Hypoxia-Induced ROS Promotes Mitochondrial Fission and Cisplatin Chemosensitivity via HIF-1α/Mff Regulation

Abstract


Background
Head and neck squamous cell carcinoma (HNSCC), an aggressive and common malignant tumor causing high morbidity and mortality, is a major health challenge worldwide.[1,2] Although great advancements in surgery, chemotherapy, and radiotherapy treatments have been achieved in the past few decades, the overall 5-years survival rate of HNSCC patients has remained approximately 50%.[3] For HNSCC patients, cisplatin (CDDP) based chemotherapy has been established as the drug of choice.[4] However, chemoresistance continues to be responsible for adverse effects from HNSCC treatments, which has led to poor outcomes.Thus, a better understanding of the molecular mechanisms underlying chemoresistance is critical for establishing the most appropriate treatment strategies.[1] The role of the tumor microenvironment and hypoxia in cancer progression has garnered much attention in recent years.[5,6] Normally, the O 2 level of tissues ranges from 1% to 11%, while cells are often cultured in a 20% O 2 environment.[7] Rapid proliferation and unmatched angiogenesis in cancer cells leads to a hypoxic condition in most solid malignant tumors.[8]A recent study has shown that HNSCC experiences hypoxic conditions, which may positively affect anti-EGFR therapies.[9] Previous studies have also shown that excessive accumulation of reactive oxygen species (ROS) could promote cancer progression, [10] and hypoxia induces increased ROS.[8,11] To alter cellular adaptive responses to hypoxia, the activity of hypoxia-inducible factor 1 α-subunit (HIF-1α) acts as a key mechanism.[12] HIF-1α, a transcription factor, could transactivate various target genes to participate in malignant tumor biological behavior.[13,14] Previous studies have shown that abnormal mitochondrial dynamics are correlated with diseases via the regulation of apoptosis.[15,16] Tumor cells respond to microenvironment alteration through the dynamic balance of mitochondrial ssion and fusion.[17] Accumulated evidence has revealed that mitochondrial ssion promotes tumor cell CDDP sensitivity in tongue squamous cell carcinoma.[18,19] However, how mitochondrial ssion contributes to CDDP sensitivity in hypoxia-cultured HNSCC cells is still unclear.
Furthermore, the molecular mechanism of mitochondrial ssion and fusion in modulating CDDP sensitivity in hypoxic-cultured HNSCC cells has not been elucidated.
In the present study, we aimed to investigate the effect of hypoxia on CDDP-cultured HNSCC cells and mitochondrial ssion.This study revealed that hypoxia-induced ROS accumulation promotes mitochondrial ssion and CDDP chemosensitivity via the regulation of HIF-1α/Mff in HNSCC cells.

Hypoxia facilitates CDDP chemosensitivity in HNSCC cells
Hypoxia can regulate malignant tumor vascularization, invasion, chemoresistance, and metastasis in various types of cancer.[8,20,21] To understand whether the HNSCC microenvironment was hypoxic, hypoxia markers[8] (solute carrier family 2 member 1 (SLC2A1), platelet-derived growth factor B (PDGFB), vascular endothelial growth factor A (VEGFA), and HIF-1α) were detected in 72 samples of tumor tissues paired with adjacent normal tissues.The expression levels of hypoxia markers were signi cantly higher in tumor tissues than in the normal tissues (Fig. 1A).The levels of PDGFB were related to patient age (≥ 60 years and < 60 years), sex (male and female), smoking history (yes or no), and alcohol history (yes or no) in our study (Supplementary Table 2).An analysis of The Cancer Genome Atlas (TCGA) database further showed that these hypoxia markers were also signi cantly upregulated in HNSCC samples (Supplementary Fig. 1A).These data con rm the hypoxic environment in HNSCC tumors.
To determine the effect of hypoxia on CDDP sensitivity in HNSCC, Cal27, HN6, and HN30 cells were cultured in 20% O 2 or a hypoxic environment (1% O 2 ).The IC50 value of CDDP, identi ed by MTT assay in Cal27, HN6, and HN30 cells, was 11.05, 15.91, and 9.2 μM, respectively (Supplementary Fig. 1B).Cal27, HN6, and HN30 cells were treated with the IC50 of CDDP for apoptosis assays.The proportion of apoptotic cells (positive for Annexin V FITC and propidium iodide staining) in hypoxia-cultured HNSCC cells was higher than that in 20% O 2 -cultured HNSCC cells.Moreover, the apoptotic effect of hypoxia was augmented in HNSCC cells after incubation with CDDP.The proportion of apoptotic cells in hypoxia-CDDP-cultured HNSCC cells was dramatically higher than that in the 20% O 2 -CDDP-cultured HNSCC cells after 24 h and 48 h (Fig. 1B).These ndings showed that hypoxia enhances CDDP sensitivity in HNSCC cells.

