Point Mutations in the DNA Binding Domain of p53 Contributeto Glioma Progression and Poor Prognosis1

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Point Mutations in the DNA Binding Domain of p53 Contribute to Glioma Progression and Poor Prognosis

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P. P. Sarma^a, D. Dutta^b, Z. Mirza^c, K. Kr. Saikia^a^, *, and B. Kr. Baishya^b

aDepartment of Bioengineering and Technology, Gauhati University, Guwahati, 781014 India

bDepartment of Neurosurgery, Gauhati Medical College and Hospital, Guwahati, 781026i India

cKing Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

*e-mail: [email protected]

Received December 31, 2015; in final form, February 16, 2016

Abstract⎯TP53 mutations play a significant role in glioma tumorigenesis. When located in in the DNA binding domain, these mutations can perturb p53 protein conformation and its function, often culminating in altered downstream signaling. Here we describe prevalent pattern of TP53 point mutations in a cohort of 40 glioma patients and show their relevance to gliomagenesis. Point mutations in exon 5–9 of TP53 gene were detected by DNA sequencing. Possible inf luence of identified mutations at the function of p53 was studied computationally and correlated with the survival. Point mutations in TP53 were detected in 10 glioma samples (25%), out of which 70% were from high grade glioma. A total of 19 TP53 point mutations were identified, out of which 42% were found to be in the DNA binding region of p53. Computational analysis predicted 87.5% of these mutations to be “probably damaging”. In three patients with tumors possessing point mutations R273H, R248Q, Y163H and R175H and poor survival times, structural analysis revealed the nature of these mutations to be disruptive and associated with high risk for cancer progression. In high grade glioma, recurrent TP53 point mutations may be the key to tumor progression, thus, emphasizing their significance in gliomagenesis.

Keywords: TP53, point mutations, high prevalence, glioma, malignancy, tumorigenesis, prognosis DOI: 10.1134/S0026893317020182

INTRODUCTION

Gliomas are most common primary adult brain tumours originating from glial cells with an incidence of 7 per 100000 people worldwide. According to the 2007 World Health Organization (WHO) malignancy grade glioma is classified into grade I–IV. Grade I tumors are benign and mostly found in children eg., pilocytic astrocytomas. Grade II consist of astrocytoma, oligodendroglioma and oligoastrocytoma and has higher tendency for malignant transformation.

Grade III glioma tumors have high cellular density and include anaplastic astrocytoma, anaplastic oligo- dendroglima and anaplastic oligoastrocytoma. Grade IV glioma are the most malignant ones and are often referred to as glioblastomas [1].

Recent large scale analysis of glioma has revealed a number of genetic alterations responsible for gliomagenesis [2, 3]. These alterations cause disruption to the key signalling pathways such as p53, Rb and RTK/

Ras/PI3K signalling and contribute towards glioma malignancy [3]. Identification of these key genetic alterations certainly improvises the therapeutic approach against the tumor [4, 5]. Mutations in TP53

are few such important molecular markers which can be frequently seen altered in glioma [6]. Wild type TP53 gene plays crucial role in various cellular process including cell-cycle arrest, apoptosis, DNA repair, autophagy etc., but often seen mutated or deleted in early stage of glioma [7]. In about 30–40% of the anaplastic astrocytoma loss of p53 function can be observed at an early stage [8]. While in glioblastomas this gene mutation can be found in 28 to 65% of the tumours [9].

In malignant glioma most of the p53 hot spot residues that frequently alter are encoded by exons 5–9 of the TP53 gene [10]. Crystal structure of p53 revealed six hotspot residues, Arg175, Arg248, Arg249, Arg273, Arg282 and Gln245 found in most of the cancers including glioma [11, 12]. Mutations in these residues can disrupt structural and functional properties of the protein to varied degrees and can affect patient prognosis. Gain of function mutations at residues Arg248 and Arg282 are seen associated with shorter patient survival [13].

Through this study we have identified and documented the prevalence pattern of TP53 point mutations in a cohort of glioma patients and established their possible clinical correlation with gliomagenesis.

