Prognostic value of pigment epithelium-derived factor for neoadjuvant radiation therapy in patients with locally advanced rectal carcinoma
- Authors:
- Published online on: July 12, 2016 https://doi.org/10.3892/ijo.2016.3620
- Pages: 1415-1426
Abstract
Introduction
Locally advanced rectal carcinoma (LARC) is the third most common cancer and the third most common cancer cause of death globally (1). The prognosis of patients with LARC has slowly improved during the past decades in many countries (2), but there is much room to improve the treatment of patients suffering from colorectal cancer. At present, the standard procedures to treat LARC include neoadjuvant radiation therapy (nRT), surgery, and adjuvant therapy (3). Neoadjuvant therapy could further downstage tumors, improve the rate of sphincter preservation, and reduce the rate of local recurrence (4). For patients with stage II and III LARC, nRT reduces rates of local recurrence and toxic effects. Though rectal cancer patients have benefited from neoadjuvant therapy, the response of patients to neoadjuvant therapy varies from pathological complete response (pCR) (9–37%) to complete resistance (5). Patients who achieved pCR have a prior prognosis compared with those who did not achieve pCR. The identification of patients at risk of being radioresistant would avoid the adverse side effects and cost of nRT and provide the patients alternative options such as surgical intervention without nRT (6). Even with nRT to achieve pCR, a substantial number of LARC patients will experience metastatic progression (7). Currently, it is inconclusive whether nRT may reduce the risk of metastatic development in rectal cancer (8). Therefore, identification of a specific molecular marker could be a target of neoadjuvant radiotherapeutic interventions. To date, however, no biomarker predicts tumor response to nRT and prevent metastatic progression.
Pigment epithelium-derived factor (PEDF; encoded by SERPINF1 and also known as EPC1), a serpin that has multiple biological actions, is a differentiation factor for retino-blastoma cells (9). PEDF is a potent inhibitor of angiogenesis (10). Many observations have shown that PEDF is implicated in several biological processes, such as neurogenesis, neuroprotection, anti-angiogenesis, retina protection, stem cell renewal, and inflammation (11). PEDF expresses in various kinds of cancers, such as lung cancer, pancreatic cancer, and breast cancer (12–14), and some research suggests that PEDF could be a new treatment to benefit cancer patients (15–17). In colorectal cancer (CRC), PEDF plays a role in inhibiting tumor growth and metastasis, anti-angiogenesis, decreasing tumor microvessel density (MVD) and tumor cell apoptosis, and recently, PEDF has also been identified as a prognostic marker for CRC (18–20). However, there are scarce data on the relationship between PEDF and sensitivity to nRT. This study evaluated the expression of PEDF after nRT and correlated the expression of PEDF with the clinicopathological parameters and prognosis of the disease.
Materials and methods
Subjects and samples
This research was approved by the ethics committee of the Health Science Center of Peking University and the Oncology Center at Peking University. Written informed consent was obtained from all the participants prior to the enrollment.
Pre-treatment biopsy tumor samples were obtained at colonoscopy or rigid sigmoidoscopy from 48 rectal cancer patients. To ensure consistent quality and tumor presence, both normal and tumor tissues were evaluated by an experienced pathologist.
We also retrospectively reviewed the data of 197 rectal cancer patients who received 30 Gy/10 fractions (10 f) nRT at Peking University Cancer Hospital between August 2003 and October 2009. Each subject conformed to the following inclusion criteria: i) diagnosis of rectal adenocarcinoma by biopsy; ii) resectable rectal cancer ≤10 cm from the anal verge; iii) evaluated by endorectal ultrasound (ERUS) or magnetic resonance imaging (MRI) before treatment; iv) presence of distant metastases excluded by imaging examinations; v) neoadjuvant radiotherapy of 30 Gy/10f; vi) radical surgery following total TME.
