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Phospholipase C delta 1 inhibits WNT/β‐catenin and EGFR-FAK-ERK signaling and is disrupted by promoter CpG methylation in renal cell carcinoma
Clinical Epigenetics volume 15, Article number: 30 (2023)
PLCD1, located at 3p22, encodes an enzyme that mediates cellular metabolism and homeostasis, intracellular signal transduction and movement. PLCD1 plays a pivotal role in tumor suppression of several types of cancers; however, its expression and underlying molecular mechanisms in renal cell carcinoma (RCC) pathogenesis remain elusive.
RT-PCR and Western blot were used to detect PLCD1 expression in RCC cell lines and normal tissues. Bisulfite treatment, MSP and BGS were utilized to explore the CpG methylation status of PLCD1 promoter. Online databases were analyzed for the association between PLCD1 expression/methylation and patient survival. In vitro experiments including CCK8, colony formation, wound-healing, transwell migration and invasion, immunofluorescence and flow cytometry assays were performed to evaluate tumor cell behavior. Luciferase assay and Western blot were used to examine effect of PLCD1 on WNT/β‐catenin and EGFR‐FAK-ERK signaling.
We found that PLCD1 was widely expressed in multiple adult normal tissues including kidney, but frequently downregulated or silenced in RCC due to its promoter CpG methylation. Restoration of PLCD1 expression inhibited the viability, migration and induced G2/M cell cycle arrest and apoptosis in RCC cells. PLCD1 restoration led to the inhibition of signaling activation of WNT/β-catenin and EGFR-FAK-ERK pathways, and the EMT program of RCC cells.
Our results demonstrate that PLCD1 is a potent tumor suppressor frequently inactivated by promoter methylation in RCC and exerts its tumor suppressive functions via suppressing WNT/β‐catenin and EGFR‐FAK-ERK signaling. These findings establish PLCD1 as a promising prognostic biomarker and treatment target for RCC.
PLCD1 is methylated and downregulated in RCC and functions as a tumor suppressor gene
Inactivation of PLCD1 contributes to RCC tumorigenesis.
PLCD1 inhibits WNT/β-catenin and EGFR-FAK-ERK signaling in RCC.
Kidney cancer, with renal cell carcinoma (RCC) accounting for 90% of the cases, is the most frequent malignancy of the urinary system . RCC is a heterogeneous disease, comprising three main subtypes: kidney renal clear cell carcinoma (KIRC, or ccRCC), renal papillary cell carcinoma (KIRP) and kidney chromophobe carcinoma (KICH). Annually, approximately 430,000 new cases and 180,000 fatalities of RCC occur globally . RCC is frequently discovered coincidentally during imaging for other health concerns, often at the late stage, with − 30% of patients already developed metastases at the time of diagnosis. RCC is primarily surgically treated and has a poor 5-year survival rate, with − 25% of patients reporting recurrence . Thus, deeper understanding of the molecular pathogenesis of RCC is critical to develop efficient diagnostic markers and treatment strategy.
Epigenetic alterations, including promoter CpG methylation and histone modifications, are critically involved in multi carcinogenesis through silencing tumor suppressor genes (TSGs) and activating oncogenes [4, 5]. Due to the frequent loss of heterozygosity (LOH) at chromosome 3p21.3 in multiple cancers including esophageal squamous cell carcinoma (ESCC), lung cancer as well as nasopharyngeal carcinoma (NPC) , the 3p22-21.3 region is considered as a classic TSG locus. We and others previously discovered that several 3p22-21.3 genes including RASSF1A, BLU, DLEC1, ZMYND10 and PLCD1 were frequently methylated in various cancers [6,7,8].
