SH3GL2 and CDKN2A/2B loci are independently altered in early dysplastic lesions of head and neck:...

Journal of PathologyJ Pathol 2009; 217: 408–419Published online 30 September 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/path.2464

Original Paper

SH3GL2 and CDKN2A/2B loci are independently altered inearly dysplastic lesions of head and neck: correlation withHPV infection and tobacco habitAmlan Ghosh,1 Susmita Ghosh,1 Guru P Maiti,1 Mohammed G Sabbir,1 Neyaz Alam,2 Nilabja Sikdar,3

Bidyut Roy,3 Susanta Roychoudhury4 and Chinmay K Panda1*1Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata 700026, India2Department of Surgery, Chittaranjan National Cancer Institute, Kolkata 700026, India3Human Genetics Unit, Indian Statistical Institute, Kolkata 700108, India4Human Genetics and Genomic Division, Indian Institute of Chemical Biology, Kolkata 700032, India

*Correspondence to:Chinmay K Panda, Departmentof Oncogene Regulation,Chittaranjan National CancerInstitute, 37, SP Mukherjee Road,Kolkata 700026, India.E-mail: [email protected];[email protected]

No conflicts of interest weredeclared.

Received: 6 May 2008Revised: 9 September 2008Accepted: 20 September 2008

Abstract

To understand the association of candidate tumour suppressor genes SH3GL2, p16INK 4a ,p14ARF , and p15INK 4b in the pathogenesis of head and neck squamous cell carcinoma(HNSCC), we studied the deletion, mutation, and methylation of these genes in 61 dysplasticlesions and 94 HNSCC samples. In mild dysplasia, SH3GL2, p16INK 4a , and p14ARF showeda higher frequency of overall alterations (60–70%) than in p15INK 4b (40%). However, insubsequent stages of tumour progression, the alteration frequency of these genes did notchange significantly. One novel mutation in common exon 2 of p16INK 4a/p14ARF and threein exon 9 of SH3GL2 were seen. Concordance was seen in the expression of these genes withtheir molecular alterations. Deletions of INK4A-ARF and p15INK 4b have a significant poorpatient outcome. The alterations of p16INK 4a , p14ARF , and p15INK 4b were positively correlatedwith tobacco and inversely with HPV, while SH3GL2 alterations were independentof these factors. Based on aetiological factors, four tumour subtypes were recognized:HPV−tobacco− (I), HPV+tobacco− (II), HPV−tobacco+ (III), and HPV+tobacco+ (IV).Groups III and IV showed a high frequency of p16INK 4a/p14ARF /p15INK 4b alterationswith significant poor patient outcome in comparison to group II. Our findings suggestthat deregulation of SH3GL2-associated signalling and p16INK4a/p14ARF/p15INK4b-mediatedG1–S/G2–M checkpoints of cell cycle are independent pathways for the development ofearly dysplastic lesions of the head and neck.Copyright 2008 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.

Keywords: head and neck squamous cell carcinoma; dysplastic lesions; tumour suppressorgene; SH3GL2 ; p16INK 4a ; p14ARF ; p15INK 4b

Introduction

Head and neck squamous cell carcinoma (HNSCC)is the sixth most common cancer worldwide andit accounts for 30–40% of all cancer types in theIndian subcontinent [1]. Despite the diverse anatomyand mixture of tissues represented by the head andneck region, all HNSCCs share common risk factors[eg tobacco, alcohol, human papilloma virus (HPV)infection, etc] and are similar in their epidemiology,treatment, and prognosis, and hence are often studiedtogether [2]. Reported gene expression studies of headand neck tumourigenesis using microarrays suggeststhat the transition of normal mucosa to premalignantlesion is the most important event, as reflected bymajor transcriptional alterations, compared with the

transition from premalignant lesion to invasive car-cinoma [3]. Among chromosomal alterations, loss ofthe 9p21–22 region appears to be an early event inthe development of premalignant lesions of the headand neck [4]. Chromosome 9p21 harbours two candi-date tumour suppressor gene (TSG) loci — CDKN2Aand CDKN2B at 10 kb apart. CDKN2A encodes twodifferent transcripts (p16 INK 4a and p14 ARF ) derivedfrom alternative splicing of upstream exons (1α and1β, respectively) with a common splice acceptor site,whereas CDKN2B encodes p15INK4b. Both p16INK4a

and p15INK4b negatively regulate cell cycle progressionat the G1–S checkpoint through the Rb–E2F pathway,while p14ARF blocks the MDM2-mediated degradationof p53, resulting in cell cycle arrest either at G1–S orat G2–M [5].

Copyright 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.www.pathsoc.org.uk

SH3GL2 and CDKN2A/2B alterations in HNSCC 409

According to a recent tumour progression model ofHNSCC, 9p21 deletion, p16 INK 4a/p14 ARF inactiva-tion, and epidermal growth factor receptor (EGFR)overexpression are essential for the transition of nor-mal mucosa to hyperplasia [6]. But Nagai [7] sug-gested that inactivation of p16 INK 4a might be neces-sary for the development of carcinoma in situ fromdysplasia. However, in these models, the involvementof p15 INK 4b in HNSCC development is not clear,although CpG island methylation of the gene promoterhas been reported in about 23% of oral squamouscell carcinomas [8]. This may be due to lack of acomparative study of these gene loci on the same setof samples including both premalignant lesions andmalignant tumours.

