Discovering the Role of FZD4 Gene in Human Cutaneous Squamous Cell Carcinoma.

BACKGROUND: Frizzled 4 (FZD4) is an important receptor for Wnt proteins that stimulate several downstream signaling pathways. It has been known that the FZD4-Wnt interaction is involved in many types of cancers. However, the role of FZD4 in cutaneous squamous cell carcinoma (CSCC) has not been well studied. AIMS: We sought to investigate the association between FZD4 expression level and tumor cell proliferation and apoptosis rates in CSCC.
METHODS: Expression of FZD4 at mRNA level in CSCC tissues and controls was measured. Colo16 cell proliferation and viability were measured by CCK-8 assay and flow cytometry respectively after siRNA and plasmid transfection.
RESULTS: We discovered a significant downregulation of FZD4 expression in CSCC tissues and cell lines compared to controls. Furthermore, our data suggested that over expression of FZD4 inhibited proliferation and promoted apoptosis of Colo16 cells.
CONCLUSION: The results indicated that FZD4 may play as a tumor suppressor gene in the pathogenesis of CSCC. Copyright:

Introduction

Cutaneous squamous cell carcinoma (CSCC) is a keratinocytes associated malignant skin tumor with an increasing prevalence around the world, especially in elderly.[1] Most primary CSCC cases are considered low risk and generally have a good prognosis. However, the high recurrence and metastasize rates in some patients often lead to unfavorable results.[2] Therefore, it is important to decipher the pathogenesis of CSCC so better treatment strategies can be developed.

Mutations in WNT/β-catenin signaling components are generally rare in CSCC, but aberrant regulation of the WNT signaling network has been associated with the development of CSCC.[3] Down-regulation of secreted frizzled-related proteins (SFRPs) and increasing WNT5A, FZD6, nuclearβ-catenin expression have been identified in CSCC.[4] Some crucial components within Wnt pathway have been proposed to be novel targets for cancer therapy.[5] FZD4 is a transmembrane receptor that belongs to the frizzled gene family that receives Wnt signals and plays important roles in cell growth and differentiation.

Increasing studies have demonstrated that the alteration of FZD4 expression affects tumor cell proliferation and invasion in various tumors such as cervical carcinoma,[6] prostate cancer,[7] lung cancer,[8] hepatocellular carcinoma,[9] glioblastoma multiforme,[10] acute myeloid Leukemia.[11] However, the effects of FZD4 on CSCC have not been well examined.

In the present study, we investigated the association between FZD4 expression level and tumor cell proliferation and apoptosis rates in CSCC. Elucidating the specific molecular and functional mechanism that underlies the development of CSCC may help us to better understand the role of the Wnt signaling pathway in the pathogenesis of CSCC.

Materials and Methods

Samples and reagents

A total of 30 CSCC biopsy samples and 30 control skin tissues were obtained from the dermatological department of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Jiangsu Provincial Key Laboratory of Molecular Biology for Skin Diseases and STIs.

FZD4 siRNAs were purchased from Thermofisher Scientific, Shanghai, China; Plasmids were purchased from Shanghai Generay Biotech Co, Ltd Shanghai, China.

The primers used in this study were as follows:

FZD4 F: 5'-CCTCGGCTACAACGTGACC-3',

R: 5'-TGCACATTGGCACATAAACAGA -3';

β-actin F: 5'-CCATCGTCCACCGCAAAT-3',

R: 5'-GCTGTCACCTTCACCGTTCC-3'.

The primers are all synthesized by Sangon Biotech, Shanghai, China.

Cell lines

Human CSCC cell line Colo16 was purchased from Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured in DMEM with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO2.

Downregulate and over-expression of FZD4

To decrease the expression of FZD4 (Accession No. NM_203401 form NCBI GenBank), Control siRNA and FZD4 siRNA were respectively transfected to the cell at 80% to 90% confluence according to Lipofectamine RNAiMAX transfection reagent manual. The sequence alignment (BLAST) was carried out to ensure no homology with others genes in human.

To upregulate the expression of FZD4, control plasmid and FZD4 plasmid encodes the open reading frame (ORF) were respectively transfected to the cell at 80% to 90% confluence according to Lipofectamine RNAiMAX transfection reagent instruction.

