Clinical significance and biological role of KLF17 as a tumour suppressor in colorectal cancer

  • Authors:
    • Xun Jiang
    • Tong‑Yi Shen
    • Helei Lu
    • Chenzhang Shi
    • Zhongchen Liu
    • Huanlong Qin
    • Feng Wang
  • View Affiliations

  • Published online on: September 18, 2019     https://doi.org/10.3892/or.2019.7324
  • Pages: 2117-2129
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

It has been reported that kruppel‑like factor 17 (KLF17) acts as a tumour suppressor in several tissues and cancer cells, however, the molecular roles, the underlying mechanisms and clinical significance of KLF17 in colorectal cancer (CRC) have not been completely elucidated. In the present study, KLF17 protein expression was detected in 140 primary CRCs and paired adjacent non‑tumour tissues using immunohistochemistry with tissue microarrays. The KLF17 mRNA expression was determined in 4 CRC cell lines and 20 pairs of the aforementioned tissues using reverse transcription quantitative polymerase chain reaction. The correlation between KLF17 expression and clinicopathologic characteristics was determined. Next, the functions of KLF17 in CRC were examined by cell proliferation, colony formation, adhesion, invasion and mouse xenograft assays. Methylation‑specific PCR and bisulfite sequencing PCR were also carried out to investigate the promoter methylation status of KLF17 in CRC cells and tissues and explore the effects of lentiviral‑mediated RNAi of UHRF1 on the methylation and expression of KLF17. The results revealed that KLF17 expression was abnormally decreased in CRC and associated with lymph node metastasis and unfavorable overall survival. Moreover, ectopic KLF17 expression suppressed CRC cell growth and invasion in vitro and in vivo. In addition, the downregulation of KLF17 was associated with the hypermethylation of the CpG nucleotides on the KLF17 promoter. The knockdown of the epigenetic regulator UHRF1 reduced the methylation level of the KLF17 promoter and inhibited CRC cell adhesion, invasion and epithelial‑mesenchymal transition by upregulating KLF17. The present findings indicated that KLF17 may act as a tumour suppressor gene in CRC and a potential independent prognostic biomarker in CRC patients. UHRF1 can suppress KLF17 expression through the hypermethylation of its promoter in CRC. These results offer insights into the KLF17 expression regulation in CRC and suggest an inhibitory effect of KLF17 on tumourigenesis.

Introduction

As one of the most prevalent types of cancer, colorectal cancer (CRC) is a considerable contributor to cancer mortality worldwide (1). In spite of recent improvements in surgical techniques, dosing and scheduling of adjuvant therapy, the overall prognosis of CRC patients varies substantially depending on tumour progression. Accordingly, elucidating the molecular mechanisms underlying tumour development, as well as identifying novel related factors, are crucial for the development of new diagnostic and therapeutic methods against CRC.

Kruppel-like factor 17 (KLF17) belongs to the KLF family of transcription factors, which consists of 17 members. The KLF family members play important roles in multifarious cellular processes, including tumour development (2). Recent studies have investigated the downregulation of KLF17 in multiple human malignancies, including breast, liver, stomach and esophageal cancer, suggesting the potential of KLF17 as a tumour suppressor in cancerous cells (36). Furthermore, KLF17 may be involved in the regulation of epithelial-mesenchymal transition (EMT), which has been identified as a critical process in the progression of cancer to the metastatic state (7). Moreover, KLF17 has been revealed to be regulated by epigenetic mechanisms such as DNA methylation, and identified as a possible prognostic biomarker for CRC (6,8). However, the clinical significance and regulatory mechanism of KLF17 remains unclear in CRC and needs to be further explored.

The aim of the present study was to explore the clinical significance of KLF17 expression in CRC and determine whether KLF17 inhibited CRC cell growth and metastasis, as well as the possible underlying molecular mechanism.

Materials and methods

Clinical samples and database

A total of 140 patients with CRC undergoing curative surgeries between 2014 and 2016 at the Tenth People's Hospital (Shanghai, China) were included in this study. No patients received preoperative chemotherapy or radiotherapy. The paired adjacent non-cancerous samples were obtained at a distance of at least 10 cm from the tumour lesion. Demographic and clinicopathological characteristics, including age, sex, tumour location, histologic differentiation and TNM stage, were collected based on the UICC criteria. Survival data were collected through follow-up visits and telephone interviews. This study was approved by The Ethical Committee of the Tenth People's Hospital.

Tissue microarray (TMA) construction and immunohistochemistry (IHC)

The TMA slides included 140 CRC and matched adjacent non-cancerous tissues. All tissues were formalin-fixed and paraffin embedded, and diverted 2 mm cores from representative areas into recipient block microarrays.

For IHC, the sections were mounted onto slides, dewaxed in xylene, and then treated for 30 min with methanol containing 1% hydrogen peroxide to block endogenous peroxidases. A primary rabbit anti-human KLF17 polyclonal antibody (dilution, 1:100; cat. no. ab84196; Abcam) was used, according to the manufacturer's instructions. For immunostaining, a peroxidase-conjugated goat anti-rabbit secondary antibody (OriGene Technologies, Inc.) was used, according the manufacturer's instructions. IHC staining was assessed by two independent investigators under light microscopy. KLF17 expression was quantified by evaluating the percentage of positive tumour cells (9). Positive expression was defined as ≥5% stained cells in a sample (10).

