TLR4 Agonist and Hypoxia Synergistically Promote the Formation of TLR4/NF-κB/HIF-1α Loop in Human Epithelial Ovarian Cancer

doi:10.21203/rs.3.rs-768984/v1

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Research Article

TLR4 Agonist and Hypoxia Synergistically Promote the Formation of TLR4/NF-κB/HIF-1α Loop in Human Epithelial Ovarian Cancer

https://doi.org/10.21203/rs.3.rs-768984/v1

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posted 13 Aug, 2021

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Inflammation and hypoxia are involved in numerous cancer progressions. Reportedly, the toll-like receptor 4 (TLR4)/nuclear factor κappaB (NF-κB) pathway and hypoxia-inducible factor-1α (HIF-1α) are activated and closely related to the chemoresistance and poor prognosis of epithelial ovarian cancer (EOC). However, the potential correlation between TLR4/NF-κB and HIF-1α remains largely unknown in EOC. In our study, the possible positive correlation among TLR4, NF-κB and HIF-1α proteins were investigated in the EOC tissues. Our in vitro results demonstrated that LPS can induce and active HIF-1α through the TLR4/NF-κB signaling in A2780 and SKOV3 cells. Moreover, hypoxia-induced TLR4 expression and the downstream transcriptional activity of NF-κB were HIF-1α dependent. The crosstalk between the TLR4/NF-κB signaling pathway and HIF-1α was also confirmed in nude mice xenograft model. Therefore, we first proposed the formation of a TLR4/NF-κB/HIF-1α loop in EOC. The positive feedback loop enhanced the susceptibility and responsiveness to inﬂammation and hypoxia, which synergistically promote the initiation and progression of EOC. The novel mechanism may act as a future therapeutic candidate for the treatment of EOC.

Oncology

Toll-like receptor 4 (TLR4)

Nuclear factor κappaB (NF-κB)

Hypoxia-inducible factor-1α (HIF-1α)

Epithelial ovarian cancer (EOC)

Lipopolysaccharide (LPS)

Hypoxia

Epithelial ovarian cancer (EOC) is aleadinglethal cancer among the gynecological malignancies occurring worldwide[1].In fact, 70–75% of EOC patients are detected at advanced stages, which converts into a high mortality rate. Although the popular treatment options currently available include surgery and chemotherapy, they are restricted by cases of frequent recurrence and thefive‑year survival rate is approximately 30%[2,3]. Therefore, it is necessaryto further elucidate the molecular mechanismsand to improve the treatment modalities forEOC.

Chronic inflammation and hypoxia are linked to thetumor microenvironment[4].Inflammation is a crucial risk stressorthat is associated withthe tumor survivaland invasion. Toll-like receptor 4 (TLR4), which is the indispensablereceptor forlipopolysaccharide (LPS), is detected in a variety of tumors, including in ovarian malignant tumor[5,6]. Past studies have demonstratedthat the activation of TLR4 signaling generates pro-inflammatory cytokines and anti-apoptotic proteins, which in turn contributes tothe growth, metastasis, and chemoresistancein ovarian cancer[7,8]. In response to LPS, TLR4 mediates both myeloid differentiation factor 88 (MyD88)-dependent and -independent signaling pathways. The MyD88-dependent pathway leads to the expression of pro-inflammatory factors by activatingthe early phase of NF-κB. The MyD88-independent signaling pathway induces the late phase activation of NF-κB, which triggers the expression of interferon-beta[9].NF-κB, a critical regulator of inflammatory, is believed to be a key link between inflammation and tumor[10]. Therefore, targeting NF-κB may have therapeutic potential in ovarian cancer treatment.

Hypoxia is an essential feature existing in mostsolid tumors. Hypoxia-inducible factor-1 (HIF-1), which is a key transcription factor of hypoxia response, is a heterodimer that comprises of an induciblesubunit,HIF-1αanda constitutivesubunit,HIF-1β. Under hypoxia, HIF-1α accumulates, forms a dimer with HIF-1β, and modulates more than 100 hypoxia-related genes[11].

Past studies have demonstrated that the TLR4/NF-κB pathway can stabilize and accumulate HIF-1αunder normoxic conditions. In macrophages and dendritic cells, LPS can mediateHIF-1αexpression by activating NF-κB[12,13].More recently, peroxiredoxin 1 (Prx1), a TLR4endogenous ligand, has also been identified to increase the interaction between NF-κBand HIF-1α promoter, which contributes to the enhancement of the HIF-1α mRNA levels along with augmentation of the HIF-1 activity[14]. Therefore, HIF-1α is a key link between hypoxia and inflammation. Kim and his colleaguesdocumented that the expressionlevel of TLR4 was upregulated through HIF-1α in macrophages under hypoxia. Furthermore, the PI3K/Akt pathway, which promotes the translocation and activation of HIF-1, contributes to hypoxia-induced TLR4 expression[15,16]. These factors suggest that the cross-talk between TLR4/NF-κB signaling pathway and HIF-1αmay form a positive feedback loop that promotes the susceptibility to inflammatory signals under hypoxia. Reportedly, the loop of TLR4/NF-κB/HIF-1αcontributes to tissue damage and tumor progressionin pancreatic ductal adenocarcinoma, oral squamous cell carcinoma, and lung ischemia-reperfusion injury[17-19].However, the presence of any association between TLR4 and HIF-1αsignaling in EOC remains unclear.