Using a microcell hybrid system, Parris et al [9]suggested the location of a new TSG telomeric tothe CDKN2A locus. In deletion mapping, the D9S157marker located 5 Mb telomeric to CDKN2A showeda high frequency of loss of heterozygosity (LOH) inhead and neck lesions [1], pituitary adenoma [10], andneuroblastoma [11]. The D9S157 marker is intragenicto a candidate TSG SH3GL2 /endophilin associatedwith receptor-mediated endocytosis and subsequentdegradation of activated EGFR from the cell surface,an essential process to avoid constitutive growthsignalling and tumourigenesis [12]. The molecularalterations of SH3GL2 have not yet been studied inHNSCC, although its reduced expression has beenreported in laryngeal carcinoma [13].

Thus, in the present study, an attempt has been madeto understand the association of SH3GL2, p16 INK 4a ,p14 ARF , and p15 INK 4b in the development of HNSCC.For this, genetic and epigenetic alterations of thesegenes were analysed in 61 dysplastic lesions of thehead and neck and 94 HNSCC samples from Indianpatients. The expression of these genes was also anal-ysed to examine any concordance with the molecu-lar data. Furthermore, the different molecular alter-ations were correlated with various clinicopathologicalparameters (including HPV status) and also with dis-ease recurrence/death.

Materials and methods

Patients, tumour tissues, and cell lines

Primary head and neck lesions and corresponding nor-mal areas were collected from Chittaranjan NationalCancer Institute and Cancer Center and Welfare Home,Kolkata after appropriate approval of the institutionalethical committee and informed consent from thepatients. A total of 155 head and neck lesions werecollected from 153 unrelated individuals between 1998and 2008. Samples were frozen immediately after col-lection at −80 ◦C until use. Part of the freshly oper-ated tissues was directly collected in TRIzol reagent(Invitrogen, USA) for RNA isolation and another partwas embedded in paraffin for immunohistochemistry.

Detailed clinicopathological histories of patients arepresented in Table 1. Among the HNSCC cell lines,Hep2 was procured from the National Centre for CellSciences, Pune, India and SCC084 was kindly pro-vided by Professor SM Gollin, University of Pitts-burgh, USA.

Microdissection and DNA extractionThe contaminant normal cells in the head and necklesions were removed by manual microdissection (seethe Supporting information, Supplementary materials).Microdissected samples containing 70–80% dysplasticepithelium/tumour cells were taken for DNA isolationaccording to a standard procedure [14].

Deletion analysis of the candidate TSGsDeletion of the candidate TSGs was analysed bymicrosatellite and exonic markers (Supporting infor-mation, Supplementary materials) in 61 dysplasticlesions and 94 HNSCC samples. Six microsatellitemarkers were selected on the basis of their map posi-tions and heterozygosity (Ensembl release 44; GenomeDatabase). The procedure is described in detail inthe Supporting information, Supplementary materials.Among the samples, 17 dysplastic lesions and 40HNSCC samples had already been mapped for dele-tion by D9S157, D9S942 and p16 INK 4a/p14 ARF exon2 markers [1,15].

PCR-based methylation-sensitive restrictionanalysis (MSRA)

The methylation status of the SH3GL2, p16 INK 4a ,p14ARF, and p15 INK 4b promoters was assessed in40 dysplastic lesions and 63 HNSCC samples byMSRA [16] (Supporting information, Supplementarymaterials). Methylation of the p16 INK 4a promoterwas screened earlier in 15 dysplastic lesions and 20HNSCC samples [15].

Mutation analysis

Mutation of p16 INK 4a , p14 ARF , and p15 INK 4b wasscreened for in a total of 145 samples and that ofSH3GL2 in 30 samples by single-strand conformationpolymorphism (SSCP) analysis using [α-32P]dCTP, asdescribed by Tripathi et al [15]. Primer sequences aregiven in the Supporting information, Supplementarymaterials. The mutation of exon 1α and common exon2 of p16 INK 4a/p14 ARF had been screened previouslyin samples, as described in deletion mapping [15].Sequencing of both strands of samples showing anabnormal band shift was performed using a 3100-Avant Genetic Analyzer (PE Applied Biosystems Inc,USA).

mRNA expression analysisTotal RNA was isolated from samples using TRI-zol reagent according to the manufacturer’s proto-col. Synthesis of cDNA was done using Superscript

J Pathol 2009; 217: 408–419 DOI: 10.1002/pathCopyright 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

410 A Ghosh et al

Table 1. Clinicopathological features of head and neck lesions

HPV 16/18 positivityClinical Patient No Median age

features (N = 155) (years) Age (years) HPV+ HPV− p value

Primary siteOrofacial 13 (8.3%) 39 22–76 6 (46.1%) 7 (53.8%)Oral cavity 133 (85.8%) 48 30–74 74 (55.6%) 59 (44.3%)Larynx 9 (5.8%) 58 50–75 7 (77.7%) 2 (22.2%) 0.163

Tumour stage∗Dysplasia (N = 61)