Cell culture

The human colorectal cancer cell lines LoVo, SW620, HT29 and Caco-2 cells were purchased from the American Type Culture Collection. The HT29 cell line was recently authenticated in Microread Gene Technology by performing a short tandem repeat (STR) profiling analysis. All cells were cultured in recommended medium supplemented with 10% fetal bovine serum. For the demethylation experiments, 2 µM 5-Aza-dC (Merck KGaA) was added to the cell medium and the exponentially growing cells were treated for 72 h.

Lentiviral infection and transient transfection

The recombinant lentiviral vector pLV.0-KLF17 overexpressing KLF17 (lenti-KLF17) and control recombinant lentivector carrying a scramble construct and green fluorescent protein (lenti-GFP) were obtained from GeneCopoeia, Inc. RNA interference recombinant lentiviral vector for UHRF1 (lenti-shF1) was described in our previous study (11). Virus packaging was performed in 293T cells, following standard procedures, and viral supernatant was used to infect LoVo and SW620 cells. KLF17 small interfering RNA (si-F17) and scramble oligonucleotide used as a negative control (si-NC) were purchased from Thermo Fisher Scientific, Inc. and transfected into the Lenti-shF1-infected LoVo cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.) to knockdown the expression of KLF17.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

The mRNA level was quantified by RT-qPCR using the Quantitect SYBR Green PCR kit (Qiagen AB). β-Actin was used as the internal control to normalize the results. Primers used in this experiment were as follows: KLF17 forward, 5′-GCTGCCCAGGATAACGAGAAC-3′ and reverse, 5′-ATCTCTGCGCTGTGAGGAAAG-3′; UHRF1 forward, 5′-CAAGATTGAGCGGCCGGGTGAAGG-3′ and reverse, 5′-TGAGGGGCGGGTCCAGGCAGTAGA-3′; E-cadherin forward, 5′-CGAGAGCTACACGTTCACGG-3′ and reverse, 5′-GGGTGTCGAGGGAAAAATAGG-3′; vimentin forward, 5′-AGTCCACTGAGTACCGGAGAC-3′ and reverse, 5′-CATTTCACGCATCTGGCGTTC-3′; β-actin forward, 5′-CCTGTACGCCAACACAGTGC-3′ and reverse, 5′-ATACTCCTGCTTGCTGATCC-3′. Amplification conditions were 94°C for 2 min followed by 40 cycles of 94°C for 45 sec, 60°C for 40 sec and 72°C for 40 sec. The 2−ΔΔCq method was used to calculate the difference in expression (12).

Western blotting

Total protein was extracted from cells and tissues with the Total Protein Extraction kit (EMD Millipore), according to the manufacturer's instructions. The protein content was determined using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad Laboratories, Inc.) with bovine serum albumin as the standard. Proteins (20 µg) were separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.05% Tween-20 at room temperature for 2 h, then probed with antibodies against KLF17 (cat. no. ab 84196; 1:1,000 dilution), UHRF1 (cat. no. ab 57083; 1:2,000 dilution), E-cadherin (cat. no. ab 40772; 1:1,000 dilution; all from Abcam), vimentin (cat. no. 5741; 1:500 dilution; Cell Signalling Technology, Inc.) and β-actin (cat. no. A2228, 1:5,000 dilution; Sigma-Aldrich; Merck KGaA) overnight at 4°C. After washing, membranes were incubated at 37°C for 1 h with the horseradish peroxidase-conjugated secondary antibody (cat. no. A0208, 1:1,000 dilution; Beyotime Institute of Biotechnology, Haimen, China). The blotted proteins were visualized with an enhanced chemiluminescence (ECLTM plus Western Blotting Detection Kit; GE Healthcare) and images were obtained by using the chemiluminescent image analyzer (LAS-4000; Fujifilm). The density of protein band was quantified using Quantity One v4.4 software (Bio-Rad Laboratories). Relevant protein expression levels were defined as the ratio of the density of the specific band for a target protein to that of the band for β-actin.

Cell proliferation assay

Cell proliferation rates were detected by MTT assay. Cells (5×103) were seeded in 96-well plates and cultured for 24, 48, 72 or 96 h. MTT (20 µl) was then added into the plates and the cells were treated for another 2 h at 37°C. Following the dissolution of the crystals using dimethyl sulphoxide (DMSO), the optical density was determined using a microplate reader (BioTek Instruments, Inc.).

Adhesion assay

CRC cells were placed on 24-well plates covered with fibronectin (BD Biosciences) and cultured for 24 h. The medium was then removed and the adherent cells were fixed and counted under a fluorescence microscope (Olympus Corp.). Five random visual fields were selected on each insert at a magnification of ×200.

Cell invasion assays

Next, 5×104 cells were seeded in a Transwell chamber (Corning, Inc.) and covered with Matrigel (BD Biosciences). The chambers were then filled with complete RPMI-1640 medium and incubated for 24 h. The cells on the top surface of the membranes were removed, whereas those invading from the upper to the lower surface were fixed and stained with 0.1% crystal violet for 1 h at room temperature. Five random visual fields were captured at a magnification of ×200.

Tumourigenicity and liver metastasis assay in nude mice

Six-week-old female athymic BALB/c nude mice (n=60; Chinese Academy of Sciences) were maintained under specific pathogen-free conditions, using a 10-h light/14-h dark cycle at a temperature of 25±1°C and relative humidity of 40–60% with free access to food and water.

