AdipoR1 Regulates Ionizing Radiation-Induced Ferroptosis in HCC cells through Nrf2/xCT Pathway

Radiotherapy has been used for decades in the treatment of liver cancer. We previously found that adiponectin receptor (AdipoR1) is a prognostic biomarker for hepatoma carcinoma (HCC) after stereotactic body radiation therapy (SBRT) and blocking AdipoR1 enhances radiation sensitivity in hepatoma carcinoma cells. In the current study, we aimed to elucidate the roles of AdipoR1 in ionizing radiation- (IR-) induced radiosensitivity by activating ferroptosis pathway in HCC cells. We found that IR upregulated the expression of AdipoR1 and furthermore promoted the protein stability of transcription factor Nrf2, Nrf2 binded to the xCT promoter and increased xCT transcription and expression, and this directly contributed to the protective function in the early stage of radiation in HCC cells. AdipoR1 knockdown significantly inhibited expression of Nrf2 and xCT and, furthermore, increased both IR- and erastin-induced ferroptosis, which could be abolished by the rescue of Nrf2 and xCT. For the first time, we found that radiation-induced ferroptosis was mediated by AdipoR1-Nrf2-xCT pathway in HCC cells. These results provide new insights to the development and application of novel therapeutic strategies for hepatoma carcinoma.


Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver cancer with poor prognosis, and it is also the third cause of global cancer-related death [1][2][3][4]. Most patients with HCC are usually diagnosed at late stage and usually receive local radiotherapy and chemotherapy [5,6]. However, radiotherapy still has some inherent limitations, such as side effects and radiation resistance. It is an urgent task to explore novel targets that improve the radiosensitivity of HCC.
Ferroptosis is regulated by a network revolving around glutathione peroxidase 4 (GPX4). Correspondingly, genetically or pharmacologically inactivating GPX4 or xCT can lead to ferroptosis [8,16,17]. The function of cystine/glutamate antiporter xCT is to import cystine for glutathione biosynthesis and antioxidant defense, and it is overexpressed in multiple human cancers. Thus, the mechanism of ferroptosis agonists (such as erastin) is to inhibit systemic xc−-mediated cystine import, resulting in depletion of intracellular GSH and subsequent iron-dependent lipid peroxidation [18,19]. Nuclear factor E2-related factor 2 (Nrf2) is a key regulator required for cells to maintain oxidative homeostasis, which is activated under conditions of high oxidative stress. Nrf2 protects cells from oxidative stress by binding to antioxidant response elements (AREs) in the nucleus, thereby promoting transcription of target genes and translation of antioxidant and anti-inflammatory proteins [20]. In addition, studies have shown that Nrf2 can inhibit ferroptosis by upregulating the expression of xCT, and knocking down Nrf2 can significantly reduce the level of xCT and promote the accumulation of lipid peroxides [21].
Adiponectin is a protein with insulin-sensitizing, antiinflammatory, and antiapoptotic functions [22]. Adiponectin needs to bind to its receptor to exert its biological function. At present, 3 kinds of adiponectin receptors were proven: AdipoR1, AdipoR2, and T-cadherin. Studies have shown that adiponectin receptors are closely related to the occurrence and development of various cancers, such as breast cancer, colorectal cancer, and renal cell carcinoma [23,24].
In our previous work, we followed up and collected blood samples from liver cancer patients with SBRT treatment. After transcriptome analysis, we found that AdipoR1 was significant increased and might be a prognostic biomarker for HCC after SBRT. However, how AdipoR1 regulates the progression of HCC and its underlying mechanisms remains unknown. In this study, we found that AdipoR1 is highly expressed in tumor cells, and its knockdown could enhance radiosensitivity of HCC cells via Nrf2-xCT signaling pathway. These findings suggested that AdipoR1-regulated ferroptosis may be an important target for enhancing the radiosensitivity of HCC, which also provides a new strategy for reducing the radioresistance of HCC in the future. 2.3. Radiation. The cells were exposed to ionizing radiation (10 Gy) using an X-ray generator (X-RAD 320 ix, Precision X-ray Inc., North Branford, CT, USA) at a dose rate of 3 Gy/min.

