Accepted Manuscript Title: Heat shock protein gp96 decreases p53 stability by regulating Mdm2 E3 ligase activity in liver cancer Author: Bo Wu, Xiaoyu Chu, Cong Feng, Junwei Hou, Hongxia Fan, Ningning Liu, Changfei Li, Xianping Kong, Xin Ye, Songdong Meng PII: DOI: Reference:
S0304-3835(15)00068-3 http://dx.doi.org/doi: 10.1016/j.canlet.2015.01.034 CAN 12259
To appear in:
Cancer Letters
Received date: Revised date: Accepted date:
16-11-2014 15-1-2015 24-1-2015
Please cite this article as: Bo Wu, Xiaoyu Chu, Cong Feng, Junwei Hou, Hongxia Fan, Ningning Liu, Changfei Li, Xianping Kong, Xin Ye, Songdong Meng, Heat shock protein gp96 decreases p53 stability by regulating Mdm2 E3 ligase activity in liver cancer, Cancer Letters (2015), http://dx.doi.org/doi: 10.1016/j.canlet.2015.01.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Heat shock protein gp96 decreases p53 stability by regulating Mdm2 E3
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ligase activity in liver cancer
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Bo Wub a, Xiaoyu Chub a, Cong Fengb a, Junwei Houa, Hongxia Fana, Ningning
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Liua, Changfei Lia, Xianping Kongc, Xin Yea, Songdong Menga*
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Authors' Affiliations a
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute
of Microbiology, Chinese Academy of Sciences (CAS), Beijing, P.R. China. b
School of Life Sciences, Anhui University, Hefei, P.R. China.
c
Transgenic Engineering Research Laboratory, Infectious Disease Center,
458th Hospital, Guangzhou, P.R. China.
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Corresponding Author: *Songdong Meng, Institute of Microbiology, CAS,
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No. 1 West Beichen Road, Chaoyang District, Beijing 100101, P.R. China;
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Tel: (86)-10-64807350; Fax: (86)-10-64807381; E-mail: [email protected].
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Highlights
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cells.
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Gp96 interacts with both p53 and Mdm2 to enhance Mdm2-mediated p53 ubiquitination and degradation.
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Gp96 inhibits cell apoptosis and decreases p53 levels in liver cancer
Gp96 knockdown greatly inhibited in vivo liver tumor growth and promoted apoptosis.
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Abstract
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The resistance to apoptosis displayed by liver cancer plays a key role in
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hepatocarcinogenesis, tumor progression, and resistance to chemo- or radio-
30
therapy. In this study, we uncovered the potential role and mechanism of heat
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shock protein gp96 in regulating liver tumor cell growth and apoptosis. P53
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protein was identified as a gp96 client protein by profiling apoptosis-related
33
proteins in gp96-knockdown liver cancer cells. Overexpression and
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knockdown studies both demonstrated that gp96 decreases p53 protein
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levels, and gp96 regulated cell apoptosis in a p53-dependent manner. We
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further provide evidence that gp96 interacts with both p53 and Mdm2 to
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enhance Mdm2-mediated p53 ubiquitination and degradation. Moreover,
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targeting gp96 with siRNA induced cell apoptosis and led to the suppression
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of liver tumor growth in vivo. In conclusion, we elucidated an underlying
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mechanism by which gp96 promotes p53 degradation via increasing Mdm2
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E3 ligase activity and provided a new therapeutic strategy to target the gp96-
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mediated anti-apoptotic characteristic of hepatocellular carcinoma.
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Key Words: gp96, apoptosis, ubiquitination, chaperone.
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1. Introduction
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As one of the most abundant chaperones in the endoplasmic reticulum (ER),
48
heat shock protein (HSP) gp96 associates with client nascent polypeptides
49
and guides their maturation and assembly into large multimeric protein
50
complexes in the secretory pathway. In contrast to its paralog, the cytosolic
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HSP90 that can bind to and activate hundreds of client proteins, gp96 has a
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relatively strict binding selectivity and is known to associate with only a
53
handful of confirmed clients, including certain Toll-like receptors [1, 2],
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integrins [3], and the platelet glycoprotein Ib-IX-V complex [4]. Like other
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HSPs, gp96 is induced by the accumulation of misfolded proteins under ER
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stress to mediate the unfolded protein response (UPR), which may lead to
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activation of activating transcription factor (ATF) 6, ATF4, and the spliced form
58
of X box binding protein 1 (XBP1) [5].
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Cancer cells are under ER stress, and the rapid growth of cancer cells
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requires increased protein synthesis that is performed by ER proteins.
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Elevated expression of gp96 is observed in multiple tumors and correlates
63
with tumor malignant phenotypes and disease progression [6-8]. Due to its
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essential role in tumor cell survival, proliferation, and apoptosis resistance,
65
gp96 serves as a novel target for cancer therapy [5, 9-11].
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Liver cancer is the sixth most common cancer in incidence and the third most
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common cause of cancer mortality worldwide. It is intrinsically resistant to
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cytotoxic chemotherapies due to potent anti-apoptotic activity and unique
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regenerative characteristics. Multiple signaling pathways and complex
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signaling networks are involved in hepatocarcinogenesis and malignant
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progression, including the NF-κB, mutant p53, JAK-STAT, and
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Raf/MAPK/ERK pathways [12-15]. As these defined signaling pathways
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possess broad functions and reciprocal crosstalk, uncovering the potential
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specific targets and blocking anti-apoptotic pathways may be a more efficient
76
way to develop liver cancer treatments.
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Elevated expression of gp96 is observed in liver tumor issues and correlates
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with tumor phenotypes and disease progression [8, 16, 17]. In this study, we
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investigated the roles of gp96 in liver cancer cells by analysis of apoptotic
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protein profiles and identified a key gp96 client protein that regulates cell
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apoptosis. We further explored the impact of interactions between gp96 and
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its client proteins on liver tumor cell growth and apoptosis both in vitro and in
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vivo. Ultimately, our results may offer a new therapeutic strategy for liver
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cancer treatment.
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2. Materials and methods
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2.1. Cell culture and transfection
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Human hepatoma cell lines (SK-Hep-1 and HepG2) were obtained from the
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ATCC (Manassas, VA, USA). The immortalized normal human hepatic cell
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line (L02) was provided by the Cell Bank of Shanghai Institute of Biochemistry
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and Cell Biology, Chinese Academy of Sciences (Shanghai, China). SK-Hep-
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1, HepG2 and L02 cells were cultured in MEM, DMEM and RPMI 1640
95
mediums, respectively. HCT116 p53-/- and HCT116 p53+/+ colorectal
96
carcinoma cells were provided to Dr. X. Ye by Prof. B. Vogelstein (Johns
97
Hopkins University, Baltimore, MD) and maintained in McCoy’s 5A medium.
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All mediums were supplemented with 10% fetal bovine serum (Gibco, NY,
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USA), 25 μg/ml streptomycin, and 100 IU/ml penicillin. All cells were
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incubated at 37 °C in an atmosphere containing 5% CO2. All transfections
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were performed with Lipofectamine 2000 (Invitrogen) as recommended by the
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manufacturers.
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2.2. Reagents and antibodies
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The gp96-specific small interfering RNA (siRNA) and control siRNA (specific
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for luciferase) were designed and synthesized by RiboBio Co, Ltd.
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(Guangzhou, China), as described previously [18]. The following reagents and
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antibodies were obtained as indicated: MG132, cycloheximide (CHX), and
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cisplatin (Beyotime, China); rat anti-gp96 ( sc-56399), mouse anti-p53 (sc-
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126), mouse anti-hsp70 (sc-32239), rabbit anti-calnexin (sc-11397), mouse
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anti-p21 (sc-6246), and rabbit anti-p27 (sc-528) (Santa Cruz Biotechnology,
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CA); rabbit anti-Mdm2 (BS1223), rabbit anti-p53 (BS3736), rabbit anti-
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ubiquitin (BS1487), and rabbit anti-cleaved caspase-3 (BS7073) (Bioworld
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Technology, Minneapolis, USA); mouse anti-human actin, mouse anti-human
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GAPDH, mouse anti-GST tag, and horseradish peroxidase-conjugated
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secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing,
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China); the ECL-Plus chemiluminescence system (Applygen Technologies,
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Beijing, China).
