stat3 activation has important implications in the cell proliferation of cutaneous squamous cell carcinoma ( scc ) .
knockdown of stat3 activation completely prevents cell proliferation and growth in cutaneous scc but is not sufficient to induce cell death     .
signaling cross talk may be responsible for the survival mechanism of scc     [ 2 , 3 ] .    
the epidermal growth factor receptor ( egfr ) is a member of the erbb family , which consists of four members : egfr , erbb2 , erbb3 , and erbb4 . in particular , egfr and erbb2
receptor activation of the egfr leads to the recruitment and phosphorylation of several downstream intracellular substrates , leading in turn to mitogenic signaling and other tumor - promoting cellular activities . over - expression of the egfr in epithelial tumors , including head and neck , lung , breast , colon , and other solid malignant tumors , has frequently been correlated with their poor prognosis     [ 69 ] . in breast cancers and head and neck scc ( hnscc ) , a strong correlation between stat3 and egfr expression has been observed , and it has been suggested that the jak / stat pathway is one of the important downstream routes for egfr signaling     .
the recruitment sites of stat3 within the egfr have been identified and characterized , indicating the direct association of these two molecules     .
in addition , the existence of egfr independent signaling for stat3 activation has been reported , enhancing the importance of stat3 activation in tumor growth     .
these data lead to the idea that stat3 activation is very important for malignant proliferation of the epithelial tumors , and that egfr activation correlates closely with stat3 activation and is one of the key regulators .    
phosphotidylinositol-3 kinase ( pi3k ) is one of the downstream signaling molecules of the egfr and plays a role in the proliferation or invasion of scc     [ 12 , 13 ] .
constitutive activation of pi3k by mutation and inactivation of its encoding gene is observed in a significant number of cancers .
antagonists of egfr , pi3k , and mek have inhibitory effects on the growth of hnscc     . however , there is little information on the role of pi3k / akt and stat3 pathways in mediating cell proliferation and the correlation between stat3 and pi3k / akt signaling in cutaneous scc .      in this study
, we aimed to investigate the activity of stat3-related activators such as egfr and pi3k / akt in the proliferation of a cutaneous malignant tumor , scc , and evaluate the therapeutic value of inhibition of the signaling pathways .
an egfr inhibitor ( ag1478 ) and a phosphotidylinositol-3 kinase ( pi3k ) inhibitor ( wortmannin ) were purchased from calbiochem ( san diego , ca ) .
samples of scc , including three metastasis cases and adjacent skin , were obtained from 32 patients , 16 males and 16 females , with an average age of 74 years ( range : 41101 years ) . all subjects provided written informed consent prior to enrollment in the study .
surgically removed tissue samples were fixed in 10% neutral buffered formaldehyde and paraffin embedded for histopathology or immediately frozen in liquid nitrogen for nuclei extracts .
three human cutaneous scc cells lines , hsc-1 , -3 , and -4 , were kindly provided by dr .
these cells were grown in dulbecco 's modified eagle 's medium ( life technologies , gaithersburg , md ) supplemented with 10% fetal bovine serum , 100  u / ml penicillin , and 100  g / ml streptomycin .
primary human epidermal keratinocytes ( heks ) were purchased from kurabo ( osaka , japan ) .
heks were grown in keratinocyte serum - free medium ( sfm ) supplemented with human keratinocyte growth supplement and psa ( penicillin , streptomycin , and amphotericin b ) solution ( gibco , grand island , ny ) .
cells were maintained in a standard culture incubator with humidified air containing 5% co2 at 37c .
skin specimens of 5  m paraffin - embedded sections were stained immunohistochemically using a streptavidin - biotin - peroxidase complex procedure provided by the dako csa system , hrp ( dako , carpinteria , ca ) , according to the manufacturer 's protocol . in brief
, the sections were incubated at room temperature for 30 minutes with an anti - phospho - egfr monoclonal antibody ( mab ) ( tyr1173 ) ( 53a5 ) , an anti - phospho - akt ( ser473 ) ( d9e ) mab , or a phospho - stat3 ( tyr705 ) antibody ( cell signaling technology , beverly , ma ) .
after three washes with pbs , the samples were incubated with biotinylated antirabbit igg and the avidin - biotin complex at room temperature for 15 minutes .
nuclear extracts from surgical tissues were purified following the method described by corsini et al .     , with slight modifications .
