ORIGINAL PAPER

Expression pattern of Notch intracellular domain (NICD)and Hes-1 in preneoplastic and neoplastic human oral squamousepithelium: their correlation with c-Myc, clinicopathologicalfactors and prognosis in Oral cancer

Ravindran Gokulan • Devaraj Halagowder

Received: 22 June 2014 / Accepted: 5 July 2014 / Published online: 19 July 2014

� Springer Science+Business Media New York 2014

Abstract Notch pathway molecules crosstalk with Wnt/

b-catenin signaling cascade in stem cells and tumors.

However, the correlation between the expression pattern of

Notch intracellular domain NICD, Hes-1 and c-Myc has

not been studied in oral squamous cell carcinoma. The aim

of this study is to investigate the correlation and prognostic

significance of NICD, Hes-1 and c-Myc in oral cancer.

Immunohistochemistry was used to study the expression

pattern of NICD, Hes-1 and c-Myc in oral preneoplastic

and neoplastic tissues. In addition, double immunofluo-

rescence was used to examine the co-localization of NICD,

Hes-1 and c-Myc in H314 cells. The expression pattern of

NICD and Hes-1 was gradually increased from normal to

dysplasia to carcinoma. Interestingly, statistically signifi-

cant correlation was not observed between NICD, Hes-1

and c-Myc in oral squamous cell carcinoma. Furthermore,

NICD?/c-Myc? and Hes-1?/c-Myc? double positive

cases showed worst survival when compared with other

cases in oral cancer. Notch signaling molecules, NICD and

Hes-1, are found to be involved in the progression of oral

squamous cell carcinoma. Interestingly, NICD, Hes-1 and

c-Myc may have independent roles in oral cancer. On the

other hand, we have demonstrated that NICD?/c-Myc?

and Hes-1?/c-Myc? double positivity might be used as

independent prognostic indicator of oral carcinoma.

Keywords Notch intracellular domain � Oral cancer �Hes-1 � Cancer stem cells � c-Myc

Introduction

Oral squamous cell carcinoma (OSCC) is the most com-

mon type of cancer of the oral cavity. The 5 year survival

rate of OSCC patients has increased marginally as a result

of extensive investigations and novel therapeutic strategies.

However, the 5 year survival rate still remains at 50–60 %

[1]. This insists the need for new therapeutic approaches

and prognostic methods for oral cancer. Despite the accu-

mulation of genetic and epigenetic alterations, reactivation

of stem cell related signaling cascades has been docu-

mented in OSCC [2]. Recently, the concept of synthetic

lethality becomes more ubiquitous among scientists to

investigate the therapeutic potential of anticancer drugs. In

synthetic lethality, either a lethal partner for a mutant gene

or two molecules of different pathways that regulate a

similar process have been targeted [3]. More recently,

EGFR was found to be a synthetic lethal partner for Notch

pathway and their inhibition results in suppressing tumor

growth of basal-like breast cancer in mice [4]. We have

partially applied this concept to find out the prognostic

significance of molecules of Notch and Wnt/b-catenin

signaling cascades which regulates proliferation of oral

carcinoma cells. The aim of this study is to investigate the

expression pattern of Notch intracellular domain (NICD)

and Hes-1 in the progressive stages of OSCC. In addition,

we have analyzed the correlation between NICD, Hes-1

and c-Myc in oral cancer. On the other hand, the prognostic

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12032-014-0126-1) contains supplementarymaterial, which is available to authorized users.

R. Gokulan � D. Halagowder (&)

Unit of Biochemistry, Department of Zoology, University of

Madras, Guindy Campus, Chennai, Tamil Nadu, India

e-mail: rhdbio7@gmail.com; hdrajum@yahoo.com

Present Address:

R. Gokulan

Department of Oral Biology and Biomedical Sciences, Faculty

of Dentistry, University of Malaya, Kuala Lumpur, Malaysia

123

Med Oncol (2014) 31:126

DOI 10.1007/s12032-014-0126-1

significance of NICD, Hes-1 and c-Myc has been studied in

oral cancer.

