
 the wnt/-catenin pathway has been implicated in the development and progression of melanoma and a wide range of cancer types , including colorectal cancer , breast cancer , esophageal carcinoma , and liver cancer [ 13 ] . under normal conditions ,
increases in expression and binding of certain wnt ligands to frizzled receptor or mutations in specific components of the -catenin degradation assembly deactivate this regulatory mechanism .
nuclear -catenin stimulates transcription of a large number of tcf/-catenin responsive genes that include cyclin d1 , c - myc [ 5 , 6 ] , and the melanocyte - specific gene , microphthalmia - associated transcription factor mitf - m .
thus , accumulation of nuclear -catenin as observed in several cancer types is considered a marker of canonical wnt/-catenin pathway deregulation and unfavorable prognosis [ 3 , 8 ] .
previous studies have reported an association between nuclear -catenin accumulation and melanoma progression and suggested nuclear -catenin to be a marker of poor prognosis [ 1 , 7 ] .
however , recent studies have shown that contrary to breast and colon cancer , metastatic progression of melanoma is associated with decreases in nuclear and cytoplasmic -catenin expression [ 9 , 10 ] . moreover , clinical , genetic , and histological studies suggest that nuclear and cytoplasmic -catenin may be used as biomarkers of good prognosis in melanoma [ 1114 ] . recently , hhr6 , a human homologue of the yeast rad6 gene and a principal component of the postreplication dna repair pathway , has been identified as an important regulator of canonical wnt/-catenin signaling [ 15 , 16 ] .
hhr6 , referred hereafter as rad6 , stabilizes -catenin by polyubiquitin modifications that render -catenin resistant to 26s proteasomal degradation .
furthermore , rad6 is a transcriptional target of -catenin , thus revealing a positive feedback loop between -catenin - mediated activation of rad6 gene expression and rad6-induced -catenin stabilization .
rad6 expression is low in normal breast tissues ; however , increases in rad6 protein expression are detected in hyperplastic , ductal carcinoma in situ ( dcis ) and invasive breast carcinomas .
we have previously demonstrated a role for rad6 in breast cancer progression through its regulatory effect on the canonical wnt/-catenin pathway . since the decrease / loss of nuclear -catenin [ 9 , 10 ] , rather than increases as in breast cancer ,
is linked to melanoma progression , it is not known whether rad6 and -catenin work in concert to promote melanoma pathogenesis .
furthermore , rad6 expression in the skin has not been investigated , and there are no data on the role of rad6 in the pathogenesis of benign ( nevi ) and malignant ( melanoma ) melanocytic lesions .
it is important to address this gap in knowledge because of the unmet medical need for new effective antimelanoma therapies and because rad6 and -catenin have been identified as therapeutic targets [ 19 , 20 ] . in this study , we examined rad6 and -catenin expressions in serial sections of nevi , primary , and metastatic melanomas to determine their potential roles in melanoma development and metastatic progression .
our data suggest that membranous relocation of -catenin and upregulation of rad6 are independent markers of melanoma development and progression .
we also offer a hypothesis that explains the role of membranous -catenin relocation and decreasing cytoplasmic -catenin in melanoma development , a phenomenon that has been linked to unfavorable prognosis [ 9 , 21 , 22 ] .
cases were retrieved from the files of the pinkus dermatopathology laboratory ( pdl ) , a private dermatopathology laboratory located in monroe , mi . preserved paraffin - embedded tissue specimens collected for each case were assigned an accession code that excluded patient identifier information .
nevus and primary melanoma cases were selected for study using random numbers generated by a uniform random number generator ( stata / mp 13.1 ) .
the study groups consisted of 30 cases of melanocytic nevi , 29 cases of primary cutaneous melanoma , and 29 cases of metastatic cutaneous melanoma .
the study includes all metastatic cutaneous melanoma samples that were archived between 2010 and 2012 .
the number of cases for each nevus and primary melanoma subtype was determined to reflect the lesion 's relative representation in cases obtained at the pdl during the above period .
