Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells

Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells Wan-xin Peng a,1, Er-meng Xiong a,1, Lu Ge a, Yan-ya Wan a,b, Chun-li Zhang a, Feng-yi Du a, Min Xu b, Reyaz Ahmed Bhat b, Jie Jin a, Ai-hua Gong a,n a b

School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 4 July 2015 Received in revised form 9 December 2015 Accepted 15 December 2015

Previous studies suggest that early growth response gene-1 (Egr-1) plays an important role in hypoxiainduced drug-resistance. However, the mechanism still remains to be clarified. Herein, we investigated the role of Egr-1 in hypoxia-induced autophagy and its resulted hypoxia-driven chemo-resistance in Hepatocellular Carcinoma (HCC) cells. Our data demonstrated that Egr-1 was overexpressed in HCC tissues and cells and conferred them drug resistance under hypoxia. Mechanistically, Egr-1 transcriptionally regulated hypoxia-induced autophagy by binding to LC3 promoter in HCC cells, which resulted in resistance of HCC cells to chemotherapeutic agents; while dominant negative Egr-1 could inhibit autophagy level, and thus enhanced the sensitivity of HCC cells to chemotherapeutic agents, indicating that hypoxia-induced Egr-1 expression enhanced drug resistance of HCC cells likely through autophagy. Accordingly, it is suggested that a mechanism of hypoxia/Egr-1/autophagy axis might be involved in drug resistance in HCC. & 2015 Published by Elsevier Inc.

Keywords: HCC Hypoxia Egr-1 Autophagy Chemo-resistance

1. Introduction Hepatocellular Carcinoma (HCC) is the fifth most common tumor in the world and the third most common cause of cancer related mortality [1,2]. Surgery that offers the greatest potential cure for HCC, but most patients have unrespectable disease at presentation [3]. Chemo-therapy has been used for over 30 years but resistance remains a significant barrier for both cytotoxic and targeted agents, and therefore has triggered great research efforts worldwide for new treatment modalities that might be applicable to this cancer [4,5]. Due to hypovascularity, the hepatic tumors are significantly more hypoxic than adjacent normal tissues (ANT) [6,7]. To adapt to this unfavorable condition, cancer cells would activate various biological behaviors, such as angiogenesis, and migration to less hypoxic and more nutritious areas [8–10]. The extent of tumor hypoxia seems to inversely correlate with patient prognosis and is often associated with resistance to conventional Abbreviations: AFP, Alpha-fetoprotein; ANT, adjacent normal tissues; CQ, chloroquine; DAPI, 4′, ,6-diamidino-2-phenylindole; Ad-DN-Egr-1, Adenovirus-dominant-negative Egr-1; Egr-1, early growth response gene-1; HCC, Hepatocellular Carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LC3, microtubule-associated protein 1 light chain 3; 3-MA, 3-methyladenine n Corresponding author. E-mail address: [email protected] (A.-h. Gong). 1 These authors contribute to this paper equally.

treatment modalities [11–13]. Previous studies suggested that autophagy can be induced in many solid tumors by hypoxia, nutrient deprivation or metabolic stress [14,15]. It has been well documented that autophagy plays an important role in the resistance of cancer cells to chemotherapy, likely associated with classic hypoxia-inducible factor-1 (HIF1)-dependent pathway [13]. Recently, growing evidence suggests that many HIF-1-independent signaling pathways are essential for induction of autophagy, such as mTOR signaling and ER stress [16]. However, molecular information about the regulation of autophagy in cancer cell is still vague. Hence, an understanding of underlying mechanisms of autophagy associated drug resistance is critical [17,18]. Egr-1 is a zinc finger transcription factor classified as an immediate-early response which is induced with many environmental signals including oxidative stress [19,20]. It has been reported that Egr-1 expression level correlates with sensitivity to chemo-drugs in cancer cells [21,22]. Furthermore, Egr-1 can control MDR1 expression at the transcriptional level and enhance drug resistance of breast cancer [23], and Egr-1 knockdown enhances chemo-sensitivity of breast cancer cells to camptothecin [22]. In previous study, we also unveiled the relationship between Egr-1 and chemo-resistance in HCC cells, but the mechanism remains largely unknown [24]. In this report, we examined the effects of hypoxia-induced Egr-1 on drug resistance in HCC cells. We investigated Egr-1 expression in

http://dx.doi.org/10.1016/j.yexcr.2015.12.006 0014-4827/& 2015 Published by Elsevier Inc.

