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Abstract

Objectives:

The aims of this study were to investigate the incidence of Krüppel-like factor 6 (KLF6) protein staining in patients with cutaneous malignant melanoma and examine its potential relevance to clinicopathological characteristics and tumour cell proliferation.

Methods:

Clinicopathological data from patients with cutaneous malignant melanoma were analysed retrospectively. Presence of KLF6 and the antigen Ki-67 in malignant melanoma and healthy tissue samples from each patient was detected by immunohistochemistry. The proliferation index was calculated on the basis of Ki-67 expression. The relationship between KLF6 and clinicopathological characteristics was also analysed.

Results:

KLF6 was detected more frequently in normal healthy skin tissue compared with cutaneous malignant melanoma lesions (n = 40). There was a negative correlation between the presence of KLF6 and the proliferation index. The presence of KLF6 was also significantly correlated with tumour diameter, lymph node metastasis, tumour–node–metastasis stage and 3-year survival rate.

Conclusions:

KLF6 protein is downregulated in human cutaneous malignant melanoma lesions compared with healthy skin tissue. KLF6 may be involved in tumour progression and may be a tumour suppressor and prognostic marker for cutaneous malignant melanoma.

Keywords

Malignant melanoma Krüppel-like factor 6 proliferation prognosis

Introduction

Malignant melanoma originates from naevus cells and melanocytes; it is associated with poor prognosis and high mortality due to its high degree of malignancy, early blood and lymphatic metastasis and resistance to chemotherapy and radiotherapy.¹ The occurrence, development and metastasis of melanoma are complex and multi-phased biological processes that are controlled by many genes.² Krüppel-like factor 6 (KLF6) is a nuclear transcription factor that inhibits tumour cell growth, promotes apoptosis and participates in the regulation of cell senescence.³ Research has shown that, consistent with its function as a tumour suppressor, KLF6 expression is downregulated or absent in astrocytomas, and in prostate, liver, colorectal and nasopharyngeal cancers.^4–8 There are no published reports on the role of KLF6 in malignant melanoma. The present study aimed to investigate the presence of KLF6 protein in patients with cutaneous malignant melanoma and to examine the relationship between KLF6, clinicopathological characteristics, cell proliferation and survival.

Materials and methods

Tissue samples and clinical data collection

Complete pathological data were retrospectively collected from consecutive patients with cutaneous malignant melanoma, treated at the Department of Dermatology and Pathology, Qilu Hospital, Shandong University, Jinan, China between January 2000 and December 2007. None of the patients had received preoperative anticancer treatment. There were no other specific inclusion or exclusion criteria for the study. Tissue samples were obtained from included cases: samples were fixed in 10% neutral formalin and embedded in paraffin wax before sections of 3-µm thickness were cut and transferred onto slides. The diagnosis of cutaneous malignant melanoma was confirmed by histological analysis, according to the World Health Organization classification of skin tumours;⁹ tumour–node–metastasis (TNM) stage was also confirmed.¹⁰ Control specimens were obtained from areas of healthy unaffected skin tissue, taken from the same patients (healthy tissue sites were 3 cm away from the melanoma tissue).

The study protocol was approved by the Ethics Review Committee of Qilu Hospital, Shandong University and all patients provided written informed consent.

Immunohistochemistry of KLF6 and Ki-67

The presence of KLF6 in malignant melanoma tissues was analysed by immunohistochemistry. The proliferative rate of tumour cells was determined by immunohistochemistry analysis of the nuclear antigen Ki-67. Sections (3 µm thick) were deparaffinized twice (10 min each) in dimethylbenzene and rehydrated through graded concentrations of ethanol. Slides were incubated in 0.001 mol/l ethylenediaminetetra-acetic acid (pH 8.0) and underwent high-pressure steam antigen retrieval for 5 min. Tissue sections were then incubated in rabbit antihuman KLF6 monoclonal antibodies (Abcam, Cambridge, UK; 1 : 100 dilution) and mouse antihuman Ki-67 ready-to-use monoclonal antibodies (ZhongShan Golden Bridge Biotechnology Company, Beijing, China) for 1 h at 37℃. The slides were then washed three times in 0.01 M phosphate-buffered saline (PBS; pH 7.3). Slides were incubated with a secondary antibody (horseradish peroxidase goat antimouse, used without dilution; supplied by DAKO, Glostrup, Denmark]) for 1 h at 37℃. The slides were then washed three times in PBS, pH 7.2. Immunohistochemistry was performed using the EnVision™ method according to the manufacturer’s instructions (DAKO). Diaminobenzidine-treated slides were counterstained with Giemsa staining, to mask yellow-pigmented granules in the melanoma cells; this was undertaken to avoid them obscuring the immunohistochemistry staining.