In the present study, we detected a positive association among TLR4, NF-κB, and HIF-1α protein levels in the EOC tissues. Further studies demonstrated the formation of TLR4/NF-κB/HIF-1αpositive feed-forward loop in EOC cells. These findings are closely associated with inflammation and hypoxia, which synergistically promote the development of ovarian cancer. We believe that our study will contribute to develop novel targeted therapies for EOC treatment.

Materials

Solutions and chemicals listed in the study: lipopolysaccharides (LPS) extracted from Escherichia coli O55:B5, cobalt chloride (CoCl₂), and pyrrolidine-dithiocarbamic acid, ammonium salt (PDTC) (Sigma-Aldrich Inc., Germany); 6R-[[(2-Chloro-4-fluorophenyl)amino]sulfonyl]-1-cyclohexene-1-carboxylic acid, ester (TAK-242) and 5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol (YC-1) (Cayman Chemical Inc., USA); anti-TLR4 antibody (1:500dilution, cat. no.35577; Signaling Antibody, Inc., USA); anti-MyD88 antibody (1:1000dilution, cat. no. ab133739; Abcam, Cambridge, UK); anti-NF-κBp65 (1:1000dilution, cat. no.8242), anti-phospher-NF-κBp65 (Ser536)(1:1000dilution, cat. no.3033) and anti-HIF-1α antibodies (1:1000dilution,; cat. no.14179) (Cell Signaling Technology Inc., USA); anti-β-actin antibody (1:8000dilution,; cat. no.RM2001), goat-anti-mousesecondary antibody (1:8000dilution,; cat. no.RM3001), and goat-anti-rabbit secondary antibody (1:8000dilution,; cat. no.RM3002) (Beijing Ray Antibody Biotech Company, Beijing, China); RPMI 1640 and DMEM-H medium (Invitrogen Co., USA); and fetal bovine serum (FBS; LONSA SCIENCE SRL., USA). All chemicals were of 99% purity.

Cell culture

Human epithelial ovarian cancer cell lines A2780, ES-2, SKOV3 and OVCAR3 were sourced from ATCC. Four cell lines were maintained in RPMI 1640 with 10% FBS or DMEM-H with 10% FBS, respectively. To establish a model of cellular hypoxia, we cultured the ovarian cancer cell lines in 1% oxygen (hypoxia group) instead of 21% (normoxia or control group). CoCl₂ dissolving in 1% FBS medium was used to simulate chemical hypoxia when incubated with cells, and others in cell experiments, like PDTC, were utilized similarly to cobalt chloride. The non-polar reagents such as TAK-242 and YC-1 were dissolved in DMSO medium to a concentrated liquid, followed by dilution to different concentrations with 1% FBS medium. In addition, the concentrations of the reagents mentioned in this paper were determined through preliminary tests.

Immunohistochemistry analysis

The levels of TLR4, NF-κBp65, and HIF-1α in human ovarian normal tissues (n=10), human benign ovarian tumor (n = 10), human borderline ovarian tumor (n= 10), and well-differentiated (n = 12) and poorly differentiated human EOC tissues(n = 14) were assessed by immunohistochemistry analysis. The tissue specimens were obtained from the Department of Pathology,Blinded for peer review, fixed in 10% formalinbuffer,and embedded in paraffin. The sections (5-μm-thick)were prepared and subjected to testing by using the SP Kit (KeyGenNanJing, China) as per the manufacturer’s instruction, followed by overnight incubation with anti-TLR4, anti-NF-κBp65, and anti-HIF-1α (1:200) antibodies at 4℃ in a wet box. After washing thrice with PBS, the biotin-labeled secondary antibody working solution was added drop-wise and incubated at 37℃ for 20 min. Next, the sample was incubated withhorseradish peroxidase-labeled streptavidin at 37℃ for 30 min (washed thrice with PBS). DAB coloring solution was added to the tissue section for color development. After the optimal coloring time was reached based on the observation under a microscope, the section was washed under running water for 2 min to terminate any further color development. The value of integrated option density (IOD) was evaluated by the Image-Pro Plus 6.0 software（Media Cybernetics, Silver Spring, MD，USA）, represented as the summational IODs of 5 random scales of each paraffin section. The Ethics Committee of Blinded for peer reviewapproved the study.