Notch was initially identified in Drosophila as a trans-

membrane receptor. There are four Notch receptors in

mammals (Notch 1, 2, 3 and 4) and their activation is

dependent on their binding to the ligands Jagged 1, Jagged

2, Delta 1, Delta 3 and Delta 4. The binding results in two

successive cleavages by ADAM type proteases and c-

secretase-presenilin complex. This leads to the release of

NICD which translocates into the nucleus. In the nucleus,

NICD interacts with DNA binding proteins like CSL/

CBF1/RBPjk to induce the expression of target genes

including Hes-1 and Hey families [2]. Notch signaling

plays an important role in maintaining stemness and self-

renewal of diverse type of stem cells [5]. However, the

function of Notch depends upon the cell type. For example,

Notch maintains proliferation of Hematopoietic stem cells,

whereas, it induces differentiation of keratinocytes [6].

Aberrant expression of Notch signaling molecules have

been reported in various cancers. Interestingly, mutations

of Notch suggest its role as a tumor suppressor in Head and

neck squamous cell carcinoma [7]. Furthermore, increase

in the copy number of its ligands JAG1 and JAG2 was

found to be associated with Notch status [8].

As seen in hematological malignancies, dysregulation of

Notch signaling has been observed in renal, lung, pancre-

atic, gastric, head and neck, endometrial, oral, gastric and

hepatocellular carcinomas [2]. Notably, Notch functions as

a tumor suppressor in skin, pancreatic and hepatocellular

carcinomas [9]. Notch 1 signaling promotes cell cycle

arrest in small cell lung cancer cells [10]. Interestingly,

Notch1 inhibits phosphorylation and transactivation of p53

[11]. Notch acts as a pro-differentiation molecule in

keratinocytes. It promotes epithelial to mesenchymal

transition (EMT) by upregulating Snail, Slug and TGF-b[2]. In OSCC, Notch induces EMT during hypoxic condi-

tions and it was prevented by c-secretase inhibitor treat-

ment [12]. More recently, clinical response to c-secretase

inhibitor MK-052 was found in phase-I trail in patients

with advanced solid tumors [13]. The Notch receptors have

been found to maintain stemness of colon cancer stem

cells. Also, Notch signaling might play a significant role in

inducing and maintaining angiogenesis of head and neck

squamous cell carcinoma [14]. Recent reports suggest that

Notch1 expression has been associated with cisplatin

resistance of nasopharyngeal carcinoma cells [15]. More-

over, c-Myc was found to be a target of Notch 1 signaling

cascade and the expression of Notch 1 was found to be

decreased in b-catenin and p63 double positive oral car-

cinoma cells [16].

Nevertheless, c-Myc oncogene is a downstream target of

b-catenin pathway and it induces proliferation of oral car-

cinoma cells. c-Myc is regulated by CNBP, FBP, TCF,

single stranded bubbles, G-quadruplexes and Z-DNA. Upon

activation, b-catenin inhibits the tumor suppressor APC

which leads to the nuclear translocation of b-catenin. In the

nucleus, b-catenin binds with TCF and activates target genes

including c-Myc [17]. Based on the function, c-Myc was

used as one of the factors to produce induced pluripotent

cells from differentiated cell types with other factors like

Sox-2, Oct-4 and Klf4 [18]. Also, c-Myc regulates the

expression of cyclins and CDKs which are essential for cell

cycle, proliferation and survival. The c-Myc gets downreg-

ulated upon activation of anti-proliferative signals generated

by TGF-b signaling. Furthermore, c-Myc represses cyclin

dependent kinase inhibitors p21CIP1/WAF1 and p27 [17].

More recently, CDK was found to have a synthetic lethal

interaction with c-Myc in lymphoma, liver cancer and breast

cancer. Particularly, CDK1 is a synthetic lethal partner for

c-Myc in breast cancer cells [19].

Eventhough, the expression of Notch signaling mole-

cules was studied in oral cancer, their expression pattern

has not been extensively studied in the multistep process of

OSCC. In addition, the correlation between NICD, Hes-1

and c-Myc was uncovered in oral cancer. Moreover, studies

on the combinatorial expression of NICD, Hes-1 and

c-Myc and their prognostic significance have not been

studied yet. Taken together, we have studied the expression

pattern of NICD and Hes-1 in the progressive stages of

OSCC. Furthermore, the correlation between NICD, Hes-1

and c-Myc was analyzed in oral cancer. On the other hand,

the prognostic significance of NICD, Hes-1 and c-Myc was

investigated in oral carcinoma cases.

