Background
Colorectal cancer (CRC) is one of the most common malignancies and is the second-leading cause of cancer-related death worldwide [
1]. It is expected that the CRC burden will substantially increase in the next two decades [
2]. Despite current progress, many patients with advanced tumors die from this malignancy [
3]. To improve the curative effect and prognosis, it is crucial to further explore the molecular mechanism of tumorigenesis and find new biomarkers for targeted treatment.
Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that regulate multiple cellular activities including proliferation, differentiation, and apoptosis [
4]. As a major axis of the MAPK pathway, the Raf/MEK/ERK signaling pathway is activated in many human cancers [
4,
5]. Raf kinases (A-Raf, B-Raf, and c-Raf) that play indispensable roles in this pathway are regulated by a network of protein-protein interactions and phosphorylation-dephosphorylation events [
6,
7]. Generally, Raf kinases are regulated by RAS proteins to activate the Raf/MEK/ERK pathway [
8,
9]. However, recent studies have shown that Raf is also regulated by different binding partners, including 14–3-3 proteins, RKIP, KSR, and CNK [
10‐
12]. Some of these binding partners even play important roles in regulating Raf function and tumorigenesis. Cancer treatments that target the c-Raf kinase-inhibitory protein, RKIP, have shown unprecedented response rates [
13]. The binding partners of Raf kinases and 14–3-3 proteins negatively regulate the activation of major survival pathways [
10,
14]. RUVBL1, which is another c-Raf-binding protein, has been reported to activate the Raf/MEK/ERK pathway and thus promote lung cancer tumorigenesis [
15]. These findings indicate that Raf-interacting proteins have an influence on cancer progression.
PDCD6, which is also known as apoptosis-linked gene-2 (ALG-2), encodes a calcium-binding protein that contains five serially repeated EF-hand motifs [
16‐
18]. PDCD6 was originally considered a pro-apoptotic protein that participates in T cell receptor-, Fas-, and glucocorticoid-induced apoptosis [
19,
20]. It has also been implicated in diverse physiological processes, including endoplasmic reticulum stress-induced cell death, neuronal apoptosis during organ formation, signal transduction, membrane trafficking, and posttranscriptional control of gene expression [
18]. Recent studies have revealed that PDCD6 overexpression can promote the progression of hepatomas, breast, and ovarian cancer, suggesting that PDCD6 might be involved in the maintenance of cellular viability [
21‐
23]. In contrast, downregulating of PDCD6 expression could accelerate gastric cancer, HeLa cells, and glioblastoma cell proliferation [
24‐
26]. These findings suggest PDCD6 has different effects on different tumor types. However, the role of PDCD6 in the pathogenesis of colorectal cancer has not been thoroughly investigated.
In this study, we aimed to identify novel therapeutic targets for CRC. We found that the PDCD6 expression level was elevated in CRC and that PDCD6 overexpression was correlated with poor survival in patients with CRC. The tumor-promoting activity of PDCD6 was characterized by in vitro and in vivo tumorigenesis assays. RNA-Seq and pathway analyses were used to investigate the molecular signaling of PDCD6, and the interaction between PDCD6 and c-Raf was also investigated. Overall, these observations indicated that targeting PDCD6 may provide a new opportunity for treating colorectal cancer.
Methods
Cell culture and reagents
HCT116 and HCT15 cells were cultured in Iscove’s Modified Dulbecco’s medium (HyClone) with 10% fetal bovine serum (FBS), while HEK293T cells were cultured in Dulbecco’s modified Eagle medium (HyClone) with 10% FBS. All cell lines were obtained from the Cell Resource Center of Peking Union Medical College. Plasmids were constructed according to a standard cloning technique. PDCD6 was cloned into PCDH-CMV-MCS-EF1-copGFP for overexpression. BAPTM, RAF709, Trametinib, and Oxaliplatin were purchased from Tsbiochem. All compounds were dissolved in DMSO to a final concentration of 10 mmol/ml and stored at − 20 °C.
Antibodies
Antibodies against PDCD6 were purchased from Proteintech. Antibodies against phospho-c-Raf, c-Raf, phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2, phospho-STAT3, Ki67 and cleaved-Caspase3 were purchased from Cell Signaling Technology. Tubulin antibodies were purchased from Santa Cruz Biotechnology, Inc.