Cell Viability by CCK-8 Assays.
Cell viability was determined by Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) according to the manufacturer's protocol. The cells were seeded in 96-well plates (2 × 10 3 cells/well) and treated with drugs. CCK-8 was added to each well, and the cells were incubated for 3 h. OD values were recorded at 450 nm using a microplate reader. The proliferation rate of the cells was calculated by the following formula: cell viability = ðOD experimental group − OD blank/OD control group − OD blankÞ × 100%.
2.5. Flow Cytometric Analysis of Cell Death. Trypan blue solution was used for observation of cell death by flow cytometry. The cells were seeded in 6-well plates (8 × 10 4 cells/well) and irradiated with 10 Gy. The cells were collected and centrifuged at 500 × g for 5 minutes at 4°C. The cell pellet was washed with PBS, stained with trypan blue for 3 minutes, and detected cell death by flow cytometry (ACEA NovoCyte 2040R, USA).
2.6. Colony Formation Assay. The cells were placed in 6-well plates and cultured in DMEM (Invitrogen) containing 10% FBS (Gibco) at 37°C and 5% CO 2 . 24 h later, the cells were irradiated with different dose, as 0, 2, 4, and 6 Gy. After incubation for 14 days, the colonies were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 30 min and stained with 0.2% crystal violet (Solarbio, Beijing, China) at room temperature for 15 min. The colonies containing ≥50 cells per dish were counted. Cell survival curves were fitted with the multitarget, single-hit model.    Table 1, and the 2 −ΔΔCt was utilized to calculate the relative expression levels.
2.12. Western Blot Analysis. The cells were collected with a cell scraper and lyzed with RIPA buffer. Total proteins (20 μg) were separated on a 12% SDS-PAGE and transferred onto PVDF membrane (Millipore). After blocking with 6% skim milk in Tris-buffered saline-tween (TBST) for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies, washing 3 times followed by incubation with secondary antibody for 1 h at room temperature. The immunoreactions were visualized by ECL solution (Thermo Fisher Scientific, USA) and analyzed using the ImageJ software (Bio-Rad).
2.13. Statistical Analysis. SPSS 22.0 software (SPSS, Chicago, IL, USA) was utilized for statistical analyses. Differences between the two groups were evaluated with Student's t -test (two-tailed). One-way ANOVA followed by Bonferroni post hoc tests was performed to analyze multiple groups. Survival curve was generated with Kaplan-Meier method. Experimental results are presented as mean ± SD. Data was considered as statistically significant when p value was less than 0.05.

IR Upregulated the Expression of AdipoR1 in HCC Cells.
Referring to the TCGA, AdipoR1 mRNA level was higher in tumor as compared with normal tissues (Fig. S1A). K-M curve showed that high expression of AdipoR1 was related to poor prognosis in HCC patients (Fig. S1B). Furthermore, the basal expression of AdipoR1 in HCC cells was tested and remarkably higher than hepatic cells (Figures 1(a) and 1(b)). Next, we analyzed the expression of AdipoR1 under IR in HCC cells; 10 Gy X-rays increased the expression of Adi-poR1 in both MHCC-97H and HepG2 cells compared with sham (Figures 1(c)-1(f)). Taken together, these results demonstrated that radiation could upregulate AdipoR1 expression in HCC cells. To further determine the effects of AdipoR1 on radiosensitivity, colony formation assay was performed in MHCC-97H (Figures 2(b) and 2(c)) and HepG2 cells (Figures 2(e) and 2(f)). Colony formation assay revealed that AdipoR1 knockdown significantly reduced the survival rate in HCC cells, compared with shControl group. Taken together, these results demonstrated that AdipoR1 knockdown increased radiosensitivity in HCC cells.