the tissues were frozen immediately in liquid nitrogen , and then 8  m slices from the tissue were cut with a cryostat .
the sections were washed with pbs and were homogenized in 1  ml of a hypotonic lysis buffer ( 10  mm hepes , ph 7.8 , 10  mm kcl , 2  mm mgcl2 , 1  mm dithiothreitol , 0.1  mm ethylenediaminetetraacetic acid , and 0.1  mm phenylmethylsulfonyl fluoride ) with a tissue homogenizer for 20 seconds .
homogenates were kept on ice for 15 minutes , then 125  l nonidet p-40 solution was added and mixed for 15 seconds , and the mixture was then centrifuged for 30 seconds at 12000  rpm . nuclear extracts from the pellets or cells were prepared and emsas were performed essentially as described earlier     .
binding reaction mixtures ( 20  l ) containing 5  g nuclear extract protein , 2  g poly ( di - dc ) ( amersham pharmacia biotech , piscataway , nj , sweden ) , p - labeled probe ( stat3 ) , 50  mm nacl , 2  mm mgcl2 , 0.2  mm na2edta , 1  mm dtt , 10% ( v / v ) glycerol , and 4  mm tris - hcl ( ph 7.9 ) were incubated for 30 minutes at room temperature .
proteins were separated by electrophoresis in native 6% polyacrylamide gels using a tris - borate - edta running buffer ( 12.5  mm tris - borate containing 0.25  mm na2edta , ph 8.0 ) , followed by autoradiography . the stat3 probe ( santa cruz biotechnology , santa cruz , ca ) 5'  gat cct tct ggg
aat tcc tag atc  3 ' ; 3'  cta gga aga ccc tta agg atc tag  5 ' was labeled with [ -p ] datp ( du pont nen , boston , ma ) using t4 polynucleotide kinase ( boehringer mannheimroche , mannheim , germany ) .
the cells were washed with ice - cold d - pbs containing 200  m sodium orthovanadate ( na3vo4 ) , frozen immediately in liquid nitrogen , and then lysed in lysis buffer ( 25  mm tris - hcl , ph 7.6 , 200  mm boric acid , 150  mm nacl , 50  mm naf , 5  mm na2edta , 1% triton x-100 , 10  mm sodium pyrophosphate , 2  mm egta , 20  mm p - nitrophenyl phosphate , 1% bsa , 20  m na3vo4 , and 2  mm dtt ) containing protease inhibitors ( 2  g / ml each of aprotinin , leupeptin , pepstain , antipain , and 100  g / ml pmsf ) .
the lysates were centrifuged at 10,000  g at 4c for 15 minutes , and the resulting supernatants were subjected to immunoprecipitation or immunoblot analysis .
protein concentrations in the supernatants were quantitated using the coomassie plus protein assay reagent ( bio - rad loboratories , tokyo , japan ) .
samples ( 20  g protein each ) for immunoblotting were separated on 8% sds - polyacrylamide gel electrophoresis gels for akt and stat3 and were blotted onto hybond - ecl nitrocellulose membranes ( amersham pharmacia biotech , piscataway , nj , usa ) , followed by blocking with 5% skim milk in tris - buffered saline for 2 hours at room temperature , and were then probed with a primary antibody to phospho - akt ( ser473 ) , phospho - stat3 ( tyr705 or ser727 ) antibody ( cell signaling technology ) , or a horseradish peroxidase ( hrp)-conjugated anti - phosphotyrosine antibody , py20 ( santa cruz biotechnology , santa cruz , ca ) , overnight at 4c .
bound antibodies were detected using the ecl western blotting detection reagent ( amersham pharmacia biotech ) .
after film exposure , the membranes were washed 4 times for 5 minutes each in pbs - tween 20 and were then incubated for 30 minutes at 50c in stripping buffer ( 62.5  mm tris hcl ph 6.8 , 2% sds , and 100  mm 2-mercaptoethanol ) .
the membranes were blocked with 5% skim milk again , and then reprobed with an antibody to akt antibody or stat3 antibody ( cell signaling technology ) or reblotted with human actin ( santa cruz ) as a loading control overnight at 4c .
