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Gang Wang - One of the best experts on this subject based on the ideXlab platform.
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small interfering rna targeting of Keratin 17 reduces inflammation in imiquimod induced psoriasis like dermatitis
Chinese Medical Journal, 2020Co-Authors: Chunying Xiao, Chen Zhang, Gang Wang, Zhenlai Zhu, Hongjiang Qiao, Erle DangAbstract:BACKGROUND Psoriasis is a common chronic inflammatory skin disease with 2% to 3% prevalence worldwide and a heavy social-psychological burden for patients and their families. As the exact pathogenesis of psoriasis is still unknown, the current treatment is far from satisfactory. Thus, there is an urgent need to find a more effective therapy for this disease. Keratin 17 (K17), a type I intermediate filament, is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis. Therefore, we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis. This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA (siRNA) on mice with imiquimod (IMQ)-induced psoriasis-like dermatitis. METHODS Eight-week-old female BALB/c mice were administered a 5% IMQ cream on both ears to produce psoriatic dermatitis. On day 3, K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days. The right ears of the mice were treated in parallel with negative control (NC) siRNA. Inflammation was evaluated by gross ear thickness, histopathology, the infiltration of inflammatory cells (CD3+ T cells and neutrophils) using immunofluorescence, and the expression of cytokine production using real-time quantitative polymerase chain reaction. The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance. RESULTS The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears, as evidenced by the alleviated ear inflammation phenotype, including decreased ear thickness, infiltration of inflammatory cells (CD3+ T cells and neutrophils), and inflammatory cytokine/chemokine expression levels (interleukin 17 [IL-17], IL-22, IL-23, C-X-C motif chemokine ligand 1, and C-C motif chemokine ligand 20) (P < 0.05 vs. the Blank or NC siRNA groups). Compared to the NC siRNA treatment, the K17 siRNA treatment resulted in increased K1 and K10 expression, which are characteristic of Keratinocyte differentiation (vs. NC siRNA, K17 siRNA1 group: K1, t = 4.782, P = 0.0050; K10, t = 3.365, P = 0.0120; K17 siRNA2 group: K1, t = 4.104, P = 0.0093; K10, t = 4.168, P = 0.0042; siRNA Mix group: K1, t = 3.065, P = 0.0221; K10, t = 10.83, P < 0.0001), and decreased K16 expression, which is characteristic of Keratinocyte proliferation (vs. NC siRNA, K17 siRNA1 group: t = 4.156, P = 0.0043; K17 siRNA2 group: t = 2.834, P = 0.0253; siRNA Mix group: t = 2.734, P = 0.0250). CONCLUSIONS Inhibition of K17 expression by its specific siRNA significantly alleviated inflammation in mice with IMQ-induced psoriasis-like dermatitis. Thus, gene therapy targeting K17 may be a potential treatment approach for psoriasis.
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Small interfering RNA targeting of Keratin 17 reduces inflammation in imiquimod-induced psoriasis-like dermatitis.
Chinese medical journal, 2020Co-Authors: Chunying Xiao, Chen Zhang, Gang Wang, Zhenlai Zhu, Hongjiang Qiao, Erle DangAbstract:BACKGROUND Psoriasis is a common chronic inflammatory skin disease with 2% to 3% prevalence worldwide and a heavy social-psychological burden for patients and their families. As the exact pathogenesis of psoriasis is still unknown, the current treatment is far from satisfactory. Thus, there is an urgent need to find a more effective therapy for this disease. Keratin 17 (K17), a type I intermediate filament, is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis. Therefore, we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis. This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA (siRNA) on mice with imiquimod (IMQ)-induced psoriasis-like dermatitis. METHODS Eight-week-old female BALB/c mice were administered a 5% IMQ cream on both ears to produce psoriatic dermatitis. On day 3, K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days. The right ears of the mice were treated in parallel with negative control (NC) siRNA. Inflammation was evaluated by gross ear thickness, histopathology, the infiltration of inflammatory cells (CD3+ T cells and neutrophils) using immunofluorescence, and the expression of cytokine production using real-time quantitative polymerase chain reaction. The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance. RESULTS The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears, as evidenced by the alleviated ear inflammation phenotype, including decreased ear thickness, infiltration of inflammatory cells (CD3+ T cells and neutrophils), and inflammatory cytokine/chemokine expression levels (interleukin 17 [IL-17], IL-22, IL-23, C-X-C motif chemokine ligand 1, and C-C motif chemokine ligand 20) (P
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Keratin 17 in disease pathogenesis: from cancer to dermatoses
The Journal of pathology, 2018Co-Authors: Luting Yang, Shaolong Zhang, Gang WangAbstract:Keratin 17 (K17) is a type I intermediate filament mainly expressed in the basal cells of epithelia. As a multifaceted cytoskeletal protein, K17 regulates a myriad of biological processes, including cell proliferation and growth, skin inflammation and hair follicle cycling. Aberrant overexpression of K17 is found in various diseases ranging from psoriasis to malignancies such as breast, cervical, oral squamous and gastric carcinomas. Moreover, genetic mutation in KRT17 is related to tissue-specific diseases, represented by steatocystoma multiplex and pachyonychia congenita. In this review, we summarize our findings concerning the regulatory mechanisms of K17 overexpression in psoriasis and compare them to the literature relating to other diseases. We discuss data that proinflammatory cytokines, including interleukin-17 (IL-17), IL-22, interferon-gamma (IFN-γ), transforming growth factor-beta (TGF-β) and transcription factors glioma-associated oncogene homolog 1/2 (Gli1/2), Nrf2 and p53 can regulate K17 by transcriptional and translational control. Moreover, post-translational modification, including phosphorylation and ubiquitination, is involved in the regulation of K17 stability and biological functions. We therefore review the current understanding of the K17 regulatory mechanism and its pathogenic role in diseases from dermatoses to cancer. Prospects for anti-K17 therapy in diagnosis, prognosis and disease treatment are also discussed. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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e3 ligase trim21 ubiquitylates and stabilizes Keratin 17 to induce stat3 activation in psoriasis
Journal of Investigative Dermatology, 2018Co-Authors: Luting Yang, Y Ke, T Zhang, Chen Zhang, Huijie Bian, Gang WangAbstract:Keratin 17 (K17), a marker of Keratinocyte hyperproliferation, is a type I intermediate filament that is overexpressed in psoriatic epidermis and plays a critical pathogenic role by stimulating T cells. However, the posttranslational modification of K17, which is reversible and targetable, has not been elucidated. Herein, we reported that K17 could be modified through ubiquitination that controlled its stability and led to the phosphorylation and nuclear translocation of its interactor signal transducers and activators of transcription 3 (STAT3), which is a key regulator of cell proliferation in psoriasis. First, we stimulated human Keratinocyte cell line HaCaT cells with psoriasis (pso)-mix, which is a cytokine pool (IL-17, IL-22, tumor necrosis factor-α, and IFN-γ) mimicking the in vitro “psoriasis-like” status and found that the ubiquitination of K17 was essential to stabilize its protein expression in pso-mix-treated HaCaT cells. Subsequently, tripartite motif-containing protein 21 was identified as the E3 ligase of K17, which ubiquitylated K17 via K63 linkage to maintain K17 stabilization. More importantly, we uncovered that K17 was a direct interactor of STAT3, and K17 ubiquitination could promote STAT3 activation in pso-mix-treated HaCaT cells. Our study demonstrated that targeting K17 ubiquitination may be a potential therapeutic approach by attenuating STAT3 signaling in psoriasis.
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nb uvb irradiation downregulates Keratin 17 expression in Keratinocytes by inhibiting the erk1 2 and stat3 signaling pathways
Archives of Dermatological Research, 2018Co-Authors: Yuchen Zhuang, Changxu Han, Erle Dang, Liang Jin, Hui Fang, Hongjiang Qiao, Gang WangAbstract:Keratin-17 (K17) is a cytoskeletal protein produced by Keratinocytes (KCs), which is overexpressed in psoriasis and may play a pivotal role in its pathogenesis. Narrow-band ultraviolet B (NB-UVB) irradiation is used as a general treatment for psoriasis, although its impact on K17 expression has yet to be determined. In this study, we aimed to investigate the effect of NB-UVB irradiation on K17 expression and its signaling pathways. After exposure to NB-UVB irradiation, immortalized human Keratinocytes (HaCaT cells) were analyzed by flow cytometry, CCK-8 assays and transmission electron microscopy to examine proliferation. Meanwhile, K17 expression in primary human epithelial Keratinocytes was detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis and immunofluorescence. HaCaT cells pre-incubated with PD-98059 and piceatannol were subjected to western blot analysis to examine ERK1/2 and STAT3 phosphorylation. The ears of mice treated with imiquimod (IMQ) and irradiated by NB-UVB were taken to examine K17 expression by qRT-PCR, western blot analysis, and immunofluorescence. Our results showed that 400 mJ/cm2 of NB-UVB irradiation was the maximum tolerable dose for HaCaT cells and could cause inhibited HaCaT cell proliferation and moderate increase of the early apoptosis. Furthermore, NB-UVB irradiation could downregulate K17 expression by inhibiting the ERK1/2 and STAT3 signaling pathways. In experiments conducted in vivo, NB-UVB irradiation with doses of MED or higher could eliminate the IMQ-induced psoriasis-like dermatitis and inhibit K17 expression. These results indicated that NB-UVB irradiation may eliminate chronic psoriatic plaques by suppressing K17 expression via the ERK1/2 and STAT3 signaling pathways.
Pierre A Coulombe - One of the best experts on this subject based on the ideXlab platform.
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Keratin 17 regulates nuclear morphology and chromatin organization.
Journal of cell science, 2020Co-Authors: Justin T. Jacob, Raji R. Nair, Brian G. Poll, Christopher M. Pineda, Ryan P. Hobbs, Michael J. Matunis, Pierre A CoulombeAbstract:Keratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor Keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wildtype K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17 null cells. Analyses of primary cultures of skin Keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization, and RNA processing. Key histone modifications and LAP2β localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells.
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Keratin 17 regulates nuclear morphology and chromatin organization
bioRxiv, 2020Co-Authors: Justin T. Jacob, Raji R. Nair, Brian G. Poll, Christopher M. Pineda, Ryan P. Hobbs, Michael J. Matunis, Pierre A CoulombeAbstract:Keratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor Keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wildtype K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17 null cells. Analyses of primary cultures of skin Keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization, and RNA processing. Key histone modifications and LAP2{beta} localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells. SummaryKeratin 17 (K17) is one of two non-lamin intermediate filament proteins recently identified to localize to and function in the cell nucleus. K17 is here shown to regulate nuclear morphology, chromatin organization, LAP2 localization, and cell proliferation.
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Keratin 17 regulates nuclear morphology and chromatin organization
2020Co-Authors: Justin T. Jacob, Raji R. Nair, Brian G. Poll, Christopher M. Pineda, Ryan P. Hobbs, Michael J. Matunis, Pierre A CoulombeAbstract:AbstractKeratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor Keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wildtype K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17 null cells. Analyses of primary cultures of skin Keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization, and RNA processing. Key histone modifications and LAP2β localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells.SummaryKeratin 17 (K17) is one of two non-lamin intermediate filament proteins recently identified to localize to and function in the cell nucleus. K17 is here shown to regulate nuclear morphology, chromatin organization, LAP2 localization, and cell proliferation.
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Loss of Keratin 17 induces tissue-specific cytokine polarization and cellular differentiation in HPV16-driven cervical tumorigenesis in vivo.
Oncogene, 2016Co-Authors: Ryan P. Hobbs, A S Batazzi, M C Han, Pierre A CoulombeAbstract:Despite preventive human papilloma virus (HPV) vaccination efforts, cervical cancer remains a leading cause of death in women worldwide. Development of therapeutic approaches for cervical cancer are hampered by a lack of mechanistic insight during tumorigenesis. The cytoskeletal protein Keratin 17 (KRT17;K17) is robustly expressed in a broad array of carcinomas, including in cervical tumors, where it has both diagnostic and prognostic value. In this study, we have established multiple functional roles for K17 in the promotion of cervical tumorigenesis in vivo using the established HPV16tg mouse model for cervical squamous cell carcinoma. In HPV16tg/+;Krt17-/-relative to HPV16tg/+ reference female mice, onset of cervical lesions is delayed and closely paralleled by marked reductions in hyperplasia, dysplasia and vascularization. In addition, loss of Krt17 is associated with a cytokine polarization and recruitment of effector immune cells to lesion-prone cervical epithelia. Further, we observed marked enhancement of terminal differentiation in HPV16tg/+;Krt17-/-cervical epithelium accompanied by a stimulation and expansion in the expression of p63, a known basal/reserve cell marker in this tissue. Altogether, the data suggest that the loss of Krt17 may foster an overall protective environment for lesion-prone cervical tissue. In addition to providing new insights into the immunomodulatory and cellular mechanisms of cervical tumorigenesis, these findings may help guide the development of future therapies including vaccines.
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Regulation of C-X-C chemokine gene expression by Keratin 17 and hnRNP K in skin tumor Keratinocytes
The Journal of cell biology, 2015Co-Authors: Byung Min Chung, Artem D. Arutyunov, Erika Ilagan, Nu Yao, Marsha Wills-karp, Pierre A CoulombeAbstract:High levels of the intermediate filament Keratin 17 (K17) correlate with a poor prognosis for several types of epithelial tumors. However, the causal relationship and underlying mechanisms remain undefined. A recent study suggested that K17 promotes skin tumorigenesis by fostering a specific type of inflammation. We report here that K17 interacts with the RNA-binding protein hnRNP K, which has also been implicated in cancer. K17 is required for the cytoplasmic localization of hnRNP K and for its role in regulating the expression of multiple pro-inflammatory mRNAs. Among these are the CXCR3 ligands CXCL9, CXCL10, and CXCL11, which together form a signaling axis with an established role in tumorigenesis. The K17–hnRNP K partnership is regulated by the ser/thr kinase RSK and required for CXCR3-dependent tumor cell growth and invasion. These findings functionally integrate K17, hnRNP K, and gene expression along with RSK and CXCR3 signaling in a Keratinocyte-autonomous axis and provide a potential basis for their implication in tumorigenesis.
Kenneth R Shroyer - One of the best experts on this subject based on the ideXlab platform.
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Keratin 17 Expression Predicts Poor Clinical Outcome in Patients With Advanced Esophageal Squamous Cell Carcinoma.
Applied immunohistochemistry & molecular morphology : AIMM, 2021Co-Authors: Kester Haye, Luisa F. Escobar-hoyos, Ali Akalin, Sruthi Babu, Lyanne Oblein, Rajarsi Gupta, Kenneth R ShroyerAbstract:The major roles of Keratin 17 (K17) as a prognostic biomarker have been highlighted in a range of human malignancies. However, its relevance to esophageal squamous cell carcinoma (ESCC) remains unexplored. In this study, the relationship between K17 expression and clinicopathologic parameters and survival were determined by RNA sequencing (RNA-Seq) in 90 ESCCs and by immunohistochemistry (IHC) in 68 ESCCs. K17 expression was significantly higher in ESCC than in paired normal tissues at both the messenger RNA and protein levels. K17 messenger RNA and staining by IHC were significantly correlated with aggressive characteristics, including advanced clinical stage, invasion depth, and lymph node metastases; and were predictive of poor prognosis in advanced disease patients. Furthermore, K17 expression was detected by IHC in high-grade premalignant lesions of the esophageal mucosa, suggesting that K17 could also be a biomarker of dysplasia of the esophageal mucosa. Overall, this study established that K17 is a negative prognostic biomarker for the most common subtype of esophageal cancer.
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Abstract PO-050: Solubilization of Keratin 17 promotes the metastatic dissemination of the most lethal form of pancreatic cancer
Poster Presentations - Proffered Abstracts, 2020Co-Authors: Ryan R Kawalerski, Taryn Boyle, Cindy V Leiton, Natasha T Snider, Kenneth R Shroyer, Lucia Roa-peña, Chun-hao Pan, Mariana Torrente Gonçalves, Luke A. Torre-healy, Luisa F. Escobar-hoyosAbstract:CLINICAL NEED & OBJECTIVES: There is an unmet need for the discovery and development of novel therapeutic approaches for PDAC. Molecular subtyping, however, has recently uncovered potential biomarkers that, subject to further investigation, may be druggable to extend PDAC patient survival. Expression of Keratin 17 (K17), an oncofetal intermediate filament (IF) protein and hallmark of the most aggressive subtype, associates negatively with survival and drives chemotherapy resistance in PDAC. This suggests a functional role of K17 in PDAC, though the underlying mechanisms are unknown. Nuclear localization, however, has been suggested as a part of the overarching processes through which K17 promotes tumor growth. In related Keratins, post-translational modifications (PTM) such as phosphorylation drive IF dissociation (solubilization) and can contribute to migration and metastatic dissemination. Thus, we hypothesized that K17 may harbor PTM- and/or solubilization-dependent oncogenic functions that could reveal new PDAC therapeutic options. METHODS & RESULTS: Using isogenic murine PDAC models, we found that K17 expression enhances tumor growth and metastatic potential. Furthermore, K17 solubility was associated with shorter survival, independent of total K17 expression or cell proliferation in both murine and human PDACs. To identify K17 PTMs potentially driving solubilization, we performed liquid chromatography mass spectrometry to sequence K17 isolated from patient PDACs and identified phosphorylation of a conserved N-terminal region (Ser 10-13) as the predominant PTMs. To determine if Ser 10-13 phosphorylation regulates K17 solubility, we generated mutants that mimic K17 Ser 10-13 phosphorylation and found that this was sufficient to solubilize K17 and promote nuclear localization. Building on these findings, we used kinase inhibitors to show that the PKC-MEK-RSK signaling pathway is involved in phosphorylation of K17 Ser 10-13. To assess the impact of K17 Ser 10-13 phosphorylation on tumors, nude mice were implanted orthotopically with isogenic human PDAC cell lines expressing either wild type (WT), loss of function (LOF) Ser 10-13 mutants, or Ser 10-13 phosphorylation gain of function (GOF) mutants. Compared to tumors expressing WT K17, GOF tumors resulted in increased metastases, and LOF mutants abrogated these characteristics. CONCLUSION: These results suggest that enhanced K17 solubility by phosphorylation at Ser 10-13 is sufficient to impact PDAC metastasis and survival and reveal novel druggable sites on K17. These results motivate future studies to uncover K17 phosphorylation targeting approaches for PDAC. If successful, they will be coupled with the identification of PDAC patients by tumor K17 expression, providing the basis of a potential precision medicine strategy. Citation Format: Ryan R. Kawalerski, Mariana T. Goncalves, Lucia Roa-Pena, Cindy V. Leiton, Chun-Hao Pan, Luke A. Torre-Healy, Taryn Boyle, Natasha T. Snider, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Solubilization of Keratin 17 promotes the metastatic dissemination of the most lethal form of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-050.
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abstract c26 oncogenic mechanism of soluble Keratin 17 offers potential therapeutic vulnerability in pancreatic cancer
Cancer Research, 2019Co-Authors: Ryan R Kawalerski, Lucia Roapena, Luke Torrehealy, Taryn Boyle, Cindy V Leiton, Natasha T Snider, Kenneth R Shroyer, Luisa F EscobarhoyosAbstract:Clinical Need and Objectives: Our previous reports suggest that Keratin 17 (K17), an intermediate filament (IF), is an oncoprotein and biomarker of the most aggressive and treatment-resistant form of pancreatic ductal adenocarcinoma (PDAC). Importantly, K17 transitions from its “insoluble” IF form to a “soluble” form that can regulate cell cycle progression and gene expression, though the mechanisms that mediate K17 solubilization are yet unknown. Determining these mechanisms may uncover new Keratin 17-specific PDAC therapeutic vulnerabilities. Methods and Results: We found that in human and murine PDAC models, the soluble fraction of K17 exists in the full-length form (flK17) and as a caspase-cleaved peptide (ccK17), each with potential roles as biomarkers and/or functional drivers of PDAC aggression. In a PDAC tumor mouse model and human samples, we show, independent of total K17 protein amount, that the percent of soluble and ccK17 in tumor cells is negatively prognostic. We sequenced both the soluble and cytoskeletal insoluble forms of K17 from primary PDACs using liquid chromatography mass spectrometry (LC-MS) and identified that K17 is phosphorylated on several N-terminal serine residues, some of which identify a semiconserved IF motif that may regulate caspase cleavage and solubilization. Serine phosphatase inhibition in vitro demonstrated that phosphorylation is necessary to trigger K17 solubilization followed by nuclear translocation. Mutagenesis of N-terminal serine sites demonstrated that phosphorylation of three serine residues is sufficient to solubilize K17 from its filamentous state, and that phosphorylation of K17 by a PKC-RSK signaling pathway is likely to induce solubilization. In vitro and in vivo experiments are ongoing to address the functional role of this mechanism in regulating PDAC pathogenesis. Conclusion: Phosphorylation is an important regulator of K17 dynamics in PDAC, promoting solubilization and, in some cases, caspase cleavage. Our results suggest that soluble K17 may have a functional role in promoting tumor aggression. Inhibiting RSK or PKC kinases that phosphorylate K17 could impact tumor growth by blocking K17 solubilization/cleavage, and this line of research could accelerate the development of more effective treatments, coupled to soluble K17 testing as a predictive biomarker for PDAC. Citation Format: Ryan R. Kawalerski, Lucia Roa-Pena, Luke A. Torre-Healy, Taryn Boyle, Cindy V. Leiton, Natasha T. Snider, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Oncogenic mechanism of soluble Keratin 17 offers potential therapeutic vulnerability in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C26.
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Abstract B50: Keratin 17 drives tumor aggression and could be targeted for treatment of pancreatic ductal adenocarcinoma
Tumor Microenvironment, 2019Co-Authors: Kenneth R Shroyer, Ryan R Kawalerski, Cindy V Leiton, Luisa F. Escobar-hoyos, Lucia Roa-peña, Chun-hao Pan, Sruthi BabuAbstract:Our aims are to uncover the molecular mechanisms through which Keratin 17 (K17), a prognostic biomarker, drives tumor aggression and to target these mechanisms to provide more effective treatment for pancreatic ductal adenocarcinoma (PDAC). In murine orthotopic xenografts, we found that K17-positve PDACs survive for a shorter interval than controls. Prompted by previous reports that post-translational modifications (PTMs) regulate intermediate filament dynamics, we established in vitro that phosphorylated K17 detaches from the cytoskeleton and enters the nucleus, where it promotes tumor growth by targeting tumor suppressor proteins, including p27, for nuclear export and degradation. To further understand the events that control K17 solubilization, we sequenced K17 from primary PDACs by liquid chromatography-mass spectrometry and identified serine sites within the N-terminus that are phosphorylated only in soluble K17. Furthermore, phosphorylation is required to maintain K17 solubility and soluble K17 accumulates in the nucleus of PDAC cells. By an unbiased screen of 80 small-molecule kinase inhibitors in PDAC, we determined that SYK kinase inhibitors, already in clinical trials for other malignancies, abrogated K17 solubilization. Prompted by our finding that K17 serves as a nuclear shuttle of p27, we identified two amino acid sequences in K17 that have similar polarity to sequences that are used by cyclins to dock to p27. Point mutations in two of these domain key residues blocked K17-mediated degradation of nuclear p27, and we identified similar effects in the background of wild-type and oncogenic KrasG12D PDAC cells. Current studies are under way to find additional protein and RNA targets for potential therapeutic intervention. Using patient-derived organoids, human and murine PDAC cells, we determined that K17-expressing PDAC cells are more than twice as resistant as isogenic K17-negative cells to gemcitabine (Gem) and 5-fluorouracil (5-FU), two key components of current chemotherapeutic regimens. By unbiased liquid chromatography-coupled tandem mass spectrometry metabolomics, RNA-sequencing analyses (TCGA), and in vivo magnetic resonance spectroscopy, we found that K17 induces metabolic reprogramming by increasing glycolysis and pyrimidine biosynthesis, pathways that have been linked to chemoresistance. We are extending this work to determine if disruption of K17-mediated metabolic rewiring by small-molecule inhibitors will resensitize tumor cells to pyrimidine analogues. In conclusion, K17 undergoes key post-translational modifications that enable solubilization and nuclear translocation, the targeting of tumor suppressor proteins, and enhanced pyrimidine biosynthesis to drive chemoresistance. Uncovering these mechanisms could ultimately lead to the identification of novel approaches to target the oncogenic functions of K17, and thereby, to enable the development of more effective treatment options for PDAC. Citation Format: Kenneth R. Shroyer, Luisa Escobar-Hoyos, Cindy Leiton, Chun-Hao Pan, Ryan Kawalerski, Lucia Roa-Pena, Sruthi Babu. Keratin 17 drives tumor aggression and could be targeted for treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B50.
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Keratin 17 is a negative prognostic biomarker in high-grade endometrial carcinomas.
Human pathology, 2019Co-Authors: Ji Dong K. Bai, Luisa F. Escobar-hoyos, Lucia Roa-peña, Ali Akalin, Sruthi Babu, Wei Hou, Kenneth R ShroyerAbstract:Summary Keratin 17 (K17) has been established as a negative prognostic biomarker in cervical and ovarian cancers but has not previously been evaluated as a prognostic biomarker in endometrial adenocarcinoma. The association of K17 with decreased patient survival may be explained in part by the discovery that K17 drives tumor aggression by serving as a nuclear shuttle of p27, leading to cell cycle progression and tumor growth. The current study tests the hypothesis that K17 mRNA and protein levels correlate with decreased survival of patients with high-grade endometrial cancer. Gene expression data (mRNA) from The Cancer Genome Atlas were analyzed for 271 high-grade endometrial carcinomas and K17 immunohistochemistry (IHC) was performed on a separate cohort of 119 high-grade endometrial cancer cases from two academic medical centers. Survival analyses were determined by Cox proportional hazards regression. High K17 mRNA and IHC correlated with decreased overall survival (HR: 1.8, P = .0101, HR: 1.8, P = .0488, respectively). K17 was positive in malignant glandular cells of the endometrium but not in other tissues, including endometrial stroma, myometrium and uterine sarcoma. These results support the conclusion that K17 is a negative prognostic biomarker in high-grade endometrial carcinoma and that K17 IHC test results could be used to inform decisions related to therapeutic intervention.
Erle Dang - One of the best experts on this subject based on the ideXlab platform.
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Small interfering RNA targeting of Keratin 17 reduces inflammation in imiquimod-induced psoriasis-like dermatitis.
Chinese medical journal, 2020Co-Authors: Chunying Xiao, Chen Zhang, Gang Wang, Zhenlai Zhu, Hongjiang Qiao, Erle DangAbstract:BACKGROUND Psoriasis is a common chronic inflammatory skin disease with 2% to 3% prevalence worldwide and a heavy social-psychological burden for patients and their families. As the exact pathogenesis of psoriasis is still unknown, the current treatment is far from satisfactory. Thus, there is an urgent need to find a more effective therapy for this disease. Keratin 17 (K17), a type I intermediate filament, is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis. Therefore, we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis. This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA (siRNA) on mice with imiquimod (IMQ)-induced psoriasis-like dermatitis. METHODS Eight-week-old female BALB/c mice were administered a 5% IMQ cream on both ears to produce psoriatic dermatitis. On day 3, K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days. The right ears of the mice were treated in parallel with negative control (NC) siRNA. Inflammation was evaluated by gross ear thickness, histopathology, the infiltration of inflammatory cells (CD3+ T cells and neutrophils) using immunofluorescence, and the expression of cytokine production using real-time quantitative polymerase chain reaction. The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance. RESULTS The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears, as evidenced by the alleviated ear inflammation phenotype, including decreased ear thickness, infiltration of inflammatory cells (CD3+ T cells and neutrophils), and inflammatory cytokine/chemokine expression levels (interleukin 17 [IL-17], IL-22, IL-23, C-X-C motif chemokine ligand 1, and C-C motif chemokine ligand 20) (P
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small interfering rna targeting of Keratin 17 reduces inflammation in imiquimod induced psoriasis like dermatitis
Chinese Medical Journal, 2020Co-Authors: Chunying Xiao, Chen Zhang, Gang Wang, Zhenlai Zhu, Hongjiang Qiao, Erle DangAbstract:BACKGROUND Psoriasis is a common chronic inflammatory skin disease with 2% to 3% prevalence worldwide and a heavy social-psychological burden for patients and their families. As the exact pathogenesis of psoriasis is still unknown, the current treatment is far from satisfactory. Thus, there is an urgent need to find a more effective therapy for this disease. Keratin 17 (K17), a type I intermediate filament, is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis. Therefore, we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis. This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA (siRNA) on mice with imiquimod (IMQ)-induced psoriasis-like dermatitis. METHODS Eight-week-old female BALB/c mice were administered a 5% IMQ cream on both ears to produce psoriatic dermatitis. On day 3, K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days. The right ears of the mice were treated in parallel with negative control (NC) siRNA. Inflammation was evaluated by gross ear thickness, histopathology, the infiltration of inflammatory cells (CD3+ T cells and neutrophils) using immunofluorescence, and the expression of cytokine production using real-time quantitative polymerase chain reaction. The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance. RESULTS The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears, as evidenced by the alleviated ear inflammation phenotype, including decreased ear thickness, infiltration of inflammatory cells (CD3+ T cells and neutrophils), and inflammatory cytokine/chemokine expression levels (interleukin 17 [IL-17], IL-22, IL-23, C-X-C motif chemokine ligand 1, and C-C motif chemokine ligand 20) (P < 0.05 vs. the Blank or NC siRNA groups). Compared to the NC siRNA treatment, the K17 siRNA treatment resulted in increased K1 and K10 expression, which are characteristic of Keratinocyte differentiation (vs. NC siRNA, K17 siRNA1 group: K1, t = 4.782, P = 0.0050; K10, t = 3.365, P = 0.0120; K17 siRNA2 group: K1, t = 4.104, P = 0.0093; K10, t = 4.168, P = 0.0042; siRNA Mix group: K1, t = 3.065, P = 0.0221; K10, t = 10.83, P < 0.0001), and decreased K16 expression, which is characteristic of Keratinocyte proliferation (vs. NC siRNA, K17 siRNA1 group: t = 4.156, P = 0.0043; K17 siRNA2 group: t = 2.834, P = 0.0253; siRNA Mix group: t = 2.734, P = 0.0250). CONCLUSIONS Inhibition of K17 expression by its specific siRNA significantly alleviated inflammation in mice with IMQ-induced psoriasis-like dermatitis. Thus, gene therapy targeting K17 may be a potential treatment approach for psoriasis.
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NB-UVB irradiation downregulates Keratin-17 expression in Keratinocytes by inhibiting the ERK1/2 and STAT3 signaling pathways.
Archives of dermatological research, 2018Co-Authors: Yuchen Zhuang, Changxu Han, Erle Dang, Liang Jin, Hui Fang, Hongjiang Qiao, Gang WangAbstract:Keratin-17 (K17) is a cytoskeletal protein produced by Keratinocytes (KCs), which is overexpressed in psoriasis and may play a pivotal role in its pathogenesis. Narrow-band ultraviolet B (NB-UVB) irradiation is used as a general treatment for psoriasis, although its impact on K17 expression has yet to be determined. In this study, we aimed to investigate the effect of NB-UVB irradiation on K17 expression and its signaling pathways. After exposure to NB-UVB irradiation, immortalized human Keratinocytes (HaCaT cells) were analyzed by flow cytometry, CCK-8 assays and transmission electron microscopy to examine proliferation. Meanwhile, K17 expression in primary human epithelial Keratinocytes was detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis and immunofluorescence. HaCaT cells pre-incubated with PD-98059 and piceatannol were subjected to western blot analysis to examine ERK1/2 and STAT3 phosphorylation. The ears of mice treated with imiquimod (IMQ) and irradiated by NB-UVB were taken to examine K17 expression by qRT-PCR, western blot analysis, and immunofluorescence. Our results showed that 400 mJ/cm2 of NB-UVB irradiation was the maximum tolerable dose for HaCaT cells and could cause inhibited HaCaT cell proliferation and moderate increase of the early apoptosis. Furthermore, NB-UVB irradiation could downregulate K17 expression by inhibiting the ERK1/2 and STAT3 signaling pathways. In experiments conducted in vivo, NB-UVB irradiation with doses of MED or higher could eliminate the IMQ-induced psoriasis-like dermatitis and inhibit K17 expression. These results indicated that NB-UVB irradiation may eliminate chronic psoriatic plaques by suppressing K17 expression via the ERK1/2 and STAT3 signaling pathways.
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nb uvb irradiation downregulates Keratin 17 expression in Keratinocytes by inhibiting the erk1 2 and stat3 signaling pathways
Archives of Dermatological Research, 2018Co-Authors: Yuchen Zhuang, Changxu Han, Erle Dang, Liang Jin, Hui Fang, Hongjiang Qiao, Gang WangAbstract:Keratin-17 (K17) is a cytoskeletal protein produced by Keratinocytes (KCs), which is overexpressed in psoriasis and may play a pivotal role in its pathogenesis. Narrow-band ultraviolet B (NB-UVB) irradiation is used as a general treatment for psoriasis, although its impact on K17 expression has yet to be determined. In this study, we aimed to investigate the effect of NB-UVB irradiation on K17 expression and its signaling pathways. After exposure to NB-UVB irradiation, immortalized human Keratinocytes (HaCaT cells) were analyzed by flow cytometry, CCK-8 assays and transmission electron microscopy to examine proliferation. Meanwhile, K17 expression in primary human epithelial Keratinocytes was detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis and immunofluorescence. HaCaT cells pre-incubated with PD-98059 and piceatannol were subjected to western blot analysis to examine ERK1/2 and STAT3 phosphorylation. The ears of mice treated with imiquimod (IMQ) and irradiated by NB-UVB were taken to examine K17 expression by qRT-PCR, western blot analysis, and immunofluorescence. Our results showed that 400 mJ/cm2 of NB-UVB irradiation was the maximum tolerable dose for HaCaT cells and could cause inhibited HaCaT cell proliferation and moderate increase of the early apoptosis. Furthermore, NB-UVB irradiation could downregulate K17 expression by inhibiting the ERK1/2 and STAT3 signaling pathways. In experiments conducted in vivo, NB-UVB irradiation with doses of MED or higher could eliminate the IMQ-induced psoriasis-like dermatitis and inhibit K17 expression. These results indicated that NB-UVB irradiation may eliminate chronic psoriatic plaques by suppressing K17 expression via the ERK1/2 and STAT3 signaling pathways.
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Effect of Calcipotriol on IFN-γ-Induced Keratin 17 Expression in Immortalized Human Epidermal Keratinocyte Cells.
Medical science monitor : international medical journal of experimental and clinical research, 2017Co-Authors: Jieyu Zhang, Erle Dang, Hui Fang, Ruoyang Wang, Man Jiang, Gang WangAbstract:BACKGROUND Calcipotriol ointment has been demonstrated to be a very safe and effective topical drug for psoriasis. This study aims to investigate the effect of calcipotriol on IFN-γ-induced Keratin 17 (K17) expression in a human Keratinocyte cell line (HaCaT), which is a widely accepted as a mimic in vitro model for psoriasis. MATERIAL AND METHODS We used Western blot, immunofluorescence staining, and luciferase reporter system assays to evaluate the expression of K17 and the possible underlying mechanisms. RESULTS Administration of IFN-γ (125-1000 U) increased K17 expression in a dose-dependent manner, and 250 U/ml IFN-γ significantly elevated K17 expression. The experimental results showed that calcipotriol at concentrations of 10^-7 M and 10^-5 M suppressed the IFN-γ-induced K17 expression by 58.10% and 70.68%, respectively. Through immunofluorescence staining and luciferase reporter assay, we found that Vitamin D Response Element (VDRE) affected IFN-activated site (Gamma-activated sequence, GAS) function at the transcriptional level and was involved in the inhibition of K17 expression. CONCLUSIONS Our data suggest that calcipotriol downregulates IFN-γ-mediated K17 expression in Keratinocytes in a dose-dependent manner via VDRE effect GAS function. The inhibitory effect of calcipotriol on K17 expression may be a potential mechanism and function in the treatment psoriasis.
Luisa F Escobarhoyos - One of the best experts on this subject based on the ideXlab platform.
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abstract c26 oncogenic mechanism of soluble Keratin 17 offers potential therapeutic vulnerability in pancreatic cancer
Cancer Research, 2019Co-Authors: Ryan R Kawalerski, Lucia Roapena, Luke Torrehealy, Taryn Boyle, Cindy V Leiton, Natasha T Snider, Kenneth R Shroyer, Luisa F EscobarhoyosAbstract:Clinical Need and Objectives: Our previous reports suggest that Keratin 17 (K17), an intermediate filament (IF), is an oncoprotein and biomarker of the most aggressive and treatment-resistant form of pancreatic ductal adenocarcinoma (PDAC). Importantly, K17 transitions from its “insoluble” IF form to a “soluble” form that can regulate cell cycle progression and gene expression, though the mechanisms that mediate K17 solubilization are yet unknown. Determining these mechanisms may uncover new Keratin 17-specific PDAC therapeutic vulnerabilities. Methods and Results: We found that in human and murine PDAC models, the soluble fraction of K17 exists in the full-length form (flK17) and as a caspase-cleaved peptide (ccK17), each with potential roles as biomarkers and/or functional drivers of PDAC aggression. In a PDAC tumor mouse model and human samples, we show, independent of total K17 protein amount, that the percent of soluble and ccK17 in tumor cells is negatively prognostic. We sequenced both the soluble and cytoskeletal insoluble forms of K17 from primary PDACs using liquid chromatography mass spectrometry (LC-MS) and identified that K17 is phosphorylated on several N-terminal serine residues, some of which identify a semiconserved IF motif that may regulate caspase cleavage and solubilization. Serine phosphatase inhibition in vitro demonstrated that phosphorylation is necessary to trigger K17 solubilization followed by nuclear translocation. Mutagenesis of N-terminal serine sites demonstrated that phosphorylation of three serine residues is sufficient to solubilize K17 from its filamentous state, and that phosphorylation of K17 by a PKC-RSK signaling pathway is likely to induce solubilization. In vitro and in vivo experiments are ongoing to address the functional role of this mechanism in regulating PDAC pathogenesis. Conclusion: Phosphorylation is an important regulator of K17 dynamics in PDAC, promoting solubilization and, in some cases, caspase cleavage. Our results suggest that soluble K17 may have a functional role in promoting tumor aggression. Inhibiting RSK or PKC kinases that phosphorylate K17 could impact tumor growth by blocking K17 solubilization/cleavage, and this line of research could accelerate the development of more effective treatments, coupled to soluble K17 testing as a predictive biomarker for PDAC. Citation Format: Ryan R. Kawalerski, Lucia Roa-Pena, Luke A. Torre-Healy, Taryn Boyle, Cindy V. Leiton, Natasha T. Snider, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Oncogenic mechanism of soluble Keratin 17 offers potential therapeutic vulnerability in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C26.
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Keratin 17 is overexpressed and predicts poor survival in estrogen receptor negative human epidermal growth factor receptor 2 negative breast cancer
Human Pathology, 2017Co-Authors: Ross D. Merkin, Luisa F Escobarhoyos, Elizabeth A. Vanner, Jamie Romeiser, Stephanie Burke, Robert S. Powers, Laurie A Shroyer, Kenneth R ShroyerAbstract:Clinicopathological features of breast cancer have limited accuracy to predict survival. By immunohistochemistry (IHC), Keratin 17 (K17) expression has been correlated with triple-negative status (estrogen receptor [ER]/progesterone receptor/human epidermal growth factor receptor-2 [HER2] negative) and decreased survival, but K17 messenger RNA (mRNA) expression has not been evaluated in breast cancer. K17 is a potential prognostic cancer biomarker, targeting p27, and driving cell cycle progression. This study compared K17 protein and mRNA expression to ER/progesterone receptor/HER2 receptor status and event-free survival. K17 IHC was performed on 164 invasive breast cancers and K17 mRNA was evaluated in 1097 breast cancers. The mRNA status of other Keratins (16/14/9) was evaluated in 113 ER-/HER2- ductal carcinomas. IHC demonstrated intense cytoplasmic and membranous K17 localization in myoepithelial cells of benign ducts and lobules and tumor cells of ductal carcinoma in situ. In ductal carcinomas, K17 protein was detected in most triple-negative tumors (28/34, 82%), some non-triple-negative tumors (52/112, 46%), but never in lobular carcinomas (0/15). In ductal carcinomas, high K17 mRNA was associated with reduced 5-year event-free survival in advanced tumor stage (n = 149, hazard ratio [HR] = 3.68, P = .018), and large (n = 73, HR = 3.95, P = .047), triple-negative (n = 103, HR = 2.73, P = .073), and ER-/HER2- (n = 113, HR = 2.99, P = .049) tumors. There were significant correlations among Keratins 17, 16, 14, and 9 mRNA levels suggesting these Keratins (all encoded on chromosome 17) could be coordinately expressed in breast cancer. Thus, K17 is expressed in a subset of triple-negative breast cancers, and is a marker of poor prognosis in patients with advanced stage and ER-/HER2- breast cancer.
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abstract b76 translating Keratin 17 status to stratify clinically relevant pancreatic cancer heterogeneity and survival
Cancer Research, 2016Co-Authors: Luisa F Escobarhoyos, Lucia Roapena, Elizabeth A. Vanner, Ali Akalin, Jela Bandovic, Shula Schechter, Jinelle M Wint, Scott Powers, Peter Bailey, David K ChangAbstract:Despite the poor long-term survival of patients with pancreatic ductal adenocarcinoma (PDA), there are significant survival probabilities in subgroups of patients that cannot be predicted by surgical, pathological, and clinical features, including stage, surgical margins and perineural invasion. Only in the last year, more than a handful papers reported advanced genomic analyses on multiple components of tumors to dissect molecular heterogeneities that may provide a better understanding of gene expression signatures to predict survival probabilities. Although gene-mutation and expression “omic” signatures hold promise, they are difficult to translate into clinical practice. Therefore, we initiated a two phase unbiased and systematic study to first identify and validate a prognostic biomarker by screening multiple independent RNASeq databases and performing survival analyses, on a total of 493 cases. Second, we performed immunohistochemical analyses on 117 cases to validate the in silico analysis results using a pathology based scoring system. We identified in the discovery dataset that Keratin 17 (K17) expression discriminated between rapid and slower progressing PDA among patients with localized disease. K17-positive cases had four times the likelihood of dying from this disease compared to stage-matched K17-negative cases (P= 0.0094; median survival 1.64 years compared to >5 years, respectively). In addition, we found that positive-K17 patients have almost twice the probability of recurrence after chemotherapy, compared to matched stage, negative-K17 counterparts. These critical observations were validated in four independent PDA patient cohorts, indicating the negative prognostic value of K17 in this devastating disease, independent of the patient’s age, sex, and tumor stage and grade. Furthermore, we found that pathology-based quantification of K17 expression by immunohistochemistry in malignant cells was associated with poor-patient outcome (P= 0.0026). Based on the recent molecular “omics” subtyping PDA identified by Bailey et al., 2016 and Moffitt et al., 2015, we found that K17 expression can be used to distinguish the “squamous” and “basal” subtypes presented in these reports, respectively. Equally important, in addition to PDA, we found that high expression of K17 is strongly associated with decreased patient survival in a number of carcinomas, including but not limited to cancers of the lung, breast, liver, stomach. Results consistently indicated that K17 expression is associated with shortened interval to death (Hazard Ratio >2). Collectively, these results suggest that activation of K17 expression across multiple primary tumors is a conserved molecular event in tumors with shared clinical behavior and not anatomic location. Based on mechanistic approaches, we know that K17 is a regulator of cell-cycle progression and directly acts to enhance pancreatic tumor growth, providing insight into the mechanisms that drive PDA tumor biology as a major cause of patient mortality. In conclusion, K17 was identified as a robust and independent clinically relevant prognostic biomarker that may be used to better stratify clinical outcome at the time of initial diagnosis. Citation Format: Luisa Escobar-Hoyos, Elizabeth A. Vanner, Shula Schechter, Lucia Roa-Pena, Jinyu Li, Jinelle Wint, Scott Powers, Ali Akalin, Jela Bandovic, Peter Bailey, David K. Chang, Richard A. Moffit, Jen Jen Yeh, Andrew Biankin, Kenneth R. Shroyer.{Authors}. Translating Keratin 17 status to stratify clinically relevant pancreatic cancer heterogeneity and survival. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B76.
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abstract 2036 Keratin 17 mediates p27kip1 nuclear export proliferative signaling and tumor growth
Cancer Research, 2015Co-Authors: Luisa F Escobarhoyos, Lucia Roapena, Ruchi Shah, Elizabeth A. Vanner, Nilofar Najafian, Anna Banach, Erik Nielsen, Ali Akalin, Ramsey Alkhalil, David A. TalmageAbstract:Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA One of the most fundamental traits of cancer cells is the ability to sustain proliferation by circumventing cell cycle regulatory programs normally controlled by tumor suppressors. Keratin 17 (K17) is not expressed in most somatic tissues but is overexpressed and is a negative prognostic marker in cervical, breast, ovarian, and gastric carcinomas. The mechanisms by which K17 contributes to cancer-related signaling, however, remain unknown. Here, we report that nuclear-localized K17 interacts with tumor suppressor p27KIP1 and mediates its nuclear export, maintaining proliferation. After validating K17 is a prognostic marker in cervical cancer, independent of tumor stage, we performed in vitro and in vivo experiments to investigate the role of K17 in proliferative signaling and tumor growth using loss- and gain- of function approaches in cervical cancer and other cancer-derived cell lines. We found that K17 expression maintains proliferation, while silencing K17 induces G1 arrest by nuclear accumulation and stabilization of tumor suppressors p27KIP1 and pRb. During G1, K17 localizes in the nucleus, mediated by a classical bipartite nuclear localization signal (NLS) (AA385-400) that was identified by in silico analysis. This NLS is specific among Keratins and present only in primates but not in other species. To our knowledge, this is the first report that a Keratin has a NLS and is capable of undergoing nuclear translocation. During G1, p27KIP1is exported from the nucleus in a CRM1-dependent manner and is subsequently degraded, triggering G1/S transition. p27KIP1 lacks the classical leucine-rich nuclear export signal (NES) and requires an adaptor for CRM1-exportin binding. After confirming the binding of K17 and p27KIP1 within the nucleus, we performed in silico analyses and identified a leucine-rich NES required for CRM1-binding in K17 (AA194-199), which was further validated by a 3-fold nuclear retention of K17 and p27KIP1 after leptomycin B treatment. We introduced point mutations within the K17-NES (mNES) and the K17-NLS (mNLS). Nuclear p27KIP1 was lost in cells expressing wild-type K17. In contrast, nuclear p27KIP1 levels were 3-fold higher in cells expressing either mNLS or mNES. Furthermore, nuclear localization of K17 was abolished in mNLS cells. Xenografts of cervical cancer cells showed that tumors derived from K17 expressing cells were 3-times larger than those derived from K17 knockdown cells, the latter showing increased nuclear p27KIP1 and decreased PCNA and Ki67 expression. Finally, we identified an inverse correlation between K17 and p27KIP1 expression in human cervical cancer specimens, intertumorally and intratumorally. These data suggest a model in which nuclear-localized K17 acts as an oncoprotein promoting G1/S transition in cancer cells, by mediating exportin binding and nuclear translocation of tumor suppressor p27KIP1, contributing to sustained proliferation, tumor aggressiveness and poor-patient outcome. Citation Format: Luisa F. Escobar-Hoyos, Ruchi Shah, Lucia Roa-Pena, Nilofar Najafian, Elizabeth Vanner, Anna Banach, Erik Nielsen, Ramsey Al-Khalil, Ali Akalin, David Talmage, Kenneth Shroyer. Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2015-2036
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Keratin 17 cervical cancer prognostic marker promotes p27 nuclear export and tumor growth
The FASEB Journal, 2015Co-Authors: Lucia Roapena, Luisa F Escobarhoyos, Ruchi Shah, Ali Akalin, Elizabeth Vanner, Kenneth ShroyerAbstract:Recently, we identified Keratin 17 (K17) as a negative prognostic marker for cervical cancer and based on preliminary data, we set out to determine if K17 is related to tumor stage and sustained proliferation. K17 and tumor suppressor p27 were detected by immunohistochemistry in cervical cancer specimens and multivariate survival analysis was conducted. Loss and gain of function approaches were performed by in vitro and in vivo by RNAi and transgenic expression of wild type and mutant K17 in cervical cancer cell lines. The five-year survival for low K17 cervical cancer cases was 90% versus 52% for high K17 cases; the differences were consistent across low and high stage groups. K17 expression promoted cell proliferation and tumor growth and silencing of K17 induced G1 arrest and decreased tumor mass by nuclear stabilization of p27, a key regulator of G1/S transition. During G1, K17 was found in the nucleus bound to nuclear p27. Nuclear p27 was lost in cells expressing wild-type K17 but nuclear p27 levels ...