Materials and methods

Tissue samples

The samples from Normal (8), Mild-moderate dysplasia

(24), Severe dysplasia (22) and OSCC (44) were obtained

from the Royapettah Hospital, Chennai. Normal samples

were obtained from the patients undergoing orthodontic

surgery. The cells were fixed with 10 % buffered formalin

before they were processed for paraffin embedding. 4 lm

sections were cut and mounted on coated glass slides. Two

Pathologists have assessed the histological grading of

dysplasia and OSCC according to the criteria illustrated by

Pindborg et al. [20] until a consensus was reached. The

range of follow up is 14–50 months with a mean follow up

period of 32.1. The clearance was obtained from the

Hospital Medical Board with permission from the Direc-

torate of Medical Education, Government of Tamil Nadu,

India. The cell-bearing slides of H314 cell line (derived

from a poorly differentiated tumour of the floor of the

mouth) [21] was obtained as gift from Dr. Angela Hague.

126 Page 2 of 10 Med Oncol (2014) 31:126

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Immunohistochemistry

Sections were deparaffinized in xylene, rehydrated through

graded alcohols and the antigen retrieval was done by

heating the slides in 0.1 M sodium citrate buffer (pH 6.0)

in a microwave oven. After cooling, the endogenous per-

oxidase activity was blocked by placing the slides in 3 %

hydrogen peroxide for 10 min. The non-specific binding

sites were blocked with 0.3 % Bovine Serum Albumin

(BSA) for 30 min at room temperature. The sections were

then incubated (overnight at 4 �C) with goat polyclonal

anti-Notch (NICD) (Santa Cruz Biotech., USA), mouse

monoclonal anti-Hes-1 (Santa Cruz Biotech., USA) and

rabbit polyclonal anti-c-Myc (a kind gift from Dr. Stephen

Hann, USA) in a dilution of 1:200 for the above mentioned

antibodies. The slides were immunostained using the

respective horseradish peroxidase conjugated secondary

antibodies (Invitrogen, USA). The chromogen 3,30 diam-

inobenzidine was then used as a substrate for localizing the

antibody binding. After counter-staining with haematoxy-

lin, the sections were mounted and viewed under micro-

scope. Negative controls without primary antibody were

included for each staining. The immunostained sections

were scored according to the percentage of stained cells in

more than three random areas of cancer tissue as examined

in 2009 magnification. Two independent observers with-

out prior knowledge on the patient’s clinicopathological

data assessed the immunostained slides. The staining was

assessed as 0 (negative), no staining or staining in less than

5 % of cells; 1? (mild), staining in 5–15 % of carcinoma

cells; 2? (moderate), staining in 16–25 % of carcinoma

cells; 3? (intense), staining in [25 % of carcinoma cells.

The staining was also graded as Negative (includes cate-

gories 0 and 1?) and Positive (includes categories 2? and

3?). In case of disagreement, the slides were re-evaluated

by the two observers and a consensus was reached after

discussion.

Immunofluorescence

The Immunofluorescence analysis was done in H314 cells.

The sections were deparaffinized in xylene, rehydrated and

fixed for 5 min. The cells were then washed with Phos-

phate Buffer Saline (PBS) and permeabilized with 0.1 %

Triton X-100. The cells were then blocked with 1 % BSA

for 30 min and incubated (1 h) with a combination of anti-

NICD/anti-Hes-1, anti-NICD/anti-c-Myc and anti-Hes-1/

anti-c-Myc antibodies. After washing with PBS, the slides

were incubated for 2 h with respective FITC conjugated

secondary antibodies for Hes-1 and c-Myc. In addition,

Alexa Fluor 594 (red) conjugated secondary antibody was

used for NICD and c-Myc respectively. After incubation,

the slides were washed with PBS, mounted and examined

under confocal fluorescence microscope (Leica TCS SP2,

Leica Microsystems, Germany).