Phospholipase C delta 1 (PLCD1), located at 3p22.2, is regarded as the basic isoform of PLC family members and belongs to the PLCδ subgroup . PLCD1 encodes an enzyme involved in calcium homeostasis, energy metabolism, intracellular transport and signal transduction. Silence of PLCD1 by promoter CpG methylation has been described in a few epithelial and hematological malignancies, including gastric cancer (GsCa), ESCC, colorectal cancer (CRC), breast cancer (BrCa), pancreatic cancer (PAAD), chronic myeloid leukemia (CML) and non-small cell lung cancer (NSCLC) [10,11,12,13,14]. However, its relevance to RCC pathogenesis is unclear.
In this study, we examined the expression level and methylation status of PLCD1 in RCC and further explored their relationship in patient tissues. Effects of ectopic PLCD1 expression on RCC cell behavior and its underlying mechanisms were also evaluated. Our results demonstrate that PLCD1 is a potent TSG disrupted by CpG methylation and closely involved in RCC pathogenesis and development.
Cell lines, normal tissue samples and RCC cell line samples
RCC cell lines including HH244, A498, Caki-2, RCC98, 786-O and HEK293T cells were obtained from American Type Culture Collection and maintained in RPMI-1640 or DMEM medium with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin at 37 ℃ in a humidified atmosphere supplemented with 5% CO2. RNA samples of human normal adult tissues and RCC cell line panel were purchased from Stratagene (La Jolla, CA) or Millipore (Chemicon, Billerica, MA).
PLCD1 mRNA expression and protein expression of tumor and adjacent normal tissues were analyzed by GENT2 (GENT2 (appex.kr)), UALCAN (UALCAN (uab.edu)) and TIMER2.0 (TIMER2.0 (comp genomics.org))databases [15,16,17]. Human Protein Atlas version 20.1. (https://www.proteinatlas.org) was used to describe the PLCD1 protein expression of various types of tissues. PLCD1 genetic alteration rate in RCC patients was studied in cBioportal database (cBioPortal for Cancer Genomics), and mutation rate in TCGA samples was evaluated in TIMER2.0 database [17, 18]. PLCD1 methylation status and its correlation with overall survival were analyzed on EWAS data hub (https://ngdc.cncb.ac.cn/ewas/datahub/index) [19, 20]. TIMER2.0 database was also employed to clarify the association between PLCD1 expression and patients’ overall survival.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis
RT-PCR analysis for gene expression was done using GoTaq (Promega, Madison, WI). For PLCD1 (F: 5’-TGTCGCTACTCAAGTGAGTC, R: 5’-AGTCCTCCTGCAACTTGTAG), the following program was conducted for 32 cycles: 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. GAPDH (GAPDH-F: 5’-GATGACCTTGCCCACAGCCT, GAPDH-R: 5’-ATCTCTGCCCCCTCTGCTGA) was used as a reference, and its reaction condition for 23 cycles was: 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s.
Bisulfite treatment and assessment of promoter methylation
DNA bisulfite modification and methylation-specific PCR (MSP), as well as bisulfite genomic sequencing (BGS), were used to detect promoter methylation. DNA extraction was achieved using DNA extraction kit (Qiagen, Hilden, Germany), and then, the extracted DNA was subjected to bisulfite treatment described previously . After bisulfite treatment, unmethylated cytosines are converted into uracil, while methylated cytosine remains unchanged. We used allele-specific methylated (M) and unmethylated (U) primers for MSP amplification, and MSP products were analyzed by electrophoresis with 1.8% agarose gel. The amplified BGS products with BGS primers were purified and cloned into pCR4-TOPO cloning vector, with 6–8 colonies randomly selected for sequencing by Beijing Genomics institution (BGI). Primers used: MSP: m11: 5’-AATGATAGGGTTCGCGGTTC, m2: 5’-CCCGAACCAACGAACGCG, u1: 5’-GTAATGATAGGGTTTGTGGTTT, u2: 5’-CTAACCCAAACCAACAAACACA; BGS1: 5’-GTATTTTTGGGGTTAGAAATT, BGS4: 5’-AAAAACAAAACTAAAAACCC.