Mild 13 (21.3%) 44 30–60 5 (38.4%) 8 (61.5%)Moderate 28 (45.9%) 45 25–52 14 (50%) 14 (50%)Severe 20 (32.7%) 50 22–70 9 (45%) 11 (55%)

HNSCC (N = 94)Stage I 9 (9.5%) 45 33–70 4 (44%) 5 (55.5%)Stage II 26 (27.6%) 60 32–74 16 (61.6%) 10 (38.4%)Stage III 29 (30.8%) 50 58–62 17 (59%) 12 (41%)Stage IV 30 (31.9%) 50 30–75 22 (73%) 8 (27%) 0.014

GenderMale 116 (74.8%) 50 22–76 61 (52.5%) 55 (47.4%)Female 39 (25.1%) 45 30–65 26 (66.6%) 13 (33.3%) 0.125

Tumour differentiationWell 45 (48%) 52 22–76 22 (49%) 23 (51%)Moderate 37 (39.3%) 50 34–65 26 (70%) 11 (30%)Poor 12 (12.7%) 50 32–75 11 (91.6%) 1 (8.4%) 0.003

Lymph nodeNode+ 33 (35%) 50 30–70 19 (57.6%) 14 (42.4%)Node− 61 (65%) 52 22–76 40 (65.6%) 21 (34.4%) 0.443

TobaccoTobacco+† 95 (61%) 48 30–75 46 (48.4%) 49 (51.6%)Tobacco− 60 (39%) 46 22–76 41 (68.3%) 19 (31.7%) 0.014

∗ According to the UICC TNM classification (excluding dysplasia).† Ten to 15 cigarettes or bidis, or equivalent amount of chewable tobacco, per day for the last 10 years.

III (Invitrogen, USA) reverse transcriptase. Semi-quantitative RT-PCR analysis was performed usingspecific primers (Supporting information, Supplemen-tary materials) for the candidate TSGs together with β-actin as an endogenous control [17]. Real-time quan-tification of the genes was performed using a powerSYBR-green assay in two HNSCC cell lines (Hep2and SCC084), 25 primary HNSCC samples, and theiradjacent normal tissues (see the Supporting informa-tion, Supplementary materials for details of the quan-titation procedure).

To confirm inactivation of candidate TSGs by pro-moter hypermethylation, the mRNA levels of thesegenes were studied in SCC084 cells in the presenceand absence of 5-aza-2′-deoxycytidine (5-aza-dC), adrug that inhibits DNA methylation. A sub-confluentculture of SCC084 was grown in the presence of10 µM, 20 µM 5-aza-dC or in the absence of 5-aza-dC(control) separately for 5 days. RNA preparation andRT-PCR analysis (semi-quantitative and quantitative)of the genes were carried out as described above.

Expression analysis of candidate TSGs byimmunohistochemistry

Immunohistochemical analysis of p16 and SH3GL2protein was done in eight dysplastic lesions and 28

HNSCC samples using an ABC staining kit accordingto the manufacturer’s protocol (Santa Cruz Biotechnol-ogy, CA, USA). The procedure is described in detailin the Supporting information, Supplementary materi-als.

Detection of HPV-16 and HPV-18

The presence of HPV in head and neck lesions wasdetected by PCR, using primers MY09 and MY11from the consensus L1 region, followed by typing ofHPV 16/18 in the L1-positive samples, as describedpreviously [18].

Statistical analysis

Fisher’s exact test was used to determine the asso-ciation between the genetic profiles of the tumoursand different clinicopathological features. Survivalcurves were calculated according to the Kaplan–Meiermethod. Probability (p) value ≤0.05 was consideredstatistically significant. All the statistical analyses wereperformed using the statistical program SPSS (SPSSInc, Chicago, IL, USA).



Figure 1. (A) Autoradiograph showing loss of heterozygosity (LOH), microsatellite size alteration of one allele (MA-I), and of bothalleles (MA-II), and loss of one allele and size alteration of the other (LOH + MA). An arrow indicates loss of the correspondingalleles and an asterisk indicates size alteration of one or both alleles. (B) Homozygous/hemizygous deletion (HD/HED) of(a) CDKN2A (p16/p14 exon 2) and (b) SH3GL2 loci. (C) Methylation analysis of (a) SH3GL2 and (b) p15INK4b gene promoters inhead and neck lesions using MSRA. Tumour and normal DNA were restriction-digested by MspI and HpaII. U = mock digestion;K1 and K2 = DNA digestion and integrity controls; T = DNA from head and neck lesions; N = corresponding normal

Results

Deletion analysis of candidate TSGs

In dysplastic lesions, p16 INK 4a and p14 ARF wereobserved to have a high frequency of deletion, atcomparable levels (42–53% and 42–48%, respec-tively), followed by SH3GL2 (25–29%) and p15 INK 4b

(13–15%), as shown in Figures 1A, 1B, and Table 2.A similar trend was noted in the HNSCC samples.The majority of samples having LOH in microsatellite-based mapping showed concordant hemizygous dele-tion in exonic markers (Supporting information, Sup-plementary material 3A, 3B). A high frequency ofmicrosatellite size alterations (MA) was seen in theD9S157 (16–21%), hMP16α (9–16%), and D9S942(11–14%) markers. Similarly, biallelic alterations suchas MA-II and LOH + MA were also prevalent in theD9S157 (10–13%) and D9S942 (9–11%) markers.However, homozygous deletion was mainly present inthe p16 INK 4a/p14 ARF locus in both dysplastic lesions(5%; 3/60) and HNSCC samples (6%; 5/86) (Table 2).