1The article is published in the original.

MOLECULAR CELL BIOLOGY

UDC 575.221.22.616.006.484

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EXPERIMENTAL Collection of Tumor Samples

Study methodology was approved by institutional ethics committee of Gauhati Medical College and Hospital, Assam (no. 233/2013/229). Non-duplicate 40 fresh tumor biopsy tissues were collected from patients with written informed consent post surgery, from Neurosurgery Department, Gauhati Medical Col- lege and Hospital in between February, 2014 to Novem- ber, 2015. Tumor biopsy samples were obtained by microsurgical tumor resection and immediately stored in RNAlater solution (Sigma Aldrich, USA) to stabilize and protect nucleic acids for molecular studies. All samples were analyzed and reviewed centrally by a sin- gle neuropathologist. 2007 WHO histopathological grading system was used to classify these samples and clinical data such as age, sex, family history, previous medical history of patient, tumor location, and status at discharge were recorded. Patients were followed-up post discharges until writing this manuscript.

Molecular Screening: PCR Amplification and Sequencing of Exon 5–9 of TP53 Gene DNA from the tumour samples were extracted using Qiagen DNeasy^® Blood and Tissue Kit (Qiagen, GmbH) and quantified in a nanondrop spectropho- tometer. PCR amplification and direct sequencing of exon 5–9 of TP53 gene were performed using previously published primers [14]. PCR reactions were car- ried out in a total volume of 25 μL, which contained 0.02 u/μL of Phusion High-Fidelity DNA Polymerase (Thermo Scientific, USA), 1× Phusion HF Buffer, 0.5 μM of each forward and reverse primer, 200 μM each dNTPs, and 100 ng/μL DNA template. PCR conditions were as follows: 98°C for 2 min, 30 cycles of 98°C for 30 s, N°C for 30 s, 72°C for 30 s and 72°C for 5 min. (N°C is primer specific annealing temperature which is 45, 48, 56, 52, and 45°C for TP53 exon 5, 6, 7, 8, and 9, respectively). PCR reactions were performed in a Veriti® Thermal Cycler, (Applied Biosystems, CA) and analyzed in 1.5% non- denaturing agarose gel. Same protocol was followed for all samples.

PCR products were sequenced on both strands using the same set of primers used for amplification.

Big Dye terminator v3.1 Cycle Sequencing kit and Applied Biosystems 3730xl DNA analyzer (Applied Biosystems, USA) were used for sequencing. Novel sequences were submitted to GenBank (GenBank’s accession No.: KM488331, KM597769).

Mutational Analysis at the Protein Structure Level Understanding the effects of mutation on the protein’s structure and function is worthwhile. Structures previously determined by X-ray crystallography were taken from Research Collaborator for Structural Bio-

informatics (RCSB’s) protein data bank (PDB) (www.rcsb.org/pdb). We selected PDB ID: 1TUP (p53 complexed with DNA) as the wild structure and PDB ID: 2BIM (p53 core domain mutant M133L- V203A-N239Y-N268D-R273H) as the mutant structure for specific alignment for R273H mutation. For the rest mutations, the iterative threading assembly refinement (I-TASSER) server for automated protein structure prediction was employed to model protein structures for specific translated mutant exon sequences [15]. I-TASSER uses multiple threading alignments and iterative structural assembly simula- tions to predict and display three dimensional structures in PDB format. The structural alignment and molecular visualization was done by using PyMol [16].

Selected residues were compared using command

“align” which performs a sequence alignment followed by a structural alignment, and then carries out zero or more cycles of refinement in order to reject structural outliers found during the fit.

Computational Analysis

Possible functional effects of detected point mutations on respective protein structure were predicted using computational tool, PolyPhen-2 (available at http://genetics.bwh.harvard.edu/pph2/). Based upon PolyPhen-2 prediction point mutations were grouped in “possible damaging” or “probably damaging” or in

“benign” category.

