Tumor Biol. (2014) 35:12361–12368 DOI 10.1007/s13277-014-2550-4
RESEARCH ARTICLE
Novel mutations and role of the LKB1 gene as a tumor suppressor in renal cell carcinoma Zübeyde Yalniz & Hulya Tigli & Hatice Tigli & Oner Sanli & Nejat Dalay & Nur Buyru
Received: 9 May 2014 / Accepted: 25 August 2014 / Published online: 2 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The tumor suppressor LKB1 gene is a master kinase and inhibits mammalian target of rapamycin (mTOR) by activating AMP-activated protein kinase (AMPK) and AMPK-related kinases. LKB1 is a critical intermediate in the mTOR signaling pathway, and mutations of the LKB1 gene have been implicated in the development of different tumor types. Recent evidence indicates that LKB1 alterations contribute to cancer progression and metastasis by modulating vascular endothelial growth factor (VEGF) production. The Ras homolog enriched in brain (RHEB) protein is a component of the mTOR pathway and functions as a positive regulator of mTOR. However, the mechanisms and effectors of RHEB in mTOR signaling are not well known. In this study, we analyzed the expression of RHEB and HIF1α genes in correlation with LKB1 gene mutations. All coding exons and exon/intron boundaries of the LKB1 gene were analyzed by direct sequencing in 77 renal cell carcinoma (RCC) tumors and 62 matched noncancerous tissue samples. In 51.6 % of the patients, ten different mutations including four novel mutations in the coding sequences and six single nucleotide substitutions in the introns were observed. Rheb and HIF1α expression levels were not statistically different between the tumor and corresponding noncancerous tissue samples. However, expression of the Rheb gene was upregulated in the tumor samples carrying the intron 2 (+24 G→T) alteration. Z. Yalniz : H. Tigli : N. Dalay Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey H. Tigli : N. Buyru (*) Cerrahpasa Medical Faculty, Department of Medical Biology, Istanbul University, Kocamustafapasa, 34098 Istanbul, Turkey e-mail: [email protected] O. Sanli Istanbul Medical Faculty, Department of Urology, Istanbul University, Istanbul, Turkey
Association between the gene expression and tissue protein levels was also analyzed for HIF1α in a subgroup of patients, and a high correlation was confirmed. Our results indicate that the LKB1 gene is frequently altered in RCC and may play a role in RCC progression. Keywords LKB1 . Rheb . HIF1α . Mutation . Expression
Introduction Renal cell carcinoma (RCC) accounts for approximately 2 % of all adult malignancies and 90–95 % of the kidney neoplasms [1]. However, the pathogenesis of renal cell cancer and the signaling pathways and genes associated with RCC are still not well defined. The most frequent tumor is clear cell carcinoma. It is well known that clear cell carcinomas are associated with mutations or inactivation of the von HippelLindau (VHL) gene. Mutations in other genes involved in different pathways have been analyzed only in very small numbers of cases. There has been some evidence in the literature indicating the relevance of the phosphoinositide 3kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in RCC [2]. Recently, two comprehensive studies have shown that a large number of important genes are implicated in the development of renal cell cancer [3, 4]. Both studies have revealed the importance of the PI3K/AKT/ mTOR pathway in renal carcinogenesis which is affected by multiple, but less frequent mutations. mTOR is a serine/ threonine kinase and functions in two multiprotein complexes TORC1 and TORC2 [5]. mTOR kinase and autokinase activities are modulated in cells by growth factors, nutrients, energy levels, and cellular stress [6]. mTOR is inhibited by the tuberous sclerosis complex (TSC)1/TSC2 complex which receives inhibitory and activating signals via the mitogenic PI3K and LKB1/AMP-activated protein kinase (AMPK)