5-Aza-2′-deoxycytidine (5-Aza) and trichostatin A (TSA) treatment
Cell lines (A498, Caki-2, RCC98, HH244, and 786-O) were freshly seeded at a density of 1 × 105 cells/ml and allowed to grow in T175 flasks overnight. Fresh medium with 10 µM 5-Aza (Sigma-Aldrich, St Louis, MO) was changed every 24 h for 3 days and then harvested; or further treated with 100 nM trichostatin A (TSA) for additional 24 h. Cells were harvested for RNA and DNA extraction. The dosages of 5-Aza and TSA were decided based on its dose–response effect on cell viability and demethylation effect previously assessed.
Colony formation assay
For colony formation analysis, A498 and HH244 cells in 6-well plates (2 × 105 cells/well) were transfected with pcDNA3.1( +)-PLCD1-V5/His plasmid or the control vector (2.5 μg/well), using Lipofectamine™ 3000 Reagent (Invitrogen). 24 h post-transfection, cells were harvested by trypsinization and centrifugation and plated on 6-well plates at a suitable density. The cells were maintained with G418 (0.4 mg/mL) and stably transfected cells selected for 10–12 days with selection media changed every 2 days. Surviving colonies (> 50 cells per colony) were stained using Gentian Violet (ICM Pharma) and counted. Assays were performed for three times, in triplicate wells.
Cell counting kit-8 (CCK-8) assay
Cell proliferation was evaluated by CCK-8 assay. A498 and HH244 cells in 6-well plates (2 × 105/well) were transfected with pcDNA3.1( +)-PLCD1-V5/His plasmid or control vector (2.5 μg each well), using Lipofectamine™ 3000 Reagent (Invitrogen). After 24 h, cells were obtained and plated into 96-well plates at a density of 2000 cells/well. Cell proliferation rates were evaluated by using CCK-8 assay (MCE, HY-K0301). Absorbance at 450 nm was measured at designated time point by spectrophotometer. All assays were conducted for three times in triplicates.
Transwell migration and invasion analyses
Cell migration and invasion were examined using transwell migration and Matrigel invasion assays. Migration assay was done on 24-well transwell inserts (8-um-pore filters, BD Biosciences). Matrigel invasion assays were done on 24-well plates containing Matrigel (8.0 Micron, Corning). After 24 h of transfection, RCC cells were harvested, resuspended in RPMI-1640 medium (serum-free), with 2.5 × 104 cells seeded into the upper chamber. Then, 10% FBS-supplemented complete medium was added into the lower chambers. After 24 h of incubation, the inserts were fixed in methanol for 25 mins, stained using crystal violet, and non-migratory and non-invasive cells were eliminated with cotton-tipped swabs. Cell numbers were counted for different fields using a microscope, with the mean number of cells per field determined. Analyses were conducted independently for 3 times.
Briefly, transfected cells (2.5–3.5 × 105) were seeded in 6-well plates. Careful scratching of the monolayer was done using sterile yellow tips followed by washing using phosphate-buffered saline (PBS). A light microscope was used to observe and measure the scratch width. All assays were independently repeated 3 times.
Cell cycle and apoptosis analyses
Cell cycle and apoptosis were evaluated by flow cytometry. RCC cells (A498 and HH244) were transfected with pcDNA3.1( +)-PLCD1-V5/His plasmid or control vector, and washed with PBS before fixed with 70% ice-cold ethanol. For apoptosis, cells were stained with propidium iodide (PI) and Annexin V-FITC (BD Biosciences, Bedford, MA). Cells were stained with PI (Sigma) for cell cycle assessment. Both were sorted by BD Accuri C6 (BD Biosciences, Bedford, MA) as instructed by the manufacturer. Cell cycle data were analyzed using Modfit 3.0, while apoptosis assay finding was evaluated using BD Accuri C6 Software. Assays were done in triplicates.