A significant correlation was observed among dele-tions in p16 INK 4a , p14 ARF , and p15 INK 4b (Supportinginformation, Supplementary material 4A).

Promoter methylation status of the candidateTSGs

The promoter region of the candidate TSGs showeddifferential methylation in the head and neck lesions

(Figure 1C). In dysplasia, SH3GL2 showed the high-est level of methylation (42%, 17/40), followed byp15 INK 4b (27%, 11/40), p14 ARF (20%, 8/40), andp16 INK 4a (17%, 7/40). A similar trend was alsoobserved in HNSCCs (Supporting information, Sup-plementary material 4B). Methylation of SH3GL2 andp15 INK 4b was also seen in 13% (8/63) and 11% (7/63)of samples, respectively, in both tumour and corre-sponding normal tissues (Supporting information, Sup-plementary material 5). A significant correlation wasobserved between methylation in p16 INK 4a , p14 ARF ,and p15 INK 4b (Supporting information, Supplementarymaterial 4B). Considering the two HNSCC cell lines,SCC084 showed methylated promoters of SH3GL2,p14 ARF , and p15 INK 4b , whereas no methylation of anyof the four genes was detected in Hep2.

Mutation analysis

In SSCP analysis, 16% (10/61) of the dysplasticlesions and 19% (16/84) of the HNSCC samplesshowed abnormal band shifts in common exon 2 ofp16 INK 4a/p14 ARF (Figure 2A). Direct sequencing ofmutation-positive samples revealed transversion fromG → C, C → A, C → G and also transition fromC → T, G → A, T → C resulting in splice junctionvariants, missense, nonsense, and silent mutations(Table 3). A C → A novel transversion was found atcodon 62 of p16 INK 4a or codon 76 of p14ARF (GeneBank Accession No EU038 281).


412 A Ghosh et al

Tab

le2.

Ove

rall

patt

ern

ofal

tera

tions

ofth

em

icro

sate

llite

and

exon

icm

arke

rsin

dysp

last

icle

sion

san

dH

NSC

Csa

mpl

es

Dys

plas

tic

lesi

ons

HN

SC

Csa

mpl

esD

yspl

asti

cle

sio

nsB

ialle

lical

tera

tio

nsH

NS

CC

sam

ples

Bia

llelic

alte

rati

ons

Dis

tanc

e

Mar

ker

Lo

cus

fro

mp-

ter

(Mb)

LO

H/d

elet

ion

MA

LO

H/d

elet

ion

MA

MA

II+

LM

AH

DT

ota

lM

AII

+L

MA

HD

To

tal

D9S

157

9p22

17.6

112

/41

(29%

)10

/48

(21%

)23

/62

(37%

)11

/68

(16%

)6/

48(1

3%)

—6/

48(1

3%)

7/68

(10%

)—

7/68

(10%

)SH

3GL2

exon

99p

2217

.713

/53

(25%

)0/

53(0

%)

24/8

6(2

8%)

0/86

(0%

)—

0/61

(0%

)0/

61(0

%)

—1/

86(1

%)

1/86

(1%

)p1

6/p1

4ex

on2

9p21

21.9

625

/60

(42%

)0/

60(0

%)

43/8

6(5

0%)

0/86

(0%

)—

3/60

(5%

)3/

60(5

%)

—5/

86(6

%)

5/86

(6%

)hM

p16α

9p21

21.9

617

/32

(53%

)7/

44(1

6%)

39/5

8(6

7%)

5/55

(9%

)1/

44(2

%)

—1/

44(2

%)

3/55

(5%

)—

3/55

(5%

)D

9S94

29p

2121

.98

17/3

8(4

4%)

4/35

(11%

)32

/69

(46%

)8/

58(1

4%)

4/35

(11%

)—

4/35

(11%

)5/

58(9

%)

—5/

58(9

%)

D9S

1748

9p21

21.9

819

/39

(48%

)5/

40(1

2%)

31/6

7(4

6%)

4/59

(7%

)4/

40(1

0%)

—4/

40(1

0%)

2/59

(3%

)—

2/59

(3%

)hM

p19A

RF9p

2121

.98

15/3

6(4

2%)

3/44

(7%

)28

/54

(52%

)3/

54(6

%)

2/44

(4%

)—

2/44

(4%

)2/

54(4

%)

—2/

54(4

%)

p15

exon

19p

2121

.99

7/55

(13%

)0/

55(0

%)

18/9

2(2

0%)

0/92

(0%

)—

0/55

(0%

)0/

55(0

%)

—1/

92(1

%)

1/92

(1%

)D

9S17

529p

2121

.99

7/46

(15%

)2/

54(4

%)

13/5

1(2

5%)

6/80

(8%

)1/

46(2

%)

—1/

46(2

%)

1/80

(1%

)—

1/80

(1%

)

MA

=m

icro

sate

llite

size

alte

ratio

n;H

D=

hom

ozyg

ous

dele

tion;

LMA

=LO

H+

MA

;−=

not

appl

icab

le.