Hypoxia increases mitochondrial ssion via Mff/Mfn2 and ROS levels in HNSCC cells
In the tumor microenvironment, ROS increases under hypoxic conditions [8,11] and is produced as a byproduct by mitochondria.[10] To explore the effect of hypoxia on ROS and mitochondrial ssion, ROS levels and mitochondrial ssion were measured in Cal27, HN6, and HN30 cells.After 24 h or 48 h cultured in hypoxic conditions, Cal27, HN6, and HN30 cells showed a dramatic increase in both the intracellular H 2 O 2 and superoxide (O 2 − ) levels compared to the 20% O 2 -cultured cells (Fig. 2A).Mitochondrial ssion in hypoxia-cultured HNSCC cells increased more dramatically than that in 20% O 2 -cultured HNSCC cells for 24 h or 48 h, indicated by decreased mitochondrial length (Fig. 2B).
Our results showed that the transcriptional expression level of Mff increased more signi cantly in hypoxia-cultured HNSCC cells than that in 20% O 2 -cultured HNSCC cells, and also showed a timedependent increasing pattern (Fig. 2C).In addition, the expression level of Mfn2 decreased more dramatically in hypoxia-cultured HNSCC cells compared to that in 20% O 2 -cultured HNSCC cells (Fig. 2C).
To further determine if Mff and Mfn2 proteins were responsible for mitochondrial ssion induced by hypoxia, Mff and Mfn2 were detected by western blotting.The post-translational level of Mff increased under hypoxia in a time-dependent manner (Fig. 2D).The post-translational level of Mfn2, however, decreased under hypoxic conditions (Fig. 2D).To con rm hypoxic conditions in HNSCC cells, the protein level of HIF-1α, detected by western blotting, was signi cantly increased in HNSCC cells under hypoxic conditions (Fig. 2D).These results indicate that hypoxic HNSCC cells undergo mitochondrial ssion via Mff upregulation and Mfn2 downregulation.

CDDP
To explore the effect of hypoxia on ROS and mitochondrial ssion in HNSCC cells treated with CDDP, ROS levels and mitochondrial ssion were measured.In hypoxia-CDDP-cultured HNSCC cells, the intracellular H 2 O 2 levels and superoxide (O 2 − ) levels were signi cantly higher than that in 20% O 2 -CDDP-cultured cells for 24 h or 48 h (Fig. 3A).The mitochondrial ssion in hypoxia-CDDP-cultured HNSCC cells were more dramatically increased than that in 20% O 2 -CDDP-cultured HNSCC cells for 24 h or 48 h (Fig. 3B).
The protein level of HIF-1α signi cantly increased in the hypoxia-CDDP-cultured HNSCC cells (Fig. 3C).To determine if Mff and Mfn2 proteins were still responsible for mitochondrial ssion induced by hypoxia in CDDP-treated cells, Mff and Mfn2 were detected by western blotting.The post-translational level of Mff increased in hypoxia-CDDP-cultured HNSCC cells and showed a time-dependent pattern, while the posttranslational level of Mfn2 was not signi cantly altered (Fig. 3C).Together, these results suggest that hypoxia-CDDP-cultured HNSCC cells undergo mitochondrial ssion through Mff upregulation.

