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087 The polycomb protein EZH2 promotes disease progression in cutaneous CD30+ lymphoproliferative disease through histone methyltransferase activity
ABSTRACTS | Carcinogenesis & Cancer Genetics 086
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Combination of retinoid and histone deacetylase inhibitor produced an anti-tumor effect in cutaneous T-cell lymphoma by restoring tumor suppressor gene, retinoic acid receptorb2, via histone acetylation Y Kato1,2, C Egusa1, T Maeda1 and R Tsuboi1 1 Dermatology, Tokyo Medical University, Shinjuku, Japan and 2 Dermatology, Tokyo Metropolitan Tama Medical Center, Fuchu, Japan Retinoids exert anti-proliferative, differentiative, and apoptosis-inducing effects through their receptors. Retinoic acid receptor (RAR) b2 behaves as a tumor suppressor gene, and its expression is suppressible by DNA methylation in many malignancies. We aimed to determine whether combining a retinoid, Am 80, with a histone deacetylase inhibitor, MS-275, could suppress tumor growth in a RARb2-negative human cutaneous T cell lymphoma (CTCL) cell lines and freshly isolated primary CTCL cells, and to elucidate the epigenetic mechanism behind the phenomena. SeAx cells were implanted subcutaneously in NOD-SCID mice which were randomly divided into four groups and treated with either Am80, MS-275 by oral gavage (five days/week), or a combination of the two agents. Cell proliferation assay, methylation-specific PCR, flow cytometric analysis of cell cycle and apoptosis and chromatin immunoprecipitation assay were employed.Quantitative PCR analysis revealed that RARb2 gene expression was restored only by this combination rather than by either of the agents singly. Restored retinoid sensitivity was observed in combining retinoid with a histone deacetylase inhibitor significantly inhibited cell growth in vitro, suppressed subcutaneously transplanted tumor growth, and prolonged survival of tumor-bearing mice in vivo by more strongly inducing apoptosis and p21 expression in CTCL cells than either agent alone. In the combination treatment, the histone H4 acetylation level at lysine 12 and 16 in the promoter region increased after restoration of RARb2 expression although the DNA methylation of RARb2 remained unchanged. This is the first report of histone acetylation as the primary event in the restoration of RARb2. Inducible RARb2 expression may serve as a reliable predictor for tumor response in patients undergoing ‘epigenetic & differentiation’ therapy.
The polycomb protein EZH2 promotes disease progression in cutaneous CD30+ lymphoproliferative disease through histone methyltransferase activity S Yi1, J Sun1, W Fu1, ME Kadin2, P Tu1 and Y Wang1 1 Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China and 2 Department of Dermatology and Skin Surgery, Boston University School of Medicine, Providence, RI Aberrant epigenetic programming is a hallmark of cancer and an attractive target for pharmacologic therapy. The enhancer of zest homolog 2 (EZH2), a histone-lysine N-methyltransferase which mediates histone H3 lysine 27 trimethylation (H3K27me3), has recently been implicated in the pathogenesis of a variety of malignancies. Here we investigate the expression pattern and pathobiology of EZH2 in primary cutaneous CD30+ lymphoproliferative disease (CD30+LPD). We found that EZH2 is highly expressed in the CD30+ anaplastic T cells in 11 of 12 cases of lymphomatoid papulosis, 16 of 20 cases of primary cutaneous anaplastic large cell lymphomas, and 4 of 4 cases of mycosis fungoides with large cell transformation, while reactive small lymphocytes demonstrated negative EZH2 expression.EZH2 silencing in CD30+LPD model cell lines Mac-1 and Mac-2A resulted in decreased nuclear H3K27me3 levels. EZH2 silencing conferred significant growth disadvantage accompanied by two-fold increase of spontaneous cell apoptosis. Moreover, specific inhibition of EZH2 enzymatic activity with 4 small molecules (GSK343, UNC1999, EPZ5687, and EPZ5676) induced cell apoptosis and cell growth arrest in Mac-1 and Mac-2A cells. Therefore, EZH2 overexpression may promote disease progression in CD30+LPD through histone methyltransferase activity. EZH2 inhibitors, which are currently under clinical trials may serve as novel therapeutic regimens for cutaneous T cell lymphomas.
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Doxycycline as potential therapy for CTCL C Alexander1, C Richardson2 and B Poligone1,3 1 Rochester General Hospital Research Institute, Rochester, NY, 2 Dermatology, University of Rochester, Rochester, NY and 3 Rochester Skin Lymphoma Center, Fairport, NY The purpose of this study was to evaluate doxycycline as a potential adjuvant therapy for cutaneous T cell lymphoma (CTCL). Current systemic therapies for CTCL have significant toxic side effects. Doxycycline is a common, inexpensive, and well-tolerated systemic antiinflammatory medication. Doxycycline has recently been discovered as a potent inhibitor of the NF-kB pathway, an important pathway in the pathogenesis of CTCL. In our current study, we analyzed the induction of cellular killing by doxycycline in both CTCL cell lines and primary CD4+ T cells from patients with Sezary Syndrome (SS). Flow cytometry and western blots were used to evaluate both NF-kB and apoptotic pathways. Doxycycline was found to inhibit TNF-induced NF-kB activation in a dose dependent manner and to reduce the expression of NF-kB dependent apoptotic proteins. Assessment of procaspase-3, caspase-8, Parp-1, FasL, cytochrome c and BCL2a indicates that doxycycline acts through both the intrinsic and extrinsic pathways of apoptosis. Doxycycline was also found to induce reactive oxygen species. In summary, our results indicate that doxycycline is able to induce apoptosis through multiple pathways in both CTCL cell lines and primary CD4+ T cells from SS patients. This suggests that doxycycline provides a potential inexpensive and safe therapy for CTCL.
Stromal re-organization in hair follicle development and cancer suppression T Sun, V Greco and P Myung Dermatology, Genetics, Cancer Center, Yale School of Medicine, New Haven, CT The tumor stroma, in particular fibroblasts, can be an active inducer of carcinoma initiation and progression, and resistance to drug therapy. However, current cancer treatments have not targeted this population or these crucial stromal-epithelial interactions. This study begins to investigate a novel modality to treat cancer by targeting the stroma itself: re-organizing the cancer stroma, specifically fibroblasts, into one that may actively suppress cancer. Basal cell carcinoma, a malignant tumor of the embryonic hair germ, distinctively lacks underlying dermal condensates/papillae in its tumor stroma in contrast to benign follicular tumors. The dermal condensate/papilla is a dense cluster of stromal fibroblasts that is required for organized differentiation of the epithelium into hair follicles during development and for hair follicle regression during the adult hair cycle. By promoting tumor stromal re-organization into dermal condensates in basal cell carcinoma, we hypothesize this will result in differentiation, and potentially, regression of the basal cell carcinoma. However, it is currently unknown how the dermal condensate is normally formed. Here we show by using live imaging of embryonic mouse skin explants that dermal condensate cells originate from the Wntactive upper dermis and that they form mostly by migration, without significant contribution from cellular proliferation. Inhibiting Sonic Hedgehog and myosin-2B activation both result in the loss of normal dermal condensate polarity and cellular density, suggesting that Sonic Hedgehog and myosin-2B activation cooperate to induce dermal condensate formation. Our results demonstrate that the dermal condensate forms by cellular migration during development. These results also suggest that activating Sonic Hedgehog signaling in dermal fibroblasts may promote dermal condensate formation in the stroma of basal cell carcinoma. Collectively, this study proposes that re-organization of the tumor stroma into one that promotes normal epithelial differentiation may be a novel effective modality to treat certain cancers.
S16 Journal of Investigative Dermatology (2016), Volume 136
Neoplasms bypass immune evasion via hair follicle residence BJ Kubick and DR Roop Dermatology, University of Colorado AMC, Aurora, CO Anti-tumor immunity exerts selective pressure on transformed clones, permitting only mutation sequences that are either non-immunogenic or include complementary, immuneevasive adaptations. This evolutionary process often includes an “equilibrium phase,” during which transformed clone outgrowth is prevented by immune effectors without complete elimination. We hypothesized that this phase could be targeted for cancer prevention. Therefore to characterize this state and test the efficacy of interventions during dormancy, we assembled a genetically-engineered mouse model (GEMM) of inducible, Kras-driven carcinogenesis coupled to a fluorescent reporter. To mimic the immunogenicity of somatic transformation, we graft dissociated epidermal keratinocytes from the GEMM onto recipient animals that do not carry the inducible mutant Kras allele. This transplant step also enables us to control the immune status of the recipient animals. After healing, transformation is induced and the grafts are imaged repeatedly via intravital confocal microscopy. In immunodeficient recipients, transformation initiates the rapid growth of labeled cells and resultant tumor formation. Whereas in immunocompetent backgrounds, we observe long-term persistence of clones originating exclusively from hair follicles. This argues that the relative immune privilege of the hair follicle provides a safe harbor for transformed cells, effectively generating a spatially-defined equilibrium phase. This also provides a mechanism by which a single source of anti-tumor activity could produce both elimination of some clones and immunemediated dormancy of others. Comparing waxed grafts to shaved grafts, we observe that the protective benefits of hair follicle residence are lost during exit from the anagen phase. This evidence, combined with past studies linking immune privilege to anagen, lead us to theorize that the hair follicle cycle is actually a potent anti-tumor mechanism. We conclude that targeting of long-lived, niche-protected neoplasms is a viable strategy for rational cancer prevention.
The role of Hmga2 translocation during skin squamous cell carcinoma development Y Li, K Boland, K Johnson, S Lad, X Pi and RJ Morris Stem cells and Cancer, University of Minnesota, Austin, MN High Mobility Group A2 (Hmga2) functions as an oncogene overexpressed in a variety of human malignant neoplasms, induced in epithelial-mesenchymal transition, and in association with squamous skin carcinoma stemness. However, little is known about the expression of Hmga2 in cutaneous epithelium and its role during skin non-melanoma tumor development. Here, we find that Hmga2 is expressed as membrane protein out of bulge region in normal skin tissue by immunofluorescence staining, as membrane/cytoplasm/nuclear protein in papilloma tissue, and as nuclear protein in the skin squamous carcinoma tissue. CD34+/ CD49f+ keratinocyte stem cells have significantly higher expression of mRNA for Hmga2 and Let-7 family members than that in CD34-/CD49f+ (p¼0.0317 for Hmga2, p¼0.026 Let7b, p¼0.0234, Let7c-1, p¼0.00013 Let7f). Hmga2 is translocated from membrane to nucleus upon keratinocyte culture and explant culture from normal untreated adult skin. The translocation is inhibited by panobinostat, a pan-histone deacetylase inhibitor. Moreover, qRT-PCR disclosed that ROCK1 and ROCK2 expression significantly decreased in keratinocytes after treated with panobinostat (ROCK1, p¼0.00003, ROCK2, p¼0.0003). The ROCK inhibitor, Y27632 also modulated the translocation as observed in keratinocytes by immunofluorescence staining. The data demonstrate that Hmga2 is located not only in the nucleus, but also in the membrane of normal epidermis, and Hmga2 translocation from membrane to nucleus involves the ROCK pathway. Our study implicates Hmga2 translocation from membrane to nucleus as a new mechanism for skin squamous cell carcinoma development.
087
Combination of retinoid and histone deacetylase inhibitor produced an anti-tumor effect in cutaneous T-cell lymphoma by restoring tumor suppressor gene, retinoic acid receptorb2, via histone acetylation Y Kato1,2, C Egusa1, T Maeda1 and R Tsuboi1 1 Dermatology, Tokyo Medical University, Shinjuku, Japan and 2 Dermatology, Tokyo Metropolitan Tama Medical Center, Fuchu, Japan Retinoids exert anti-proliferative, differentiative, and apoptosis-inducing effects through their receptors. Retinoic acid receptor (RAR) b2 behaves as a tumor suppressor gene, and its expression is suppressible by DNA methylation in many malignancies. We aimed to determine whether combining a retinoid, Am 80, with a histone deacetylase inhibitor, MS-275, could suppress tumor growth in a RARb2-negative human cutaneous T cell lymphoma (CTCL) cell lines and freshly isolated primary CTCL cells, and to elucidate the epigenetic mechanism behind the phenomena. SeAx cells were implanted subcutaneously in NOD-SCID mice which were randomly divided into four groups and treated with either Am80, MS-275 by oral gavage (five days/week), or a combination of the two agents. Cell proliferation assay, methylation-specific PCR, flow cytometric analysis of cell cycle and apoptosis and chromatin immunoprecipitation assay were employed.Quantitative PCR analysis revealed that RARb2 gene expression was restored only by this combination rather than by either of the agents singly. Restored retinoid sensitivity was observed in combining retinoid with a histone deacetylase inhibitor significantly inhibited cell growth in vitro, suppressed subcutaneously transplanted tumor growth, and prolonged survival of tumor-bearing mice in vivo by more strongly inducing apoptosis and p21 expression in CTCL cells than either agent alone. In the combination treatment, the histone H4 acetylation level at lysine 12 and 16 in the promoter region increased after restoration of RARb2 expression although the DNA methylation of RARb2 remained unchanged. This is the first report of histone acetylation as the primary event in the restoration of RARb2. Inducible RARb2 expression may serve as a reliable predictor for tumor response in patients undergoing ‘epigenetic & differentiation’ therapy.
The polycomb protein EZH2 promotes disease progression in cutaneous CD30+ lymphoproliferative disease through histone methyltransferase activity S Yi1, J Sun1, W Fu1, ME Kadin2, P Tu1 and Y Wang1 1 Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China and 2 Department of Dermatology and Skin Surgery, Boston University School of Medicine, Providence, RI Aberrant epigenetic programming is a hallmark of cancer and an attractive target for pharmacologic therapy. The enhancer of zest homolog 2 (EZH2), a histone-lysine N-methyltransferase which mediates histone H3 lysine 27 trimethylation (H3K27me3), has recently been implicated in the pathogenesis of a variety of malignancies. Here we investigate the expression pattern and pathobiology of EZH2 in primary cutaneous CD30+ lymphoproliferative disease (CD30+LPD). We found that EZH2 is highly expressed in the CD30+ anaplastic T cells in 11 of 12 cases of lymphomatoid papulosis, 16 of 20 cases of primary cutaneous anaplastic large cell lymphomas, and 4 of 4 cases of mycosis fungoides with large cell transformation, while reactive small lymphocytes demonstrated negative EZH2 expression.EZH2 silencing in CD30+LPD model cell lines Mac-1 and Mac-2A resulted in decreased nuclear H3K27me3 levels. EZH2 silencing conferred significant growth disadvantage accompanied by two-fold increase of spontaneous cell apoptosis. Moreover, specific inhibition of EZH2 enzymatic activity with 4 small molecules (GSK343, UNC1999, EPZ5687, and EPZ5676) induced cell apoptosis and cell growth arrest in Mac-1 and Mac-2A cells. Therefore, EZH2 overexpression may promote disease progression in CD30+LPD through histone methyltransferase activity. EZH2 inhibitors, which are currently under clinical trials may serve as novel therapeutic regimens for cutaneous T cell lymphomas.
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Doxycycline as potential therapy for CTCL C Alexander1, C Richardson2 and B Poligone1,3 1 Rochester General Hospital Research Institute, Rochester, NY, 2 Dermatology, University of Rochester, Rochester, NY and 3 Rochester Skin Lymphoma Center, Fairport, NY The purpose of this study was to evaluate doxycycline as a potential adjuvant therapy for cutaneous T cell lymphoma (CTCL). Current systemic therapies for CTCL have significant toxic side effects. Doxycycline is a common, inexpensive, and well-tolerated systemic antiinflammatory medication. Doxycycline has recently been discovered as a potent inhibitor of the NF-kB pathway, an important pathway in the pathogenesis of CTCL. In our current study, we analyzed the induction of cellular killing by doxycycline in both CTCL cell lines and primary CD4+ T cells from patients with Sezary Syndrome (SS). Flow cytometry and western blots were used to evaluate both NF-kB and apoptotic pathways. Doxycycline was found to inhibit TNF-induced NF-kB activation in a dose dependent manner and to reduce the expression of NF-kB dependent apoptotic proteins. Assessment of procaspase-3, caspase-8, Parp-1, FasL, cytochrome c and BCL2a indicates that doxycycline acts through both the intrinsic and extrinsic pathways of apoptosis. Doxycycline was also found to induce reactive oxygen species. In summary, our results indicate that doxycycline is able to induce apoptosis through multiple pathways in both CTCL cell lines and primary CD4+ T cells from SS patients. This suggests that doxycycline provides a potential inexpensive and safe therapy for CTCL.
Stromal re-organization in hair follicle development and cancer suppression T Sun, V Greco and P Myung Dermatology, Genetics, Cancer Center, Yale School of Medicine, New Haven, CT The tumor stroma, in particular fibroblasts, can be an active inducer of carcinoma initiation and progression, and resistance to drug therapy. However, current cancer treatments have not targeted this population or these crucial stromal-epithelial interactions. This study begins to investigate a novel modality to treat cancer by targeting the stroma itself: re-organizing the cancer stroma, specifically fibroblasts, into one that may actively suppress cancer. Basal cell carcinoma, a malignant tumor of the embryonic hair germ, distinctively lacks underlying dermal condensates/papillae in its tumor stroma in contrast to benign follicular tumors. The dermal condensate/papilla is a dense cluster of stromal fibroblasts that is required for organized differentiation of the epithelium into hair follicles during development and for hair follicle regression during the adult hair cycle. By promoting tumor stromal re-organization into dermal condensates in basal cell carcinoma, we hypothesize this will result in differentiation, and potentially, regression of the basal cell carcinoma. However, it is currently unknown how the dermal condensate is normally formed. Here we show by using live imaging of embryonic mouse skin explants that dermal condensate cells originate from the Wntactive upper dermis and that they form mostly by migration, without significant contribution from cellular proliferation. Inhibiting Sonic Hedgehog and myosin-2B activation both result in the loss of normal dermal condensate polarity and cellular density, suggesting that Sonic Hedgehog and myosin-2B activation cooperate to induce dermal condensate formation. Our results demonstrate that the dermal condensate forms by cellular migration during development. These results also suggest that activating Sonic Hedgehog signaling in dermal fibroblasts may promote dermal condensate formation in the stroma of basal cell carcinoma. Collectively, this study proposes that re-organization of the tumor stroma into one that promotes normal epithelial differentiation may be a novel effective modality to treat certain cancers.
S16 Journal of Investigative Dermatology (2016), Volume 136
Neoplasms bypass immune evasion via hair follicle residence BJ Kubick and DR Roop Dermatology, University of Colorado AMC, Aurora, CO Anti-tumor immunity exerts selective pressure on transformed clones, permitting only mutation sequences that are either non-immunogenic or include complementary, immuneevasive adaptations. This evolutionary process often includes an “equilibrium phase,” during which transformed clone outgrowth is prevented by immune effectors without complete elimination. We hypothesized that this phase could be targeted for cancer prevention. Therefore to characterize this state and test the efficacy of interventions during dormancy, we assembled a genetically-engineered mouse model (GEMM) of inducible, Kras-driven carcinogenesis coupled to a fluorescent reporter. To mimic the immunogenicity of somatic transformation, we graft dissociated epidermal keratinocytes from the GEMM onto recipient animals that do not carry the inducible mutant Kras allele. This transplant step also enables us to control the immune status of the recipient animals. After healing, transformation is induced and the grafts are imaged repeatedly via intravital confocal microscopy. In immunodeficient recipients, transformation initiates the rapid growth of labeled cells and resultant tumor formation. Whereas in immunocompetent backgrounds, we observe long-term persistence of clones originating exclusively from hair follicles. This argues that the relative immune privilege of the hair follicle provides a safe harbor for transformed cells, effectively generating a spatially-defined equilibrium phase. This also provides a mechanism by which a single source of anti-tumor activity could produce both elimination of some clones and immunemediated dormancy of others. Comparing waxed grafts to shaved grafts, we observe that the protective benefits of hair follicle residence are lost during exit from the anagen phase. This evidence, combined with past studies linking immune privilege to anagen, lead us to theorize that the hair follicle cycle is actually a potent anti-tumor mechanism. We conclude that targeting of long-lived, niche-protected neoplasms is a viable strategy for rational cancer prevention.
The role of Hmga2 translocation during skin squamous cell carcinoma development Y Li, K Boland, K Johnson, S Lad, X Pi and RJ Morris Stem cells and Cancer, University of Minnesota, Austin, MN High Mobility Group A2 (Hmga2) functions as an oncogene overexpressed in a variety of human malignant neoplasms, induced in epithelial-mesenchymal transition, and in association with squamous skin carcinoma stemness. However, little is known about the expression of Hmga2 in cutaneous epithelium and its role during skin non-melanoma tumor development. Here, we find that Hmga2 is expressed as membrane protein out of bulge region in normal skin tissue by immunofluorescence staining, as membrane/cytoplasm/nuclear protein in papilloma tissue, and as nuclear protein in the skin squamous carcinoma tissue. CD34+/ CD49f+ keratinocyte stem cells have significantly higher expression of mRNA for Hmga2 and Let-7 family members than that in CD34-/CD49f+ (p¼0.0317 for Hmga2, p¼0.026 Let7b, p¼0.0234, Let7c-1, p¼0.00013 Let7f). Hmga2 is translocated from membrane to nucleus upon keratinocyte culture and explant culture from normal untreated adult skin. The translocation is inhibited by panobinostat, a pan-histone deacetylase inhibitor. Moreover, qRT-PCR disclosed that ROCK1 and ROCK2 expression significantly decreased in keratinocytes after treated with panobinostat (ROCK1, p¼0.00003, ROCK2, p¼0.0003). The ROCK inhibitor, Y27632 also modulated the translocation as observed in keratinocytes by immunofluorescence staining. The data demonstrate that Hmga2 is located not only in the nucleus, but also in the membrane of normal epidermis, and Hmga2 translocation from membrane to nucleus involves the ROCK pathway. Our study implicates Hmga2 translocation from membrane to nucleus as a new mechanism for skin squamous cell carcinoma development.