Tissue analysis
A tissue microarray including tumor tissues and their corresponding adjacent normal tissues from 93 cases of CRC was obtained from Shanghai Biochip. A tissue microarray with tumor tissues from 423 cases of CRC was analyzed using paraffin-embedded tumor samples, which were histopathological diagnosed at the Cancer Hospital, Chinese Academy of Medical Sciences. Patient consent and approval from the Institutional Research Ethics Committee were obtained for the use of these clinical materials for research purposes. The clinical information regarding the samples is collected and summarized in Table
1 and Supplementary Table S
1. Paraffin-embedded tissue sections (4 mm) were prepared according to standard methods, and the expression of PDCD6 (1:300 dilution) was detected using immunoperoxidase. Slides were assessed by pathologists who were blinded to the experimental results and patient outcomes. The PDCD6 expression was evaluated by an immunostaining score, which was calculated as the sum of the proportion and intensity of the stained tumor cells. Briefly, a proportion score, which represented the estimated proportion of positively stained tumor cells (0, none; 1, 0 ~ 25%; 2, 25 ~ 50%; 3, 50 ~ 75%; and 4, 75 ~ 100%.), was first assigned. Next, an intensity score, which indicated the average intensity of positively stained tumor cells (0, none; 1, weak; 2, intermediate; and 3, strong) was obtained. The proportion and intensity scores were then added to obtain a total score, which ranged from 0 to 12.
Table 1
Correlation of the expression of PDCD6 in colorectal cancer with clinicopathologic parameters
Gender | | | | 0.7875 |
Male | 238 | 180 | 58 | |
Female | 185 | 142 | 43 | |
Age | | | | 0.9413 |
< 60 | 229 | 174 | 55 | |
≥ 60 | 194 | 148 | 46 | |
Differentiation | | | | 0.601 |
Low | 47 | 33 | 14 | |
Moderate | 354 | 272 | 82 | |
High | 22 | 17 | 5 | |
Tumor size (cm) | | | | 0.2589 |
< 5 | 282 | 210 | 72 | |
≥ 5 | 141 | 112 | 29 | |
pTNM | | | | 0.0020** |
I | 31 | 23 | 8 | |
II | 187 | 154 | 33 | |
III | 182 | 134 | 48 | |
IV | 23 | 11 | 12 | |
pT | | | | 0.0305* |
T1 | 1 | 1 | 0 | |
T2 | 47 | 37 | 10 | |
T3 | 334 | 261 | 73 | |
T4 | 41 | 23 | 17 | |
pN | | | | 0.2374 |
N0 | 236 | 187 | 49 | |
N1 | 132 | 93 | 39 | |
N2 | 53 | 40 | 13 | |
N3 | 2 | 2 | 0 | |
KRAS | | | | 0.6311 |
Negative | 360 | 272 | 88 | |
Positive | 63 | 50 | 13 | |
BRAF | | | | 0.5766 |
Negative | 419 | 318 | 101 | |
Positive | 4 | 4 | 0 | |
Immunofluorescence staining
Dissect tissue as fast as possible, then immerse in fixative (Servicebio. G1101) immediately. Trim tissue sample appropriately after fixation (at least 24 h). Immerse sample in 15% sugar (Sinopharm. 57–50-1) solution at 4 °C until sink down to the bottom, then transfer to 30% sugar solution at 4 °C. Take out a tissue sample from 30% sugar solution and remove the redundant solution. Mount sample in OCT compound (Sakura. 4583) and freeze at − 20 °C to − 80 °C. Cut 8-10 μm sections in cryostat and mount on histological slides. The tissues were washed with PBS. The tissues were fixed with 4% formaldehyde and permeabilized with PBS containing 0.3% Triton X-100. The tissues were blocked with 5% BSA at room temperature for 1 h and then incubated with PDCD6 at 4 °C overnight. Alexa Flour 488 FITC-conjugated secondary antibodies were added and incubated for 30 min at 37 °C.
Live cells were washed with PBS. The cells were fixed with 4% formaldehyde and permeabilized with PBS containing 0.3% Triton X-100. The cells were blocked with 5% BSA at room temperature for 1 h and then incubated with PDCD6 and c-Raf antibodies at 4 °C overnight, respectively. Alexa Flour 488/594 FITC-conjugated secondary antibodies were added and incubated for 30 min at 37 °C. The slides were stained with DAPI, mounted, and observed under a microscope. As a negative control, the specific primary antibodies were replaced with a control mouse or rabbit IgG antibody.