AdipoR1 Is Involved in IR-Induced Ferroptosis in HCC
Cells. It has been reported that IR induces ferroptosis in cancer cells [8]. We further investigated whether AdipoR1 regulates IR-induced ferroptosis in HCC. The cell viability of MHCC-97H with shAdiopR1 was lower than shControl following IR treatment. To investigate the types of cell death AdipoR1 involved, MHCC-97H cells with shAdipoR1 or shControl were pretreated by such inhibitors of cell death before irradiation as 3-MA, ZVAD, Rapan, and Fer-1. Compared to DMSO group, the IR-induced inhibition of cell viability was significantly reversed by Z-VAD and Fer-1 in AdipoR1 positive cells (shControl group) but failed to change in shAdipoR1 cells, indicating IR-induced apoptosis and ferroptosis in MHCC-97H cells in an AdipoR1 dependent manner (Figure 3(a)). Moreover, the hallmarks of ferroptosis include the accumulation of lipid peroxidation and ferroptosis-relative genes such as PTGS2 [8]. Simultaneously, the increase of lipid peroxidation and PTGS2 expression was also induced in shAdipoR1 cells compared with shControl cells after IR treatment (Figures 3(b)-3(f)).

Oxidative Medicine and Cellular Longevity
AdipoR1 silencing also increased the expression of PTGS2 (Figure 3(g)). These data suggested that ferroptosis occupied a very important position in IR-induced cell death and Adi-poR1 acted as a key regulator of this effect. Erastin-induced ferroptosis might also involve other signals. To test this hypothesis, we examined the expression of several key ferroptosis-relative proteins in response to erastin. As shown in Figures 4(j) and 4(k), after erastin treatment, the expression of AdipoR1 and xCT was induced,  3.5. Effect of AdipoR1 on Ferroptosis-Related Proteins after IR. As previously pointed out, AdipoR1 knockdown could increase IR-induced ferroptosis in HCC cells. To study the potential mechanisms, we examined the expression levels of several key protein involved in ferroptosis after IR. Transferrin is the main iron-containing protein in plasma, responsible for carrying the iron absorbed by the digestive tract and the iron released by the degradation of red blood cells. Transferrin receptor 1 (CD71) is a type II transmembrane receptor and carrier protein responsible for the uptake of cellular iron through receptor-mediated endocytosis [25]. As shown in Figures 5(a)

AdipoR1 Protected Cells from IR-Induced Ferroptosis by
Upregulating xCT Expression. To investigate the relationship between the protein expression of AdipoR1 and xCT, we analyzed publicly available gene expression datasets and found that the AdipoR1 expression level positively correlated with the xCT level in two cohorts of hepatoma carcinoma patients (Figure 6(a)). Compared with the cells in shControl group, the xCT protein levels were dramatically decreased in the shAdipoR1group (Figures 6(b) and 6(c)). This correlation is of significance given that xCT overexpression found in lung adenocarcinoma [26], colorectal cancer [27], and HCC cells [28] has been shown to promote the HepG2 (e and f) cells was evaluated through colony formation assay, respectively. The sensitization of radiation was measured using the multitarget, single-hit model. Data is presented as mean ± SD. * p < 0:05, * * p < 0:01, and * * * p < 0:001.

Oxidative Medicine and Cellular Longevity
growth of the tumors. To further verify whether AdipoR1 regulates ferroptosis via xCT after IR, xCT knockdown increased IR-induced lipid peroxidation and cell death, which could be significantly rescued by Fer-1 (Figures 6(d)-6(h)). Subsequently, xCT was overexpressed through transfection with pcDNA3.1Flag-xCT; overexpression of xCT successfully suppressed IR-induced lipid peroxidation and cell death in HCC cells (Figures 6(i)-6(m)). These results suggested that AdipoR1 protected cells from IR-induced ferroptosis by promoting the expression of xCT, which may be its key downstream target.