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2.3. Plasmids and proteins purification
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The mammalian expression vector pcDNA3.1-gp96 and bacterial expression
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vectors for GST-tagged gp96 and histidine-tagged gp96 were described
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previously [18, 19]. The p21 promoter-luciferase reporter plasmid was
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generously provided by Prof. Xin Ye (IMCAS, Beijing, China). To obtain GST-
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tagged p53, GST-tagged Mdm2, and GST-tagged segments of gp96 (N355,
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M163 and C243), the PCR-amplified coding sequences were cloned into the
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bacterial expression vector pGEX-6p-1. GST-fused or histidine-tagged
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proteins were expressed in E. coli and purified by passing the lysates through
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a glutathione Sepharose 4 Fast Flow column or a Ni Sepharose 6 Fast Flow
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column.
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2.4. Stable gp96 knock-down cell lines
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Stable knock-down of gp96 was performed in the liver cancer cell lines SK-
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hep-1 and HepG2 using a shRNA lentiviral expression system.
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Oligonucleotides, encoding short hairpin transcripts directed against the gp96
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mRNA were synthesized. Luciferase shRNA was selected as a mock
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transfection control. The procedures to generate stable shRNA cell lines were
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according to the manufacturer’s protocol and have been described previously
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[13].
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2.5. Apoptotic protein profile analysis
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A Human Apoptosis Antibody Array kit (AAH-APO-1-2) was obtained from
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Raybiotech (Guangzhou, China). The hybridization was performed following
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the instructions from the user manual. The intensities of signals in the linear
145
range were quantified and analyzed using a Licor-Odyssey system. Signals
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from the negative control spots were averaged and then subtracted from each
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of the other spots. A signal was considered valid if its value exceeded both its
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average local background and the average of all valid negative control values.
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Valid signals were normalized using the positive control spots. The fold
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changes in signals for each spot were quantified by dividing by the valid
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signals for each corresponding spot on the mock membrane.
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2.6. Apoptosis, cell cycle, and cell proliferation assays
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Apoptosis assays were performed using an Alexa Fluor® 488 Annexin
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V/Dead Cell Apoptosis Kit (Invitrogen) according to the manufacturer’s
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protocol. Cell cycle and cell proliferation assays were described previously
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[20].
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2.7. Co-immunoprecipitation
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Cell lysates were incubated with an optimum concentration of primary
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antibody. The immunocomplex was then precipitated by Protein G Sepharose
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beads (GE Healthcare). Beads were collected by brief centrifugation and
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washed three to five times with lysis buffer. Washed beads were boiled in
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SDS-PAGE sample loading buffer for immunoblotting.
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2.8. Real-time PCR
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Total RNA was extracted with Trizol Reagent and quantified by real-time PCR
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using the SYBR Green Premix Reagent (Takara Bio Inc., Shiga, Japan) with a
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GAPDH internal control for normalization. The primers used in real-time PCR
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are indicated in Supplementary Table S1.
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2.9. In vitro ubiquitination assay
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In vitro ubiquitination reactions were performed as described [21]. The
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reaction mixture (20 µl) contained 10 ng GST-p53, 24 ng E1 (Boston
175
Biochem), 20 ng GST-UbcH5C (Boston Biochem), 150 ng GST-Mdm2, 10 µg
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His-ubiquitin (Boston Biochem), and 0–800 ng his-gp96 protein. After
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incubation at 37 °C for 60 min, the reactions were terminated with stop buffer
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(Boston Biochem). Ubiquitinated and un-ubiquitinated p53 were detected by
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immunoblotting using a p53 antibody (DO-1).
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2.10. Mouse xenograft
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All mouse experiments were performed in strict accordance with the
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regulations of the Institute of Microbiology, Chinese Academy of Sciences of
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Research Ethics Committee. Four-week-old female BALB/C nude mice were
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purchased from Beijing HFK Bio-technology Co. Ltd. (Beijing, China) for tumor
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cell implantation and maintained under pathogen-free conditions. SK-Hep-1
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cells (1×107) or HepG2 cells (5×106) suspended in 200 μl PBS were
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subcutaneously injected (6 mice/group). After developing palpable tumors,
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tumor volumes were monitored twice per week with a microcaliper and were
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calculated with the formula: (width) 2 × length/2. Approximately 30 days after
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injection, mice were scarified, and all tumor xenografts were excised and
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weighed. Half of the excised tumor tissue was fixed in 10% formalin and
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embedded in paraffin for immunohistochemical staining. The other half was
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quick-frozen in liquid nitrogen for further use.
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2.11. Statistical analyses
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All data are presented as means ± SD, and significance was determined by
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two-tailed Student’s t test unless otherwise specified. P < 0.05 was
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considered significant.
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3. Results
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3.1. Gp96 inhibits cell apoptosis and decreases p53 levels in liver cancer
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cells
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The intrinsic anti-apoptotic property of liver cancer plays a critical role in
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pathogenesis and drug resistance [11]. To investigate if gp96 affects
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hepatocarcinoma apoptosis, we generated a stable gp96 knockdown cell line,
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SK-Hep-1-gp96 KD (Fig. 1a). Knockdown of gp96 led to significantly
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decreased cell proliferation (Fig. 1b) and induced accumulation of cells in
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G0/G1 phase compared to mock cells (Fig. 1c). Notably, gp96 depletion
211
resulted in increased (~2-fold; P < 0.01) cell apoptosis (Fig. 1d).
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We hypothesized that gp96, which usually acts as an important molecular
214
chaperon, might indirectly regulate apoptosis by influencing other apoptosis-
215
related proteins. Thus, the expression levels of 43 different apoptosis-related
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proteins were compared in gp96 knockdown and mock cells using a Human
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Apoptosis Antibody Array kit (Fig. 2a and 2b). The expression levels of 14
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proteins were significantly changed (mostly increased) by gp96 knockdown
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(Fig. 2c). Among these 14 differently expressed proteins, nine are pro-
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apoptotic, and four are anti-apoptotic. The anti-apoptotic HSP27 was the only
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protein with decreased expression. In contrast to the relatively small changes
222
in anti-apoptotic proteins between gp96 knockdown and mock cells, the
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expression of the pro-apoptotic proteins p53 and IGFBP-5 were markedly
224
increased (both > 2-fold) in gp96 knockdown cells (Fig. 2c).
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Among these differentially expressed proteins under gp96 depletion, p53
227
(which plays a key role in the regulation of hepatocarcinoma cell apoptosis)
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was chosen for further study in SK-Hep-1 cells (p53 wild-type). Increased
229
expression of p53 (~2-fold) and its target gene p21 (~1.6-fold) under gp96
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depletion was confirmed by western blotting analysis (Fig. 3a). As expected,
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gp96 knockdown significantly increased p21-luciferase activity (Fig. 3b) and
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mRNA levels of the p53 target gene p21 and downstream molecule TRAIL
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receptor 2 (Fig. 3c). However, no significant change in p53 mRNA levels was
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observed under gp96 knockdown, indicating that gp96 does not affect p53 at
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the transcriptional level. In addition, we established another gp96 stable
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knock-down cell line by using the liver carcinoma cell line HepG2. No
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significantly increased apoptosis was found after gp96 knocked down in
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HepG2 cells (data not show), but cells become more susceptible to apoptosis
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inducer cisplatin compared to the mock by detecting cleaved caspase-3, a
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critical executioner of apoptosis (Fig. 3d), and by flow cytometric analysis (Fig.
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3e). Similar to SK-Hep-1 cells, p53 levels also increased in HepG2 cells upon
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gp96 knockdown (Fig. 3d). Conversely, overexpression of gp96 in the normal
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hepatocyte line L02 (p53 wt) caused a dramatic decrease in p53 and cleaved
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caspase-3 levels by approximately 70% and 90% (Fig. 3f), and reduced cell
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apoptosis by 75% (Fig. 3g).
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HCT116 p53 wild-type (+/+) and null (-/-) isogenic colorectal cancer cell lines
248
were used to further explore the role of p53 in gp96-mediated apoptosis and
249
cell cycle regulation. As seen in Figure 3h, 3i and S1, the effects of gp96 on
250
p53 and its downstream molecules p21 and caspase-3, as well as its effect on
251
cell apoptosis and cell-cycle arrest, were largely abolished in the p53 null
252
cells, indicating that gp96 inhibits cell apoptosis and cell-cycle G1 arrest in a
253
p53-dependent manner.