RESULTS

Clinical Data on Patients’ Samples

Clinical features of patients’ samples used in this study are shown in Table 1. Amongst 40 patient samples, 21 belong to high grade glioma (astrocytoma with degeneration, AD, n = 9; glioblastoma, GBM, n = 5;

anaplastic astrocytoma, AA, n = 7) while 19 were low grades (diffuse astrocytoma, DA, n = 1; pilocytic astrocytoma, PA, n = 3; moderately differentiated astrocytoma, MDA, n = 12; pleomorphic xantho astrocytoma, PXA, n = 1; oligodendroglioma, OG, n= 1; gemitocytic astrocytoma, GA, n = 1) as revealed by histopathology. More number of males (n = 24) then females (n = 16) were seen effected in this cohort.

Health statuses of patients were good, with conscious oriented at the time of discharge. Except 3, all patients were alive till writing this manuscript (Table 1). No post surgical complications were recorded in these 3 patients.

Exonic TP53 Point Mutations Are Frequently Observed in High Grade Glioma Samples

Mutations in TP53 gene were detected in 10 out of 40 samples (25%) as confirmed by DNA sequencing and most of these samples belonged to high grade glioma (7/10, 70%). A total of 19 different point muta-

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Table 1. Clinical features and TP53 gene status of different glioma patient’s samples used in the study

a AA—anaplastic astrocytoma; AD—astrocytoma with degeneration; GA—gemitocytic astrocytoma; GBM—glioblastoma; DA—diffuse astrocytoma; MDA—moderately differentiated astrocytoma; OG—oligodendroglioma; PA—pilocytic astrocytoma; PXA—pleomorphic xantho astrocytoma. ^bRT—right side; LT—left side. ^cWT—wild type. Apart from point mutations in TP53 leading to amino acid substitutions in the encoded protein, all indicated SNPs were detected in the intronic regions of TP53 according to the reference sequence NC_000017.9 (NCBI36/hg18, Chr17:7512445…7531642) obtained from IARC TP53 database. All amino acid substitutions presented in this table corresponds to mutated p53. ^dNA—Not applicable.

Sample number

Age,

years Sex Histopathological grading of tumour^a

Tumor site

in the brain^b TP53 gene status^c Present status

Overall survival^d

1 10 F GBM RT WT Alive NA

2 40 M MDA LT WT Alive NA

3 11 F MDA RT WT Alive NA

4 35 F AD RT WT Alive NA

5 27 F MDA LT WT Alive NA

6 30 F AA RT Mutated (1 SNP) Alive NA

7 8 F PA RT Mutated (3SNPs, R158H) Alive NA

8 35 M PA LT WT Alive NA

9 58 M AD LT WT Alive NA

11 34 M AA RT WT Alive NA

12 21 F AD RT Mutated (2 SNPs) Alive NA

13 42 M AA RT Mutated (R273H) Expired 4 months

14 20 M AD RT Mutated (1 SNP, H168Q) Alive NA

15 52 M GBM RT WT Alive NA

16 30 M GBM RT Mutated (R248Q) Expired 1 month

17 50 M PXA RT Mutated (2 SNP) Alive NA

18 45 M GA LT WT Alive NA

19 18 M OG LT WT Alive NA

20 50 M GBM LT WT Alive NA

21 35 F AA LT Mutated (Y163C; R175H) Expired 4 months

22 45 M MDA RT Mutated (K305N; Y205C;

R158G; 1 SNP)

Alive NA

23 48 M AD RT WT Alive

24 40 M GBM LT WT Alive NA

25 13 F AA RT WT Alive NA

26 40 M AD RT WT Alive NA

28 46 M AD RT WT Alive NA

29 44 M AD RT Mutated (I255T) Alive NA

30 10 F AA LT WT Alive NA

31 30 M MDA RT WT Alive NA

34 11 F AD LT WT Alive NA

36 27 F DA RT WT Alive NA

37 10 F PA LT WT Alive NA

38 30 M AA RT WT Alive NA

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Fig. 1. Chromatograms of wild type and mutated TP53 gene sequences and structural analysis of p53 proteins bearing the R273H (a), R248Q (b), R175H (c) or Y163C (d) mutation.