For the tumourigenicity assay, 30 mice were randomly divided into three groups on average and subcutaneously injected in the right flank with 1.5×106 LoVo (blank), lenti-KLF17- or lenti-GFP-infected cells in 100 µl PBS. The length and width of the tumours was measured daily and the volume was calculated using the formula V=(LxW2)/2. Tumour volumes were observed for 28 days and the growth curves of the tumours for each group were plotted. The mice were then euthanized by inhalation of a lethal dose of isoflurane (concentration of 5%) in a closed chamber and the resected tissues were collected for RNA detection and IHC.

A nude mouse model with CRC cells described previously was used for the liver metastasis assay (13). First, a total of 30 nude mice were randomly divided into three groups on average. After the mice were anesthetised by inhalation of isoflurane, a small left abdominal incision was performed to expose the spleen. Next, 1×106 LoVo (blank), lenti-KLF17- or lenti-GFP-infected cells in 100 µl PBS were injected into the spleen of the mice from the three groups. Splenectomy was performed 10 min later, and the abdomen was closed. The mice were euthanized 6 weeks later, their livers were examined for metastasis and the resected tissues were collected for RNA detection and IHC.

All animal experiments were performed according to protocols approved by the Ethics Committee of Animal Experiments of the Tenth People's Hospital Affiliated to Tongji University.

DNA extraction and methylation analysis

The CGI analysis software (www.urogene.org) was used to find typical CGIs in the promoter region of KLF17. Two different methods were used to determine the DNA methylation status of the KLF17 CGI in CRC. For bisulfite sequencing PCR (BSP), the genomic DNA was bisulfite-converted using the EZ DNA methylation kit (Zymo Research, Corp.) and amplified using the following bisulfite PCR primers: 5′-TTTGTTGTTTAGGTTGGAGTGTAAT-3′ and 5′-AATCACTTAAAATCAAAAATTTAAAACC-3′. The PCR products were cloned into pMD-18T (Takara Bio, Inc.) and the sequencing was performed in 10 positive clones. QUMA analyser software was used to detect and analyse the sequencing data. For methylation-specific PCR (MSP), following treatment with the DNA methylation kit, the genomic DNA was amplified using the following primers: Methylated, 5′-TTTAGGTTGGAGTGTAATGGC-3′ and 5′-ATTAACCAAACGTAATAACGCGTA-3′; Unmethylated, 5′-GTTGTTTAGGTTGGAGTGTAATGGT-3′ and 5′-AATTAACCAAACATAATAACACATA-3′.

Statistical analysis

Data are presented as the mean ± SD of at least three independent experiments. Comparisons between the two groups were performed by Student's t-test, Dunnett's t-test, or the Mann-Whitney U test, where appropriate, and comparisons among multiple groups were performed by a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The relationship between the expression of KLF17 and clinicopathological features was calculated using χ2 and Fisher's exact tests. Kaplan-Meier curves and log-rank analysis evaluated the effects of KLF17 expression on overall survival. Multivariate cox regression survival analysis with the stepwise backwards (Wald) method was used to discover independent prognostic factors. P<0.05 was considered to indicate a statistically significant difference. SPSS version 13 (SPSS, Inc.) was used for statistical analysis.

Results

Downregulation of KLF17 in CRC tissue samples and highly metastatic cells

Positive KLF17 staining by IHC was observed in the nucleus of the CRC (Fig. 1A-a and e) and adjacent non-cancerous cells (Fig. 1A-b and -f). The IHC staining scores of 140 CRC and matched adjacent non-cancerous tissues are included in Table SI. Among the 140 paired tissues, the percentage of KLF17 positive cells in CRC (25.7%, 36/140) was statistically lower than that in the adjacent non-cancerous samples (72.9%, 102/140; P<0.001).

Next, RT-qPCR was performed to detect the KLF17 mRNA expression in 20 paired CRC and adjacent non-tumour tissues; the results revealed a significantly decreased KLF17 expression in CRC tissues, as compared to the adjacent normal tissues (Fig. 1B). In addition, the highly metastatic CRC LoVo and SW620 cells exhibited a lower KLF17 expression, as compared to the lowly metastatic Caco- 2 and HT29 cells using RT-qPCR detection (Fig. 1C).

KLF17 expression is inversely correlated with lymph node metastasis and adverse prognosis in CRC patients

The relationship between KLF17 expression and patient characteristics is presented in Table I. The low expression of KLF17 was correlated with lymph node metastasis (P=0.036; Table I) in CRC patients.

Table I.

Association of KLF17 expression with clinicopathological features in CRC patients.

Table I.

Association of KLF17 expression with clinicopathological features in CRC patients.

KLF17 expression

Clinicopathological featuresNo. of patients (%)Positive (n=36)Negative (n=104)P-value
Age (years)
  <6036729
  ≥6010429750.381
Sex
  Female561840
  Male8418640.244
Tumour location
  Right colon491435
  Left colon481038
  Rectum4312310.632
Differentiation
  Well-Moderate1042579
  Poor3611250.508
Tumor size
  <5821765
  ≥55819390.12
Depth of invasion
  T0-T21239
  T3-T412833950.953
Nodal status
  N0682345
  N1-N27213590.036
Liver metastasis
  Absent1323597
  Present8170.68
TMN stage
  0-II651848
  III–IV7518560.703

[i] KLF17, kruppel-like factor 17; CRC, colorectal cancer.

