oral squamous cell carcinoma ( oscc ) is the sixth largest group of malignancies globally and represents one of the leading causes of mortality .
it remains a major cancer in the indian subcontinent , comprising more than 40% of all cancer cases .
the most commonly involved sites of tumor development in the indian population are buccal mucosa and tongue . despite the advances in treatment and therapeutic modalities the five year survival rate of oscc
the possible reasons for poor survival rates are late detection and/or local recurrence / regional lymph node metastasis .
several studies have focused on defining tumor - specific molecular markers that can either detect cancer at an early stage or can predict patient 's outcome
. however , clinicopathological factors and molecular biomarkers that could identify patients at early stage or patients at high risk of recurrence / lymph node metastasis are still undefined . keratins ( k ) are epithelia predominant intermediate filament proteins which are expressed in a differentiation dependent and site specific manner . on one hand keratins
are connected to the nuclear envelope while on the other hand , they are connected to the junctional complexes like desmosomes and hemidesmosomes through desmoplakin and plectin-4 integrin respectively .
desmosomes form cell - cell contacts while hemidesmosomes attach the cells to the basal lamina .
previous results from our laboratory have shown deregulation of paired expression of keratins in oral cancer .
changes in keratin and associated proteins like that of the desmosomes ( desmoplakin ) and hemidesmosomes ( 64 integrin ) are also documented in oscc . to our knowledge
there is no report to date about the alterations of keratins and associated proteins together during sequential stages of oral carcinogenesis .
it is important to understand these molecular alterations during oral cancer development , which can help in development of early diagnostic and/or prognostic markers . in patients ,
the difficulty in procuring the normal tissues and tissues of all tumor stages , hampers the analysis of molecular markers
. however , animal models of carcinogenesis allow the reproducible isolation of all tumor stages , including the normal tissues . to this end ,
4nqo induced rat model of carcinogenesis remains the preferred model for studies related to oral carcinogenesis . in the present communication ,
we have studied the alterations in keratins and associated proteins during sequential stages of experimental oral carcinogenesis .
our results have shown alterations in keratins , desmoplakin as well as 64 integrin at different stages of rat oral carcinogenesis , which show a potential to be used as diagnostic / prognostic markers .
fourty five days old male sprague dwaley rats ( weighing150 - 200 g ) were fed with standard diet and water ad libitum .
they were randomly divided into three groups , 4nqo treated group ( n=56 ) , propane diol ( vehicle ) control group ( n=56 ) and untreated control group ( n=56 ) ( 8 animals in each of these groups were treated / untreated for 10 , 20 , 40,80,120,160,200 days ) .
12l of 0.25% 4nqo ( sigma ) was applied on each side of buccal mucosa , thrice a week for induction of tumors in buccal mucosa ( 60g of 4nqo ) .
intramuscular atropine injections ( 0.04mg / kg ) were given prior to 4nqo application to prevent salivation and the animals were also kept inaccessible to drinking water after treatment for 2 hrs .
4nqo was also given in drinking water ( 0.001% 4nqo ) for induction of tumors in tongue .
animals were weighed after every 15 days . after each time point rats were fed with normal drinking water for another 15 days .
2de for keratins was carried out using ipg strips for first dimention ( biorad ) . for second dimention 3.9% stacking and 8 - 10% gradient separating gels
sds page for desmoplakin was performed using 3.9% stacking gel and a combination of 6% top and 10% bottom gel .
the primary antibodies used were ae1 ( zymed , usa , cat no [ x ] 18 - 0153)/ae3 ( zymed , usa , cat no [ x ] 18 - 0154 ) , desmoplakin ( serotec , uk , cat .
a 5316 ) cell lysates were prepared in sds lysis buffer ( 5 mm egta , 5 mm edta , 0.4% sds and protease inhibitor cocktail ( cat no .
protein estimation for total cell lysates was carried out according to modified lowry 's method .
total rna was isolated by using tri reagent ( sigma ) from frozen tongue tissues .