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3.2. Gp96 interacts with p53 and Mdm2 to stimulate p53 ubiquitination
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and degradation
257
Next, we explored the mechanism of gp96-induced p53 reduction. As seen in
258
Figure 3, gp96 down-regulates p53 at the posttranscriptional level. Thus, we
259
then investigated whether the decrease in p53 protein by gp96 was due to
260
increased ubiquitination. As seen in Figure 4a and 4b, knockdown of gp96 by
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RNAi caused a dramatic increase in p53 stability, whereas overexpression of
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gp96 caused an obvious reduction in p53 stability. In addition, the effect of
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gp96 on p53 stability was abolished in the presence of proteasome inhibitor
264
MG132, indicating that gp96 decreases p53 level by promoting its
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ubiquitination and subsequent proteasome-mediated degradation. As expect,
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a significant decrease in the ubiquitination of p53 was observed in gp96 RNAi-
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treated cells, whereas increased p53 ubiquitination was observed upon gp96
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overexpression (Fig. 4c and Fig. 4d).
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As Mdm2 serves as the major E3 ubiquitin ligase for p53 proteasomal
271
degradation, we decided to investigate the modulation of the ubiquitin ligase
272
activity of Mdm2 by gp96. As seen in Figure 5a and 5b, endogenous gp96
273
associated with p53 and Mdm2 by co-immunoprecipitation assays with either
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anti-gp96, anti-p53, or anti-Mdm2 antibodies. The interaction between gp96
275
and p53 or Mdm2 was also confirmed by GST-pull down assays (Fig. 5c). To
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identify the interaction regions of gp96 involved in the gp96-p53 and gp96-
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Mdm2 interactions, we expressed a variety of truncated fragments of gp96
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(Fig. 5d). We found that the gp96 N-terminal domain (aa 22-376) was able to
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interact with Mdm2, whereas the middle domain of gp96 (aa 377-539)
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interacted with p53 (Fig. 5e). To determine whether gp96 directly interacts
281
with p53 or Mdm2, in vitro GST-pull down assays were performed using GST–
282
p53, GST-Mdm2, and His-gp96 fusion protein expressed in E. coli (Fig. 6a).
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As shown in Figure 6b, in vitro GST-pull down assays demonstrated a direct
284
interaction between gp96 and p53 or Mdm2. Next, the ubiquitination of p53 by
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Mdm2 in the presence of gp96 was assayed in vitro. As shown in Figure 6c
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and 6d, in the presence of Mdm2, gp96 enhanced p53 ubiquitination in a
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dose-dependent manner, whereas no p53 ubiquitination was observed in the
288
absence of Mdm2. Moreover, overexpression and knockdown studies both
289
showed that gp96 did not affect the interaction of Mdm2 with p53 (data not
290
shown). These results indicate that gp96 enhances p53 ubiquitination by
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affecting the ubiquitin ligase activity of Mdm2.
292
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In addition, the cytoplasmic form of the normally ER-resident gp96 in liver
294
cells was confirmed by western blotting of cytoplasmic extracts from digitonin-
295
permeabilized L02 cells (Fig. 6e) and SK-Hep-1 cells (data not shown), as
296
well as by partial co-localization of gp96 with a known cytosolic protein HSP70
297
(Fig. 6f), supporting the notion that gp96 interacts with p53 and Mdm2 to
298
target p53 for ubiquitination and proteasomal degradation in the cytoplasm.
299
Together, these data indicate that gp96 and p53/Mdm2 co-exist in a complex,
300
and gp96 promotes Mdm2-mediated p53 ubiquitination and degradation.
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3.3. Gp96 promotes liver tumor growth in nude mice
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Finally, we examined the growth-promoting and apoptosis-resisting function of
304
gp96 in vivo. In concert with the in vitro results described above, tumor growth
305
was significantly slowed in SK-Hep-1-gp96 KD- and HepG2-gp96 KD-
306
xenografted nude mice compared to mock-treated mice (Fig. 7a). Gp96
307
knockdown resulted in approximate 60 and 50% decreases in tumor weight in
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SK-hep-1 and HepG2 cells, respectively (both P < 0.05) (Fig. 7b). Depletion of
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gp96 by RNAi, as well as upregulation of p53 and the apoptosis marker
310
cleaved caspase-3 in tumors, was verified by IHC detection (Fig. 7c). Taken
311
together, these in vivo results demonstrate the anti-apoptotic and tumor
312
promoting properties of gp96 in hepatocarcinoma.
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4. Discussion
315
The anti-apoptotic characteristic of hepatocarcinoma is one of the most
316
significant factors for hepatocarcinogenesis, tumor progression, and
317
resistance to chemo- or radio-therapy. In the present study, we found that
318
gp96 induces liver cancer cell cycle arrest and significantly reduces apoptosis.
319
Next, we identified p53 as a gp96 client protein by profiling apoptosis-related
320
proteins and demonstrated that gp96 acts as a scaffolding protein to enhance
321
Mdm2 E3 ligase activity, thereby increasing p53 ubiquitination and
322
degradation. In addition, gp96 knockdown greatly inhibited in vivo liver tumor
323
growth and promoted apoptosis. These results suggest that gp96 is a
324
potential therapeutic target for liver cancer.
325
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The p53-mediated pathways play critical roles in cell cycle control,
327
senescence, apoptosis, transcriptional regulation, and DNA repair. As a tumor
328
suppressor, altered p53 expression or mutation is observed in about half of all
Page 16 of 32
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human cancers and is correlated with poor prognosis in patients, including
330
those with liver cancer [14, 22, 23]. P53 mutants are known to gain additional
331
functions that enable continuous cell proliferation, apoptosis resistance, and
332
genomic instability, thereby promoting cell malignancy and cancer metastasis.
333
Whereas in tumors retaining wild-type p53, the p53 functions are likely
334
inactivated or antagonized by multiple mechanisms and pathways [24]. For
335
instance, Twist reduces the ARF tumor-suppressor protein, leading to the
336
ubiquitin-mediated proteolysis of p53 through modulation of the
337
ARF/Mdm2/p53 pathway [25]. ATK kinase directly phosphorylates Mdm2,
338
promoting Mdm2-mediated p53 degradation [26]. A developmental
339
transcription factor Pax3 has recently been shown to stimulate Mdm2-
340
mediated p53 ubiquitination and degradation [21]. More importantly, as an
341
oncogene that is overexpressed in a wide variety of human cancers, Mdm2
342
binds to and promotes p53 ubiquitination for degradation and inhibits the
343
transcriptional activity of p53 [27-29]. In this study, we found that the HSP
344
gp96, whose expression is elevated in a variety of human cancers [6, 16, 17,
345
30], interacts with both p53 and Mdm2, and this interaction increases Mdm2-
346
mediated p53 ubiquitination and degradation. Gp96 therefore inhibits
347
hepatocarcinoma apoptosis and enhances cell proliferation. As ribosomal
348
proteins (RPs), Pax3 and ARF play a complex role in the regulation of the E3
349
ubiquitin ligase activity of Mdm2, but the exact role of gp96 in this context
350
awaits further investigation. In addition, it will be worthwhile to investigate how
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gp96 regulates mutant p53, contributes to tumorigenesis, and whether gp96
352
regulation of p53 is a common feature of liver cancer.
353
354
Emerging evidence points to the critical roles of gp96 in the regulation of
355
cancer initiation, progression, and metastasis. Gp96 interacts with MesD, an
356
important chaperon for the Wnt co-receptor LRP6, and regulates human
357
multiple myeloma (MM) cell growth by attenuating the Wnt-LRP-survivin
358
pathway [9, 31]. Additionally, gp96 plays an essential role in the dimerization
359
of integrin αL and β2, which is involved in cancer metastasis [3]. Moreover,
360
because gp96 activates macrophages through its interaction with toll-like
361
receptors and induces the secretion of pro-inflammatory cytokines, gp96 may
362
contribute to inflammatory colon tumorigenesis [32]. Our work further
363
identified that gp96 acts as a negative regulator of p53 protein stability and
364
demonstrated that targeting gp96 in hepatocarcinoma cells by RNAi inhibits
365
tumor growth in a xenograft mouse model, suggesting that the gp96 may be a
366
potential drug target for liver cancer.