Patients with the following characteristics were excluded: i) cases with familial adenomatous polyposis LARC and hereditary non-polyposis colorectal carcinoma; ii) previous history (within 5 years) of malignant tumor; iii) presence of unresectable cancer; iv) death due to complications or other non-cancer related causes.
Tumor regression grade
The histology of all surgical specimens was reviewed and confirmed by an independent element and was classified based on the Mandard tumor regression grade (TRG) system (21), as follows: i) complete regression (fibrosis without detectable tissue of tumor); ii) fibrosis with scattered tumor cells; iii) fibrosis and tumor cells with preponderance of fibrosis; iv) fibrosis and tumor cells with preponderance of tumor cells; and v) tumor tissue lacking changes related to regression.
Tissue microarray and immunohistochemistry
Formalin-fixed and paraffin-embedded specimens were microdissected to perform tissue microarrays (TMAs) with a tissue-arraying instrument (Beecher Instruments, Silver Spring, MD, USA). The TMAs were analyzed by immunohistochemistry as described previously (22). Sections were incubated with monoclonal antibodies against PEDF (1:100, Santa Cruz, Dallas, TX, USA). All images were examined by two experienced pathologists independently. PEDF immunohistochemical staining of a tissue sample was graded as either negative (patchy and weak or negative immunoreactivity) or positive (uniformly intense immunoreactivity) (Fig. 1).
Subcutaneous tumor xenografts
Ten NOD/SCID mice were randomly divided into LoVo-PEDF and LoVo-control groups (n=5 per group). Equal amounts of corresponding cells (2×106) were subcutaneously implanted on the right buttock of each mouse. After three weeks, mice were sacrificed and xenografts were harvested and weighed. All animal experiments were reviewed and approved by the Ethics Review Committee at the Peking University School of Oncology.
Cell culture and cell lines
Human CRC cell lines (LoVo) from the American Type Culture Collection were cultured in DMEM medium with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) in a humidified atmosphere of 5% CO2 at 37°C. The cell lines have been tested and authenticated by STR profiling.
Animal
Female NOD-SCID mice (4–6-week-old) were provided by Beijing HFK Bio-technology Co., Ltd. (Beijing, China). Mice were maintained in a pathogen-free facility and used under the institutional guidelines for animal care.
Plasmid construction and cell transfection
PEDF-over-expressing cells were generated by using a ribozyme transgene system. Ribozyme transgenes specific for PEDF were synthesised and cloned into pEF6/V5-His TOPO TA expression plasmids in line with the manufacturer’s protocol (Invitrogen, Shanghai, China). PEDF ribozyme transgene plasmids and control pEF6 plasmids were subsequently transfected into LoVo cells. Cells were placed in normal medium overnight to allow recovery and then in selection medium (normal medium containing blasticidin 5 μg/ml) for eight days to select for cells containing the plasmid. Following selection, cells were placed into a maintenance medium (0.5 μg/ml blasticidin). Following selection, the cells were tested, at the translational level, for the efficacy of the expression plasmid.
Irradiation
Irradiation was delivered by linear accelerator at room temperature. The dose rate was 400 cGy/min. The irradiation doses were 0, 2, and 4 Gy. After irradiation, the medium with drugs was adsorbed and replaced with fresh DMEM medium containing 10% FBS.
Growth curve
LoVo-TR and LoVo-PEDF Cells (3×104 cells/well) were plated in a 96-well plate (Corning Life Science, MA, USA). After 24 h, the medium was replaced. Growth curves were determined by placing cells in the IncuCyte ZOOM system (Essen BioScience), which allows an automated and non-invasive method of monitoring live cells in culture. A growth curve is constructed automatically from data points acquired during round-the-clock kinetic imaging.
Clonogenic survival assay
The LoVo cells were plated at 200 cells per well in 6-well tissue culture plates and allowed to attach for 24 h. The cells were incubated for 24 h. Immediately after exposure of the cells to 0, 2, and 4 Gy of radiation, the medium was replaced with fresh DMEM supplemented with 10% FBS. Cells were incubated under standard growth conditions for 14 days, and the resultant colonies were stained with Giemsa. Colonies containing ≥50 were scored manually.
