No Tumor Suppressor Role for LKB1 in Prostate Cancer

Abstract

To elucidate the pathogenesis of prostate diseases, following in silico analysis, the LKB1 gene was selected for further investigation. The LKB1 gene has been associated with poor prognosis and is frequently mutated in different types of cancers. In this study, 50 benign prostatic hyperplasia (BPH) and 57 prostate cancer (PCa) tissues, including matched normal tissue for the patients, were analyzed by qRT-PCR and DNA sequencing for LKB1 expression and the mutation profile, respectively. Expression of LKB1 was increased in 60.7% of the PCa tissues compared with noncancerous tissue samples (p ≤ 0.001). However, LKB1 expression was lower when compared with normal tissues in BPH (p = 0.920). Four coding sequence alterations were detected in BPH. Three silent mutations were located in codons 9, 32, and 275 and a missense mutation was observed in codon 384. Six alterations were identified in the intronic regions of the LKB1 gene in both PCa and BPH. Five mutations were observed in both patient groups. A new alteration in intron 6 was observed in a patient with PCa. The LKB1 gene may be associated with benign transformations rather than the tumors in prostate pathogenesis when its expression and mutation status are considered. However, the mechanism of LKB1 in PCa needs further studies.

Introduction

Prostate cancer (PCa) is the second most common cancer in men and accounts for 15% of all cancers diagnosed (Ferlay et al., 2015). It is the second most common reason of death related to cancer after lung cancer with a rate of 10% (Siegel et al., 2014). Therefore, early diagnosis of PCa is of uttermost importance. An important marker used for screening of PCa is the prostate-specific antigen (PSA). Still, routine biopsy for levels of PSA ≥4 ng/mL in older men has a low diagnostic efficiency of 30% for PCa and provides an unreliable diagnostic marker (Catalona et al., 1994). PSA expression is increased during infections and inflammation in benign prostatic hyperplasia (BPH), and management of patients with high PSA levels has a higher rate of false negativity, resulting in repeated prostatic Tru-cut biopsies with transrectal ultrasonography and an increased rate of complications related to this invasive procedure (Loeb et al., 2013).

Some novel markers such as PCA3; prostate-specific membrane antigen (PSMA); and the TPMRSS2-ERG gene fusion have been suggested as possible biomarkers, but the utility of these as diagnostic and prognostic parameters is still not established and no single biomarker has ideal characteristics for detection and evaluation of PCa (Fiella et al., 2018). Therefore, for the diagnosis and management of prostatic diseases, new biomarkers that can selectively distinguish patients with BPH and PCa are essential. Identification of genes displaying differential expression in BPH may provide new insights into the mechanisms of prostatic disease and these genes may serve as potential candidates for future targets of therapy.

To figure out the principal genes and related pathways playing a role in the pathogenesis of prostate diseases, PubMed was text-mined using the Pathway Studio 9 program (Elsevier, Amsterdam, the Netherlands) and its internal ResNet 9 Mammalian database (Nikitin et al., 2003). Using gene set enrichment analyses, various interacting genes in curated pathways related to both PCa and benign prostate hyperplasia were determined. After the subnetwork enrichment analysis, genes were regrouped as those with known or unknown roles in PCa and benign prostate hyperplasia. Among these candidate genes with unclear regulatory function in prostate diseases, LKB1 was chosen for further analysis.

The LKB1 gene is frequently mutated in different types of cancers. Originally defined in patients with Peutz–Jeghers syndrome, LKB1 functions in multiple cellular processes, including regulation of the cell cycle, apoptosis, and metabolism (Hemminki et al., 1998a). Recent evidence indicates that LKB1 expression is associated with poor prognosis in cancer (He et al., 2014; Sun et al., 2016; Xiao et al., 2016). Although mutations of LKB1 have been investigated in different tumors, they have not been analyzed in human PCa. On the other hand, LKB1 expression in human prostate tumors has been investigated only in a single study (Lu et al., 2015).

In this study, we aimed to determine the differential expression and sequence alterations of the LKB1 gene in both PCa and BPH to clarify its role, if any, in the pathophysiology of PCa and to investigate its potential role as a diagnostic marker.

Methods

Samples of 50 BPH and 57 PCa tissues, including matched normal tissue controls, were obtained from BPH and clinically localized PCa patients. The study was approved by the Cerrahpasa Medical Faculty Ethics Committee of Istanbul University-Cerrahpasa (Approval No.: 83045809-604.01.02-161870), in accordance with ethical standards laid down in the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

Tissue samples for BPH were taken from patients by either transurethral resection of the prostate (TUR-P) or open prostatectomy. Tissue samples for PCa were obtained by open radical retropubic prostatectomy. The mean ages of the patients with BPH and PCa at the time of surgery were 69.26 ± 8.93 and 59.96 ± 6.19, respectively. Bipolar TUR-P was performed in 40%, monopolar TUR-P in 30%, and open prostatectomy in 30% of the patients with BPH. In patients with PCa, the pathological T stages were pT2 in 95% and pT3 in 5%. Patients who underwent open prostatectomy had significantly higher prostate volumes and PSA levels compared with TUR-P and radical prostatectomy groups (p < 0.001). Diabetes mellitus (DM) was present in 48% and 51% of patients with BPH and PCa, respectively. The respective rates for intake of oral metformin were 61% and 59%. Seventeen percent and 23% of patients with BPH and PCa, respectively, were smokers. All patients with PCa had adenocarcinoma. The mean preoperative PSA level was 6.78 ± 2.97 ng/mL and the postoperative mean level was 0.065 ± 0.19 ng/mL (medians were 6 and 0.001, respectively). The mean volume ratio of cancerous tissue within the prostate gland was 24.96% ± 17.45% (median 23%) in patients with malignant tumors. Clinicopathological characteristics of patients with localized PCa are shown in Table 2.