atypical nevi were diagnosed using criteria originally proposed by clark and lesion architecture as reviewed by roth et al . .
primary antibodies used in the study are as follows : ( i ) anti--catenin ( is702 ) was purchased from dako ( glostrup , denmark ) and used in an undiluted form ; and ( ii ) anti - rad6 ( ab31917 ) was purchased from abcam ( cambridge , ma ) and used at a 1  :  500 dilution . in humans , the yeast homologous rad6 gene is duplicated and the proteins encoded by the two genes hhr6a ( or rad6a ) and hhr6b ( rad6b ) from chromosomes xq24-q25 and 5q23-q31 , respectively , share 95% identical amino acid residues .
neither ab31917 , our own rad6 antibody , nor any other commercially available anti - rad6 antibody is currently able to distinguish between rad6a and rad6b proteins .
therefore , rather than referring as rad6a or rad6b , we refer to the protein detected by the antibody as rad6 .
briefly , five - micrometer sections were deparaffinized in xylene and rehydrated in graded ethanol . for antigen retrieval ,
sections were microwaved in citrate buffer ph 6.0 ( biogenex , san ramon , ca , usa ) for 12  min at 95c and cooled for 30  min prior to immunostaining .
sections were incubated with 3% hydrogen peroxide for 15  min , followed by incubation with primary antibody for 60  min .
an automated immunostainer ( i6000 ; biogenex ) was utilized for subsequent incubation steps : sections were incubated in multilink biotinylated anti - igg for 20  min , horseradish peroxidase conjugated secondary antibody for 20  min , followed by development with 3-amino-9-ethyl - carbazole for 10  min ( biogenex ) .
all incubation steps were performed at room temperature , and sections were washed with tris - buffered saline between incubations .
lung and colon cancer tissues were included as positive controls for immunostaining with anti--catenin antibody , and breast cancer tissues were included as positive controls for staining with anti - rad6 antibody .
stained sections were independently enumerated by two coauthors ( d. r. mehregan and m. campbell ) , who were blinded to patient medical records for each case .
blinded enumeration was performed under light microscopy at 400x magnification , and an ocular grid consisting of a simple square lattice of 100 test points was utilized to count the number of positively and negatively stained melanomas or nevus cells for each section . per section , the total number of positively and negatively stained cells was counted for each of three sequential horizontal fields .
the mean value of the three fields was used to estimate the relative density of cells in a specimen . to increase assessment accuracy , all positively and negatively stained melanomas or nevus cells in a visual field
when independent readings for positively stained cells differed by 20% for a given section , both evaluators reviewed the section together to establish a consensus reading .
a specimen was considered negative if less than 4% of the cells were immunostained for rad6 or -catenin .
a tumor was considered to be stained with high intensity if > 50% of the cells in a specimen expressed rad6 or -catenin , similar to the criteria used by mineta et al . .
kruskal - wallis tests were used to compare groups on the basis of continuous variables such as age and percent positive cells .
chi - square tests for differences in proportions were used to compare groups on the basis of categorical variables such as gender and -catenin localization .
spearman 's rank correlation was used to assess the pairwise association between age and percent of rad6 positive and -catenin positive cells .
multinomial logistic regression was used to assess the simultaneous association of rad6 and age with diagnostic group .
adjustments were made for multiple comparisons using wilcoxon rank sum tests with bonferroni correction for pairwise comparisons .
our analysis included 30 individuals diagnosed with nevi , 29 with primary melanoma , and 29 with metastatic melanoma ( table 1 ) .
these groups differed marginally with respect to gender ( p = 0.08 ) and significantly in age ( p = 0.0001 ) .
significant age differences were observed between individuals with nevi and those with either primary melanomas ( p = 0.02 ) or metastatic melanomas ( p = 0.0001 ) .
in contrast , no age difference is observed between individuals with primary and metastatic melanomas ( p = 0.27 ) .