supernatants for immunoprecipitation were incubated at 4c with the anti - egfr ( 1005 ) antibody ( santa cruz ) bound to g - sepharose beads ( 2  g ab per 20  l beads ; amersham pharmacia biotech ) for 4 hours , after which the beads were washed twice with 1  ml wg buffer ( 50  mm hepes - naoh , ph 7.6 , 150  mm nacl , 0.1% triton x-100 ) and resuspended in sds sample buffer .
quantification of protein expression was done using imagej analysis software ( version 1.44b ) ( bethesda , md , usa ) .
cell growth was determined using the mts cell proliferation assay ( promega , madison , wi ) .
twenty - four hours after seeding , the cells were treated with ag1478 ( 0.25 or 2.5  m ) for the indicated time and the medium and substrates were exchanged every 24 hours , after which the mts reagent was added and allowed to react for 2 hours .
absorbance at 490  nm was measured using a microplate reader ( emax ; molecular devices , sunnyvale , ca ) .
cell growth is expressed as a percentage of that in nontreated cells at each indicated time point .
dmso was used as a control for ag1478 treatment . to assess the effect of serum on cell growth
, the cells were seeded at a density of 1  10 cells / well in the medium with serum in 96 well plates .
cell growth was evaluated at 0 hour , 36 hours , and 48 hours after the first exchange of medium .
the results are expressed as a percent ratio of serum - free to serum conditions for hacat and hsc-1 cells .
early in apoptosis , membrane phospholipid phosphatidylserine ( ps ) is translocated to the outer leaflet of the plasma membrane .
annexin v is a 35 - 36  kd ca dependent , phospholipid - binding protein that has a high affinity for ps . staining with annexin v - fitc ( bd pharmingen , san diego , ca ) in conjunction with the vital dye propidium iodide ( pi ) ( sigma )
hsc-1 cells were treated with control solvent or with ag1478 ( 0.25 or 2.5  m ) for 12 hours .
cells were scraped , then spun down , and washed twice with 5  ml cold pbs .
the cells were resuspended in binding buffer ( 0.01  m hepes , ph 7.4 ; 0.14  m nacl ; 2.5  mm cacl2 ) at a concentration of 1  10  cells / ml and then stained according to the manufacturer 's protocol .
cells were analyzed using a facs caliber flow cytometer ( bd , san jose , ca ) .
apoptotic cells exclude all those dyes which are used in cell viability assays , such as pi , while necrotic cells do not . in cells with a damaged cell membrane , pi induces a red fluorescence of the dna , while it is excluded from cells with a preserved plasma membrane .
the lower left quadrant ( ll ) of the cytograms shows the viable cells , which exclude pi and are negative for annexin v - fitc binding .
the upper right quadrant ( ur ) represents nonviable , necrotic cells , positive for annexin v - fitc binding and pi uptake .
the lower right quadrant ( lr ) represents apoptotic cells , annexin v - fitc is positive but pi is negative .
statistical analyses were performed using prism 5 software ( graphpad software , san diego , ca ) .
thirty - five paraffin - embedded sections of scc including 2 metastatic lesions were examined immunohistochemically for the expression of phosphorylated ( p)-egfr , p - akt , and p - stat3 , and the cases in which they were expressed in more than 20% of the cells were defined as positive ( figure 1 , table 1 ) .
twenty - five of the 35 sccs showed positive expression of p - egfr in tumor cells .
the reactivity of p - egfr was seen mainly in the cytoplasm and partially in the nucleus ( figure 1(a ) ) .
p - akt was expressed in the cytoplasm and nucleus of atypical cells ( figure 1(b ) ) .
stronger p - akt expression was seen in atypical cells surrounding the invasive lesions into the dermis .
we evaluated stat3 activation by the expression of both phospho - tyr705 and phospho - ser727 .
most of the cases showed phosphorylation on both sites , mainly in the nuclei , and partially in the cytoplasm , though the expression at each site differed in degree .
overall , expression of tyr705 was considered to be positive for p - stat3 ( figure 1(c ) ) .
actinic keratosis , a premalignant lesion of scc , also over - expressed p - stat3 if the adjacent scc was also positive for p - stat3 . none of p - egfr , p - akt , or p - stat3 were seen in tissue adjacent to the tumor or normal skin ( figures 1(d ) , 1(e ) , and 1(f ) ) .
twenty - seven of 30 p - stat3 positive cases over - expressed both or either of p - egfr and p - akt , and three of the 30 cases showed negative for both p - egfr and p - akt expression .
