Identification of h-TERT Promoter Mutations in Germline DNA from North Indian Lung Carcinoma Patients

Abstract

Lung cancer is a severe and the leading cause of cancer related deaths worldwide. The recurrent h-TERT promoter mutations have been implicated in various cancer types. Thus, the present study is extended to analyze h-TERT promoter mutations from the North Indian lung carcinoma patients. Total 20 histopathologically and clinically confirmed cases of lung cancer were enrolled in this study. The genomic DNA was extracted from venous blood and subjected to amplification using appropriate h-TERT promoter primers. Amplified PCR products were subjected for DNA Sanger sequencing for the identification of novel h-TERT mutations. Further, these identified h-TERT promoter mutations were analysed for the prediction of pathophysiological consequences using bioinformatics tools such as Tfsitescan and CIIDER. The average age of patients was 45 ± 8 years which was categorized in early onset of lung cancer with predominance of male patients by 5.6 fold. Interestingly, h-TERT promoter mutations were observed highly frequent in lung cancer. Identified mutations include c. G272A, c. T122A, c. C150A, c. 123 del C, c. C123T, c. G105A, c. 107 Ins A, c. 276 del C corresponding to −168 G>A, −18 T>A, −46 C>A, −19 del C, −19 C>T, −1 G>A, −3 Ins A, −172 del C respectively from the translation start site in the promoter of the telomerase reverse transcriptase gene which are the first time reported in germline genome from lung cancer. Strikingly, c. −18 T>A [C.T122A] was found the most prevalent variant with 75% frequency. Notwithstanding, other mutations viz c. -G168A [c. G272A] and c. −1 G>A [c. G105A] were found to be at 35% and 15% frequency respectively whilst the rest of the mutations were present at 10% and 5% frequency. Additionally, bioinformatics analysis revealed that these mutations can lead to either loss or gain of various transcription factor binding sites in the h-TERT promoter region. Henceforth, these mutations may play a pivotal role in h-TERT gene expression. Taken together, these identified novel promoter mutations may alter the epigenetics and subsequently various transcription factor binding sites which are of great functional significance. Thereby, it is plausible that these germline mutations may involve either as predisposing factor or direct participation in the pathophysiology of lung cancer through entangled molecular mechanisms.

Introduction

Lung Cancer is one of most common cancers in the world. This cancer was found mostly in men over 50 years of age, notwithstanding, most often at the age of 60–75 years. Strikingly, it is diagnosed very rarely under 40 years of age [1, 2]. Lung cancer is a malignant tumor characterized by uncontrolled cell growth in the lung tissues. The lung cancer is usually classified into two types such as small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) on the basis of their appearance under microscope. SCLC is a distinct clinical pathological entity, characterized by neuro-endocrine differentiation, early metastatic spread and initial responsiveness to cytotoxic therapy [3]. On other hand, NSCLC is a group of lung cancers that behave similarly such as squamous cell carcinoma and adenocarcinoma [4]. Lung carcinoma patients are associated with coughing, loss of weight, shortness of breath and chest illness. Notably, 85% of lung cancer is due to long term tobacco smoking [5]. Additionally, other factors including random gas, asbestos, some forms of air pollution and genetic components have also been implicated in the pathophysiology of lung carcinoma [6].

Telomerase and telomere biology have been implicated in the pathogenesis and progression of various cancers [7]. Telomeres are indispensable structural elements that protect the ends of chromosomes from recombination and end to end fusion. Therefore, maintenance of telomere length is important to avoid genomic instability. In fact, telomerase comprises of a multiple proteins including telomerase reverse transcriptase (h-TERT) and its integral RNA subunit. In normal somatic cells, telomeres gradually shorten after successive rounds of cell division and lead to senescence. Strikingly, tumor cells vanquish restraint by re-activating telomerase. Notwithstanding, the underlying molecular mechanism is still not clear.

For the first time, the presence of mutations within the regulatory regions of 24,667 human reference sequence [8] disclosed that insertion type mutations can significantly alter the predicted transcription factor binding sites [TFBSs]. The TFBSs includes 25 families in the promoter region which involved in intracellular signalling, cell fate, morphogenesis of organs and epithelium, development of urogenital system and neuron fate commitment [9]. A number of closely regulated genes have been found to possess promoters with a high frequency of G and C residues. For instance, SP-1 is often fraternized with near binding sites for other transcription factors viz. CTF/NF1, API, NF-kB, CIEBP and AP-2 etc.