Statistical analysis

The relationship between clinicopathological parameters

and the expression of proteins were analyzed using Chi

square test. The correlation analysis was done using

Spearman’s correlation analysis. The overall and disease-

free survival curves were constructed by the Kaplan–Meier

method and the log-rank test was used to calculate the

difference between resulting curves. The duration of dis-

ease-free survival was between the date of treatment to the

date of recurrence or metastasis or disease related death.

The cases were censored either at the date of death for

patient who died during the trial or at the date of last

examination for patient who lost the follow-up. The Uni-

variant and Multivariant survival analyses were performed

using Cox proportional hazards regression model. The

statistical analyses were performed using Acastat Statisti-

cal software, version 6.1 [Acastat Software, USA] and the

survival analysis was performed using SPSS software

(SPSS for windows 14.0, SPSS Inc., Chicago, Illinois). The

p \ 0.05 was considered significant for all the statistical

analyses. Due to the exploratory nature of the study, we did

not adjust for multiple tests.

Results

NICD

The expression pattern of Nestin was presented in Table 1.

NICD staining was observed in the cytoplasm of dysplastic

and carcinoma cells (Fig. 1). The normal oral epithelium

Table 1 Expression of NICD and Hes-1 in precancerous and can-

cerous tissues of oral squamous epithelium

No. of

patients

NICD Hes-1

0 1? 2? 3? 0 1? 2? 3?

Normal 8 6 2 0 0 7 1 0 0

Mild-

moderate

24 6 8 7 3 8 7 6 3

Severe 22 5 6 5 6 6 3 6 7

Carcinoma 44 3 10 14 17* 5 8 11 20**

0 (negative), no staining or \5 % positive cells; 1? (mild), 5–15 %

positive cells; 2? (moderate), 16–25 % positive cells and 3? (intense),

[25 % positive cells

* Expression of NICD shows significant difference between different

groups (p \ 0.05)

** Expression of Hes-1 shows significant difference between differ-

ent groups (p \ 0.05)

Med Oncol (2014) 31:126 Page 3 of 10 126

123

predominantly showed negative staining for NICD. Inter-

estingly, the expression of NICD was gradually increased

from mild-moderate dysplasia to severe dysplasia to

OSCC. Moreover, the expression of NICD showed a sig-

nificant difference between mild-moderate dysplasia,

severe dysplasia, OSCC and normal cases (p \ 0.05)

(Table 1). Notably, NICD staining was higher in stage III–

IV when compared with stage I–II. Also, a statistically

significant difference was found between NICD expression

and lymph node metastasis of OSCC. In particular, the

expression of NICD was higher in lymph node positive

cases when compared with lymph node negative cases.

Fig. 1 Immunostaining of NICD in normal (a), dysplasia (c) and

carcinoma (e) cells of oral squamous epithelium. Immunostaining of

Hes-1 in normal (b), dysplasia (d) and carcinoma (f) (Inset nuclear

staining of c-Myc in oral carcinoma cells) cells of oral squamous

epithelium. Bar 100 lm

126 Page 4 of 10 Med Oncol (2014) 31:126

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However, a significant correlation was not found between

age, sex, location of tumour, histological grade and NICD

expression (Table 2).

Hes-1

Hes-1 expression was predominantly found in the nucleus of

oral carcinoma cells (Fig. 1). However, some cells showed

cytoplasmic expression of Hes-1. The expression pattern of

Hes-1 varies between precancerous and cancerous tissues.

Hes-1 expression was higher in carcinoma tissues when

compared with dysplastic tissues. The expression pattern of

Hes-1 showed a significant difference between normal, mil-

moderate dysplasia, severe dysplasia and carcinoma tissues

(Table 1). Statistically significant difference was found

between the stage and Hes-1 expression. Particularly, the

expression of Hes-1 was higher in stage III–IV compared

with stage I–II of OSCC. The expression of Hes-1 was found

in higher levels in lymph node metastasis positive cases

when compared with lymph node metastasis negative cases.

Whereas, the association between age, sex, histological

grade, location of tumour and Musashi-1 expression was not

reached (Table 2).

c-Myc

Nuclear expression of c-Myc was predominantly observed

in oral carcinoma tissues. Out of 44 cases of oral cancer, 6

cases showed negative staining for c-Myc. Whereas, 3 and

13 cases showed mild and moderate staining respectively.