Kaplan-Meier analysis revealed that the 5-year overall survival rate was higher in patients with a high KLF17 expression, as compared to patients with a low KLF17 expression (75 vs. 56.7%; P=0.019; Fig. 1D). Moreover, univariate and multivariate analysis revealed that KLF17 expression is an independent prognostic biomarker for CRC (RR: 0.444, 95% CI: 0.216–0.912, P=0.027; Table II).

Table II.

Cox proportional hazard regression model analysis (n=140)

Table II.

Cox proportional hazard regression model analysis (n=140)

VariablesCategoriesRR (95% CI)Wald χ2P-value

Univariate analysis
Age (years)≥60 vs. <600.900 (0.496–1.633)0.1210.728
SexMale vs. female0.840 (0.491–1.438)0.4040.84
DifferentiationPoor vs. well-moderate1.273 (0.709–2.289)0.6530.419
Tumor size≥5 vs. <51.056 (0.614–1.814)0.0380.845
Depth of invasionT3-T4 vs. T0-T21.161 (0.419–3.217)0.0820.775
Nodal statusN1-N2 vs. N02.140 (1.212–3.779)6.8720.009
Metastasis to other organsPresent vs. absent5.191 (2.309–11.670)15.876<0.001
TNM stageIII–IV vs. 0-II2.257 (1.267–4.019)7.6390.006
KLF17 expressionPositive vs. negative0.432 (0.211–0.887)5.2230.022

Multivariate analysisa

KLF17 expressionPositive vs. negative0.444 (0.216–0.912)4.8890.027
Metastasis to other organsPresent vs. absent3.785 (1.634–8.771)9.640.002
TNM stageIII–IV vs. 0-II1.925 (1.058–3.505)4.5960.032

a Forward LR stepwise elimination procedure. KLF17, kruppel-like factor 17; LR, linear regression.

Ectopic KLF17 expression inhibits cell growth, invasion and EMT in CRC cells

Considering that lower KLF17 levels were associated with worse outcomes in CRC patients, the function of KLF17 in CRC cells was subsequently evaluated. In accordance with the KLF17 mRNA level in different CRC cell lines (Fig. 1C), stable LoVo and SW620 cell lines were established through lentiviral infection that ectopically expressed KLF17.

As revealed in Fig. 2A-C, the expression levels of KLF17 were significantly increased in LoVo and SW620 cells following lentivirus-mediated KLF17 infection, as compared to cells infected with lenti-GFP control.

The MTT assays then revealed that the upregulation of KLF17 inhibited cell proliferation in LoVo and SW620 cells (Fig. 2D and E). Similarly, the potential of colony formation was substantially reduced in the KLF17-upregulated CRC cells (Fig. 2F and G).

To detect the effects of KLF17 expression on the invasion and metastasis capacities of these cells, adhesion and Transwell assays were performed and the results revealed that increased KLF17 expression significantly reduced the ability of adhesion and invasion in LoVo and SW620 cells (Fig. 2H-K).

KLF17 has been demonstrated to be a negative regulator of metastasis and EMT. It has been revealed to band directly to the promoters of genes such as E-cadherin and vimentin, which are involved in EMT, and inhibit their expression. Therefore, the expression of certain EMT biomarkers was next investigated in stable LoVo and SW620 cells with KLF17 overexpression to evaluate the effects of KLF17 on EMT in CRC cells. As revealed in Fig. 3, ectopic KLF17 expression led to the upregulation of epithelial biomarker E-cadherin and the downregulation of mesenchymal biomarker vimentin, indicating that KLF17 represses EMT in CRC cells.

Ectopic KLF17 expression suppressed the tumour growth and metastasis of CRC in nude mice

In the following experiment, a nude tumour model was established to verify the in vitro findings as aforementioned. As revealed in Fig. 4A, the mouse group subcutaneously injected with the lenti-KLF17-infected LoVo cells exhibited a lower proliferation capacity, as compared to the control groups. The tumour volume in mice injected with lenti-KLF17-infected LoVo cells was considerably smaller than that in mice injected with LoVo cells or lenti-GFP-infected LoVo cells. At the end of the experiment, the tumour weight was significant lower in the group with lenti-KLF17-infected LoVo cells (0.118±0.021 g), as compared to the groups with LoVo (0.304±0.032 g) and lenti-GFP-infected LoVo (0.282±0.051 g) cells (Fig. 4B). Using antibodies against Ki-67, a standard biomarker for cellular proliferation, IHC was further performed, and the results revealed that lenti-KLF17-transfected LoVo cells displayed increased levels of KLF17 and decreased levels of Ki-67, as compared to the control groups (Fig. 4C).

Figure 4.

Ectopic KLF17 expression suppresses CRC cell proliferation and metastasis in nude mice. (A) The tumour volume growth curve of LoVo cells, lenti-GFP- or lenti-KLF17-infected LoVo cells. Over 18 days, lenti-KLF17 infection significantly inhibited tumour growth, as compared with the control groups. *P<0.05 and **P<0.01 vs. the blank group; #P<0.05 and ##P<0.01 vs. the lenti-GFP group. (B) Decreased tumour weights were observed in the lenti-KLF17 group, as compared with the other two groups. *P<0.05 vs. the blank group; #P<0.05 vs. the lenti-GFP group. (C) IHC analysis revealed increased KLF17 and decreased Ki-67 expression in the tumours of lenti-KLF17-infected LoVo cells, as compared to the other two groups. (D-F) Quantification of KLF17 expression via (D) RT-qPCR and (E and F) western blotting in the lenti-KLF17-infected group, as compared to other control groups. **P<0.01 vs. the blank group; ##P<0.01 vs. the lenti-GFP group. (G-a) Ultrasonic image and (G-b-d) gross liver samples from LoVo liver metastatic nude mice. (b) Liver specimen in vivo. (c) Diaphragmatic surface of isolated liver specimen. (d) Visceral surface of isolated liver specimens. (H) Number of metastatic hepatic nodules in three mice groups. *P<0.05 vs. the blank group; ##P<0.01 vs. the lenti-GFP group. (I) Relative KLF17 mRNA levels in three mice groups. **P<0.01 vs. the blank group; ##P<0.01 vs. the lenti-GFP group. KLF17, kruppel-like factor 17; CRC, colorectal cancer; GFP, green fluorescent protein.

