Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation
© Feng et al.; licensee BioMed Central Ltd. 2014
Received: 10 May 2014
Accepted: 28 September 2014
Published: 1 October 2014
A remolded microenvironment in hepatocellular carcinoma (HCC) caused by abnormally expressed matricellular proteins could promote HCC progression. The cell-matrix interactions mediated by integrins play an important role in tumor microenvironment. Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), an extracellular matrix (ECM) protein with angiogenic and anti-inflammatory effects, is abnormally highly expressed in HCC. Here we aim to analyze its expression in liver and HCC tissues, investigate the underlined mechanisms accounted for HCC progression.
EDIL3 expression level is examined in normal liver, cirrhotic liver and HCC at both mRNA and protein level. The association between EDIL3 and clinical outcomes is analyzed. The pattern of EDIL3 expression and location is examined using Immunofluorescence and ELISA. Overexpression or knock-down of EDIL3 in a panel of cell lines are subjected to assays related to proliferation, invasion, and anoikis to investigate the mechanisms of this matrix protein in HCC progression. Recombinant EDIL3 treatment is applied to confirm the results.
Compared with normal liver and cirrhotic liver, EDIL3 is elevated in HCC. High level of EDIL3 protein is much more commonly in patients with larger tumor or portal vein tumor thrombus (PVTT) formation, associated with poor prognosis. EDIL3 is abundantly expressed in HCC cells and secreted by cancer cells. In vitro and in vivo studies indicate that EDIL3, probably in an autocrine manner, inhibits anoikis and promotes anchorage-independent growth of HCC cells. Further mechanistic studies suggest integrin ligation by EDIL3 and thus that the sustained activation of the FAK-Src-AKT signal is responsible for the anoikis resistance and anchorage independence. Both the administration of cilengitide, a RGD-containing integrin antagonist, and silencing of integrin αV, an important RGD-binding integrin, results in the blockade of anoikis-resistance induced by EDIL3.
Our study suggests that high levels of autocrine EDIL3 may contribute to a receptive microenvironment for the survival of detached HCC cells and may involve in cancer cell spreading. We also highlight the importance of interaction between EDIL3 and integrin αV and suggest disrupting the ligation of EDIL3 to integrins via RGD-blocking in selected patients may bear potential therapeutic value.
Hepatocellular carcinoma (HCC) is a lethal disease with high mortality due to the high rate of postoperative recurrence and metastasis . Both genetic and epigenetic alterations  within the HCC and a remolded microenvironment [3, 4] surrounding HCC affect the biological behaviors of HCC and thus result in different outcomes of HCC patients. Extracellular matrix (ECM) proteins are the main non-cellular components of the tumor microenvironment; many of them can interact with the tumor cell through direct binding to specific receptors or by modulating the activation of other cytokines [5, 6]. Some reports have suggested that microenvironmental changes caused by abnormally expressed matricellular proteins may modulate HCC progression by affecting cell growth, migration, invasion, anoikis and metastasis; thus, targeting ECM proteins may have therapeutic value [3, 7–10].
Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), also known as DEL-1, is a secreted ECM protein that was firstly characterized in vascular morphogenesis . Structurally, EDIL3 contains 3 EGF-like repeats and 2 discoidin-like repeats, with an RGD motif located in the second EGF-like repeat . Three-dimensional crystal structures reveal a unique RGD finger, which is believed to be advantageous for its ligation with integrins over other RGD-containing ECM proteins . EDIL3 has been intensively studied in vascularization, exhibiting strong angiogenic or vascularizing function through integrin αvβ3 modulation [13, 14]. Moreover, this protein plays a role in modulating immunocyte adhesion through interactions with leukocyte-specific integrins . The role of EDIL3 in cancer is also revealed in several malignancies, albeit in observational level. For example in pancreatic carcinoma, EDIL3 is one of the SHH-dependent stromal factors that predict poor prognosis ; another study focusing on carcinogenesis of ulcerative colitis-associated colorectal cancer suggests EDIL3 may add some power in this process ; interestingly, EDIL3 could also be detected in exosomes of bladder cancer cells and facilitate cancer progression trough EGFR signal . In the field of HCC, high-throughput genomic data suggests elevated EDIL3 in HCC compared with adjacent liver , and a clinical analysis reveals that EDIL3 might affect the prognosis of HCC . However, no study has addressed how EDIL3 is involved in HCC development and progression.