2g of rna was reverse transcribed using mbi fermentas cdna synthesis kit . for rt - pcr
the following pcr primers were used : for 6 , sense 5-gtgaggtgtgtgaacatcag-3 and antisense 5-catggtatcggggaatgct-3 ( gene accession number - xm_001059465 ) ; for 4 , sense 5-gcagccctgggcccaaca-3 and antisense 5-cccaggcaggctttgcag- 3 ( gene accession number - nm_013180 ) ; for 18s sense 5-atggccgttcttagttggtg-3 and antisense 5-aacgccacttgtccctctaa-3 ( gene accession number - v01270.1 ) for immunohistochemistry paraffin embedded sections were deparaffinized , dehydrated and the antigen was retrieved using 0.1 m citrate buffer at ph 6.0 by microwave treatment .
frozen sections were fixed with methanol and immunocytochemistry was performed using k8 ( abcam , uk , cat.no .
sprague dwaley rats were sacrificed after treatment with 4nqo at different time points and dorsal tongue / buccal mucosa were dissected .
figure 1a2 to a4 shows gross changes in the tongue and figure 1a6 shows gross changes in buccal mucosa after 4nqo treatment .
the tumour sizes for papillomas / carcinomas were 38.91 + 3.94/62.43 + 9.11 mm respectively [ figure 1b ] .
it was not possible to decipher the weights of the tumours , as the tumors have different invasive areas in the lingual tissue which makes them difficult to separate .
the histopathological analysis of posterior dorsal tongue epithelium revealed no alterations in vehicle control and untreated control groups , 10 , 20 and 40 days 4nqo treated lingual tissues .
the dysplastic tissues showed increase in basal cell and single cell keratinization with abnormal nuclei [ figure 1c2 ] .
160 days treated animals showed papillary growth of the squamous epithelium with marked parakeratosis[figure 1c3 ] . at the end of 200 days all the animals demonstrated well differentiated scc with disordered and infiltrative growth of squamous cells [ figure 1c4 ] [ table 1 ] .
the analysis of buccal mucosal tissues revealed no histological alterations in vehicle treated tissue and tissues treated with 4nqo upto 160 days [ figure 1c5 ] .
papillomatous lesions were observed at the end of 200 days of 4nqo treatment [ figure 1c6 ] .
the study could not be further extended beyond 200 days because the animals became moribund .
for isolation of earlier stages , the experiment was repeated with higher doses of 4nqo , with 80/100g of oral application , for 120/160 days however , the earlier stages could not be obtained .
a. morphology of ( 1 ) vehicle treated tongue , ( 2 ) tongue treated for 80/120 days , ( 3 ) 160 days and ( 4 ) 200 days with 4nqo .
note white patch in the posterior dorsal tongue after 80/120 days , and exophytic growth in both 160 and 200 days after 4nqo treatment as indicated by the arrow , ( 5 ) control buccal mucosa and ( 6 ) buccal mucosa treated for 200 days showing small papillomatous growth indicated by the arrow .
( c ) h and e sections of ( 1 ) vehicle treated tongue , ( 2 ) tongue treated for 80/120 days ( dysplasia ) , ( 3 ) 160 days ( papillomatous lesion ) and ( 4 ) 200 days ( carcinoma ) with 4nqo ( 5 ) vehicle treated buccal mucosa and ( 6 ) papillomatous growth of buccal mucosa obtained after 200days of 4nqo treatment .
bar = 100m time points and number of animals utilized in the study keratins are identified based on the molecular weight and their isoelectric point which can be separated on a 2-dimensional electrophoretic gel .
hence , in order to establish the pattern of normal rat lingual keratins we performed mass spectrometry of the spots isolated after 2-de of enriched keratins .
the ms analysis showed presence of k5,6a , k14 and k17 [ figure 2a ] . after establishing the keratin pattern of lingual tissues western
dysplastic , papillomatous lesions and carcinoma tissues demonstrated consistent up regulation of k5 and k6a as compared to normal tissue [ figure 2b244 ] respectively ] .
further , there were no major consistent changes in other keratins on the 2-de gel .
( a ) 2de separation and colloidal coomassie staining of enriched keratins from normal lingual epithelium , for identification by mass spectrometry .
( b ) western blotting of keratins separated on 2de at various stages of lingual carcinogenesis .
( 1 ) normal , ( 2 ) dysplastic , ( 3 ) pappiloma and ( 4 ) scc tissue .
note increase in k5/6a in ( 2 ) dysplastic , ( 3 ) papillomatous lesion and ( 4 ) carcinoma tissues .
numbers indicate keratin numbers . c. k8 expression at various stages of oral carcinogenesis using ihc .
note absence of k8 in normal tissue , however , k8 is present in suprabasal layers of ( 2 ) dysplastic tissues as indicated by arrow .
further , very high levels of k8 can be observed in ( 3 ) papillomatous lesion and ( 4 ) carcinoma tissues .