Please cite this article as: W.-x. Peng, et al., Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.12.006i

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clinical samples from HCC patients and HCC cells cultured in hypoxic condition. We found that inhibition of Egr-1 transcriptional activity prevented autophagy-induced drug resistance under hypoxic condition. The results detail a mechanism of hypoxia/Egr-1/ autophagy axis involving in drug resistance in HCC.

2. Materials and methods 2.1. Materials Adenoviruses expressing green fluorescent protein (Ad-GFP) or dominant-negative-Egr-1 (Ad-DN-Egr-1) were kindly provided by Prof. Chao-jun Li in Nanjing University. 3-Methyladenine (3-MA) was purchased from Sigma. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum and trypsin were purchased from Gibco (Invitrogen, CA, USA). Antibodies against Beclin-1, LC3, Egr-1 and β-tubulin were purchased from Cell Signaling (CA, USA), antibody against AFP was purchased from Santa Cruz (CA, USA) and antibody against SQSTM1/p62 was purchased from Proteintech (IL, USA). HRP-conjugated secondary antibodies were purchased from Pierce (IL, USA). All the other chemicals were of high purity from commercial sources. 2.2. Cell lines and culture conditions The HCC cell lines HepG2 and SMMC-7721 were obtained from ATCC (USA) and Cancer Cell Repository (Shanghai, China). Cells were maintained in DMEM with 10% FBS at standard cell culture conditions (37 °C, 5% CO2 in humidified incubator). 2.3. Immunohistochemically staining and scoring tissues samples Samples of tumor and adjacent non-tumor (ANT) liver tissues were obtained from patients who had undergone primary HCC curative hepatic resection at the Shanghai Changzheng Hospital of China. IHC staining was performed as Lee et al. previously described [25]. Briefly, 4 μm sections of paraffin-embedded tissue microarrays were deparaffinized and rehydrated in xylene and degradation alcohol. Antigen unmasking was performed by pretreatment of the slides in 0.01-M citrate buffer (pH 6.0) at 98 °C for 5 min using a microwave oven. The slides were then cooled to room temperature. Endogenous peroxidase was cleaned by incubating the slides in 3% hydrogen peroxide for 10 min. After washed in 0.01 M PBS (pH 7.4), the sections were incubated for 10 min at room temperature with normal goat serum, followed by incubation with anti-Egr-1 antibody (dilution: 1:50), and anti-AFP antibody (dilution: 1:40,) overnight at 4 °C. The S-P kit (KIT-9710; MAIXIN, Fuzhou, China) was used to visualize antibody binding on the slides. Counterstaining was performed with hematoxylin. AFP and Egr-1 protein expression in these specimens was evaluated under an Olympus CX31 microscope (Olympus, Center Valley, PA). A mean percentage of positive tumor cells were determined in at least five areas at
400 magnifications (50–250 cancer cells per area). The intensity score representing the estimated staining intensity was scored as follows: negative, 0; weak, 1; moderate, 2; and strong, 3. 2.4. Western blotting Following treatments, both adherent and floating cells were collected, and then western blot analysis was carried out by the method as described previously [26].