Hypoxia-induced HIF-1α facilitates Mff expression level in HNSCC
To examine whether Mff accounts for hypoxia-induced chemosensitivity and malignant transformation in HNSCC, the expression levels of HIF-1α, Mff, and Mfn2 were detected in HNSCC tissues.The mRNA expression level of Mff was upregulated more in CDDP-sensitive HNSCC patients than in CDDP-resistant HNSCC patients, while Mfn2 expression was not signi cantly altered (Fig. 4A).Further analysis revealed that HIF-1α expression was correlated with the Mff levels (r = 0.9456, P 0.0001) but not correlated with Mfn2 (Fig. 4B).Moreover, both Mff and Mfn2 expression were signi cantly upregulated in HNSCC tissues (Fig. 4C).In addition, Mff was correlated with the extracapsular spread of metastatic lymph nodes, while Mfn2 was related to tumor size, prior radiotherapy, and pathological differentiation (Fig. 4D).Together, these data revealed that hypoxia-induced mitochondrial ssion events in HNSCC are correlated with chemosensitivity.
In the present study, a signi cantly positive correlation between HIF-1α and Mff was observed.This result indicated that HIF-1α may interact and bind directly with Mff in HNSCC cells.To verify this hypothesis, co-IP, luciferase analysis, and immuno uorescence staining were performed.The endogenous interaction between HIF-1α and Mff was con rmed by Co-IP (Fig. 4E).Moreover, hypoxia-induced HIF-1α dramatically increased the Mff transcriptional activity in 293T cells, and the knockdown of HIF-1α partly decreased Mff activity (Fig. 4F).In addition, immuno uorescent staining showed that Mff was localized to the cytoplasm and that part of HIF-1α was also localized to the cytoplasm (Fig. 4G).These results suggest that HIF-1α binds directly and regulates Mff in the cytoplasm of HNSCC cells.

ROS promote Mff upregulation via HIF-1α in hypoxic-CDDP-cultured HNSCC cells
In the present study, the ndings showed that hypoxia increases ROS and HIF-1α levels in HNSCC cells.
To determine the effect of hypoxia-induced ROS on mitochondrial ssion, H 2 O 2 was treated exogenously to 20% O 2 -cultured HNSCC cells and the free radical scavenger NAC was applied to hypoxic-CDDPcultured HNSCC cells.

Knockdown of Mff suppresses hypoxia-induced mitochondrial ssion and CDDP chemosensitivity in HNSCC cells
To evaluate the biological function of Mff knockdown in hypoxia-CDDP-cultured HNSCC cells, Cal27, HN6, and HN30 cells were stably transfected with LV-Mff and LV-NC.The protein expression level of Mff in HNSCC cells with LV-Mff was notably low (Supplementary Fig. 3A).Moreover, there was no statistically signi cant difference in the proportion of LV-Mff and LV-NC transfected HNSCC cells that were apoptotic in 20% O 2 for 24 h (Supplementary Fig. 3B).Sensitivity to CDDP was higher in the LV-NC transfected HNSCC cells than that in the LV-Mff transfected HNSCC cells under hypoxia for 24 h (Fig. 6A) and 48 h (Fig. 6B).Meanwhile, mitochondrial ssion in hypoxia-CDDP-cultured conditions was dramatically decreased in HNSCC cells with LV-Mff for 24 or 48 h (Fig. 6C).Therefore, inhibition of hypoxia-induced mitochondrial ssion could attenuate the sensitivity of hypoxic HNSCC cells to CDDP.

Mff regulates CDDP sensitivity in HNSCC xenografts
Since Mff expression facilitates the chemosensitivity of HNSCC cells under hypoxia, we evaluated the effect of Mff on tumorigenicity in a xenograft model.For this purpose, Cal27 cells stably transfected with LV-Mff or LV-NC were injected subcutaneously into the dorsal anks of nude mice.The results showed that Cal27 cells with a stable expression of LV-Mff had enhanced tumor growth and tumor weight in the presence of CDDP (Fig. 7A, B).Moreover, apoptosis was attenuated in CDDP-treated xenografts of Cal27 cells stably transfected with LV-Mff (Fig. 7C).Conclusively, our ndings reveal that hypoxia-induced ROS promote mitochondrial ssion and CDDP chemosensitivity through HIF-1α/Mff regulation in HNSCC cells (Figure 7D).