Real-time fluorescence quantitative PCR

We used TRIZOL(Invitrogen, Carlsbad, CA, USA) to extract the total RNA in the cells according to the manufacturer’s protocol, and then detectedmRNA byTransStart®TopGreen qPCR SuperMix (TransGen Biotech, China). Real-timefluorescencequantitative PCR(qRT-PCR) was executedby CFX96 Connect Real-Time PCR Systems (Bio-Rad, USA). The cycling program was as follows:initial denaturation step at 95°C for 3 min, followed by denaturation at 95°C for 10 s, annealing at 60°C for 15 s, and extension at 72°C for 10 s for 40 cycles. All qRT-PCR reactions were performed in triplicate, and the test gene was normalized to the β-actin.The relative expression of gene was evaluated based on the 2⁻^△△^Ct method. The primer sequences used in the experiment were as follows: TLR4(218 bp), (F)5’-GCTCGGCAGACGGTGATAG-3’ and (R)5’-AAGCTCTGGGTTTCATGCCA-3’; MyD88(170bp), (F)5’- TGGCACCTGTGTCTGGTCTA-3’ and (R)5’-ACTTGATGGGGATCAGTCGC-3’; NF-κBp65(240 bp), (F)5’- GCGAGAGGAGCACAGATACC-3’ and (R)5’- AGGGGTTGTTGTTGGTCTGG-3’; HIF-1α(290 bp), (F)5’- ACAAGCCACCTGAGGAGAGG-3’ and (R)5’- TGGCTGCATCTCGAGACTTT-3’ ; β-actin(150 bp), (F)5’- GCACTCTTCCAGCCTTCCTT-3’ and (R)5’-AATGCCAGGGTACATGGTGG-3’.

Western Blotting

We lysed cells with lysis buffer(NanJingKeyGen, China) on an ice bath, and then centrifuged the resultant at 12000 rpm at 4°C for 30 min, then used the BCA kit (Pierce Biochemicals, USA) to detect the protein concentration. 30–80μg protein were separated on 10-12%SDS-PAGE, and then transferred onto a polyvinylidene fluoride membrane. After blocking the antigen, the membrane was incubated overnight at 4°C with primary antibodies, and thenwashed with TBST and incubated with secondary antibodies for 1 h.We detected the target protein levels using enhanced chemiluminescence reagents (Millipore, USA). The values of target protein were normalized to the corresponding β-actin values.

Cell viability assays

MTT assay was performed to detect the cell viability following hypoxia. Briefly, A2780 or SKOV3 cells (4 × 10³ cells per well) were inoculated into 96-well plates overnight, followed by replacement with a medium with 1% FBS, and then culturedfor 24 h. The resultant cells were treated with 1% O₂ or50, 100, 150, 200, 300 μM CoCl₂, respectively. After 24 h, the cells were incubated with 0.5 mg/mL MTT solution (PBS) for 4 h. After dissolvingwith acid-isopropano, the plates weredetected at wavelength of 490 nm.

Plasmids

The kind gifts ofthe pCMVh-HA-ssHIF-1α and pCMVh-HA plasmids were obtained from Dr. Andrew L. Kung (Dana-Farber Cancer Institute, USA). HIF-1α shRNA sequences were obtained from Suzhou Genepharma Co., Ltd. (homo 1614; SuZhou, China). To construct the pGPU6-HIF-1α shRNA plasmid, the HIF-1α shRNA sequences were synthesized, pair annealed, and subcloned into the eukaryotic expression vector pGPU6 vector. The recombinant vectors were transiently transfected into A2780 and SKOV3 cells by Lipofectamin^TM2000 (Invitrogen, Carlsbad, CA, USA). pGL3-HRE6-luc and pGL3-NF-κB-Luc reporter plasmid, a 516-bp fragment harboring 6 copies of the hypoxia-response element (HRE) sequences or NF-κB sequences were amplified from HEC-1-B genomic DNA and cloned into the pGL3-basic vectoras per the manufacturer's instructions.

Transient transfection

The HIF-1α knockdown or overexpression, and the luciferase-expressing cells were established by transfecting with the Lipofectamin^TM2000. Cells were plated into 6-well plates until a confluence of 90-95% was reached. The cells were transiently transfected with 4μg of DNA containing the target gene fragment, for instance, ssHIF-1α, HIF-1α shRN, HRE6-luc, and NF-κB-Luc reporter, among others.