RNA isolation and cDNA preparation

Total cellular RNA was obtained from both tumor and nontumor samples using the PureLink^® RNA Mini Kit (Thermo Fisher Scientific, Inc., Waltham, MA). Nucleic acid yields were measured spectrophotometrically using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.) and 300 ng of total RNA in a reaction volume of 20 μL. The G6PDH (glucose-6-phosphate dehydrogenase) gene was used as the reference gene for normalization. Five microliters of cDNA was used for each real-time PCR, which was performed in a 20-μL reaction volume containing 1 × PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Inc.) and gene-specific primers [LKB1 (forward 5′-AACCGGCCAAGAGGTTCT-3′ and reverse 5′-GATGGGCACTGGTGCTTC-3′) and G6PDH (forward 5′-ATGCCTTCCATCAGTCGGATACA-3′and reverse 5′-ATAGCCCACGATGAAGGTGTTTTC-3′)].

The target and reference genes were coamplified in the same reaction in different wells. Amplification was performed at 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s for 40 cycles with an initial denaturation step at 95°C for 10 min, using the LightCycler 480 II platform (Roche Diagnostics, Basel, Switzerland). The expression levels were calculated by the 2^−ΔΔCt method (Schmittgen and Livak, 2008).

LKB1 coding sequence analysis

Genomic DNA isolated from the tumor and normal tissues was used to amplify all exons and exon–intron boundaries of the LKB1 gene. Amplification reactions were performed using primers previously described (Onozato et al., 2007). The reaction contains 200–300 ng of genomic DNA, 10 pmol of each primer, 2.5 mM of MgCl₂, 200 μM of dNTP mix, 1 × PCR buffer, and 1 U of Taq polymerase (Thermo Fisher Scientific, Inc.) in a final volume of 50 μL. Following an initial denaturation step at 95°C for 10 min, all exons of the LKB1 gene were amplified for 35 cycles at 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s, with a final elongation step at 72°C for 10 min. After electrophoresis on 2% agarose gels and visualizing under ultraviolet light for confirmation of the products, sequencing was performed using an Applied Biosystems 3100 automated DNA sequencer (ABI, Pleasanton, CA) and the ABI Prism BigDye Terminator cycle sequencing ready reaction kit. Sequencing was performed twice in both forward and reverse directions for the mutation-positive samples.

Statistical analyses

Statistical analyses were performed using the IBM SPSS Statistics software (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, version 21.0. Armonk, NY: IBM Corp.). We used the unpaired χ² test to analyze the differences in expression levels in the tumor and normal tissues for both genes. The correlation of LKB1 expression with clinicopathologic parameters such as histologic grade, age, gender, and prostate volume was analyzed using Spearman's rho and chi-square tests. ROC analyses were performed for diagnostic discrimination of BPH and PCa. p < 0.05 was considered statistically significant.

Results

When we evaluated the expression level of the LKB1 gene in PCa tumor tissues compared with matched noncancerous tissues, we observed highly significant differences. The LKB1 gene expression was upregulated in tumor tissues compared with normal tissues (Table 1, p ≤ 0.001). LKB1 overexpression was observed in 60.7% of tumor tissues compared with noncancerous tissue samples. The average increase in the LKB1 mRNA level in tumor tissues was 40% compared with corresponding normal tissues. When the association of LKB1 expression levels with clinicopathological parameters was analyzed, the LKB1 gene expression levels did not show any significant difference according to age, DM, metformin use, smoking status, prostate volume, PSA levels, Gleason group, and lymphatic/vascular/perineural invasion (Table 2).

Table 1.

Expression Level of the LKB1 Gene in Tissue Samples with Prostate Cancer and Benign Prostatic Hyperplasia

	LKB1 Ct Mean ± SD	G6PD Ct Mean ± SD	ΔCt Mean ± SD	ΔΔCt Mean ± SD	2^−ΔΔCt	p ^a
PCa
Tumor	29.22 ± 2.49	30.78 ± 2.7	−1.56 ± 1.38	−0.49 ± 1.33	1.4	0.009
Normal	29.91 ± 2.48	30.98 ± 3.18	−1.07 ± 1.37	0	1	0.009
BPH
Tumor	30.95 ± 2.77	31.88 ± 4.29	−0.93 ± 3.19	0.27 ± 2.67	0.82	0.920
Normal	30.87 ± 3.02	32.07 ± 4.09	−1.2 ± 2.66	0	1	0.920

Statistical analysis was performed by using the Wilcoxon signed rank test.

BPH, benign prostatic hyperplasia; PCa, prostate cancer; SD, standard deviation.

Table 2.