Discussion
The present study shows that Mff promotes mitochondrial ssion and consequently CDDP sensitivity.
Through exploring the mechanism of Mff expression regulation, we found that HIF-1α binds directly to and regulates Mff in the HNSCC cell cytoplasm.Through further experiments, the results revealed that hypoxia-induced ROS increased HIF-1α/Mff levels, promoting mitochondrial ssion and CDDP sensitivity (Figure 7D).
Emerging evidence suggests that abnormal mitochondrial morphology is relevant in various diseases.[23,24] Moreover, the biological mechanisms of the response of mitochondrial ssion to CDDP sensitivity in malignant tumors are still unclear.Previous studies have revealed that an increase in mitochondrial ssion in cancer cells enhances apoptosis and CDDP sensitivity in gastric cancers, [25] gynecological cancers,[26] and tongue squamous carcinomas.[18,19] Due to the hypoxic microenvironment of HNSCCs, the cells were cultured under hypoxic conditions in the present study.A recent study showed that hypoxiainduced mitochondrial ssion causes CDDP resistance in ovarian cancer cells.[8] In the present study, our ndings indicated that hypoxia-induced mitochondrial ssion promotes CDDP sensitivity in HNSCC cells.The opposite effects of tumor cell mitochondrial ssion on CDDP sensitivity may be explained by differences in the cancer cells.
Carefully orchestrated interactions between mitochondrial proteins that facilitate tumor cell death remain an immediate focus of research.Mff, a key molecule that determines mitochondrial size and shape and the regulation of mitochondrial cell death, is a novel therapeutic target in cancer.[27,28] LATS2 facilitates the apoptosis of non-small cell lung cancer by regulating Mff-induced mitochondrial ssion.[29] Furthermore, Mff-induced mitochondrial ssion was attributed to decreased colorectal cancer cell viability.[30] Our ndings showed that Mff was a key molecule participating in mitochondrial ssion, contributing to CDDP sensitivity in hypoxia-cultured HNSCC cells.Both Mff and Mfn2 expression were upregulated in HNSCC tissues, indicating that mitochondrial dynamics in tumor tissues are more active than that in normal tissues.Moreover, the expression of Mff and HIF-1α in HNSCC patients showed that hypoxia-induced mitochondrial ssion events in HNSCC are correlated with chemosensitivity.Recent evidence has shown that HIF-1α regulates the participation of mitochondrial dynamics in various biological behaviors.[31][32][33] Under hypoxic conditions, HIF-1α modulated mitochondrial ssion by direct regulation of the expression of Drp1.[34] Due to the markedly positive correlation between HIF-1α and Mff, further experiments in the present study revealed that HIF-1α participates in mitochondrial ssion by regulating Mff.
Previous studies have revealed that hypoxic conditions could promote an increased level of ROS in the tumor microenvironment.[8,11] The ROS are generated by mitochondria and other cellular elements and participate in malignant tumor biological behavior through the regulation of HIF-1α.[10,35,36] Hypoxiainduced mitophagy is associated with cancer aggressiveness via the interplay of ROS/HIF-1α in gastric cancer cells.[37] Our results demonstrated that hypoxia-induced ROS increases the expression of HIF-1α and Mff, while NAC attenuates these ROS effects.

Conclusions
In summary, we demonstrated that hypoxia-induced ROS promote mitochondrial ssion and CDDP

RNA extraction and real-time PCR analysis
The RNA was extracted from the tumor samples and cells using TRIzol reagent (Takara, Japan), and cDNA was synthesized with a PrimeScript RT Reagent Kit (Takara, Japan).A real-time PCR (RT-PCR) was performed using the SYBR Premix Ex Taq Reagent Kit (Takara, Japan) with the StepOne RT-PCR System (Life Technologies, USA) according to the manufacturer's instructions.The mRNA levels were normalized to β-actin levels.The primer sequences used in the present study are listed in Supplementary Table 1.

