Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells
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], [9], [10]. The extent of tumor hypoxia seems to inversely correlate with patient prognosis and is often associated with resistance to conventional treatment modalities [11], [12], [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 (HIF-1)-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 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.
Section snippets
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)
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+), moderate (2+), or strong (3+).
Discussion
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 hypoxia-inducible 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
Disclosure statement
The authors have no conflict of interest.
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
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These authors contribute to this paper equally.