eight of 35 sccs showed over - expression of p - egfr and p - stat3 without p - akt over - expression .
in contrast , only two of the 35 cases showed over - expression of p - akt and p - stat3 without p - egfr over - expression .
two of the 3 metastatic lesions over - expressed p - stat3 as well as p - egfr and p - akt ( table 1 ) .   next , to evaluate the functional activity of p - stat3 protein expression in vivo , the stat3 dna binding activity of scc tumors and normal skin was investigated .
four of 5 human scc tissues showed strong stat3 dna binding activity while no dna binding activity was seen in normal skin samples ( figure 2 ) .
the results of stat3 dna binding activity were consistent with those of the immunohistochemical study ; that is , all of the 4 tissues with positive dna binding activities were positively stained for p - stat3 by immunohistochemistry and one case without dna binding activity was negative for protein expression ( table 1 ) .
the status of egfr and stat3 phosphorylation was investigated in hek , hacat , and human scc cell lines ( hsc-1 , -3 , and -4 ) .
cells were cultured in serum - free medium for 24 hours before harvest , and then lysates were utilized for immunoprecipitation or immunoblotting .
hsc-1 , -3 , and -4 cells showed strong expression of p - egfr while hek , and hacat cells showed little p - egfr expression ( figure 3(a ) ) .
although the medium for hek contains egf ( 1~2  ng / ml ) , no detectable egfr phosphorylation was observed in hek ( figure 3(a ) ) .
constitutive activation of stat3 was observed in hacat , hsc-1 , -3 , and -4 cells under conventional culture conditions ( figure 3(b ) ) .
hacat cells showed little stat3 dna binding activity when they were cultured in serum - free medium while the addition of fetal calf serum activated stat3 within 24 hours ( figure 3(c ) ) .
in contrast , cultivation of hsc-1 cells in serum - free medium reduced little amount of the stat3 dna binding activity ( figure 3(c ) ) .
the basal activity of stat3 in hek was very low ( figures 3(b ) and 3(c ) ) .
the correlation between stat3 activation and egfr activation was examined using the egfr - specific inhibitor tyrphostin ag1478 .
expression of p - egfr was reduced by treatment with ag1478 ( figure 4(a ) ) .
constitutive activation of stat3 in hsc-1 cells was also downregulated by ag1478 in a dose - dependent manner ( figure 4(d ) ) .
however , the dna binding activity of stat3 was still observed in hsc-1 cells after treatment with 2.5  m of ag1478 , indicating that alternative pathways are involved in stat3 activation in hsc-1 cells . to explore the distinct signal that activates stat3 in scc
, akt activation was investigated by immunoblot analysis . low expression of p - akt and p - stat3 protein was observed in hek and hacat cells in serum - free conditions ( data not shown ) . over - expression of p - akt protein as well as p - stat3 protein
p - akt protein expression was slightly reduced after 24-hour serum starvation in hsc-1 cells , but the akt activity was still maintained even under serum - free conditions .
in contrast , the dramatically reduced level of p - akt expression was observed in hacat cells in serum - free conditions ( figure 4(b ) ) .
we investigated the role of pi3k / akt signaling on stat3 activation in hsc-1 cells .
the activation of akt was abrogated by treatment with a pi3k inhibitor , wortmannin , but not by treatment with ag1478 ( figure 4(c ) ) .
the effect of treatment with wortmannin on stat3 phosphorylation and dna binding activity was examined by emsa in hsc-1 cells . each treatment , respectively , suppressed the activity of stat3 to some degree , and the combination of ag1478 and wortmannin showed an additive effect on the suppression of stat3 dna binding activity ( figure 4(d ) ) .
next , the link between stat3 activation and akt activation was examined by immunoblotting with anti - phospho - antibody to stat3 or akt .
after preincubation in the absence of serum for 24 hours , hsc-1 cells were incubated in the presence or absence of 100  nm insulin for 15 minutes and then lysed .
immunoblotting with the anti - phospho - antibody of akt ( ser 473 ) , stat3 ( tyr705 ) , or stat3 ( ser 727 ) and the total antibody of akt or stat3 showed synchronized upregulation of both proteins , and a stimulator of akt activation , insulin , resulted in significant increase on phosphorylation of both tyr705 and ser727 ( figure 4(e ) ) .