In the case of exon 10 of SH3GL2, an abnormalband shift was seen in 1/10 dysplastic lesions and2/20 HNSCC samples (Figure 2A). Direct sequenc-ing showed a novel A → C transversion at codon 321in one sample and in the other two, single or doublenucleotide deletion at codon 330 and 334, respectively(Table 4) (Gene Bank Accession Nos EF988093,EF988094, and EF988095, respectively). However, nomutation was detected in the two HNSCC cell linesHep2 and SCC084 for either of the genes.

mRNA expression of the candidate TSGs

Expression of the candidate TSGs was readily detect-able in head and neck tissues by RT-PCR analysis.Specific PCR products were obtained, as demonstratedby gel electrophoresis (Figure 2B). In quantitative RT-PCR, 68% (17/25) of the samples showed more thana two-fold reduction in SH3GL2 expression, with amean of 46.97 ± 6.49 (SEM); 64% (16/25) of thesamples in p16INK4a and p14ARF, each with a mean of32.13 ± 7.08 and 24.51 ± 5.55, respectively; and 48%(12/25) of the samples in p15INK4b, with a mean of20.57 ± 3.35 compared with their normal counterparts.Reduced expression of SH3GL2 (64-fold), p15 INK 4b

(15-fold), and p14 ARF (10-fold) was also detected inthe SCC084 cell line (Figure 3). However, 8- to16-fold activation of the SH3GL2, p15 INK 4b , and p14 ARF

genes was seen in SCC084 in the presence of 5-aza-dC, compared with only 1.5-fold in p16 INK 4a

(Supporting information, Supplementary material 6).

Immunohistochemical analysis

Immunohistochemical analysis of normal epitheliumlocalized SH3GL2 protein mainly in the membraneand p16 in the nucleus. In tumours, diffuse cytoplas-mic staining was seen for SH3GL2, while nuclearlocalization of p16 remained unaffected (Figure 4).Low-level expression of SH3GL2 was seen inSCC084; among the head and neck lesions, a low/intermediate level of expression of both p16 andSH3GL2 was seen in 61% (22/36) of the samples,for each protein. Due to the scarcity of samples, RNAexpression in the dysplastic lesions and some of theHNSCC samples could not be analysed. There wasconcordance between molecular alterations with RNAand protein expression (Table 5).

Detection of HPV

Using L1 primers, HPV DNA was detected in 56%(87/155) of the head and neck lesions. Among HPV-positive samples, 94% (82/87) were HPV-16-positiveand 6% (5/87) HPV-18-positive. HPV infection wasfound to be positively correlated with tumour stage andtumour differentiation, and inversely correlated withtobacco consumption (Table 1).



Figure 2. (A) Representative example of SSCP analysis of (a) p16INK4a/p14ARF exon 2 and (b) SH3GL2 exon 10. An arrow indicatesan abnormal band shift. Representative chromatograms showing C > A and C > G transversions in the 62nd and 63rd codonsof p16INK4a and a single nucleotide (G) deletion in the 330th codon of SH3GL2. (B) Representative agarose gel photographs ofRT-PCR analysis of (a) the SH3GL2, (b) p14ARF , (c) p16INK4a, and (d) p15INK4b genes in HNSCC cell lines, primary tumours, andcorresponding normal head and neck tissues. β-actin was taken as a housekeeping gene. Samples were separated by 2% agarosegel electrophoresis and stained with ethidium bromide. HNSCC cell lines: Hep2 and SCC084; primary HNSCC samples: 5165,4271, 6817, and 6329. T = RNA from tumour samples; N = corresponding normal

Table 3. Compilation of the mutations of p16INK4a/p14ARF exon 2 in head and neck lesions

p16INK4a p14ARF

No ofsamples Codon

Nucleotidechange

Amino acidaltered Codon

Nucleotidechange

Aminoacid

altered

−4position −4position5 intron 1/of exon G to C — intron 1/of exon G to C —

2 splice junction 2 splice junction2 58 CGA to TGA Arg to Term 72 CCG to CTG Pro to Leu2 108 GAT to AAT Asp to Asn 122 CGA to CAA Arg to Gln2 113 CTG to CCG Leu to Pro 127 TCT to TCC No change2 114 CCC to CTC Pro to Leu 128 GCC to GCT No change1 118 GCT to GTT Ala to Val 132 GGC to GGT No change3 122 GGC to GAC Gly to Asp 136 GGG to GGA —1 133 GCT to GCC No change 147 TGC to CGC —1 143 GCC to ACC Ala to Thr 157 TGC to TAC —3 148 GCG to ACG Ala to Thr 162 CGC to CAC —3 62 CTG to ATG Leu to Meth 76 GCT to GAT Ala to Asp

63 CTG to GTG Leu to Val 77 GCT to GGT Ala to Gly1 114 CCC to CTC Pro to Leu 169 GCC to GCT —

148 GCG to ACG Ala to Thr 162 CGC to CAC —

= not applicable.