2.5. Cell migration assay Cells were infected with Ad-GFP or Ad-DN-Egr-1 for 24 h and then trypsinized, resuspended in serum-free DMEM, and transferred to the upper chamber (5
103 cells in 500 μl) transferred on the top of chambers (24-well insert, 8-μm pore size, BD Biosciences, San Jose, United States). DMEM containing 10% fetal calf serum was added to the lower chamber. After incubation for 24 h under hypoxia or normoxia condition, non-invaded cells were removed from the upper well with cotton swabs, while the invaded cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed (
100) in five independent fields for each well. Each test was repeated in triplicate. 2.6. Immunofluorescence and image analysis Cells were seeded on glass cover slips in 6-well plates and grown to subconfluence, then infected with Ad-GFP or Ad-DN-Egr1 for 24 h. Next the plates were incubated for 4 h under hypoxic conditions. Routinely, cells were washed with PBS, fixed with icecold 100% methanol for 15 min at  20 °C, and blocked with 3% BSA in PBS for 1 h. Cells were incubated with primary antibody to LC3 (1:100 dilution,) overnight at 4 °C, and then incubated with anti-rabbit cy3-conjugated secondary antibodies (Cwbio, Beijing, China) for 1 h at room temperature in the dark, DAPI (1 μg/ml, Pierce, IL, USA) counter stain was used for nuclear staining. After extensive washing, the cover slips were then mounted on glass slides, and the fluorescent images were captured with a fluorescent microscope and a SPOT CCD camera. 2.7. Real time PCR Total RNA extracting, cDNA amplifying and quantifying as described previously [28]. The followings were primers used in this investigation: LC3: Fwd: 5′-GGACAACAGTCAGTTCTGCTT-3′; Rev: 5′CACAGGGCTATCAGGG AGC-3′; GAPDH: Fwd-5′-GGGTGTGAACCATGAGAAGT-3′ and Rev-5′-GTAGAGGCAGGGATGATGTT-3′. 2.8. Cell counting kit-8 for the drug sensitivity The measurement of viable cell mass was performed with Cell Counting Kit-8 (Beyotime, Shanghai China) according to manufacturer's instructions. Briefly, 3000 cells/well were seeded in a 96-well plate, grown at 37 °C for 24 h, and then the cells were exposed to different concentrations of cisplatin and epirubicin in hypoxia or normoxia for a further 24 h. Subsequently, 10 μl WST-8 dye was add to each well, cells were incubated at 37 °C for 1 h and the absorbance was finally determined at 450 nm. 2.9. Chromatin immunoprecipitation (ChIP) assay Chromatin immunoprecipitation (ChIP) was performed using the Simple Chip enzymatic chromatin immunoprecipitation kit (catalog #9002) from CST according to the manufacturer's protocol. Briefly, for one chromatin preparation, 1
107 cells were cross-linked with 1% (v/v) formaldehyde for 10 min at room temperature. Subsequently, nuclei were isolated by lysis of the cytoplasmic fraction, and chromatin was digested into fragments of 150–900 bp by micrococcal nuclease (400 gel units) for 20 min at 37 °C, followed by ultrasonic disruption of the nuclear membrane using a standard microtip and a Branson W250D Sonifier (four pulses, 60% amplitude, duty cycle 40%). For immunoprecipitation, 5–15 μg of total chromatin was incubated overnight at 4 °C with 5 μg of the respective antibodies and Normal Rabbit IgG (catalog number 2729) as negative control. After incubation with 30 μl of ChIP grade protein G-agarose beads for

Please cite this article as: W.-x. Peng, et al., Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.12.006i