Dual-luciferase reporter assay
This assay was conducted to evaluate transcriptional activity. To identify PLCD1-modulated signaling pathways, several luciferase reporters for signaling pathways were assessed in HEK293T, A498 and HH244 cells, following exogenous expression of PLCD1. CCND1p, p53-bs, MMP7p, C-mycp, PAI-1p, NF-κB-bs, AP1-bs, GRR5, SRE (Stratagene), as well as TOPflash plasmids (gifts from Prof. Christof Niehrs, DKFZ, Heidelberg), were individually co-transfected into cells along with Renilla and pcDNA3.1( +)-PLCD1-V5/His or pcDNA3.1( +). Cells were harvested after 48 h, and luciferase assay was performed using the Dual-Luciferase® reporter assay system (Promega). At least 3 independent experiments with triplicate wells were done.
Western blot assay
Cell lysis was done in ice-cold RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Then, proteins were resolved by SDS-PAGE and electroblotted onto Hybond-P membranes (Amersham). Membranes were blocked using Blotting-Grade Blocker (Bio-Rad, cat# 1,706,404) followed by incubation with primary antibody overnight (4℃), and then secondary antibody for 1 h at room temperature. Signal was then developed by ECL™ Detection Reagents (Amersham Biosciences) and quantitated with ImageJ. Antibodies used are: anti-mouse IgG-HRP (DAKO, P0161), anti-rabbit IgG-HRP (DAKO, P0448), GAPDH (Millipore, MAB374), Anti-V5-Tag (Invitrogen, R96025), E-cadherin (CST, 4065), vimentin (Sigma: V6630), Twist (Santa Cruz, sc-15393), MMP7 (Thermo Fisher, MS-813-P0), Total EGFR (CST: 54,359), p-EGFR (CST: 3777), Total FAK (CST: 71,433), p-FAK (Tyr397) (CST: 8556), Total SRC (CST: 2191), p-SRC (CST: 59,548), Total β-catenin (CST: 59,548), active β-catenin (Millipore, 05,665), p-β-catenin (Ser552) (CST: 5651), c-Myc (CST: 18,583), cyclinD1 (CST: 55,506), p-ERK1/2 (CST: 9101), Total ERK1/2 (CST: 4695), Cleaved-Caspase3 (CST: 9661), Cleaved-PARP (CST: 9541), Caspase 3 (CST:9504), PARP (CST: 9532), Bax (CST:2772), Bcl-2 (CST: 2872).
Cells were plated on cover slips in 6-well plates (80,000 cells/well). After transfection for 48 h, cells were fixed in paraformaldehyde (PFA) (4%), permeabilized using Triton X-100 (0.1%), followed by blocking with normal goat serum (DAKO, X0907). Cells on cover slips were incubated with primary and secondary antibodies (Anti-rabbit IgG-Alexa Fluor 488-F(ab')2 antibody: Thermo Fisher, A-11070) successively, and nucleus was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). For phalloidin staining, cells were incubated with rhodamine phalloidin for 1 h at room temperature after permeabilization. Cover slips were mounted with DABCO and then observed by immunofluorescence microscopy.
Data analyses were performed using SPSS version 22.0. Fisher exact test, two-tailed t test or chi-square test were used to evaluate p values. p < 0.05 denoted significance.