Luciferase assays

The luciferase activities weredetectedby reagents purchased from Promega (Madison, WI, USA). The activity of β-galactosidase was measured using the β-galactosidase Enzyme Assay System (Sigma, St. Louis, MO, USA). The relative luciferase activitieswere normalized to the corresponding β-galactosidase activities.

Xenograft mouse model

4-week-old female BALB/c nude mice (weight: 18-22 g, n=32) were obtained from the Institute of Materia Medica at Blinded per Author Guidelines and bred under SPF condition.After1 week, a total of 5 × 10⁶ SKOV3 cells were collected, washed thrice with saline, and subcutaneously implanted into the right-sided flanks of the nude mice. At 7 days of implantation, the tumor became observable. Tumor dimensions were measured every alternate day. A total of 32 mice showed a tumor diameter of approximately 9 mm at 21 days after implantation, and were randomly divided into 8 groups. Using the intratumoral injection technique, TAK-242, PDTC, or YC-1 or their common vector DMSO/saline were injected into the xenograft tumors, and LPS or saline was injected 1 h later in the same inlet. After 24h, mice were sacrificed and the tumors were excised, weighed, and snap-frozen for RNA and protein extractions or paraffin-embedded for H&E dye (Solarbio, Science and Technology Co., Ltd.). The animal procedure was approved by The Blinded for peer reviewAnimal Experimentation Ethics Committee.

Statistical analyses

Data were analyzed with IBM SPSS21.0 software package (SPSS, Statistical Product and Service Solution Chicago). Differences of multiple sets of measurement data were performed using one-way or two-way ANOVA. The LSD-t test was used for the post-hoc analysis when the variances were equal, and Dunnett’s T3 test for unequal variances. Univariate association among clinical samples was assessed by the Chi-square test. P < 0.05wasset as statisticalsignificant.

Positive correlation exsisted between TLR4 level and the progression of EOC, and the levels of NF-κBp65 and HIF-1α in clinical specimens.

To confirm the presence of a correlation among TLR4, NF-κBp65, and HIF-1α, we first determined the status of the abovementioned proteins in human normal ovary tissues and the clinical specimens of EOC by immunohistochemistry. The representative images (inset in Fig. 1a) illustrated that TLR4 was mainly expressed on the cell surface and cytoplasm, while NF-κBp65 and HIF-1α existed in the cytoplasm and nucleus, respectively. The normal ovary tissues, benign ovarian tumors and borderline ovarian tumors were studied to reveal that expressions of TLR4, NF-κBp65, and HIF-1α were absent or at a low level, while as the cancer progressed, the proportion of the TLR4^+ve, NF-κBp65^+ve, and HIF-1α^+ve EOC cells were significantly increased (Fig. 1a). As illustrated in Figure. 1b, the integrated optical density (IOD) value analysis of positively stained settings indicated that the increased levels of TLR4, NF-κBp65 and HIF-1α were observed in the well or poorly differentiated EOC specimens in comparison with the normal ovarian tissues (all P< 0.05). Meanwhile, compared with the well differentiated EOC, IOD values of TLR4^+ve, NF-κBp65^+ve, and HIF-1α^+vecells obtained from poorly differentiated EOC were significantly higher (all P< 0.05). More importantly, further analysis revealed that, in the clinical specimens of EOC, the TLR4 level was significantly positively related to the levels of NF-κBp65 and HIF-1α, respectively (Fig. 1c). Among the TLR4 strongly positive specimens (++/+++, IODs ＞3.0 × 10⁵), the percent of low NF-κBp65 or HIF-1α expression (-/+, IODs ≤ 3.0 × 10⁵) was only approximately 11.1% or 33.3%, whereas high NF-κBp65 or HIF-1α expression was approximately 88.9% or 66.7%. In addition, we also found that positive correlation existed with the NF-κBp65 levels and HIF-1α. Based on the abovementioned clinical outcomes involving IHC staining, we speculated the possibility of a positive interaction among TLR4, NF-κBp65, and HIF-1α expression during EOC progression.

The constitutive expression of TLR4/MyD88/NF-κBp65/HIF-1α signals in human EOC cell lines.

MyD88, the critical adaptor protein, contributed to the development and immune escape of various tumors. Considering the active role of MyD88, the TLR4/NF-κBp65 signaling pathway can be divided into MyD88-dependent or MyD88-independent pathways. To investigate the exact role of TLR4/MyD88/NF-κBp65/HIF-1α pathway in the progression of EOC, we first examined the respective constitutive mRNA and protein levels of TLR4, MyD88, NF-κBp65, and HIF-1α in A2780, SKOV3, OVCAR3, and ES-2 cell lines. As shown in Figure 2b, the broad expressions of the abovementioned proteins were recorded in the 4 EOC cell lines, except for MyD88 in the A2780 cells. No MyD88 expression could be detected even after repeat experimentations. Notable, as compared with the A2780 cells, the SKOV3 cells showed extraordinarily high levels of MyD88, NF-κBp65, and HIF-1α. To elucidate whether the MyD88-dependent and MyD88-independent TLR4/NF-κBp65 signaling pathways had similar effects on the HIF-1α activity of EOC, we used the A2780 and SKOV3 cells as the study cell models for the subsequent analysis.