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signaling pathways, respectively [5, 7]. The major cellular function of the TSC1/TSC2 complex is to inhibit phosphorylation of the S6 kinase (S6K) and 4EBP1 [8]. The Ras homolog enriched in brain (RHEB) protein is a component of the PI3K pathway that positively regulates mTOR signaling. Effects of RHEB on the cell cycle are mediated by its action on the mTOR signaling pathway, and overexpression of RHEB induces phosphorylation of mTOR substrates [6, 9]. LKB1 is the causal gene of Peutz-Jeghers syndrome (PJS), and mutations of the LKB1 gene have also been implicated in several cancers [10]. LKB1 is a serine/threonine kinase and has the ability to phosphorylate at least 14 downstream proteins [11]. The main canonical target of LKB1 is the energyregulated AMPK. When cellular energy levels are depleted, LKB1 activates AMPK to restore the ATP/AMP ratio through promotion of ATP production and blocking its consumption (Fig. 1). Hence, in concert with AMPK, LKB1 restricts cell growth by activation of the TSC1/TSC2 complex and subsequent blocking of Rheb and mTOR [12]. On the other hand, experimental animal models indicate that LKB1 may negatively regulate vascular endothelial growth factor (VEGF)
Tumor Biol. (2014) 35:12361–12368
production and vascular development [13]. In hypoxic tissue, the hypoxia-inducible factor 1 (HIF1) induces expression of VEGF. HIFs are transcription factors and regulate biological processes by facilitating both oxygen delivery and adaptation of the cells to oxygen deprivation. HIF1α together with HIF1β binds to the hypoxia response elements (HREs) in the gene promoters and regulates expression of these genes which are involved in angiogenesis, energy metabolism, cell proliferation, apoptosis, and other biological processes [14]. Several reports indicate that primary and metastatic RCC is an angiogenesis-dependent and hypoxia-driven malignancy [15]. Activation of the mTOR/p70S6K signaling pathway in RCC was reported by several studies and has been associated with TSC1 and TSC2 mutations and RCC progression [3, 4, 16]. Functional importance of the mutations in the mTOR pathway in the development and therapy of RCC was recently revealed by two genomic profiling studies which indicate that the majority of these mutations have activating effects on mTOR signaling [17, 18]. However, there is no report in the literature investigating the LKB1 gene as an upstream activator of the TSC1/TSC2 complex or RHEB and as a negative regulator of VEGF in RCC. Therefore, in this study, we investigated LKB1 mutations and HIF1α and RHEB expression in association with tumor progression in RCC patients.
Materials and methods Seventy-seven tumors and 62 matched noncancerous tissue samples were obtained from patients (mean age 61.6±11.5, 57 men and 20 women) diagnosed with renal cell carcinoma and undergoing surgery in the Department of Urology at the Istanbul Medical Faculty. Immediately after surgery, samples were divided into two portions and frozen in liquid nitrogen until nucleic acid isolation. The data on gender, clinical stage, histological type, and pathological stage are shown in Table 1. The study was approved by the Istanbul Medical Faculty Ethics Committee, and a signed informed consent was obtained from all patients. DNA was obtained using a DNA isolation kit (Invitek, Berlin, Germany) according to the manufacturer’s instructions and was kept at −80 °C until used.
Fig. 1 The role of the LKB1 signaling in the mTOR signaling pathway
LKB1 mutation analysis LKB1 gene mutations were analyzed in 62 patients. PCR amplification was performed using previously described primers [16]. The Reaction conditions were as follows: 200–300 ng of genomic DNA, 10 pmol of each primer, 2 μM MgCl2, 200 μM dNTP mix, 1× PCR buffer, and 1 U Taq polymerase (Fermentas, Lithuania) in a final volume of 50 μl. The amplification consisted of an initial denaturation for 10 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 40 s with a final extension for
Tumor Biol. (2014) 35:12361–12368
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Table 1 Clinicopathological characteristics and LKB1 coding region mutations of the patients
n (%)
Exon 1 S19X
Exon 3 E145V
Exon 4/5 E199V
Exon 4/5 D194V
Exon 4/5 T212S
Exon 6 S271C
Exon 9 S404F
17 (27) 6 (10) 39 (63)
7 0 16
1 0 10
4 0 4
1 0 1
1 0 1
0 0 2
0 0 1
16 (26)
7
3
5
2
1
1
1
26 (42)
12
4
2
0
1
1
0
20 (32)
4
4
1
0
0
0
0
25 (40) 22 (36) 15 (24)
11 9 3
3 4 4
4 4 0
0 2 0
0 2 0
1 1 0
1 0 0
>10 cm 37 (60) 0.05). The level of RHEB expression was not
Tumor Biol. (2014) 35:12361–12368
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Table 2 Distribution of frequent coding region alterations and their correlation with RHEB and HIF1α expression N (%)