p - akt appeared to be a candidate as an activator of stat3 in hsc-1 cells .
the effects of ag1478 on the growth of hsc-1 cells and hek were evaluated using the mts assay .
cell growth is expressed as a percentage of that in untreated cells at each indicated time point .
treatment with ag1478 significantly suppressed the growth of hsc-1 cells ( p < .01 or  .05 ) ( figure 5(a ) ) .
after 72 hours of ag1478 treatment at a concentration of 2.5  m , the cell density was suppressed significantly to 38% of untreated hsc-1 cells ( p <
, the same treatment did not affect the growth of hek for at least 72 hours under culture conditions containing 1 or 2  ng / ml egf ( figure 5(b ) ) .
hacat cells grew slowly in the serum - free medium . to examine the effect of serum in the culture medium on the growth of hsc-1 or hacat cells , they were seeded in medium with 10% fetal calf serum , after which the medium was exchanged for serum - free medium or medium with 10% fetal calf serum every 24 hours .
the growth of hsc-1 and hacat cells was monitored using the mts assay and was expressed as the percent ratio ( serum - free / serum ) ( figure 5(c ) ) .
the percent ratio ( serum - free / serum ) of cell growth at 48 hours was 38% and 71% in hacat cells and hsc-1 cells , respectively .
the proliferation of hacat cells under serum - free conditions was significantly decreased at 48 hours ( p < .05 ) ( figure 5(c ) ) .
these values were significantly different , indicating that the proliferation of hacat cells is stimulated more by serum . to elucidate the role of egfr activation on apoptosis , double staining for annexin v - fitc binding and for cellular dna using pi
apoptotic cell populations were increased by treatment with ag1478 in a dose - dependent manner .
ag1478 at doses of 0.025 , 0.25 , or 2.5  m increased the apoptotic cell population ( data not shown ) .
the apoptosis was also induced in hsc-1 cells by treatment with a pi3k inhibitor , wortmannin ( p < .01 , when compared with untreated ) ( figure 6(a ) ) .
the percentage of apoptotic cells induced by wortmannin at 100  nm was significantly ( p < .05 ) lower than that by ag1478 at 2.5  m in hsc-1 cell lines ( figure 6(b ) ) .
the greatest induction of apoptosis ( 48.2% ) ( p < .05 , when compared with ag1478 ) among the treatments tested in this study was obtained by the combined treatment of ag1478 at 2.5  m and wortmannin at 100  nm in hsc-1 cells ( figure 6(b ) ) .
pi staining showed that cell viability ( ur ) was not affected by these treatments ( figure 6(a ) ) .
although a similar trend was observed in hsc-3 and 4 cells , the difference between apoptosis rates induced by each treatment in hsc-3 and 4 cells was not as big as in hsc-1 cells ( figure 6(b ) ) .
   this study demonstrated the crucial role of stat3 in the proliferation of cutaneous scc and the important contribution of the dual signaling of egfr and pi3k / akt on the activation of stat3 . in vivo analysis using immunohistochemistry and emsa reveals that stat3 activation is frequently observed in association with egfr and/or akt activation in human cutaneous scc .
nearly 86% of human cutaneous sccs expressed p - stat3 as shown in table 1 .
no preferential regions for p - stat3 expression were found in this study . in accordance with in vivo results , we easily found three human cutaneous scc cell lines , which showed constitutive stat3 activation .
although stat3 activation was not shown in all scc cell lines , stat3 appears to be activated in most cases of cutaneous sccs .    in agreement with studies emphasizing the critical role of egfr on stat3 activation in the carcinogenesis of mouse or human hnscc     [ 22 , 23 ]
, we also demonstrated the importance of egfr activation in stat3 activation in human cutaneous scc .
it has been suggested that the molecular mechanisms for egfr activation are autocrine regulation or gene amplification in other organ malignancies such as breast or brain tumors     [ 2426 ] .
however , the autocrine mechanism may not be relevant in hsc-1 cells since the addition of a neutralizing antibody to egf did not affect activation of the egfr in the cells ( data not shown ) .    
a different reaction to serum regarding growth and stat3 activation between hacat cells and hsc-1 cells was observed in our study .
the stat3 activity of hacat cells was markedly stimulated by serum , while that of hsc-1 cells was slightly stimulated ( figure 3(c ) ) .