PLCD1 is silenced by promoter CpG methylation in RCC cell lines
We first evaluated the expression profile of PLCD1 using RT-PCR and bioinformatics analysis. RT-PCR analysis conducted in a panel of normal adult tissues showed that PLCD1 was widely expressed in multiple normal adult tissues (Fig. 1a). Likewise, Protein Atlas analysis showed that PLCD1 protein was expressed among an array of human normal tissues, including kidney (Additional file 1: Fig. S1a) . We then detected PLCD1 mRNA expression in RCC cell lines and found that PLCD1 was robustly expressed in HEK293 and RHEK-1, which were immortalized normal kidney cell lines, but silenced or downregulated in 6/9 RCC cell lines (Fig. 1b). According to the criteria in the Database of CpG islands and Analytical Tool (DBCAT): minimal island size > 200 bp, CG content > 50% and Obs/Exp > 0.6, a CpG island (CGI) was identified at the PLCD1 promoter (Fig. 1c). Thus, the role of promoter CpG methylation in PLCD1 silencing raised our interest. We performed MSP with methylation-specific and unmethylation-specific primers and found that PLCD1 was methylated in 7/9 RCC cell lines, correlated with its mRNA suppression (Fig. 1b). To verify the MSP findings, we carried out BGS to evaluate the methylation status of 37 CpG sites within the PLCD1 promoter. There were densely methylated alleles in PLCD1-silenced cells, such as A498 (99% CpG sites methylated), Caki-2 (99% CpG sites methylated) and RCC98 (90% CpG sites methylated). Consistently, more unmethylated alleles were detected in RHEK-1 and ACHN cells (Fig. 1d). RCC cell lines with PLCD1 methylation/downregulation were further treated with DNA methyltransferase inhibitor, 5-Aza, either alone or in combination with a histone deacetylase (HDAC) inhibitor, trichostatin A. Cell viability of 80–90% was observed after exposure to this concentration (10 µM) of 5-Aza for 3 days. Results showed that PLCD1 mRNA level could be restored by demethylation treatment, accompanied by promoter allele demethylation (Fig. 1e).
CpG methylation predominantly accounts for PLCD1 downregulation, and correlated with survival in patients
Next, we investigated the relationship between expression and methylation of PLCD1 in patient tissues. By analyzing theTIMER2.0 database, we found differential expression of PLCD1 between tumor and adjacent tissues across TCGA tumors. Relative to adjacent normal tissues, PLCD1 exhibited significant lower expression in many types of cancers, including RCC (Fig. 2a, Additional file 1: S1b). This distinctive expression model could be observed in different subtypes of RCC, both KIRC and KIRP (Fig. 2b). Additionally, compared with adjacent normal tissues, PLCD1 displayed a reduced protein expression in primary KIRC analyzed by UALCAN database (Fig. 2c). With the consistent decrease of PLCD1 expression in RCC, we proceeded to examine the contribution of its genetic alteration. Data from TIMER2.0 database showed that PLCD1 manifested rare mutations across a spectrum of TCGA cancers, with only 0.54% (2/370) in KIRC patients (Additional file 1: Fig. S1c). Apart from TCGA patients, we analyzed PLCD1 alteration rate in patients involved in other reported studies in the cBioportal database. Similar low mutation rates of PLCD1 was found, with 0.4% (1/249) in KIRC and 0.5% (1/207) in non-KIRC renal carcinoma (Fig. 2d). Therefore, methylation appears to be the main driver of PLCD1 disruption in cancer patients. This hypothesis is also supported by information from EWAS Data Hub, which revealed that PLCD1 methylation level in tumors was significantly higher than that in adjacent normal tissues (Fig. 2e). By further analyzing 3611 patient samples in 17 kidney related studies from the cBioportal database, we found an inverse correlation between PLCD1 methylation and its mRNA expression in 124 cases (Fig. 2f). Moreover, PLCD1 downregulation is of clinical significance, substantially associated with tumor stage and distant metastasis of KIRC and KIRP patients in TCGA database (Tables 1, 2). Moreover, we found that RCC patients with higher PLCD1 expression, including KIRC and KIRP, had a better prognosis in the TIMER2.0 database (Fig. 2g). Furthermore, PLCD1 methylation was linked with worse overall survival of RCC patients (Fig. 2h). Taken together, our findings suggest that PLCD1 methylation was the main cause of its silencing and could be a prognostic biomarker.
PLCD1 restoration inhibits RCC cell proliferation, migration and invasion
We moved on to examine the biological roles of PLCD1 in RCC cells. Ectopic PLCD1 expression was confirmed by Western Blot (Fig. 3a). Analysis of RCC cells viability using CCK-8 and colony formation assays revealed that ectopic PLCD1 expression dramatically reduced the proliferation rate of RCC cells. The numbers and size of colonies formed in PLCD1-expressing cells were reduced, relative to the control group (Fig. 3b–c).