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2 h at 4 °C, antibody-DNA complexes were eluted from the beads and digested by 40 μg of Proteinase K for 2 h at 65 °C, followed by spin column-based purification of the DNA. Transcription factor binding was finally assessed by PCR using the following primers: Fwd: 5′-CTGACCCCTCCCTCAAG-AGT-3′ Rev: 5′-CCCTGAGGTGACGGTTGT-3′ to amplify a 149 bp DNA fragment ( 272 to 124) of the LC3B promoter. 2.10. Promoter reporters and dual-luciferase assay The human LC3 promoter was amplified by PCR using the following primers: Fwd: 5′-CCGCTCGAGGCGCCCAGAATGAAGGCTCGGGAC3′ Rev: 5′-CCCAAGCTTGGTGCAGGGATCTGGGCGGCGG-3′. PCR products were digested and then cloned into the multiple cloning site of the pGL3-Basic vector (Promega). The LC3 mutant (M) promoter constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene, Jolla, CA). In the dual-luciferase assays, cells were cultured for 36 h after transfection, and the activities of firefly luciferase and Renilla luciferase were quantified using the dual-luciferase reporter assay system (Promega). All experiments were conducted at least twice, in triplicate. 2.11. Cell cycle analysis Cells were cultured in six-well plates to 70–80% confluence. Next they were treated with cisplatin or epirubicin as indicated concertation for 24 h under hypoxia. Apoptosis was analyzed by measuring sub-G1 peaks on a flow cytometer (BD Instruments, CA,

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USA) after cells were fixed with ethanol and stained with propidium iodide [29]. 2.12. Ethics statement Human samples were obtained from all patients with written informed consent. Both written informed consent and study were approved by the Institutional Review Board of the Changzheng Hospital, Second Military Medical University. All procedures were done according to protocols approved by the Institutional Review Board of the Changzhen Hospital, Second Military Medical University and conformed to the NIH guidelines on the ethical use of animals.

3. Results 3.1. Egr-1 is over-expressed in HCC tissues To evaluate the correlation of Egr-1 with HCC, we first employed a human tissue microarray containing 96 normal and 102 malignant HCC samples using IHC. We found that HCC samples showed strong immunostaining of Egr-1 and AFP compared to adjacent tissues, representative images of both normal and malignant HCC of different scores are shown in Fig. 1A. Cores were blindly and semiquantitatively scored for Egr-1 and AFP staining intensity as negative (0), weak (1 þ), moderate (2 þ), or strong (3 þ). The quantization results of Egr-1 and AFP immunostaining

Fig. 1. Egr-1 is over-expressed in human HCC tissues compared to ANT tissue samples. (A) Immunohistochemical staining of Egr-1 and AFP. A tissue microarray was stained for Egr-1; staining is shown for Egr-1 in ANT and tumor tissue. Large panel sare 10
images; inset windows show 100
zoom images. (B) Quantitation of Egr-1 and AFP immunostaining. Cores were blindly and semiquantitatively scored for Egr-1 and AFP staining intensity as negative (0), weak (1þ ), moderate (2 þ ), or strong (3 þ ). Tissue types were grouped into either negative or weak staining compared to moderate or strong for statistical analysis. Numbers represent the total number of cores in each scoring intensity group. Numbers in parentheses are percentage of cores within each tissue type (normal or melanoma) in each of the four scoring categories. (C) Graphical representation of the differences of Egr-1 and AFP staining in ANT (N) and cancer tissues (T) (*Po 0.05, ***Po 0.01).

Please cite this article as: W.-x. Peng, et al., Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.12.006i

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Fig. 2. Egr-1 confers drug resistance in liver cancer under hypoxia. (A) Egr-1 expression was rapidly induced by hypoxia. HepG2 and SMMC-7721 cells were cultured in normoxic (0 h) or hypoxic (0.5, 1, 4 h) condition. (B) Growth inhibition in the presence or absence of transcriptional activity of Egr-1. HepG2 and SMMC-7721 cells were infected with Ad-GFP or Ad-DN-Egr-1s for 24 h, and then treated with different concentrations of cisplatin for 24 h at normoxia or hypoxia. (C) Immunoblot analysis of total and cleaved caspase3 (C-caspase3) in HepG2 and SMMC-7721 cells after Ad-GFP or Ad-DN-Egr-1 infection and treatment with 5 μM cisplatin under hypoxic condition for 24 h. (D) Flow cytometry of PI staining in cisplatin-treated HepG2 and SMMC-7721 cells. HepG2 and SMMC-7721 cells were performed after propidium iodide staining. The cells were incubated for 48 h in the presence of cisplatin (5 μM) under normoxia or hypoxia condition. Figure shows the representative mean 7SD of percent population of cells in each phase of cell cycle of three experiments (**Po 0.01 vs. hypoxia Ad-GFP).