For the knockdown and overexpression of human PDCD6, the PDCD6-specific shRNAs and the PDCD6 sequence were cloned into the vectors pLKO.1 puro and pCDH-CMV-MCS-EF1-copGFP, respectively. The pCMV-VSV-G and psPAX2 were used as helper plasmids to produce the lentivirus. The sequences are shown in Supplementary Table S
2. The virus was harvested 48 h after transfecting in HEK293T cells. The target cells were infected by the viral supernatants, which were diluted fourfold in a fresh medium. To isolate the cells that expressed GFP, the cells were dissociated into a single-cell suspension using trypsin and aggregates were removed by a 40-μm cell strainer. FACS was performed using a BD Aria II sorter, which was gated for a moderate level of GFP expression.
qRT-PCR
Total RNA was isolated from the different cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Equal amounts of RNA were reverse transcribed into cDNA using a Revert Aid First Strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer’s instructions. Quantitative PCR was performed using an ABI Step One Plus system. The PCR reactions were carried out in 10 μL reactions using SYBR Green PCR master mix (Invitrogen) and 0.5 μM specific primers. The primers used for PCR are shown in Supplementary Table S
3.
Cell proliferation assay and colony formation assays
Cell proliferation was assessed using a CCK-8 assay (Dojindo Molecular Technologies). Briefly, HCT116 and HCT15 cells were seeded in 96-well plates. Ten microliters of CCK-8 solution was added to each well containing 100 μL culture medium and incubated for 2 h at 37 °C. The absorbance was measured at a wavelength of 450 nm using an ELISA plate reader. For the cell proliferation assays, cell growth was analyzed once per day for 6 days. For the colony formation assays, 200 cells per well were seeded in six-well plates and cultured at 37 °C for 2 weeks. At the end of the incubation, the cells were fixed with 1% paraformaldehyde for 30 min and stained with 0.1% (w/v) crystal violet for 30 min. Cell colonies were counted.
In vivo tumorigenesis assays
Animal experiments were performed with the approval of the Peking Union Medical College Animal Care and Use Committees. Five million tumor cells were resuspended in 0.2 ml phosphate-buffered saline and inoculated into the flanks of 6-week old male athymic nude mice (6 mice in each group). Tumor growth was monitored every 3 days by measuring tumor diameters. Tumor width (W) and length (L) were measured, and the tumor volume was calculated using the following formula: volume = (W × L)2/2. The mice were sacrificed at 25 days after inoculation. The tumors were removed, photographed, and weighed and the average weights of the tumors were obtained (*P < 0.05).
Total RNA was isolated, as previously described. Novogene Technology Co., Ltd. (Tianjin, China) prepared the libraries and performed the sequencing. RNA-seq was performed to detect the mRNA expression profiles of PDCD6 knockdown colorectal cancer cells using HiSeq3000 (Illumina). LifeScope v2.5.1 was used to align the reads to the genome, generate raw counts corresponding to each known gene, and calculate the FPKM (fragments per kilobase million) values. Differentially expressed genes with a fold change > 2 were selected, and gene ontology (GO) analysis was used for pathway enrichment using Cytoscape (ClueGo) with a P-value < 0.05.
Mass spectrometry (MS)
The HCT116 cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5 mM Ca2+, 0.5% NP-40 and protease inhibitor cocktails) and subjected to affinity purification with anti-PDCD6 antibody. The purified protein complex was resolved on SDS-PAGE and Coomassie brilliant blue stained. The gel bands of interest were excised from the gel. Peptides were analyzed by Thermo Scientific Q Exactive mass spectrometer.
The MS/MS spectra from each LC-MS/MS run were searched against the raf1.fasta from UniProt using an in-house Proteome Discoverer (Version PD1.4, Thermo-Fisher Scientific, USA). The false discovery rate (FDR) was also set to 0.01 for protein identifications.
Co-IP assay
Immunoprecipitation assays were performed, as previously described [
27]. For coimmunoprecipitation assays, HCT116 cells were harvested and lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5 mM Ca
2+, 0.5% NP-40 and protease inhibitor cocktails). Immunocomplexes were solubilized in 5 × SDS loading buffer and immunoblotted with the indicated antibodies.
