This article is mentioned in:

Introduction

Renal cell carcinoma (RCC) is the most common primary renal malignancy, and accounts for 2–3% of adult malignancies (1). In China, the incidence of RCC is ~540 cases per million individuals every year (2). It has been shown that RCC has a high mortality rate, and the 5-year survival rate of metastatic RCC patients is <10% (3). RCC can be divided into several subtypes according to the morphological and microscopic features, and clear cell RCC (ccRCC) is the most predominant subtype, which accounts for 75–80% of all RCCs (4).

The tumor suppressor serine-threonine kinase 11 (STK11), also termed liver kinase B1 (LKB1), was first identified as a germline-mutated gene in Peutz-Jeghers Syndrome in 1996 (5). The product of the STK11 gene is a 50-kDa serine-threonine kinase involved in various biological functions, including cell polarity, cell detachment and adhesion, cell structure and energy metabolism (6). Germline mutations of the STK11 gene are found in a variety of cancer types, including lung cancer (7), hepatocellular carcinoma (8) and breast cancer (9). In addition, functional studies showed that STK11 heterozygous knockout mice would develop tumors in several organs (10,11).

Somatic mutations of the STK11 gene have also been found in several tumors, including pancreatic cancer (12), biliary cancer (12), hepatocellular carcinoma (8) and testicular tumor (13); however, the frequency of mutations is relatively rare, with a range of 0–6%. In lung cancer, a geographically variable incidence was observed. Mutational inactivation of the STK11 gene is frequently detected in Caucasian, but not in Asian, lung cancer patients (14). As for RCC, a study by Avizienyte et al (15) detected no somatic mutations in the STK11 gene in 19 RCC specimens, whereas a controversial result was observed by Yalniz et al (16), in which 51.6% of RCC patients were found to have somatic mutations in the STK11 gene. Decreased expression of STK11 has also been shown to occur in several cancer types, such as non-small cell lung cancer (8), breast carcinoma (17) and ccRCC. Duivenvoorden et al (18) showed that under-expression of STK11 was a common event in all 10 examined ccRCC samples. However, the mechanism of reduced expression of STK11 in ccRCC remains to be elucidated.

Although inactivation of STK11 gene was found in several cancers, its somatic mutations appear rare. This indicates that the under-expression of the STK11 gene may be also mediated by other mechanisms. In addition to mutation, the expression of STK11 can also be regulated through epigenetic modification, transcriptional regulation and post-translational modification (19). Epigenetic alterations that suppress the activity of tumor suppressor genes is an alternative mechanism for tumor development and progression (20). The methylation status of the STK11 promoter has been investigated in colorectal cancer (21), non-small cell lung cancers (22), and breast, gastric, pancreatic, thyroid, bladder and testicular carcinomas (23). These studies reported that frequency of hypermethylation of the STK11 promoter in the described tumors is low (0–13%) (21–23); however, this indicates that STK11 promoter methylation contributes to the inactivation of the STK11 gene and STK11-mediated functions.

At present, the methylation status of the STK11 promoter in RCC cells remains unclear. In addition, the role of the methylation status of the STK11 promoter in the pathogenesis of RCC remains to be investigated. In order to determine the possible inactivation of STK11 by epigenetic mechanisms, the present study aimed to investigate the methylation status of the STK11 promoter in ccRCC and its association with tumor disease stage and survival of ccRCC patients. The methylation status of the STK11 promoter in RCC was determined by analysis of 42 cases of ccRCC Paraffin-embedded specimens were assessed using methylation-specific polymerase chain reaction (MSP) and the association with RCC progression was analyzed.

Patients and methods

Patients

The present study was reviewed and approved by the Institutional Review Board of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). Paraffin-embedded tumor specimens were obtained and prepared from 42 patients with ccRCC (29 men and 13 women) admitted to the First Affiliated Hospital of Sun Yat-sen University between February 1999 and August 2009. Patients pathologically diagnosed with ccRCC were included, and patients that did not comply with follow-up visits were excluded from the study. Clinical data, including the tumor-node-metastasis (TNM)/American Joint Committee on Cancer (AJCC) staging (9), hematological parameters and post-operative follow-up were documented for further analyses. Written informed consent was obtained from all patients prior to inclusion in the present study.

