Loss of syndecan-1 is associated with malignant conversion in skin carcinogenesis

Abstract

Syndecan-1 (sdc-1) is a cell surface proteoglycan that mediates the interaction of cells with their matrix, influencing attachment, migration, and response to growth factors. In keratinocytes, loss of sdc-1 delays wound healing, reduces migration, and increases Transforming growth factor beta (TGFbeta) 1 expression. In this study we show that sdc-1 expression is significantly reduced in basal cell, squamous cell, and metastatic human skin cancers compared to normal human skin. In experimental mouse skin tumor induction, compared to wildtype (wt) BALB/c mice, papilloma formation in sdc-1 null mice was reduced by 50% and the percent of papillomas converting to squamous cell carcinoma (SCC) was enhanced. sdc-1 expression on wt mouse papillomas decreased as they converted to SCC. Furthermore, papillomas forming on sdc-1 null mice expressed suprabasal alpha3 and beta4 integrins; suprabasal beta4 integrin is a marker of a high risk for progression. While the proliferative response to phorbol-12-myristate-13-acetate (TPA) did not differ among the genotypes, sdc-1 null mice had an enhanced inflammatory response and retained higher levels of total TGFbeta1 within their skin after TPA treatment. sdc-1 null keratinocytes, transduced in vitro by oncogenic ras(Ha), expressed higher levels of beta4 integrin and had enhanced pSmad2 signaling and reduced senescence when compared to wt ras(Ha)-transduced keratinocytes. When ras(Ha)-transduced cells of both genotypes were grafted onto nude mice, null tumors converted to SCC with higher frequency confirming the skin painting experiments. These data indicate that sdc-1 is important both early in the development of skin tumors and in progression of skin cancers suggesting that reduced expression of sdc-1 could be a useful marker for progression in neoplastic skin lesions.

Figure 1. Sdc-1 is reduced in human skin tumors of several phenotypes

A human tumor array was used to assess the expression of sdc-1 in human cancers. A. Shown are representative IHC images showing sdc-1 in normal skin and in tumors identified as SCC, BCC, and metastatic adenocarcinomas. B. Values assigned to sdc-1 positive tumors were averaged for the human tumor macroarray. Sdc-1 is significantly reduced in SCCs, basal cell carcinomas, and metastatic adenocarcinomas. Bar = 60 μm.

Figure 2. Sdc-1 null mice develop fewer tumors than wildtype mice after chemical carcinogenesis

Age matched (8 wk old) mice of sdc-1 null and wildtype genotypes were used for skin painting studies. A. The numbers of mice with tumors and the total numbers of tumors generated for both genotypes of mice were determined and the percentages of mice with tumors shown for 2 independent experiments. Experiment 1 utilized 30 wt and 23 sdc-1 null mice; experiment 2 utilized 33 wt and 43 sdc-1 null mice. Statistical significance was determined by the Chi-square test and found to be not quite significant at p=0.06 for experiment 1 and highly significant for experiment 2 at p=0.01.B. The tumor burden per mouse was determined by dividing the total numbers of tumors formed by the number of mice with tumors. C. The percentage of tumors that converted from papilloma to either CIS or SCC was determined by dividing the number of CIS carcinoma in situ/SCC tumors as determined by histopathology by the total number of tumors; too few SCCs were generated and results were not significant D. The percentage of tumors that were positive for either K1 or K13 was determined by immunohistochemistry.

Figure 3. Integrin and laminin localization is altered in sdc-1 papillomas

Unfixed frozen sections from wt and sdc-1 null mouse tumors were evaluated by immunoflourescence microscopy. Images from serial frozen sections of representative papillomas are presented in A and B whereas C and D show representative wt and sdc-1 null SCCs. The asterisks in the merged images in A and B indicate the region shown below after 3-fold magnification. Bars in A–D = 60 μm.

Figure 4. Response of wildtype and sdc-1 mice after 3 weekly treatments to topical TPA

Shown are 3 timepoints after the last application of TPA. A. BrdU incorporation (2 hours IP), n=6. B. MPO activity in 8mm skin punch biopsies, n=6 C. Unfixed frozen sections were used to localize α3 integrin and TGFβ1. White dashed white lines demarcate the apical margin of the epidermis and arrows indicate TGFβ1 localization within the epidermis. D. Total epidermal and dermal TGFβ1 levels determined by ELISA assay at 3 timepoints after the third TPA treatment or 7 days after the second TPA treatment, n=6. Bar in C = 15 μm.

Figure 5. Integrin and laminin localization in tumors developing from grafts of oncogenic ras transduced sdc-1 null and wildtype keratinocytes

Images from serial frozen sections of representative tumors from nude mice grafted with wt or sdc-1 null ras^Ha-transduced keratinocytes. Indirect immunofluorescence for α3 and β4 integrins (A) and LN332 (B) are presented. Asterisks indicate areas shown below after 3-fold magnification and arrows indicate suprabasal localization in sdc-1 null tumors. C. IHC showing the loss of sdc-1 in tumors from grafted ras^Ha transduced wildtype keratinocytes as they progress from benign to malignant D. Typical wt and sdc-1 null SCCs from nude mouse grafts stained for α3 and β4 integrins or LM332 and β4 integrin Bar in A and B = 60 μm, Bar in C = 100 μm and the Bar in D = 15 μm.

Figure 6. Comparative analysis of cultured ras^Ha-transduced primary sdc-1 null and wildtype keratinocytes

Isolated primary keratinocytes from both genotypes were cultured in 0.05 mM Ca²⁺-medium to select for basal cells, mock or ras^Ha transduced on day 3, and analyzed 5 days later. A. Immunoblots of integrin expression before and after ras^Ha transduction. B. Time lapse studies to quantify relative migration in the presence or absence of functional α6 integrin using a blocking antibody. C. Immunoblot for pSmad2 and total Smad2 in the presence or absence of TGFβ1 for the indicated time. D. Senescence associated β-galactosidase positive cells were counted as a function of time. Shown are results for wt and sdc-1 null mock and ras^Ha-transduced primary keratinocytes at day 15 in culture.