Immunohistochemical (IHC) staining
5 μm longitudinal sections of the paraffin-embedded femurs were kept at 60 °C for 24 h in the oven and then followed by deparaffinized with xylene and hydrated with an ethanol gradient (100–70%). After successively incubating with antigen retrieval solution (Shanghai Shunbai Biotechnology Company; Shanghai, China) and 3% H2O2 for 30 min, the slides were rinsed with water and incubated with the primary antibody (IGF-1 (1: 50)) overnight at 4 °C. For negative controls, the primary antibody was replaced by nonimmunized serum. The next day, the slides were rinsed and incubated with the corresponding secondary antibody (Beijing Biosynthesis Biotechnology Co. Ltd.; Beijing, China) for 30 min followed by 3,3′-diaminobenzidine (DAB) and hematoxylin staining, respectively.
Hematoxylin-eosin (HE) staining
After deparaffinization and rehydration, 5 μm longitudinal sections were stained with hematoxylin solution for 5 min followed by 5 dips in 1% acid ethanol (1% HCl in 70% ethanol) and then rinsed in distilled water. Then the sections were stained with eosin solution for 3 min and followed by dehydration with graded alcohol and clearing in xylene.
Statistical analysis
Comparisons between PDCD6 expression in CRC tissues and adjacent normal tissues were analyzed by the Wilcoxon signed-rank test. Associations of the relationship between PDCD6 expression and the clinicopathological parameters of 93 CRC patients were analyzed using the χ2 test . We estimated the expression of PDCD6 for each patient and categorized the patients into PDCD6-low and PDCD6-high groups according to the optimal cutoff (5) obtained in Xtile software. Disease-free survival was analyzed by the Kaplan-Meier method and tested by the log-rank test. For the cytological studies, the data are presented as means ± SDs of independent experiments which were repeated three times. The significance of differences between experimental groups was analyzed using a Student’s t-test. P < 0.05 was considered statistically significant. All the analyses were performed by GraphPad Prism 7.0. software.
Discussion
Previous studies have revealed that PDCD6 was involved in cancer development; The expression of PDCD6 varies in different tumors, indicating that PDCD6 plays different roles in different cancer types [
21,
24]. The MAPK/ERK pathway stimulates cellular proliferation and invasion; however, its activation can also increase cellular apoptosis or antagonize pro-oncogenic input from other signals. The effect that predominates depends on the intensity of the signal and the context or tissue in which the signal is aberrantly activated [
39]. The downstream MAPK/ERK signaling is predominantly activated by upstream Raf signaling. Our study found that PDCD6 promotes tumor growth by interacting with c-Raf and regulating downstream the MAPK kinase pathway. So, the opposite effect of PDCD6 might due to the opposite role of raf in different tumor types, in which the tissue-specific tumor microenvironment and the intensity of which signal activated are different.
In the present study, we explored the role of PDCD6 in the development and progression of colorectal cancer and the prognostic value of PDCD6 in patients with CRC. We found that PDCD6 was overexpression in CRC tissues. The functional experiments showed that PDCD6 depletion significantly inhibited colorectal cancer tumorigenesis and PDCD6 overexpression enhanced the proliferation and tumor growth of CRC cells. These findings and findings from clinical studies suggest the PDCD6 plays an oncogenic role in the pathogenesis of colorectal cancer. Moreover, PDCD6 overexpression tends to positively correlate with the tumor stage in patients with CRC, indicating a potential correlation between PDCD6 and malignancies. Furthermore, a Kaplan-Meier analysis revealed that PDCD6 overexpression in tumor cells had a significantly worse prognostic impact on the disease-free survival of patients with CRC, indicating that PDCD6 is a predictor of the survival of patients with colorectal cancer. The function of PDCD6 in CRC was consistent with previously reported results regarding ovarian cancer [
23], and PDCD6 overexpression has been reported in hepatomas and lung cancer tissue, suggesting that this protein plays an oncogenic role in the progression of these types of tumors [
21]. Overall, our clinical findings demonstrated that PDCD6 is closely associated with the progression of human CRC, and indicated that PDCD6 could serve as a useful biomarker for the prognosis of patients with CRC.
