Keratin 17 Expression Research Articles

Validation of Keratin 17 as a Tissue Biomarker in the Diagnosis of Upper Tract Urothelial Carcinoma.

Upper tract urothelial carcinoma (UTUC) has a relatively low incidence but presents significant surveillance and treatment challenges. Therefore, novel biomarkers for the accurate detection of upper tract urothelial tumors are urgently needed. We evaluated the expression of Keratin 17 (KRT17), an oncoprotein implicated in the cell cycle progression of multiple human cancers and previously studied in bladder urothelial carcinoma, by immunohistochemistry in 139 UTUC cases, including noninvasive, invasive papillary urothelial carcinoma and urothelial carcinoma in situ. KRT17 expression pattern (basal/negative vs. nonbasal) and H-score were evaluated. The expression pattern was significantly different in normal (NL) compared to malignant urothelium. Nonbasal KRT17 expression was significantly higher in pTa (p<0.001) and invasive (pTinv) (p=0.0023) urothelial carcinoma compared to NL, and in pTinv compared to pTa (p=0.0391). Sensitivity and specificity for distinguishing benign from malignant tumors were 85% and 82, respectively, with an area under the curve of 0.83 (p <0.001). The KRT17 H-score was significantly higher in pTa and pTinv compared to NL (p < 0.001 and p=0.0035, respectively). Sensitivity and specificity for distinguishing benign from malignant carcinoma were 91% and 69%, respectively, with an AUC of 0.81 (p=0.0010). KRT17 was not associated with tumor site, grade, or stage. In summary, K17 is a sensitive and specific marker of neoplastic upper tract urothelium, and its potential use in routine diagnostics should be explored in larger studies.

Abstract B012: TP53 missense mutations and keratin 17 are negative prognostic biomarkers in pancreatic ductal adenocarcinoma

Abstract Background: Cancer patients with similar clinical and pathologic characteristics may have different responses to treatments and survival, which may be attributable to distinct molecular properties of their tumors. In pancreatic ductal adenocarcinomas (PDACs), there is a pressing need to identify prognostic biomarkers that could help us to predict disease progression and patient survival. Our group previously reported that keratin17 (K17) expression is a hallmark of PDACs of patients with the shortest patient survival. Furthermore, there is some evidence suggesting that missense mutations in TP53 are associated with worse survival in patients with PDAC. To date, however, no studies have addressed the differential prognostic value of molecular characterization of TP53 coupled with K17 expression for predicting survival in patients with PDAC. Objective and methods: To fill this gap, we assessed the effect of different TP53 mutations and K17 expression on patient survival, using a clinically annotated cohort of 128 patients containing molecular profiling on patient tumors. K17 Immunohistochemistry (IHC) staining on patient’s formalin-fixed, paraffin-embedded (FFPE) tissue blocks were performed to assess the expression of this marker in tumor cells. K17 IHC scoring was conducted based on a subjective assessment of the overall proportion of stained cells relative to the overall number of tumor cells present in a single representative section from each case. Kaplan-Meier and Cox proportional hazards regression models were analyzed to determine survival differences of patients with PDACs harboring different TP53 mutations and levels of K17 IHC expression, adjusting for patient demographics (age at diagnosis, sex, race/ethnicity) and tumor characteristics (presence of distant metastasis at diagnosis). Results: Tumors were classified as high for K17 when ≥90% of tumor cells showed strong staining. High-K17 cases (n=16;12.50%) had shorter survival compared with low-K17 cases (n=112; 87.50%) (26.80 vs. 16.77 months; P=0.035). The majority of cases harbored missense mutations in TP53 (42.19%), while about one-third harbored truncating TP53 (27.34%) mutations. We found that PDACs with missense TP53 mutations and high K17 were associated with worse patient survival compared to tumors with missense TP53 and low K17 expression (HR=5.27; 95% CI, 1.76-15.82; P=0.003). Similarly, compared with patients with wild-type TP53 and low K17, patients with both missense TP53 mutations and high K17 had significantly worse survival (HR=3.51, 95% CI, 1.45-8.51; P=0.005). Conclusions: Our findings suggest that molecular characterization of specific TP53 mutations coupled with K17 IHC staining may help to determine differences in patient prognosis and identify subgroups with worse patient survival, therefore, potentially guiding future clinical management of patients. Citation Format: Carlos Mauricio Mejia Arbelaez, Gabriella Baraks, Lyanne Oblein, Michael Horowitz, Lynn Matrisian, Kenneth Shroyer, Luisa Fernanda Escobar-Hoyos. TP53 missense mutations and keratin 17 are negative prognostic biomarkers in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B012.