Clinicopathological Characteristics and LKB1 Expression in Patients with Prostate Cancer

Clinical parameters	LKB1 expression n (%)
Clinical parameters	Increase	Decrease	No change	p ^a
Age, years
≤60	14 (25)	8 (14.3)	3 (5.4)	0.663
>60	17 (30.4)	7 (12.5)	4 (7.1)
Unknown	3 (5.4)	0 (0)	0 (0)
Smoker
No	17 (30.4)	10 (17.9)	5 (8.9)	0.546
Yes	14 (25)	5 (8.9)	2 (3.6)
Unknown	3 (5.4)	0 (0)	0 (0)
DM
No	16 (28.6)	8 (14.3)	3 (5.4)	0.683
Yes	15 (26.8)	7 (12.5)	4 (7.1)
Unknown	3 (5.4)	0 (0)	0 (0)
Metformin
No	6 (10.7)	2 (3.6)	3 (5.4)	0.327
Yes	8 (14.3)	5 (8.9)	0 (0)
Unknown	20 (35.7)	8 (14.3)	4 (7.1)
Gleason group
1	14 (25)	8 (14.3)	4 (7.1)	0.809
2	13 (23.2)	6 (10.7)	2 (3.6)
3	4 (7.1)	1 (1.8)	1 (1.8)
Unknown	3 (5.4)	0 (0)	0 (0)
Perineural invasion
Absent	16 (28.6)	8 (14.3)	3 (5.4)	0.683
Present	15 (26.8)	7 (12.5)	4 (7.1)
Unknown	3 (5.4)	0 (0)	0 (0)
Vascular invasion
Absent	29 (51.8)	14 (25)	6 (10.7)	0.627
Present	2 (3.6)	1 (1.8)	1 (1.8)
Unknown	3 (5.4)	0 (0)	0 (0)
Lymphatic invasion
Absent	24 (42.9)	12 (21.4)	5 (8.9)	0.688
Present	7 (12.5)	3 (5.4)	2 (3.6)
Unknown	3 (5.4)	0 (0)	0 (0)
Capsule invasion
Absent	26 (46.4)	13 (23.2)	6 (10.7)	0.714
Present	5 (8.9)	2 (3.6)	1 (1.8)
Unknown	3 (5.4)	0 (0)	0 (0)
Pre-op. PSA
≤6	14 (25)	10 (17.9)	2 (3.6)	0.245
>6	17 (30.4)	5 (8.9)	5 (8.9)
Unknown	3 (5.4)	0 (0)	0 (0)
Prostate volume, mL
≤47	19 (33.9)	5 (8.9)	4 (7.1)	0.250
>47	12 (21.4)	10 (17.9)	3 (5.4)
Unknown	3 (5.4)	0 (0)	0 (0)

Statistical analyses were performed by using the chi-square test.

DM, diabetes mellitus; PSA, prostate-specific antigen.

In contrast to PCa, expression of the LKB1 gene was lower in BPH tissues compared with their matched normal counterparts. The decrease in expression of LKB1 was 20%. Subgroup analyses of patients with BPH, considering the presence of age, DM, metformin use, smoking status, prostate volume, and PSA levels, did not reveal any significant difference with regard to the LKB1 gene expression (Table 3).

Table 3.

Clinicopathological Characteristics and LKB1 Expression in Patients with Benign Prostatic Hyperplasia

Clinical parameters	LKB1 expression n (%)
Clinical parameters	Increase	Decrease	No change	p ^a
Age, years
≤69	12 (25.5)	10 (21.3)	1 (2.1)	0.922
>69	13 (27.7)	9 (19.1)	1 (2.1)
Unknown	1 (2.1)	0 (0)	0 (0)
Smoker
No	18 (38.3)	12 (25.5)	0 (0)	0.268
Yes	7 (14.9)	7 (14.9)	2 (4.3)
Unknown	1 (2.1)	0 (0)	0 (0)
DM
No	15 (31.9)	9 (19.1)	0 (0)	0.429
Yes	10 (21.3)	10 (21.3)	2 (4.3)
Unknown	1 (2.1)	0 (0)	0 (0)
Metformin
No	4 (8.5)	3 (6.4)	1 (2.1)	0.407
Yes	5 (10.6)	6 (12.8)	1 (2.1)
Unknown	17 (36.2)	10 (21.3)	(0)
Pre-op. PSA
≤3.8	15 (31.9)	7 (14.9)	0 (0)	0.161
>3.8	9 (19.1)	12 (25.5)	2 (4.3)
Unknown	2 (4.3)	0 (0)	0 (0)
Prostate volume, mL
≤67	16 (34)	6 (12.8)	0 (0)	0.120
>67	9 (19.1)	13 (27.7)	2 (4.3)
Unknown	1 (2.1)	0 (0)	0 (0)

Statistical analyses were performed by using the chi-square test.

To determine the mutation status of the LKB1 gene in PCa or BPH patients, we screened the LKB1 gene by direct sequencing. Although we identified four different coding sequence alterations in samples from patients with BPH, no mutations were detected in any PCa tissue samples (Table 4). Three of these alterations were silent mutations occurring as a result of G to A, C to G, and G to A transitions at the third position of codons 9, 32, and 275, respectively. The remaining mutation was a C to G transition at the first nucleotide of codon 384 and resulted in a tryptophan to arginine substitution. In addition to coding sequence alterations, we detected six different alterations in intronic sequences of the LKB1 gene in PCa and BPH. Five of these alterations were common in both patient groups with different frequencies. Additionally, we observed a T to A transition in intron 6 of the gene in the tumor from a patient with PCa (Table 5).

Table 4.