Real-time quantity PCR (RT-qPCR)

Total RNA was extracted using TrIzol reagent (Life Technologies, USA) according to the manufacturer's instruction and reverse transcribed into cDNA samples with Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany). 480 SYBR Green I Master (Roche, Germany) was used for RT-qPCR. The mRNA of FZD4 was detected by forward primer, 5'-CCTCGGCTACAACGTGACC-3', reverse primer: 5'-TGCACATTGGCACATAAACAGA-3'. The procedure was as follows: reverse transcription at 50°C for 2 min; pre-denaturation at 95°C for 5 min; follow by 40 cycles of at 95°C for 20 s, 60°C for 30 s, and 5°C for 15 s. The experiment was triplicated and the relative gene expression was calculated according to the formula of 2-ΔΔCt.[12]

Western blotting

Colo16 cells (untreated and transfected for 48 h) were collected and total protein was extracted using the RIPA lysis buffer (Beyotime, Jiangsu, China). The protein was then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to polyvinylidine difluoride filter (PVDF) membranes. The membranes were incubated with mouse-anti-human FZD4 (1:1000 dilutions, Abcam, USA) and mouse-anti-human β-actin (1:1000 dilution, Santa Cruz, USA) as internal control. After rinsing with TBST, the membrane was incubated with secondary antibody conjugated to horseradish peroxidase (HRP) (donkey anti-goat IgG-HRP with 1:5,000 dilution for FZD4; goat anti-mouse IgG-HRP with 1:5,000 dilution for β-actin) for 1.5 h at room temperature followed by rinsing in TBST for 5 min. The relative protein expression quantity was analyzed by Gene Tools.

Cell proliferation analysis

Colo16 Cells were cultured for 24, 48, 72 h after transfection before harvest and the cell growth rate was measured by CCK-8 assay. Cells were cultured in the equivalent fresh medium containing 10% CCK-8 at 37°C for 4 h before the optical density (OD) was measured at 450 nm using a Fluorescence Spectrophotometer (HITACHI, Japan).

Cell apoptosis assay

Apoptosis of the Colo16 cells treated by siRNAs and plasmid was determined by Annexin V-FITC/PI double-positive staining on flow cytometry (FCM) analysis. Briefly, 1 × 106 cells were cultured for 48 h after different treatments before harvest. The cells were collected and washed with PBS before resuspended in Binding Buffer and stained with Annexin V-FITC antiboty and PI for 15 min at room temperature. Cells were analyzed by FCM analysis using BD CELLQuest software (BD Biosciences, USA).

Results

FZD4 expression in CSCC tissues and cell lines

To investigate the role of FZD4 in CSCC, we measured the expression of FZD4 at mRNA level in CSCC lesions and perilesion tissues. We found that the mRNA expression of FZD4 was lower in CSCC lesions compared to perilesions [Figure 1].

Figure 1

Expression of FZD4 in Colo16 cells was lower in CSCC lesions compared to perilesions

Inhibition of FZD4 by siRNA

Next, we down regulated the expression of FZD4 in Colo16 cells by transfecting the cells with pre-constructed siRNAs. The efficacy was determined by RT-qPCR after treatments with siRNA for 72 h. As shown in Figure 2, the mRNA [Figure 2a] and protein [Figure 2b] levels of FZD4 were both significantly inhibited by FZD4_siRs, among which FZD4_ siR2 being the most effectively [Figure 3].

Figure 2

Expression of FZD4 in Colo16 cells was effectively down-regulated by specific FZD4 siRNAs. (a) FZD4 knockdown efficiency at mRNA level was detected by RT-qPCR, (b) FZD4 knockdown efficiency at protein level was detected by Western blot. (P < 0.05) compared with NC_siR* and the blank group. *NC: Negative control, siR: siRNA

Figure 3

Expression of FZD4 in Colo16 cells was effectively down-regulated by specific FZD4_ siR2

Effect of FZD4 overexpression by plasmid transfection

To upregulate FZD4 in the Colo16 cells, Colo16 cells were transfected with pre-designed plasmid. As shown in Figure 4, the mRNA [Figure 4a] and protein [Figure 4b] level of FZD4 were both upregulated significantly by FZD4 plasmid.