Abstract C067: Correlation of keratin17 expression with tumor cell proliferation and invasion in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) shows variable degrees of differentiation, with both intratumoral, regional, and case to case heterogeneity, including glandular elements, sheet-like aggregates, cell clusters, and single diffusely infiltrative tumor cells. Prior research from our lab and others have established keratin 17 (K17) to be a defining hallmark of the basal molecular subtype of PDAC. In addition to its role as a component of the cytoskeleton, soluble forms of K17 undergo nuclear translocation to drive cell cycle progression and chemoresistance to first line chemotherapy. While K17 is heterogeneously expressed in the gland-forming elements of PDACs, it is most consistently expressed in tumor cells which are either in small groups or are single diffusely infiltrative. The aims of the current study are to define the correlation between K17 expression and tumor cell differentiation, with the focus on glandular versus single diffusely infiltrative components of PDAC, and to relate these findings to established biomarkers of cell proliferation and invasion. Methods: Archival human PDACs were provided by the Stony Brook Cancer Center Biobank and K17 immunohistochemistry was performed by indirect immunoperoxidase method, as previously described, to select cases with both glandular and diffusely infiltrative cell components and heterogeneous K17 expression. Multiplex immunofluorescence with a panel of antibodies to K17 (Cell Signaling Technology), pan-cytokeratin, Ki-67 and vimentin [KS1] (Miltenyi Biotec) was performed on 5-micron FFPE sections using the MACSima system (Miltenyi Biotec). Distribution patterns of K17 were evaluated in glandular and diffusely invasive components and were related to the Ki-67 status. Results: Cytoplasmic staining for K17 was found in the minority of tumor cells in glands and displayed intraglandular heterogeneity but was found in most diffusely infiltrative tumor cells. Also, nuclear K17 was rarely detected in the glandular components but was found in a high proportion of the diffusely infiltrative cells. Ki-67 was detected mainly in glandular cells, unrelated to K17 status, and rarely in single diffusely infiltrative cells, never co-localizing with nuclear K17. Of note, only a small fraction of tumor cells co-expressed K17 and vimentin, suggesting that majority of diffusely infiltrative tumor cells were not engaged in epithelial to mesenchymal transition. Conclusion: While cytoplasmic K17 is heterogeneously expressed in the glandular components of PDAC, strong staining for K17 in both the cytoplasm and the nucleus is a hallmark of diffusely infiltrative, Ki-67-negative tumor cells. These observations support the paradigm that tumor cells are committed to either cell division in the glandular components, or to diffuse single cell invasion, highlighted by increased cytoplasmic and nuclear K17 expression. Therefore, the results emphasize the importance of understanding the expression of PDAC biomarkers in both gland-forming elements and diffusely infiltrative tumor cells. Citation Format: Shayan Sarkar, Lyanne Oblein, Michael Horowitz, Luisa Escobar-Hoyos, Natalia Marchenko, Kenneth Shroyer. Correlation of keratin17 expression with tumor cell proliferation and invasion in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C067.

KRT17 From Keratinocytes With High Glucose Stimulation Inhibit Dermal Fibroblasts Migration Through Integrin α11.

To investigate the effects of overexpressed keratin 17 (KRT17) on the biology of human dermal fibroblasts (HDFs) and to explore the mechanism of KRT17 in diabetic wound healing. KRT17 expression was tested in diabetic keratinocytes, animal models, and patient skin tissues (Huazhong University of Science and Technology Ethics Committee, [2022] No. 3110). Subsequently, HDFs were stimulated with different concentrations of KRT17 in vitro. Changes in the proliferation and migration of HDFs were observed. Then, identification of KRT17-induced changes in dermal fibroblast of RNA sequencing-based transcriptome analysis was performed. KRT17 expression was upregulated under pathological conditions. In vitro stimulation of HDFs with different concentrations of KRT17 inhibited cell migration. RNA-seq data showed that enriched GO terms were extracellular matrix components and their regulation. KEGG analysis revealed that the highest number of enriched genes was PI3K-Akt, in which integrin alpha-11 (ITGA11) mRNA, a key molecule that regulates cell migration, was significantly downregulated. Decreased ITGA11 expression was observed after stimulation of HDFs with KRT17 in vitro. Increased expression of KRT17 in diabetic pathological surroundings inhibits fibroblast migration by downregulating the expression of ITGA11. Thus, KRT17 may be a molecular target for the treatment of diabetic wounds.

MicroRNA-485-5p targets keratin17 to regulate pancreatic cancer cell proliferation and invasion via the FAK / SRC / ERK pathway.