Grading standards of immunohistochemistry

The presence of KLF6 and Ki-67 was determined by light microscopy (BX51, Olympus Optical, Tokyo, Japan) and was observed by yellow-to-brown staining in the cytoplasm and nucleus. KLF6 staining was evaluated in five randomly-selected high-power (× 400) microscope fields with the most intense staining, in which the total number of cells and the number of positively-stained cells were counted. The total number of cells from five fields was ≥ 200; the percentage of cells staining positive was calculated and the results were graded as follows: 1, ≤ 5%; 2, 6−25%; 3, 26−50%; 4, > 50% of cells stained positive. Grade 1 was defined as negative for staining and grades 2–4 were defined as positive. The proliferation index was calculated according to the method of Bologna-Molina et al.¹¹ Briefly, the percentage of cells staining positive for Ki-67 was calculated using photomicrographs (one per case) obtained using a high-power objective (× 100) of 10 randomly-selected fields of view, where tumour cells were predominant; the proliferation index = cells staining positive for Ki-67/total number of tumour cells × 100.

Statistical analyses

All statistical analyses were performed using SPSS® statistical software, version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Relative levels of KLF6 in malignant melanoma lesions and healthy skin tissue, and the relationship between KLF6 and clinicopathological parameters, were analysed using the χ²-test. The association between KLF6 and the proliferation index was analysed using Spearman’s rank correlation coefficient. Cumulative survival rate was determined using the Life Table method.¹² Univariate survival analysis was performed by the Kaplan–Meier, method with the log-rank test applied for comparison; a survival curve was used to represent the accumulated survival rate. Multivariate survival analysis was performed using logistic regression. A P-value of < 0.05 was considered statistically significant.

Results

Data from 40 patients (12 women and 28 men) with a mean age of 49.2 years (range: 25–70 years) were analysed. A total of 30 patients had lymph node metastasis while the remaining 10 patients had no evidence of metastasis.

The protein KLF6 was found in normal tissues and malignant melanoma lesions: positive staining of a yellow-to-brown colour was mainly observed in the epithelial prickle and basal layers in normal tissues; in malignant melanoma lesions, yellow staining was observed in the cuticular layer. Malignant melanoma tissue from 11 patients was positive for KLF6, giving a positive rate of 27.5%, which was significantly lower compared with control tissues (χ²= 45.5, P < 0.001, Table 1).

Table 1.

Immunohistochemical staining of Krüppel-like factor 6 (KLF6) protein in cutaneous malignant melanoma lesions and healthy skin (control) tissue samples taken from the same patients.

	Staining score
Tissue sample	0	1	2	3	4	Positive rate^a
Healthy skin, n = 40	0	0	0	6	34	100
Malignant melanoma, n = 40	10	19	11	0	0	27.5

Staining for KLF6 was graded as follows: 1, ≤ 5% of cells stained positive; 2, 6−25%; 3, 26−50%; 4, > 50%. Grade 1 was defined as negative staining and grades 2–4 as positive.

Staining for Ki-67 was mainly detected in the nucleus. Sporadic staining was observed in normal tissues compared with a more diffuse staining in malignant melanoma tissues: the proliferation index was significantly higher in malignant melanoma compared with normal tissues (72.7 ± 11.31 versus 3.06 ± 1.02; t = −38.79, P < 0.001). Spearman’s rank correlation coefficient analysis demonstrated a negative correlation between the presence of KLF6 and the proliferation index (r = −0.7, P < 0.01).

The presence of KLF6 in cutaneous malignant melanoma was correlated with tumour diameter, lymph node metastasis and TNM stage¹⁰ (P < 0.05, Table 2). There was no significant correlation between KLF6 and other clinicopathological features such as age or sex (Table 2).

Table 2.

Relationship between the Krüppel-like factor 6 (KLF6) protein level and clinicopathological parameters in 40 patients with cutaneous malignant melanoma.

Parameter	KLF6-positive, n = 11	KLF6-negative, n = 29	Statistical significance^a
Age, years
≤50	6 (54.6)	14 (48.3)	NS
>50	5 (45.4)	15 (51.7)
Sex
Male, n = 28	8 (72.7)	20 (69.0)	NS
Female, n = 12	3 (27.3)	9 (31.0)
Lymph node metastasis
Present, n = 30	2 (18.2)	28 (96.6)	P < 0.05
Absent, n = 10	9 (81.8)	1 (3.4)
TNM stage¹⁰
I/II, n = 11	9 (81.8)	2 (6.9)	P < 0.05
III/IV, n = 29	2 (18.2)	27 (93.1)
Tumour diameter, cm
≤2.5, n = 26	5 (45.4)	21 (72.4)	P < 0.05
>2.5, n = 14	6 (54.6)	8 (27.6)

Data presented as n (%) of patients.