In addition, RT-qPCR and western blotting were performed to detect the mRNA and protein levels in tumour specimens when the mice were euthanized (Fig. 4D-F). The increased KLF17 expression in lenti-KLF17-infected mice confirmed the successful transmission of KLF17 by lentivirus-mediated infection.

The tumour metastasis potential of lenti-KLF17-infected LoVo cells was analyzed using a nude mice model of metastasis. As revealed in Fig. 4G and H, more hepatic metastatic nodules were found in the lenti-GFP and blank control groups than in the lenti-KLF17 group. In addition, the higher KLF17 expression level in the lenti-KLF17-infected group confirmed that synthetic KLF17 was successfully delivered into the LoVo cells (Fig. 4I).

UHRF1-regulated promoter methylation suppresses KLF17 expression in CRC

It has been reported that KLF17 can be silenced by the methylation of its promoter (6), as the methylation of CpG islands (CGIs) in promoters suppresses reciprocation with transcription factors and inhibits gene expression (14). Therefore, it was investigated whether promoter methylation in CRC is associated with the downregulation of KLF17. Using CGI analysis software (www.urogene.org), a typical CGI in the promoter region of KLF17 was revealed (Fig. 5A).

To determine whether promoter methylation directly downregulated KLF17, LoVo cells were treated with methylation inhibitor 5-aza-dc, and then BSP analysis was performed. Following demethylation, it was observed that the methylation of KLF17 promoter decreased (Fig. 5B) and the expression of KLF17 was restored (Fig. 5C). MSP was next used to analyze the CGI methylation level of KLF17 in human CRC. As revealed in Fig. 5D, methylated PCR products were detected in 93.3% (28/30) and 53.3% (16/30) of CRC and paired normal mucosa specimens, respectively. The methylation level of CRC tissue samples was evidently higher than that of corresponding normal tissue samples (P=0.001).

In our previous study, UHRF1 was highly expressed in CRC and revealed to play an essential role in CRC carcinogenesis (11). Given the potential of UHRF1 as a DNA methylation regulator and that CGI exists on the KLF17 promoter, we questioned whether the overexpression of UHRF1 is an underlying mechanism of KLF17 DNA methylation in CRC. Lentiviral-mediated RNAi of UHRF1 was then carried out to knock down the UHRF1 expression of LoVo cells (lenti-shF1), and BSP analysis was performed to detect the CGI methylation status. As revealed in Fig. 5E and F, the depletion of UHRF1 decreased CpG methylation and elevated expression of KLF17 in CRC.

Rescue experiments were then performed to inspect whether UHRF1 promotes CRC progression through the downstream KLF17 gene using lenti-shF1-infected LoVo cells co-transfected with KLF17 small interfering RNA. It was revealed that the decreased adhesion, invasion and EMT of CRC cells induced by UHRF1 inhibition could be partially rescued by KLF17 silencing (Fig. 6). Collectively, the present results indicated that KLF17 may be a potent downstream gene of UHRF1 and its downregulation by UHRF1 can be caused by DNA methylation.

Discussion

The KLF transcription factor family proteins have vital functions in many physiological processes and cancer development, including proliferation, invasion and metastasis. Increasing evidence has revealed that KLF4, KLF6 and KLF9 were downregulated (1517), but KLF5 upregulated in CRC specimens, as compared to normal epithelium specimens (18). Moreover, decreased KLF4 expression was correlated with lymph node metastasis and poor survival in CRC patients (19).

As an inhibitor of EMT and a potential tumour suppressor gene in several types of cancer (6,2022), KL17 has been reported to be negatively correlated with a poor outcome in lung (23), liver (3), gastric (4) and papillary thyroid cancer (24). However, the impact of KLF17 on CRC development has not been fully elucidated. In the present study, it was demonstrated that KLF17 downregulation was associated with lymph node metastasis and may be an independent prognostic factor for CRC. In addition, a lower KLF17 expression was observed in the highly metastatic LoVo and SW620 CRC cell lines than that in the lowly metastatic CRC cell lines, indicating that KLF17 could play a negative regulatory role in CRC progression and metastasis.

Considering its higher transfection efficiency and more sustained long-term gene expression, lentivirus-mediated ectopic expression of KLF17 in 2 CRC cell lines was used in this study to further investigate the biological potential of KLF17 in the development of CRC (25). Our in vitro functional experiments revealed that KLF17 overexpression inhibited the proliferation and colony formation capacity of CRC cells. The in vivo tumourigenicity assay in nude mice verified that KLF17 overexpression led to a significant decrease in tumour growth in CRC. In an earlier study, Cai et al revealed that forced KLF17 expression in lung cancer cells inhibited the growth rate and colony formation in a time-dependent manner (23). In a later study, Ye et al reported that the loss of KLF17 enhances the proliferation of papillary thyroid cancer cells (24). Furthermore, Ali et al observed that ectopic KLF17 expression in breast cancer cells containing mutant p53 reduced cell proliferation, while the depletion of KLF17 promoted cell growth and decreased the apoptotic level of adriamycin-treated breast cancer cells (20).