Anoikis is a special apoptotic process resulting from the loss of or inappropriate cell adhesion. Upon detaching from the primary lesion, the lack of cell-ECM adhesion fails to maintain the pro-survival signals within cancer cells, leading to decreased anti-apoptotic signals, thus activating anoikis . Gaining anoikis resistance is a prerequisite for intra-hepatic spreading and extra-hepatic metastasis of HCC. In addition to adaptive alterations, such as switch in integrin expression patterns or the hyperactivation of receptor tyrosine kinases, the abnormal microenvironment also helps the cancer evading anoikis . Integrins are the key mediators of the cell-ECM interaction, sending pro-survival signals in the presence of ECM ligands, e.g., collagens and laminins, and inducing apoptosis in their unligated status . EDIL3 is an important ligand for αV-coupled integrins via RGD recognition and has been shown to be able to bind these groups of integrins  and inhibit anoikis in the endothelium .
In the present study, we focus on the role of EDIL3 in HCC and demonstrate that EDIL3 is highly expressed in HCC patients. Moreover, the accumulation of tumor-derived EDIL3 in the microenvironment promotes anoikis resistance and anchorage independent growth advantage through the activation of integrin signal pathways.
The EDIL3 level is significantly elevated in HCC tissue and is associated with adverse biological behaviors
Correlation between EDIL3 and key clinicopathological parameters
EDIL3 (n = 202)
EDIL3 is abundantly expressed in HCC cell lines and exhibits unique expression pattern
Autocrine EDIL3 promotes anoikis resistance and anchorage-independent growth in HCC cells in vitro
We tested whether knocking down EDIL3 would cause the opposite effects. Using two shRNA sequences, we significantly suppressed the mRNA level of EDIL3, leading to a decrease in secreted EDIL3 in Huh-7 cells but no changes in intracellular EDIL3 in control cells (Additional file 5: Figure S5A). In agreement with the result from overexpression experiments, the decrease in EDIL3 in CM increased the caspase3/7 activity, lowered the WST-8 value and inhibited the colonies formed in soft agar, while adding recombinant EDIL3 partly rescued these inhibitory effects (Additional file 5: Figure S5B-D).
Taken together, these results indicate that EDIL3, especially secreted by cancer cells in our current study, is effective in protecting them from anoikis after suspension, thus supporting anchorage independent growth.
EDIL3 promotes HCC tumorigenesis in vivo
Activation of FAK-Src-AKT signaling by EDIL3 ligation is associated with anoikis resistance
Disrupting integrin ligation restores anoikis susceptibility to HCC
This study provides a clear expression pattern of one ECM protein, EDIL3, in clinical samples and highlights the role of autocrine EDIL3 in the integrin-mediated interaction between HCC and ECM, which resulted in an anti-anoikis and anchorage-independent growth advantage. Our results also suggest a potential therapeutic strategy for personalized medicine by targeting ECM-integrin interactions for cancer cells in a subgroup of HCC patients.
In tissue level, EDIL3 is dominantly expressed and secreted by the endothelium, acting as a regulator of vascularization, immunocyte-endothelium adhesion and platelet microparticle clearance [14, 15, 26], or by a subset of macrophages to mediate engulfment . Our data reveal that EDIL3 expression is turned on in adult human normal liver (cells) and is amplified during malignant transformation, which is different from mouse data, wherein EDIL3 is undetectable in the liver . The cause of the up-regulation of EDIL3 in HCC is not clear; however, there is evidence suggesting that VEGF induces EDIL3 expression in malignant cells  and some inflammatory cytokines in endothelium . Therefore it is possible that EDIL3 is elevated in response to cytokines in HCC. However, other events, such as HBV incorporation or epigenetic modifications, may also affect EDIL3 expression. Meaningfully, by analyzing an adequately sized sample, we demonstrated that when EDIL3 protein is accumulated to a high level, it is associated with larger tumor size and more PVTT formation, as well as greater relapse risk and shorter overall survival, which is consistent with a previous study, although the previous report only reached a positive result in overall survival , most likely due to the sample size and difference in patients selection.