Tumor histology Clear cell Non-clear cell Not specified Grade Low grade (1 and 2) High grade (3 and 4) Not specified Stage 1 and 2 3 and 4 Unknown Tumor size >10 cm
RESEARCH ARTICLE
Novel mutations and role of the LKB1 gene as a tumor suppressor in renal cell carcinoma Zübeyde Yalniz & Hulya Tigli & Hatice Tigli & Oner Sanli & Nejat Dalay & Nur Buyru
Received: 9 May 2014 / Accepted: 25 August 2014 / Published online: 2 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The tumor suppressor LKB1 gene is a master kinase and inhibits mammalian target of rapamycin (mTOR) by activating AMP-activated protein kinase (AMPK) and AMPK-related kinases. LKB1 is a critical intermediate in the mTOR signaling pathway, and mutations of the LKB1 gene have been implicated in the development of different tumor types. Recent evidence indicates that LKB1 alterations contribute to cancer progression and metastasis by modulating vascular endothelial growth factor (VEGF) production. The Ras homolog enriched in brain (RHEB) protein is a component of the mTOR pathway and functions as a positive regulator of mTOR. However, the mechanisms and effectors of RHEB in mTOR signaling are not well known. In this study, we analyzed the expression of RHEB and HIF1α genes in correlation with LKB1 gene mutations. All coding exons and exon/intron boundaries of the LKB1 gene were analyzed by direct sequencing in 77 renal cell carcinoma (RCC) tumors and 62 matched noncancerous tissue samples. In 51.6 % of the patients, ten different mutations including four novel mutations in the coding sequences and six single nucleotide substitutions in the introns were observed. Rheb and HIF1α expression levels were not statistically different between the tumor and corresponding noncancerous tissue samples. However, expression of the Rheb gene was upregulated in the tumor samples carrying the intron 2 (+24 G→T) alteration. Z. Yalniz : H. Tigli : N. Dalay Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey H. Tigli : N. Buyru (*) Cerrahpasa Medical Faculty, Department of Medical Biology, Istanbul University, Kocamustafapasa, 34098 Istanbul, Turkey e-mail: [email protected] O. Sanli Istanbul Medical Faculty, Department of Urology, Istanbul University, Istanbul, Turkey
Association between the gene expression and tissue protein levels was also analyzed for HIF1α in a subgroup of patients, and a high correlation was confirmed. Our results indicate that the LKB1 gene is frequently altered in RCC and may play a role in RCC progression. Keywords LKB1 . Rheb . HIF1α . Mutation . Expression
Introduction Renal cell carcinoma (RCC) accounts for approximately 2 % of all adult malignancies and 90–95 % of the kidney neoplasms [1]. However, the pathogenesis of renal cell cancer and the signaling pathways and genes associated with RCC are still not well defined. The most frequent tumor is clear cell carcinoma. It is well known that clear cell carcinomas are associated with mutations or inactivation of the von HippelLindau (VHL) gene. Mutations in other genes involved in different pathways have been analyzed only in very small numbers of cases. There has been some evidence in the literature indicating the relevance of the phosphoinositide 3kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in RCC [2]. Recently, two comprehensive studies have shown that a large number of important genes are implicated in the development of renal cell cancer [3, 4]. Both studies have revealed the importance of the PI3K/AKT/ mTOR pathway in renal carcinogenesis which is affected by multiple, but less frequent mutations. mTOR is a serine/ threonine kinase and functions in two multiprotein complexes TORC1 and TORC2 [5]. mTOR kinase and autokinase activities are modulated in cells by growth factors, nutrients, energy levels, and cellular stress [6]. mTOR is inhibited by the tuberous sclerosis complex (TSC)1/TSC2 complex which receives inhibitory and activating signals via the mitogenic PI3K and LKB1/AMP-activated protein kinase (AMPK)
12362