Background: It is crucial to probe into the biological effect and mechanism of miRNA-485-5p regulating keratin 17 (KRT17) in pancreatic cancer (PC) to understand its pathogenesis and identify potential biological targets. Methods: The bioinformatics means were used to evaluate the clinical significance of KRT17 expression in the Cancer Genome Atlas (TCGA) database. TargetScan database analysis in conjunction with dual luciferase and RNA Immunoprecipitation (RIP) experiments was used to probe the interaction relationship of miRNA-485-5p with KRT17. The expression of miRNA-485-5p and KRT17 in PC tissue and cancer cell lines was detected by Q-PCR paired with western blot assay. The biological function of miRNA-485-5p in regulating KRT17 was investigated in the PC cell line via gene silencing/overexpression technique. A western blot experiment was utilized to investigate the regulatory effect of KRT17 on cell cycle-related proteins and the FAK/Src/ERK signal pathway. Results: The level of KRT17 was increased in PC tissues and this significantly decreased the survival rate of PC patients. TargetScan in combination with dual luciferase and RIP experiments verified the miRNA-485-5p target KRT17. The expression of KRT17 was high in the PC cell line, although the expression of miRNA-485-5p was low. Silencing KRT17 or overexpression of miRNA-485-5p significantly inhibited PC cell viability, proliferation, invasion, and colony formation, while promoting apoptosis. Overexpression of KRT17 drastically reversed the function of miRNA-485-5p. The silenced KRT17 remarkably downregulated the expression of cyclinD1, Cyclin Dependent Kinase 1 (CDK1), CDK2, Phospho-Focal Adhesion Kinase (p-FAK), p-Src, and p-ERK proteins in the PC cells. Conclusion: Generally, an essential signaling cascade of miRNA-485-5p/KRT17/FAK/Src/ERK influences the biological functions of PC cells.

KRT17 serves as an oncogene biomarker of poor survival in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death from worldwide. Thus, it is imperative to clarify valuable diagnostic and prognostic biomarkers for early-stage HCC. The expression of Keratin 17 (KRT17) has been reported to be associated with certain cancer types previously. However, its role in the development of HCC has not been fully clarified. Here, we assessed the expression of KRT17 in the cancer genome atlas liver hepatocellular carcinoma (TCGA LIHC) database and in 44 pairs of samples collected from patients with HCC. In addition, Kaplan-Meir curves were used to assess the prognostic relevance of KRT17. The essential cancer- and KRT17-related biological processes were defined through gene set enrichment analysis (GSEA). Finally, the functional association between KRT17 expression and tumor cell proliferation/survival was evaluated and the signaling pathways associated with KRT17 expression in HCC were identified. Overall, we found that increased KRT17 levels were associated with lower survival, more aggressive disease, and altered immune cell infiltration in HCC patients. KRT17 may potentially act as a prognostic biomarker in HCC.

KRT17 Accelerates Cell Proliferative and Invasive Potential of Laryngeal Squamous Cell Carcinoma (LSCC) through Regulating AKT/mTOR and Wnt/β-Catenin Pathways.

Background Laryngeal squamous cell carcinoma (LSCC) is a prevalent malignant tumor of the head and neck with a dismal prognosis. Keratin17 (KRT17) has been proven to serve as an oncogene in various cancers, but it has never been explored in LSCC. We proposed to assess the impact and possible mechanisms of KRT17 in the development of LSCC. Methods Quantitative reverse transcription-PCR (qRT-PCR) was utilized to examine the mRNA levels. The Kaplan–Meier method was used to calculate the relationship between KRT17 expression and survival curves in LSCC patients. Cell counting kit-8 (CCK-8), colony formation, and flow cytometry assays were utilized to estimate LSCC cell proliferation. The migration and invasion abilities of LSCC cells were ascertained by wound-healing and transwell assays. Immunohistochemical and western blot assays were utilized to appraise protein levels. The xenograft tumor model was used to determine the effect of KRT17 on tumor growth. Results In the present study, KRT17 was extremely high in LSCC tissues and cells and correlated with a poor prognosis. Inhibition of KRT17 weakens cell proliferative, migratory, and invasive abilities in LSCC and contributes to cell cycle arrest. Besides, we approved that knockdown of KRT17 extraordinarily restrained the xenograft tumor growth in vivo. We preliminarily investigated the role of KRT17 on the AKT/mTOR and Wnt/β-catenin signaling axes and found that these signaling pathways were largely blocked by KRT17 deletion. Conclusion Collectively, we uncovered that exhaustion of KRT17 suppresses LSCC progression through coordinating AKT/mTOR and Wnt/β-catenin signaling axes, illustrating KRT17 as a promising biomarker for making strides in LSCC treatment.

MicroRNA-485-5p targets keratin 17 to regulate oral cancer stemness and chemoresistance via the integrin/FAK/Src/ERK/β-catenin pathway

BackgroundThe development of drug resistance in oral squamous cell carcinoma (OSCC) that frequently leads to recurrence and metastasis after initial treatment remains an unresolved challenge. Presence of cancer stem cells (CSCs) has been increasingly reported to be a critical contributing factor in drug resistance, tumor recurrence and metastasis. Thus, unveiling of mechanisms regulating CSCs and potential targets for developing their inhibitors will be instrumental for improving OSCC therapy.MethodssiRNA, shRNA and miRNA that specifically target keratin 17 (KRT17) were used for modulation of gene expression and functional analyses. Sphere-formation and invasion/migration assays were utilized to assess cancer cell stemness and epithelial mesenchymal transition (EMT) properties, respectively. Duolink proximity ligation assay (PLA) was used to examine molecular proximity between KRT17 and plectin, which is a large protein that binds cytoskeleton components. Cell proliferation assay was employed to evaluate growth rates and viability of oral cancer cells treated with cisplatin, carboplatin or dasatinib. Xenograft mouse tumor model was used to evaluate the effect of KRT17- knockdown in OSCC cells on tumor growth and drug sensitization.ResultsSignificantly elevated expression of KRT17 in highly invasive OSCC cell lines and advanced tumor specimens were observed and high KRT17 expression was correlated with poor overall survival. KRT17 gene silencing in OSCC cells attenuated their stemness properties including markedly reduced sphere forming ability and expression of stemness and EMT markers. We identified a novel signaling cascade orchestrated by KRT17 where its association with plectin resulted in activation of integrin β4/α6, increased phosphorylation of FAK, Src and ERK, as well as stabilization and nuclear translocation of β-catenin. The activation of this signaling cascade was correlated with enhanced OSCC cancer stemness and elevated expression of CD44 and epidermal growth factor receptor (EGFR). We identified and demonstrated KRT17 to be a direct target of miRNA-485-5p. Ectopic expression of miRNA-485-5p inhibited OSCC sphere formation and caused sensitization of cancer cells towards cisplatin and carboplatin, which could be significantly rescued by KRT17 overexpression. Dasatinib treatment that inhibited KRT17-mediated Src activation also resulted in OSCC drug sensitization. In OSCC xenograft mouse model, KRT17 knockdown significantly inhibited tumor growth, and combinatorial treatment with cisplatin elicited a greater tumor inhibitory effect. Consistently, markedly reduced levels of integrin β4, active β-catenin, CD44 and EGFR were observed in the tumors induced by KRT17 knockdown OSCC cells.ConclusionsA novel miRNA-485-5p/KRT17/integrin/FAK/Src/ERK/β-catenin signaling pathway is unveiled to modulate OSCC cancer stemness and drug resistance to the common first-line chemotherapeutics. This provides a potential new therapeutic strategy to inhibit OSCC stem cells and counter chemoresistance.

Promoter Methylation Changes in KRT17: A Novel Epigenetic Marker for Wool Production in Angora Rabbit.

Wool production is an important economic trait of Angora rabbits. Exploring molecular markers related to wool production is one of the essentials of Angora rabbits’ breeding. KRT17 (Keratin 17) is an important gene of hair follicle development, which must be explored for genetic/epigenetic variation to assess its effect on wool production. Based on the effective wool production data of 217 Angora rabbits, the high and low yield groups were screened with 1.5 standard deviations of the population mean. The full-length sequence of KRT17 was obtained by rapid amplification of cDNA ends technology, and the polymorphism was analyzed in the promoter, exon, and intron regions by direct sequencing. KRT17, SP1 over-expression plasmids, and siRNA were constructed and transfected into dermal papilla cells. The mRNA expressions of relevant genes were analyzed by RT-qPCR. The methylation level of the KRT17 promoter was determined by Bisulfite Sequencing PCR. Dual-luciferase system, site-directed mutagenesis, and electrophoretic mobility shift assays were used to analyze the binding relationship between SP1 and the promoter of KRT17. The structure map of KRT17 was drawn, and no SNPs were found in the promoter, exon, and intron, indicating a relatively conserved structure of KRT17. Expression of KRT17 was significantly higher in cutaneous tissues than in other tissues and was significantly upregulated in the high-yield group compared to the low-yield group (p < 0.05). Furthermore, the overall high methylation levels of KRT17 CpG I and CpG III showed significant association with low wool yield; the methylation levels of 5 CpG locus (CpG I site 4 and CpG III site 2–5) were significantly different between the high and low yield groups (p < 0.05). The methylation levels of 3 CpG locus (CpG I site 4 and CpG III site 4, 14) showed a significant correlation with KRT17 expression (p < 0.05). Overall, CpG III site 4 significantly affects wool production and KRT17 expressions (p < 0.05). This site promotes SP1 binding to the KRT17 promoter region (CGCTACGCCC) to positively regulate the KRT17 expression. KRT17 CpG III site 4 can be used as candidate epigenetic markers for the breeding of high wool-producing Angora rabbits.

Co‐expression of Zeb‐1 and K17 provides new insights into the role of EMT in pancreatic cancer progression

INTRODUCTIONDespite improvements in screening and therapeutic options, pancreatic ductal adenocarcinoma (PDAC) has the third‐worst mortality rate, highlighting the need for better diagnostic and therapeutic approaches. Epithelial‐mesenchymal transition (EMT) has been conferred with a greater likelihood of metastasis in multiple cancers, specifically identified through markers like Zeb‐1 and Twist‐1. Zeb‐1 (Zinc Finger E‐Box Binding Homeobox 1) was shown to repress E‐cadherin expression, consequently inducing EMT.Our lab has previously identified K17 (Keratin 17) as an independent prognostic biomarker of poor outcomes in PDAC patients. While increasing evidence has suggested that EMT drives pancreatic cancer migration and metastasis, there have not been any well‐established connections between K17 expression and EMT. This study aims to provide new insights into the role of K17 in EMT by evaluating the co‐expression of K17 and Zeb‐1 in human PDAC biopsies. We hypothesized that tumor cells co‐express K17 and Zeb‐1 as a hallmark of EMT in PDAC. In addition, our study aims to establish a novel method of scoring Zeb‐1 expression in stromal fibroblasts via immunofluorescence to remove variability in the stromal constituency.METHODSPDAC tissues obtained from patients who underwent surgical resection between 2013 and 2017 at Stony Brook University (n=20) were selected for serial sectioning. Double immunofluorescence and single immunohistochemical staining were achieved by incubation with Zeb‐1 and K17 antibodies followed by incubation of fluorescent or biotin conjugated secondary antibodies, respectively. Histologic examination was performed using immunofluorescence staining along with H&amp;E and immunohistochemical staining (IHC) to determine concordance.RESULTS● IHC for Zeb‐1 was detected predominantly in the nuclei of centroacinar cells, benign proliferative ducts, and stromal fibroblasts. Focal nuclear staining in tumor epithelial was also detected in some cases.● High levels of K17 and but decreased expression of Zeb‐1 were detected in diffusely infiltrative cells with morphologic features of EMT.● Both high Zeb‐1 and high K17 protein expression were associated with poor survival of PDAC patients.● Using confocal immunofluorescence microscopy, we detected co‐localization of Zeb‐1 and K17 in PDAC.● We quantified Zeb‐1 protein expression in PDAC samples from 20 patients and confirmed significantly higher expression in the tumor‐associated stroma of high‐grade tumors compared to stromal fibroblasts in areas devoid of tumor.CONCLUSIONOur results provide evidence that K17 is upregulated in tumor epithelial cells that show morphologic immunohistochemical properties of EMT. The loss of Zeb‐1 in diffusely infiltrative, K17‐positive tumor cells suggests that EMT upregulates K17, driving tumor aggression. Our results suggest that K17 may enhance PDAC aggression by mediating EMT. Hence, novel therapeutic agents targeting K17 may improve clinical care for this biologically distinct patient subgroup.

A pan-cancer analysis of the oncogenic role of Keratin 17 (KRT17) in human tumors.

BackgroundAlthough new evidence from cells or animals suggests a relationship between Keratin 17 (KRT17) and cancer, no pan-cancer analysis is currently available.MethodsThe expression level of KRT17 in generalized carcinoma was detected by the Tumor Immune Estimation Resource, version 2 (TIMER2) database, and then verified the protein expression of KRT17 in different cancer species in UALCAN database, and analyzed the relationship between the expression level of KRT17 and the clinical stage and survival of different cancers. We further explored the genetic variation of KRT17 in different tumor types included in The Cancer Genome Atlas (TCGA) and the specific mutations in each domain. The changes of KRT17 protein phosphorylation levels and protein expression levels at different phosphorylation sites in different tumors were explored. TIMER2 database was used to explore the potential relationship between the infiltration level of different immune cells and KRT17 gene expression in different TCGA cancer types. Finally, the protein binding to KRT17 and genes related to KRT17 expression were explored by STRING database and TCGA database.ResultsKRT17 is overexpressed in most malignancies, and we observed a distinct relationship between KRT17 expression and tumor patient prognosis. Enhanced phosphorylation levels of S13, S24, S32, and S39 were observed in several tumors, such as lung adenocarcinoma (LUAD), colon and ovarian cancers, and uterine corpus endometrial carcinoma (UCEC). Intermediate filament cytoskeleton and keratinization may be simultaneously acting with KRT17 on tumor pathogenesis.ConclusionsOur pan-cancer analysis provides relatively complete information on the oncogenic functions of KRT17 in various cancers.

KRT17 Promotes the Activation of HSCs via EMT in Liver Fibrosis

Background and AimsAlthough activation of hepatic stellate cells (HSCs) plays a central role in the development of liver fibrosis, the mechanism underlying the activation of HSCs remains unclear. Keratin 17 (KRT17), a member of the intermediate filament family, can regulate tumor cell proliferation and migration. The current study aimed to elucidate the role of KRT17 in the activation of HSCs and the mechanisms underlying liver fibrosis.MethodsThe expression of KRT17 was determined using immunohistochemistry in tissue microarray. Western blotting and qRT-PCR assays were used to determine the KRT17 expression in fibrotic liver tissues obtained from human subjects and mice. LX-2 cells were treated with TGF-β1 recombinant protein and adipocyte differentiation mixture (MDI) mix to induce and reverse LX-2 cell activation, respectively, in order to explore the correlation between KRT17 and HSC activation. Additionally, cell proliferation and migration abilities of LX-2 cells transfected with KRT17-overexpressing plasmid or small interfering RNA were determined using CCK-8, flow cytometry, Transwell, and wound healing assays. Finally, rescue assay was used to explore the role of KRT17 in HSC activation and epithelial-mesenchymal transition (EMT).ResultsThe expression of KRT17 was higher in the human and mouse fibrotic liver tissues than in healthy liver tissues, and it was positively correlated with HSC activation. Upregulated KRT17 enhanced proliferation, migration, HSC activation and EMT in LX-2 cells, while knockdown of KRT17 reversed these effects. TGF-β1 recombinant protein accelerated KRT17-mediated EMT, HSC activation and proliferation, while TGF-β1 inhibitor counteracted the effect of KRT17 in vitro.ConclusionsKRT17 promoted HSC activation, proliferation and EMT in hepatic fibrosis probably via TGF-β1 signaling, and KRT17 might serve as a therapeutic target for the treatment of liver fibrosis.

The Gain-of-Function Mutation p53R248W Suppresses Cell Proliferation and Invasion of Oral Squamous Cell Carcinoma through the Down-Regulation of Keratin 17

Keratin 17 (KRT17) expression promotes the proliferation and invasion of oral squamous cell carcinoma (OSCC), and mutations in TP53 have been reported in 65% to 85% of OSCC cases. We studied the correlation between KRT17 expression and TP53 mutants. Ca9-22 cells, which exhibit low KRT17 expression, carried mutant p53 (p53R248W) and p53R248W knockdown promoted KRT17 expression. p53R248W knockdown in Ca9-22 cells promoted migration and invasion activity. In contrast, in HSC3 cells, which have p53 nonsense mutations and exhibit high KRT17 expression, the overexpression of p53R248W decreased KRT17 expression, cell size, proliferation, and migration and invasion activities. In addition, p53R248W significantly suppressed MMP2 mRNA expression and enzyme activity. Moreover, s.c. and orthotopic xenografts were generated from p53R248W- or p53R248Q-expressing HSC3 cells. Tumors formed from p53R248W-expressing HSC3 cells grew more slowly and had a lower Ki-67 index than those derived from the control or p53R248Q-expressing HSC3 cells. Finally, the survival rate of the mice inoculated with p53R248W-expressing HSC3 cells was significantly higher than that of the control mice. These results indicate that the p53R248W mutant suppresses proliferation and invasion activity through the suppression of KRT17 expression. We propose that OSCC with p53R248W-expressing cells may be classified as a new OSCC type that has a good prognosis.

Molecular Mechanism of KLF4 Up-regulating Keratin 17 Expression in Keratinocytes

To explore the role of Kruppel-like factor 4 (KLF4) in the regulation of Keratin 17 (KRT17) expression, and to reveal the molecular mechanism of overexpression of KRT17 in psoriatic lesions. The skin lesions of 18 patients with psoriasis vulgaris were taken as experimental group and 10 healthy persons as control group. Real time-PCR and Western blot were used to detect the expression of KLF4 in psoriasis and normal skin samples, and the changes of KRT17 expression in HaCat cells after transfection of KLF4 overexpression and EP300 interfering plasmid. ChIP-qPCR was used to detect KLF4 binding and histone H3 acetylation levels in the promoter region of KRT17 in psoriasis and normal skin samples, and the changes of KLF4 binding and histone H3 acetylation levels in the promoter region of KRT17 in HaCat cells after transfection of KLF4 overexpression and EP300 interfering plasmid. Co-IP detects the interaction between KLF4 and EP300. The expression level of KLF4, KLF4 binding level and histone H3 acetylation level in the promoter region of KRT17 in psoriasis group were significantly higher than those in normal group ( P<0.01). Compared with the control group, the expression level of KRT17 was significantly higher after KLF4 overexpression ( P<0.01). After KLF4 overexpression combined with EP300 interference, the expression level of KRT17 was significantly lower than that of KLF4 overexpression group ( P<0.01), slightly lower than that of control group ( P<0.05). Compared with the control group, the histone H3 acetylation level in KRT17 promoter region in KLF4 over-expression group was increased significantly ( P<0.01). After KLF4 over-expression combined with EP300 interference, the acetylation level of histone H3 in KRT17 promoter region was significantly lower than that in KLF4 overexpression group ( P<0.01) and control group ( P<0.01). Co-IP confirmed that KLF4 and EP300 could form protein complexes. Excessive KLF4 increases the level of histone H3 acetylation in KRT17 promoter region by synergistic EP300, and mediates the over-expression of KRT17 in psoriatic lesions.

Silencing of keratin 17 by lentivirus-mediated short hairpin RNA inhibits the proliferation of PANC-1 human pancreatic cancer cells.

Keratin 17 (KRT17) has been demonstrated to be a potential biological marker for the prediction of prognosis in particular types of cancer. The aim of the present study was to investigate the molecular mechanisms underlying the function of KRT17 in the pancreatic cancer (PAC) cell line PANC-1 and the potential of KRT17 as a therapeutic target for PAC. KRT17 expression levels were analyzed using quantitative PCR and compared with histological data using bioinformatics tools in PAC samples and three human PAC cell lines. Cell proliferation was determined using an MTT assay, in addition to cell cycle distribution and apoptosis analysis using flow cytometry, colony formation assay using Giemsa staining and cell motility analysis using a Transwell migration assay. Tumor growth was evaluated in vivo in nude mice. The expression levels of a number of signaling molecules were measured to establish the potential mechanism by which silencing KRT17 expression affected PAC PANC-1 cells. Increased levels of KRT17 expression were observed in human PAC compared with normal tissues, as well as in three human PAC cell lines (MIA PaCa-2, PANC-1 and KP-3 cells) compared with the H6c7 human immortal pancreatic duct epithelial cell line. High expression levels of KRT17 in PAC samples were associated with poor overall survival (P=0.036) and disease-free survival (P=0.017). Lentivirus-mediated KRT17 silencing inhibited cell proliferation, colony formation and migration, but promoted apoptosis and resulted in cell cycle arrest in the G0/G1 phase in PANC-1 cells. In addition, KRT17 knockdown inhibited in vivo tumor growth. KRT17 knockdown induced dysregulation of ERK1/2 and upregulation of the pro-apoptotic Bcl-2 protein Bad. In conclusion, the present study demonstrated that elevated KRT17 levels are positively associated with pancreatic cancer progression; KRT17 knockdown suppressed cell growth, colony formation, migration and tumor growth, and induced apoptosis and cell cycle arrest, affecting ERK1/2/Bad signaling. Therefore, the results of the present study suggested that KRT17 may be a potential target for the treatment of pancreatic cancer.

KRT17 Functions as a Tumor Promoter and Regulates Proliferation, Migration and Invasion in Pancreatic Cancer via mTOR/S6k1 Pathway.

BackgroundPancreatic cancer (PC) is one of the most well-known malignancies with high mortality, but the underlying mechanism of PC remains unknown. Keratin17 (KRT17) expression has been reported in many malignancies, but its functions in PC are not clear. The aim of our study was to evaluate KRT17 expression and its potential role in PC.MethodsThe online databases GEPIA and THPA were used to identify KRT17 expression in tissues. Quantitative real-time PCR (qRT-PCR) was used to determine KRT17 expression in cell lines. Ki67 and ROS levels were detected by immunofluorescence assay and a 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. KRT17 downregulation was induced by the small interfering RNA (siRNA) technique. Proliferation function was evaluated by colony formation assay and RTCA. Migration and invasion were evaluated by transwell migration assay. A Western blot assay was used to detect protein levels.ResultsKRT17 was overexpressed in PC tissues compared to that in normal tissues. The results showed that Ki67 and ROS levels were decreased in pancreatic cancer cells after transfection with siKRT17. After KRT17 downregulation in PC cell lines, cell viability functions, including proliferation, migration and invasion, and mTOR/S6K1 phosphorylation levels were attenuated.ConclusionKRT17 knockdown significantly inhibited proliferation, migration and invasion in pancreatic cancer cells.

Overexpression of KRT17 promotes proliferation and invasion of non-small cell lung cancer and indicates poor prognosis.

PurposeKeratin 17 (KRT17) is a 48 KDa type I intermediate filament, which is mainly expressed in epithelial basal cells. KRT17 has been shown to be overexpressed in many malignant tumors and play an important role in the occurrence and development of tumors. Therefore, this study explored the role and underlying mechanism of KRT17 in non-small cell lung cancers (NSCLC).MethodsKRT17 expression and its correlations with clinicopathological factors were examined in lung cancer tissues by immunohistochemistry. The prognosis value of KRT17 in NSCLCs was retrieved from The Cancer Genome Atlas (TCGA) online databases. The expression level of KRT17 was increased or decreased by KRT17 gene transfection or small RNA interference in lung cancer cells, respectively. Further, proliferation and invasiveness of lung cancer cells were determined by cell proliferation and invasion assays, respectively. Finally, expression levels of proteins related to Wnt signaling pathways and epithelial mesenchymal transition (EMT) were detected by Western blot.ResultsThe expression level of KRT17 in NSCLCs was significantly higher than normal lung tissues. High expression of KRT17 predicted poor prognosis of patients with NSCLCs, especially lung adenocarcinomas, and was correlated with poor differentiation and lymphatic metastasis. Overexpression of KRT17 enhanced, while KRT17 knockdown inhibited, the proliferation and invasiveness of lung cancer cells. Overexpression of KRT17 up-regulated β-catenin activity and levels of Wnt target genes, such as cyclin D1, c-Myc, and MMP7. Moreover, KRT17 promoted EMT by up-regulating Vimentin, MMP-9, and Snail expression and down-regulating E-cadherin expression.ConclusionOverexpression of KRT17 is common in NSCLCs and indicates poor prognosis. Overexpression of KRT17 enhances the proliferation and invasiveness of NSCLC cells by activating the Wnt signaling pathway and EMT process. KRT17 is a potential indicator of NSCLC progression and poor survival.

NB-UVB irradiation downregulates keratin-17 expression in keratinocytes by inhibiting the ERK1/2 and STAT3 signaling pathways.

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 400mJ/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.

Abstract 2021: Ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis through upregulation of keratin-17

Abstract Solar UV irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancerr and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic ablation of caspase-7 are significantly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and tumor numbers were increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation biomarkers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. 2-DE gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. Furthermore the expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study suggests that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17. Citation Format: Mee-Hyun Lee, Do Young Lim, Sung Young Lee, Jung Hyun Shim, Xuejiao Liu, Ran Zhao, Hai Huang, G.Timothy Bowden, Young-Joon Surh, Yong-Yeon Cho, Ann M. Bode, Zigang Dong. Ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis through upregulation of keratin-17 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2021. doi:10.1158/1538-7445.AM2017-2021

GLI-mediated Keratin 17 expression promotes tumor cell growth through the anti-apoptotic function in oral squamous cell carcinomas.

Keratin 17 (KRT17) has been suggested as a potential diagnostic marker of squamous cell carcinoma including oral squamous cell carcinoma (OSCC). The current study was conducted to clarify the function of KRT17 and its expression mechanism in OSCC. Immunohistochemical analyses were carried out to examine the expression of KRT17, GLI family zinc finger (GLI)-1, GLI-2, or cleaved caspase-3 in OSCCs. The expression of KRT17, GLI-1, or GLI-2 was investigated among OSCC cell lines, and the effects of loss-of-function of KRT17 or GLI, using siRNA or inhibitor, on the cell growth of the OSCC cell line HSC-2 particularly with respect to apoptosis were examined. Immunohistochemical analyses of tissue specimens obtained from 78 OSCC patients revealed that KRT17 was not observed in non-tumor regions but was strongly expressed at high frequencies in tumor regions. Knockdown of KRT17 increased the number of cleaved caspase-3-positive cells, leading to the reduction of cell number. Loss-of-function of GLI-1 or GLI-2 also increased the cell numbers of apoptotic cells positive for staining of Annexin-V and propidium iodide (PI) and the terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling (TUNEL) method, and induced DNA fragmentation. This inhibitory effect on cell growth was partially rescued by exogenous KRT17 expression. In the KRT17-positive regions in OSCCs, GLI-1 or GLI-2 was frequently detected, and the number of cells with cleaved caspase-3 positive was decreased. KRT17 promotes tumor cell growth, at least partially, through its anti-apoptotic effect as a result of the KRT17 overexpression by GLIs in OSCC.