Moreover, 22 oral carcinoma cases showed intense staining

of c-Myc.

Correlation between NICD, Hes-1 and c-Myc in oral

carcinoma

The association between the expression patterns of NICD,

Hes-1 and c-Myc was investigated in OSCC (Tables 3, 4).

Table 2 Correlation of NICD, Hes-1 and c-Myc expression with clinicopathological factors

No. of

patients

NICD Hes-1 c-Myc

Negative

(13)

Positive

(31)

p value Negative

(13)

Positive

(31)

p value Negative

(9)

Positive

(35)

p value

Age

\60 24 5 19 6 18 5 19

C60 20 8 12 p \ 0.16 7 11 p \ 0.33 4 16 p \ 0.94

Gender

Male 25 7 18 6 17 7 18

Female 19 6 13 p \ 0.79 7 14 p \ 0.59 2 17 p \ 0.15

Stage

I–II 24 11 13 12 12 8 16

III–IV 20 2 18 p \ 0.009** 1 19 p \ 0.001** 1 19 p \ 0.02*

Lymph node metastasis

Negative 23 10 13 11 12 8 15

Positive 21 3 18 p \ 0.03* 2 19 p \ 0.005** 1 20 p \ 0.01*

Histological grade

Well

differentiated

13 5 8 7 6 4 9

Moderately

differentiated

14 5 9 p \ 0.38 3 11 p \ 0.07 3 11 p \ 0.43

Poorly

differentiated

17 3 14 3 14 2 15

Location of the tumours

Tongue 12 5 7 4 8 4 8

Buccal 9 3 6 4 5 2 7

Palate 4 1 3 p \ 0.58 1 3 p \ 0.78 1 3 p \ 0.65

Gingiva 9 3 6 2 7 1 8

Floor of mouth 10 1 9 2 8 1 9

Negative (includes category 0 and 1?) and positive (includes category 2? and 3?)

* Statistically significant

** Statistically highly significant

Med Oncol (2014) 31:126 Page 5 of 10 126

123

Statistically significant correlation was not found between

the expression of NICD and c-Myc in oral carcinoma

(p [ 0.05) (Table 3). In addition, significant association

was not reached between Hes-1 and c-Myc in Oral cancer

(Table 4). Moreover, co-immunolocalization of NICD and

Hes-1 was observed in H314 cells. Interestingly, few H314

cells showed NICD/c-Myc and Hes-1/c-Myc co-immuno-

staining patterns (Fig. 2).

Survival analysis

The survival curves were constructed using Kaplan–Meier

method and they are compared by log-rank test. NICD,

Hes-1 and c-Myc positive cases had poorer survival when

compared with negative cases in both overall and disease

free survival (Table 5). Interestingly, NICD/c-Myc and

Hes-1/c-Myc double positive cases had a significant shorter

survival time than other cases (Figs. 3, 4). The poor dif-

ferentiation status, extent of lymph node metastasis and

clinical stage were significantly correlated with worst

survival in oral carcinoma (Table 5) (Supplement figure 1

and 2).

The Univariate Cox Proportional Hazards Regression

model revealed that high clinical stage and lymph node

metastasis had prognostic significance in overall survival

Fig. 2 Co-immunolocalization of NICD, Hes-1 and c-Myc in H314

cells. A.H314 cells showing staining for Hes-1 (a), c-Myc (b) and co-

localization of Hes-1 and c-Myc (c). B.H314 cells showing staining

for c-Myc (a), NICD (b) and co-localization of c-Myc and NICD (c).