367
368
Considering the key role of p53 in liver tumor invasion and metastasis, as well
369
as the emerging roles of gp96 in the regulation of cancer development and
370
metastasis, the regulation of p53 expression by gp96 presented here has
371
several implications. First, our study represents an effort to address the
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underlying mechanism of altered p53 levels in liver cancer. Second, our
373
results demonstrated that gp96 may function as a scaffolding protein to
374
increase Mdm2 E3 ligase activity for p53 ubiquitination and degradation. More
375
importantly, knockdown of gp96 enhanced liver cancer cell apoptosis and
376
greatly inhibited tumor growth. It would be worthwhile to investigate whether
377
elevated expression of gp96 contributes to the intrinsic drug-resistant and
378
potent regenerative properties of liver cancer, which are the major obstacles
379
to the development of efficient therapies. Given that gp96 expression is
380
elevated in hepatocarcinoma cells but not in normal hepatocytes, our study
381
therefore supports the notion that the inhibition of gp96 may provide a novel
382
therapeutic target for liver cancer.
383
384
5. Acknowledgments
385
We thank Libo Zhao for technical support in animal tests and Tao Niu of
386
Raybiotech for technical help with the apoptotic protein profile assay. This
387
work was supported by grants from Major State Basic Research Development
388
Program of China (973 Program) (No.2014CB542602, 2012CB519003),
389
grants from the National Natural Science Foundation of China (31230026,
390
81321063, 81102018, 81471960), and grants from Key Projects in the
391
National Science & Technology Program (2013ZX10002001-003-003,
392
2012ZX10004503).
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Conflict of Interest Statement: The authors declare no conflict of interest
394
Figure legends
395
Figure S1. Gp96 knock-down induces cell-cycle G1 arrest through
396
promoting p53 activity. (a) HCT116 p53 wild type (+/+) and null (-/-) cells
397
were transfected with gp96 siRNA or control siRNA (mock) for 72 h.
398
Expression of p21 and p27 were examined by western blotting. Actin was
399
used as an internal loading control. (b) The cell cycle was assessed by
400
propidium iodide staining and flow cytometry.
401
Supplementary Table S1. Oligonucleotide sequences for gene expression analysis by real time PCR
402 403
404
405
6. References
406
[1] Y. Yang, B. Liu, J. Dai, P.K. Srivastava, D.J. Zammit, L. Lefrancois, Z. Li,
407
Heat shock protein gp96 is a master chaperone for toll-like receptors and is
408
important in the innate function of macrophages, Immunity, 26 (2007) 215-
409
226.
410
[2] S. Wu, F. Hong, D. Gewirth, B. Guo, B. Liu, Z. Li, The molecular
411
chaperone gp96/GRP94 interacts with Toll-like receptors and integrins via its
412
C-terminal hydrophobic domain, The Journal of biological chemistry, 287
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Cancer research, 74 (2014) 446-459.
521
522
523
524
525
526
527
528
529
530
531
532
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533
534
535
536
Figure legends
537
538
Figure 1. Knockdown of gp96 suppresses cell growth and induces
539
apoptosis in hepatic carcinoma cells. (a) Real-time PCR and western blot
540
analysis of gp96 expression in SK-Hep-1 cells stably transfected with gp96
541
siRNA (gp96 knock-down or gp96 KD) or luciferase siRNA (mock). (b) Cell
542
growth was detected by CCK-8 assays. (c) The cell cycle of mock and gp96
543
KD SK-Hep-1 cells was assessed by propidium iodide staining and flow
544
cytometry. (d) Flow cytometric analysis of cellular apoptosis. Mock and gp96
545
KD SK-Hep-1 cells were stained with Annexin V/fluorescein isothiocyanate
546
and PI. The apoptotic cells were analyzed by FACS. The percentage of
547
apoptotic cells (Annexin V single positive and Annexin V/PI double positive)
548
was assessed. The values shown are the mean ± SD from three independent
549
experiments. *p < 0.05 and **p < 0.01 compared to the mock.
550
551
Figure 2. Profile analysis of apoptosis-related proteins in gp96-
552
knockdown hepatocarcinoma cells. (a) Template showing the location of
Page 27 of 32
553
apoptosis-related antibodies spotted on the Human Apoptosis Array Kit. (b)
554
Equal amounts of protein extracts from mock and gp96 KD SK-Hep-1 cells
555
were analyzed using the antibody array. The chemiluminescent intensities
556
were quantified by densitometry. A positive control was used to normalize the
557
results from different membranes. (c) Fold changes in signals > 1.5 (gp96 KD
558
vs. mock) were selected and divided into three groups (pro-apoptotic, anti-
559
apoptotic, and context-dependent).
560
561
Figure 3. Gp96 mediates cell apoptosis through regulation of p53. (a)
562
Western blotting analysis of p53, p21 and p27 expression in mock and gp96
563
KD SK-Hep-1 cells. GAPDH was used as an internal loading control. (b) Mock
564
and gp96 KD SK-Hep-1 cells were transfected with the p21 promoter reporter
565
p21-luc. After 48 h, p21-luc and renilla luciferase activities were measured
566
using a dual luciferase assay kit. (c) Total RNA was isolated from mock and
567
gp96 KD SK-Hep-1 cells, and the mRNA levels of p53, p21, and TRAILR-2
568
were determined by real-time PCR. (d) HepG2 cells stably transfected with
569
gp96 siRNA (gp96 knock-down or gp96 KD) or luciferase siRNA (mock) were
570
treated with 20 μM cisplatin for 24 h. Gp96, p53 and cleaved caspase-3 levels
571
were analyzed by western blotting. (e) Apoptotic cells were analyzed by flow
572
cytometry in mock and gp96 KD HepG2 cells. (f) Human hepatic cells (L02)
573
were transfected with the gp96 expression vector pcDNA3.1-gp96 (gp96), a
574
GFP expression vector pEGFP-C1 (mock), or the empty vector pcDNA3.1 as
Page 28 of 32
575
a control. The protein levels of gp96, p53 and cleaved caspase-3 were
576
examined by western blotting 48 h after transfection. (g) Cell apoptosis was
577
analyzed by flow cytometry in L02 cells transfected with pcDNA3.1-gp96
578
(gp96), pEGFP-C1 (mock), or pcDNA3.1 (control). (h) HCT116 p53 wild type
579
(+/+) and null (-/-) cells were transfected with gp96 siRNA or control siRNA
580
(mock) for 72 h and then treated with 20 μM cisplatin for 24 h. Expression of
581
gp96, p53 and cleaved caspase-3 were examined by western blotting. (i)
582
Cells were stained with Annexin V/fluorescein isothiocyanate and PI and
583
apoptosis was analyzed by flow cytometry in HCT116 p53 wild type (+/+) and
584
null (-/-) cells transfected with gp96 siRNA or control siRNA (mock). Results
585
are presented as the mean ± SD from three independent experiments. *p
S0304-3835(15)00068-3 http://dx.doi.org/doi: 10.1016/j.canlet.2015.01.034 CAN 12259
To appear in:
Cancer Letters
Received date: Revised date: Accepted date:
16-11-2014 15-1-2015 24-1-2015
Please cite this article as: Bo Wu, Xiaoyu Chu, Cong Feng, Junwei Hou, Hongxia Fan, Ningning Liu, Changfei Li, Xianping Kong, Xin Ye, Songdong Meng, Heat shock protein gp96 decreases p53 stability by regulating Mdm2 E3 ligase activity in liver cancer, Cancer Letters (2015), http://dx.doi.org/doi: 10.1016/j.canlet.2015.01.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Heat shock protein gp96 decreases p53 stability by regulating Mdm2 E3
2
ligase activity in liver cancer
3
4
Bo Wub a, Xiaoyu Chub a, Cong Fengb a, Junwei Houa, Hongxia Fana, Ningning
5
Liua, Changfei Lia, Xianping Kongc, Xin Yea, Songdong Menga*
6
7
8 9
10
11 12
Authors' Affiliations a
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute
of Microbiology, Chinese Academy of Sciences (CAS), Beijing, P.R. China. b
School of Life Sciences, Anhui University, Hefei, P.R. China.
c
Transgenic Engineering Research Laboratory, Infectious Disease Center,
458th Hospital, Guangzhou, P.R. China.
13
14
Corresponding Author: *Songdong Meng, Institute of Microbiology, CAS,
15
No. 1 West Beichen Road, Chaoyang District, Beijing 100101, P.R. China;
16
Tel: (86)-10-64807350; Fax: (86)-10-64807381; E-mail: [email protected].
17
18
Highlights
Page 1 of 32
19
cells.
20 21
24
Gp96 interacts with both p53 and Mdm2 to enhance Mdm2-mediated p53 ubiquitination and degradation.
22 23
Gp96 inhibits cell apoptosis and decreases p53 levels in liver cancer
Gp96 knockdown greatly inhibited in vivo liver tumor growth and promoted apoptosis.
25
26
27
Abstract
28
The resistance to apoptosis displayed by liver cancer plays a key role in
29
hepatocarcinogenesis, tumor progression, and resistance to chemo- or radio-
30
therapy. In this study, we uncovered the potential role and mechanism of heat
31
shock protein gp96 in regulating liver tumor cell growth and apoptosis. P53
32
protein was identified as a gp96 client protein by profiling apoptosis-related
33
proteins in gp96-knockdown liver cancer cells. Overexpression and
34
knockdown studies both demonstrated that gp96 decreases p53 protein
35
levels, and gp96 regulated cell apoptosis in a p53-dependent manner. We
36
further provide evidence that gp96 interacts with both p53 and Mdm2 to
37
enhance Mdm2-mediated p53 ubiquitination and degradation. Moreover,
38
targeting gp96 with siRNA induced cell apoptosis and led to the suppression
39
of liver tumor growth in vivo. In conclusion, we elucidated an underlying
Page 2 of 32
40
mechanism by which gp96 promotes p53 degradation via increasing Mdm2
41
E3 ligase activity and provided a new therapeutic strategy to target the gp96-
42
mediated anti-apoptotic characteristic of hepatocellular carcinoma.
43
44
Key Words: gp96, apoptosis, ubiquitination, chaperone.
45
46
1. Introduction
47
As one of the most abundant chaperones in the endoplasmic reticulum (ER),
48
heat shock protein (HSP) gp96 associates with client nascent polypeptides
49
and guides their maturation and assembly into large multimeric protein
50
complexes in the secretory pathway. In contrast to its paralog, the cytosolic
51
HSP90 that can bind to and activate hundreds of client proteins, gp96 has a
52
relatively strict binding selectivity and is known to associate with only a
53
handful of confirmed clients, including certain Toll-like receptors [1, 2],
54
integrins [3], and the platelet glycoprotein Ib-IX-V complex [4]. Like other
55
HSPs, gp96 is induced by the accumulation of misfolded proteins under ER
56
stress to mediate the unfolded protein response (UPR), which may lead to
57
activation of activating transcription factor (ATF) 6, ATF4, and the spliced form
58
of X box binding protein 1 (XBP1) [5].
59
Page 3 of 32
60
Cancer cells are under ER stress, and the rapid growth of cancer cells
61
requires increased protein synthesis that is performed by ER proteins.
62
Elevated expression of gp96 is observed in multiple tumors and correlates
63
with tumor malignant phenotypes and disease progression [6-8]. Due to its
64
essential role in tumor cell survival, proliferation, and apoptosis resistance,
65
gp96 serves as a novel target for cancer therapy [5, 9-11].
66
67
Liver cancer is the sixth most common cancer in incidence and the third most
68
common cause of cancer mortality worldwide. It is intrinsically resistant to
69
cytotoxic chemotherapies due to potent anti-apoptotic activity and unique
70
regenerative characteristics. Multiple signaling pathways and complex
71
signaling networks are involved in hepatocarcinogenesis and malignant
72
progression, including the NF-κB, mutant p53, JAK-STAT, and
73
Raf/MAPK/ERK pathways [12-15]. As these defined signaling pathways
74
possess broad functions and reciprocal crosstalk, uncovering the potential
75
specific targets and blocking anti-apoptotic pathways may be a more efficient
76
way to develop liver cancer treatments.
77
78
Elevated expression of gp96 is observed in liver tumor issues and correlates
79
with tumor phenotypes and disease progression [8, 16, 17]. In this study, we
80
investigated the roles of gp96 in liver cancer cells by analysis of apoptotic
Page 4 of 32
81
protein profiles and identified a key gp96 client protein that regulates cell
82
apoptosis. We further explored the impact of interactions between gp96 and
83
its client proteins on liver tumor cell growth and apoptosis both in vitro and in
84
vivo. Ultimately, our results may offer a new therapeutic strategy for liver
85
cancer treatment.
86
87
2. Materials and methods
88
89
2.1. Cell culture and transfection
90
Human hepatoma cell lines (SK-Hep-1 and HepG2) were obtained from the
91
ATCC (Manassas, VA, USA). The immortalized normal human hepatic cell
92
line (L02) was provided by the Cell Bank of Shanghai Institute of Biochemistry
93
and Cell Biology, Chinese Academy of Sciences (Shanghai, China). SK-Hep-
94
1, HepG2 and L02 cells were cultured in MEM, DMEM and RPMI 1640
95
mediums, respectively. HCT116 p53-/- and HCT116 p53+/+ colorectal
96
carcinoma cells were provided to Dr. X. Ye by Prof. B. Vogelstein (Johns
97
Hopkins University, Baltimore, MD) and maintained in McCoy’s 5A medium.
98
All mediums were supplemented with 10% fetal bovine serum (Gibco, NY,
99
USA), 25 μg/ml streptomycin, and 100 IU/ml penicillin. All cells were
100
incubated at 37 °C in an atmosphere containing 5% CO2. All transfections
Page 5 of 32
101
were performed with Lipofectamine 2000 (Invitrogen) as recommended by the
102
manufacturers.
103
104
2.2. Reagents and antibodies
105
The gp96-specific small interfering RNA (siRNA) and control siRNA (specific
106
for luciferase) were designed and synthesized by RiboBio Co, Ltd.
107
(Guangzhou, China), as described previously [18]. The following reagents and
108
antibodies were obtained as indicated: MG132, cycloheximide (CHX), and
109
cisplatin (Beyotime, China); rat anti-gp96 ( sc-56399), mouse anti-p53 (sc-
110
126), mouse anti-hsp70 (sc-32239), rabbit anti-calnexin (sc-11397), mouse
111
anti-p21 (sc-6246), and rabbit anti-p27 (sc-528) (Santa Cruz Biotechnology,
112
CA); rabbit anti-Mdm2 (BS1223), rabbit anti-p53 (BS3736), rabbit anti-
113
ubiquitin (BS1487), and rabbit anti-cleaved caspase-3 (BS7073) (Bioworld
114
Technology, Minneapolis, USA); mouse anti-human actin, mouse anti-human
115
GAPDH, mouse anti-GST tag, and horseradish peroxidase-conjugated
116
secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing,
117
China); the ECL-Plus chemiluminescence system (Applygen Technologies,
118
Beijing, China).
119
120
2.3. Plasmids and proteins purification
Page 6 of 32
121
The mammalian expression vector pcDNA3.1-gp96 and bacterial expression
122
vectors for GST-tagged gp96 and histidine-tagged gp96 were described
123
previously [18, 19]. The p21 promoter-luciferase reporter plasmid was
124
generously provided by Prof. Xin Ye (IMCAS, Beijing, China). To obtain GST-
125
tagged p53, GST-tagged Mdm2, and GST-tagged segments of gp96 (N355,
126
M163 and C243), the PCR-amplified coding sequences were cloned into the
127
bacterial expression vector pGEX-6p-1. GST-fused or histidine-tagged
128
proteins were expressed in E. coli and purified by passing the lysates through
129
a glutathione Sepharose 4 Fast Flow column or a Ni Sepharose 6 Fast Flow
130
column.
131
132
2.4. Stable gp96 knock-down cell lines
133
Stable knock-down of gp96 was performed in the liver cancer cell lines SK-
134
hep-1 and HepG2 using a shRNA lentiviral expression system.
135
Oligonucleotides, encoding short hairpin transcripts directed against the gp96
136
mRNA were synthesized. Luciferase shRNA was selected as a mock
137
transfection control. The procedures to generate stable shRNA cell lines were
138
according to the manufacturer’s protocol and have been described previously
139
[13].
140
141
2.5. Apoptotic protein profile analysis
Page 7 of 32
142
A Human Apoptosis Antibody Array kit (AAH-APO-1-2) was obtained from
143
Raybiotech (Guangzhou, China). The hybridization was performed following
144
the instructions from the user manual. The intensities of signals in the linear
145
range were quantified and analyzed using a Licor-Odyssey system. Signals
146
from the negative control spots were averaged and then subtracted from each
147
of the other spots. A signal was considered valid if its value exceeded both its
148
average local background and the average of all valid negative control values.