Upregulating effects of LPS on HIF-1α activity in human EOC cells were inducedthroughthe TLR4/NF-κB pathway.

The close association between hypoxia and inflammation is already well-known. In the present study, we verified whether TLR4/NF-κBp65 pathway is involved in mediatingthe upregulating effects of LPS on the expression of HIF-1αin EOC cells.

First, LPS, the inflammation inducer and the proven natural ligand of TLRs, was used as an activator of the TLR4/NF-κB pathway in the subsequent analysis. As shown in Figure 3a, a time-effect study by western blotting analysis showed that the HIF-1α level along with the levels of TLR4, NF-κBp65, and p-NF-κBp65 in the A2780 and SKOV3 cellswere significantly enhanced by treatment with 1 μg/mL LPS for 30 min, 2 h, or 6 h, except for the p-NF-κBp65 levels in SKOV3 cells (for 5 or 15 min). Notably, the MyD88 expression in A2780 cells could not be detected either with or without LPS treatment for less than 2 h, but could be induced by LPS treatment exceeding 6 h. Meanwhile, the level of MyD88 in the SKOV3 cells was slightly enhanced after LPS treatment for 2 h and 6 h. These results indicate that in EOC cells the resultant inflammation may activate and enhance the MyD88-dependent TLR4/NF-κB pathway. For exploring the exact mechanisms underlying these events, the treatment time of LPS was fixed to 6 h in the subsequent analysis.

Next, the TLR4 inhibitor TAK-242 and the NF-κB inhibitor PDTC were used to determine whether upregulating effects of LPS on the HIF-1α level in EOC cells were mediated via TLR4/NF-κB signaling. As illustrated in Figure 3b, the levels of TLR4, NF-κBp65, p-NF-κBp65, and HIF-1α in A2780 and SKOV3 cells treated with LPS were obviously greater than those in the control cells. Pretreatment with 25 μM TAK-242 (TLR4 inhibitor) or 25 μM PDTC (NF-κB inhibitor) for 1 h followed by LPS treatment could remarkably block the abovementioned effects, indicating that LPS may enhance the HIF-1αexpression in EOC cells via TLR4/NF-κB-mediated pathway.

Third, a luciferase assay was conducted to observe the HIF-1αtranscriptional activity. Our results suggested that HIF-1αtranscriptional activity (indicated by the relative HRE-luc activity inFigure 3c) after treatment of LPS were significantly higher than the control group for A2780 and SKOV3 cells. While pretreatment with TAK-242 or PDTC could remarkably block the upregulation effects of LPS. Therefore, our cumulative findings suggest that LPS may promote the HIF-1α activity in EOC via the TLR4/NF-κBsignaling pathway.

LPS and hypoxia stimuli possess the synergistic effects on HIF-1α activity in human EOC cells.

Accumulating evidence has demonstrated that chronic inflammation and hypoxia stimuli are the two key factors involved in tumor development. After confirming the upregulating effects of LPS on the HIF-1α activity in EOC cells, we next investigated the presence of synergetic effects of LPS and hypoxia stimuli.

For this purpose, we first determined the mimic dose-effect and time-effect of CoCl₂ similar to that of 1% O₂ by the MTT and western blotting methods. InFigure 4a,low-dose CoCl₂ treatment (50 and 100 μM) for 24 h had no effect on EOC cell proliferation, similar to that by 1% O₂.The HIF-1α level in EOC cells increased by different intensities after CoCl₂ treatment for 24 h (50, 100, 150, 200, 300 μM), and the maximum effect of CoCl₂ occurred at the 50 μM dosefor A2780 cells and 100 μM dose for SKOV3 cells. Consequently, the working concentration of 50 μM and 100 μM CoCl₂ were respectively used for A2780 and SKOV3 in the subsequent experiments. Meanwhile, a time-effect study by western blotting showed that the HIF-1αexpression and the TLR4, MyD88, NF-κBp65, and p-NF-κBp65 levels were significantly enhanced by CoCl₂ treatment for different designated durations. As shown in Figure 4b, the obvious upregulating effects of CoCl₂ accrued mainly at the treatment time of 2 h, 6 h, and 12 h. Therefore, we used the LPS treatment time of 6 h (Fig. 3a) as the CoCl₂ treating time in the subsequent teststo investigate the synergetic effect of LPS and hypoxia stimuli on EOC cells,.