C.H314 cells showing staining for Hes-1 (a), NICD (b) and co-

localization of Hes-1 and NICD (c)

Table 5 Kaplan–Meier and log-rank analysis for clinicopathological

variables, NICD, Hes-1 and c-Myc in relation to overall and disease-

free survival of 44 patients with OSCC

Variables Overall

survival

Disease-free

survival

p value p value

1. Stage (I, II/III, IV) 0.023* 0.022*

2. Grade (well/moderate, poor) 0.033* 0.010**

3. LN metastasis (?/-) \0.001** 0.006**

4. NICD (?/-) 0.004** 0.031*

5. Hes-1 (?/-) 0.004** 0.033*

6. c-Myc (?/-) 0.032* 0.160

7. NICD ? c-Myc (N?/M? vs others) \0.001** \0.001**

8. Hes-1 ? c-Myc (H?/M? vs others) \0.001** \0.001**

- = includes category 0 and 1?, ? = includes category 2? and 3?,

LN = lymph node, N?/M? = NICD and c-Myc double positive

cases, H?/M? = Hes-1 and c-Myc double positive cases

* Statistically significant; ** Statistically highly significant

Table 3 Relationship between the expression of NICD and c-Myc in

oral cancer

Expression of NICD Expression of c-Myc

(No. of patients = 44) 0 (6) 1? (3) 2? (13) 3? (22)

0 3 2 0 1 0

1? 10 1 0 2 7

2? 14 2 1 6 5

3? 17 1 2 4 10*

0 (negative), no staining or \5 % positive cells; 1? (mild), 5–15 %

positive cells; 2? (moderate), 16–25 % positive cells and 3? (intense),

[25 % positive cells

* Significant correlation was not found between the expression of

NICD and c-Myc in oral carcinoma samples (p [ 0.05)

Table 4 Relationship between the expression of Hes-1 and c-Myc in

oral cancer

Expression of Hes-1 Expression of c-Myc

(No. of patients = 44) 0 (6) 1? (3) 2? (13) 3? (22)

0 5 1 1 0 3

1? 8 2 0 1 5

2? 11 0 1 7 3

3? 20 3 1 5 11*

0 (negative), no staining or \5 % positive cells; 1? (mild), 5–15 %

positive cells; 2? (moderate), 16–25 % positive cells and 3? (intense),

[25 % positive cells

* Significant positive correlation was not found between the expres-

sion of hes-1 and c-Myc in oral carcinoma samples (p [ 0.05)

126 Page 6 of 10 Med Oncol (2014) 31:126

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of patients with OSCC. Whereas, high clinical stage, lymph

node metastasis, Nestin and Hes-1 had prognostic signifi-

cance in disease-free survival of patients with OSCC.

Interestingly, NICD/c-Myc and Hes-1/c-Myc double posi-

tivity had high prognostic value in overall and disease free

survival of patients with OSCC (Table 6). Moreover, the

Multivariate Cox Proportional Hazards Regression model

indicate that clinical stage, NICD/c-Myc double positivity

and Hes-1/c-Myc positivity were independent prognostic

factors in overall and disease-free survival of oral carci-

noma patients (Table 7).

Discussion

Oral carcinogenesis is a multistep process with accumula-

tion of series of mutations, aberrant signaling events,

reactivation of stem cells and increased epithelial to mes-

enchymal transition. Besides stem cell markers, upregula-

tion of b-catenin and Notch signaling molecules has been

demonstrated in OSCC [22, 23]. However, studies on the

prognostic significance of Notch signaling molecules are

limited in oral cancer. Interestingly, Notch pathway mol-

ecules were reported to crosstalk with proliferative markers

Fig. 3 Overall (a) and disease free (b) survival of patients with OSCC according to NICD/c-MYC double positivity calculated by the Kaplan–

Meier method

Fig. 4 Overall (a) and disease free (b) survival of patients with OSCC according to Hes-1/c-MYC double positivity calculated by the Kaplan–

Meier method

Med Oncol (2014) 31:126 Page 7 of 10 126

123

in keratinocytes and OSCC [16, 24]. In this study, we have

investigated the expression pattern of NICD and Hes-1 in

the precancerous lesions of oral squamous epithelium. We

have also analyzed the prognostic significance of NICD,

Hes-1 and c-Myc in oral cancer.

Notch 1 signaling cascade regulates several biological

processes such as proliferation, differentiation and cell

survival. Dysregulation of Notch signaling has been

observed in various cancers including OSCC [2]. In line

with the previous report of Zhang et al. [25] in gastric

cancer, the expression of NICD was predominantly

observed in the cytoplasm of oral precancerous and can-

cerous cells. However, cytoplasmic and nuclear expression

pattern of NICD also persists in few cells of the oral car-

cinoma tissue. In this study, Hes-1 showed a strong nuclear

staining in preneoplastic and neoplastic tissues which was

consistent with the report of Yoshida et al. [26]. Further-

more, the expression pattern of NICD and Hes-1 was

gradually increased from normal to dysplasia to carcinoma.