significant differences in age were also observed between groups defined by -catenin localization ( p = 0.007 ) .
individuals with -catenin localized in the cytoplasm were significantly younger than individuals with -catenin localization at the cell membrane ( p = 0.02 ) and marginally younger than individuals with -catenin localization at both the cytoplasm and the cell membrane ( p = 0.05 ) . when age was categorized as < 50 , 5060 , and > 60 years , there were statistically significant differences in rad6 expression between the groups ( p = 0.0008 ) , although there is substantial variability ( table 2 ) .
median rad6 is greater in the group of people older than 60 years compared to the 5060 years old group ( p = 0.04 ) and < 50 years old group ( p = 0.001 ) .
a 20% discrepancy of positively stained cells between the two evaluators was observed in fewer than 5% of the cases , and these cases were evaluated together to establish a consensus reading .
melanoma development and progression were not associated with significant changes in percentage of specimens expressing -catenin .
-catenin staining was observed in 97% of nevi and in all primary and metastatic melanomas .
also , the percentages of nevi ( 93% ) , primary melanoma ( 97% ) , and metastatic melanoma ( 93% ) that expressed -catenin in more than 50% of the cells did not differ significantly ( figures 1 , 2 , and 3 ) .
however , significant differences were observed between the percentages of nevi ( 59% ) , primary melanoma ( 90% ) , and metastatic melanoma ( 56% ) that expressed -catenin in more than 90% of the cells ( p = 0.02 ; figure 2 ) .
these differences were greatly impacted by the percentage of primary melanomas ( 48% ) that expressed -catenin in 100% of the cells , which was approximately twofold higher than the percentages of nevi ( 21% ) or metastatic melanoma ( 26% ) ( data not shown ) .
none of the nevi or melanomas expressed -catenin in the nucleus ( figure 1 ) .
the percentage of tumors that expressed membranous -catenin increased dramatically from nevi ( 10% ) to primary and metastatic melanomas ( 83% and 93% , resp . ;
concurrently with this increase , the percentage of tumors that expressed cytoplasmic -catenin decreased from nevi ( 90% ) to primary and metastatic melanomas ( 45% and 38% , resp . ;
in contrast , no significant differences were observed between the percentages of primary and metastatic melanomas that expressed -catenin at either the membrane ( 83% and 93% , resp . ;
p = 0.289 ) or the cytoplasm ( 45% and 38% , resp . ; p = 0.633 ; figure 4 ) . while all four nevi types ( junctional , intradermal , compound , and atypical ) expressed -catenin in the cytoplasm , only junctional and atypical nevi expressed -catenin at the plasma membrane .
as opposed to nevi types , the three primary melanoma types examined ( superficial spreading , nodular , and lentigo maligna ) did not differ in -catenin localization as -catenin was localized at the plasma membrane , in the cytoplasm , or in both ( figure 4 ) .
conversely , all primary melanomas ( 100% ) and the majority of metastatic melanomas ( 96% ) exhibited greater than 50% rad6 expression .
the increase in tumor populations expressing rad6 from 37% of nevi to 100% of primary and metastatic melanomas was significant ( p = 0.0001 ; figures 1 , 2 , and 3 ) .
melanoma progression from primary to metastatic disease was not associated with changes in the ( i ) percentage of melanomas expressing rad6 ( 100% of primary and metastatic melanomas ) , or ( ii ) percentage of melanomas expressing rad6 in more than 50% of the tumor cells ( 100% and 96% of primary and metastatic melanomas , resp . ) .
the increase in proportion of tumor populations , expressing rad6 in more than 50% of the cells in primary melanoma ( 67% ) versus metastatic melanoma ( 79% ) was not significant ( p = 0.37 ; figure 2 ) .
this study was not designed to test whether the distribution of the tumor cells positive for rad6 is the same between the subtypes of nevi .
however , the percentages of benign tumors that lacked rad6 were similar between atypical nevi ( 62% ) and the group of other three nevi types ( 59% ) .