A major cause of cancer mortality, metastasis, is a complex process consisting of multiple steps (26). EMT is considered the key process for cancer metastasis. During this cellular process, the epithelial features are lost and mesenchymal features are acquired for the epithelial cells, leading to the elimination of cell connection and an increase in cell migration and invasion (27). Using both mouse and cell models, Gumireddy et al (7) observed that KLF17 knockdown resulted in EMT and spindle-like and fibroblastic morphology of breast cancer cells. Furthermore, KLF17 has been revealed to inhibit EMT and cancer metastasis by controlling related genes (28). The lower KLF17 expression was associated with the alteration of EMT-related gene expression in HCC patients. Specifically, the depletion of KLF17 altered the expression of E-cadherin, vimentin and ZO-1 in HepG2 cells (29). Sun et al (3) revealed that KLF17 directly binds to the promoter of vimentin, ZO-1 and fibronectin, suggesting that KLF17 is an upstream regulator of those EMT-related genes.

Following lentivirus-mediated forced KLF17 expression, two CRC cell lines exhibited reduced adhesion and invasion capacities. In addition, an in vivo liver metastasis model further confirmed the in vitro results, in which ectopic KLF17 expression significantly depressed LoVo cell metastasis to the liver. Major EMT markers were then analysed in CRC cells by RT-qPCR and western blotting. As anticipated, ectopic KLF17 expression suppressed vimentin (the mesenchymal marker) expression but promoted that of E-cadherin (the epithelial marker) in CRC cell lines. Collectively, these results indicated that KLF17 may act as a negative tumour regulator by suppressing EMT progression in CRC.

Given the potential diagnostic and prognostic value of KLF17 and its function as a tumour suppressor during CRC tumourigenesis, the mechanisms controlling KLF17 expression should be fully elucidated. In a previous study, Sachdeva et al observed the 5-Aza-dC-mediated induction of KLF3, a member of the KLF family, in soft tissue sarcomas. As the DNA methylation inhibitor, 5-Aza-dC induced KLF3 expression by three-fold. Subsequently, experiments detected conserved CGI in both mouse and human KLF3 promoters and discovered that the downregulation of KLF3 in sarcoma occurs due to promoter hypermethylation (30). Aberrant DNA methylation patterns are commonly observed in many types of cancer, including CRC. Cancer-specific CGI methylation blocks the initiation of gene transcription and affects many genes in CRC; these modifications were considered to be a key component of tumorigenesis (31). In the present study, a typical CGI was revealed in the promoter region of KLF17, and its methylation led to the suppression of KLF17 expression. In accordance with our findings, another study demonstrated that the methylation of CGI in the promoter of KLF17 by UHRF1 decreased KLF17 expression in breast cancer (6). These results suggested that the CGI in the promoter of KLF17 may be a cancer-related CGI, and the regulation of UHRF1 to the expression of KLF17 by methylation may be a crucial component in the mechanism underlying tumourigenesis not limited to CRC.

As a vital regulator of DNA methylation, UHRF1 has been revealed to be overexpressed and play an important role in the carcinogenesis of several types of cancer (3234). In our previous study, UHRF1 expression was correlated with CRC progression and promoted the growth and metastasis of CRC (11). In the present study, it was further revealed that UHRF1 silences KLF17 expression through the methylation of CGI in the promoter of KLF17, and mediates cellular EMT and invasion in a KLF17-dependent manner in CRC cells. The results additionally elucidated the underlying mechanism of KLF17 downregulation and the regulation of UHRF1 in CRC carcinogenesis, invasion and metastasis.

Despite the considerable number of clinical specimens used in the present study, larger-scale prospective studies are required to further evaluate or confirm the potential of KLF17 as a novel biomarker for CRC. Moreover, additional extensive mechanistic studies are required to elucidate how KLF17 promoter methylation occurs and how KLF17 regulates downstream targets in CRC carcinogenesis and progression.

In summary, these data indicated that KLF17 is frequently silenced in CRC and may serve as a potential independent prognostic CRC biomarker. KLF17 reduced CRC EMT and suppressed tumour cell proliferation, adhesion, invasion and metastasis. Moreover, the knockdown of UHRF1 decreased the methylation level of the KLF17 promoter, triggered the expression of KLF17 and reduced cell adhesion and invasion by inhibiting EMT in CRC. In combination, the present study offered insights into KLF17 expression regulation in CRC progression and suggested that making changes to this mechanism may represent a new therapeutic approach to blocking CRC development.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was funded by the National Natural Science Foundation of China (nos. 81301753, 81230057, 81730102 and 81472262).

Availability of data and materials

The data and materials used in the present study are available for research purposes.