On the other hand, co-immunolocalization of NICD and

Hes-1 was observed in H314 cells. These results suggest a

significant role for activated Notch 1 signaling in oral

carcinogenesis.

Epithelial to mesenchymal transition occurs in precan-

cerous, early and advanced stages of OSCC. As a result of

EMT, the carcinoma cells acquire the ability to invade

through basal lamina into the blood stream which results in

metastasis. Recently, it was found that the cell obtained

through EMT has stem cell like characteristics [27].

Notably, hypoxia induces EMT of OSCC cells by activat-

ing Notch signaling cascade [12]. In the present study, the

expression of NICD and Hes-1 are significantly correlated

with advanced stages of OSCC. Particularly, the expression

of NICD and Hes-1 are higher in stage III–IV when com-

pared with stage I–II. Interestingly, both NICD and Hes-1

correlates with lymph node metastasis of oral carcinoma

cells as reported by Luo et al. [28] in gastric cancer. These

results clearly indicate the role of NICD and Hes-1 in

invasion and aggressiveness of oral carcinoma cells.

It is well known that the function of Notch 1 depends

upon the cell type and it induces differentiation of kerati-

nocyte/epidermal stem cells [29]. Reduction of Notch 1

was found to induce neoplasms of squamous epithelial

cells of oral, esophageal and cervical tissues [30]. Recently,

the activated Notch pathway was found to maintain stem-

ness of lung cancer cells [31]. Notch also controls self-

renewal of cancer stem cells and thereby regulates metas-

tasis, invasion and chemoresistance [2]. In addition, Notch

1 signaling regulates keratinocyte stem/progenitor cell

markers including p63 in differentiating keratinocytes.

Furthermore, Notch activation inhibits proliferation and

induces differentiation of keratinocyte [24]. More recently,

it was found that the growth of CD133 positive A549 cells

is inhibited by blocking Notch pathway and it sensitizes

A549 cells to chemotherapeutic response [31]. Notch

suppresses the downstream effectors of b-catenin in

Table 6 Univariate Cox

proportional hazards regression

analysis (Cox Method) for

variables in relation to overall

and disease-free survival of 44

patients with OSCC

- = includes category 0 and

1?, ? = includes category 2?

and 3?, LN = lymph node

* Statistically significant;

** Statistically highly

significant

Variables Overall survival Disease-free survival

p value Hazards

ratio

95 % CI p value Hazards

ratio

95 % CI

1. Stage (I, II/III, IV) 0.036* 3.877 1.093–13.750 0.029* 2.648 1.104–6.352

2. Grade (well/

moderate, poor)

0.067 0.150 0.020–1.140 0.019* 0.233 0.069–0.784

3. LN metastasis (?/-) 0.003** 21.971 2.877–67.786 0.009** 2.962 1.312–6.689

4. NICD (?/-) 0.100 41.111 0.492–437.665 0.042* 3.049 1.042–8.918

5. Hes-1 (?/-) 0.100 43.015 0.540–565.530 0.044* 3.014 1.030–8.823

6. c-Myc (?/-) 0.191 30.467 0.182–5,090.190 0.177 2.297 0.687–7.686

7. NICD ? c-Myc \0.001** 0.061 0.017–0.224 \0.001** 0.070 0.026–0.192

8. Hes-1 ? c-Myc \0.001** 0.081 0.025–0.255 \0.001** 0.176 0.077–0.403

Table 7 Multivariate Cox proportional hazards regression analysis (Cox Method) for variables in relation to overall and disease-free survival of

44 patients with OSCC

Variables overall survival Disease-free survival

p value Hazards ratio 95 % CI p value Hazards ratio 95 % CI

1. NICD ? c-Myc 0.001** 0.098 0.026–0.366 0.008** 0.153 0.039–0.607

2. Hes-1 ? c-Myc 0.032* 0.282 0.089–0.896 0.012* 0.176 0.045–0.679

* Statistically significant; ** Statistically highly significant

126 Page 8 of 10 Med Oncol (2014) 31:126

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colorectal cancer [32]. However, b-catenin regulates Hes-1

expression and it co-ordinate with Notch 1 pathways in

maintaining tumorigenic potential of intestinal cells [33].