χ²-test.

NS, not statistically significant (P ≥ 0.05).

Detailed follow-up information was available for all 40 patients. Kaplan–Meier survival analysis demonstrated that the 3-year survival rate in patients with low levels of KLF6 protein was significantly lower than that in patients with high levels of KLF6 (χ²= 4.871, P < 0.05).

Discussion

The mammalian KLF gene family includes 15 proteins (KLF 1–15), each with a similar Cys2/His2 zinc-finger structure at their C-termini, that are involved in the regulation of transcription.^13,14 KLF6 (also known as zinc-finger transcription factor-9 or core promoter-element binding protein) is a ubiquitously expressed nuclear transcription factor, located on chr10p15, consisting of four exons: exon two of KLF6 encodes the domain with transcription activity.¹⁵ Unlike the other members of the KLF family, the N-terminal domain of KLF6 has abundant proline and serine residues in addition to the C-terminal Cys2/His2 zinc-finger structure.¹⁶ KLF6 binds specifically to GC box-promoter motifs and upregulates or downregulates the expression of target genes, thereby controlling cell proliferation and differentiation, apoptosis and signal transduction.^17–19

Many important transcription factors are KLF6 targets.^4,18,19 These include placenta glycoprotein, collagen α(I), transforming growth factor (TGF)-β1, TGF-β1 receptor-I and receptor-II, urokinase-type plasminogen activator and p21/WAFl. Activation of key cell-cycle regulator genes by the transcription factor E2F is inhibited by phosphorylation of the retinoblastoma (Rb) gene. By blocking phosphorylation of Rb, p21 regulates expression of E2F and thereby arrests the cell cycle by preventing G₁/S transition.²⁰ KLF6 is a p53-independent activator of p21 and may have an effect similar to p53 in the regulation of p21 expression, through which it suppresses cell proliferation.^4,21–24 Tumour cell growth is also inhibited by KLF6 through other mechanisms. An antiproliferative effect of KLF6 through upregulation of the cyclin-dependent kinase inhibitor p27 has been demonstrated in lung cancer.²⁵ In addition, KLF6 has been shown to interact with c-Src protein, leading to the inactivation of Erk and Akt (which are critical for cell proliferation and survival) and the suppression of oestrogen receptor-mediated cell growth in breast cancer.²⁶ Research has also suggested that a KLF6-related epigenetic mechanism may be involved in tumour cell proliferation.²⁷

Data from the present study indicate that fewer cutaneous malignant melanoma lesions had evidence of KLF6 immunohistochemical staining compared with healthy control tissues, taken from the same patient. In addition, the presence of KLF6 protein was shown to be negatively correlated with tumour cell proliferation. The present study also demonstrated that KLF6 was associated with tumour diameter, TNM stage and lymph node metastasis. Therefore, KLF6 expression levels might reflect the degree of malignancy of cutaneous malignant melanoma. More patients with positive KLF6 staining had tumours in TNM I/II stages compared with TNM III/IV, and more had no evidence of lymph node metastasis. Furthermore, shorter survival rates were determined in patients with no evidence of KLF6 staining. Taken together, these data support an essential role for loss or downregulation of KLF6 in the development and progression of cutaneous malignant melanoma.

The authors acknowledge that there were some limitations to the present study. First, because malignant melanoma is rare in China, the number of patients included in the study was small. Secondly, this was a retrospective analysis of archived patient data; larger, prospective studies are needed to clarify these initial study findings.

In conclusion, the present study revealed that KLF6 expression is downregulated in tissue samples of cutaneous malignant melanoma compared with healthy control samples from the same patients. These data indicate that KLF6 may be involved in tumour progression and may be a tumour suppressor and prognostic marker for cutaneous malignant melanoma.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This project was supported by a grant from Shandong Province Science and Technology Development Projects (No. 2011GSF11804).

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Krüppel-like factor 6 in the progression and prognosis of malignant melanoma

Abstract

Objectives:

Methods:

Results:

Conclusions:

Keywords

Introduction

Materials and methods

Tissue samples and clinical data collection

Immunohistochemistry of KLF6 and Ki-67

Grading standards of immunohistochemistry

Statistical analyses

Results

Discussion

Footnotes

Declaration of conflicting interest

Funding

References