Telomerase gene expression is regulated by h-TERT promoters through binding of transcription factors [10]. Recently, promoter mutations in h-TERT on chromosome 5 have been reported in melanoma [11]. Recurrent somatic mutations in the h-TERT promoter are associated in cancers of the central nervous system (43%), bladder (59%), thyroid (10%) and skin [12, 13]. Two hotspots at −124 and −146 base pairs upstream of h-TERT ATG start site (hereafter referred as C228T and C250T) respectively create binding for transcription factors ETS2 and increase TERT transcriptional activity [14]. Surprisingly, there are no reports available on h-TERT promoter mutations in lung carcinoma from Indian population. It is noteworthy that leukocyte telomere length has been suggested as a biomarker of cancer prognosis [15]. Therefore, the present study was executed for the identification of novel mutations in h-TERT promoter from the North Indian lung carcinoma patients for the prediction of pathophysiological consequences. Additionally, identified h-TERT promoter mutations were also analysed using bioinformatics tools.

Materials and Methods

Patients

It is a preliminary study, therefore 20 histopathologically and clinically confirmed cases of lung carcinoma from Pulmonary Medicine unit of Maharishi Markandeshwar Institute of Medical Sciences and Research (MMIMSR), Maharishi Markandeshwar University (MMU), Mullana, Ambala, India were enrolled after taking Informed consent. 20 sex and age matched controls were also recruited in the study. This study was approved by Institute ethics committee of Institution. Fine Needle Aspiration Cytology (FNAC) was done for cytopathological studies.

DNA Isolation

5 ml of venous blood from 20 cases and control subjects was collected in K₂ ethylenediaminetetraacetic acid (EDTA) vial for genetic analysis. Collected blood was stored at −20 °C till further use. Genomic DNA of high quality was isolated from whole blood by well standardized rapid extraction procedure [13]. DNA quantity was measured by spectrophotometry and isolated DNA quality was assessed by both 260/280 ratio and agarose gel electrophoresis.

Identification of TERT Promoter Mutations

A standard protocol for polymerase chain reaction (PCR) of genomic DNA was used for the genetic sequencing to identify h-TERT promotor mutations. Briefly, a fragment of h-TERT promoter was amplified using primers 5′-AGTGGATT CGCGGGCACACA-3′ (sense) and 5′-CAGCGCTACCTGAAACTC-3′ (antisense) as described earlier [16]. The resulted product was 235 bp. About 40–50 ng of genomic DNA was used in the PCR with an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of 95 °C denaturation for 10 s, annealing at 68 °C for 10 s and extension at 72 °C for 10 s. Notably, the final extension was carried out at 72 °C for 5 min. The quality of amplified PCR was checked by resolving PCR products on 2.5% agarose gel electrophoresis. The purified PCR product was further subjected to automated DNA Sanger sequencing with forward and reverse primers on ABI Prism (Model 31000, Perkin Elmer, Waltham MA, USA) using the Big Dye Terminator Cycle sequencing ready reaction kit (Perkin Elmer, Waltham MA, USA).

Bioinformatics Tools

The identified h-TERT promoter mutations were analysed for the prediction of pathophysiological consequences using bioinformatics tools such as Tfsitescan (http://www.ifti.org/Tfsitescan/) and CIIIDER (http://ciiider.com/).

Results

Clinical-Pathological Data Characteristics of the Patients

In this prospective study, 20 histopathological and clinical confirmed cases of lung cancer and 20 sex as well as age matched controls were included. Among 20 patients, 17 were males and 3 were females, with males to female ratio of 5.6:1. All the patients ranged from 45 to 65 years of age with the median age was 45 ± 8 years. Among them, 17 patients were smokers and 12 patients were alcoholic and smokers. Most of the patients were admitted with coughing, short of breathlessness and high fever.

The cytopatholgical analysis of lung biopsy of all patients demonstrated that three cases were of small cell lung carcinoma, seven cases were associated with bronchogenic lung carcinoma and three cases belonged to squamous cell carcinoma whilst seven cases were found of non-bronchoalveolar (adenocarcinoma). All the patients were accompanied with either third or fourth stage as well as poorly differentiated grade of lung carcinoma. The detailed clinical and laboratory parameters are shown in Table 1. All the 20 patients were analysed for the mutations in h-TERT promoter region.