AdipoR1
Regulated IR-Induced Ferroptosis by AdipoR1-Nrf2-xCT Pathway. Previously, Nrf2 exert its antioxidant role in cellular protection by regulating xCT expression [21]. In addition, we further explored the molecular mechanism of AdipoR1 in regulating xCT. Firstly, we determined whether AdipoR1 knockdown influenced the expression of    (Figures 7(b) and 7(c)), indicating that AdipoR1 knockdown decreased NRF2 protein stability. Moreover, luciferase reporter assay determined that Nrf2 could bind to xCT promoter and improve the transcription of xCT   Oxidative Medicine and Cellular Longevity

Discussion
Radiotherapy has been used for decades in the treatment of liver cancer. For early stage liver cancer, radiotherapy is mainly used for patients who cannot be surgically removed or ablated; for intermediate and advanced liver cancer, radiotherapy is mainly combined with other therapies. However, the problem of radioresistance in current radiotherapy leads to poor prognosis.
AdipoR1 is an adiponectin receptor, which participates in regulation of glucose and lipid metabolism through stimulation of fatty acid oxidation, suppression of hepatic glucose output, and increased insulin sensitivity in liver [29]. In our previous work, we found that AdipoR1 is a prognostic biomarker for HCC after SBRT in primary liver cancer and blocking AdipoR1 enhances radiation sensitivity in hepa-toma carcinoma cells both in vitro and in vivo [30]. In this study, we aimed to understand the mechanisms regulating the radioresistance and figured out what type of cell death did AdipoR1 participate in. We found that knockdown of AdipoR1 increased IR-induced increased cell death and that Fer-1, a ferroptosis inhibitor, significantly reduced cell death. We then examined ferroptosis indicators lipid ROS and PTGS2, both of which we found were also significantly increased in the AdipoR1 knockdown group after IR. These results were also consistent with what we detected after treatment of HCC cells with erastin (an inducer of ferroptosis). Taken together, AdipoR1 is involved in ionizing radiation-induced ferroptosis in HCC cells.
Ferroptosis is a nonapoptotic form of cell death that can be induced by metabolic stress such as GSH depletion [8].
Many studies have shown that ferroptosis can selectively target aggressive cancer stem cells and is also expected to enhance the efficacy of radiotherapy and overcome the resistance [11,12,[31][32][33][34]. However, the mechanism by which AdipoR1 regulated IR-induced ferroptosis remains unclear. Recent studies revealed that xCT overexpression inhibits ferroptosis through importing cystine, promoting GSH biosynthesis, and subsequently facilitating GPX4-mediated detoxification of lipid peroxides [9]. GPX4, a glutathione peroxidase, utilizes reduced glutathione to convert lipid   11 Oxidative Medicine and Cellular Longevity hydroperoxides to lipid alcohols, thereby mitigating lipid peroxidation and inhibiting ferroptosis [16,17,35]. We then examined the expression levels of several key proteins involved in ferroptosis. The datas showed that AdipoR1 knockdown significantly inhibited expression of xCT in HCC cells. We also examined the expression of ferroptosisrelated protein CD71 and transferrin. The results showed that the expression of CD71 and transferrin was reduced in shAdipoR1 MHCC-97H cells but had no significant effect on the expression of both proteins in HepG2 cells. This indicated that AdipoR1 mainly regulated IR-induced ferroptosis in HCC cells by regulating the expression of xCT. And overexpression of xCT could decrease the lipid ROS and cell death caused by AdipoR1 knockdown. Therefore, we suspected that AdipoR1 might regulate ferroptosis through xCT in HCC cells.
Recent studies have shown that AdipoR1 could maintain the integrity of the blood-brain barrier through the APPL1/ AMPK/Nrf2 signaling pathway [36]. And Nrf2 exerts its antioxidant role in cellular protection by regulating xCT expression [21]. Therefore, we speculated that AdipoR1 could regulate the expression of xCT via Nrf2. The results showed that overexpression of Nrf2 could decrease the lipid ROS and cell death caused by AdipoR1 knockdown. And AdipoR1 could mediate the expression of Nrf2 by regulating the protein stability of Nrf2. Meanwhile, Nrf2 could regulate transcription activity of xCT. This result was consistent with the previous reports [21].
In summary, our results demonstrated that AdipoR1 knockdown could enhance radiosensitivity of HCC cells via Nrf2-xCT signaling pathway (Figure 8). These findings suggest that AdipoR1-regulated ferroptosis may be an important target for enhancing the radiosensitivity of HCC, which also provides a new strategy for reducing the radioresistance of HCC in the future.