( c ) the upper panel shows k8 staining of normal and various stages of carcinogenesis of lingual mucosa .
bar = 100m k8 is expressed in various malignant tissues and it plays an important role in the process of transformation .
none of the normal tissues of any time points showed k8 expression [ figure 2c1 ] , however , dysplastic tissues demonstrated presence of k8 in the suprabasal layers [ figure 2 c2 ] .
further , papillomatous lesions and carcinomas showed very high levels of k8 [ figure 2c3 and c4 ] .
non reactivity of antibody both in immunohistochemistry and western blotting hindered k18 analysis of rat tissues .
however , it is possible that k18 is expressed in these tissues . to further substantiate our results on aberrant k8 expression we performed immunofluorescence using the same k8 antibody .
as shown in the figure 2c , the normal tissue does not show k8 expression whereas dysplastic , papilloma and scc tissues showed vivid presence of k8 .
keratins on one hand are connected to the nuclear envelope while on the other hand , they are connected to the junctional complexes like desmosomes and hemidesmosomes through desmoplakin and plectin-4 integrin respectively .
it was of our interest to understand the levels and localization of keratin associated proteins .
hence , to study the alterations in desmoplakin levels in these tissues , western blot analysis was performed .
no changes in desmoplakin levels were observed in histologically normal tissues treated upto 40 days with 4nqo [ figure 3b1 ] .
dysplastic tissues showed 4.1 fold increase in desmoplakin levels [ figure 3b2 ] , whereas papillomatous and carcinoma tissues demonstrated 2.8 and 4.6 fold decrease in desmoplakin levels respectively [ figure 3b3 and b4 ] .
-actin was used as a loading control in normal , dysplasia and papillomatous tissues , whereas cooma c blue staining was used for equal loading as the carcinoma tissues demonstrated alterations in -actin levels [ figure 3 b4 ] .
( a ) immunohistochemical analysis of desmoplakin during oral carcinogenesis . in ( 1 ) normal tissue , desmoplakin was detected as diffused cytoplasmic and intense membranous staining in the suprabasal layers .
while , in ( 2 ) dysplasia increase cytoplasmic and membranous staining of desmoplakin is evident in the suprabasal layers ( arrowheads and arrow respectively ) . in ( 3 ) papillomatous lesion , decreased staining of desmoplakin is present in the suprabasal layers and only membranous staining can be seen ( arrow ) . in ( 4 ) scc decreased intensity of desmoplakin was observed .
n1 , n2 , n3 indicate vehicle treated and t1 , t2 , t3 indicate 4nqo treated lingual tissues . ( 1 ) desmoplakin levels in 4nqo treated histologically normal tissues of 40 days , ( 2 ) normal and dysplastic tissues ( 3 ) normal and papillomatous tissues of 160 days ( 4 ) normal and scc tissues of 200 days .
note increase in desmoplakin levels in ( 2 ) dysplastic tissues and decrease in ( 3 ) papillomatous lesion and ( 4 ) scc tissues further , to analyse the alterations in desmoplakin localization at various stages of rat lingual carcinogenesis , immunohistochemical analysis was carried out .
it was found that in histologically normal tissues desmoplakin was detected as diffused cytoplasmic and intense membranous staining in the suprabasal layers [ figure 3a1 ] . while , in dysplastic tissues increase cytoplasmic and membranous staining of desmoplakin was evident in the suprabasal layers [ figure 3 a2 ] . in papillomatous lesion ,
decreased staining of desmoplakin was seen in the suprabasal layers and only membranous staining could be observed [ figure 3a3 ] , whereas in scc , decreased intensity of desmoplakin was observed with diffuse cytoplasmic staining and loss of cell membrane staining [ figure 3a4 ] .
inspite of repeated efforts , and using various commercially available antibodies towards rat 64 integrin none of the antibodies reacted in western blot .
therefore rt - pcr was carried out for both 6 and 4 integrin at different stages of lingual carcinogenesis .
the dysplastic and papillomatous lesions did not show any significant change in the levels of 6 and 4 integrin mrna [ figure 4 a1and a2 ] .
however , scc tissues demonstrated significant increase in 6 and 4 integrin mrna ( p<0.05 , n=4 ) [ figure 4 a3 ] .
( a ) rt - pcr analysis of 6 and 4 integrin . ( 1 ) normal and dysplastic tissues , ( 2 ) normal and papillomatous tissues , ( 3 ) normal and carcinoma tissues .
6 integrin staining in ( 1 ) normal tissues , ( 2 ) dysplasia ( 3 ) papilloma and ( 4 ) scc
. please note suprabasal expression in dysplastic and papillomatous tissues and increased staining intensity in scc . to understand the cellular localization of 6 integrin ,
6 integrin staining was detected only in the basal cells of vehicle treated tissues [ figure 4b1 ] however , in dysplastic tissues 6 integrin was seen in the basal as well as in the suprabasal layers [ figure 4 b2 ] . in papillomatous lesions ,
further , weak staining of integrin 6 was observed in the basal layer as compared to that seen in the control tissues [ figure 4b3 ] . intense but diffused staining of 6 integrin was observed in the cytoplasm and the cell membrane of scc [ figure 4b4 ] .
our rt - pcr analysis of integrin 6 showed no alterations in papillomas however , increase staining intensity of integrin 6 was observed in ihc of papilloma tissues .