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(b)

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R273H

Normal

R273H Mutant

H273

R273

G G G G G

G G G G G A G

A A

T T

T C

C

T

Aligned focusing on the specific residue 273.

Grey = wild; Black = mutant

R248Q

Normal

R248Q Mutant

R248

Q248

G G

G A A A

A A C C A A

C C

Black = wild; Grey = mutant

R175H

Normal

R175H Mutant

R175

G G H175

G

G G

A C

C

C C

T T

T

T Y163C

Normal

Y163C Mutant

C163

G Y163

A A A A

A A A

C

С С

C

tions were detected in these TP53 mutated samples. Nine among them were detected in the intronic regions while other 10 resulted to amino acid substitutions (R158G, Y163C, H168Q, R175H, Y205C, R248Q, I255T, R273H

and K305N) occurred in the DNA binding domain of the protein. Patients with gliomas bearing Y163C, R175H, R248Q and R273H mutations in the p53 died within 4 months post surgery (Table 1).

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p53 Mutations in the DNA Contact Domain Disrupt the Protein’s Tumor Suppressive Activity

R273H. Structural analysis revealed R273H to be a hotspot mutation resulting in the disruption of DNA contact points and can alter the overall structure of the protein to prevent DNA binding (Fig. 1a). The partic- ular substitution Arg (wild type) to His (mutant) at position 273 occurs within the core of DNA-binding domain which contains the sequence-specific DNA binding activity of the p53 protein (residues 102–292), will potentially result in loss of DNA binding (Fig. 1a).

R248Q. Residue R248 interacts directly with the DNA helix minor groove as revealed by crystallo- graphic studies and any mutation on this position (reported hotspot mutations are R248Q and R248W) is known as DNA contact mutation, the other prime contact mutation residue being R273. The mutational change from basic to an acidic amino acid might be perturbing and destabilizing the loop region (Fig. 1b).

R175H. The residue R175 is the third most frequently mutated p53 codon in human cancers. The mutation of arginine to histidine at position 175 of p53 affects the protein’s structural conformation as seen in Fig. 1c.

Y163C. The Y163C is a substitution missense mutation [17]. Structural analysis revealed that cysteine at position 163 might form a disulfide bond with the other neighboring cysteine residues present. This might contribute to the domain stability and overall p53 protein’s stability. Also as the tyrosine side chain is bigger than that of cysteine, the mutation of tyrosine 163 into cysteine may disrupt the DNA binding (Fig. 1d).

DISCUSSION

Since past few years the importance of molecular markers in glioma research has seen gradual increase.

These molecular markers can not only help in deci- phering the mechanism behind tumorigenesis but could also predict survival benefit associated with the same. TP53 is one of those markers which are diag-

nostically or prognostically proved to be important in glioma tumorigenesis [6]. In our study the prevalence pattern of TP53 gene mutation and its clinical correlation with respect to tumor progression and patient’s prognosis was evaluated.

Through sequencing TP53 gene we could able to identify nine different amino acid substitutions in the p53 protein, namely: R158G, Y163C, H168Q, R175H, Y205C, R248Q, I255T, R273H and K305N. These mutations affect the DNA binding domain of the protein. Some of these mutations are classified as DNA contact mutations (e.g. mutation in R248 and R273) or structural mutations (e.g. mutations in Y163 and R175) [9]. Most of these mutations were identified in the high grade glioma samples indicating a possible role in tumor progression. Computational analysis also revealed nature of most of the identified p53 mutations to be “probably damaging” (Table 2).

Literature citing poor clinical outcome associated with TP53 gene mutations in anaplastic astrocytoma is limited. In this study, shorter patient survival associated with R248Q, R273H, Y163H and R175H were documented among high grade glioma patients. Ear- lier poor prognostic effects of TP53 hotspot mutations were shown in low grade astrocytomas and oligodendroglioma [18]. Structural analysis of these p53 mutants revealed their possible inf luence in tumor progression by disrupting protein structure.