Past studies have shown that hypoxia could trigger inflammation and even further aggravate the inflammatory response. Our results confirmed that both 1% O₂ and CoCl₂ treatment could enhance the upregulating influence of LPS on the HIF-1α activity in human EOC cells via the TLR4/MyD88/NF-κBpathway. As compared to the control group, 1% O₂ alone, CoCl₂ alone, or LPS alone treatment could increase the HIF-1αexpression and the TLR4/MyD88/NF-κBp65 signaling. Meanwhile, treatment with 1% O₂ or CoCl₂together with LPS, in comparison with treatment with LPS alone, could significantly increase the effects (Fig. 5a). We further detected the transcriptional activity of HIF-1α after LPS or (and) hypoxia treatment. In Figure 5b, hypoxia or LPS alone could obviously increase the HRE luciferase activity in both the groups of EOC cells. Furthermore, treatment with 1% O₂ or CoCl₂could upregulate the transcriptional activity of HIF-1α in comparison with that of the corresponding cells treated with LPS alone in A2780 cells. A similar increasing trend was recorded for SKOV3 cells, albeit there was no statistical significance between the treatments with 1% O₂ together with LPS and with LPS alone. These results indicate that hypoxia, in the presence of LPS, could further enhance the TLR4/MyD88/NF-κBp65 signaling and the expression and activity of HIF-1α.

LPS and hypoxia stimuli induced the formation of TLR4/NF-κB/HIF-1α signaling loop in human EOC cell lines.

HIF-1αplaysa vital part in the initial and developing stages of tumor metastasis. As confirmed by other researchers and based on our results, both hypoxia and LPS stimuli contribute to the activation of HIF-1α via TLR4/NF-κB signaling pathway. We therefore investigated whether hypoxia could regulate the activation of TLR4/NF-κB signaling viaHIF-1α. First, pretreatment with YC-1 (10 μM), the commonly used specific blocker of HIF-1α, for 1 h could remarkably block the upregulating effects of 1% O₂ or CoCl₂on the TLR4/NF-κB signals with the co-concurrent blockage of HIF-1α, indicating that hypoxia could indirectly affect the TLR4/NF-κB signals via HIF-1α (Fig. 6a). Moreover, pretreatment with YC-1 for 1 h could remarkably block the transcriptional activity of NF-κB induced by 1% O₂ or CoCl₂, respectively (Fig. 6b). Second, sense HIF-1α vector (pCMVh-HA-ssHIF-1α), small hairpin RNA targeting HIF-1α (shRNA-HIF-1α) vector, and the corresponding empty vectors were respectively used to increase or decrease the HIF-1α expression in A2780 and SKOV3. As illustrated in Figure. 6c, the overexpression or knockdown of HIF-1α led to the enhancement or attenuation of the TLR4/NF-κB signaling in A2780 and SKOV3 cells, respectively. Based on the abovementioned results (Figs. 3,5and 6), we speculated that the LPS and hypoxia stimuli induced the TLR4/NF-κB/HIF-1α signaling loop and that this signaling loop may exist and contribute to the EOC development.

In vivo study of the TLR4/NF-κB/HIF-1α signaling loop induced by LPS.

To explore the impact of LPS on the TLR4/NF-κB/HIF-1α signaling loop of EOC in vivo, SKOV3 cell suspension was injected into the flanks of nude mice. After 3 weeks of injecting, TAK-242 (TLR4 inhibitor, 20 μg/mouse), PDTC (NF-κB inhibitor, 60 μg/mouse), or YC-1 (HIF-1α inhibitor, 20 μg/mouse) were injected to the subcutaneous tumor basement, and, after an hour, LPS (5 μg/mouse) was injected by the same method. After 24 h, the mice were sacrificed to evaluate the expression levels of the TLR4/NF-κB/HIF-1αin tumor tissues. As illustrated in Figure 7a, the morphological results of solid EOC tumors and HE staining suggested the successful construction of murine EOC model. A large number of EOC cells and a small number of epithelial-mesenchymal cells appeared in the exacted tumor tissues. Statistical analysis on the weight of tumor-bearing mice and the diameter of tumors showed no difference (Fig. 7b) among the groups. Notably, western blotting analysis was conducted on protein extraction from tumor samples and revealed significant blockage caused by TAK-242, PDTC, as well as YC-1 on the TLR4/NF-κB/HIF-1α signaling loop (Fig. 7c).

TLR4, which recognizes LPS, initiates the innate immune and inflammatory responses. The transcription factor,HIF-1α plays akey roleinhypoxia-relateddisease. It is well known that TLR4 and HIF-1α contribute to the progression of numerous inflammatory diseases, including cancer[15,17]. In this study, we revealedthat TLR4 could induce the expression and activation of HIF-1αvia NF-κB and that HIF-1α could also directly regulate the TLR4/NF-κB pathway. The cross-talk between TLR4/NF-κB and HIF-1α signaling pathway may form a positive feedback loop and contribute to tumor initiation,malignant progression and prognosis in EOC.