were presented in Fig. 1B. Fig. 1C was the graphical representation of the differences of Egr-1 and AFP staining in ANT (N) and liver cancer tissues. 3.2. Egr-1 expression enhances the chemoresistance of hepatocellular carcinomacells under hypoxia Egr-1, a transcriptional regulator, which is known to be rapidly induced by several microenvironmental stimulants, including hypoxia [30]. To determine whether Egr-1 expression could be upregulated in hepatocellular carcinoma cells in response to hypoxia stimuli, we determined Egr-1 expression in SMMC-7721 and HepG2 cells cultured in complete medium at normoxia (20% O2) or hypoxia (1% O2) condition for 0.5 h, 1 h and 4 h. The results showed that Egr-1 expression was rapidly up-regulated under hypoxic condition for 1 h (Fig. 2A). Recently, Egr-1 was shown to mediate chemo-resistance in breast tumor cells [21]. Therefore, we asked whether the upregulated Egr-1 might protect hepatomic cells from chemotherapeutic agents-induced cell death. To test this hypothesis, we infected SMMC-7721 and HepG2 cells with adenoviruses-Ad-GFP or dominant-negative Egr-1 (Ad-DN-Egr-1) for 24 h, and then treated cells with different concentrations of chemotherapeutic agents including cisplatin and epirubicin for 48 h in normoxic or hypoxic condition. Cell viability assay by CCK-8

showed that 23.7% SMMC-7721 and 39.5% HepG2 cells were killed in Ad-GFP group, compared with 30.8% and 46.8% in Ad-DN-Egr-1 group under hypoxia by cisplatin at 5 μM (Fig. 2B). Treatment with other chemotherapeutic agents such as epirubicin also showed that cell mortality in Ad-DN-Egr-1 group was higher than that in Ad-GFP group under hypoxia (Supplementary Fig. S1). Moreover, we also found that Egr-1 knockdown increased the activation of the pro-apoptotic protein caspase-3 (Fig. 2C). These results were further confirmed by PI staining assay with flow cytometry (Fig. 2D). Taken together, the above results suggested that Egr-1 might confer HCC cells the chemo-resistance induced by hypoxia. 3.3. Egr-1 promote migration of HCC cells under hypoxia in vitro Another function of the hypoxia is an enhancement of migration of HCC, which was previously demonstrated by many studies [31–33]. Meanwhile, it is proved that migration can facilitate chemoresistance in many kinds of cancer such as ovarian cancer, breast cancer and pancreatic cancer [34–36]. To characterize the effects of Egr-1 on migration in HCC under hypoxia, we infected SMMC-7721 and HepG2 cells with Ad-GFP or dominant-negative Egr-1 (Ad-DN-Egr-1) for 24 h. Next, transwell experiment was done on infected cells. We observed that Ad GFP cells migrated 2or 3-fold faster than parental cells in normoxic and hypoxic

Please cite this article as: W.-x. Peng, et al., Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.12.006i

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Fig. 3. Egr-1 induces the migration of HCC cells. (A, B) HepG2 and SMMC-7721 cells were infected with Ad-GFP or Ad-DN-Egr-1 for 24 h, and the metastasis changes was measured by transwell assay (*P o0.05 vs. Hypoxia Ad-GFP); (C) the morphological change of SMMC-7721 cells after Ad-DN-Egr-1 infection under hypoxic condition; (D) the protein levels of Collagen-I, N-cadherin, Snail and vimentin were measured by Western blot.