The highly conserved core DNA-binding p53 domain structure consists of a β-sandwich that serves as a scaffold for 2 large β-turn loops stabilized by a tet- rahedrally coordinated zinc atom and a loop-sheet- helix motif which forms the DNA binding surface of p53 [19, 20]. Substitutions in the core region comprise the majority of the p53 mutations identified in tumors.

Residues from the loop-sheet-helix motif interact in the major groove of DNA, while an arginine residue from one of the two large loops interacts in the minor groove. The structure supports the hypothesis that DNA binding is critical for the biological activity of p53, and provides a framework for understanding how Table 2. Possible effects of p53 mutations as predicted by PolyPhen-2

Protein Substitution PolyPhen-2 prediction score Effect

p53 R158G 0.942 Probably damaging

Y163C 0.995 Probably damaging

H168Q 0.805 Possibly damaging

R175H 0.089 Benign

Y205C 0.992 Probably damaging

R248Q 0.976 Probably damaging

I255T 0.978 Probably damaging

R273H 0.96 Probably damaging

K305N 0.979 Probably damaging

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mutations inactivate it [11]. The mutation R273H is located around the DNA-binding face of the protein.

In the wild structure, the arginine (pI 10.76) goes into the minor groove of the DNA, forming a strong stabi- lizing interaction. When mutated to aromatic amino acid histidine, this interaction gets lost. The residue 273 contacts the DNA directly and also seems to be involved in positioning other DNA-binding amino acids. The cancer hot spot mutation R273H simply removes an arginine involved in DNA binding without causing significant structural distortions in neigh- bouring residues as seen in PDB ID 2BIM [21]. Alter- ations at hotspot Arg248 is classified as DNA contact mutations, i.e. Arg248 is involved directly in binding to DNA; also, leads to gain of novel oncogenic func- tions and reportedly has significant association with shorter patient survival [13]. Likewise, M. Brazdova et al. [22] described a molecular mechanism of gene regulation where mutant p53 (R273H), can contribute towards oncogenesis. These p53 mutants can target and transcriptionally activates many genes that help in malignant transformation of cancer cells [23].

The mutations of residues that are not in direct contact with DNA but function to stabilize the DNA binding structure are referred to as ‘structural mutants’. In contrast to contact mutants, structural mutants affect the overall architecture of the DNA- binding surface and change the protein conformation [11]. Human cancer hotspot mutations R175H, Y220C, G245S, R249S and R282W belong to this category. In R175H, mutation to His residue causes distortions that directly interfere with zinc binding. How- ever, substitutions introducing smaller residues at this position evince less destabilization with partial reten- tion of function [24]. Friedlander et al. [25] examined DNA binding activity of p53 proteins at 25 or 37°C using labelled oligonucleotides and electrophoretic mobility shift assay and reported significantly lesser DNA binding by p53 His175-mutant at 25°C, display- ing only ~5% of wild type p53 activity. They also observed that fresh preparations of His175-p53 bound very well to DNA but then rapidly lost activity, indicating its conformational instability [25].

Wild type p53 protein acts as a tumor suppressor due to its DNA-binding activity. But in silico analysis suggests that missense mutations at the core DNA- binding domain may disrupt the protein’s DNA-binding ability and results in loss of its tumor suppressive property.

Thus, we investigated a prevalence pattern of TP53 mutations among adult patients with glioma and underscore their possible association role with poor prognosis. But further studies of p53 mutations involved in DNA binding are required to establish a strong clinical-molecular relation between these mutations and glioma tumorigenesis.

ACKNOWLEDGMENTS

Partha Pratim Sarma has received INSPIRE Fel- lowship (no. DST/INSPIRE Fellowship/2014/219) from Department of Science and Technology, Minis- try of Science and Technology of India as a financial support to carry out this study.

Conflict of interest: None declared.

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