In recent years, enhanced TLR4 expression was identified in several tumors, including pancreatic cancer, colorectal cancer, and esophageal cancer, among others[20-22]. Moreover, the TLR4 expression was correlated with increased levelsof NF-κB and HIF-1α, higher clinical staging, and dramatically reduced patient survival[17]. Kelly et al[7]. first reported that the expression of TLR4 in EOC cells and the chemoresistance to paclitaxel are mediated by the TLR4/MyD88 signaling. Furthermore, the levelof TLR4 was positively associated with the key markers of NF-κB signaling pathway, while the co-expression of TLR4/MyD88/NF-κB was correlatedwith poor prognosis in EOC patients[23,24]. Recent investigation demonstrated that HIF-1α level is markedly enhanced in ovarian cancer in comparison with that in normal ovarian tissues and that the enhanced expression of HIF-1α is related to markedly shorter overall patient survival. Furthermore, the expression level of HIF-1αis associated with chemoresistance in metastatic and aggressiveovarian tumor. Thus, the high level of HIF-1α may be taken for a prognostic marker in ovarian cancer[25,26]. However, the potential associations with TLR4/NF-κB and HIF-1α remain largely unknown in EOC. In this study, we noted elevated expressions of TLR4, NF-κBp65, and HIF-1α in EOC specimens, and their expressions were significantly correlated with the histological differentiation degree of the tumor. We further demonstrated a positive correlation among TLR4, NF-κBp65, and HIF-1α, indicating a possible crosstalk between TLR4 signaling and HIF-1 pathway, which may act synergistically to promotethe progression of EOC.

MyD88, a key adaptor molecule, plays critical roles in TLR4 signaling. The expression of MyD88 was detected in approximately 70% of EOC patients, and it has been consideredas a significantly poor prognostic indicator of tumor[27,28]. In this study, we selected the MyD88(-) A2780 cells and MyD88(+) SKOV3 cells for further analysis. Our results indicated that LPS couldactivate the TLR4/NF-kB signaling pathway in both A2780 and SKOV3 cells. Both MyD88-dependent and -independent pathways are known to mediate TLR4/NF-Kbsignaling pathwayfollowing LPS response. Although TLR4 signaling through MyD88 activates the early phase of NF-κB, MyD88-independent pathway also triggers TLR4-mediated late phase activation of NF-κB[9]. This finding is consistent with our observation of earlier NF-κB P65 phosphorylate peak in SKOV3 cells than in the A2780 cells. Unexpectedly, our results showed that LPS could induce MyD88 protein expression in MyD88(-) A2780 cells by exceeding 6 h of stimulation. It has beendemonstrated that MyD88, an essential signalingcomponent of TLR4pathway,mediates paclitaxel resistance[7]. Accordingly, we speculated that inflammation may be involved in the acquisition of chemoresistance through the MyD88 expression in ovarian cancer cells. However, further research is warranted to confirm this speculation. Meanwhile, our results indicatedthat in both A2780 and SKOV3 cells, the expression levels of TLR4 decreased 24 h after LPS treatment. It is possible that the endocytosis of receptor following the ligand-receptor interaction results in lysosomal degradation, which leads to controlled duration of TLR4 signaling[29]. We further revealedHIF-1α expression and activation in ovarian cancer cells followingLPSstimulation, which also involves the TLR-induced NF-κB activation. Previous studies have indicated that LPS-induced HIF-1α stabilization is mediated by the TLR4/NF-κB signaling pathway under normoxia in immune cells and oral squamous cell carcinoma[18,30,31]. Indeed, NF-κBcoulddirectly bind withthe promoter of HIF-1αlocated at position 197/188 upstream of the transcription start site and modulate the expression of HIF-1α. Mutation of this site resulted in a significantreduction in response to p65, conﬁrming that this site is functional[32,33]. Within the present study, when TLR4 or NF-κB was inhibited pharmacologically, our results showed that the protein levels and transcriptional activity of HIF-1αwere downregulated after LPS-induction. Therefore, the present study suggests for the first time the existence of TLR4-NF-κB-HIF-1α signaling pathway in ovarian cancer cells, which implicates an important association between the inﬂammatory pathway and hypoxia-response pathway.