Wound healing assay
Approximately 5×105 cells per well were seeded into 6-well culture plates and an incision was made in the central area of the confluent cells 24 h later. Cell migration near the wound area in confluent monolayers was monitored under a microscope (Leica, Germany) at the time of scratching, and after 24, 48, and 72 h. The experiment was performed in triplicate with three independent repeats.
Transwell migration and invasion assay
To further evaluate the effect of PEDF on cell migration and invasion, 2.5×104 cells in 100 μl DMEM with 1% FBS were plated into the upper chamber of the Transwell chamber (8-μm pore size; Corning Inc.). A total of 500 μl of DMEM with 10% FBS was loaded into the lower chamber to serve as a chemoattractant for the cells. After 24 h, cells migrated to or invaded the other side of the membrane and were counted and imaged under a microscope (Leica), after fixing with 2% methanol and staining with 1% crystal violet solution. The experiments were repeated three times.
Protein extraction and western blotting
Cell proteins were extracted using RIPA buffer containing complete protease. Extracted protein (20 μg) was separated by 10% SDS-PAGE and blotted onto PVDF membranes (Millipore, Billerica, MA, USA). Rabbit anti-PEDF (1:1,500 dilution; Abcam, Cambridge, MA, USA), mouse anti-P53 (1:2,000 dilution; Cell Signaling Technology), and mouse anti-GAPDH (1:10,000 dilution; Bioworld Biotechnology, GA, USA) were used as primary antibodies. Horseradish-peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:50,000 dilution, cwBiotech, Beijing, China) was used as secondary antibody. Signals were detected with a chemiluminescence (ECL) kit (Millipore).
TCGA datasets
One of the largest publicly available cancer datasets, the Cancer Genome Atlas (TCGA), was used to make the analysis (23). We downloaded the normalized gene expression data generated using the Illumina-GA RNA-seq platform data and Illumina-Hiseq RNA-seqV2 platform data and the DNA methylation data generated using Illumina Infinium Human Methylation 450 platform from the cBioPortal through the cgdsr R package (version 2.7.1). Gene expression values were transformed as X‘=log2ðX +1Þ, where X represents the normalized fragments per kilobase transcript per million mapped reads values. Altogether, we collected 171 rectum adenocarcinoma patients for analysis from TCGA.
Statistical analysis
Association
Comparison of PEDF expression between the subgroups of various clinicopathological parameters was evaluated by χ2 test or Fisher’s exact test. P<0.05 was considered significant. Pearson correlation coefficient was used to estimate the strength and significance of the association between two continuous variables, such as gene expression and DNA methylation.
Prognosis
Categorical variables were analysed with Pearson’s χ2 or Fisher’s exact test, and the level of significance was set at 0.05. The DFS was compared by the log-rank test using a Kaplan-Meier survival curve. Multivariate Cox proportional hazards regression was used to determine the independent factors affecting DFS, with the level of significance set at 0.05.
Gene ontology/pathway analysis
To identify functional clusters of genes co-expressed with PEDF, we performed Functional Annotation Clustering using Reactome Pathway database (24) and SRTING online database (25).
Gene set enrichment analysis
To identify the pathways that are significantly enriched in genes associated with the PEDF, we computed the association of gene sets with PEDF calls using the following logistic regression model: C =β0 + β1X described before (26).