Table 1.

Clinical, pathological and epidemiological characteristics of lung carcinoma patients

Variable	Characteristics	No. of patients
Age (years)	45–50 50–55 55–60 60–65 Median 45 ± 8	7 4 5 4
Gender	Male	17
	Female	03
	Male to Female ratio	5.6:1
Alcohol consumption	Alcoholic	12
	Non-alcoholic	08
Cigarette smoking	Smokers	17
	Non-smokers	03
Cell types	Adenocarcinoma (non bronchoalveolar)	07
	Broncho alveolar cancer	07
	Squamous cell	03
	Others small cell	03
Pathological type	Stage I
	Stage II
	Stage III or IV	All 20
Pathological grade	Well differentiated
	Moderate differentiated
	Poorly differentiated	All 20

Identification of Mutations in h-TERT Promoter from Lung Carcinoma Patients

Eight novel mutations in h-TERT promoter region using DNA Sanger sequencing are given in Fig. 1 and Table 2. The identified novel mutations are described as: c. -G168A [c. G272A] mutation, insertion of nucleotide A at position −168 of promoter region of AP2 binding site, thereby it alters the transcription binding sites onwards after −168 nucleotide sequences. This mutation occurs at 35% frequency in this study. c. −18 T>A [c. T122A] mutation was found as the most common mutation at 75% frequency. This mutation was located between transcription start sites (TSS) and ATG in AP-2 binding site region. c. −46 C>A [c. C150A] mutation was identified in AP-4 binding site of the promoter with the frequency of 10% in this study. c. −19 del C [c. 123 del C] mutation was found with 10% frequency in AP-2 binding sites of the promoter. c. −19 C>T [c. C123T] mutation was identified in AP-2 binding site of promoter region with 10% frequency. c. −1 G>A [c. G105A] mutation was identified with the substitution of A at G at position −1 of h-TERT promoter. This mutation was noticed at 15% frequency. c. −3 Ins A [C. 107 Ins A] mutation was noticed at −3 nucleotide position of promoter with the frequency of 5%. This mutation was located in NF1 binding sites of promoter region. c. −172 del C [c. 276 del C] mutation was accompanied with deletion of C nucleotide from −172 position which results in other aberrant alteration in transcription binding sites of the promoter onwards. This mutation was found with 5% frequency in this study.

Fig. 1.

Sanger DNA sequencing analysis shows different novel mutations in h-TERT promoter region of lung carcinoma cases

Table 2.

Identified h-TERT promoter mutations in lung cancer patients

Nucleotide change	Nucleotide position	Mutations identified	Common name of mutation	De novo ETS binding sites	Frequency (%)
G → A	−168	c. −168G → A	c. G272A	W:TGGGGA P: TGGAGA	35
T → A	−18	c. −18 T → A	c. T122A	W:TCCTCGC P: TCCACGC	75
C → A	−46	c. −46 C → A	c. C150A	W:TCCCCTT P:TCCACTT	10
del C	−19	c. −19 del C	c. 123 del C	W:TCTCCTCG P: TCTC*TCG	10
C → T	−19	c. −19 C → T	c. C123T	W:TCTCCTCG P:TCTCTTCG	10
G → A	−1	c. −1 G → A	c. G105A	W:TTCAGCA P: TTCAACA	15
Ins A	−3	c. −3 Ins A	c. 107 Ins A	W:TCAGGC P: TCAAGGC	5
del C	−172	c. −172 del C	c. 276 del C	W:GGACTG P: GGA*TG	5

The bold values represent nucleotides which indicates mutations followed by conversion of wild type nucleotide to patient type nucleotide

Pathological Predictions of Novel Identified Mutations

The results of pathological predictions of identified mutations in promoter region from lung cancer cases using bioinformatics tools such as Tfsitescan and CIIIDER are given in Tables 3 and 4. All the identified mutations were categorized into two categories on the basis of alteration in transcription factor binding sites in the promoter region. Bioinformatics analysis revealed that the identified mutations can cause either loss or gain of several transcription factor binding sites in the h-TERT promoter site.

Table 3.