signaling pathways, respectively [5, 7]. The major cellular function of the TSC1/TSC2 complex is to inhibit phosphorylation of the S6 kinase (S6K) and 4EBP1 [8]. The Ras homolog enriched in brain (RHEB) protein is a component of the PI3K pathway that positively regulates mTOR signaling. Effects of RHEB on the cell cycle are mediated by its action on the mTOR signaling pathway, and overexpression of RHEB induces phosphorylation of mTOR substrates [6, 9]. LKB1 is the causal gene of Peutz-Jeghers syndrome (PJS), and mutations of the LKB1 gene have also been implicated in several cancers [10]. LKB1 is a serine/threonine kinase and has the ability to phosphorylate at least 14 downstream proteins [11]. The main canonical target of LKB1 is the energyregulated AMPK. When cellular energy levels are depleted, LKB1 activates AMPK to restore the ATP/AMP ratio through promotion of ATP production and blocking its consumption (Fig. 1). Hence, in concert with AMPK, LKB1 restricts cell growth by activation of the TSC1/TSC2 complex and subsequent blocking of Rheb and mTOR [12]. On the other hand, experimental animal models indicate that LKB1 may negatively regulate vascular endothelial growth factor (VEGF)
Tumor Biol. (2014) 35:12361–12368
production and vascular development [13]. In hypoxic tissue, the hypoxia-inducible factor 1 (HIF1) induces expression of VEGF. HIFs are transcription factors and regulate biological processes by facilitating both oxygen delivery and adaptation of the cells to oxygen deprivation. HIF1α together with HIF1β binds to the hypoxia response elements (HREs) in the gene promoters and regulates expression of these genes which are involved in angiogenesis, energy metabolism, cell proliferation, apoptosis, and other biological processes [14]. Several reports indicate that primary and metastatic RCC is an angiogenesis-dependent and hypoxia-driven malignancy [15]. Activation of the mTOR/p70S6K signaling pathway in RCC was reported by several studies and has been associated with TSC1 and TSC2 mutations and RCC progression [3, 4, 16]. Functional importance of the mutations in the mTOR pathway in the development and therapy of RCC was recently revealed by two genomic profiling studies which indicate that the majority of these mutations have activating effects on mTOR signaling [17, 18]. However, there is no report in the literature investigating the LKB1 gene as an upstream activator of the TSC1/TSC2 complex or RHEB and as a negative regulator of VEGF in RCC. Therefore, in this study, we investigated LKB1 mutations and HIF1α and RHEB expression in association with tumor progression in RCC patients.
Materials and methods Seventy-seven tumors and 62 matched noncancerous tissue samples were obtained from patients (mean age 61.6±11.5, 57 men and 20 women) diagnosed with renal cell carcinoma and undergoing surgery in the Department of Urology at the Istanbul Medical Faculty. Immediately after surgery, samples were divided into two portions and frozen in liquid nitrogen until nucleic acid isolation. The data on gender, clinical stage, histological type, and pathological stage are shown in Table 1. The study was approved by the Istanbul Medical Faculty Ethics Committee, and a signed informed consent was obtained from all patients. DNA was obtained using a DNA isolation kit (Invitek, Berlin, Germany) according to the manufacturer’s instructions and was kept at −80 °C until used.
Fig. 1 The role of the LKB1 signaling in the mTOR signaling pathway
LKB1 mutation analysis LKB1 gene mutations were analyzed in 62 patients. PCR amplification was performed using previously described primers [16]. The Reaction conditions were as follows: 200–300 ng of genomic DNA, 10 pmol of each primer, 2 μM MgCl2, 200 μM dNTP mix, 1× PCR buffer, and 1 U Taq polymerase (Fermentas, Lithuania) in a final volume of 50 μl. The amplification consisted of an initial denaturation for 10 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 40 s with a final extension for
Tumor Biol. (2014) 35:12361–12368
12363
Table 1 Clinicopathological characteristics and LKB1 coding region mutations of the patients
n (%)
Exon 1 S19X
Exon 3 E145V
Exon 4/5 E199V
Exon 4/5 D194V
Exon 4/5 T212S
Exon 6 S271C
Exon 9 S404F
17 (27) 6 (10) 39 (63)
7 0 16
1 0 10
4 0 4
1 0 1
1 0 1
0 0 2
0 0 1
16 (26)
7
3
5
2
1
1
1
26 (42)
12
4
2
0
1
1
0
20 (32)
4
4
1
0
0
0
0
25 (40) 22 (36) 15 (24)
11 9 3
3 4 4
4 4 0
0 2 0
0 2 0
1 1 0
1 0 0
>10 cm 37 (60) 0.05). The level of RHEB expression was not
Tumor Biol. (2014) 35:12361–12368
12365
Table 2 Distribution of frequent coding region alterations and their correlation with RHEB and HIF1α expression N (%)
Tumor histology Clear cell Non-clear cell Not specified Grade Low grade (1 and 2) High grade (3 and 4) Not specified Stage 1 and 2 3 and 4 Unknown Tumor size >10 cm