Results
The association between PEDF expression level and clinicopathological features in LARC patients
To determine the relationship between the expression level of PEDF and clinical pathological variables, we examined PEDF expression by immunohistology in 197 human LARC tissues received nRT. As shown in Table I, the number of positively and negatively stained PEDF was counted. Consequently, we found 110 patients had positive PEDF expression. On analysis, low expression of PEDF was significantly correlated with tumor differentiation (P<0.016), ypT stage (P<0.037), and ypTNM stage (P<0.033). Of note, the PEDF expression level was significantly negatively correlated with ypN stage (P=0.006). Clinicopathological characteristics of all patients are summarized in Table I.
 | Table IAssociation between clinicopathological features and pigment epithelium-derived factor status in the cohort received neoadjuvant radiotherapy. |
Positive PEDF expression is associated with prior disease-free survival and overall survival of LARC patients with nRT
For tumor specimens, Kaplan-Meier curve analysis revealed that positive expression of PEDF predicted prior patient disease-free survival (DFS) and over survival (OS). Fig. 2A and B showed that the DFS time (77.1 vs 49.0%, P<0.001) and OS time (87.1 vs 56.3%, P<0.001) for patients with positive PEDF expression were significantly longer than those for patients with negative PEDF expression. According to a univariate analysis (Table II), several clinicopathological parameters, including ypN stage, ypTNM stage, and PEDF, were important prognosistic factors for both DFS and OS. Cox proportional hazard regression analysis showed only PEDF, was significant, as an independent factor for DFS [P=0.001; HR, 0.422 (95% CI, 0.249–0.717)] and OS [P=0.003; HR, 0.418 (95% CI, 0.234–0.749)] (Table III). These data indicated that positive PEDF expression predicted prior prognosis for LARC patients received nRT.
| Table IIUnivariate log-rank analysis for important clinicopathological features and pigment epithelium-derived factor status in the cohort received neoadjuvant radiotherapy. |
| Table IIIMultivariate analysis for important cliniopathological features and pigment epithelium-derived factor status in the cohort received neoadjuvant radiotherapy. |
PEDF may predict the radiosensitivity and prognosis for LARC patients
To determine the relationship between the expression level of PEDF and radiosensitivity, we examined PEDF expression by immunohistology in 48 pre-treatment biopsy tissues (Table IV). We found a trend that PEDF had a negative correlation with TRG, suggesting its role in sensitivity to nRT (correlation coefficient=−0.290, P=0.046) (Table V). The same as TRG, positive PEDF expressing patients had a prior DFS time (81.3 vs 50.0%, P=0.061) than those with negative PEDF expressing (Fig. 2C and D). However, we did not find the correlation between clinicopathological characteristics and PEDF status. All these data indicate that PEDF may be a factor to predict the radiosensitivity and prognosis for LARC patients.
| Table IVAssociation between clinicopathological features and PEDF status in the pre-treatment biopsy specimens. |
| Table VSpearman correlation test between tumor regression grade and PEDF status in the pre-treatment biopsy specimens. |
PEDF suppresses LoVo cell proliferation, spread, migration, and invasion and enhances the radiation sensitivity
The relationship between increased expression of PEDF and cell invasion of LARC drove us to explore the possible biological functions of PEDF in cancer cells; therefore, we overexpressed PEDF in the highly metastatic LoVo cells. As shown in Fig. 3A, the expression of PEDF increased markedly in LoVo-PEDF cells (Fig. 3A). The survival rate and cell growth curve (Fig. 3B) showed PEDF played an important role in inhibiting cell proliferation. Clonogenic survival assay showed PEDF displayed a radiation-enhancing effect at the irradiation doses of 2 and 4 Gy in LoVo cells (Fig. 3C and D). LoVo cells that overexpressed PEDF showed a significant decrease in cell spreading (Fig. 3F), cell migration, and invasion (Fig. 3E), compared with control cells. In vivo, we confirmed PEDF decreased the ability of LoVo cells to form and grow tumors (Fig. 3G and H). Collectively, overexpression of PEDF suppresses cell proliferation, spreading, migration, and invasion and enhances the radiation effect.