It is well-known that hypoxia stabilizes the inducible subunitHIF-1α, which is rapidly degraded via the ubiquitin-proteasome pathway undernormoxia. Interestingly, short-term hypoxia increases the HIF-1α levels not only by stabilizing HIF-1α protein but also by inducing HIF-1α genetranscription in the pulmonary artery smooth muscle cells.This mechanism involves the activation of NF-κB and its binding to the HIF-1α promoter located at position 197/188[34]. In contrast, as per a diverse range of studies, hypoxia has no influence on the levels of HIF-1α mRNA. Given the transient nature of this response, differences in the exposure time to hypoxia and the cell types may explain the controversial results. In addition, the NF-κBbasal activity is a prerequisite to accumulate HIF-1α protein under hypoxia in cultured cells or tissue. The complete block of the NF-κB activation pathway prevented hypoxia-induced HIF-1α[35,36]. Therefore, NF-κBmaybe essential for the extent and speed of HIF-1α activation following hypoxic insult. In accordance with our results, CoCl₂ treatment (a hypoxia mimetic) activated NF-κB and induced HIF-1α protein expression in both A2780 and SKOV3 ovarian cancer cells. As per these results, we speculated that hypoxia may active the NF-κB signaling pathway and contribute to the regulation of inﬂammation viain EOC.

In addition to activating NF-κB, hypoxia could enhance the response of susceptibility to inﬂammatory signals by upregulating TLR4. In macrophages, HIF-1α transcriptionally regulates the TLR4 level under hypoxia via binding to the TLR4 promoter. The mouse TLR4 promoter contains a hypoxia-responsive element (HRE) located at position -407 to -404, while the human TLR4 promoter containingat least two HREs is located at the positions -2811 to -2814 and -1185 to -1188. Therefore, hypoxia augmented the cell responsiveness to bacterial components owing to the increased TLR4 expression in macrophages[15]. In addition, further investigation demonstrated that PI3K/Akt at least partially contributes to hypoxia-induced TLR4 expression via regulating HIF-1α accumulation and transcriptional activation[23]. Our results also showedthat the expression levels of TLR4 were augmented on exposure to hypoxic stress. Moreover, hypoxia treatment augmented the responsiveness of ovarian cancer cells to LPS, including the TLR4 expression level and the downstream NF-κB and HIF-1α activities. Here, we further demonstrated that the hypoxia-mediatedTLR4 expression and downstream NF-κBtranscriptional activity were HIF-1α dependent by blockingthe HIF-1α accumulation with YC-1. Meanwhile, the ability of HIF-1α expression plasmid to enhance the TLR4 expression and the ability of HIF-1α shRNA to inhibit the TLR4 expression provided additional evidence to sustain the role of HIF-1α on regulating the expression of TLR4 in ovarian cancer cells. In accordance with our results, recent evidences have showed that HIF-1α could mediate the expression of TLR4 in pancreatic cancer, oral squamous cell carcinoma, and hepatocellular carcinoma under hypoxia. These studiesrevealed the critical effectof HIF-1α-mediatedTLR4-NF-κB signaling pathway in tumor growth and progression[18,37,38].

In the present study, we noted a strong positive correlation among TLR4, NF-κBp65, and HIF-1α in EOC specimens, indicating the existence of a crosstalk between TLR4/NF-κB and HIF-1α signaling pathway. Our in vitro results showedthat TLR4 signaling pathway induced the HIF-1α protein expression and enhanced HIF-1α transcription activity via NF-κB. Moreover, HIF-1α could activated the TLR4/NF-κB signaling pathway by upregulating the expression of TLR4. The in vivo transplantation model confirmed that TLR4 and HIF-1α mutually regulated each other in EOC. Combined with the results, we believe that TLR4-mediated HIF-1α activation and HIF-1α-induced TLR4 expression form a positive feedback loop that is closely associated with inflammation and hypoxia, which in turn can synergistically promote the development of EOC. Our findings may provide morepromising therapeutic strategies for epithelial ovarian cancer based on the inhibition of TLR4/NF-κB/HIF-1 loop.

Funding

This work was supported by the National Natural Science Foundation of China (No.81572852, 81502256)and the Great Program of Science Foundation of Tianjin (No. 18JCZDJC33200).

Competing interests

The authors have no conﬂicts of interest.

Data Availability

All data in this published article are available from the corresponding author on reasonable request.

Authors’ contributions

XG and YW contributed to the study design and provided the funding acquisition, BZ, XN and SH conducted the study and analyses, JY and YW were involved in data validation, XW provided resources, XG, XN and SH wrote the original draft, YW and BZ refiningthe manuscript.

Ethics approval

The clinical study was approved by the Ethics Committee of the Tianjin Tumor Hospital. The animal experiments were approved by The Tianjin Medical University Animal Experimentation Ethics Committee.

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TLR4 Agonist and Hypoxia Synergistically Promote the Formation of TLR4/NF-κB/HIF-1α Loop in Human Epithelial Ovarian Cancer

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