condition, respectively. (Fig. 3A and B). In contrast, little movement of cells was seen in Ad-DN-Egr-1 group under hypoxic condition, indicating that cell migration was Egr-1 dependent. Furthermore, we observed the morphology of SMMC-7721 cells, compared with Ad-GFP group, all the cells infected with Ad-DNEgr-1 loss their scattering, spindle-shaped morphology and presented epithelial characteristics (Fig. 3C). To probe the molecular basis for Egr-1-enhanced cell motility, we next examined some cell migration biomarkers such as vimentin, collagen-I and N-cadherin. Knockdown of Egr-1 activity in SMMC-7721 and HepG2 cells decreased N-cadherin, vimentin, collagen-I and snail levels (Fig. 3D) compared with control cells. 3.4. Hypoxia-induced autophagy depends on Egr-1 expression Previous study proved that Egr-1 regulated autophagy in cigarette smoke-induced chronic obstructive pulmonary disease [37,38]. To test whether hypoxia-induced Egr-1 also regulates autophagy in HCC cells, we utilized the Ad-DN-Egr-1 to inhibit the function of Egr-1 and proceeded to detect the procedure of autophagy. As observed in Fig. 4A, hypoxia treatment robustly induced LC3II transformation and expression of Beclin-1. Interestingly, competitive inhibition of Egr-1 transcriptional function by Ad-DN-Egr-1 significantly blocked the induction of autophagosomes as indicated by the immunofluorescence of LC3 punctate structures (Fig. 4B). We further confirmed the above data by

western blot (Fig. 4D), indicating that Egr-1 was required for hypoxia-induced autophagy. 3.5. Egr-1 transcriptionally up-regulates LC3 expression To test whether Egr-1 directly regulates LC3 expression, we first investigated the influence of Egr-1 expression on LC3B mRNA levels in hypoxia-treated cells. Infection of HCC cells with Ad-DNEgr-1 decreased LC3 mRNA transcription during 24 hours continuous hypoxia in HepG2 cells and SMMC-7721 cells, compared with Ad-GFP group (Fig. 5A). We further analyzed the sequence of the LC3 promoter by using the Egr-1 consensus binding sequences and identified four candidate transcription factor binding. Next, we generated wild type and various mutants of LC3 promoter (Supplementary Fig. S2). Luciferase assays revealed that Egr-1 was rapidly induced to bind the LC3 promoter region ( 223 to  214) under stimulation by hypoxia in SMMC-7721 cells. Nevertheless, the mutant containing mutations in binding site exhibited lower promoter activity than wild-type LC3 promoter in (Fig. 5B and C). To provide direct evidence that Egr-1 binds to the endogenous LC3 promoter during transcription, we performed chromatin immunoprecipitation (ChIP) assays using SMMC-7721 cells. The result demonstrated that the binding site of Egr-1 in LC3 promoter ( 223 to  214) bound specifically to Egr-1 protein, and Egr-1 transcriptional function knockdown by Ad-DN-Egr-1 strikingly inhibited the Egr-1 binding to this region (Fig. 5D). Taken together,

Please cite this article as: W.-x. Peng, et al., Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.12.006i

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Fig. 4. Hypoxia induced autophagy is depended on Egr-1 in HCC cells. (A) Western blot analysis of LC3II/LC3I ratio and Beclin1expression level under normoxic and hypoxic condition. (B, C) Immunofluorescence staining of LC3. HepG2 and SMMC-7721 cells were infected by Ad-GFP or Ad-DN-Egr-1 for 24 h before cultured in normoxia or hypoxia for 4 h. Magnification,
40. White arrows, punctuate LC3 structure. (D) Western blot analysis of LC3II/LC3I ratio, p62 expression. HepG2 and SMMC-7721 cells were infected by Ad-GFP or Ad-DN-Egr-1 for 24 h before cultured in normoxia or hypoxia for 4 h.

these results clearly indicated that Egr-1 regulated LC3 gene transcription by binding to the GC element on the LC3 promoter.

data particularly demonstrated that the inhibition of autophagy could enhance the chemosensitivity of HCC cells.