PEDF regulates the double-strand breaks (DSBs) repair pathway and activates G protein pathway
Until now, the exact pathways that PEDF may regulate in LARC remain unclear. To probe the PEDF-associated pathways on an unbiased basis, we performed GSEA using the Illumina-GA RNA-seq platform data of the rectal cancer cohort of The Cancer Genomic Atlas project (TCGA, 171 patients), GSEA is designed to detect coordinated differences in expression of predefined sets of functionally related genes (26). Among all the 674 predefined ‘Reactome’ gene sets, the DSBs repair pathway and G protein activation pathways were identified with a strongly negative and positive association, respectively, with PEDF expression in the TCGA rectal carcinoma dataset (Fig. 4A and B). By the analysis of LARC cohort of TCGA, parts of key genes correlated with the DSBs repair pathway (Fig. 4C) on mRNA levels were found to be negatively associated with PEDF (Fig. 5A). For genes correlated with the G protein activation pathway (Fig. 4D), parts of key genes were found to have positive association with PEDF (Fig. 5B). These findings consistently suggest that PEDF may be involved in the regulation of the DSBs repair pathway and enhancement of G protein activation pathway.
PEDF activates P53 to perform functions
To validate how PEDF is involved in the DSBs repair and G protein activation pathways, the online database STRING (25) was used to investigate interactions between PEDF and associated genes. By protein interaction analysis, we found P53 could be the downstream target gene of PEDF, and interaction network was constructed (Fig. 5A). We then used The Cancer Cell Line Encyclopedia database (27) to confirm that P53 is wild-type in LoVo cells and has anticancer function (data not shown). We further validated that P53 was more highly expressed in LoVo cells with PEDF overexpression compared with the control cells (Fig. 5B). These findings consistently suggest that P53 is a downstream gene of PEDF.
PEDF expression is controlled by promoter methylation
The relationship between PEDF expression level and PEDF promoter methylation status was analyzed using Illumina-Hiseq RNA-seqV2 platform data and Illumina Infinium Human DNA Methylation 450 beadchip data.
To investigate whether PEDF methylation status affects the mRNA level in rectal cancer, we used the TCGA date-base (23) to make the correlation between methylation of PEDF cg11692409 and PEDF cg27102649 and PEDF mRNA expression level. The data showed that methylation of the two genomic coordinates significantly negatively correlated with PEDF mRNA expression (correlation coefficient=−0.38, P<0.001 and correlation coefficient=−0.44, P<0.001) (Fig. 6C).
Discussion
PEDF has been found to enhance the antitumor effects of radiotherapy on nasopharyngeal carcinoma by downregulating VEGF expression and inhibiting angiogenesis (28), but the prognostic value of PEDF in response to nRT prognosis for LARC patients is rarely reported. To the best of our knowledge, this is the first time that PEDF expression was found to be different between the long-term DFS group and the short-term DFS group. LARC patients with high levels of PEDF had prior progression-free survial and tumor regression. The research demonstrated that PEDF could be a biomarker to predict the response of the LARC patients to nRT; in vitro and in vivo data showed that PEDF could be a target for developing novel therapeutic strategies.
The nRT has become a standard treatment for LARC patients, and the nRT regimen we adopted in this study was 30 Gy in 10 fractions, which is recommended by the Chinese Anti-Cancer Association. Our previous data revealed prior response rates and clinical efficiency compared with traditional and published nRT regimens (29). However, not all patients are sensitive to it. Several studies have suggested TRG can be used to monitor treatment and as a prognostic parameter for patients who received nRT (30). The response to nRT varies from sensitivity to resistance, and some LARC patients suffer side effects and risk of disease progression during therapy (6). We found a new marker to predict the TRG before nRT, providing a new therapeutic strategy for LARC patients. In accordance with our data, PEDF contributed to radiosensitivity in melanoma therapy. Using quantitative reverse transcription PCR and immunohistochemical staining, we previously detected that low PEDF levels were associated with liver metastasis and disease-free survival in a large corhort of LARC patients (20).