To further assess the functions of PLCD1, we performed wound-healing, transwell migration and invasion assays. PLCD1 was found to have an inhibitory effect on both migration and invasion ability of RCC cells. RCC cells transfected with PLCD1 migrated and invaded at a slower rate than those transfected with empty vector (Fig. 3d–e). The efficiency of closing the open wound was greatly suppressed in PLCD1-expressing cells, compared to vector group (Fig. 3f, g).
PLCD1 re-expression attenuates EMT in A498 and HH244 cells
Epithelial–mesenchymal transition (EMT) is critical in cancer metastasis, a process in which epithelial cells obtain mesenchymal characteristics like cell motility and migration . Based on the influence of PLCD1 expression on distant metastasis of RCC patients, we examined the effect of PLCD1 expression on EMT. We conducted immunofluorescence assay to check the cellular location and expression of E-cadherin and vimentin. Results showed that compared to control group, PLCD1-transfected cells had higher E-cadherin and lower vimentin fluorescence (Fig. 4a–b). Consistent with this, we discovered that PLCD1 expression was positively associated with CDH1 expression in KIRC patients in TCGA database (Fig. 4c). Western blot assays showed that PLCD1 ectopic expression upregulated the epithelial marker E-cadherin and suppressed the expression of mesenchymal markers (vimentin, Twist, MMP7) in A498 and HH244 cells, indicating that PLCD1 inhibited EMT (Fig. 4d). In accordance with this, after transfection with PLCD1-expressing plasmid, A498 cell morphology changed from a long and spindly to a fried-egg morphology (Fig. 4e). Confocal microscopy showed that PLCD1 is mainly localized in microtubules in U-2 OS cells, which is an important part of cytoskeleton and provides shape maintenance (Fig. 4f). The subcellular localization of PLCD1 could partly explain its functions in cytoskeleton remodeling and epithelial morphology regulation. This was further supported by fluorescence staining with rhodamine phalloidin in A498 cells, in which actin filaments distributed along the elongated and well-spread cells in vector group, while clustered in PLCD1-expressing cells. These results demonstrate that PLCD1 suppresses the EMT program and further inhibits the migration and invasion ability of RCC cells.
Ectopic PLCD1 induces G2/M cell cycle arrest and RCC cell apoptosis
Next, we used flow cytometry to analyze cell cycle and apoptosis status of RCC cells. We found that PLCD1-expressing cells exhibited significant increase in cells at G2/M phases post-transfection (A498: control: 8.01%, PLCD1: 15.20%, p = 0.0072; HH244: control: 10.34%; PLCD1: 15.69%, p = 0.0030), relative to control cells, but with no statistic difference in S phase cells (A498: control: 42.07%, PLCD1: 45.66%, p = 0.9316; HH244: control: 42.75%; PLCD1: 29.44%, p = 0.0605) and G0/G1 phase (A498: control: 49.64%, PLCD1: 41.08%, p = 0.1211; HH244: control: 46.91%; PLCD1: 54.86%, p = 0.1943) (Fig. 5a). Apoptosis analysis revealed that in contrast to vector control, PLCD1-transfected cells had a higher proportion of early apoptotic cells (A498: 11.6 ± 1.78% vs 16.4 ± 1.93%, p < 0.05, HH244: 1.73 ± 0.42% vs 3.5 ± 0.36%, p < 0.05) (Fig. 5b). Western blot of apoptosis biomarkers revealed that relative to controls, A498 and HH244 cells transfected with PLCD1 plasmids had significantly higher ratio of cleaved-Caspase 3/caspase 3 and cleaved-PARP/PARP levels (Fig. 5c). Consistently, pro-apoptotic Bax level was elevated and anti-apoptotic protein Bcl-2 was decreased, accompanied by a significantly upregulated Bax/Bcl-2 ratio in PLCD1-expressed cells (Fig. 5c). These findings showed that PLCD1 triggered RCC cell cycle arrest in G2/M phase and induced apoptosis.