Figure 4

Expression of FZD4 in Colo16 cells was effectively up-regulated by specific FZD4 plasmid. (a) FZD4 mRNA expression levels in Colo16 cells transfected with FZD4 plasmid. (b) FZD4 protein expression levels in Colo16 cells transfected with FZD4 plasmid. (P < 0.05)

Upregulation of FZD4 inhibits Colo16 cell proliferation

The CCK-8 cell proliferation experiment result showed no significant difference of cell proliferation between the untreated group and the siRNA group (P > 0.05). However, the CCK-8 assay suggested that the proliferation of Colo16 cells was inhibited by FZD4 plasmid, which indicates that the promotion of FZD4 expression can inhibit Colo16 cell proliferation [Figure 5].

Figure 5

The absorbance (OD) values of the Colo16 cells at 0,24, 48 and 72 h post-transfected with FZD4 plasmid and FZD4 siR

Upregulation of FZD4 induces Colo16 cell apoptosis

Next, cell viability was measured by Annexin V and PI double staining by flow cytometry after siRNA transfection. As shown in Figure 6, treatment with plasmid resulted significant increase of apoptosis compared to untreated cells and cells transfected with FZD4 siRNA (P < 0.05). The results showed that FZD4 might play an antioncogenic role in CSCC.

Figure 6

Colo16 cell apoptosis was inhibited by FZD4_plasmid (C) detected by FCM analysis. (a. Blank, b. FZD4-plasmid control, c. FZD4-plasmid, d. FZD4-Sir control, e. FZD4-Sir)

Discussion

CSCC is characterized by local proliferation of moderate or poorly differentiated keratinocytes.[13] Surgical resection of locally advanced or metastatic disease is associated with functional morbidity and disfigurement. Despite a variety of techniques have been employed when removing NMSC such as Mohs micrographic surgery, tangential shave removal, curettage, and electrodessication, there is still an urgent need for a novel therapeutic target for suppressing tumor invasion and recurrence.[14]

FZD4 is a receptor for Wnt proteins and coupled to the β-catenin canonical signaling pathway and non-canonical pathways. The FZD4 can be activated by Wnt-3a, Wnt-5a, Wnt-7a, Wnt-10b,[15] Wnt-5a[16] and Norrin. Wnt signaling pathways such as Wnt-beta-catenin, Wnt-PCP and Wnt-Ca2+ have been reported to be associated with cancer progression and poor prognosis.[17] Mutations in FZD4 pathway components have been correlated in many types of disease including retinal vascular disorders[18] and brain diseases.[19] Furthermore, Erin A Bassett found that down-regulation of Norrin/Fzd4 axis created a tumor-permissive stroma that promoted the formation of preneoplastic lesions facilitated their progression. The interaction between FZD4 and Norrin was found to mediate tumor-suppressive effects and regulate cancer stem cell biology.[20] A recent study supports that the selectivity of FZD4 ligand for Norrin is enhanced by TSPAN12, a tetraspanin that combines with the FZD4-Norrin complex via its extracellular loops,[21] which provides a potential therapeutic target for malignant diseases.

Wnt signaling plays a pivotal role in the genesis and maintenance of cancer stem cells in many types of malignancies. However, in this study, we showed for the first time that FZD4 is down-regulated in CSCC specimens. Our data suggested that upregulation of FZD4 significantly promoted Colo-16 cell apoptosis and inhibit its proliferation. However, the detailed mechanisms underlying the mechanism of FZD4 mediated cell proliferation and apoptosis remain unknown. CSCC has lower remote metastatic potential compared to other malignant cancers with increased FZD4 expression.[22] Therefore, our data indicated that the role of FZD4 in tumor pathogenesis is more complicated than we thought. Nevertheless, whether a similar network signaling, responsible for the regulatory effect of FZD4 on Wnt signaling pathway needs to be evidenced by further studies.

In summary, our results indicated that FZD4 may be involved in cancer cell proliferation and apoptosis in CSCC. Our study provided new insights for FZD4. Further investigation should be done to better understand the role of FZD4 in pathogenesis of CSCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.