interestingly , only one of the 30 nevi ( atypical nevus , 3% ) expressed rad6 in > 80% of the cells , and none of the primary and metastatic melanomas expressed rad6 in < 40% of the cells ( figure 2 ) .
these results prompted us to examine whether rad6 expression can serve as a marker for histological diagnosis of melanoma . using a multiple logistic regression model
, we found that the strength of rad6 expression is a strong predictor of melanoma ( p < 0.001 ) even when age group ( p = 0.65 ) and gender ( p = 0.24 ) are included in the model .
the model predicts that every 1% increase in rad6 expression results in a 9% increase in the probability that a lesion is melanoma .
if we assume that a predicted probability of > 0.5 indicates melanoma , the model with only rad6 has sensitivity of 93% and specificity of 80% .
these results are very encouraging ; however , they need to be validated in a larger study . the expression profiles of -catenin and rad6 differed considerably in nevi .
approximately 93% of nevi expressed -catenin in more than 50% of the cells , whereas only 27% of the same population of nevi expressed rad6 ( figure 2 ) .
-catenin and rad6 expressions in nevi were not significantly correlated ( r = 0.06 ; p = 0.77 ) .
there is a 2.7-fold difference in the percentage of primary melanomas ( 100% ) expressing rad6 compared to nevi ( 37% ) and virtually no difference in -catenin expression between primary and metastatic melanomas ( 100% ) .
accordingly , rad6 and -catenin expressions in primary melanoma were not correlated ( r < 0.001 , p > 0.99 ) . a significant correlation between rad6 and -catenin positive cells
however , this association diminished ( r = 0.40 , p = 0.05 ) following the exclusion of two observations which are disproportionally influential ( one with < 50% positive rad6 and one with < 50% positive -catenin ) .
this is the first study to characterize rad6 expression in cutaneous benign and malignant melanocytic tumors . in this study
, we examined the association between rad6 and -catenin expressions in benign and malignant melanocytic tumors to determine whether rad6 works in concert with -catenin to influence melanoma development and progression .
rad6 and -catenin positively regulate each other in breast cancer [ 15 , 18 ] . however , while -catenin has been implicated in the pathogenesis of melanoma and other cancer types , data about the role of rad6 in cancer pathogenesis are mostly limited to breast cancer .
therefore , we hypothesized that comparison of rad6 and -catenin expressions in the same nevi and melanoma tumors would help determine whether these two signals collaborate to promote melanoma development and progression as they do in breast cancer [ 15 , 28 ] .
accumulation of nuclear and cytoplasmic -catenin has been implicated in driving the development and progression of several cancer types ( e.g. , colon and ovarian cancers ) [ 2931 ] . however , our results show that the expression levels of -catenin do not contribute to melanoma initiation and progression since no difference in -catenin levels was found between nevi , primary melanoma , and metastatic melanoma ( 93%97% of all samples expressed -catenin in > 50% of the tumor cells ) .
the high expression levels of -catenin are in line with the crucial role of -catenin in differentiation and proliferation of both normal melanocytes and metastatic melanoma cells . also , our findings are in agreement with previous reports of positive -catenin staining in nevi ( 100% ) and primary melanoma ( 95% , 94% ) but are higher than reported in metastatic melanoma ( 75% , 68% ) [ 9 , 21 ] . the variation in expression of -catenin levels in metastatic melanomas between the studies
while we studied only melanoma metastases to the skin , other studies either obtained 58% of their specimens from lymph nodes , tonsil , and liver or did not identify the anatomical site of their metastases [ 9 , 21 ] .
furthermore , different anatomical sites may regulate dissimilar antigen expressions in metastases that originate from the same primary tumor in the same patient [ 33 , 34 ] .
previous studies have shown higher percentages of nuclear -catenin in nevi than in melanoma ( 84% versus 33% , and 44% versus 15% ) [ 9 , 22 ] .