MSP

A total of 42 paraffin-embedded tissues were sectioned into 5 µm thick slices, then dried for 2 h at 60°C or overnight at 37°C. The slices were immersed in 2X xylene for 15 min, and then dehydrated through a graded series of ethanol (70, 80, 90 and 95%; 5 min each). DNA from the tissue samples was isolated using QIAamp DNA FFPE Tissue Kit (Qiagen, Inc., Valencia, CA, USA), according to the manufacture's protocol. EZ DNA Methylation kit (Zymo Research Corp., Irvine, CA, USA) was used for bisulfite conversion to assess the DNA methylation status. The bisulfite-modified DNA was then used as a template, together with primers specific for methylated and unmethylated sequences for MSP. Polymerase chain reaction (PCR) was performed with DNA polymerase (Beijing Sunbiotech, Beijing, China) at a final volume of 25 µl. The primers used are as previously reported (10): Primers specific for methylated sequence were STK11 forward 5′-ACGAAGTTGATTTTGATCGGGTC-3′ and reverse 5′-CGATACAAAATCTACGAACCGACG-3′, whereas those for the unmethylated sequence were STK11 forward, 5′-GGATGAAGTTGATTTTGATTGGGTT-3′, and reverse, 5′-ACCCAATACAAAATCTACAAACCAACA-3′; GAPDH forward 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′. All primers were synthesized and purchased from Zymo Research (USA). PCR fragments were 122 bp in length. The reaction consisted of initialization at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 30 sec, cooling at 57°C for 59 sec and extension at 72°C for 30 sec, and final extension at 72°C for 10 min. The PCR products were analyzed on a 1% agarose gel (Beijing Hengao Biotechnology, Beijing, China); DNA bands were captured using a UV gel imaging system (EC3 Imaging system, UVP LLC, Upland, CA, USA). The presence of methylated and unmethylated bands in the PCR product indicated partial methylation, the presence of only a methylated band indicated methylation, and the presence of only an unmethylated band indicated unmethylation.

Statistical analysis

Statistical analysis was performed using R 3.0.2. All data are presented as the mean ± standard deviation. Significance was assessed using analysis of variance followed by Tukey Honestly Significant Difference test for all baseline characteristics and hematological parameters, with the exception of the TNM stage, AJCC stage, blood type and sex, which were analyzed using Fisher's exact test. Survival curves of patients in the three groups were plotted and the differences between the three curves were estimated by log-rank test. P<0.05 was considered to indicate a statistically significant difference.

Results

Methylation status of STK11 promoter in ccRCC

The methylation status of the STK11 promoter in 42 ccRCC paraffin-embedded tissue samples was determined using MSP. The data showed that, among the 42 samples, there were 12 (28.6%), 18 (42.9%) and 12 (28.6%) samples in the methylation group (M group), partial methylation group (P group) and unmethylation group (U group), respectively, based on the status of the STK11 promoter (Fig. 1).

Figure 1. Methylation status of serine-threonine kinase 11 in clear cell renal cell carcinoma tissues was determined by methylation-specific polymerase chain reaction. 1–42 indicates the patient number. U, unmethylated, M, methylated.

Patient demographic data

To investigate the effect of the methylation status of the STK11 promoter on ccRCC, the 42 enrolled patients were grouped into the M, P and U groups, according to the methylation status of the STK11 promoter. The patient demographic data of the 3 groups is shown in Table I. In general, with the exception of the follow-up time, there were no significant differences in the clinical characteristics among the 3 groups. Comparison of hematological parameters among the three groups also showed no significant difference (Table II). These results revealed the equivalence of demographic and clinical characteristics of the three groups.

Table I. Demographic data of clear cell renal cell carcinoma patients in the M, P and U groups with regard to serine-threonine kinase 11 promoter status (n=42).

Table I.

Demographic data of clear cell renal cell carcinoma patients in the M, P and U groups with regard to serine-threonine kinase 11 promoter status (n=42).

	Group
Characteristic	M	P	U	P-value
Total, n	12	18	12
Sex, n (%)				0.1783
Male	10 (83.3)	9 (50.0)	9 (75.0)
Female	2 (16.7)	9 (50.0)	3 (25.0)
Age, years (SD, range)	50.8 (13.4, 33–82)	44.4 (15.5, 14–68)	49.9 (12.9, 27–67)	0.413
Height, cm (SD, range)	165.1 (8.7, 152–176)	160.2 (9.5, 150–178)	163.8 (7.8, 147–171)	0.292
Body weight, kg (SD, range)	62.4 (10.6, 47–86.5)	59.4 (13.8, 41–85)	65.2 (12.6, 47–82)	0.474
BMI, kg/m2 (SD, range)	22.7 (2.2, 20–28)	23.0 (4.2, 17–31)	24.2 (3.6, 17–29)	0.565
Tumor diameter, cm (SD, range)	6.9 (2.98, 3.5–13)	6.7 (2.74, 2.5–11)	6.71 (2.71, 2.9–11.5)	0.981
Follow-up time, months (SD, range)	47.17 (22.64, 20–94)	95.25 (51.13, 14–153)	92.56 (45.53, 19–150)	0.010

[i] SD, standard deviation; M, methylated; P, partially methylated; U, unmethylated; BMI, body mass index.

Table II. Hematological parameters of clear cell renal cell carcinoma patients in the M, P and U groups with regard to serine-threonine kinase 11 promoter status (n=42).

Table II.

Hematological parameters of clear cell renal cell carcinoma patients in the M, P and U groups with regard to serine-threonine kinase 11 promoter status (n=42).

	Group
Parameter	M	P	U	P-value
K+, mmol/l (SD, range)	4.43 (0.64, 3.7–5.9)	4.15 (0.39, 3.5–4.9)	4.43 (0.34, 3.9–4.9)	0.382
Na+, mmol/l (SD, range)	140.1 (2.9, 136–145)	139.9 (2.3, 135–142)	141.7 (5.5, 131–149)	0.554
Cl+, mmol/l (SD, range)	105.5 (5.1, 96–115)	103.9 (4.1, 99–114)	105.6 (7.8, 90–114)	0.772
Ca2+, mmol/l (SD, range)	2.31 (0.47, 1.03–2.91)	2.38 (0.12, 2.09–2.55)	2.12 (0.46, 1.02–2.5)	0.219
AST, U/l (SD, range)	23.6 (13.5, 13–57.2)	22.1 (9.4, 6–41)	22.8 (12.1, 8–51)	0.934
ALT, U/l (SD, range)	28.1 (25.9, 6–89.9)	21.1 (14.2, 7–56)	23.4 (12.4, 3–41)	0.584
TBA, mmol/l (SD, range)	4.0 (1.9, 1.1–7.1)	5.7 (2.9, 2.2–11.5)	6.6 (5.1, 1.7–20.8)	0.226
ALP, U/l (SD, range)	73.1 (21.1, 42–117.1)	67.5 (35.2, 35–190)	73.8 (27.8, 35–136)	0.811
GGT, U/l (SD, range)	36.2 (33.4, 10–132)	30.6 (26.5, 2.4–93)	37.8 (31.8, 1–99)	0.787
LDH, U/l (SD, range)	156 (31.3, 114–208)	202 (48.8, 133–296.3)	187 (91.8, 82–346)	0.142
AFU, nmol/ml·h (SD, range)	12.9 (5.97, 5–26)	10.2 (4.92, 5–21)	8.78 (5.53, 2–19.4)	0.191
ALB, g/l (SD, range)	40.3 (3.99, 34.6–47.4)	43.1 (3.6, 37.5–48.6)	40.5 (5.16, 30.3–48.6)	0.116
GLO, g/l (SD, range)	28.1 (5.9, 22.1–38)	31.5 (4.72, 22.2–40.8)	30.7 (5.75, 22.5–43.9)	0.223
DBIL, µmol/l (SD, range)	3.37 (2.39, 1.1–10.1)	3.34 (1.88, 0.22–8.87)	3.37 (2.23, 0.76–8.94)	0.999
IBIL, µmol/l (SD, range)	7.27 (3.02, 3.1–11.72)	11.5 (8.6, 3.4–40.3)	10.7 (5.4, 0.81–20.83)	0.249
BUN, mmol/l (SD, range)	5.03 (2.53, 2.13–10.11)	5.1 (1.61, 2.82–8.01)	5.11 (1.71, 2.49–8)	0.994
CRE, µmol/l (SD, range)	99.3 (26.1, 67.29–141)	92.5 (21.1, 51.3–134)	93.2 (28.7, 29–133)	0.747
UA, µmol/l (SD, range)	326 (89.8, 172.6–454)	323 (94.1, 118–551)	375 (144.5, 87–597)	0.403
CHO, mmol/l (SD, range)	4.34 (0.97, 2.97–5.4)	4.91 (1.10, 3.61–7.52)	4.39 (0.85, 3.14–6)	0.233
TG, mmol/l (SD, range)	1.15 (0.62, 0.54–2.51)	1.52 (0.92, 0.53–3.96)	2.02 (1.42, 0.65–5.19)	0.126
GLU, mmol/l (SD, range)	4.92 (0.77, 3.22–5.94)	5.08 (0.73, 4.16–6.89)	5.07 (0.58, 3.68–5.96)	0.819
HDL, mmol/l (SD, range)	1.17 (0.42, 0.59–2.26)	1.25 (0.33, 0.82–1.87)	1.09 (0.22, 0.71–1.47)	0.413
LDL, mmol/l (SD, range)	2.67 (0.82, 1.47–4.12)	3.0 (0.83, 1.46–4.53)	2.72 (0.93, 1.64–4.46)	0.532
WBC, 109/l (SD, range)	8.04 (1.92, 4.6–11)	8.11 (2.52, 5.1–13.5)	8.73 (2.44, 5–14)	0.725
RBC, 1012/l (SD, range)	4.64 (0.61, 3.35–5.71)	4.48 (0.99, 2.79–6.76)	4.77 (0.72, 3.79–6.32)	0.647
Hb, g/l (SD, range)	132 (17.4, 95.3–156)	129 (25.3, 79–165)	135 (21.8, 93–171)	0.750
PLT, 109/l (SD, range)	285 (91.6, 179–475)	272 (124.6, 128–553)	235 (33.8, 198–286)	0.420

[i] SD, standard deviation; M, methylated; P, partially methylated; U, unmethylated.

Association between STK11 promoter methylation and TNM/AJCC staging

To investigate whether the STK11 promoter methylation status is associated with the disease stage of RCC, the distributions of TNM and AJCC stages among the three groups were investigated. As shown in Table III, all stage distributions were significantly different between the 3 groups. There was a statistically significant difference in the distribution of the T (P=0.036), N (P=0.007) and AJCC (P<0.001) stages among the M, P, and U groups. In addition, significant or marginally significant trends were observed that the M group had more patients with advanced stage disease than the P and U groups (P<0.10 for T and N stages, P<0.05 for M and AJCC stages; residual analysis). The data suggested that the methylation status of the STK11 promoter was associated with T, N and AJCC stages in RCC.

Table III. TNM and AJCC staging based on the methylation status of the serine-threonine kinase 11 promoter (n=42).

Table III.

TNM and AJCC staging based on the methylation status of the serine-threonine kinase 11 promoter (n=42).

	Group, n (%)
Variable	M	P	U	P-value
Total, n	12	18	12
T stage				0.036
T1	4 (33.3)	8 (44.4)	8 (66.7)
T2	3 (25.0)	10 (55.6)	3 (25.0)
T3	3 (25.0)	0 (0.0)	1 (8.3)
T4	2 (16.7)	0 (0.0)	0 (0.0)
N stage				0.007
N0	5 (41.7)	15 (83.3)	11 (91.0)
N1	3 (25.0)	3 (16.7)	0 (0.0)
N2	4 (33.3)	0 (0.0.)	1 (9.0)
M stage				0.154
M0	9 (75.0)	17 (94.4)	12 (100.0)
M1	3 (25.0)	1 (5.6)	0 (0.0)
AJCC stage				<0.001
I	0 (0.0)	7 (38.9)	8 (66.7)
II	3 (25.0)	9 (50.0)	3 (25.0)
III	3 (25.0)	0 (0.0)	0 (0.0)
IV	6 (50.0)	2 (11.1)	1 (8.3)

[i] Fishers exact test showed methylation status was associated with T, N and AJCC stages. TNM, tumor-node-metastasis; AJCC, American Joint Committee on Cancer; M, methylated; P, partially methylated; U, unmethylated.

STK11 promoter methylation and survival

Since the association between methylation status and tumor stage was observed, whether the methylation status has an effect on the survival of RCC patients was then investigated. The results of Kaplan-Meier survival analysis showed that there was a significant survival difference among the three groups (log-rank test, P<0.05; Fig. 2A). Additional analysis revealed that the survival times of patients in the P (P=0.021) and U (P=0.048) groups were significantly increased compared with the M group (Fig. 2B and C). However, there was no significant difference in survival time between the U and P groups (P=0.640; Fig. 2D). The data suggest that the methylation status of the STK11 promoter has an impact on the survival of RCC patients.

Figure 2. (A) Kaplan-Meier survival curves for overall survival in ccRCC patients with different STK11 promoter methylation status. The (B) P and (C) M groups had significantly better survival than the U group. (D) Survival in the M and P groups was not significantly different.

Discussion

In the present study, STK11 promoter methylation was analyzed using specimens from 42 ccRCC patients and found an association between methylation status and cancer stage. The results showed that 28.6 and 42.9% of ccRCC samples had methylation and partial methylation at the STK11 promoter, respectively. Additional analyses found the methylation status of the STK11 promoter was associated with the T, N and AJCC stages in RCC. In addition, the M group had an increased number of patients at an advanced stage compared with the P and U groups. Furthermore, survival analyses among three groups showed that the survival time was significantly longer in both P and U groups compared with the in M group, indicating that the methylation status of the STK11 promoter has an impact on the survival of RCC patients. To the best of our knowledge, the present study is the first to report the methylation frequency of the STK11 promoter in ccRCC and its impact on the tumor stage and survival of ccRCC patients.