In cell lines, however, EDIL3 demonstrates a unique expression. By examining multiple HCC cell lines and normal hepatocyte derived cell line, we find EDIL3 is expressed by both the normal and malignant cell lines, suggesting EDIL3 may not be directly linked to the relative tumorigenicity of HCC cell lines. Interestingly, we reveal that intracellular EDIL3 protein is observed at almost the same level despite the large differences in transcription. This inconsistency in transcription and translation was explained by the difference in secreted EDIL3. Moreover, either overexpressing or knocking-down the transcription level of EDIL3 only affected the secreted EDIL3. Based on these observations in cell lines and human samples, we postulate that EDIL3 is maintained in a standby status by many types of cells in quiescent (or normal) conditions and is secreted upon elevated transcription in physiologic or pathophysiological conditions; notably, the secretion is probably key to its biological functions. However, it will also be interesting to see the effects of EDIL3 knock-out in malignancies, and the potential regulation of EDIL3 secretion.
EDIL3 is well documented as an integrin ligand, modulating a wide range of biological processes that require integrins, such as integrin αV in angiogenesis and integrin αL in the neutrophil adhesion cascade [13, 29]. Because integrins such as integrin αV are also abnormally expressed in HCC , we postulated that EDIL3 may affect HCC cells through integrin ligation when it is secreted and anchored on the cell membrane, or deposited in the ECM. By overexpressing EDIL3 or recombinant EDIL3 treatment, we observed that secreted EDIL3 reduced the likelihood of anoikis in cancer cells and promoted anchorage-independent growth, both of which are indispensable for HCC spreading and PVTT formation, whereas do not affect proliferation or invasion. Indeed, all of the fresh PVTT samples in our study exhibited a very high level of EDIL3 protein, so it is highly possible that tumor cells bring this protective protein when leaving primary lesion, thus assisting PVTT formation. Similar strategies to overcome anoikis have been reported in other types of cancer, in which another ECM protein, collagen IV, is expressed .
The anoikis is a complex apoptosis process resulted from cell detachment from ECM or inappropriate cell-cell adhesion. The lack of ECM contact or the engagement with inappropriate ECM leads to the activation of anoikis from death receptors (extrinsic pathway) or mitochondria (intrinsic pathway) . Integrins on the cell surface are crucial for anoikis modulation by modulating both the extrinsic and intrinsic pathway, passing opposite signals in their ligated or unligated status [21, 23, 33] or even independent of their ligation status . The 397FAK-416Src-473AKT axis is a well-documented pro-survival pathway in anoikis resistance [32, 35–37]. Our results suggest that this axis is sustained when cancer cells are located in an environment rich with EDIL3, whereas phosphorylation of all three effectors was partly inhibited when RGD binding sites were blocked by cilengitide, suggesting that RGD recognition is critical for this pro-survival signal. Another interesting finding is that the suppression of integrin αV (or α5) expression led to a strong anoikis resistance, suggesting that unligated integrin may trigger anoikis, which is supported by other studies [38, 39]. However, intriguingly enough, integrin αV or α5 expression by cancer cells is required for invasion and spreading [40–42]. This seemly contradictory role of integrins reflects the importance of a suitable interaction between cancer cells and the microenvironment, or ECM, at the suitable stage of tumor progression; in this study, an EDIL3-integrin alliance may be exploited by detached HCC cells to escape anoikis when cells detach from the primary lesion, which resulted into a higher success rate of metastasis.