those observations provided the basis for the currently held concept that loss of nuclear and cytoplasmic -catenin suggest poor prognosis and decreased overall survival of melanoma patients [ 12 , 22 ] . in light of these data , the absence of nuclear -catenin in all the nevi and melanomas analyzed in our study was surprising .
usage of different anti--catenin antibodies may explain in part the discrepancy in nuclear -catenin expression observed between the studies .
however , our results are consistent with the lack of nuclear -catenin reported in four studies which comprised 57 nevi , more than 55 primary melanomas , and 20 metastatic melanomas [ 21 , 3537 ] .
moreover , nuclear -catenin was not found in either the nevus portion or the melanoma portion of 15 cutaneous lesions and was absent in additional 42 primary melanomas . in another study of 70 primary melanomas , nuclear -catenin was reported in only 6.4% of the melanomas .
finally , in a study of 230 primary and metastatic melanomas , nuclear -catenin was reported in only 13 cases ( 5.6% ) and therefore those cases were excluded from analysis . taken together , the absence or negligible amount of nuclear -catenin detected in the aforementioned studies as well as ours suggests possible extranuclear roles for -catenin in nevi and melanoma .
this notion is supported by a role for cytoplasmic -catenin to execute functions that do not require nuclear translocation ( e.g. , activation of map kinase p38 and nf - kb ) [ 37 , 40 ] .
a major finding of this study is the association between melanoma development and intracellular redistribution of -catenin .
the percentage of cases that expressed -catenin on the cell membrane increased dramatically from 10% in nevi to 83% and 93% in primary and metastatic melanomas , respectively .
concurrently , the percentage of cases that expressed cytoplasmic -catenin decreased from 90% in nevi to 45% and 39% in primary and metastatic melanomas , respectively ( figure 4 ) .
we hypothesize that the relocation of -catenin from the cytoplasm to the cell membrane may serve as a deactivating mechanism of canonical wnt/-catenin signaling and that the resulting reduction in cytoplasmic -catenin level may contribute to the malignant transformation of melanocytic nevi .
the proposed hypothesis is supported by the following observations : ( i ) as in our study , bachmann et al . also reported an association between nevus to melanoma development and relocation of -catenin to the cell membrane .
nevertheless , the authors of that study did not offer an explanation for their observation ; ( ii ) our analysis of the data of kagashita et al .
showed -catenin decrease in the cytoplasm and increase at the cell membrane and that these changes in -catenin distribution corresponded with the malignant transition of nevi ; ( iii ) wnt4 signal has been identified as a mechanism that can drive -catenin relocation from cytoplasm to cell membrane ; and ( iv ) -catenin relocation from cytoplasm to cell membrane has been reported to block -catenin signaling in a human embryonic kidney ( hek293 ) cell line .
of note , this hypothesis can explain how despite the abundant -catenin expression in melanoma [ 1 , 7 ] , cytoplasmic -catenin is selectively decreased , a phenomenon that has been associated with unfavorable melanoma prognosis [ 9 , 21 , 22 ] .
our current efforts are directed towards determining if the increases in membranous -catenin observed in primary and metastatic melanomas result from relocation of existing molecules in the cytoplasm or deposition of newly generated -catenin at the membranous site .
rad6 has been implicated in early breast cancer development since an increase in rad6 levels is observed in adenosis and benign hyperplasias as compared to normal tissue .
in contrast , our findings do not support rad6 to play a similar role in nevus formation as in benign breast neoplasia , since 63% of the nevi were negative for rad6 .
rad6 has also been implicated in breast cancer progression because rad6 levels increase with progression from ductal carcinoma in situ to invasive primary carcinoma and metastatic cancer [ 15 , 28 ] . in accordance with the upregulation of rad6 in early stages of breast cancer development as compared to benign hyperplasia [ 15 , 17 ] , we observed a striking increase in rad6 expression in primary melanoma when compared to nevi .
while all primary melanomas displayed strong rad6 staining ( > 50% of the tumor cells ) , rad6 was negative in 63% of the nevi .
