation of DUSP1 in nasopharyngeal carcinoma cells. Phytother Res. 2023;37:2979–2994. 10.1002/ptr.7793.

(Not answered)

Download PDF

Reviewer #1 agreed at journal
31 Oct, 2024
Reviewers invited by journal
24 Oct, 2024
Submission checks completed at journal
05 Sep, 2024
Editor assigned by journal
05 Sep, 2024
First submitted to journal
05 Sep, 2024

You are reading this latest preprint version

Targeting the FOXA1/BMI1 Axis to Overcome Chemoresistance and Suppress Tumor Progression in Nasopharyngeal Carcinoma

Status:

Version 1

Abstract

Figures

Introduction

Results

FOXA1 silencing enhances proliferation, migration and invasion of NPC cells

FOXA1 silencing diminishes Cisplatin chemosensitivity in NPC Cells

FOXA1 transcriptionally inhibits the expression of BMI1 in NPC cells

BMI1 downregulation abrogates the malignant phenotypes conferred by FOXA1 suppression in NPC cells

BMI1 downregulation reverses FOXA1-silencing-induced cisplatin resistance in NPC cells

Discussion

Materials and methods

Patient specimens

Cell lines and culture

Stable cell line generation via lentiviral transduction

Western blotting

Cisplatin treatment

CCK-8 and Plate colony‑forming assays

Wound healing

Transwell migration and invasion assays

Flow cytometry

Chromatin immunoprecipitation (ChIP) and PCR

Dual luciferase reporter assay

Generation of the subcutaneous xenograft model in mice

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1