Authors' contributions

FW and HQ designed the experiments and executed laboratory analysis. XJ, TYS, HL, CS and ZL conducted the experiments and contributed to the data collection. XJ, FW and HQ wrote the manuscript. All authors have approved the final version of the publication and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

The research protocol was assessed and permitted by the Ethical Committee of the Tenth People's Hospital Affiliated to Tongji University. Written informed consent was obtained from all patients. All animal experiments were performed according to protocols approved by the Ethics Committee of Animal Experiments of the Tenth People's Hospital Affiliated to Tongji University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester RGS, Barzi A and Jemal A: Colorectal cancer statistics, 2017. CA Cancer J Clin. 67:177–193. 2017. View Article : Google Scholar : PubMed/NCBI

2 

Kim C, He P, Bialkowska A and Yang V: SP and KLF transcription factors in digestive physiology and diseases. Gastroenterology. 152:1845–1875. 2017. View Article : Google Scholar : PubMed/NCBI

3 

Sun Z, Han Q, Zhou N, Wang S, Lu S, Bai C and Zhao R: MicroRNA-9 enhances migration and invasion through KLF17 in hepatocellular carcinoma. Mol Oncol. 7:884–894. 2013. View Article : Google Scholar : PubMed/NCBI

4 

Peng JJ, Wu B, Xiao XB, Shao YS, Feng Y and Yin MX: Reduced Krüppel-like factor 17 (KLF17) expression correlates with poor survival in patients with gastric cancer. Arch Med Res. 45:394–399. 2014. View Article : Google Scholar : PubMed/NCBI

5 

Li S, Qin X, Cui A, Wu W, Ren L and Wang X: Low expression of KLF17 is associated with tumor invasion in esophageal carcinoma. Int J Clin Exp Pathol. 8:11157–11163. 2015.PubMed/NCBI

6 

Gao SP, Sun HF, Li LD, Fu WY and Jin W: UHRF1 promotes breast cancer progression by suppressing KLF17 expression by hypermethylating its promoter. Am J Cancer Res. 7:1554–1565. 2017.PubMed/NCBI

7 

Gumireddy K, Li A, Gimotty PA, Klein-Szanto AJ, Showe LC, Katsaros D, Coukos G, Zhang L and Huang Q: KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer. Nat Cell Biol. 11:1297–1304. 2009. View Article : Google Scholar : PubMed/NCBI

8 

Gao HL, Zhou N, Sun Z, Dou XL, Guan M and Bai CM: KLF17 expression in colorectal carcinoma and its clinical significance. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 38:69–72. 2016.PubMed/NCBI

9 

Zlobec I, Terracciano L, Jass J and Lugli A: Value of staining intensity in the interpretation of immunohistochemistry for tumor markers in colorectal cancer. Virchows Arch. 451:763–769. 2007. View Article : Google Scholar : PubMed/NCBI

10 

Crnogorac-Jurcevic T, Gangeswaran R, Bhakta V, Capurso G, Lattimore S, Akada M, Sunamura M, Prime W, Campbell F, Brentnall T, et al: Proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma. Gastroenterology. 129:1454–1463. 2005. View Article : Google Scholar : PubMed/NCBI

11 

Wang F, Yang YZ, Shi CZ, Zhang P, Moyer MP, Zhang HZ, Zou Y and Qin HL: UHRF1 promotes cell growth and metastasis through repression of p16(ink(4)a) in colorectal cancer. Ann Surg Oncol. 19:2753–2762. 2012. View Article : Google Scholar : PubMed/NCBI

12 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

13 

Yamamoto Y, Hirakawa E, Mori S, Hamada Y, Kawaguchi N and Matsuura N: Cleavage of carcinoembryonic antigen induces metastatic potential in colorectal carcinoma. Biochem Biophys Res Commun. 333:223–229. 2005. View Article : Google Scholar : PubMed/NCBI

14 

Koch A, Joosten SC, Feng Z, de Ruijter TC, Draht MX, Melotte V, Smits KM, Veeck J, Herman JG, Van Neste L, et al: Analysis of DNA methylation in cancer: Location revisited. Nat Rev Clin Oncol. 15:459–466. 2018. View Article : Google Scholar : PubMed/NCBI

15 

Zhao W, Hisamuddin IM, Nandan MO, Babbin BA, Lamb NE and Yang VW: Identification of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene. 23:395–402. 2004. View Article : Google Scholar : PubMed/NCBI

16 

Reeves HL, Narla G, Ogunbiyi O, Haq AI, Katz A, Benzeno S, Hod E, Harpaz N, Goldberg S, Tal-Kremer S, et al: Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene frequently inactivated in colorectal cancer. Gastroenterology. 126:1090–1103. 2004. View Article : Google Scholar : PubMed/NCBI

17 

Kang L, Lu B, Xu J, Hu H and Lai M: Downregulation of Kruppel-like factor 9 in human colorectal cancer. Pathol Int. 58:334–338. 2008. View Article : Google Scholar : PubMed/NCBI

18 

Nakaya T, Ogawa S, Manabe I, Tanaka M, Sanada M, Sato T, Taketo MM, Nakao K, Clevers H, Fukayama M, et al: KLF5 regulates the integrity and oncogenicity of intestinal stem cells. Cancer Res. 74:2882–2891. 2014. View Article : Google Scholar : PubMed/NCBI

19 

Xu J, Lu B, Xu F, Gu H, Fang Y, Huang Q and Lai M: Dynamic down-regulation of Kruppel-like factor 4 in colorectal adenoma-carcinoma sequence. J Cancer Res Clin Oncol. 134:891–898. 2008. View Article : Google Scholar : PubMed/NCBI

20 

Ali A, Shah AS and Ahmad A: Gain-of-function of mutant p53: Mutant p53 enhances cancer progression by inhibiting KLF17 expression in invasive breast carcinoma cells. Cancer Lett. 354:87–96. 2014. View Article : Google Scholar : PubMed/NCBI