these findings suggest that rad6 may play a role in malignant transformation of nevi as in breast cancer .
progression of melanoma from primary to metastatic disease was not significantly associated with further changes in the percentage of tumors expressing rad6 or rad expression intensity as > 50% of tumor cells stained positively in 100% and 96% of primary melanomas and metastatic melanomas , respectively .
these findings suggest that rad6 may play a sustained role in melanoma metastasis as it does in melanoma development . in benign and malignant breast tumors ,
rad6 stabilizes -catenin , and , in turn , -catenin positively upregulates rad6 transcription [ 1517 ] .
however , this direct positive correlation between -catenin and rad6 expression does not appear to be conserved in melanoma as the expression profiles of -catenin and rad6 differed considerably in nevi .
approximately 93% of nevi expressed -catenin compared to only 27% of nevi that expressed rad6 in more than 50% of their cells ( figure 2 ) .
these observations suggest that the high -catenin expression in nevi is likely driven by regulators other than rad6 . at first glance
, it would appear that -catenin and rad6 expressions are correlated in primary melanoma because these proteins were coexpressed in approximately all primary melanomas . also , these findings correspond to the 80% correlation between rad6 and -catenin expressions in primary breast cancer .
however , it is unlikely that the high rad6 expression in primary melanoma is driven by the concurrent high -catenin expression , because rad6 expression is low in nevi despite the presence of high cytoplasmic -catenin expression that is comparable to primary melanoma .
this notion is confirmed by lack of statistical correlation between rad6 and -catenin expressions in primary melanoma .
-catenin is not the only activator of rad6 ; for instance , rad6 is activated by nerve growth factor in nervous tissue . therefore , it is conceivable that , in primary melanoma , rad6 expression is regulated by yet unidentified activators .
we also demonstrated that further progression of melanoma from primary to metastatic disease is not associated with a correlation between the -catenin and rad6 expressions .
taken together , our study does not support a direct positive interaction between -catenin and rad6 in either benign or malignant melanocytic tumors .
we characterized for the first time rad6 expression in cutaneous benign and malignant melanocytic tumors .
we are showing a striking upregulation of rad6 from a negative expression in most benign melanocytic tumors to 100% of primary and metastatic melanomas .
these findings strongly suggest a role for rad6 in the development of primary melanoma and metastatic disease .
we show that in contrast to rad6 , -catenin is expressed in more than 50% of the tumor cells in almost all nevi and melanoma tumors . taken together , in contrast to the rad6 -catenin positive relationship in breast cancer [ 1517 ] , our study does not support a similar positive interaction between -catenin and rad6 in benign or malignant melanocytic tumors .
finally , our findings suggest a role for the cytoplasmic to membrane translocation of -catenin in the development of primary melanoma .
future studies will determine whether newly generated -catenin at the membranous site coincide with -catenin translocation from the cytoplasm .
 we have previously demonstrated that rad6 and -catenin enhance each other 's expression through a positive feedback loop to promote breast cancer development / progression . while -catenin has been implicated in melanoma pathogenesis , rad6 function has not been investigated . here 
 , we examined the relationship between rad6 and -catenin in melanoma development and progression . 
 eighty - eight cutaneous tumors 
 , 30 nevi , 29 primary melanoma , and 29 metastatic melanomas , were immunostained with anti--catenin and anti - rad6 antibodies . strong expression of rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas . 
 -catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas , and unlike rad6 , in 93% of nevi . 
 none of the tumors expressed nuclear -catenin . 
 -catenin was exclusively localized on the cell membrane of 55% of primary , 62% of metastatic melanomas , and only 10% of nevi . 
 cytoplasmic -catenin was detected in 90% of nevi , 17% of primary , and 8% of metastatic melanoma , whereas 28% of primary and 30% of metastatic melanomas exhibited -catenin at both locations . 
 these data suggest that melanoma development and progression are associated with rad6 upregulation and membranous redistribution of -catenin and that -catenin and rad6 play independent roles in melanoma development . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion