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Loss of the p53/p63 regulated desmosomal protein Perp promotes tumorigenesis.

Beaudry VG, Jiang D, Dusek RL, Park EJ, Knezevich S, Ridd K, Vogel H, Bastian BC, Attardi LD - PLoS Genet. (2010)

Bottom Line: Tumor development is associated with inactivation of both of Perp's known functions, in apoptosis and cell-cell adhesion.Interestingly, Perp-deficient tumors exhibit widespread downregulation of desmosomal constituents while adherens junctions remain intact, suggesting that desmosome loss is a specific event important for tumorigenesis rather than a reflection of a general change in differentiation status.Using gene expression profiling, we show further that Perp loss induces a set of inflammation-related genes that could stimulate tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University School of Medicine, Stanford, California, USA.

ABSTRACT
Dysregulated cell-cell adhesion plays a critical role in epithelial cancer development. Studies of human and mouse cancers have indicated that loss of adhesion complexes known as adherens junctions contributes to tumor progression and metastasis. In contrast, little is known regarding the role of the related cell-cell adhesion junction, the desmosome, during cancer development. Studies analyzing expression of desmosome components during human cancer progression have yielded conflicting results, and therefore genetic studies using knockout mice to examine the functional consequence of desmosome inactivation for tumorigenesis are essential for elucidating the role of desmosomes in cancer development. Here, we investigate the consequences of desmosome loss for carcinogenesis by analyzing conditional knockout mice lacking Perp, a p53/p63 regulated gene that encodes an important component of desmosomes. Analysis of Perp-deficient mice in a UVB-induced squamous cell skin carcinoma model reveals that Perp ablation promotes both tumor initiation and progression. Tumor development is associated with inactivation of both of Perp's known functions, in apoptosis and cell-cell adhesion. Interestingly, Perp-deficient tumors exhibit widespread downregulation of desmosomal constituents while adherens junctions remain intact, suggesting that desmosome loss is a specific event important for tumorigenesis rather than a reflection of a general change in differentiation status. Similarly, human squamous cell carcinomas display loss of PERP expression with retention of adherens junctions components, indicating that this is a relevant stage of human cancer development. Using gene expression profiling, we show further that Perp loss induces a set of inflammation-related genes that could stimulate tumorigenesis. Together, these studies suggest that Perp-deficiency promotes cancer by enhancing cell survival, desmosome loss, and inflammation, and they highlight a fundamental role for Perp and desmosomes in tumor suppression. An understanding of the factors affecting cancer progression is important for ultimately improving the diagnosis, prognostication, and treatment of cancer.