The outcome prediction of novel mutations identified in h-TERT promoter from lung carcinoma patients using Tfsitescan tool (http://www.ifti.org/Tfsitescan/)

Mutation	TFSitescan prediction
> c-168 G>A	No change
> c-18 T>A	Formation of New site TF binding site: ETS2_S/S2_site
> c-46 C>A	Loss of TF binding site: Sp1-KDR/flk-1-IV
> c-19 del C	No change
> c-19 C>T	No change
> c-1 G>A	Loss of TF binding site: IL5-CLE1
> c-3 Ins A	Loss of TF binding site: IL5-CLE1
> c-172 del C	No change

Table 4.

The prediction of novel mutations identified in h-TERT promoter from lung carcinoma patients using CIIIDER bioinformatics tool (http://www.ciiider.com)

Mutation	CIIIDER prediction
> c-168 G>A	Formation of new site TF binding site: Hand1::Tcf3; Loss of TF binding site: MZF1, PLAGL2
> c-18 T>A	Formation of new site TF binding site: Ahr::Arnt, Arnt::HIF1A, HEES2, HEY1, ZNF354C; Loss of TF binding site: TFAP2B
> c-46 C>A	Formation of new site TF binding site: ELF1, ETV6, NKX2-3, NKX3-1, NKX2-8, SPI1, SPIB, ZNF354C; Loss of TF binding site: Klf1, Klf6, Klf4, Klf5, SP8, SP9
> c-19 del C	Formation of New site TF binding site: ZBTB33; Loss of TF binding site: TFAP2B
> c-19 C>T	Loss of TF binding site: TFAP2B
> c-1 G>A	Formation of new site TF binding site: LIN54
> c-3 Ins A	Formation of new site TF binding site: HLTF, LIN54, NR5A1
> c-172 del C	Formation of new site TF binding site: MZF1(Var.2), TEAD3

Discussion

Lung cancer is a paramount cause of cancer associated mortality for more than 1.5 million deaths per year in both men and women in developed as well as developing countries [16]. Cumulating lines of evidence suggest that lung cancer represents a group of histopathologically and molecularly heterogeneous disease [17]. Similarly, miscellaneous types of lung cancer are bronchoalveolar cancer, non-bronchalveolar adenocarcinoma, small cell and squamous cell lung carcinoma. Average age of onset of lung cancer was about 45 years. In European population, the onset of lung cancer was found at age of 50 years in men, most often at the age of 60–75 years [2]. Strikingly, the diagnosis of lung cancers was usually established in the advanced stage of disease. Earlier onset of lung cancer in present study could be associated with various factors importantly smoking, alcohol consumption, dietary and environmental and genetic factors [5, 6].

Basically, telomerase gradually shorten after successively cycles of cell division in somatic cells. Notably, various cell types overcome this limitation by re-activation of telomerase [17]. However, the underlying molecular mechanism for re-activation is not yet clear. Telomerase, a ribonucleoprotein complex responsible for maintaining telomere length at the end of chromosomes plays a key role in the cellular immortality and tumorigenesis. h-TERT gene expression is aberrantly regulated by TERT promoter mutations due to the generation of de novo consensus binding sites (T/A) TCC for E-twenty-six [ETS] family of transcription factors which in turn upregulated by MAPK signaling cascade [18, 19]. Therefore, the present preliminary study was extended to find out the spectrum of TERT promoter mutations from lung carcinoma patients from Northern region of India. Strikingly, total 8 novel mutations viz c. -G168A [c. G272A], c. −18 T>A [c. T122 A], c. −46 C>A [c. C150A], c. −19 del C [c. 123 del C], c. −19 C>T [c. C123T], c. −1 G>A [c. G105A], c. −3 Ins A [c. 107 Ins A,], c. −172 del C [c. 276 del C] were identified (Fig. 1). Two somatic mutations at −124 and −146 base pairs upstream of the h-TERT ATG start site, hereafter, commonly referred as C228T and C250T respectively which are associated with about 70% of melanoma tumors. Moreover, h-TERT promoter mutations are not only restricted to melanoma whilst these were frequently observed in other tumor types viz. liposarcomas [12], urothelial carcinomas [20, 21], hepatocellular carcinomas [22] and gliomas [12]. Importantly, recurrent h-TERT promoter mutations have been reported in lung cancers. However, some studies demonstrated a low frequency of h-TERT promoter mutation in non-small cell lung carcinoma. The known h-TERT promoter mutations in lung carcinomas include C228T, C250T, C216T, C228A, G267C, C295T and G233C [23]. It is noteworthy here that the identified h-TERT promoter mutations in this study are novel.