414 A Ghosh et al

Table 4. Compilation of the mutations of SH3GL2 exon 10 in head and neck lesions

No of samples Codon Nucleotide change Coding change Result

1 321 AAC to ACC Asn to Thr Missense mutation1 330 GGG to GG 1 bp deletion Frameshift mutation1 334 GGC to G 2 bp deletion Frameshift mutation

Figure 3. Quantitative RT-PCR analysis of mRNA expression of (A)SH3GL2, (B) p14ARF , (C) p16INK4a, and (D) p15INK4b inthe HNSCC cell lines, C1 and C2 (C1 = Hep2; C2 = SCC084) and primary tumour samples (T1–T25). The bars representfold-reduction of mRNA expression of the candidate TSGs (calculated by the 2−ddCt method)

Clinicopathological association and patient survival

In mild dysplastic lesions, the order of deletionfrequency of the genes was p14 ARF (46%, 6/13)> p16 INK 4a (38%, 5/13) > SH3GL2 (23%, 3/13)> p15 INK 4b (15%, 2/13), with the methylationfrequency of SH3GL2/p15 INK 4b (30%, 4/13) >

p14ARF /p16 INK 4a (15%, 2/13) and the mutationfrequency of p14 ARF /p16 INK 4a common exon 2 was15% (2/13). A similar trend was seen in the subsequentstages of tumour progression (Figure 5A).

Overall, alterations of SH3GL2, p16 INK 4a , p14 ARF ,and p15 INK 4b were considerably high in mild dys-plastic lesions and did not change significantly alongwith tumour progression (Figure 5B). The alterationsof p16 INK 4a , p14 ARF , and p15 INK 4b were positivelycorrelated with tobacco habit and inversely correlatedwith HPV infection, whereas these aetiological fac-tors had no effect on SH3GL2 alterations (Table 6).Considering these two aetiological factors together,alterations of the three candidate TSGs increasedsignificantly from tobacco−HPV−/tobacco−HPV+ totobacco+HPV+/tobacco+HPV− cases (Table 7). Log-rank testing revealed a significantly poorer survival inthe tobacco+HPV− and tobacco+HPV+ cases than inthe tobacco−HPV+ cases, and in cases with deletion

at CDKN2A and CDKN2B loci than in those withoutdeletion (Figure 6).

Discussion

The aim of our study was to understand the associationof the candidate TSGs SH3GL2, p16 INK 4a , p14 ARF ,and p15 INK 4b in the development of HNSCC. In milddysplastic lesions, a higher frequency of overall alter-ations (60–70%) was seen in SH3GL2, p16 INK 4a , andp14 ARF than in p15 INK 4b (40%) (Figure 5B). In thesubsequent stages of tumour progression, the alter-ation frequency did not change significantly. Therewas concordance between molecular alterations andreduced expression (RNA/protein) of the genes. Itseems that alterations of SH3GL2, p16 INK 4a , andp14 ARF occurred first for the development of earlydysplastic lesions, followed by p15 INK 4b alterations.The association of p16/p14 alterations with the devel-opment of hyperplastic lesions of the head andneck was suggested by Ordonez et al [6]. How-ever, the association of SH3GL2 and p15 INK 4b inthe development of early dysplastic lesions of thehead and neck has not been reported previously.Interestingly, alterations of p16 INK 4a , p14 ARF , and



Figure 4. Immunohistochemical staining patterns of p16INK4a and SH3GL2 in (A) the HNSCC cell lines (HE-p2 and SCC084)and (B) head and neck tissues. T = HNSCC samples; N = corresponding normal tissue; L = leukoplakia/dysplastic lesions ofhead and neck. A black arrow indicates the expression pattern of the corresponding proteins. Original magnification ×40; scalebars = 10 µm in the cell lines and 30 µm in the head and neck tissues

p15 INK 4b were inversely associated with HPV infec-tion. This suggests that p16 INK 4a/p14 ARF/p15 INK 4b

inactivation and HPV infection could independentlylead to HNSCC development, and in HPV-positivesamples, these genes might be in the wild-type form.Similarly, rare occurrence of homozygous deletionof p16 INK 4a/p14 ARF /p15 INK 4b was reported by Per-rone et al [19], and rare p53 mutation by Mitra et al[20] in HPV-positive HNSCC samples. Based onthe association of aetiological factors, a model hasbeen proposed for HNSCC development (Figure 7).In our samples, the frequency of HPV−tobacco−was low (12%: 19/155), but the frequency of alter-ations of the candidate TSGs in this group wascomparable to that of HPV+tobacco−. This indi-cates that besides HPV infection and tobacco habit,other aetiological factors may have a role in thedevelopment of this tumour. Significantly more com-mon p16 INK 4a/p14 ARF /p15 INK 4b alterations in eitherHPV−tobacco+ or HPV+tobacco+ groups indicatesthat tobacco-induced genotoxic stress may influencethe genetic/epigenetic alterations of these genes. Simi-lar to us, Weinberger et al [21] proposed two pathwaysof HNSCC development: one with tobacco-associatedp16 inactivation/p53 mutation, and the other through

HPV infection and low frequency of p16 inactiva-tion/p53 mutation. But in that model the tumourswith absence of HPV infection and tobacco habitwere not discussed. The poor survival of patients withtobacco habit compared with HPV-infected patientswith no tobacco habit suggests that tobacco-associatedgenetic/epigenetic alterations accelerate the diseaseprogression. On the other hand, the significantly pooroutcome of patients with deletion of CDKN2A andCDKN2B in the same set of samples has not beenreported before and indicates its prognostic signifi-cance. In other studies, better survival of patients withoverexpression of p16INK4a, p14ARF, and HPV infec-tion was reported [21,22].