Previous studies have reported that PDCD6 promotes breast cancer growth and metastasis by regulating the cytoskeleton and mediating the proapoptotic activity of cisplatin and TNF-α through the downregulation of NF-κB expression in different biological process [
22,
40]. However, by using RNA sequencing, we found that the MAPK signaling pathway is the main pathway regulated by PDCD6 in CRC. As the MAPK signaling pathways serves as a central node that regulates cell proliferation and survival [
41,
42], our data indicated that PDCD6 is important for MAPK pathway activation and the growth of CRC cells. For the first time, our study reported that PDCD6 affects the MAPK signaling pathway in CRC, supplementing of the mechanistic investigation of PDCD6 in cancer. Proteomics analysis and immunoprecipitation analyses indicated that PDCD6 is physically associated with c-Raf and subsequent assays revealed that PDCD6 promotes colorectal cancer development and progression by binding c-Raf and increasing its phosphorylation level, which is consistent with previous reports on the tumor-promoting role of c-Raf [
13,
43,
44], indicating that c-Raf is an executor of PDCD6 in colorectal cancer. Our results further revealed that the PDCD6/c-Raf complex activates the Raf/MEK/ERK pathway to promote the CRC progression. However, the mechanism of how the PDCD6/c-Raf complex regulates the phosphorylation levels of c-Raf requires further investigation.
Calcium ions are the secondary intracellular messenger that regulate numerous biological processes [
45,
46]. Increasing evidence has suggested the role of PDCD6 as a Ca
2+-responsive adaptor protein [
47‐
49]. Upon binding to Ca
2+, PDCD6 undergoes a conformational change that facilitates its interaction with various proteins [
50,
51]. This conformational change enables PDCD6 to interact with various proteins [
51‐
53]. It was experimentally verified that the interaction between PDCD6 and c-Raf requires the presence of Ca
2+, suggesting that the interaction between PDCD6 and c-Raf is possibly due to the conformational change caused by the combination of PDCD6 and Ca
2+, thus affecting the phosphorylation of downstream signaling pathways. Changes in the levels of intracellular Ca
2+ provide dynamic and highly versatile signals that regulate cell proliferation [
46]. The Ca
2 + −dependence can be excluded by the fact that Ca
2+ is essential for the effect of PDCD6 on colorectal cancer. Therefore, the effect of Ca
2+ on cell proliferation is likely partially attributed to PDCD6.
RAF709 and Trametinib, which are effective MAPK pathway effective inhibitors, have been shown to be anticancer agents in multiple tumor types [
54,
55]. To further show that PDCD6 affects the MAPK signaling pathway, RAF709 and Trametinib were used to stimulate PDCD6-OE HCT-116 cells and to observe the changes in the MAPK signaling pathway. Although PDCD6 was overexpressed, the MAPK signaling pathway was strongly inhibited after adding these two inhibitors. These results suggested that the effect of PDCD6 on CRC growth is mediated by its regulating effect on the MAPK signaling pathway. Moreover, these findings suggest the potential for antagonizing Raf/MEK/ERK signaling as a strategy to inhibit the growth of tumors hyperactivated by PDCD6.
The results from the cell lines were validated in xenografts tumor tissues to confirm their reliability. We proved that PDCD6 and c-RAF/MEK/ERK were positively correlated at the protein level by IHC in tumor tissues from xenografts and patients with CRC, which will further facilitate translational medicine research on PDCD6. In addition, this cascade mediates its function mainly through the regulation of several vital genes including MYC and JUN [
28]. Consistent with the IHC analysis, the positive correlation of PDCD6 and JUN and MYC mRNA levels indicated that PDCD6 effects on CRC have a closely clinicopathological relevance.
PDCD6 has a higher expression in colorectal cancer. On one hand, PDCD6 could be used as a drug target for screening PDCD6 specific colorectal cancer treatment drug. On the other hand, PDCD6 is also a Ca2+ binding protein, our results show that the PDCD6 play its role to promote cancer only when it combined with Ca2+, so we can also inhibit Ca2+ as targeting PDCD6 treatment project for the colorectal cancer patients. Moreover, combination therapy of conventional drugs with PDCD6-targeted specific drugs or Ca2+ inhibitor drugs would be a good direction for the PDCD6-overexpressed patients. Further studies on drug exploration targeted PDCD6 could help to establish the true significance in clinical therapy. The CRC patients will benefit from the elimination of PDCD6.
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