Genetic Alterations in the Coding Regions of the LKB1 Gene in Benign Prostatic Hyperplasia

Position	Genetic alteration	Patient (%)	LKB1
Codon 9 CTG>CTA (c.27 G>A, p.Leu9Leu)	Homozygous	0 (0)	Exon 1
Codon 9 CTG>CTA (c.27 G>A, p.Leu9Leu)	Heterozygous	2 (4.1)	Exon 1
Codon 32 ACC>ACG (rs791752120) (c.96 C>G, p.Thr32Thr)	Homozygous	0 (0)	Exon 1
Codon 32 ACC>ACG (rs791752120) (c.96 C>G, p.Thr32Thr)	Heterozygous	1 (2)	Exon 1
Codon 275 CCG>CCA (c.825 G>A, p.Pro275Pro)	Homozygous	0 (0)	Exon 6
Codon 275 CCG>CCA (c.825 G>A, p.Pro275Pro)	Heterozygous	1 (2)	Exon 6
Codon 384 CGG>TGG (rs752015385) (c.1150 C>T, p.Arg384Trp)	Homozygous	0 (0)	Exon 9
Codon 384 CGG>TGG (rs752015385) (c.1150 C>T, p.Arg384Trp)	Heterozygous	2 (4.1)	Exon 9

Table 5.

Genetic Alterations in the Noncoding Regions of the LKB1 Gene

Position	Genetic alteration	Patient (%)	LKB1
PCa
+36 G → T (rs3764640) (c.290 + 36 G>T)	Homozygous	2 (3.7)	Intron 1
+36 G → T (rs3764640) (c.290 + 36 G>T)	Heterozygous	24 (44.4)	Intron 1
+24 G → T (rs2075604) (c.374 + 24 G>T)	Homozygous	1 (1.9)	Intron 2
+24 G → T (rs2075604) (c.374 + 24 G>T)	Heterozygous	3 (5.5)	Intron 2
7 bp dup +40–46 (rs58579265) (c.464 + 40_46dup7)	Homozygous	2 (3.7)	Intron 3
7 bp dup +40–46 (rs58579265) (c.464 + 40_46dup7)	Heterozygous	20 (37.7)	Intron 3
−51 T → C (rs2075606) (c.597–51 T>C)	Homozygous	2 (4)	Intron 3
−51 T → C (rs2075606) (c.597–51 T>C)	Heterozygous	23 (44.2)	Intron 3
+45 T → A (c.862 + 45 T>A)	Homozygous	0 (0)	Intron 6
+45 T → A (c.862 + 45 T>A)	Heterozygous	1 (2)	Intron 6
+7 G → C (rs2075607) (c.920 + 7 G>C)	Homozygous	0 (0)	Intron 7
+7 G → C (rs2075607) (c.920 + 7 G>C)	Heterozygous	2 (3.7)	Intron 7
BPH
+36 G → T (rs3764640) (c.290 + 36 G>T)	Homozygous	3 (6.1)	Intron 1
+36 G → T (rs3764640) (c.290 + 36 G>T)	Heterozygous	22 (45)	Intron 1
+24 G → T (rs2075604) (c.374 + 24 G>T)	Homozygous	0 (0)	Intron 2
+24 G → T (rs2075604) (c.374 + 24 G>T)	Heterozygous	6 (12.2)	Intron 2
7 bp dup +40–46 (rs58579265) (c.464 + 40_46dup7)	Homozygous	3 (6.1)	Intron 3
7 bp dup +40–46 (rs58579265) (c.464 + 40_46dup7)	Heterozygous	18 (36.7)	Intron 3
-51 T → C (rs2075606) (c.597–51 T>C)	Homozygous	3 (6.25)	Intron 3
-51 T → C (rs2075606) (c.597–51 T>C)	Heterozygous	21 (43.75)	Intron 3
+7 G → C (rs2075607) (c.920 + 7 G>C)	Homozygous	2 (4.1)	Intron 7
+7 G → C (rs2075607) (c.920 + 7 G>C)	Heterozygous	13 (26.5)	Intron 7

Discussion

Early diagnosis of PCa is essential as the disease is the second most common cancer in men as well as the second most common cause of cancer deaths after lung cancer (Siegel et al., 2014; Ferlay et al., 2015). Currently, PSA is an important marker used for screening of PCa. However, especially for low PSA levels, clinically critical PCa can be missed when using the PSA strategy (Thompson et al., 2004). In addition, some tumors showing neuroendocrine differentiation produce low levels of PSA (Okotie et al., 2007). Thus, better and more specific markers other than PSA and PSA density are needed to identify PCa with low false negativity rates (Mettlin et al., 1994; Wever et al., 2010). In this study, we investigated the utility of the LKB1 gene in PCa and BPH.

One of the main characteristics of cancer cells is unlimited replication potential, which results in high energy requirement. The energy sensor of the cell is adenosine monophosphate-activated kinase (AMPK) and its activation protects cells against physiological and pathological stress. Upon activation, it switches on the metabolism to generate ATP while blocking energy expenditure (Oakhill et al., 2009). Therefore, AMPK plays an important role in regulating energy hemostasis, cell proliferation, senescence, and differentiation (Hardie et al., 2012). LKB1 activates AMPK through phosphorylation (Carling, 2006).

LKB1 has been accepted as a tumor suppressor gene because it is frequently mutated in the cancer-prone PJS (Hemminki et al., 1998b). LKB1 mutation frequency has been investigated in various types of cancers. However, the results are controversial and mutation types and rates are different in various cancers (Avizienyte et al., 1998; Su et al., 1999; Kim et al., 2004; Kenanli et al., 2010; Tigli et al., 2013; Yalniz et al., 2014). The mutation rate of the LKB1 gene in PCa has not yet been investigated. In a recent study using comprehensive genomic profiling, 1356 formalin-fixed, paraffin-embedded tissue sections of PCa samples were analyzed among other solid tumors. Alterations were identified in 18 PI3K pathway-associated genes, including LKB1. Although genetic alterations were detected in 46% of PCa samples, consistent with our results, none of these alterations were located in the LKB1 gene (Milis et al., 2019).

