Inhibition of the Tumor Suppressor Gene SPINK5 via EHMT2 Induces the Oral Squamous Cell Carcinoma Development.

Serine protease inhibitor Kazal-type 5 (SPINK5) has been revealed as a significant prognostic biomarker in oral squamous cell carcinoma (OSCC). However, there is little information regarding the detailed epigenetics mechanism underlying its dysregulation in OSCC. Using the Gene Expression Omnibus database, we identified SPINK5 as a significantly downregulated gene in OSCC tissues. Moreover, SPINK5 inhibited the malignant aggressiveness of HSC3 and squamous cell carcinomas (SCC)9 cells, whereas depletion of SPINK5 using shRNAs led to the opposite trend. The euchromatic histone lysine methyltransferase 2 (EHMT2) was found to bind to the SPINK5 promoter, and EHMT2 repressed the SPINK5 expression. SPINK5 reversed the stimulating effects of EHMT2 on the aggressiveness of HSC3 and SCC9 cells by impairing the Wnt/ß-catenin pathway. Wnt/ß-catenin inhibitor IWR-1 treatment reverted the malignant phenotype of OSCC cells in the presence of short hairpin RNA (sh)-SPINK5. Silencing of EHMT2 inhibited tumor growth and blocked the Wnt/ß-catenin signaling in OSCC, which was reversed by SPINK5 knockdown. Our study shows that SPINK5, mediated by the loss of EHMT2, can inhibit the development of OSCC by inhibiting Wnt/ß-catenin signaling and may serve as a treatment target for OSCC.

SPINK5 is a Tumor-Suppressor Gene Involved in the Progression of Nonsmall Cell Lung Carcinoma through Negatively Regulating PSIP1.

This study aimed to elucidate how SPINK5 affects the malignant phenotypes of NSCLC and the molecular mechanism. NSCLC and adjacent normal tissues were collected to detect the differential level of SPINK5. The influence of SPINK5 on pathological indicators of NSCLC was analyzed. Cellular functions of NSCLC cells overexpressing SPINK5 were assessed by CCK-8, EdU, and transwell assay. By confirming the downstream target of SPINK5, its molecular mechanism on regulating NSCLC was finally explored through rescue experiments. SPINK5 was lowly expressed in NSCLC tissues, and it predicted tumor staging and lymphatic metastasis. In vitro overexpression of SPINK5 declined proliferative and migratory rates in NSCLC cells. PSIP1 was verified as the target gene binding SPINK5, and they displayed a negative correlation in NSCLC tissues. Overexpression of PSIP1 was able to reverse the inhibited proliferative and migratory potentials in NSCLC cells overexpressing SPINK5. SPINK5 level has a close relation to tumor staging and lymphatic metastasis in NSCLC. It serves as a tumor-suppressor gene that inhibits proliferation and migration of NSCLC through negatively regulating PSIP1.

A novel SPINK5 donor splice site variant in a child with Netherton syndrome.

BACKGROUND: Netherton syndrome (NS) is a genodermatosis caused by loss-of-function mutations in SPINK5, resulting in aberrant LEKTI expression. METHOD: Next-generation sequencing of SPINK5 (NM_001127698.1) was carried out and functional studies were performed by immunofluorescence microscopy of a lesional skin biopsy using anti-LEKTI antibodies. RESULTS: We describe a novel SPINK5 likely pathogenic donor splice site variant (NM_001127698.1:c.2015+5G>A) in a patient with NS and confirm its functional significance by demonstrating complete loss of LEKTI expression in lesional skin by immunofluorescence analysis. CONCLUSION: The 2015+5G>A is a novel, likely pathogenic variant in NS. Herein we review and assimilate documented SPINK5 pathogenic variants and discuss possible genotype-phenotype associations in NS.

Otitis media susceptibility and shifts in the head and neck microbiome due to SPINK5 variants.

BACKGROUND: Otitis media (OM) susceptibility has significant heritability; however, the role of rare variants in OM is mostly unknown. Our goal is to identify novel rare variants that confer OM susceptibility. METHODS: We performed exome and Sanger sequencing of >1000 DNA samples from 551 multiethnic families with OM and unrelated individuals, RNA-sequencing and microbiome sequencing and analyses of swabs from the outer ear, middle ear, nasopharynx and oral cavity. We also examined protein localisation and gene expression in infected and healthy middle ear tissues. RESULTS: A large, intermarried pedigree that includes 81 OM-affected and 53 unaffected individuals cosegregates two known rare A2ML1 variants, a common FUT2 variant and a rare, novel pathogenic variant c.1682A>G (p.Glu561Gly) within SPINK5 (LOD=4.09). Carriage of the SPINK5 missense variant resulted in increased relative abundance of Microbacteriaceae in the middle ear, along with occurrence of Microbacteriaceae in the outer ear and oral cavity but not the nasopharynx. Eight additional novel SPINK5 variants were identified in 12 families and individuals with OM. A role for SPINK5 in OM susceptibility is further supported by lower RNA counts in variant carriers, strong SPINK5 localisation in outer ear skin, faint localisation to middle ear mucosa and eardrum and increased SPINK5 expression in human cholesteatoma. CONCLUSION: SPINK5 variants confer susceptibility to non-syndromic OM. These variants potentially contribute to middle ear pathology through breakdown of mucosal and epithelial barriers, immunodeficiency such as poor vaccination response, alteration of head and neck microbiota and facilitation of entry of opportunistic pathogens into the middle ear.

Genetic activation of Nrf2 reduces cutaneous symptoms in a murine model of Netherton syndrome.

Netherton syndrome is a monogenic autosomal recessive disorder primarily characterized by the detachment of the uppermost layer of the epidermis, the stratum corneum It results from mutations in the SPINK5 gene, which codes for a kallikrein inhibitor. Uncontrolled kallikrein activity leads to premature desquamation, resulting in a severe epidermal barrier defect and subsequent life-threatening systemic infections and chronic cutaneous inflammation. Here, we show that genetic activation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2/Nrf2) in keratinocytes of Spink5 knockout mice, a model for Netherton syndrome, significantly alleviates their cutaneous phenotype. Nrf2 activation promoted attachment of the stratum corneum and concomitant epidermal barrier function, and reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α and thymic stromal lymphopoietin. Mechanistically, we show that Nrf2 activation induces overexpression of secretory leukocyte protease inhibitor (Slpi), a known inhibitor of kallikrein 7 and elastase 2, in mouse and human keratinocytes in vivo and in vitro, respectively. In the Spink5-deficient epidermis, the upregulation of Slpi is likely to promote stabilization of corneodesmosomes, thereby preventing premature desquamation. Our results suggest pharmacological NRF2 activation as a promising treatment modality for Netherton syndrome patients.This article has an associated First Person interview with the first author of the paper.

A proinflammatory role of KLK6 protease in Netherton syndrome.

BACKGROUND: Netherton syndrome (NS) is a rare but severe type of ichthyosis characterized by atopy, allergies, and potentially lethal skin overdesquamation associated with highly elevated proteolytic activities in LEKTI-deficient epidermis. NS symptoms are recapitulated in Spink5-/- mouse where the gene encoding Lekti has been invalidated. Spink5-/- mice die within 5h from birth due to their severe skin barrier defect leading to dehydration. Spink5-/- mice also serve as a model for atopic dermatitis. The KLK6 protease is expressed by epidermal keratinocytes and shown in vitro to cleave desmosomal components. OBJECTIVE: To investigate in vivo whether KLK6 is implicated in epidermal overdesquamation and/or inflammation associated with NS. METHODS: The role of KLK6 was evaluated by generating Spink5-/-Klk6-/- double knockout mice. The phenotype was assessed by macroscopic observation, immunohistochemistry for differentiation markers, in situ zymography for proteolysis, and quantification of proinflammatory cytokines. RESULTS: Elimination of Klk6 in Spink5-/- remarkably suppresses the expression of Tslp, a major itching-inducing factor and driver of allergic reactions. Tnfα and the Th17 promoting cytokine Il-23 were also suppressed. Spink5-/-Klk6-/- mice display normalized keratinocyte differentiation, nevertheless, epidermal proteolytic activities and the associated overdesquamation were not ameliorated, and Spink5-/-Klk6-/- still died from a severe epidermal barrier defect as the Spink5-/-. CONCLUSIONS: Ablation of Klk6 largely suppresses epidermal inflammation but cannot rescue overdesquamation leading to the lethal NS phenotype. Nonetheless, our findings demonstrate for the first time that KLK6 is implicated in skin inflammation and may represent a novel druggable target for NS and other inflammatory conditions e.g. atopic dermatitis.