149
Valid signals were normalized using the positive control spots. The fold
150
changes in signals for each spot were quantified by dividing by the valid
151
signals for each corresponding spot on the mock membrane.
152
153
2.6. Apoptosis, cell cycle, and cell proliferation assays
154
Apoptosis assays were performed using an Alexa Fluor® 488 Annexin
155
V/Dead Cell Apoptosis Kit (Invitrogen) according to the manufacturer’s
156
protocol. Cell cycle and cell proliferation assays were described previously
157
[20].
158
159
2.7. Co-immunoprecipitation
160
Cell lysates were incubated with an optimum concentration of primary
161
antibody. The immunocomplex was then precipitated by Protein G Sepharose
Page 8 of 32
162
beads (GE Healthcare). Beads were collected by brief centrifugation and
163
washed three to five times with lysis buffer. Washed beads were boiled in
164
SDS-PAGE sample loading buffer for immunoblotting.
165
166
2.8. Real-time PCR
167
Total RNA was extracted with Trizol Reagent and quantified by real-time PCR
168
using the SYBR Green Premix Reagent (Takara Bio Inc., Shiga, Japan) with a
169
GAPDH internal control for normalization. The primers used in real-time PCR
170
are indicated in Supplementary Table S1.
171
172
2.9. In vitro ubiquitination assay
173
In vitro ubiquitination reactions were performed as described [21]. The
174
reaction mixture (20 µl) contained 10 ng GST-p53, 24 ng E1 (Boston
175
Biochem), 20 ng GST-UbcH5C (Boston Biochem), 150 ng GST-Mdm2, 10 µg
176
His-ubiquitin (Boston Biochem), and 0–800 ng his-gp96 protein. After
177
incubation at 37 °C for 60 min, the reactions were terminated with stop buffer
178
(Boston Biochem). Ubiquitinated and un-ubiquitinated p53 were detected by
179
immunoblotting using a p53 antibody (DO-1).
180
181
2.10. Mouse xenograft
Page 9 of 32
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All mouse experiments were performed in strict accordance with the
183
regulations of the Institute of Microbiology, Chinese Academy of Sciences of
184
Research Ethics Committee. Four-week-old female BALB/C nude mice were
185
purchased from Beijing HFK Bio-technology Co. Ltd. (Beijing, China) for tumor
186
cell implantation and maintained under pathogen-free conditions. SK-Hep-1
187
cells (1×107) or HepG2 cells (5×106) suspended in 200 μl PBS were
188
subcutaneously injected (6 mice/group). After developing palpable tumors,
189
tumor volumes were monitored twice per week with a microcaliper and were
190
calculated with the formula: (width) 2 × length/2. Approximately 30 days after
191
injection, mice were scarified, and all tumor xenografts were excised and
192
weighed. Half of the excised tumor tissue was fixed in 10% formalin and
193
embedded in paraffin for immunohistochemical staining. The other half was
194
quick-frozen in liquid nitrogen for further use.
195
196
2.11. Statistical analyses
197
All data are presented as means ± SD, and significance was determined by
198
two-tailed Student’s t test unless otherwise specified. P < 0.05 was
199
considered significant.
200
201
3. Results
Page 10 of 32
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203
3.1. Gp96 inhibits cell apoptosis and decreases p53 levels in liver cancer
204
cells
205
The intrinsic anti-apoptotic property of liver cancer plays a critical role in
206
pathogenesis and drug resistance [11]. To investigate if gp96 affects
207
hepatocarcinoma apoptosis, we generated a stable gp96 knockdown cell line,
208
SK-Hep-1-gp96 KD (Fig. 1a). Knockdown of gp96 led to significantly
209
decreased cell proliferation (Fig. 1b) and induced accumulation of cells in
210
G0/G1 phase compared to mock cells (Fig. 1c). Notably, gp96 depletion
211
resulted in increased (~2-fold; P < 0.01) cell apoptosis (Fig. 1d).
212
213
We hypothesized that gp96, which usually acts as an important molecular
214
chaperon, might indirectly regulate apoptosis by influencing other apoptosis-
215
related proteins. Thus, the expression levels of 43 different apoptosis-related
216
proteins were compared in gp96 knockdown and mock cells using a Human
217
Apoptosis Antibody Array kit (Fig. 2a and 2b). The expression levels of 14
218
proteins were significantly changed (mostly increased) by gp96 knockdown
219
(Fig. 2c). Among these 14 differently expressed proteins, nine are pro-
220
apoptotic, and four are anti-apoptotic. The anti-apoptotic HSP27 was the only
221
protein with decreased expression. In contrast to the relatively small changes
222
in anti-apoptotic proteins between gp96 knockdown and mock cells, the
Page 11 of 32
223
expression of the pro-apoptotic proteins p53 and IGFBP-5 were markedly
224
increased (both > 2-fold) in gp96 knockdown cells (Fig. 2c).
225
226
Among these differentially expressed proteins under gp96 depletion, p53
227
(which plays a key role in the regulation of hepatocarcinoma cell apoptosis)
228
was chosen for further study in SK-Hep-1 cells (p53 wild-type). Increased
229
expression of p53 (~2-fold) and its target gene p21 (~1.6-fold) under gp96
230
depletion was confirmed by western blotting analysis (Fig. 3a). As expected,
231
gp96 knockdown significantly increased p21-luciferase activity (Fig. 3b) and
232
mRNA levels of the p53 target gene p21 and downstream molecule TRAIL
233
receptor 2 (Fig. 3c). However, no significant change in p53 mRNA levels was
234
observed under gp96 knockdown, indicating that gp96 does not affect p53 at
235
the transcriptional level. In addition, we established another gp96 stable
236
knock-down cell line by using the liver carcinoma cell line HepG2. No
237
significantly increased apoptosis was found after gp96 knocked down in
238
HepG2 cells (data not show), but cells become more susceptible to apoptosis
239
inducer cisplatin compared to the mock by detecting cleaved caspase-3, a
240
critical executioner of apoptosis (Fig. 3d), and by flow cytometric analysis (Fig.
241
3e). Similar to SK-Hep-1 cells, p53 levels also increased in HepG2 cells upon
242
gp96 knockdown (Fig. 3d). Conversely, overexpression of gp96 in the normal
243
hepatocyte line L02 (p53 wt) caused a dramatic decrease in p53 and cleaved
Page 12 of 32
244
caspase-3 levels by approximately 70% and 90% (Fig. 3f), and reduced cell
245
apoptosis by 75% (Fig. 3g).
246
247
HCT116 p53 wild-type (+/+) and null (-/-) isogenic colorectal cancer cell lines
248
were used to further explore the role of p53 in gp96-mediated apoptosis and
249
cell cycle regulation. As seen in Figure 3h, 3i and S1, the effects of gp96 on
250
p53 and its downstream molecules p21 and caspase-3, as well as its effect on
251
cell apoptosis and cell-cycle arrest, were largely abolished in the p53 null
252
cells, indicating that gp96 inhibits cell apoptosis and cell-cycle G1 arrest in a
253
p53-dependent manner.
254
255
3.2. Gp96 interacts with p53 and Mdm2 to stimulate p53 ubiquitination
256
and degradation
257
Next, we explored the mechanism of gp96-induced p53 reduction. As seen in
258
Figure 3, gp96 down-regulates p53 at the posttranscriptional level. Thus, we
259
then investigated whether the decrease in p53 protein by gp96 was due to
260
increased ubiquitination. As seen in Figure 4a and 4b, knockdown of gp96 by
261
RNAi caused a dramatic increase in p53 stability, whereas overexpression of
262
gp96 caused an obvious reduction in p53 stability. In addition, the effect of
263
gp96 on p53 stability was abolished in the presence of proteasome inhibitor
264
MG132, indicating that gp96 decreases p53 level by promoting its
Page 13 of 32
265
ubiquitination and subsequent proteasome-mediated degradation. As expect,
266
a significant decrease in the ubiquitination of p53 was observed in gp96 RNAi-
267
treated cells, whereas increased p53 ubiquitination was observed upon gp96
268
overexpression (Fig. 4c and Fig. 4d).
269
270
As Mdm2 serves as the major E3 ubiquitin ligase for p53 proteasomal
271
degradation, we decided to investigate the modulation of the ubiquitin ligase
272
activity of Mdm2 by gp96. As seen in Figure 5a and 5b, endogenous gp96
273
associated with p53 and Mdm2 by co-immunoprecipitation assays with either
274
anti-gp96, anti-p53, or anti-Mdm2 antibodies. The interaction between gp96
275
and p53 or Mdm2 was also confirmed by GST-pull down assays (Fig. 5c). To
276
identify the interaction regions of gp96 involved in the gp96-p53 and gp96-
277
Mdm2 interactions, we expressed a variety of truncated fragments of gp96
278
(Fig. 5d). We found that the gp96 N-terminal domain (aa 22-376) was able to
279
interact with Mdm2, whereas the middle domain of gp96 (aa 377-539)
280
interacted with p53 (Fig. 5e). To determine whether gp96 directly interacts
281
with p53 or Mdm2, in vitro GST-pull down assays were performed using GST–
282
p53, GST-Mdm2, and His-gp96 fusion protein expressed in E. coli (Fig. 6a).
283
As shown in Figure 6b, in vitro GST-pull down assays demonstrated a direct
284
interaction between gp96 and p53 or Mdm2. Next, the ubiquitination of p53 by
285
Mdm2 in the presence of gp96 was assayed in vitro. As shown in Figure 6c
286
and 6d, in the presence of Mdm2, gp96 enhanced p53 ubiquitination in a
Page 14 of 32
287
dose-dependent manner, whereas no p53 ubiquitination was observed in the
288
absence of Mdm2. Moreover, overexpression and knockdown studies both
289
showed that gp96 did not affect the interaction of Mdm2 with p53 (data not
290
shown). These results indicate that gp96 enhances p53 ubiquitination by
291
affecting the ubiquitin ligase activity of Mdm2.
292
293
In addition, the cytoplasmic form of the normally ER-resident gp96 in liver
294
cells was confirmed by western blotting of cytoplasmic extracts from digitonin-
295
permeabilized L02 cells (Fig. 6e) and SK-Hep-1 cells (data not shown), as
296
well as by partial co-localization of gp96 with a known cytosolic protein HSP70
297
(Fig. 6f), supporting the notion that gp96 interacts with p53 and Mdm2 to
298
target p53 for ubiquitination and proteasomal degradation in the cytoplasm.
299
Together, these data indicate that gp96 and p53/Mdm2 co-exist in a complex,
300
and gp96 promotes Mdm2-mediated p53 ubiquitination and degradation.
301
302
3.3. Gp96 promotes liver tumor growth in nude mice
303
Finally, we examined the growth-promoting and apoptosis-resisting function of
304
gp96 in vivo. In concert with the in vitro results described above, tumor growth
305
was significantly slowed in SK-Hep-1-gp96 KD- and HepG2-gp96 KD-
306
xenografted nude mice compared to mock-treated mice (Fig. 7a). Gp96
307
knockdown resulted in approximate 60 and 50% decreases in tumor weight in
Page 15 of 32
308
SK-hep-1 and HepG2 cells, respectively (both P < 0.05) (Fig. 7b). Depletion of
309
gp96 by RNAi, as well as upregulation of p53 and the apoptosis marker
310
cleaved caspase-3 in tumors, was verified by IHC detection (Fig. 7c). Taken
311
together, these in vivo results demonstrate the anti-apoptotic and tumor
312
promoting properties of gp96 in hepatocarcinoma.
313
314
4. Discussion
315
The anti-apoptotic characteristic of hepatocarcinoma is one of the most
316
significant factors for hepatocarcinogenesis, tumor progression, and
317
resistance to chemo- or radio-therapy. In the present study, we found that
318
gp96 induces liver cancer cell cycle arrest and significantly reduces apoptosis.
319
Next, we identified p53 as a gp96 client protein by profiling apoptosis-related
320
proteins and demonstrated that gp96 acts as a scaffolding protein to enhance
321
Mdm2 E3 ligase activity, thereby increasing p53 ubiquitination and
322
degradation. In addition, gp96 knockdown greatly inhibited in vivo liver tumor
323
growth and promoted apoptosis. These results suggest that gp96 is a
324
potential therapeutic target for liver cancer.
325
326
The p53-mediated pathways play critical roles in cell cycle control,
327
senescence, apoptosis, transcriptional regulation, and DNA repair. As a tumor
328
suppressor, altered p53 expression or mutation is observed in about half of all
Page 16 of 32
329
human cancers and is correlated with poor prognosis in patients, including
330
those with liver cancer [14, 22, 23]. P53 mutants are known to gain additional
331
functions that enable continuous cell proliferation, apoptosis resistance, and
332
genomic instability, thereby promoting cell malignancy and cancer metastasis.
333
Whereas in tumors retaining wild-type p53, the p53 functions are likely
334
inactivated or antagonized by multiple mechanisms and pathways [24]. For
335
instance, Twist reduces the ARF tumor-suppressor protein, leading to the
336
ubiquitin-mediated proteolysis of p53 through modulation of the
337
ARF/Mdm2/p53 pathway [25]. ATK kinase directly phosphorylates Mdm2,
338
promoting Mdm2-mediated p53 degradation [26]. A developmental
339
transcription factor Pax3 has recently been shown to stimulate Mdm2-
340
mediated p53 ubiquitination and degradation [21]. More importantly, as an
341
oncogene that is overexpressed in a wide variety of human cancers, Mdm2
342
binds to and promotes p53 ubiquitination for degradation and inhibits the
343
transcriptional activity of p53 [27-29]. In this study, we found that the HSP
344
gp96, whose expression is elevated in a variety of human cancers [6, 16, 17,
345
30], interacts with both p53 and Mdm2, and this interaction increases Mdm2-
346
mediated p53 ubiquitination and degradation. Gp96 therefore inhibits
347
hepatocarcinoma apoptosis and enhances cell proliferation. As ribosomal
348
proteins (RPs), Pax3 and ARF play a complex role in the regulation of the E3
349
ubiquitin ligase activity of Mdm2, but the exact role of gp96 in this context
350
awaits further investigation. In addition, it will be worthwhile to investigate how
Page 17 of 32
351
gp96 regulates mutant p53, contributes to tumorigenesis, and whether gp96
352
regulation of p53 is a common feature of liver cancer.
353
354
Emerging evidence points to the critical roles of gp96 in the regulation of
355
cancer initiation, progression, and metastasis. Gp96 interacts with MesD, an
356
important chaperon for the Wnt co-receptor LRP6, and regulates human
357
multiple myeloma (MM) cell growth by attenuating the Wnt-LRP-survivin
358
pathway [9, 31]. Additionally, gp96 plays an essential role in the dimerization
359
of integrin αL and β2, which is involved in cancer metastasis [3]. Moreover,
360
because gp96 activates macrophages through its interaction with toll-like
361
receptors and induces the secretion of pro-inflammatory cytokines, gp96 may
362
contribute to inflammatory colon tumorigenesis [32]. Our work further
363
identified that gp96 acts as a negative regulator of p53 protein stability and
364
demonstrated that targeting gp96 in hepatocarcinoma cells by RNAi inhibits
365
tumor growth in a xenograft mouse model, suggesting that the gp96 may be a
366
potential drug target for liver cancer.
367
368
Considering the key role of p53 in liver tumor invasion and metastasis, as well
369
as the emerging roles of gp96 in the regulation of cancer development and
370
metastasis, the regulation of p53 expression by gp96 presented here has
371
several implications. First, our study represents an effort to address the
Page 18 of 32
372
underlying mechanism of altered p53 levels in liver cancer. Second, our
373
results demonstrated that gp96 may function as a scaffolding protein to
374
increase Mdm2 E3 ligase activity for p53 ubiquitination and degradation. More
375
importantly, knockdown of gp96 enhanced liver cancer cell apoptosis and
376
greatly inhibited tumor growth. It would be worthwhile to investigate whether
377
elevated expression of gp96 contributes to the intrinsic drug-resistant and
378
potent regenerative properties of liver cancer, which are the major obstacles
379
to the development of efficient therapies. Given that gp96 expression is
380
elevated in hepatocarcinoma cells but not in normal hepatocytes, our study
381
therefore supports the notion that the inhibition of gp96 may provide a novel
382
therapeutic target for liver cancer.
383
384
5. Acknowledgments
385
We thank Libo Zhao for technical support in animal tests and Tao Niu of
386
Raybiotech for technical help with the apoptotic protein profile assay. This
387
work was supported by grants from Major State Basic Research Development
388
Program of China (973 Program) (No.2014CB542602, 2012CB519003),
389
grants from the National Natural Science Foundation of China (31230026,
390
81321063, 81102018, 81471960), and grants from Key Projects in the
391
National Science & Technology Program (2013ZX10002001-003-003,
392
2012ZX10004503).
Page 19 of 32
393
Conflict of Interest Statement: The authors declare no conflict of interest
394
Figure legends
395
Figure S1. Gp96 knock-down induces cell-cycle G1 arrest through
396
promoting p53 activity. (a) HCT116 p53 wild type (+/+) and null (-/-) cells
397
were transfected with gp96 siRNA or control siRNA (mock) for 72 h.
398
Expression of p21 and p27 were examined by western blotting. Actin was
399
used as an internal loading control. (b) The cell cycle was assessed by
400
propidium iodide staining and flow cytometry.
401
Supplementary Table S1. Oligonucleotide sequences for gene expression analysis by real time PCR
402 403
404
405
6. References
406
[1] Y. Yang, B. Liu, J. Dai, P.K. Srivastava, D.J. Zammit, L. Lefrancois, Z. Li,
407
Heat shock protein gp96 is a master chaperone for toll-like receptors and is
408
important in the innate function of macrophages, Immunity, 26 (2007) 215-
409
226.
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[2] S. Wu, F. Hong, D. Gewirth, B. Guo, B. Liu, Z. Li, The molecular
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chaperone gp96/GRP94 interacts with Toll-like receptors and integrins via its
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C-terminal hydrophobic domain, The Journal of biological chemistry, 287
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[3] F. Hong, B. Liu, G. Chiosis, D.T. Gewirth, Z. Li, alpha7 helix region of
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alphaI domain is crucial for integrin binding to endoplasmic reticulum
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chaperone gp96: a potential therapeutic target for cancer metastasis, The
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Journal of biological chemistry, 288 (2013) 18243-18248.
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[4] M. Staron, S. Wu, F. Hong, A. Stojanovic, X. Du, R. Bona, B. Liu, Z. Li,
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Heat-shock protein gp96/grp94 is an essential chaperone for the platelet
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glycoprotein Ib-IX-V complex, Blood, 117 (2011) 7136-7144.
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L.Y. Lee, Y.L. Li, Y.J. Chen, A.J. Cheng, J.T. Chang, GP96 is over-expressed
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in oral cavity cancer and is a poor prognostic indicator for patients receiving
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radiotherapy, Radiation oncology (London, England), 6 (2011) 136.
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clinical significance of glucose regulated proteins GRP78 (BiP) and GRP94
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Wang, L.Y. Lee, A.J. Cheng, Differentially expressed genes in radioresistant
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nasopharyngeal cancer cells: gp96 and GDF15, Molecular cancer
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Eilber, L.D. Montejo, A.Y. Chen, B. Hu, J.A. Glaspy, W.H. McBride, J.S.
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Economou, p53 selective and nonselective replication of an E1B-deleted
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Meng, Abnormal expression of HSP gp96 associated with HBV replication in
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human hepatocellular carcinoma, Hepatobiliary & pancreatic diseases
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international : HBPD INT, 5 (2006) 381-386.
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Shao, Dynamic expression of rat heat shock protein gp96 and its gene during
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Increased expression of Gp96 by HBx-induced NF-kappaB activation
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feedback enhances hepatitis B virus production, PloS one, 8 (2013) e65588.
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[19] Z. Liu, X. Li, L. Qiu, X. Zhang, L. Chen, S. Cao, F. Wang, S. Meng, Treg
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suppress CTL responses upon immunization with HSP gp96, European
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journal of immunology, 39 (2009) 3110-3120.
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downregulation of uPAR synergizes with targeting of HER2 through the ERK
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pathway in breast cancer cells, International journal of cancer. Journal
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international du cancer, 127 (2010) 1507-1516.
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ubiquitination and degradation independent of transcription, PloS one, 6
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metastasis, Cancer discovery, 4 (2014) 405-414.
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that inhibits apoptosis, Genes & development, 13 (1999) 2207-2217.
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gastroenterological cancers, Clinica chimica acta; international journal of
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522
523
524
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529
530
531
532
Page 26 of 32
533
534
535
536
Figure legends
537
538
Figure 1. Knockdown of gp96 suppresses cell growth and induces
539
apoptosis in hepatic carcinoma cells. (a) Real-time PCR and western blot
540
analysis of gp96 expression in SK-Hep-1 cells stably transfected with gp96
541
siRNA (gp96 knock-down or gp96 KD) or luciferase siRNA (mock). (b) Cell
542
growth was detected by CCK-8 assays. (c) The cell cycle of mock and gp96
543
KD SK-Hep-1 cells was assessed by propidium iodide staining and flow
544
cytometry. (d) Flow cytometric analysis of cellular apoptosis. Mock and gp96
545
KD SK-Hep-1 cells were stained with Annexin V/fluorescein isothiocyanate
546
and PI. The apoptotic cells were analyzed by FACS. The percentage of
547
apoptotic cells (Annexin V single positive and Annexin V/PI double positive)
548
was assessed. The values shown are the mean ± SD from three independent
549
experiments. *p < 0.05 and **p < 0.01 compared to the mock.
550
551
Figure 2. Profile analysis of apoptosis-related proteins in gp96-
552
knockdown hepatocarcinoma cells. (a) Template showing the location of
Page 27 of 32
553
apoptosis-related antibodies spotted on the Human Apoptosis Array Kit. (b)
554
Equal amounts of protein extracts from mock and gp96 KD SK-Hep-1 cells
555
were analyzed using the antibody array. The chemiluminescent intensities
556
were quantified by densitometry. A positive control was used to normalize the
557
results from different membranes. (c) Fold changes in signals > 1.5 (gp96 KD
558
vs. mock) were selected and divided into three groups (pro-apoptotic, anti-
559
apoptotic, and context-dependent).
560
561
Figure 3. Gp96 mediates cell apoptosis through regulation of p53. (a)
562
Western blotting analysis of p53, p21 and p27 expression in mock and gp96
563
KD SK-Hep-1 cells. GAPDH was used as an internal loading control. (b) Mock
564
and gp96 KD SK-Hep-1 cells were transfected with the p21 promoter reporter
565
p21-luc. After 48 h, p21-luc and renilla luciferase activities were measured
566
using a dual luciferase assay kit. (c) Total RNA was isolated from mock and
567
gp96 KD SK-Hep-1 cells, and the mRNA levels of p53, p21, and TRAILR-2
568
were determined by real-time PCR. (d) HepG2 cells stably transfected with
569
gp96 siRNA (gp96 knock-down or gp96 KD) or luciferase siRNA (mock) were
570
treated with 20 μM cisplatin for 24 h. Gp96, p53 and cleaved caspase-3 levels
571
were analyzed by western blotting. (e) Apoptotic cells were analyzed by flow
572
cytometry in mock and gp96 KD HepG2 cells. (f) Human hepatic cells (L02)
573
were transfected with the gp96 expression vector pcDNA3.1-gp96 (gp96), a
574
GFP expression vector pEGFP-C1 (mock), or the empty vector pcDNA3.1 as
Page 28 of 32
575
a control. The protein levels of gp96, p53 and cleaved caspase-3 were
576
examined by western blotting 48 h after transfection. (g) Cell apoptosis was
577
analyzed by flow cytometry in L02 cells transfected with pcDNA3.1-gp96
578
(gp96), pEGFP-C1 (mock), or pcDNA3.1 (control). (h) HCT116 p53 wild type
579
(+/+) and null (-/-) cells were transfected with gp96 siRNA or control siRNA
580
(mock) for 72 h and then treated with 20 μM cisplatin for 24 h. Expression of
581
gp96, p53 and cleaved caspase-3 were examined by western blotting. (i)
582
Cells were stained with Annexin V/fluorescein isothiocyanate and PI and
583
apoptosis was analyzed by flow cytometry in HCT116 p53 wild type (+/+) and
584
null (-/-) cells transfected with gp96 siRNA or control siRNA (mock). Results
585
are presented as the mean ± SD from three independent experiments. *p