, the commercially available antibodies to 4 integrin did not react with rat tissues , we were not able to carry out immunohistochemical analysis for the same . taken together our analysis of keratin expression demonstrated , increase in k5/6a levels , and ectopic k8 expression during various stages of lingual carcinogenesis .
increased desmoplakin levels were observed in dysplastic tissues ( 80 and 120 days ) while its level decreased in papilloma and carcinoma tissues .
further , increase in suprabasal expression of 6 integrin was observed in dysplastic lesions of rat lingual epithelium which persisted in papillomas and scc .
experimental animal models have proved to be an important tool , to study the molecular alterations during the process of oral carcinogenesis , because of difficulties in obtaining sequential stages of carcinogenesis in human system . in the present study , we have isolated different stages of lingual carcinogenesis .
one of the goals of our study was to establish a model for buccal mucosal carcinogenesis , which is a predominant cancer , along with scc of tongue in the indian subcontinent .
inspite of prevention of salivation and higher doses of 4nqo we were unable to obtain all the stages of buccal mucosal carcinogenesis .
one of the possible reasons could be that the papillomatous lesion development is a rapid process and the earlier stages remained undetected .
the probable reason for not obtaining scc in buccal mucosa could be that the buccal mucosa may not have prolonged access to the carcinogen .
hence , the development of a proper model of buccal mucosal carcinogenesis still remains an area of investigation , and all the analysis was carried out only on lingual tissues .
our analysis of keratins expression at various stages of lingual carcinogenesis showed , increased levels of k5/k6a [ figure 2b2b4 ] and aberrant expression of k 8 in dysplastic , papillomatous lesions and carcinoma tissues of lingual epithelium [ figure 2c2c4 ] .
previous reports have demonstrated suprabasal expression of k5/14 and k6/16 in premalignant and malignant lesions of oral cavity .
the presence of k5/14 along with cd44 has also been shown to have cancer stem cell properties in hnscc .
thus , it is possible that the elevated levels of k5 observed in rat lingual carcinogenesis model could be indicative of retention of stem cell like properties of the premalignant and malignant cells .
previous results from our laboratory have shown down regulation of k5 expression in tobacco related oral cancer .
these differences possibly can be explained based on the fact that although 4nqo mimics many molecular changes that occur in human oral carcinogenesis , there could be subtle differences between tumor induced by a pure carcinogen like 4nqo and the tumor induced by tobacco .
further , the keratin gene regulation is not yet well understood and the partial differences observed in keratin expression during murine and human oral carcinogenesis , could be because of the complexity of keratin gene expression and their differential regulation .
keratin 6 is a marker of cell proliferation and its overexpression has been observed in hyperproliferative disorders .
suprabasal expression of k6/16 has been shown in oral cancer . moreover , in mouse epidermal carcinogenesis induction of k6/16 was observed in premalignant as wll as malignant skin tissues .
further , cadmium and arsenite treatment to bladder epithelial cells resulted into their transformation with concomitant induction of k6a .
these results indicate that k6 is closely associated with proliferation of precancerous tissue and may have a role in transformation .
the ectopic expression of k8 has also been shown previously by our laboratory and others .
further , our laboratory has demonstrated that forced expression of k8 in fetal buccal mucosal cells leads to cell transformation .
we have recently shown that downregulation of k8 results in down regulation of 64integrin mediated signaling and decreased cell motility and tumorogenicity .
these reports suggest that k8 is associated with transformation and increased malignant potential of squamous epithelial cells .
we noted increase in desmoplakin levels in dysplastic tissues [ figure 3a2 and figure 3b2 ] while we observed its downregulation in papillomatous lesions and scc [ figure 3a3 , a4 and figure 3b3 , 3b4 ] .
this increase in desmoplakin in dysplastic stages may be required for efficient carcinogenesis , further , intact cell - cell adhesion through desmoplakin may be essential in supporting tumor development .
however , this hypothesis needs to be confirmed in tissue specific knockout animal models of desmoplakin .
further , desmoplakin has been shown to be down regulated in a number of cancers including oral , and pharangeal cancers .
our rt - pcr analysis of integrin 6 showed no alterations in papillomas , however , increase staining intensity of integrin 6 was observed in ihc of papilloma tissues .