The pathological prediction of identified mutations in h-TERT promoter from lung carcinoma patients using bioinformatics Tfsitescan tool analysis documented that majority of mutations are fraternized with either formation of new transcription factor sites in h-TERT promoter or loss of transcription factor binding sites in h-TERT promoter. Both of these factors may cause in aberrant expression of h-TERT gene which in turns re-activation of telomerase activity which is the utmost important to maintain telomere length during continuous cell proliferation in course of cancers.

Identified novel mutations may generate ETS binding motifs via the formation of new transcription factors binding factors as well as loss of transcription factor binding sites (Table 3), as predicted by using Tfsitescan bioinformatics tool. Generation of ETS binding motifs may cause GABP transcription factors recruitment and consequently h-TERT gene expression. Functionally, h-TERT promoter mutations are fraternized with the formation of consensus binding sequence (CCGGAA). The E-twenty six/ ternary Complex (ETS/TCF) transcription factors provide mechanism for cancer specific upregulation of telomerase [23]. Mechanically, the binding of ETS transcription factors to the motifs generated by the mutations results in recruitment of multimeric ETS h-TERT transcription [12]. It is noteworthy here that most common mutations were associated with older and tobacco smokers in this study which are in agreement with the findings of others [24]. In the similar way, the binding of transcriptional activator (c-myc) and represssors (WT and CTCF) to h-TERT promoter may be controlled by DNA methylation in which methylated CPGs prevent their binding to the target sites and thereby lead to h-TERT activation.

In the present study, all the promoter region identified mutations demonstrates either formation or loss of transcription factor binding sites in AP-2 and SP-1 promoter region, as predicted by TESitescan and Ciider bioinformatics analysis. Several studies have reported that AP-2 acts positively to increase the transcription in a tissue specific manner, however, AP-2 can function as a repressor of human ACHE transcription since AP-2 can compete with SP-1 for overlapping binding sites located within a limited portion of promoter [25]. This notion was supported by the findings that mutations in the human Mn SOD promoter in cancer cells may lead to alteration in the binding pattern for AP-2 but not for SP-1. The binding of multiple SP-1 and AP-2 proteins to their recognition sites may  drive in such a way to generate a specific structure which is essential for the cooperative activation of other regulatory proteins and the recruitment of RNA polymerase II to activate synergistic transcription of the genes [26]. It is tempting to speculate that the mutations in h-TERT promotor may change the binding patterns of the activator and interfere with DNA protein interactions of the specific structure which lead to inhibition of protein–protein interactions in the initiation complex. In this study, alterations in various transcription factors binding sites may have impact of various important pathways which are warranted to be investigated.

Germline variation near or within h-TERT gene is equated with telomere length in peripheral blood leukocytes and risk of h-TERT promoter mutant [26] as well as non-mutant cancer [27]. It is noteworthy that h-TERT promoter polymorphism (rs2853669) modulates the prognostic value of h-TERT promoter mutations in various tumor types. It could generate a binding site for the ETS/TCF factor ETS 299 bp and 21 bp upstream of C250T and C228T hot spot mutations respectively [28]. The presence of somatic h-TERT promoter mutations in tumor patients with the rs2853669 common allele showed a decrease in overall survival and increase in tumor recurrence rate in bladder cancer [29] as well as decrease in mean survival in glioma.

Conclusion

Taken together, all these findings suggest that inherited germ-line h-TERT promoter mutations may have an important role in lung cancer risk and susceptibility. The better understanding of these germ-line mutations can lead to the development of preventive measures to reduce the likelihood of developing cancer.

Abbreviations

TERT

Telomerase reverse transcriptase

A

Adenine

C

Cytosine

T

Thymine

G

Guanine

TLC

Total leukocyte count

Hb

Haemoglobin

GABP

GA binding protein transcription factor subunit

Author contributions

RP: Conceptualization; Supervision; Writing; Project administration, Validation, SP: Investigation; Methodology, IR: Writing, review & editing, diagrams, Tables, Formal analysis; JK: Samples and Data curation, Formal analysis; GP: Software; Visualization.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationship that could have appeared to influence the work reported in this study.

Ethical approval

The study was approved by the ethical Committee of M.M. Institute of Medical Sciences and Research (MMIMSR), Maharishi Markandeshwar University (MMU), Mullana, Ambala, Haryana, India.