MTT assays
Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay.Tumor cells were seeded in a 96-well plate at a density of 2000 cells per well with 100 μL of medium at 37°C.The cells were incubated with 100 μL of 0.5 mg/mL MTT (Sigma-Aldrich, USA) in Dulbecco's modi ed Eagle's medium for 4 h.Twelve hours later, the cells were cultured with a gradient concentration of CDDP for 48 h.The formazan was solubilized in 150 μL dimethyl sulfoxide.Absorbance was measured using a multi-well plate reader (Bio-Rad Laboratories, Hercules, CA, USA) at 490 nm.The half maximal inhibitory concentration (IC50) was used to evaluate the degree of tumor cell response to CDDP.

Apoptosis analysis
Apoptosis was assessed by ow cytometric analysis and terminal deoxynucleotidyl trans-ferasemediated dUTP nick end labeling (TUNEL).TUNEL assays were performed using the Click-iT TM Plus TUNEL assay kit (Invitrogen, USA) according to the manufacturer's instructions.Flow cytometric analysis was performed using the Annexin V FITC Apop Dtec Kit (BD Pharmingen, USA) according to the manufacturer's instructions.The cells were trypsinized and centrifuged at 300 × g for 10 min at 4°C.Afterward, the cell pellets were resuspended in binding buffer and stained with Annexin V FITC and propidium iodide for 15 min on ice.Apoptotic cells were analyzed by ow cytometry (BD FACS Calibur, USA).

Western blot analysis
Tumor cells were harvested at the indicated times in SDS lysis buffer (Beyotime, China).Protein samples were separated using 10% polyacrylamide gels and transferred to 0.45-μm poly (vinylidene uoride) membranes (Merck Millipore, USA).The membranes were soaked in 10% skim milk for 1 h at room temperature and incubated overnight at 4°C with the primary antibody.Subsequently, the membranes were probed with horseradish peroxidase-labeled secondary rabbit antibodies (Beyotime, China) for 1 h.The electrochemiluminescence (New Cell Molecular Biotech, China) developing solution was used to detect signals on a chemiluminometer (Amersham Imager 600).β-Actin was used as a control.The antibodies (1:1000) against Mff, HIF-1α, Mfn2 and β-actin were purchased from Cell Signaling Technology (USA).

Detection of relative ROS levels
Intracellular ROS levels were detected using 2,7-dichloro-dihydro uorescein diacetate (Sigma-Aldrich, USA) and dihydroethidium (Sigma-Aldrich, USA) by ow cytometry (BD FACS Calibur, USA).This ROS analysis technique has been described in a previous study.[8] Mitochondrial staining and analysis of mitochondrial ssion The cells were cultured on coverslips and stained with MitoSpy TM Red CMXRos (BioLegend, USA) for 30 min at 37°C.Afterward, the cells were xed with 4% paraformaldehyde and stained with 4'-6-diamidino-2phenylindole (DAPI; Sangon Biotech, China).Cells were viewed using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany).Mitochondrial morphology was then quanti ed and assessed as described previously. [8]

Analysis of immuno uorescence
The cells grown on coverslips were xed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 10% goat serum, and incubated with the Mff antibody (CST, 1:500) and the HIF-1α antibody (CST, 1:500) at 4°C overnight then incubated with the Alexa Fluor 488-cojugated secondary antibody (Invitrogen, USA) or Alexa Fluor 549-cojugated secondary antibody (Invitrogen, USA) at room temperature in the dark.Cells were then stained with DAPI (Sangon Biotech, China) to detect the nuclei and viewed using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany).

Co-immunoprecipitation analysis
A co-immunoprecipitation (Co-IP) analysis was performed in our previous study.[38] In brief, the cells were lysed with IP buffer and incubated with the speci c antibodies previously mentioned at 4°C overnight, followed by incubation with protein A/G Magbeads (Merck) for 6 h at 4°C.The proteins were separated from the beads for 10 min at 105°C.The supernatants were then used for subsequent immunoblotting analyses.