3.6. Egr-1-induced autophagy confers hypoxia-mediated resistance to chemotherapy in HCC cells

4. Discussion

Recent data indicate that inhibition of autophagy may enhance chemo-sensitivity in human cancer cells. Thus, we determined whether Egr-1-induced autophagy also contributes to chemoresistance of hepatomic cells under hypoxia. We utilized the class III phosphatidylinositol 3-kinase (PI3K) inhibitor 3-MA or CQ (a specific inhibitor of lysosome which prevents the fusion of autophagosome to lysosome) to inhibit autophagy in SMMC-7721 and HepG2 cells. To confirm the effects of two inhibitors on autophagy under hypoxia, we then examined LC3 conversion and p62 degradation, two selective markers of autophagy. As shown in Fig. 6A, we observed the conversion of the LC3 (LC3-I to LC3-II) and the degradation of p62 after 24 h exposure to the indicated concentrations of 3-MA and CQ in both cells. To further confirm our results, cells were transfected with siRNA targeting Atg5 to suppress autophagy. The results were same as above (Fig. 6B). Flow cytometry assay results indicated that 3-MA or CQ combination with cisplatin results in an increased percentage of cells than cisplatin treatment alone under hypoxia (Fig. 6C). Consistent with above results, immunoblot analysis showed that the level of Bax was significantly increased in 3-MA/cisplatin or CQ/cisplatintreated cells compared to control group, while the level of Bcl-2 was decreased in combination treatment group (Fig. 6D). These

It is now widely accepted that the hypoxia microenvironment of HCC contributes significantly to chemotherapy failure and drug resistance [13,39,40]. It has been reported that Egr-1, a transcription factor and an immediate early gene, can be induced in a transient manner by hypoxia, which is compared with hypoxiainducible factor-1α (HIF-1α) in parallel experimental sets. Egr-1 expression increases two-fold 10 min after UV irradiation, and rises to a maximum (eight-fold induction) after 2 h [41]. In this study, we also find that Egr-1 expression is rapidly upregulated after 1 h hypoxia treatment. Egr-1 has been termed an immediate-early response protein based on the brisk kinetics of its induction within minutes of a stimulus and on its rapid decay, often within hours. It is a controversial topic on the roles of Egr-1 in HCC. Hao and his colleagues reported that Egr-1 expression was down-regulated in hepatocellular carcinoma, and re-expression of Egr-1 decreased cell growth and tumorigenicity in nude mice [42]. Conversely, Tian et. al addressed that Egr-1 repressed RRM2B transcription and promote the HCC migration through a negative feedback loop [43]. Of note, HGF, a cytokine involved in the progression of hepatocellular carcinoma, up-regulates Egr-1 expression and increases cell scattering and migration through Egr-1-mediated up-regulation of

Please cite this article as: W.-x. Peng, et al., Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.12.006i

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Fig. 5. Egr-1 directly binds to LC3 promoter to regulate its expression in vitro. (A) Overexpression of Egr-1 could significantly enhance LC3 expression under hypoxia. Total were collected and subjected to qPCR. HepG2 cells were infected with Ad-GFP or Ad-DN-Egr-1 then treated with hypoxia for 4 h. Expression of LC3 was determined by realtime PCR with GAPDH as the standard. (B) The cloned DNA fragments were sequenced and BLAST searched to identify a fragment that was located upstream of the LC3 promoter. An Egr-1 consensus binding site (  223 to  214) was found at the promoter region of LC3 (  272 to  124). (C) HepG2 cells were transfected with the luciferase reporter that contained the wild-type or mutant LC3 promoter. The Renilla luciferase plasmid was used as an internal control. After hypoxia stimulation for 4 h, luciferase activity was determined. The data were normalized by the Renilla luciferase activity and expressed as a fold induction with respect to control cells. (*Po 0.05: compared the vector group with LC3-wt; †Po 0.05: compared the LC3-wt-luc with LC3-mut-luc3). (D) HepG2 cells were infected with Ad-GFP or Ad-DN-Egr-1 for 24 h before cultured with hypoxia for 4 h. Chromatin samples were immunoprecipitated with anti-Egr-1 and evaluated for factor binding to the LC3B promoter region. The PCR primers amplify a fragment flanking the proximal GC element in the LC3 promoter.