In another study, Ikediobi et al. (2006) investigated mutations of 24 known cancer genes in the NCI-60 cell line set and a frame-shift deletion (p.K178fs*86) has been identified in the LKB1 gene in the Du145 PCa cell line. Except those database analyses, there is no study in the literature analyzing mutations of the LKB1 gene in PCa. Therefore, we investigated the LKB1 mutation frequency in prostate tissue samples from patients with BPH or PCa. Although four coding sequence alterations were identified in BPH samples, we did not observe any alteration in PCa tumor samples. Previous reports indicate that LKB1 may function as a tumor promoter or tumor suppressor in a context and/or tissue-dependent manner (Baas et al., 2003; Boudeau et al., 2003; Dorfman and Macara, 2008; Zhan et al., 2012). In accordance with this, in previous studies, we have detected the M22I mutation in 20% of HNSCC tumor samples, while this mutation was never observed in bladder or renal cancers (Kenanli et al., 2010; Tigli et al., 2013; Yalniz et al., 2014).

The most frequent alteration was S19X in renal and bladder tumor samples with a frequency of 97.1% and 81%, respectively (Tigli et al., 2013; Yalniz et al., 2014). The absence of LKB1 coding sequence alterations in PCa tumor tissues suggests that it does not function as a tumor suppressor in prostate carcinogenesis. This is in accordance with a previous study that has associated LKB1 with Akt activation in head and neck cancers (Zhong et al., 2008). The PI3K/Akt pathway is known to promote tumorigenicity and progression of PCa, and more than 90% of prostate carcinoma cases display Akt activation (Graff et al., 2000; Malik et al., 2002; Kreisberg et al., 2004; Cao et al., 2013). On the other hand, alterations at the exon/intron boundaries are common in all tumor types with different frequencies.

The strength of the present study lies in simultaneous analysis of the mutations and expression of the LKB1 gene in PCa. Most of the mutations identified by our study are located within the catalytic domain or the binding region for SMARCA4/BRG1, which is an essential member of chromatin remodeling complexes and has been shown to play an important role in prostate carcinogenesis (Marignani et al., 2001; Muthuswami et al., 2019; Cyrta et al., 2020).

We also investigated LKB1 mRNA expression levels in association with LKB1 mutations in both PCa and BPH samples. Our results show that expression of LKB1 is repressed in similar fractions of the PCa and BPH tissues (40% and 42.5%, respectively). However, the mean mRNA expression level of LKB1 was higher in PCa tumor tissues than their matched normal counterparts, while lower in BPH samples. In accordance with the absence of mutation, high expression rates of LKB1 mRNA support the notion that LKB1 does not function as a tumor suppressor in PCa development and/or progression. In accordance with these results, recent evidence suggests that LKB1 may also exert an oncogenic effect (Martínez-López et al., 2012; Delgado et al., 2019). Activation of LKB1 has been found to be essential for cancer cell survival (Lee et al., 2015; Peart et al., 2015; Trapp et al., 2017). Knockdown of LKB1 leads to loss of cell viability and clonogenicity in breast cancer (Ng et al., 2012) and inhibits cell proliferation, while promoting apoptosis in hepatocellular cancer (Martínez-López et al., 2012). Upregulation of LKB1 has been reported in hepatocellular cancer and was associated with malignant characteristics, recurrence, and worse survival (Martínez-López et al., 2010). Similar data have also been reported for breast cancer (Chen et al., 2016), and LKB1 expression has been associated with poor prognostic and clinical factors (Syed et al., 2019). In line with these data in the literature, it has been suggested that LKB1 may exert different functions in different types of cancers (Tan et al., 2018).

Recently, a study investigating TCGA data from 498 PCa patients suggested that PCa can be divided into six subgroups displaying different types of genetic alterations involving the MSH1, FOXA1, ATM, BRCA1, RB1, LKB1, P53, and AR genes and leading to different metabolic expression profiles (Srihari et al., 2018). Among these, the C3 (LKB1, RB1, and HOXB13) subgroup displayed the highest deregulation for multiple pathways, was associated with worse prognosis, and had decreased LKB1 gene expression. However, this group did not represent the whole cohort with aberrant LKB1 gene expression, and no data were presented in the study on LKB1 gene expression levels in all patients with PCa. Interestingly, about one-third of the active surveillance patients were reassigned in subgroup C3 and this subgroup was found to have a 30–40% relapse rate within the first 72 months.

There is only one study in the literature investigating LKB1 mRNA expression in human prostate tumor tissues (Lu et al., 2015). In contrast to our results, this study reports a significant decrease in the LKB1 mRNA expression in PCa tissues. This may be due to using different calculation methods and ethnic origins of the patients. They also reported associations between low expression and creatinine level, advanced clinical stage, and PSA concentration. We were not able to detect any association between LKB1 expression and the clinicopathological characteristics.

On the other hand, there is another report in the literature investigating LKB1 expression in PCa at the protein level (Xu et al., 2014). According to this report, LKB1 protein levels were much lower in prostate tumor tissues than in normal tissue. The authors also reported that LKB1 mRNA was downregulated in two PCa cell lines (Du-145 and PC-3) compared with a control cell line (RWEP-1). However, it is known that the LKB1 gene is homozygously deleted in the Du-145 cell line. It is also well known that LKB1 shuttles between the nucleus and cytoplasm and its cellular location plays an important role in its catalytic activity. However, Xu et al.'s report evaluates the total cellular level of LKB1 protein. Therefore, it is not possible to evaluate its tumorigenic effect in that context.