Consistent with our study, the region harbouringSH3GL2 has been found to be deleted in severalentities including pituitary adenoma, neuroblastoma,etc [10,11]. But the high frequency of methylationin the promoter region of SH3GL2 detected in ourstudy has not been reported earlier in any tumours. Itsactivation in the SCC084 cell line after treatment with5-aza-dC confirms that promoter methylation is oneof the inactivating mechanisms of this gene. Missenseand frameshift mutations present in the SH3 domainin exon 10 of SH3GL2 in 10% (3/30) of the samplesindicate that mutation may have an important role


416 A Ghosh et al

Table 5. Comparison of p16INK4A and SH3GL2 expression with respect to their alterations

SH3GL2 p16INK4A

RNA (Q-PCR) Protein ALT RNA (Q-PCR) Protein ALT

HNSCC cell linesHE-p2 −1.6 High — −1.2 High —SCC084 −64 Low M+ −1.5 High —

Dysplastic lesions5265 ND Low D+ ND Low D+92 ND High — ND High —L154 ND Intermediate M+ ND High —L117 ND Intermediate D+ ND High —L142 ND High — ND Low D+L127 ND Low D+ ND Intermediate D+L149 ND Intermediate M+ ND Low D+L143 ND Low M+ ND High —

HNSCC samples5165 −42 Intermediate D+ −32 Intermediate D+4271 −48.5 Low D+ −39 Low D+6329 −55.7 Low D+ −40.2 Low D+6678 −55.7 Low M+ −1.1 Intermediate —3150 −1.17 High — −28 Intermediate —944 −1.23 High — −1.18 High —1049 −42 Low M+ −39 Low M+6907 −1.56 Intermediate — −1.2 High —1004 −39 Low M+ −18 Intermediate D+872 −1.8 Intermediate M+ −37 Low D+51 −1.4 High — −1.3 High —219 −50.32 Low M+ −1.38 High —2333 −40.95 Intermediate D+ −38.99 Low D+3371 −45.4 Low D+ −37.63 Low D+5111 −40 Intermediate M+ −30.5 Intermediate M+3484 −52 Low M+ −1.43 High —3941 −1.56 High — −31.7 Intermediate D+3127 −45 Low M+ −29.3 Intermediate D+4283 −39.5 Intermediate D+ −38 Low D+6835 −1.54 High — −31.94 Low D+

Q-PCR = quantitative real-time RT-PCR; each value represents fold-reduction of the relative RNA level in tumour versus correspondingnon-tumour tissues.ALT = alterations; ND = not done; D+ = deletion-positive; D− = deletion-negative; M+ = methylation-positive; M− = methylation-negative;− = no alterations.

in inactivation of this gene. However, the potentialrole of mutations requires to be explored further byscreening other regions of the gene and on additionalsamples. Similar to our findings, a significant reductionof SH3GL2 was seen in the laryngeal carcinomas of aChinese population [13]. It seems that inactivation ofSH3GL2 leads to the stabilization of EGFR, resultingin activation of an EGFR-mediated signal transductionpathway. Thus, the overexpression of EGFR seenin the hyperplastic lesions of the head and neckby Ordonez et al [6] might be due to its reduceddegradation and therefore was not correlated with theEGFR gene status, as reported by Perrone et al [19],in oropharyngeal carcinoma.

In mild dysplastic lesions, deletion and mutationwere prevalent in p16 INK 4a and p14 ARF loci, andmethylation in p15 INK 4b . The persistence of a similartrend in the subsequent stages of tumour progressionindicates that the differential genetic and epigeneticalterations in the two closely associated TSG locimay be due to some difference in the chromosomal

conformation. Like SH3GL2, the activation of p14 ARF

and p15 INK 4b in SCC084 after treatment with 5-aza-dC suggests their inactivation by promoter hyperme-thylation. The low activation of p16 INK 4a in SCC084after treatment with 5-aza-dC might be due to absenceof promoter methylation as seen by MSRA. To the bestof our knowledge, no study has been done to analysethe alterations of these three genes during the progres-sion of mild dysplasia to stage III/IV tumours in thesame set of samples. In dysplastic lesions, Kresty et al[23] showed comparable frequencies of deletion (76%)and mutation (11–15%) in the p16 INK 4a/p14 ARF

locus, but the methylation frequency in p16 INK 4a washigher (57%) than in p14 ARF (3.8%) in their samples.In HNSCC, Viswanathan et al [8] reported compara-ble frequencies of p16 INK 4a and p15 INK 4b methyla-tion (23%), but unlike us, Shintani et al [24] reportedhigh frequencies of homozygous deletion (56–28%)and methylation (50–44%), and a low frequency ofmutation (8%) in the p16 INK 4a/p14 ARF locus. Thismight be due to differences in the methodologies used,



Figure 5. Histogram showing (A) deletion, methylation, and mutation, and (B) the overall alterations in respective tumoursuppressor genes in dysplastic lesions and HNSCC samples

Table 6. Association of alterations of the candidate TSGs with aetiological factors (tobacco habit and HPV infection) in head andneck lesions