snail [44]. Interestingly, recent studies indicate that Egr-1 confers cancer cells a multidrug resistance (MDR) phenotype through activating MDR1 gene expression [21]. Herein, we at the first time report that hypoxia-induced Egr-1 is critical to autophagy, which contributes to drug resistance in HCC cells. Previous studies have reported that autophagy can serve as a mechanism of adaptation in cancer cells to induce resistance to apoptosis for survival under drug therapy [45–47]. In this study, we confirm that hypoxia induces autophagy in HCC cells, and find that Egr-1 is required for hypoxia-induced autophagy, as evidenced by autophagosomal marker LC3 conversion, increased Beclin1 protein levels and accumulation of punctate formation of LC3. Our further data from inhibition of autophagy using 3-MA, CQ or siRNA targeting ATG5 also support that autophagy contributes to hypoxia-induced drug resistance in HCC cells. Actually, Lee et al.'s studies show that hypoxia-induced Egr-1 may play a role in hypoxia-induced pulmonary hypertension through regulating autophagy [48]. In current study, Egr-1 has been identified to be expressed at higher levels in hepatocellular carcinoma (including cancer cells and tumor tissues) in response to hypoxia. In this context, we thus questioned whether Egr-1 could induce autophagy under hypoxic condition, and in turn

resulted in the drug resistance in HCC. Mechanically, we found that Egr-1 could directly bind to the proximal region (  272/  124) of the LC-3 promoter to transcriptionally regulate LC3 expression under hypoxia, and inhibition of Egr-1 transcriptional activity by dominant negative Egr-1 abrogates the binding of Egr-1 to LC3 promoter, and repress the formation of autophagosomes. Furthermore, inhibition of Egr-1 increases the cisplatin-sensitivity of HCC cells. Accordingly, it is suggested that Egr-1-induced autophagy might contribute to drug resistance. In conclusion, our results provide new preliminary evidence for the role of Egr-1 as a molecular factor involved in the chemosensitivity of HCC cells. Our data demonstrated that Egr-1 is required for induction of autophagy, which confers the chemo-resistance of HCC cells under hypoxia. Therefore, the hypoxia/Egr-1/ autophagy axis might represent a novel target for effective HCC chemo-therapy.

Disclosure statement The authors have no conflict of interest.

Please cite this article as: W.-x. Peng, et al., Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.12.006i

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Fig. 6. Inhibition of autophagy enhanced cell death after cisplatin treatment under hypoxia. (A and B) HepG2 and SMMC-7721 cells were treated with 2 mM 3-MA, 30 μM CQ or siRNA-ATG5 for 24 h under hypoxic condition. Whole cell lysates were then subjected to immunoblotting with antibodies against p62, LC3 and β-tubulin. Differential p62 degradation levels and the relative ratio between LC3II and LC3I isoforms after normalized to β-tubulin were analyzed to confirm the inhibition of autophagy. (C, D) HepG2 and SMMC-7721 cells were treated with indicated concentrations of cisplatin and/or 3-MA, CQ or siRNA-ATG5 for 24 h. Cell apoptosis was analyzed by FACS analysis (C) or immunobloting of Bax and Bcl-2 (D) (*P o0.05 vs. hypoxia).

Acknowledgment We thank Prof. Chao-jun Li for providing us the Egr-1 and dominant negative Egr-1 adenovirus. This study was supported by National Natural Science Foundation of China (Nos. 31100964, 81372718 and 81301316) and Specialized Research Fund for Senior Personnel Programe of Jiangsu University (10JDG45), Postdoctoral Science Foundation Funded Project by Jiangsu province (1402102C) and the Grants from the State Key Laboratory of Oncogenes and Related Genes (No. 90-13-05). We thank Pratirodh Koiralu at University of Mississippi Medical center for the critical reading of the paper.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2015.12.006.

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Please cite this article as: W.-x. Peng, et al., Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.12.006i