At the same time, the LKB1 protein can be modified post-translationally by phosphorylation, acetylation, or binding to different isoforms of the pseudokinase STRADα, etc. (Marignani et al., 2007). Although investigating expression of the LKB1 protein to confirm mRNA expression levels would corroborate our results, unfortunately most of the samples made available for this study were not sufficient to analyze LKB1 protein expression by Western blotting in matched pairs of tissue specimens. However, for LKB1, a high degree of agreement between the mRNA and protein expression has been shown in PCa (Lu et al., 2015) as well as in cervical (Lao et al., 2014), pancreatic (Yang et al., 2015), and breast (Chen et al., 2016) cancers. A relevant study compared LKB1 protein expression in six benign prostatic tissue samples from cystoprostatectomy surgical specimens and 22 PCa tissue samples from radical retropubic prostatectomy surgical specimens (Grossi et al., 2015). Full-length LKB1 protein was shown in all benign samples and an immunoblotting analysis detected lower LKB1 expression in PCa. More than half of the PCa tissues showed no staining of LKB1, irrespective of grade (Grossi et al., 2015). On the other hand, we also investigated the LKB1 expression level in BPH patients. In contrast to PCa, the LKB1 mRNA expression was downregulated in these samples.

Conclusion

In conclusion, our data suggest that LKB1 is associated with a benign transformation rather than tumor formation. This is the first report on the LKB1 gene in PCa, which investigated its expression and alterations in association with clinicopathologic characteristics. We believe that our data may act as a reference, providing valuable information for future research. However, the activity of LKB1 is controlled by too many different factors, and there is a need for further functional studies to analyze the LKB1 gene in PCa in larger patient cohorts.

Footnotes

Authors' Contributions

H.K. carried out the expression analyses and participated in tissue collection and analysis and interpretation of data. A.C. and G.G. participated in the genotyping assays and expression analyses. N.D. contributed to the design and coordination of the study and to the final manuscript. H.O. provided tissue samples and clinical data. N.B. conceived the study, participated in its design and coordination, interpreted the data, and wrote the manuscript. All authors have read and approved the final manuscript.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Istanbul University Research Fund (TDK-2016-22213).

References

Avizienyte

, Roth

, Loukola

, Hemminki

, Lothe

R.A.

, Stenwig

A.E.

, et al. (1998). Somatic mutations in LKB1 are rare in sporadic colorectal and testicular tumors. Cancer Res, 58, 2087–2090.

Baas

A.F.

, Boudeau

, Sapkota

G.P.

, Smit

, Medema

, Morrice

, et al. (2003). Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J, 22, 3062–3072.

Boudeau

, Baas

A.F.

, Deak

, Morrice

N.A.

, Kieloch

, Schutkowski

, et al. (2003). MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm. EMBO J, 22, 5102–5114.

Cao

J.P.

, Zhu

, Zhou

, Li

, Liu

, Xuan

H.Q.

, et al. (2013). PLZF mediates the PTEN/AKT/FOXO3a signaling in suppression of prostate cancer. PLoS One 8, e77922.

Carling

(2006). LKB1: a sweet side to Peutz-Jegherssyndrome?. Trends Mol Med, 12, 144–147.

Catalona

W.J.

, Richie

J.P.

, deKernion

J.B.

, Ahmann

F.R.

, Ratliff

T.L.

, Dalkin

B.L.

, et al. (1994). Comparison of prostate specific antigen concentration versus prostate specific antigen density in the early detection of prostate cancer: receiver operating characteristic curves. J Urol, 152(6 Pt 1), 2031–2036.

Chen

I.C.

, Chang

Y.C.

, Lu

Y.S.

, Chung

K.P.

, Huang

C.S.

, Lu

T.P.

, et al. (2016). Clinical relevance of liver kinase B1 (LKB1) protein and gene expression in breast cancer. Sci Rep 6, 21374.

Cyrta

, Augspach

, De Filippo

M.R.

, Prandi

, Thienger

, Benelli

, et al. (2020). Role of specialized composition of SWI/SNF complexes in prostate cancer lineage plasticity. Nat Commun 11, 5549.

Delgado

T.C.

, Lopitz-Otsoa

, and Martínez-Chantar

M.L.

(2019). Post-translational modifiers of liver kinase B1/serine/threonine kinase 11 in hepatocellular carcinoma. J Hepatocell Carcinoma, 6, 85–91.

10.

Dorfman

, and Macara

I.G.

(2008). STRADalpha regulates LKB1 localization by blocking access to importin-alpha, and by association with Crm1 and exportin-7. Mol Biol Cell, 19, 1614–1626.

11.

Ferlay

, Soerjomataram

, Dikshit

, Eser

, Mathers

, Rebelo

, et al. (2015). Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer, 136, E359–E386.

12.

Filella

, Fernández-Galan

, Fernández Bonifacio

, and Foj

(2018). Emerging biomarkers in the diagnosis of prostate cancer. Pharmgenomics Pers Med, 11, 83–94.

13.

Graff

J.R.

, Konicek

B.W.

, McNulty

A.M.

, Wang

, Houck

, et al. (2000). Increased AKT activity contribute to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression. J Biol Chem, 275, 24500–24505.

14.