Tobacco+ Tobacco− p value HPV+ HPV− p value

Dysplastic lesions SH3GL2 ALT+ 23 (85%) 4 (15%) 15 (56%) 12 (44%)ALT− 10 (77%) 3 (23%) 0.51949 5 (38%) 8 (62%) 0.31118

p16INK4a ALT+ 23 (92%) 2 (8%) 9 (36%) 16 (64%)ALT− 10 (67%) 5 (33%) 0.04120 11 (73%) 4 (27%) 0.02224

p14ARF ALT+ 25 (96%) 1 (4%) 10 (38%) 16 (62%)ALT− 8 (57%) 6 (43%) 0.00195 10 (71%) 4 (29%) 0.04670

p15INK4b ALT+ 14 (100%) 0 (0%) 4 (29%) 10 (71%)ALT− 19 (73%) 7 (27%) 0.03256 16 (62%) 10 (38%) 0.04670

HNSCC samples SH3GL2 ALT+ 22 (54%) 19 (46%) 29 (71%) 12 (29%)ALT− 9 (41%) 13 (59%) 0.33456 13 (59%) 9 (41%) 0.35010

p16INK4a ALT+ 19 (54%) 16 (46%) 22 (63%) 13 (37%)ALT− 4 (22%) 14 (78%) 0.02571 16 (89%) 2 (11%) 0.04632

p14ARF ALT+ 21 (60%) 14 (40%) 21 (60%) 14 (40%)ALT− 2 (11%) 16 (89%) 0.00067 17 (94%) 1 (6%) 0.00838

p15INK4b ALT+ 19 (70%) 8 (30%) 13 (48%) 14 (52%)ALT− 12 (33%) 24 (67%) 0.00361 29 (81%) 7 (19%) 0.00692

ALT = alteration.

Table 7. The combined effect of tobacco and HPV on the alterations of candidate TSGs

SH3GL2 p16INK4a p14ARF p15INK4b

ALT+ ALT− ALT+ ALT− ALT+ ALT− ALT+ ALT−

Tobacco− HPV− 7 (64%) 4 (36%) 5 (50%) 5 (50%) 5 (50%) 5 (50%) 2 (18%) 9 (82%)Tobacco− HPV+ 16 (57%) 12 (43%) 13 (48%) 14 (52%) 10 (37%) 17 (63%) 6 (21%) 22 (79%)Tobacco+ HPV+ 28 (82%) 6 (18%) 18 (58%) 13 (42%) 21 (68%) 10 (32%) 11 (32%) 23 (68%)Tobacco+ HPV− 17 (57%) 13 (43%) 24 (96%) 1 (4%) 25 (100%) 0 (0%) 22 (73%) 8 (27%)

p value 0.96551 0.00070 0.00001 0.00004

ALT = alteration; + = positive for the criteria; − = negative for the criteria.


418 A Ghosh et al

Figure 6. Kaplan–Meier 5-year survival probability curves with cumulative survival of HNSCC patients by deletion status inCDKN2A (A) and CDKN2B (B) loci and by the combined status of two aetiological factors, ie tobacco habit and HPV infection (C,D). Post-operative overall survival was measured from the date of surgery to the date of last follow-up, known recurrence ordeath (up to 5 years). N = total number of HNSCC samples

Figure 7. Proposed model for the development of head and neck lesions. Head and neck lesions were subdivided into four groupsdepending on HPV (H) and tobacco (T) association. The frequency of genetic alterations is indicated as (i) <30%: ↓; (ii) 30–50%:↓↓; (iii) 50–70%: ↓↓↓; and (iv) >70%: ↓↓↓↓. The dotted arrows indicate possible pathways of transformation of one group toother. ND = not done

aetiology, and probably ethnicity. The significant asso-ciation of deletion/methylation in p16 INK 4a , p14 ARF ,and p15 INK 4b with each other suggests that alterations(deletion/methylation) of any one of the candidateTSGs may impose selective pressure on the others foralterations.

Thus, it is evident from our study that dysregulationof two independent pathways, ie SH3GL2-associatedEGFR stabilization and p16INK4a/p14ARF/p15INK4b-associated G1–S/G2–M checkpoints, is involved inthe development of early dysplastic lesions. Thealterations of SH3GL2 are independent of HPV



infection and tobacco habit, whereas p16 INK 4a /p14 ARF /p15 INK 4b alterations show a differential asso-ciation with these aetiological factors.

Acknowledgements

We are grateful to the Director, Chittaranjan NationalCancer Institute and Cancer Center and Welfare Home,Kolkata, India. We are also grateful to Professor HZ Hausenand EM de Villiers for their generous gift of HPV-16/18 plas-mids, and to Professor SM Gollin for her kind gift of theSCC084 cell line. Financial support for this work was providedby grants from DST (SR/SO/BB-22/2003 dt. 02.11.04) andDBT (BT/PR/5524/Med/14/649/2004 of dt. 29.11.2005), Gov-ernment of India, to Dr CK Panda and Dr S Roychoudhury;a CSIR–JRF/NET Fellowship grant [F No 2-56/2002(I) EU.II]to Mr A Ghosh; and a UGC–NET Fellowship grant [F.2-3/2000(SA-I)] to Mrs S Ghosh

Supporting information

Supporting information may be found in the onlineversion of this article.

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