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Adhesion junction analysis in tumors from K14CreER;Perpfl/fl and control mice.A) Representative Hematoxylin and Eosin (H&E) staining and immunofluorescence images of desmosome and adherens junction component protein expression in skin from tamoxifen-treated K14CreER;Perpfl/fl mice demonstrating that Perp loss itself does not disrupt membrane expression of other adhesion proteins. B) Western blot analysis showing both the Triton X-100-soluble and urea-only soluble fractions of mouse epidermal lysates from control and K14CreER;Perpfl/fl mice. Desmoglein 1/2 (Dsg 1/2) and Plakoglobin (Pg) solubility were examined. Gapdh serves as a loading control for the Triton X-100-soluble pool, while Keratin 14 serves as the loading control for the urea fraction. C, D) Tumors from K14CreER;Perpfl/fl mice were stained with antibodies against various desmosomal and adherens junction components, and both the percentage of epithelial cells staining positive for each marker and the intensity of staining were measured. Staining for each antigen was categorized as high (>70%), medium (30–70%), or low (<30%) level expression. C) Representative H&E and immunofluorescence images of a K14CreER;Perpfl/fl tumor sample demonstrating desmosomal component loss with retention of adherens junction components. Green signal indicates antigen staining, and blue signal indicates DAPI-marked nuclei. D) (Left) Graph showing the percentages of K14CreER;Perpfl/fl tumors categorized into respective groups based on quantitative immunofluorescence staining for Plakoglobin and Desmoglein 1/3. (Right) Graph depicting the percentages of tumors categorized into respective groups based on immunofluorescence staining for E-cadherin and Beta-catenin. (E) Tumors from control wild-type mice were stained for desmosomal and adherens junction components, and each component was categorized as displaying high, medium, or low level expression, as described above. (Left) Graph showing the percentages of control tumors categorized into respective groups based on quantitative immunofluorescence staining for Perp, Plakoglobin, and Desmoglein 1/3. (Right) Graph depicting the percentages of tumors categorized into respective groups based on immunofluorescence staining for E-cadherin and Beta-catenin.
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pgen-1001168-g003: Adhesion junction analysis in tumors from K14CreER;Perpfl/fl and control mice.A) Representative Hematoxylin and Eosin (H&E) staining and immunofluorescence images of desmosome and adherens junction component protein expression in skin from tamoxifen-treated K14CreER;Perpfl/fl mice demonstrating that Perp loss itself does not disrupt membrane expression of other adhesion proteins. B) Western blot analysis showing both the Triton X-100-soluble and urea-only soluble fractions of mouse epidermal lysates from control and K14CreER;Perpfl/fl mice. Desmoglein 1/2 (Dsg 1/2) and Plakoglobin (Pg) solubility were examined. Gapdh serves as a loading control for the Triton X-100-soluble pool, while Keratin 14 serves as the loading control for the urea fraction. C, D) Tumors from K14CreER;Perpfl/fl mice were stained with antibodies against various desmosomal and adherens junction components, and both the percentage of epithelial cells staining positive for each marker and the intensity of staining were measured. Staining for each antigen was categorized as high (>70%), medium (30–70%), or low (<30%) level expression. C) Representative H&E and immunofluorescence images of a K14CreER;Perpfl/fl tumor sample demonstrating desmosomal component loss with retention of adherens junction components. Green signal indicates antigen staining, and blue signal indicates DAPI-marked nuclei. D) (Left) Graph showing the percentages of K14CreER;Perpfl/fl tumors categorized into respective groups based on quantitative immunofluorescence staining for Plakoglobin and Desmoglein 1/3. (Right) Graph depicting the percentages of tumors categorized into respective groups based on immunofluorescence staining for E-cadherin and Beta-catenin. (E) Tumors from control wild-type mice were stained for desmosomal and adherens junction components, and each component was categorized as displaying high, medium, or low level expression, as described above. (Left) Graph showing the percentages of control tumors categorized into respective groups based on quantitative immunofluorescence staining for Perp, Plakoglobin, and Desmoglein 1/3. (Right) Graph depicting the percentages of tumors categorized into respective groups based on immunofluorescence staining for E-cadherin and Beta-catenin.

Mentions: As a key desmosomal constituent in the epidermis, Perp loss could also promote cancer through effects on cell-cell adhesion. In constitutive Perp knockout mice, Perp loss does not abrogate desmosome formation, but leads to decreased numbers of desmosomes and reduced stability of desmosomal components [43]. Simple immunofluorescence analysis of desmosome proteins in Perp−/− newborn skin or K14CreER;Perpfl/fl adult mouse skin does not show perturbed membrane localization of desmosomal components ([43], Figure 1A, Figure 3A), and thus it is necessary to use a biochemical assay to show that desmosomes are functionally compromised upon Perp loss. This solubility assay is based on the fact that properly formed desmosomal complexes can only be solubilized by chaotropic agents, whereas improperly assembled desmosomal components can be solubilized by the nonionic detergent Triton X-100 [51]. We found that the desmosomal constituents Desmoglein 1/2 and Plakoglobin displayed enhanced Triton X-100-solubility in skin from K14CreER;Perpfl/fl mice compared to skin from control mice (Figure 3B), confirming that acute deletion of Perp leads to impaired desmosome function similar to that observed in constitutive Perp−/− mice [43].