A novel tumor suppressor SPINK5 targets Wnt/ß-catenin signaling pathway in esophageal cancer.

Esophageal cancer is one of the most common tumor in the world, and the morbidity rate is as high as 100/100 000 in some parts of China. Therefore, it is important and urgent to explore the pathogenesis of esophageal cancer and find new therapeutic targets for esophageal cancer. In this study, we found that a novel tumor suppressor SPINK5 is significantly reduced in the development of esophageal cancer, and is closely related to the pathological differentiation and lymph node metastasis of esophageal cancer via bioinformatics analysis and esophageal cancer tissue array. Further studies have found that SPINK5 is closely related to Wnt/ß-catenin signaling pathway by bioinformatics analysis and western blot. In esophageal cancer cells, SPINK5 overexpression can inhibit Wnt/ß-catenin signaling pathway. Combined with LiCl or MG-132 treatment, SPINK5 can inhibit GSK3ß phosphorylation and promote ß-catenin protein degradation, thus inhibit Wnt/ß-catenin signaling pathway. In vivo study, SPINK5 overexpression can significantly inhibit the growth of esophageal cancer cells. Our study shows that SPINK5 can inhibit the proliferation, migration, and invasion of esophageal cancer cells by inhibiting Wnt/ß-catenin signaling pathway, and thus plays an important role in the development of esophageal cancer, and may serve as a treatment target of esophageal cancer.

Toll-like receptor signaling induces the expression of lympho-epithelial Kazal-type inhibitor in epidermal keratinocytes.

BACKGROUND: Lympho-epithelial Kazal-type inhibitor (LEKTI) tightly controls the activities of serine proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 in the epidermis. LEKTI is known to be an essential molecule for the epidermal skin barrier, as demonstrated by SPINK5 nonsense mutation, which results in Netherton syndrome. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns or damage-associated molecular patterns and produce inflammatory cytokines, chemokines, and antimicrobial peptides. However, the effect of TLR signaling on the expression of LEKTI is not clear. OBJECTIVE: To investigate whether TLR signaling can affect expression of LEKTI in epidermal keratinocytes. METHODS: We stimulated a panel of TLR ligands and investigated the expression of LEKTI in normal human epidermal keratinocytes (NHEKs). We further measured trypsin or chymotrypsin-like serine protease activity in NHEK cultured media under stimulation with TLR3 ligand, poly (I:C). Immunostaining for LEKTI was performed using skin samples from skin infectious diseases. RESULTS: TLR1/2, 3, 5, and 2/6 ligands induced the expression of LEKTI in NHEKs. The trypsin or chymotrypsin-like serine protease activity in NHEKs was up-regulated with the stimulation of poly (I:C). The gene expressions of KLK6, KLK10, KLK11, and KLK13 were also increased by poly (I:C). An immunohistochemical analysis demonstrated that the expression of LEKTI was up-regulated in the lesions of varicella, pyoderma, and rosacea. CONCLUSIONS: TLR signaling induces the expression of LEKTI in epidermal keratinocytes, which might contribute to the control of aberrant serine protease activities in inflammatory skin diseases.

Síndrome de Netherton: el caso de un recién nacido con insuficiencia respiratoria / Netherton syndrome: A neonatal case with respiratory insufficiency

El síndrome de Netherton (SN) es una enfermedad autosómica recesiva, muy poco frecuente, que se caracteriza por la presencia de eritrodermia ictiosiforme congènita, anomalías capilares y manifestaciones atópicas. Este síndrome es consecuencia de una mutación recesiva en el gen SPINK5. Las manifestaciones del síndrome de SN varían considerablemente entre las personas que lo padecen. Aquí informamos el caso de un recién nacido que presentaba insuficiencia respiratoria grave, hipotermia y eritrodermia, al que se le diagnosticó SN, confirmado mediante pruebas genéticas moleculares.

Netherton syndrome (NS) is a rare, autosomal recessive disease characterized with congenital ichthyosiform erythroderma, hair abnormality and atopic manifestations. This syndrome is caused by recessive mutation in the SPINK5 gene. Disease manifestations vary considerably among NS individuals. We report a newborn presented with severe respiratory insufficiency, hypothermia and erythroderma, was diagnosed as having NS and confirmed with molecular genetic testing.

The antiprotease SPINK7 serves as an inhibitory checkpoint for esophageal epithelial inflammatory responses.

Loss of barrier integrity has an important role in eliciting type 2 immune responses, yet the molecular events that initiate and connect this with allergic inflammation remain unclear. We reveal an endogenous, homeostatic mechanism that controls barrier function and inflammatory responses in esophageal allergic inflammation. We show that a serine protease inhibitor, SPINK7 (serine peptidase inhibitor, kazal type 7), is part of the differentiation program of human esophageal epithelium and that SPINK7 depletion occurs in a human allergic, esophageal condition termed eosinophilic esophagitis. Experimental manipulation strategies reducing SPINK7 in an esophageal epithelial progenitor cell line and primary esophageal epithelial cells were sufficient to induce barrier dysfunction and transcriptional changes characterized by loss of cellular differentiation and altered gene expression known to stimulate allergic responses (for example, FLG and SPINK5). Epithelial silencing of SPINK7 promoted production of proinflammatory cytokines including thymic stromal lymphopoietin (TSLP). Loss of SPINK7 increased the activity of urokinase plasminogen-type activator (uPA), which in turn had the capacity to promote uPA receptor-dependent eosinophil activation. Treatment of epithelial cells with the broad-spectrum antiserine protease, α1 antitrypsin, reversed the pathologic features associated with SPINK7 silencing. The relevance of this pathway in vivo was supported by finding genetic epistasis between variants in TSLP and the uPA-encoding gene, PLAU We propose that the endogenous balance between SPINK7 and its target proteases is a key checkpoint in regulating mucosal differentiation, barrier function, and inflammatory responses and that protein replacement with antiproteases may be therapeutic for select allergic diseases.

Analysis of All 34 Exons of the SPINK5 Gene in Japanese Atopic Dermatitis Patients.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a large multidomain serine protease inhibitor that is expressed in epidermal keratinocytes. Nonsense mutations of the SPINK5 gene, which codes for LEKTI, cause Netherton syndrome, which is characterized by hair abnormality, ichthyosis, and atopy. A single nucleotide polymorphism (SNP) of SPINK5, p.K420E, is reported to be associated with the pathogenesis of atopic dermatitis (AD). We studied all 34 exons of the SPINK5 gene in Japanese 57 AD patients and 50 normal healthy controls. We detected nine nonsynonymous variants, including p.K420E; these variants had already been registered in the SNP database. Among them, p.R654H (n=1) was found as a heterozygous mutation in the AD patients, but not in the control. No new mutation was detected. We next compared the data of the AD patients with data from the Human Genetic Variation Database provided by Kyoto University; a significant difference was found in the frequency of the p.S368N genotype distribution. PolyPhen-2 and SIFT, two algorithms for predicting the functional effects of amino acid substitutions, showed significant scores for p.R654H. Therefore, R654H might be a risk factor for epidermal barrier dysfunction in some Japanese AD patients.