we have further shown over expression of 6 integrin using both rt pcr and ihc in scc tissues .
our results of ihc have demonstrated suprabasal expression of 6 integrin in premalignant and malignant lesions .
previous reports have demonstrated that suprabasal localization of 6 integrin in premalignant and malignant tissues of human oral cavity . and
64 integrin protein overexpression has been shown in murine as well as human cancers . in hnscc ,
the analysis of keratins and associated proteins in experimental oral carcinogenesis showed that the alterations predominantly begin after the dysplastic changes in the lingual epithelium .
these changes were observed in inbred animal system where carcinogen treatment results in definite conversion of premalignant lesions to carcinoma .
this is indicative of the fact that the molecular changes observed can prove as useful early biomarkers if similar alterations are observed in human system .
the ongoing work in our laboratory has demonstrated ectopic expression of k8 , increase desmoplakin levels and suprabasal localization of 4 integrin in human premalignant lesions ( data not shown ) .
thus , similar results were obtained in human system and hence , some of these alterations either singly or in combination can prove useful biomarkers for early diagnosis of human oral cancer .
we have also seen alterations in keratins , desmoplakin and 64 integrin in rat lingual scc .
downregulation of desmoplakin and overexpression of 64 integrin separately have been shown to have prognostic value .
studies from our lab and others have suggested that k8/18 can also be used as prognostic markers for human oral cancer .
hence , a correlation of alterations in all these proteins , with the clinical parameters can help in the development of reliable diagnostic / prognostic markers for human oral cancer .
work in this direction is already underway in our laboratory . in summary , the 4nqo model of oral carcinogenesis
, reproduces majority of the changes that are seen in human oral carcinogenesis and it can be exploited for the development of biomarkers for oral cancer .
dr . deepak kanojia , department of biological science , university of south carolina , columbia , sc 29208 dr .
anita m borges , department of histopathology , asian institute of oncology , s.l . raheja hospital , mahim , mumbai - 400 016 , india .
arvind d ingle , laboratory animal facility , advanced centre for treatment , research and education in cancertata memorial centre sector 22 , kharghar , navi mumbai - 410 210 , india .
sharada s sawant , vaidya lab , advanced centre for treatment , research and education in cancer ( actrec ) , tata memorial centre , 
 kharghar , navi mumbai- 410210 , maharashtra india dr .
milind m vaidya , vaidya lab , advanced centre for treatment , research and education in cancer ( actrec ) , tata memorial centre , kharghar , navi mumbai- 410210 , maharashtra india
 background : oral squamous cell carcinoma ( oscc ) is the sixth largest group of malignancies globally and the single largest group of malignancies in the indian subcontinent . despite the advances in treatment and therapeutic modalities the five year survival rate of oscc has not changed in the last few decades , and remains less than 40% . 
 several studies have focused on defining molecular markers that can either detect cancer at an early stage or can predict patient 's outcome 
 . however , such markers are still undefined . 
 keratins ( k ) are epithelia predominant intermediate filament proteins which are expressed in a differentiation dependent and site specific manner . 
 keratins are being used as biomarkers in different epithelial disorders including cancer . 
 they are associated with desmoplakin and 64 integrin which are components of desmosomes and hemidesmosomes respectively.materials and methods:4-nitroquinoline 1-oxide ( 4nqo ) was used as a carcinogen for the development of various stages of oral carcinogenesis in rat lingual mucosa . 
 two - dimentional gel electrophoresis was performed for the separation of keratins followed by western blotting for their specific identification . 
 western blotting and rt pcr was carried out for desmoplakin and 64 integrin respectively to understand their levels . 
 immunohistochemical analysis was carried out to further study the localization of desmoplakin and 6 integrin.results:in this study we have analysed the alterations in keratins and associated proteins during sequential stages of 4nqo induced rat oral carcinogenesis . our results showed that the alterations primarily begin after the dysplastic changes in the lingual epithelium like the elevation of keratins 5/6a , ectopic expression of keratin 8 , increase in suprabasal expression of 6 integrin and increase in desmoplakin levels . 
 most of these alterations persisted till the development of scc except desmoplakin , the levels of which were downregulated in papillomatous lesions and scc . 
 many of these alterations have also been documented in human oral carcinogensis.conclusion:thus , 4nqo model of rat lingual carcinogenesis reproduces majority of the changes that are seen in human oral carcinogenesis and it can be exploited for the development of biomarkers . 
BACKGROUND
MATERIALS AND METHODS
RESULTS
DISCUSSION
AUTHOR'S PROFILE