PLCD1 antagonizes WNT/β-catenin signaling in RCC cells
To investigate the molecular mechanisms underlying the role of PLCD1 in RCC development, we employed dual-luciferase reporter assays to determine its influence on various oncogenic pathways. Western blot was used to evaluate PLCD1 expression in HEK239T cells (Fig. 6a). Ectopic PLCD1 expression significantly suppressed transcriptional activities for AP1-bs, NF-κB-bs, TOPflash, GRR5 and SRE elements in 293 T cells (Fig. 6b). TOPflash (TCF reporter plasmid), a reporter containing TCF binding sites, was used to detect canonical Wnt/β-catenin signaling . Inspired by decreased TOPflash activity, we then mainly focused on Wnt/β-catenin signaling. MMP7, c-Myc as well as CCND1 are the downstream target genes of Wnt/β-catenin signaling. Next, we tested the reporter activities of MMP7p, c-Mycp and CCND1p after transfection with PLCD1-plasmid. Data showed that PLCD1 markedly suppressed the reporter activities as well as expressions of c-Myc, Cyclin D1 and MMP7 in RCC cells (Fig. 6c–d). Furthermore, active β-catenin and phospho-β-catenin (ser552) levels were decreased (Fig. 6d). Together, these findings suggest that PLCD1 inhibited the activation of β-catenin and antagonized WNT/β-catenin signaling.
PLCD1 suppresses EGFR-FAK-ERK signaling in RCC cells
Epidermal growth factor receptor (EGFR), a member of ErbB family of receptor tyrosine kinase (RTK), is closely involved in human carcinogenesis . Additionally, focal adhesion kinase (FAK) and steroid receptor coactivator (Src) often function as complex to mediate signaling required for tumor initiation and progression [25, 26]. Extracellular signal regulated kinase (ERK) cascade is a well-known downstream of FAK/Src-activated phosphorylation events, following the stimulation of vascular endothelial growth factor (VEGF), which leads to tumor angiogenesis . In dual-luciferase reporter assays, we noted that the activity of SRE reporter was blocked after PLCD1 restoration, suggesting that PLCD1 inhibits ERK signaling (Fig. 6b). By Western blot analysis, we found that relative to vector control, transfection with PLCD1 plasmid reduced the levels of p-EGFR, p-Src, p-FAK (Tyr397) and p-ERK, but did not significantly change the level of their total proteins (Fig. 6e), demonstrating that PLCD1 negatively regulates EGFR-FAK-ERK signaling in RCC cells.
RCC carcinogenesis is a complex and multi-step process, involving aberrant alterations of multiple cancer genes and cell signaling pathways which confers a serials of cell physiologic responses with regard to proliferation, migration, invasion and apoptosis. In the present study, we demonstrate that PLCD1 exhibited low protein and mRNA levels in RCC. PLCD1 downregulation was validated in RCC cell lines and tumors, correlated with promoter CpG methylation. We also validated the tumor suppressive roles of PLCD1 with multiple biological assays, which is achieved by suppressing Wnt/β-catenin and EGFR-FAK-ERK signaling in RCC cells.
We detected PLCD1 expression profile and found that it was downregulated or silenced in 6/9 of RCC cell lines. After demethylation treatment, PLCD1 downregulated cell lines demonstrated considerable increase in PLCD1 transcription, which confirms dominant role of promoter CpG methylation in PLCD1 silencing. On the other hand, only rare mutations of PLCD1 were observed in RCC tumor tissues, indicating that PLCD1 silencing is the predominant type of PLCD1 inactivation in RCC.
Our results demonstrate that PLCD1 functions as a tumor suppressor in RCC cells, consistent with previous reports in other types of carcinomas. Previously, our laboratory identified the tumor inhibitory function of PLCD1 in breast cancer by inducing cell cycle G2/M arrest . In addition, PLCD1 restoration was found to impede the proliferation, migration and invasion abilities of ESCC cells through modulating the Wnt/β-catenin pathway . WNT/β-catenin signaling regulates the expression of numerous target genes, including Axin-2, c-Myc, TCF-1, CCND1, MMP7 and CD44 , thereby regulating stem cells pluripotency and controlling cell destiny during development and diseases. During tumorigenesis, WNT signaling is frequently inappropriately activated to facilitate malignant behavior, such as proliferation, migration and invasion . Approximately half of human malignancies have been linked to aberrant activation of WNT/β-catenin signaling . PLCD1 has been shown to function as a TSG by inhibiting WNT/β-catenin in ESCC, BrCa and CRC [14, 30, 31], consistent with our findings that PLCD1 inhibits active-β-catenin and downstream target gene transcription, including MMP7, c-Myc and CCND1, hence suppressing proliferation, migration and invasion of RCC cells.
EGFR is a critical regulator of a variety of cell biological processes, such as proliferation, division, differentiation and survival . EGFR signaling is also explored for drug development and clinical management . Erlotinib, an EGFR inhibitor, exhibits promising effect in advance RCC patients in undergoing clinical trials . PLC family has been reported to display a negative correlation with EGFR expression in breast, pancreatic and ovarian cancers [35,36,37,38]. Herein, we found that ectopic PLCD1 expression negatively regulated EGFR activation in RCC cells. Thus, we speculated the combination of epigenetic inhibitors and EGFR antagonists might achieve better therapeutic effects in RCC treatment. More clinical data should be collected to validate this hypothesis.
FAK/Src complex acts as intracellular nexus, transducing signals from integrin or tyrosine kinase receptors (TKRs), involving in different steps of neoplastic initiation and development [23, 38]. When activated by upstream stimulation, phosphorylation occurs at the tyrosine residue 397 (Y397) of FAK [39,40]. Then, Src is recruited to phospho-FAK and get phosphorylated and activate each other exponentially. ERK is one of the downstream effectors of FAK/Src signaling, which modulates EMT-related metastasis and VEGF-associated angiogenesis [41,42]. Currently, VEGFR inhibitors, such as bevacizumab, have become an important targeted therapy for malignant diseases . In general, our results demonstrate that PLCD1 expression inhibited EGFR phosphorylation and both activated form of FAK/Src and ERK. The intervention of PLCD1 in EGFR-FAK-ERK signaling indicates its potential value as a treatment target for RCC patients in clinical practice.
In this study, we identified PLCD1 as a functional TSG downregulated by promoter CpG methylation in RCC, and it exerts tumor suppressor effects by blocking WNT/β-catenin and EGFR-FAK-ERK signaling. These findings establish PLCD1 as a prognostic biomarker and treatment target for RCC.
Availability of data and materials
All datasets generated for this study are included in the article and supplementary materials.
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This work was supported by China National Key Research and Development Program of China MOST [#2017YFE0191700]; the General Project of Hunan Natural Science Foundation [2019JJ40254]; and the Key projects of Hunan Provincial Department of Education [18A229].
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Additional file 1
: Overview of PLCD1 bioinformatics analysis. a Protein expression of PLCD1 in 45 kinds of tissues through Human Protein Atlas. b Differential expression of PLCD1 between tumor and adjacent tissues across TCGA tumors in TIMER2.0 database. c PLCD1 mutation rates across TCGA tumors analyzed by TIMER 2.0 database.
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Xie, J., Zhou, J., Xia, J. et al. Phospholipase C delta 1 inhibits WNT/β‐catenin and EGFR-FAK-ERK signaling and is disrupted by promoter CpG methylation in renal cell carcinoma. Clin Epigenet 15, 30 (2023). https://doi.org/10.1186/s13148-023-01448-2
- CpG methylation