Cell transfection
SiRNA (Ribobio, China) was transfected into 293T cells using Lipofectamine RNAiMAX (Invitrogen, USA) according to the manufacturer's instructions.Twenty-four hours later, the cells were harvested and the corresponding experiments were performed.The siRNAs used in the present study were designed and synthesized by Guangzhou RiboBio Co., Ltd.(Guangzhou, China).

Luciferase analysis
In brief, the 293T cells (2×10 5 ), which were cultured on a 6-well plate, were co-transfected with Mff response luciferase plasmid and Renilla luciferase vector using the Lipofectamine 3000 reagent (Invitrogen, USA), according to the manufacturer's instructions.After 48 h, the dual luciferase system (Promega, USA) was used to detect luciferase activity.

Lentiviral transduction and stable strain screening
The Mff interfering lentiviral vector [39] (LV-Mff) and interfering control lentiviral vector (LV-NC), which were constructed by HanYin Biotechnology Co, Ltd (China), conferred puromycin resistance.Lentiviral transduction was performed according to the manufacturer's instructions.After 72 h of transfection, the culture medium with puromycin at a nal concentration of 10 μg/mL was used to screen for stably stained cells.

Xenograft mouse experiments
The animal experiments, using BALB/C nude mice (Shanghai Laboratory Animal Center) aged 4 weeks old, were performed in accordance with the ethical standards and national guidelines.Cal27 cells (1×10 6 ) stably expressing LV-Mff or LV-NC were resuspended in 100 μl of phosphate buffered saline and subcutaneously injected into the dorsal anks of mice (n = 6 in each group).Tumor sizes were measured using a caliper every three days.CDDP and saline were intraperitoneally injected at 5 mg/kg body weight in every 3 days from day 7 to 33.After day 33, the mice were sacri ced to collect tumor samples and measure tumor weights.

Statistical analysis
To compare the differences in expression between HNSCC and matched adjacent normal tissue, paired ttests were performed.The Kruskal-Wallis tests and Mann-Whitney U tests were used to analyze the association between mRNA (SLC2A1, PDGFB, VEGFA and HIF-1α) levels and clinical parameters.The comparison between the groups was analyzed using one-way ANOVA or Student's t-test.The correlation between two variables was analyzed using Pearson's correlation.All statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, IL, USA).All values were two-sided, and P < 0.05 was considered statistically signi cant. Figures

Figure 6 B
Figure 6

Figure 7 and
Figure 7

Figure 7 and
Figure 7 and Specimens All HNSCC samples were obtained from the Department of Oral and Maxillofacial Surgery at the Second Xiangya Hospital of Central South University.More speci cally, 72 pairs of tumor and adjacent normal tissues were collected from primary HNSCC patients who underwent an initial surgical treatment between July 2017 and June 2019.Patients who received preoperative treatment and had a malignant history were excluded.Clinical parameters were obtained from medical records.Moreover, clinical stage and pathological differentiation were performed according to the World Health Organization Classi cation of Tumors and the tumor node metastasis staging system from the Union for International Cancer Control.Cell cultures and reagents HN6 and HN30 were kindly provided by the University of Maryland Dental School, USA.Cal27 and 293T cells were purchased from the American Type Culture Collection (MD, USA).Cal27, HN6, HN30, and 293T cells were cultured in Dulbecco's modi ed Eagle's medium (Gibco-BRL, CA, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, CA, USA), streptomycin (100 μg/mL), and penicillin (100 units/mL) at 37°C in a humidi ed 5% CO 2 atmosphere.For hypoxia, tumor cells were cultured in a tri-gas incubator (ESCO, Singapore), consisting of 5% CO 2 , 94% N 2 , and 1% O 2 .Hydrogen peroxide (H 2 O 2 ), CDDP, and N- chemosensitivity via HIF-1α/Mff regulation in HNSCC cells.Moreover, Mff may serve as a new biomarker for the prediction of neoadjuvant chemosensitivity and is important for overcoming chemoresistance, which requires further exploration.enrollment.All experimental methods abided by the Helsinki Declaration.All animal studies were performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the Second Xiangya Hospital Animal Ethical and Welfare Committee (Approval No. 2020489, 2020/9/9).acetyl-L-cysteine (NAC) were obtained from Sigma-Aldrich (USA).