these results indicate a close correlation between cell growth and stat3 activation . in hacat cells ,
stat3 activity was low in serum - free conditions and the addition of serum induced stat3 activity .
hsc-1 cells also showed a small decrease in stat3 activity under serum - free conditions and accordingly there was a small additional activation following the addition of serum . in parallel to stat3 activity , the addition of serum to cells cultured in serum - free conditions stimulated the proliferation of hacat cells to a greater extent than hsc-1 cells .
a recent study by quadros et al .       has shown that egfr - dependent stat3 activation is observed only in scc cell lines but not in hacat cells , because egf did not activate stat3 in hacat cells .
since we did not add egf but only serum and found stat3 activation in hacat cells , other ligands or growth factors might have activated stat3 in hacat cells . regarding the stat3 activation in normal keratinocytes , quadros et al
.       showed its activation by egf in mouse keratinocytes , indicating a species difference .
stat3 activation may be regulated in various ways according to species and cell type .     in our analysis of hsc-1 cells ,
the dna binding activity of stat3 was downregulated by ag1478 in a dose - dependent manner . however , in spite of the nearly complete reduction of egfr expression following ag1478 treatment , stat3 activation was still observed in hsc-1 cells .
this suggests the presence of an egfr - independent activation of stat3 in hsc-1 cells .
it was shown that gp130/jak and src - like kinase contribute slightly to the egfr - independent pathway of stat3 activation in scc cell lines     .
alternatively , the pi3k , catalytic , alpha gene is somatically mutated in several kinds of human cancer     .
the response of stat3 and akt activation to serum stimulus between hacat and hsc-1 cells provided a clue .
in contrast , akt activation was observed in hsc-1 cells under serum - free condition , suggesting persistent activation of pi3k / akt signaling in cancer cells .
maximum suppression was achieved by a combination of ag1478 and wortmannin ( figure 4(d ) ) , indicating that egfr and pi3k / akt signaling directly or indirectly activate stat3 through independent pathways in hsc-1 cells .    
combined treatment of wortmannin and ag1478 showed an additive effect on the induction of apoptosis as well as on the suppression of stat3 activity in all scc cell lines ( figure 6 ) .
the induction of apoptosis in human scc cells also appeared to be inversely proportional to stat3 activity ( figures 3(b ) and 6(b ) ) .
however , we have demonstrated that stat3 inhibition is not sufficient to induce apoptosis , but stat3 activation is required for cell proliferation and tumorigenesis of scc     .
shih et al . reported that stat3 works as a strong antiapoptotic agent of tgf--induced apoptosis in cooperation with pi3k / akt     .
in addition , corney et al . revealed that akt regulates the sensitivity to tgf-    through interaction with smad3     .
these recent data may support the proapoptotic effect of the combination of inhibitors used in this study .   
we conclude that the proliferation and survival of scc cells is mostly maintained by stat3 activation and that stat3 activation is mediated through egfr and pi3k / akt activation in cutaneous scc cell lines
. this study could provide clues to elucidate the carcinogenesis of cutaneous scc and lead to a novel treatment and therapeutic strategies .
 recent studies have emphasized the important role of stat3 activation in a number of human tumors from the viewpoint of its oncogenic and antiapoptotic activity . in this study , we examined the role and related signaling molecules of stat3 in the carcinogenesis of human cutaneous squamous cell carcinoma ( scc ) . in 35 human cutaneous scc samples , 
 86% showed overexpression of phosphorylated ( p)-stat3 , and most of those simultaneously overexpressed p - egfr or p - akt . 
 constitutive activation of egfr and stat3 was observed in three scc cell lines and four of five scc tissues . 
 ag1478 , an inhibitor of the egfr , downregulated stat3 activation in hsc-1 human scc cells . 
 ag1478 inhibited cell proliferation and induced apoptosis of hsc-1 cells but did not inhibit the growth of normal human epidermal keratinocytes that did not show stat3 activation . 
 furthermore , a pi3k inhibitor also suppressed stat3 activation in hsc-1 cells to some degree . 
 combined treatment with the pi3k inhibitor and ag1478 strongly suppressed stat3 activity and dramatically induced apoptosis of hsc-1 cells . 
 these data suggest that stat3 activation through egfr and/or pi3k / akt activation plays a critical role in the proliferation and survival of human cutaneous scc . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion