Repeated stress to the skin amplifies neutrophil infiltration in a keratin 17- and PKCα-dependent manner.

Neutrophils are the first immune cells to reach inflamed sites and contribute to the pathogenesis of chronic inflammatory skin diseases. Yet, little is known about the pattern of neutrophil infiltration in inflamed skin in vivo and the mechanisms mediating their recruitment. Here, we provide insight into the dynamics of neutrophil infiltration in skin in response to acute or repeated inflammatory stress, highlighting a novel keratinocyte- and keratin 17 (K17)-dependent mechanism that regulates neutrophil recruitment to inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12 h and resolves within 24 h. A subsequent TPA treatment or a UVB challenge, when applied 24 h but not 48 h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of chemoattractants by stressed keratinocytes. K17 binds RACK1, a scaffold protein essential for PKCα activity. The N-terminal head domain of K17 is crucial for its association with RACK1 and regulation of PKCα activity. Analysis of RNAseq data reveals a signature consistent with TAR and PKCα activation in inflammatory skin diseases. These findings uncover a novel, keratin-dependent mechanism that amplifies neutrophil recruitment in skin under stress, with direct implications for inflammatory skin disorders.

Stress keratin 17 and estrogen support viral persistence and modulate the immune environment during cervicovaginal murine papillomavirus infection.

A murine papillomavirus, MmuPV1, infects both cutaneous and mucosal epithelia of laboratory mice and can be used to model high-risk human papillomavirus (HPV) infection and HPV-associated disease. We have shown that estrogen exacerbates papillomavirus-induced cervical disease in HPV-transgenic mice. We have also previously identified stress keratin 17 (K17) as a host factor that supports MmuPV1-induced cutaneous disease. Here, we sought to test the role of estrogen and K17 in MmuPV1 infection and associated disease in the female reproductive tract. We experimentally infected wild-type and K17 knockout (K17KO) mice with MmuPV1 in the female reproductive tract in the presence or absence of exogenous estrogen for 6 mon. We observed that a significantly higher percentage of K17KO mice cleared the virus as opposed to wild-type mice. In estrogen-treated wild-type mice, the MmuPV1 viral copy number was significantly higher compared to untreated mice by as early as 2 wk postinfection, suggesting that estrogen may help facilitate MmuPV1 infection and/or establishment. Consistent with this, viral clearance was not observed in either wild-type or K17KO mice when treated with estrogen. Furthermore, neoplastic disease progression and cervical carcinogenesis were supported by the presence of K17 and exacerbated by estrogen treatment. Subsequent analyses indicated that estrogen treatment induces a systemic immunosuppressive state in MmuPV1-infected animals and that both estrogen and K17 modulate the local intratumoral immune microenvironment within MmuPV1-induced neoplastic lesions. Collectively, these findings suggest that estrogen and K17 act at multiple stages of papillomavirus-induced disease at least in part via immunomodulatory mechanisms.

Dual and triple gene combinations of KRT5, KRT17, and S100A2 identify basal-like subtype of pancreatic ductal adenocarcinoma and correlate with survival outcome.

There is a significant difference in prognosis and response to chemotherapy between basal and classical subtypes of pancreatic ductal adenocarcinoma (PDAC). Further biomarkers are required to identify subtypes of PDAC. We selected candidate biomarkers via review articles. Correlations between these candidate markers and the PDAC molecular subtype gene sets were analyzed using bioinformatics, confirming the biomarkers for identifying classical and basal subtypes. Subsequently, 298 PDAC patients were included, and their tumor tissues were immunohistochemically stratified using these biomarkers. Survival data underwent analysis, including Cox proportional hazards modeling. Our results indicate that the pairwise and triple combinations of KRT5/KRT17/S100A2 exhibit a higher correlation coefficient with the basal-like subtype gene set, whereas the corresponding combinations of GATA6/HNF4A/TFF1 show a higher correlation with the classical subtype gene set. Whether analyzing unmatched or propensity-matched data, the overall survival time was significantly shorter for the basal subtype compared with the classical subtype (p < .001), with basal subtype patients also facing a higher risk of mortality (HR = 4.017, 95% CI 2.675-6.032, p < .001). In conclusion, the combined expression of KRT5, KRT17, and S100A2, in both pairwise and triple combinations, independently predicts shorter overall survival in PDAC patients and likely identifies the basal subtype. Similarly, the combined expression of GATA6, HNF4A, and TFF1, in the same manner, may indicate the classical subtype. In our study, the combined application of established biomarkers offers valuable insights for the prognostic evaluation of PDAC patients.

Keratin 17 in psoriasis: Current understanding and future perspectives.

Keratin 17 (K17) is a multifaceted cytoskeletal protein that is not commonly expressed in the epidermis under normal physiological conditions. However, in psoriasis, K17 is overexpressed in the suprabasal layer of the epidermis and plays an important role in the pathogenesis of the disease. In this review, we have summarized our findings and those reported in other studies concerning the pathogenic functions of K17, as well as the mechanisms underlying the increase in K17 expression in psoriasis. K17 exerts both pro-proliferative and pro-inflammatory effects on keratinocytes. Moreover, K17 peptides trigger autoreactive T cells and promote psoriasis-related cytokine production. In turn, these cytokines modulate the expression, stability, and protein-protein interactions of K17 through transcriptional and translational regulation and post-translational modification of K17 in keratinocytes. Thus, a K17/T-cell/cytokine autoimmune loop is implicated in the pathogenesis of psoriasis, which is supported by the fact that therapies targeting K17 have achieved good outcomes in psoriasis-like mouse models. Future perspectives of K17 in psoriasis have also been discussed to provide potential directions for further studies.

Loss of mutant p53 in HaCaT keratinocytes promotes cadmium-induced keratin 17 expression and cell death.

BACKGROUND: Cadmium exposure induces dermatotoxicity and epidermal barrier disruption and leads to the development of various pathologies. HaCaT cells are immortalized human keratinocytes that are widely used as alternatives to primary human keratinocytes, particularly for evaluating cadmium toxicity. HaCaT cells bear two gain-of-function (GOF) mutations in the TP53 gene, which strongly affect p53 function. Mutant forms of p53 are known to correlate with increased resistance to various stimuli, including exposure to cytotoxic substances. In addition, keratin 17 (KRT17) was recently shown to be highly expressed in HaCaT cells in response to genotoxic stress. Moreover, p53 is a direct transcriptional repressor of KRT17. However, the impact of TP53 mutations in HaCaT cells on the regulation of cell death and keratin 17 expression is unclear. In this study, we aimed to evaluate the impact of p53 on the response to Cd-induced cytotoxicity. METHODS AND RESULTS: Employing the MTT assay and Annexin V/propidium iodide staining, we demonstrated that knockout of TP53 leads to a decrease in the sensitivity of HaCaT cells to the cytotoxic effects of cadmium. Specifically, HaCaT cells with TP53 knockout (TP53 KO HaCaT) exhibited cell death at a cadmium concentration of 10 µM or higher, whereas wild-type cells displayed cell death at a concentration of 30 µM. Furthermore, apoptotic cells were consistently detected in TP53 KO HaCaT cells upon exposure to low concentrations of cadmium (10 and 20 µM) but not in wild-type cells. Our findings also indicate that cadmium cytotoxicity is mediated by reactive oxygen species (ROS), which were significantly increased only in TP53 knockout cells treated with 30 µM cadmium. An examination of proteomic data revealed that TP53 knockout in HaCaT cells resulted in the upregulation of proteins involved in the regulation of apoptosis, redox systems, and DNA repair. Moreover, RT‒qPCR and immunoblotting showed that cadmium toxicity leads to dose-dependent induction of keratin 17 in p53-deficient cells but not in wild-type cells. CONCLUSIONS: The connection between mutant p53 in HaCaT keratinocytes and increased resistance to cadmium toxicity was demonstrated for the first time. Proteomic profiling revealed that TP53 knockout in HaCaT cells led to the activation of apoptosis regulatory circuits, redox systems, and DNA repair. In addition, our data support the involvement of keratin 17 in the regulation of DNA repair and cell death. Apparently, the induction of keratin 17 is p53-independent but may be inhibited by mutant p53.

Keratin 17 modulates the immune topography of pancreatic cancer.

BACKGROUND: The immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival. METHODS: Multiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs. RESULTS: K17 expression had profound effects on the exclusion of intratumoral CD8+ T cells and was also associated with decreased numbers of peritumoral CD8+ T cells, CD16+ macrophages, and CD163+ macrophages (p < 0.0001). The differences in the intratumor and peritumoral CD8+ T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations. CONCLUSIONS: Thus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer.

Diagnosis of verruciform acanthotic vulvar intra-epithelial neoplasia (vaVIN) using CK17, SOX2 and GATA3 immunohistochemistry.

AIMS: Verruciform acanthotic vulvar intra-epithelial neoplasia (vaVIN) is an HPV-independent, p53 wild-type lesion with distinct morphology and documented risk of recurrence and cancer progression. vaVIN is rare, and prospective distinction from non-neoplastic hyperplastic lesions can be difficult. CK17, SOX2 and GATA3 immunohistochemistry has emerging value in the diagnosis of HPV-independent lesions, particularly differentiated VIN. We aimed to test the combined value of these markers in the diagnosis of vaVIN versus its non-neoplastic differentials in the vulva. METHODS AND RESULTS: CK17, SOX2 and GATA3 immunohistochemistry was evaluated on 16 vaVINs and 34 mimickers (verruciform xanthoma, lichen simplex chronicus, lichen sclerosus, psoriasis, pseudo-epitheliomatous hyperplasia). CK17 was scored as 3+ = full-thickness, 2+ = partial-thickness, 1+ = patchy, 0 = absent; SOX2 as 3+ = strong staining ≥ 10% cells, 2+ = moderate, 1 + =weak, 0 = staining in < 10% cells; and GATA3 as pattern 0 = loss in < 25% basal cells, 1 = loss in 25-75% basal cells, 2 = loss in > 75% basal cells. For analysis, results were recorded as positive (CK17 = 3+, SOX2 = 3+, GATA3 = patterns 1/2) or negative (CK17 = 2+/1+/0, SOX2 = 2+/1+/0, GATA3 = pattern 0). CK17, SOX2 and GATA3 positivity was documented in 81, 75 and 58% vaVINs, respectively, versus 32, 17 and 22% of non-neoplastic mimickers, respectively; ≥ 2 marker positivity conferred 83 sensitivity, 88 specificity and 86% accuracy in vaVIN diagnosis. Compared to vaVIN, SOX2 and GATA3 were differentially expressed in lichen sclerosus, lichen simplex chronicus and pseudo-epitheliomatous hyperplasia, whereas CK17 was differentially expressed in verruciform xanthoma and adjacent normal mucosa. CONCLUSIONS: CK17, SOX2 and GATA3 can be useful in the diagnosis of vaVIN and its distinction from hyperplastic non-neoplastic vulvar lesions. Although CK17 has higher sensitivity, SOX2 and GATA3 are more specific, and the combination of all markers shows optimal diagnostic accuracy.

A role for keratin 17 during DNA damage response and tumor initiation.

High levels of the intermediate filament protein keratin 17 (K17) are associated with poor prognoses for several human carcinomas. Studies in mouse models have shown that K17 expression is positively associated with growth, survival, and inflammation in skin and that lack of K17 delays onset of tumorigenesis. K17 occurs in the nucleus of human and mouse tumor keratinocytes where it impacts chromatin architecture, gene expression, and cell proliferation. We report here that K17 is induced following DNA damage and promotes keratinocyte survival. The presence of nuclear K17 is required at an early stage of the double-stranded break (DSB) arm of the DNA damage and repair (DDR) cascade, consistent with its ability to associate with key DDR effectors, including γ-H2A.X, 53BP1, and DNA-PKcs. Mice lacking K17 or with attenuated K17 nuclear import showed curtailed initiation in a two-step skin carcinogenesis paradigm. The impact of nuclear-localized K17 on DDR and cell survival provides a basis for the link between K17 induction and poor clinical outcomes for several human carcinomas.

Activation of the C3a anaphylatoxin receptor inhibits keratinocyte proliferation by regulating keratin 6, keratin 16, and keratin 17 in psoriasis.

Emerging evidence suggests that signaling through the C3a anaphylatoxin receptor (C3aR) protects against various inflammation-related diseases. However, the role of C3aR in psoriasis remains unknown. The purpose of this study was to investigate the possible protective role of C3aR in psoriasis and to explore the underlying molecular mechanisms. We initially found that the psoriatic epidermis exhibited significantly decreased C3aR expression. C3aR showed protective roles in mouse models of imiquimod (IMQ)- and interleukin-23-induced psoriasis. Furthermore, increased epidermal thickness and keratin 6 (K6), K16, and K17 expression occurred in the ears and backs of C3aR-/- mice. Pharmacological treatment with a C3aR agonist ameliorated IMQ-induced psoriasiform lesions in mice and decreased the expression of K6, K16, and K17. Additionally, the signal transducer and activator of transcription 3 (STAT3) pathway participated in the protective function of C3aR. More importantly, the expression levels of K6, K16, and K17 in keratinocytes were all restored in HaCaT cells transfected with a C3aR-overexpression plasmid after treating them with colivelin (a STAT3 activator). Our findings demonstrate that C3aR protects against the development of psoriasis and suggest that C3aR confers protection by negatively regulating K6, K16, and K17 expression in a STAT3-dependent manner, thus inhibiting keratinocyte proliferation and helping reverse the pathogenesis of psoriasis.

Keratin 17 regulates nuclear morphology and chromatin organization.

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 wild-type 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 LAP2ß (an isoform encoded by TMPO) 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.This article has an associated First Person interview with the first author of the paper.

Stress keratin 17 enhances papillomavirus infection-induced disease by downregulating T cell recruitment.

High-risk human papillomaviruses (HPVs) cause 5% of human cancers. Despite the availability of HPV vaccines, there remains a strong urgency to find ways to treat persistent HPV infections, as current HPV vaccines are not therapeutic for individuals already infected. We used a mouse papillomavirus infection model to characterize virus-host interactions. We found that mouse papillomavirus (MmuPV1) suppresses host immune responses via overexpression of stress keratins. In mice deficient for stress keratin K17 (K17KO), we observed rapid regression of papillomas dependent on T cells. Cellular genes involved in immune response were differentially expressed in the papillomas arising on the K17KO mice correlating with increased numbers of infiltrating CD8+ T cells and upregulation of IFNγ-related genes, including CXCL9 and CXCL10, prior to complete regression. Blocking the receptor for CXCL9/CXCL10 prevented early regression. Our data provide a novel mechanism by which papillomavirus-infected cells evade host immunity and defines new therapeutic targets for treating persistent papillomavirus infections.

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.

Effects of PPAR-γ and RXR-α on mouse meibomian gland epithelial cells during inflammation induced by latanoprost.

The purpose of this study is to investigate the effects of latanoprost on the secretion of cytokines and chemokines from meibomian gland epithelial cells, and to evaluate the modulation of peroxisome proliferator-activated receptor γ (PPAR-γ) and retinoid X receptor α (RXR-α) during latanoprost-induced inflammation. Mouse meibomian gland epithelial cells were cultured in proliferation and differentiation medium, respectively. Cells were exposed to latanoprost, rosiglitazone (PPAR-γ agonist), or LG100268 (RXR-α agonist), respectively. The expression of IL-6, IL-1ß, TNF-α, MMP-9, MCP-1, and CCL-5 were detected by real-time PCR and ELISA. The effect of latanoprost, rosiglitazone, LG100268, and inflammatory cytokines on the differentiation of meibocyte were evaluated by related gene expression and lipid staining. The expression of Keratin-1, 6, 17 protein was detected by western immunoblotting. The results showed that the above cytokines could be induced by latanoprost in meibomian gland epithelial cells. LG100268 and rosiglitazone could inhibit the production of IL-6 and TNF-α induced by latanoprost, respectively. Latanoprost suppressed the expression of differentiation-related mRNA through a positive feedback loop by enhancement of COX-2 expression via FP receptor-activated ERK signaling. The expression of Keratin-17 was upregulated by rosiglitazone and suppressed by LG100268. The application of IL-6 and TNF-α showed negative effects on lipid accumulation in meibomian gland epithelial cells. These results demonstrated that latanoprost could induce inflammation and suppress differentiation of mouse meibomian gland epithelial cells. The activation of PPAR-γ and RXR-α showed an anti-inflammatory effect, showing a potential role to antagonize the effect of latanoprost eyedrops on meibomian gland epithelial cells.

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

BACKGROUND: The 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. METHODS: siRNA, 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. RESULTS: Significantly 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 ß4/α6, increased phosphorylation of FAK, Src and ERK, as well as stabilization and nuclear translocation of ß-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 ß4, active ß-catenin, CD44 and EGFR were observed in the tumors induced by KRT17 knockdown OSCC cells. CONCLUSIONS: A novel miRNA-485-5p/KRT17/integrin/FAK/Src/ERK/ß-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.

A unique skin phenotype resulting from a large heterozygous deletion spanning six keratin genes.

The phenotypic spectrum of genodermatoses is continuously expanding. Three siblings were referred because of a highly unusual phenotype comprising alopecia, dystrophic nails, palmoplantar keratoderma and trauma-induced skin blistering. Whole-exome sequencing analysis identified a heterozygous large genomic alteration of around 116 0000 bp resulting in the deletion of the KRT9, KRT14, KRT15, KRT16 and KRT19 genes, as well as part of KRT17. This genomic change leads to the generation of a truncated keratin 17 (KRT17) protein encoded by the first three exons of the gene and part of intron 3. The three patients were found to carry the heterozygous genomic deletion while their healthy parents did not, indicative of germline mosaicism. The genomic alteration was found to result in reduced KRT17 expression in patient skin. More importantly, the abnormal truncated KRT17 was found to exert a deleterious effect on keratinocyte cytoskeleton formation, leading to keratin aggregation. Coexpression of wildtype and truncated KRT17 proteins also caused keratin aggregation, demonstrating that the deletion exerts a dominant negative effect. In conclusion, we are reporting on a novel clinical phenotype that was found to result from germline mosaicism for a large genomic deletion spanning six keratin genes, thus expanding the spectrum of clinical manifestations associated with keratin disorders. What is already known about this topic? Various conditions known as keratinopathies have been shown over recent years to be associated with dominant or recessive variants in several individual keratin genes. What does this study add? We report three patients presenting with a unique clinical phenotype that was found to result from germline mosaicism for a large genomic deletion spanning six keratin genes. The genomic variant is predicted to result in a truncated form of keratin 17, which was found in an in vitro assay to disrupt keratinocyte cell cytoskeleton formation.

Coexistence of pachyonychia congenita and hidradenitis suppurativa: more than a coincidence.

BACKGROUND: The coexistence of pachyonychia congenita (PC) and hidradenitis suppurativa (HS) has been described in case reports. However, the pathomechanism underlying this association and its true prevalence are unknown. OBJECTIVES: To determine the genetic defect underlying the coexistence of PC and HS in a large kindred, to delineate a pathophysiological signalling defect jointly leading to both phenotypes, and to estimate the prevalence of HS in PC. METHODS: We used direct sequencing and a NOTCH luciferase reporter assay to characterize the pathophysiological basis of the familial coexistence of HS and PC. A questionnaire was distributed to patients with PC registered with the International Pachyonychia Congenita Research Registry (IPCRR) to assess the prevalence of HS among patients with PC. RESULTS: Direct sequencing of DNA samples obtained from family members displaying both PC and HS demonstrated a missense variant (c.275A>G) in KRT17, encoding keratin 17. Abnormal NOTCH signalling has been suggested to contribute to HS pathogenesis. Accordingly, the KRT17 c.275A>G variant resulted in a significant decrease in NOTCH activity. To ascertain the clinical importance of the association of HS with PC, we distributed a questionnaire to all patients with PC registered with the IPCRR. Seventy-two of 278 responders reported HS-associated clinical features (25·9%). Disease-causing mutations in KRT17 were most prevalent among patients with a dual phenotype of PC and HS (43%). CONCLUSIONS: The coexistence of HS and KRT17-associated PC is more common than previously thought. Impaired NOTCH signalling as a result of KRT17 mutations may predispose patients with PC to HS. What is already known about this topic? The coexistence of pachyonychia congenita (PC) and hidradenitis suppurativa (HS) has been described in case reports. However, the pathomechanism underlying this association and its true prevalence are unknown. What does this study add? A dual phenotype consisting of PC and HS was found to be associated with a pathogenic variant in KRT17. This variant was found to affect NOTCH signalling, which has been previously implicated in HS pathogenesis. HS was found to be associated with PC in a large cohort of patients with PC, especially in patients carrying KRT17 variants, suggesting that KRT17 variants causing PC may also predispose to HS. What is the translational message? These findings suggest that patients with PC have a higher prevalence of HS than previously thought, and hence physicians should have a higher level of suspicion of HS diagnosis in patients with PC.

Keratin 17 as a Marker of UVB-Induced Stress in Human Epidermis and Modulation by Vitis vinifera Extract.

Human epidermis responds to ultraviolet (UV)B-induced damage by tolerating it, restoring it, or undergoing programmed cell death when the damage is massive. Recently, compounds rich in polyphenols, such as Vitis vinifera L. leaf extract (VVLe), have attracted a lot of interest for skin protection. We investigated the effect of VVLe pre-treatment (1 h) in a 2D model of HaCaT cells and in 3D organotypic cultures of normal human skin exposed to a single UVB dose to study the immediate specific events 1 h and the response orchestrated in the epidermal layer 24 h after irradiation, respectively. In both models, transmission electron microscopy analysis was carried out. The expression of the inducible keratin K17, the activation of both pSTAT3 and Nuclear Factor (NF)-κB signalling pathways, and the epidermal distribution of Toll-Like Receptor (TLR) 4 were assessed by immunofluorescence in the 2D and 3D model. In 3D organotypic cultures, thanks to the preservation of a multi-layered structure, the epidermal distribution of the differentiation biomarkers K10 and K14 as well as of K16 was analysed by immunofluorescence, while the release of interleukin (IL)-8 was evaluated by ELISA. In skin bioptic fragments, cytotoxicity and genotoxicity were investigated by LDH assay and Alkaline Comet assay, respectively, and then compared to cell proliferation. The epidermal distribution of the histone γ-H2AX, indicating the fragmented DNA, was analysed by immunofluorescence. In both experimental models, VVLe tuned UVB-induced K17 expression to a different extent in HaCaT cells and in the skin. In HaCaT cells, pSTAT3 activation was induced by UVB and reverted by VVLe pre-treatment. TLR4 expression was triggered by UVB in both models, but VVLe pre-treatment abolished this event only in HaCaT cells. NF-κB immunostaining increased both in the nucleus and in the cytoplasm only in HaCaT cells after UVB irradiation. In all irradiated skin samples, VVLe pre-treatment was not able to revert the inhibition of epidermal proliferation, K16 expression, and IL-8 secretion. The effectiveness of VVLe in contrasting the irradiation-induced genotoxicity still remains unclear. In conclusion, our study clearly shows that K17 is a robust marker induced in keratinocytes upon UVB stimulation and that this event can be reverted by a pre-treatment with VVLe. On the whole, these observations represent a novelty in the scenario of the complex relationships between the effects exerted by UVB rays on human skin and significantly improve the knowledge regarding the modulation of the early epidermal response induced by a single exposure to UVB in the presence of VVLe.

Use of Cytokeratin 17 in the Differentiation Between Desmoplastic Trichilemmoma and Cutaneous Basal Cell Carcinoma.

ABSTRACT: Desmoplastic trichilemmoma (DTL) is a variant of trichilemmoma characterized by a prominent desmoplastic stroma that may mimic invasive carcinoma. These lesions typically show features of a conventional trichilemmoma at the periphery, surrounding dense hyalinized stroma with entrapped cords of tumor cells. On a small or superficial biopsy, DTL may pose a diagnostic challenge in distinguishing this benign adnexal neoplasm from invasive carcinoma, particularly basal cell carcinoma (BCC). We aimed to investigate whether the immunohistochemical expression of cytokeratin 17 (CK17) would be useful in the differentiation between DTL and BCC. CK17 is expressed in normal adnexal structures and has been shown to demonstrate strong staining in BCCs. Expression of CK17 was examined in 23 cases of DTL and 23 BCCs. An immunoreactivity score was assigned using the percentage of tumor cells staining with scoring as follows: 0, complete negativity; 1, < 15% tumor cells staining; 2, 15%-84% tumor cells staining; and 3, >85% staining. All cases of BCC scored as 3, whereas 18% of DTL scored as 3. The mean percent staining for CK17 was significantly higher for BCCs (97% of tumor cells) than DTLs (57% of tumor cells); P < 0.001 in the unpaired t test. The pattern of CK17 staining may also help differentiate between cases scoring 3. All BCCs showed strong diffuse staining throughout, whereas for those cases of DTL with a score of 3, the peripheral basaloid rim in the tumor lobules did not stain. CK17 is a useful adjunct in distinguishing DTL from BCC in small or superficial biopsy specimens.

Cell Adhesion Molecules Affected by Ionizing Radiation and Estrogen in an Experimental Breast Cancer Model.

Cancer develops in a multi-step process where environmental carcinogenic exposure is a primary etiological component, and where cell-cell communication governs the biological activities of tissues. Identifying the molecular genes that regulate this process is essential to targeting metastatic breast cancer. Ionizing radiation can modify and damage DNA, RNA, and cell membrane components such as lipids and proteins by direct ionization. Comparing differential gene expression can help to determine the effect of radiation and estrogens on cell adhesion. An in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line MCF-10F to low doses of high linear energy transfer α particle radiation and subsequent growth in the presence of 17ß-estradiol. The MCF-10F cell line was analyzed in different stages of transformation that showed gradual phenotypic changes including altered morphology, increase in cell proliferation relative to the control, anchorage-independent growth, and invasive capability before becoming tumorigenic in nude mice. This model was used to determine genes associated with cell adhesion and communication such as E-cadherin, the desmocollin 3, the gap junction protein alpha 1, the Integrin alpha 6, the Integrin beta 6, the Keratin 14, Keratin 16, Keratin 17, Keratin 6B, and the laminin beta 3. Results indicated that most genes had greater expression in the tumorigenic cell line Tumor2 derived from the athymic animal than the Alpha3, a non-tumorigenic cell line exposed only to radiation, indicating that altered expression levels of adhesion molecules depended on estrogen. There is a significant need for experimental model systems that facilitate the study of cell plasticity to assess the importance of estrogens in modulating the biology of cancer cells.

Pseudogene KRT17P3 drives cisplatin resistance of human NSCLC cells by modulating miR-497-5p/mTOR.

Chemoresistance is a major obstacle in non-small cell lung cancer (NSCLC) treatment. The pseudogene keratin 17 pseudogene 3 (KRT17P3) has been previously shown to be upregulated in lung cancer tissues of patients with cisplatin resistance. In the present study, RT-qPCR was performed to evaluate KRT17P3 levels in plasma samples collected from 30 cisplatin-resistant and 32 cisplatin-sensitive patients. We found that the plasma level of KRT17P3 is upregulated in cisplatin-resistant patients, and the increased expression of plasma KRT17P3 is associated with poor chemotherapy response. Functional studies demonstrated that KRT17P3 overexpression in cultured NSCLC cells increases cell viability and decreases apoptosis upon cisplatin treatment in vitro and in vivo, while KRT17P3 knockdown has the opposite effect. Mechanistically, bioinformatics analysis, RNA immunoprecipitation, and dual luciferase reporter assay indicated that KRT17P3 acts as a molecular sponge for miR-497-5p and relieves the binding of miR-497-5p to its target gene mTOR. Rescue experiments validated the functional interaction between KRT17P3, miR-497-5p, and mTOR. Taken together, our findings indicate that KRT17P3/miR-497-5p/mTOR regulates the chemosensitivity of NSCLC, suggesting a potential therapeutic target for cisplatin-resistant NSCLC patients. KRT17P3 may be a potential peripheral blood marker of NSCLC patients resistant to cisplatin.