Loss of the p53/p63 regulated desmosomal protein Perp promotes tumorigenesis.

Beaudry VG, Jiang D, Dusek RL, Park EJ, Knezevich S, Ridd K, Vogel H, Bastian BC, Attardi LD - PLoS Genet. (2010)

Adhesion junction analysis in tumors from K14CreER;Perpfl/fl and control mice.A) Representative Hematoxylin and Eosin (H&E) staining and immunofluorescence images of desmosome and adherens junction component protein expression in skin from tamoxifen-treated K14CreER;Perpfl/fl mice demonstrating that Perp loss itself does not disrupt membrane expression of other adhesion proteins. B) Western blot analysis showing both the Triton X-100-soluble and urea-only soluble fractions of mouse epidermal lysates from control and K14CreER;Perpfl/fl mice. Desmoglein 1/2 (Dsg 1/2) and Plakoglobin (Pg) solubility were examined. Gapdh serves as a loading control for the Triton X-100-soluble pool, while Keratin 14 serves as the loading control for the urea fraction. C, D) Tumors from K14CreER;Perpfl/fl mice were stained with antibodies against various desmosomal and adherens junction components, and both the percentage of epithelial cells staining positive for each marker and the intensity of staining were measured. Staining for each antigen was categorized as high (>70%), medium (30–70%), or low (<30%) level expression. C) Representative H&E and immunofluorescence images of a K14CreER;Perpfl/fl tumor sample demonstrating desmosomal component loss with retention of adherens junction components. Green signal indicates antigen staining, and blue signal indicates DAPI-marked nuclei. D) (Left) Graph showing the percentages of K14CreER;Perpfl/fl tumors categorized into respective groups based on quantitative immunofluorescence staining for Plakoglobin and Desmoglein 1/3. (Right) Graph depicting the percentages of tumors categorized into respective groups based on immunofluorescence staining for E-cadherin and Beta-catenin. (E) Tumors from control wild-type mice were stained for desmosomal and adherens junction components, and each component was categorized as displaying high, medium, or low level expression, as described above. (Left) Graph showing the percentages of control tumors categorized into respective groups based on quantitative immunofluorescence staining for Perp, Plakoglobin, and Desmoglein 1/3. (Right) Graph depicting the percentages of tumors categorized into respective groups based on immunofluorescence staining for E-cadherin and Beta-catenin.
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pgen-1001168-g003: Adhesion junction analysis in tumors from K14CreER;Perpfl/fl and control mice.A) Representative Hematoxylin and Eosin (H&E) staining and immunofluorescence images of desmosome and adherens junction component protein expression in skin from tamoxifen-treated K14CreER;Perpfl/fl mice demonstrating that Perp loss itself does not disrupt membrane expression of other adhesion proteins. B) Western blot analysis showing both the Triton X-100-soluble and urea-only soluble fractions of mouse epidermal lysates from control and K14CreER;Perpfl/fl mice. Desmoglein 1/2 (Dsg 1/2) and Plakoglobin (Pg) solubility were examined. Gapdh serves as a loading control for the Triton X-100-soluble pool, while Keratin 14 serves as the loading control for the urea fraction. C, D) Tumors from K14CreER;Perpfl/fl mice were stained with antibodies against various desmosomal and adherens junction components, and both the percentage of epithelial cells staining positive for each marker and the intensity of staining were measured. Staining for each antigen was categorized as high (>70%), medium (30–70%), or low (<30%) level expression. C) Representative H&E and immunofluorescence images of a K14CreER;Perpfl/fl tumor sample demonstrating desmosomal component loss with retention of adherens junction components. Green signal indicates antigen staining, and blue signal indicates DAPI-marked nuclei. D) (Left) Graph showing the percentages of K14CreER;Perpfl/fl tumors categorized into respective groups based on quantitative immunofluorescence staining for Plakoglobin and Desmoglein 1/3. (Right) Graph depicting the percentages of tumors categorized into respective groups based on immunofluorescence staining for E-cadherin and Beta-catenin. (E) Tumors from control wild-type mice were stained for desmosomal and adherens junction components, and each component was categorized as displaying high, medium, or low level expression, as described above. (Left) Graph showing the percentages of control tumors categorized into respective groups based on quantitative immunofluorescence staining for Perp, Plakoglobin, and Desmoglein 1/3. (Right) Graph depicting the percentages of tumors categorized into respective groups based on immunofluorescence staining for E-cadherin and Beta-catenin.
Mentions: As a key desmosomal constituent in the epidermis, Perp loss could also promote cancer through effects on cell-cell adhesion. In constitutive Perp knockout mice, Perp loss does not abrogate desmosome formation, but leads to decreased numbers of desmosomes and reduced stability of desmosomal components [43]. Simple immunofluorescence analysis of desmosome proteins in Perp−/− newborn skin or K14CreER;Perpfl/fl adult mouse skin does not show perturbed membrane localization of desmosomal components ([43], Figure 1A, Figure 3A), and thus it is necessary to use a biochemical assay to show that desmosomes are functionally compromised upon Perp loss. This solubility assay is based on the fact that properly formed desmosomal complexes can only be solubilized by chaotropic agents, whereas improperly assembled desmosomal components can be solubilized by the nonionic detergent Triton X-100 [51]. We found that the desmosomal constituents Desmoglein 1/2 and Plakoglobin displayed enhanced Triton X-100-solubility in skin from K14CreER;Perpfl/fl mice compared to skin from control mice (Figure 3B), confirming that acute deletion of Perp leads to impaired desmosome function similar to that observed in constitutive Perp−/− mice [43].

Bottom Line: Tumor development is associated with inactivation of both of Perp's known functions, in apoptosis and cell-cell adhesion.Interestingly, Perp-deficient tumors exhibit widespread downregulation of desmosomal constituents while adherens junctions remain intact, suggesting that desmosome loss is a specific event important for tumorigenesis rather than a reflection of a general change in differentiation status.Using gene expression profiling, we show further that Perp loss induces a set of inflammation-related genes that could stimulate tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University School of Medicine, Stanford, California, USA.

ABSTRACT
Dysregulated cell-cell adhesion plays a critical role in epithelial cancer development. Studies of human and mouse cancers have indicated that loss of adhesion complexes known as adherens junctions contributes to tumor progression and metastasis. In contrast, little is known regarding the role of the related cell-cell adhesion junction, the desmosome, during cancer development. Studies analyzing expression of desmosome components during human cancer progression have yielded conflicting results, and therefore genetic studies using knockout mice to examine the functional consequence of desmosome inactivation for tumorigenesis are essential for elucidating the role of desmosomes in cancer development. Here, we investigate the consequences of desmosome loss for carcinogenesis by analyzing conditional knockout mice lacking Perp, a p53/p63 regulated gene that encodes an important component of desmosomes. Analysis of Perp-deficient mice in a UVB-induced squamous cell skin carcinoma model reveals that Perp ablation promotes both tumor initiation and progression. Tumor development is associated with inactivation of both of Perp's known functions, in apoptosis and cell-cell adhesion. Interestingly, Perp-deficient tumors exhibit widespread downregulation of desmosomal constituents while adherens junctions remain intact, suggesting that desmosome loss is a specific event important for tumorigenesis rather than a reflection of a general change in differentiation status. Similarly, human squamous cell carcinomas display loss of PERP expression with retention of adherens junctions components, indicating that this is a relevant stage of human cancer development. Using gene expression profiling, we show further that Perp loss induces a set of inflammation-related genes that could stimulate tumorigenesis. Together, these studies suggest that Perp-deficiency promotes cancer by enhancing cell survival, desmosome loss, and inflammation, and they highlight a fundamental role for Perp and desmosomes in tumor suppression. An understanding of the factors affecting cancer progression is important for ultimately improving the diagnosis, prognostication, and treatment of cancer.

Show MeSH
Related in: MedlinePlus