Finally, a variety or integrin inhibitors has entered clinical trial of different types of cancers [33, 43]. However, the outcomes have fallen short of expectations, revealing the complexity of integrin function as discussed above. Therefore, refined patient selection is required. Cilengitide was initially documented as an angiogenesis and invasion inhibitor . However, our preliminary study suggests a potential role of cilengitide in inhibiting anoikis resistance in EDIL3-overexpressing HCC, thus pointing to a new potential indication of integrin inhibitors in HCC and providing a reference for patient selection.
Our study suggests that high levels of autocrine EDIL3 may contribute to a receptive microenvironment for the survival of HCC cells and therefore assist cancer cell progression, which lead to adverse clinical results. Although other explanations may be concealed in our current effort, the anti-anoikis conferred by high level of EDIL3 is an important mechanism for through which EDIL3 provide protection for HCC cells. Disrupting the ligation of EDIL3 to integrins via RGD-blockers or inhibitors in selected patients with high EDIL3 may bear potential therapeutic value.
Methods and materials
The HCC cell line Sk-Hep1 was purchased from the American Type Cell Culture Collection (ATCC), HuH-7 was purchased from RIKEN Resource Centre, Japan, SMMC-7721 was purchased from the Cell Bank of the Chinese Academy of Sciences Cell Bank of Type Culture Collection. The metastatic HCC cell lines MHCC-97 L, MHCC-97H and MHCC-LM3 were obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University. The HCC cell line CSQT-2 derived from a portal vein tumor thrombus (PVTT) has been described elsewhere . Human liver epithelial cell line THLE-3 was purchased from ATCC;. All cell lines except THLE-3 were cultivated in DMEM (Dulbecco’s modified Eagle medium) supplemented with 10% (v/v) fetal calf serum at 37°C in a humidified incubator under 5% CO2 condition. THLE-3 was cultivated in BEGM (Lonza) with the addition of BEGM bullet kit according to the instruction on ACTT.
All samples were collected in Department of Liver Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine. Fresh samples, including tumor tissues and PVTTs, were obtained from HCC patients during tumor resection. Normal livers and cirrhotic livers were collected from healthy liver donors and cirrhosis patients, respectively, during liver transplantation. Approximately 202 HCC samples were collected from 2004 to 2010 and were constructed into tissue microarray (TMA). The median age of this cohort of patients was 50 years (range 17–73 years). The majority of patients are HBV-positive (187/202). The follow-up was ended in December 2012, and the median period was 33 months (range 2–90 months). All human samples were obtained with informed consent, and protocols were approved by the ethical review committee of the World Health Organization Collaborating Center for Research in Human Production (authorized by the Shanghai Municipal Government).
Quantitative real-time PCR
Total RNA was extracted using Trizol reagent (Takara) and reverse transcribed using PrimeScript qRT-PCR kit (Takara) according to the protocol. Quantitative real-time PCR analyses were performed with SYBR Premix Ex Taq (Takara) on a 7300 Real-time PCR system (Applied Biosystems Inc.), and the primer for EDIL3 was as follows: forward, GTGAACTGTCGGGTTGTTCTGAG; and reverse, 5′-GGTTCCCAAGTGAACATGTCCAT-3′. The primers for ACTB were as follows: forward, 5′-TCACCCACACTGTGCCCATCTACGA-3′; and reverse, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′. The relative expression of EDIL3 was analyzed by the comparative cycle threshold method (ΔΔCt method), which was normalized to ACTB.
Protein collection, Western blot and antibodies
The HCC samples were handled with T-Per tissue protein extraction reagent (Thermo Scientific) according to the manufacturer’s protocol. Total cell protein was obtained by IP lysis buffer (Beyotime) for further assays. The secreted proteins in conditional media were collected by ethanol precipitation. Briefly, when cells grew to approximately 80% confluence, normal media were replaced by serum-free media. After 24 h, the media were collected, and 95% ethanol was added and kept overnight. The precipitated proteins were collected with SDS loading buffer and underwent standard western blot immediately. Western blot was performed using SDS-PAGE gel for proteins separation and nitrocellulose membrane for proteins blotting. The total volume of protein used in WB is 50 μg for tissue sample assays, and 30-60 μg in cell line assays. The signal was detected by the Odyssey infrared system (LI-COR).
The antibodies used were the following: EDIL3 (ProteinTech), ITGAV (Abcam), p-FAK397 (Cell Signal Technology), FAK (Abcam), p-Src416 (Epitomics), Src (Cell Signal Technology), p-AKT473 and AKT (Cell Signal Technology), StrepII (Abcam), GAPDH (ProteinTech) and β-actin (Abcam).
Immunohistochemistry stain and analysis
A total of 202 samples of HCC on 2 TMA slices, 3 NLs, 10 HCCs and 6 PVTTs are subjected to IHC. Paraffin-embedded sections 5 μm thick and TMA were stained according to standard Immunohistochemistry (IHC) protocols. Heat-mediated antigen retrieval in pH 6.0 citric acid was performed. The antibodies used here were EDIL3 (ProteinTech). The scoring of EDIL3 was performed according to the ratio and intensity of positive-staining cells: 0-5% scored 0; 6-35% scored 1; 36-70% scored 2; and more than 70% scored 3. The final score was designated as negative (score 0), positive (score 1 or 2) and high positive (score 3). These scores were determined independently by two experienced pathologists in a blinded manner.
Enzyme-linked immunosorbent assay
HCC cell lines were planted on 10 cm petri dish until 90% confluence, then were incubated in 3 ml serum free media for 48 hour. At the end of the incubation period, the conditional media were harvested and stored at -80C until use. The secreted EDIL3 in media were quantified using ELISA kits according to the manufacturer’s instructions (CUSABio).
In cell assay, all the HCC cell lines under tests were seeded on cover slides in 24-well plates and incubated overnight. For F-actin staining, cells were incubated with phalloidin-FITC (Sigma-Aldrich) for 75 minutes at room temperature. For EDIL3 staining, cells were incubated with primary antibodies against EDIL3 (Abnova) for 75 minutes at room temperature, followed by an Alexa Fluor 594-conjugated secondary antibody. In tissue staining, two samples embedded in paraffin were subjected to heat-mediated antigen retrieval in pH 6.0 citric acid, and blocked by 10% BSA. The primary antibodies used are EDIL3 (Abnova) and CD31 (Abcam) with Alexa Fluor 594-conjugated secondary antibody against EDIL3 and Fluor 488 against CD31. Immunofluorescence signals were captured using confocal micro-scopy (LSM 510, METALaser Scanning Microscope, Zeiss).
Recombinant EDIL3 expression, purification and characterization
Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere . Briefly, the expression vector was transfected into human embryonic kidney 293/Epstein-Barr nuclear antigen cells (EBNA-293) by Xetrem (Roche). The cells were screened with puromycin, and the surviving cells were cultured on a large scale. The culture medium was collected and centrifuged, and the supernatant was subjected to the StrepTactin sepharose column (IBA) for purification. The collected proteins were quantified and validated by Coomassie Brilliant Blue (CBB) staining and western blot. The empty vector was applied as control proteins.
Establishment of stable overexpression or knock-down cell lines
For the overexpression of EDIL3 in cell lines, the full-length cDNA of EDIL3 (NM_005711 Origene) was subcloned into pCDH-CMV-MCS-EF1-Puro vector (System Biosciences). Lenti-virus was packaged in 293 T cells using Lipofectamine2000 (Invitrogen), and the virus DNA was transduced into cell lines. For knock-down of EDIL3, short hairpin RNA (shRNA) sequences (Sh1:5′-CCGGCCCAAGTTTGTCGAAGACATTCTCGAGAATGTCTTCGACAAACTTGGGTTTTTG- 3′; Sh2: 5′-CCGGGGAGGTTGCATCAGATGAAGACTCGAGTCTTCATCTGATGCAACCTCCTTTTTG- 3′) targeting EDIL3 were cloned into pLKO.1-puro vectors (Roche). ShRNA-containing plasmids were packaged into lenti-viruses and transduced into target cell lines as above. The efficiency of over-expression or knock-down was tested by qRT-PCR and western blot.
Anoikis was induced by culturing cells in poly-HEMA coated plates as described by others . Briefly, poly-HEMA were prepared as a 10 mg/ml solution in ethanol, which covered completely the petri-dish or plates, then dried and repeated once. Cells in serum-free medium were seeded into the coated plates with or without adding the recombinant EDIL3 at the corresponding concentration. To avoid survival effects caused by the clumping of cells, 0.5% methyl cellulose (Sigma-Aldrich) was added into the medium. At the designated time points, the suspended cells were collected and subjected to cell viability assays by WST-8 kit (Dojindo), apoptosis assays by Caspase3/7 Glo kit (Promega), and Annexin V/PI staining on FACS (BD) according to the protocols from the respective manufacturers.
Anchorage-independent growth assay
Colony formation in soft agar was tested to assay anchorage-independent growth. Stable overexpressing EDIL3 cell lines SMMC-7721 and MHCC-LM3 or their control cells were suspended in the upper layer consisting of culture medium with 1% FBS and 0.35% agar, which is above a basal layer of 0.6% agarose in 6-well plates in a triplicate manner. The cell density was 2,500 cells per well. The soft agars were fed twice a week with 0.3 ml of culture medium. In recombinant EDIL3 treatment assay, the soft agars were fed every 2 days with serum-free culture medium containing EDIL3 or empty vector. Colonies were stained with 0.05% crystal violet, and all the visible colonies were counted by microscopy after 21–28 days.
Tumorigenesis in vivo
A total of 1.0 × 106 of lenti-vector or lenti-EDIL3 SMMC-7721 cells were implanted subcutaneously into the right flank of 5 BALB/c (nu/nu) mice in each group. Tumor sizes were measured once a week, and mice were sacrificed for the analysis of tumor burden after 6 weeks. All procedures were performed in accordance with The Animal Care and Use Committee of Jiaotong University. The resected mice tumors were fixed in methanol and embedded in paraffin before being subjected to IHC using anti-EDIL3 (ProteinTech), anti-PCNA (Abcam) and anti-TUNEL (Millipore).
RGD-blocking assay and knock-down of ITGAV
Cyclic RGD-containing peptide cilengitide (Selleckchem) or control peptide RGE was used to block the ligation of EDIL3 to integrins on the surface of HCC cells at a final concentration of 10 μM. Briefly, the cells was suspended in Poly-HEMA coated plates and exposed to the peptides about 3 hours after suspension. Then, the cells were subjected to the assays as described above. SiRNA targeting ITGAV (Si2: 5′-GCCAGCCAAUUGAAUUUGATT- 3′; Si3: 5′-CCCAGUUGUAUCUCACAAATT - 3) was used to knock down the protein level of this integrin and was confirmed by western blot. After 72 h of transfection, the cells were subjected to the assays as above.
Statistical analyses were performed with SPSS 16.0 software. The association between EDIL3 expression and clinicopathological parameters was analyzed by Pearson Chi-Square test. Overall survival and relapse risk was calculated by the Kaplan-Meier method and compared by the log-rank test. Statistical significance was determined by two-tailed Student’s t test for any differences observed (P < 0.05).
The study was supported by the Training Program for Superb Academic Leaders in Shanghai Health System (No. XBR2011029); the Special Fund for Building of Leading Talent Teams in Shanghai; Doctoral Innovation Fund Projects of Shanghai Jiao Tong University School of Medicine (BXJ201222); National Key Sci-Tech Special Projects of Infectious Diseases (2013ZX10002-007-006) and National Science Foundation of China (81472678; 81101600; 81201624; 81372368). We are grateful to Drs Xiao-Jin Liu, Xiao-Mei Yang, Ya-Hui Wang, Guang-Dong Yang and Shu-Heng Jiang for their assistance with the experiments. We also received constructive comments on the manuscript from other members in our lab.
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