21 

Ali A, Bhatti MZ, Shah AS, Duong HQ, Alkreathy HM, Mohammad SF, Khan RA and Ahmad A: Tumor-suppressive p53 signaling empowers metastatic inhibitor KLF17-dependent transcription to overcome tumorigenesis in non-small cell lung cancer. J Biol Chem. 290:21336–21351. 2015. View Article : Google Scholar : PubMed/NCBI

22 

Ali A, Zhang P, Liangfang Y, Wenshe S, Wang H, Lin X, Dai Y, Feng XH, Moses R, Wang D, et al: KLF17 empowers TGF-β/Smad signaling by targeting Smad3-dependent pathway to suppress tumor growth and metastasis during cancer progression. Cell Death Dis. 6:e16812015. View Article : Google Scholar : PubMed/NCBI

23 

Cai XD, Zhou YB, Huang LX, Zeng QL, Zhang LJ, Wang QQ, Li SL, Feng JQ and Han AJ: Reduced expression of Kruppel-like factor 17 is related to tumor growth and poor prognosis in lung adenocarcinoma. Biochem Biophys Res Commun. 418:67–73. 2012. View Article : Google Scholar : PubMed/NCBI

24 

Ye WC, Gao L, Huang J, Fang XM and Xie G: Suppressed Krüppel-like factor 17 expression induces tumor proliferation, metastasis and a poor prognosis in papillary thyroid carcinoma. Mol Med Rep. 10:2087–2092. 2014. View Article : Google Scholar : PubMed/NCBI

25 

Milone MC and O'Doherty U: Clinical use of lentiviral vectors. Leukemia. 32:1529–1541. 2018. View Article : Google Scholar : PubMed/NCBI

26 

Stuelten CH, Parent CA and Montell DJ: Cell motility in cancer invasion and metastasis: Insights from simple model organisms. Nat Rev Cancer. 18:296–312. 2018. View Article : Google Scholar : PubMed/NCBI

27 

Campbell K: Contribution of epithelial-mesenchymal transitions to organogenesis and cancer metastasis. Curr Opin Cell Biol. 55:30–35. 2018. View Article : Google Scholar : PubMed/NCBI

28 

Zhou S, Tang X and Tang F: Krüppel-like factor 17, a novel tumor suppressor: Its low expression is involved in cancer metastasis. Tumour Biol. 37:1505–1513. 2016. View Article : Google Scholar : PubMed/NCBI

29 

Liu FY, Deng YL, Li Y, Zeng D, Zhou ZZ, Tian DA and Liu M: Down-regulated KLF17 expression is associated with tumor invasion and poor prognosis in hepatocellular carcinoma. Med Oncol. 30:4252013. View Article : Google Scholar : PubMed/NCBI

30 

Sachdeva M, Dodd RD, Huang Z, Grenier C, Ma Y, Lev DC, Cardona DM, Murphy SK and Kirsch DG: Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182. Cancer Lett. 369:202–211. 2015. View Article : Google Scholar : PubMed/NCBI

31 

Feinberg AP: The key role of epigenetics in human disease prevention and mitigation. N Engl J Med. 378:1323–1334. 2018. View Article : Google Scholar : PubMed/NCBI

32 

Kim KB, Son HJ, Choi S, Hahm JY, Jung H, Baek HJ, Kook H, Hahn Y, Kook H and Seo SB: H3K9 methyltransferase G9a negatively regulates UHRF1 transcription during leukemia cell differentiation. Nucleic Acids Res. 43:3509–3523. 2015. View Article : Google Scholar : PubMed/NCBI

33 

Taniue K, Kurimoto A, Sugimasa H, Nasu E, Takeda Y, Iwasaki K, Nagashima T, Okada-Hatakeyama M, Oyama M, Kozuka-Hata H, et al: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1. Proc Natl Acad Sci U S A. 113:1273–1278. 2016. View Article : Google Scholar : PubMed/NCBI

34 

Mudbhary R, Hoshida Y, Chernyavskaya Y, Jacob V, Villanueva A, Fiel M, Chen X, Kojima K, Thung S, Bronson RT, et al: UHRF1 overexpression drives DNA hypomethylation and hepatocellular carcinoma. Cancer Cell. 25:196–209. 2014. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

November-2019
Volume 42 Issue 5

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Jiang X, Shen TY, Lu H, Shi C, Liu Z, Qin H and Wang F: Clinical significance and biological role of KLF17 as a tumour suppressor in colorectal cancer. Oncol Rep 42: 2117-2129, 2019
APA
Jiang, X., Shen, T., Lu, H., Shi, C., Liu, Z., Qin, H., & Wang, F. (2019). Clinical significance and biological role of KLF17 as a tumour suppressor in colorectal cancer. Oncology Reports, 42, 2117-2129. https://doi.org/10.3892/or.2019.7324
MLA
Jiang, X., Shen, T., Lu, H., Shi, C., Liu, Z., Qin, H., Wang, F."Clinical significance and biological role of KLF17 as a tumour suppressor in colorectal cancer". Oncology Reports 42.5 (2019): 2117-2129.
Chicago
Jiang, X., Shen, T., Lu, H., Shi, C., Liu, Z., Qin, H., Wang, F."Clinical significance and biological role of KLF17 as a tumour suppressor in colorectal cancer". Oncology Reports 42, no. 5 (2019): 2117-2129. https://doi.org/10.3892/or.2019.7324