STK11 has multiple biological and physiological functions in cells, since knockout mice studies have shown that inactivation of STK11 has severe consequences, including tumorigenesis. Although STK11 was identified almost two decades ago (5), the studies focusing on its roles in the pathogenesis of RCC remain rare. In 1999, Avizienyte et al (15) detected no mutation in 19 RCC specimens. In 2014, Yalniz et al (16) reported an overall mutation frequency of 51.6% (32/62) in RCC patients. In 2013, Duivenvoorden et al (18) conducted a study to investigate the tumor suppressor function of STK11 in ccRCC in vitro and in vivo. Knockdown of STK11 in the ccRCC 786-O cell line increased the cell proliferation, invasion and vascular endothelial growth factor secretion. In addition, the growth of STK11 knockdown cell xenografts was significantly increased compared with the control. These results suggested a tumor suppressor function of STK11 in ccRCC. In addition, this study also investigated the expression of STK11 at the mRNA and protein levels, as well as performing immunohistochemistry staining. It was found that under-expression of STK11 in ccRCC is a comment event. However, this study did not further investigate the mechanism for the under-expression of STK11 in ccRCC (16).

A previous study has already shown that mutation of the STK11 gene may contribute to the inactivation of STK11 (24). In the present study, to determine if epigenetic alteration may also contribute to inactivation of STK11, the methylation status of the STK11 promoter region in 42 ccRCC specimens was investigated. Hypermethylation of the STK11 promoter has been demonstrated in previous studies. In a cell line study, Esteller et al (23) showed that three colorectal and one cervical carcinoma cell lines were methylated at STK11. As for sporadic primary tumors, studies showed the methylation frequency of the STK11 promoter in various tumors is rare. In the study by Esteller et al (23), a series of primary tumors were also investigated. Among colorectal, breast, gastric, pancreatic, thyroid, bladder and testicular carcinomas, only colorectal carcinoma (7.7%; 1/13) and testicular tumor (10.7%; 3/28) exhibited methylated at STK11 (23). In another study by Trojan et al (21), an overall methylation frequency of 8% (4/48) was observed in colorectal cancer. Lee et al (22) reported that promoter methylation was detected in 13.2% (21/159) of Korean patients with non-small cell lung cancer. Notably, in contrast to these studies, the present study showed a significantly increased methylation rate (28.6%; 12/42) in patients with ccRCC. This finding may indicate that epigenetic alteration plays a more important role in the pathogenesis of RCC compared with other cancer types. However, whether this relatively high methylation frequency of STK11 in ccRCC is a general phenomenon or may be attributed to the enrollment bias of the present study should be further verified in a subsequent study.

In the present study, the correlation between the methylation status of the STK11 promoter and the tumor stage and survival of ccRCC patients was further analyzed. The results showed that the methylation status of the STK11 promoter was associated with the tumor progress in RCC patients. Patients in the M group (with methylated at STK11) had a increased percentage of patients with advanced stages, using either the TNM or AJCC staging systems, compared with patients in the P and U groups. It is notable that the results of the survival analysis further support this observation. The survival time of patients with methylated STK11 (M group) was significantly lower than those in the U and P groups. The follow-up time of the M group was also significantly shorter than those of the U and P groups, which may be due to the fact that M group had a shorter survival time. These findings indicated that the methylation of STK11 may be important in the pathogenesis of RCC and may be a risk factor for the prognosis of RCC. However, this conclusion should be further verified in a subsequent study with a large sample size to exclude the possibility of enrollment bias.

There are certain limitations in the present study. The expression level of mRNA and protein was not further investigated in these tumor samples to confirm the epigenetic inactivation of STK11. Secondly, the sample size of the present study was small. Thirdly, the surrounding normal tissues of the ccRCC tumor specimens were not simultaneously analyzed to identify the methylation difference in STK11 between normal and tumor tissues. These limitations should be addressed in subsequent studies.

In summary, the present study investigated the methylation status of STK11 and its association with tumor stage and survival of ccRCC patients. The methylation frequency of STK11 was 28.4% in 42 ccRCC specimens. Patients in the M group had an increased percentage of patients with advanced stage RCC and a decreased survival time compared with the P and U groups. The present findings suggested the methylation status of STK11 may be important in the tumorigenesis of ccRCC.

Acknowledgements

The present study was supported by the Science and Technology Foundation of Guangzhou (grant no. 2014A020212580).

Glossary

Abbreviations

Abbreviations:

References