Grossi

, Lucarelli

, Forte

, Peserico

, Matrone

, Germani

, et al. (2015). Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38. Autophagy, 11, 2102–2113.

15.

Hardie

D.G.

, Ross

F.A.

, and Hawley

S.A.

(2012). AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol, 13, 251–262.

16.

T.Y.

, Tsai

L.H.

, Huang

C.C.

, Chou

M.C.

, and Lee

(2014). LKB1 loss at transcriptional level promotes tumor malignancy and poor patient outcomes in colorectal cancer. Ann Surg Oncol 21 Suppl, 4, S703–S710.

17.

Hemminki

, Avizienyte

, Roth

, Loukola

, Aaltonen

L.A.

, Järvinen

, et al. (1998a). A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Duodecim, 114, 667–668.

18.

Hemminki

, Markie

, Tomlinson

, Avizienyte

, Roth

, Loukola

, et al. (1998b). A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature, 391, 184–187.

19.

Ikediobi

O.N.

, Davies

, Bignell

, Edkins

, Stevens

, O'Meara

, et al. (2006). Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol Cancer Ther, 5, 2606–2612.

20.

Kenanli

, Karaman

, Enver

, Ulutin

, and Nuyru

(2010). Genetic alterations of the LKB1 gene in head and neck cancer. DNA Cell Biol, 29, 735–738.

21.

Kim

C.J.

, Cho

Y.G.

, Park

J.Y.

, Kim

T.Y.

, Lee

J.H.

, Kim

H.S.

, et al. (2004). Genetic analysis of the LKB1/STK11 gene in hepatocellular carcinomas. Eur J Cancer, 40, 136–141.

22.

Lee

S.W.

, Li

C.F.

, Jin

, Cai

, Han

, Chan

C.H.

, et al. (2015). Skp2-dependent ubiquitination and activation of LKB1 is essential for cancer cell survival under energy stress. Mol Cell, 57, 1022–1033.

23.

Kresiberg

J.I.

, Malik

S.N.

, Prihoda

T.J.

, Bedolla

R.G.

, Troyer

D.A.

, Kresiberg

, et al. (2004). Phosphorylation of Akt(Ser473) is an excellent predictor of por clinical outcome in prostate cancer. Cancer Res, 64, 5232–5236.

24.

Lao

, Liu

, Wu

, Zhang

, Liu

, Yang

, et al. (2014). Mir-155 promotes cervical cancer cell proliferation through suppression of its target gene LKB1. Tumour Biol, 35, 11933–11938.

25.

Loeb

, Vellekoop

, Ahmed

H.U.

, Catto

, Emberton

, Nam

, et al. (2013). Systematic review of complications of prostate biopsy. Eur Urol, 64, 876–892.

26.

, Sun

, and Wang

(2015). Low LKB1 expression results in unfavorable prognosis in prostate cancer patients. Med Sci Monit, 21, 3722–3727.

27.

Malik

S.N.

, Brattain

, Ghosh

P.M.

, Troyer

D.A.

, Prihoda

, Bedolla

, et al. (2002). Immunohistochemical demonstration of phospho-Akt in high Gleason prostate cancer. Clin Cancer Res, 8, 1168–1171.

28.

Marignani

P.A.

, Kanai

, and Carpenter

C.L.

(2001). LKB1 associatesd with Brg1 and is necessary for Brg1-induced growth arrest. J Biol Chem, 276, 32415–32418.

29.

Marignani

P.A.

, Scott

K.D.

, Bagnulo

, Cannone

, Ferrari

, Stella

, et al. (2007). Novel splice isoforms of STRADα differentially affect activity, complex assembly and subcellular localization. Cancer Biol Ther, 6, 1627–1631.

30.

Martínez-López

, García-Rodríguez

J.L.

, Varela-Rey

, Gutiérrez

, Fernández-Ramos

, Beraza

, et al. (2012). Hepatoma cells from mice deficient in glycine N-methyltransferase have increased RAS signaling and activation of liver kinase B1. Gastroenterology, 143, 787–798.e13.

31.

Martínez-López

, Varela-Rey

, Fernández-Ramos

, Woodhoo

, Vázquez-Chantada

, Embade

, et al. (2010). Activation of LKB1-Akt pathway independent of phosphoinositide 3-kinase plays a critical role in the proliferation of hepatocellular carcinoma from nonalcoholic steatohepatitis. Hepatology, 52, 1621–1631.

32.

Mettlin

, Littrup

P.J.

, Kane

R.A.

, Murphy

G.P.

, Lee

, Chesley

, et al. (1994). Relative sensitivity and specificity of serum prostate specific antigen (PSA) level compared with age-referenced PSA, PSA density, and PSA change. Data from the American Cancer Society National Prostate Cancer Detection Project. Cancer, 74, 1615–1620.

33.

Millis

S.Z.

, Jardim

D.L.

, Albacker

, Ross

J.S.

, Miller

V.A.

, Ali

S.M.

, et al. (2019). Phosphatidylinositol 3-kinase pathway genomic alterations in 60,991 diverse solid tumors informs targeted therapy opportunities. Cancer, 125, 1185–1199.

34.

Muthuswami

, Bailey

L.A.

, Rakesh

, Imbalzano

A.N.

, Nickerson

J.A.

, and Hockensmith

J.W.

(2019). BRG1 is a prognostic indicator and a potential therapeutic target for prostate cancer. J Cell Physiol, 234, 15194–15205.

35.

T.L.

, Leprivier

, Robertson

M.D.

, Chow

, Martin

M.J.

, Laderoute

K.R.