In this study, we not only revealed that PEDF was an independent prognostic factor for DFS and OS in Cox regression analysis but also found that low expression of PEDF in LARC was associated with a high degree of ypN stage. Many researchers have found that lymph node metastases are significantly correlated with decreased survival in several solid tumors (31), and reduced PEDF levels in lung cancer tissues significantly correlated with lymph node metastasis (32). In addition to the anti-angiogenesis effect, PEDF plays a role in apoptosis, autophagy, and many other antitumor processes in various malignancies (14,15). All of these suggest that PEDF may decrease lymph node metastasis in cancers.
Because of the association between PEDF and clinicopathological factors, we hypothesized that PEDF could increase the sensitivity of the tumor to nRT. DNA damage and hypoxia are important biological processes in chemoradiosensitivity (33,34), and PEDF could induce cancer cell death by apoptosis and anti-angiogenesis during therapy (18–20). To confirm this hypothesis, we overexpressed PEDF in LoVo cells. Compared to the control, LoVo cells that overexpressed PEDF reduced tumorigenesis, with fewer colonies after exposure to the radiation. Metastasis and recurrence are the determining factors affecting DFS time of LARC patients, and angiogenesis has an indispensable implication on the invasive property of cancer cells (35–37). Simultaneous treatment with PEDF and radiation has an additive effect on the downregulation of VEGF expression and on angiogenesis inhibition (28). Therefore, our in vitro and in vivo results suggested that the higher level of PEDF expression promoted the DFS time by suppressing tumor growth and metastasis in LARC patients.
To further study the mechanism by which PEDF regulates the radiosensitivity and cell proliferation, we used the TCGA database to identify the PEDF-associated genes. The GSEA indicated that PEDF suppresses the DSBs repair pathway and activates the G protein activation pathway and thus may provide useful information for targeted therapy. Demonstrating the effect of PEDF on regulating the pathways by STRING online database, we provide a more complete network that PEDF activates P53.
Gene expression is regulated by DNA methylation at CpG island promoter regions (38). Ha et al found that the methylation status of genes is associated with radiosensitivity to nRT (39). Our data demonstrated negative correlation between PEDF expression and its methylation status in LARC patients. The patients with low PEDF expression usually possess high-level status of methylation, and our results indicate that the hypermethylation of PEDF could decrease its mRNA expression, generating angiogenesis and cancer cell proliferation, which induces tumor metastasis in LARC patients.
A number of limitations need to be noted regarding this study. The most important limitation lies in the fact that the limited pre-treatment sample size could not confirm the trend between the expression level of PEDF and TRG before nRT, and we will accumulate more specimens to confirm the relationship. In addition, there is a variety of CRC cell lines available to us; we only used LoVo cells to identify the relationship between the level of PEDF expression and radiosensitivity signatures. The LoVo cell was used for overexpressed PEDF since it was exclusively lower expression compared to the other eight LARC cell lines. Researching ATCC, we determined that the LoVo cell line is derived from metastatic sites, which is suitable for our purpose. Next, we plan to validate the direct interaction between P53 and PEDF. Methylation status is different in CRC cell lines that are derived from diverse stages of CRC. Epigenetics signatures in LARC patients are also affected by various factors (38). Much more research is needed to prove the hypermethylation status of PEDF suppresses its mRNA transcription.
In concusion, that PEDF has a prognostic value to LARC patients after nRT. According to the level of PEDF expression, LARC patients may be stratified into risk subgroups and allow personalized therapeutic strategies. In addition to prolonging the DFS of patients, PEDF enhances the sensitivity of a tumor to nRT. Moreover, PEDF, as an anti-angiogenesis factor, possesses the potential to be an independent therapeutic agent for LARC patients.
Acknowledgements
This study was supported by the National Natural Science Foundation (81372593, 81201965), Beijing Natural Science Foundation (7132052), and the National High Technology Research and Development Program of China (863 Program) (no. 2012AA02A506, 2014AA020801). The authors would like to thank Dr Bin Dong, the Department of Pathology, Peking University Cancer Hospital and Institute for her technical assistance.
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