Therefore, we have selected one of the b-catenin down-

stream effectors c-Myc and studied its correlation with

NICD and Hes-1 in OSCC.

In this study, the nuclear expression of c-Myc was

considered as positive. In line with the previous report of

Shah et al. [34], the expression of c-Myc was significantly

correlated with advanced stages. In addition, c-Myc

expression was higher in lymph node metastasis positive

cases when compared with lymph node metastasis negative

cases. These results indicate the role of c-Myc in main-

taining tumorigenic potential of oral carcinoma cells. In the

present study, NICD and Hes-1 positive H314 cells showed

mild expression of c-Myc. Interestingly, statistically sig-

nificant correlation was not observed between NICD,

Hes-1 and c-Myc in OSCC. This suggests that NICD, Hes-

1 and c-Myc might have independent roles in oral cancer.

The prognostic significance of Notch receptors have been

demonstrated in solid tumors including OSCC [16]. How-

ever, studies on the prognostic significance of NICD and

Hes-1 were limited. In the present study, NICD and Hes-1

positive cases showed worst survival when compared with

negative cases in both overall and disease free survival. The

result was in line with the previous findings of Zhang et al.

[25] for NICD in gastric cancer and Hassan et al. [35] for

Hes-1 in lung adenocarcinoma. This suggests that impaired

Notch signaling may be related to the progression of OSCC.

Moreover, cases with increased c-Myc expression showed

poor survival when compared with c-Myc negative cases in

overall survival analysis. However, statistically significant

association between c-Myc and poor survival was not

reached in disease free survival analysis. Interestingly,

NICD?/c-Myc? and Hes-1?/c-Myc? double positive

cases showed worst survival than other cases in OSCC. This

indicates that NICD?/c-Myc? and Hes-1?/c-Myc? double

positivity might be used to identify high risk patients to

whom immediate attention is required for therapy.

In the present study, the Univariate analysis showed

prognostic significance of NICD and Hes-1 in disease free

survival of oral carcinoma patients. Statistically significant

correlation was not found between c-Myc expression and

prognosis which was in concurrent with the previous report

of Shah et al. [34]. Notably, the association between NICD,

Hes-1, c-Myc and prognosis was not significant in Multi-

variant analysis of oral carcinoma cases. Interestingly,

NICD?/c-Myc? and Hes-1?/c-Myc? double positivity

showed high prognostic significance in both overall and

disease free survival of OSCC in Univariate and Multi-

variate analyses. This suggests that NICD?/c-Myc? and

Hes-1?/c-Myc? double positivity might be used as inde-

pendent prognostic indicator of OSCC.

In conclusion, a gradual increase in the expression pat-

tern of NICD and Hes-1 from normal to dysplasia to car-

cinoma suggests their role in the progression of oral cancer.

In addition, NICD, Hes-1 and c-Myc might have inde-

pendent roles in OSCC as the significant correlation was

not reached. Moreover, NICD?/c-Myc? and Hes-1?/c-

Myc? double positivity might be used to identify high risk

patients for rapid and effective therapy. On the other hand,

NICD, Hes-1 and c-Myc do not have prognostic signifi-

cance in oral cancer. Conversely, NICD?/c-Myc? and

Hes-1?/c-Myc? double positivity might be used as inde-

pendent prognostic indicator of OSCC. However, further

investigations are needed with larger sample size, longer

follow up period and molecular crosstalk between Notch

and b-catenin pathways to elucidate their prognostic sig-

nificance and therapeutic strategies in OSCC.

Acknowledgments We acknowledge ‘UGC Meritorious Research

Fellowship Programme’ for financial assistance. We would like to

thank Dr. Stephen Hann, Vanderbilt University, USA for his generous

gift of c-Myc antibody used in this study. We also thank Dr. Angela

Hague for providing H314 cell line used in this study.

Conflict of interest None.

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