, et al. (2012). The AMPK stress response pathway mediates anoikis resistance through inhibition of mTOR and suppression of protein synthesis. Cell Death Differ, 19, 501–510.

36.

Nikitin

, Egorov

, Daraselia

, and Mazo

(2003). Pathway studio—the analysis and navigation of molecular networks. Bioinformatics, 19, 2155–2157.

37.

Oakhill

J.S.

, Scott

J.W.

, and Kemp

B.E.

(2009). Structure and function of AMP-activated protein kinase. Acta Physiol (Oxf), 196, 3–14.

38.

Okotie

O.T.

, Roehl

K.A.

, Han

, Loeb

, Gashti

S.N.

, and Catalona

W.J.

(2007). Characteristics of prostate cancer detected by digital rectal examination only. Urology, 70, 1117–1120.

39.

Onozato

, Kosaka

, Achiwa

, Kuwano

, Takahashi

, Yatabe

, et al. (2007). LKB1 gene mutations in Japanese lung cancer patients. Cancer Sci, 11, 1747–1751.

40.

Peart

, Valdes

Y.R

, Correa

R.J.M.

, Fazio

, Bertrand

, McGee

, et al. (2015). Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids. Oncotarget, 6, 22424–22438.

41.

Schmittgen

T.D.

, and Livak

K.J.

(2008). Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc, 3, 1101–1108.

42.

Siegel

, Ma

, Zou

, and Jemal

(2014). Cancer statistics, 2014. CA Cancer J Clin, 64, 9–29.

43.

Srihari

, Kwong

, Tran

, Simpson

, Tattam

, and Smith

(2018). Metabolic deregulation in prostate cancer. Mol Omics, 14, 320–329.

44.

G.H.

, Hruban

R.H.

, Bansal

R.K.

, Bova

G.S.

, Tang

D.J.

, Shekher

M.C.

, et al. (1999). Germline and somatic mutations of the STK11/LKB1 Peutz-Jeghers gene in pancreatic and biliary cancers. Am J Pathol, 154, 1835–1840.

45.

Sun

, Ling

, Xu

, Ma

, Li

, Cao

, et al. (2016). Decreased expression of tumor-suppressor gene LKB1 correlates with poor prognosis in human gastric cancer. Anticancer Res, 36, 869–875.

46.

Syed

B.M.

, Green

A.R.

, Morgan

D.A.L.

, Ellis

I.O.

, and Cheung

K.L.

(2019). Liver kinase B1-A potential therapeutic target in hormone-sensitive breast cancer in older women. Cancers (Basel) 11, 149.

47.

Tan

, Liao

, Liang

, Chen

, Zhang

, and Chu

(2018). Upregulation of liver kinase B1 predicts poor prognosis in hepatocellular carcinoma. Int J Oncol, 53, 1913–1926.

48.

Thompson

I.M.

, Pauler

D.K.

, Goodman

P.J.

, Tangen

C.M.

, Lucia

M.S.

, Parnes

H.L.

, et al. (2004). Prevalence of prostate cancer among men with a prostate-specific antigen level < or = 4.0ng per milliliter. N Engl J Med, 350, 2239–2246.

49.

Tigli

, Seven

, Tunc

, Sanli

, Basaran

, Ulutin

, et al. (2013). LKB1 mutations and their correlation with LKB1 and Rheb expression in bladder cancer. Mol Carcinog, 52, 660–665.

50.

Trapp

E.K.

, Majunke

, Zill

, Sommer

, Andergassen

, Koch

, et al. (2017). LKB1 pro-oncogenic activity triggers cell survival in circulating tumor cells. Mol Oncol, 11, 1508–1526.

51.

Wever

E.M.

, Draisma

, Heijnsdijk

E.A.M.

, Roobol

M.J.

, Boer

, Otto

S.J.

, et al. (2010). Prostate-specific antigen screening in the United States vs in the European Randomized Study of Screening for Prostate Cancer-Rotterdam. J Natl Cancer Inst, 102, 352–355.

52.

Xiao

, Zou

, Chen

, Gao

, Xie

, Lu

, et al. (2016). The prognostic value of decreased LKB1 in solid tumors: a meta-analysis. PLoS One 11, e0152674.

53.

, Cai

, Liu

, and Guo

(2014). LKB1 suppresses proliferation and invasion of prostate cancer through hedgehog signaling pathway. Int J Clin Exp Pathol, 7, 8480–8488.

54.

Yalniz

, Tigli

, Sanli

, Dalay

, and Buyru

(2014). Novel mutations and role of the LKB1 gene as a tumor suppressor in renal cell carcinoma. Tumour Biol, 35, 12361–12368.

55.

Yang

J.Y.

, Jiang

S.H.

, Liu

D.J.

, Yang

X.M.

, Huo

Y.M.

, Li

, et al. (2015). Decreased LKB1 predicts poor prognosis in pancreatic ductal adenocarcinoma. Sci Rep 5, 10575.

56.

Zhan

Y.Y.

, Chen

, Zhang

, Zhuang

J.J.

, Tian

, Chen

H.Z.

, et al. (2012). The orphan nuclear receptor Nur77 regulates LKB1 localization and activates AMPK. Nat Chem Biol, 8, 897–904.

57.

Zhong

, Liu

, Khuri

F.R.

, Sun

S.Y.

, Vertino

P.M.

, and Zhou

(2008). LKB1 is necessary for Akt-mediated phosphorylation pf pro-apoptotic proteins. Cancer Res, 68, 7270–7277.