Identification of Small-Molecule Inhibitors Targeting Different Signaling Pathways in Cancer-Associated Fibroblast Reprogramming Under Tumor-Stroma Interaction.

Cancer-associated fibroblasts (CAFs) interact reciprocally with tumor cells through various signaling pathways in many cancer types including cutaneous squamous cell carcinoma (cSCC). Among normal fibroblast (NF) subtypes, papillary fibroblasts (PFs) and reticular fibroblasts (RFs) respond distinctly to tumor cell signaling, eventuating the differentiation of RFs, rather than PFs, into CAFs. The regulation of subtype differentiation in fibroblasts remains poorly explored. In this study, we assessed the differences between PFs, RFs, and CAFs, and examined the effects of small-molecule inhibitors targeting the TGFß, PI3K/AKT/mTOR, and NOTCH pathways on the tumor-promoting property of CAFs and CAF reprogramming in 2D and 3D cultures. Blocking TGFß and PI3K strongly deactivated and concurrently induced a PF phenotype in RFs and CAFs. 3D co-culturing a cSCC cell line MET2 with RFs or CAFs led to enhanced tumor invasion, "RF-CAF" transition and cytokine production, which were further repressed by blocking TGFß and PI3K/mTOR pathways, but not NOTCH pathway. In conclusion, the study identified biomarkers for PFs, RFs and CAFs, and displayed different effects of blocking key signaling pathways in CAFs and tumor cell-CAF interplay. These findings prompted a "CAF to PF" therapeutic strategy, and provided perspectives of using included inhibitors in CAF-based cancer therapy.

Application of Doxorubicin-loaded PLGA nanoparticles targeting both tumor cells and cancer-associated fibroblasts on 3D human skin equivalents mimicking melanoma and cutaneous squamous cell carcinoma.

Nanoparticle (NP) use in cancer therapy is extensively studied in skin cancers. Cancer-associated fibroblasts (CAFs), a major tumor microenvironment (TME) component, promote cancer progression, making dual targeting of cancer cells and CAFs an effective therapy. However, dual NP-based targeting therapy on both tumor cells and CAFs is poorly investigated in skin cancers. Herein, we prepared and characterized doxorubicin-loaded PLGA NPs (DOX@PLGA NPs) and studied their anti-tumor effects on cutaneous melanoma (SKCM)(AN, M14) and cutaneous squamous cell carcinoma (cSCC) (MET1, MET2) cell lines in monolayer, as well as their impact on CAF deactivation. Then, we established 3D full thickness models (FTM) models of SKCM and cSCC using AN or MET2 cells on dermis matrix populated with CAFs respectively, and assessed the NPs' tumor penetration, tumor-killing ability, and CAF phenotype regulation through both topical administration and intradermal injection. The results show that, in monolayer, DOX@PLGA NPs inhibited cancer cell growth and induced apoptosis in a dose- and time-dependent manner, with a weaker effect on CAFs. DOX@PLGA NPs reduced CAF-marker expression and had successful anti-tumor effects in 3D skin cancer FTMs, with decreased tumor-load and invasion. DOX@PLGA NPs also showed great delivery potential in the FTMs and could be used as a platform for future functional study of NPs in skin cancers using human-derived skin equivalents. This study provides promising evidence for the potential of DOX@PLGA NPs in dual targeting therapy for SKCM and cSCC.

MKK3 K329 Mutation Attenuates Diabetes-Associated Cognitive Dysfunction by Blocking the MKK3-RAGE Interaction and Inhibiting Neuroinflammation.

The receptor for advanced glycation end products (RAGE) contributes to diabetes-associated cognitive dysfunction (DACD) through the interaction of its C-terminal AAs 2-5 with mitogen-activated protein kinase kinase 3 (MKK3). However, the associated MKK3 binding site is unknown. Here, db/db mice were used as a model for type 2 diabetes. GST pull-down assays and AutoDock Vina simulations were conducted to identify the key RAGE binding site in MKK3. This binding site was mutated to investigate its effects on DACD and to elucidate the underlying mechanisms. The interaction of MKK3 and RAGE, the levels of inflammatory factors, and the activation of microglia and astrocytes were tested. Synaptic morphology and plasticity in hippocampal neurons were assessed via electrophysiological recordings and Golgi staining. Behavioral tests were used to assess cognitive function. In this study, MKK3 bound directly to RAGE via its lysine 329 (K329), leading to the activation of the nuclear factor-κB (NF-κB) signaling pathway, which in turn triggered neuroinflammation and synaptic dysfunction, and ultimately contributed to DACD. MKK3 mutation at K329 reversed synaptic dysfunction and cognitive deficits by downregulating the NF-κB signaling pathway and inhibiting neuroinflammation. These results confirm that neuroinflammation and synaptic dysfunction in the hippocampus rely on the direct binding of MKK3 and RAGE. We conclude that MKK3 K329 binding to C-terminal RAGE (ct-RAGE) is a key mechanism by which neuroinflammation and synaptic dysfunction are induced in the hippocampus. This study presents a novel mechanism for DACD and proposes a novel therapeutic avenue for neuroprotection in DACD.

IL-22-Induced Ubiquitin-Specific Protease 15 Promotes Proliferation and Inflammation of Keratinocytes through Stabilization of Squamous Cell Carcinoma Antigen 2.

Ubiquitin-specific protease 15 (USP15) plays a significant role in regulating various biological processes in several autoimmune diseases and cancers. However, its role in psoriatic keratinocytes (KCs) has not been extensively studied. In this study, we described that USP15 promotes proliferation and inflammation in KCs by stabilizing squamous cell carcinoma antigen 2. We discovered that the expression of USP15 and squamous cell carcinoma antigen 2 was elevated in lesions from patients with clinical psoriasis and an imiquimod-induced psoriatic dermatitis mouse model. USP15 was able to bind, deubiquitinate, and stabilize squamous cell carcinoma antigen 2. Knocking down USP15 resulted in reduced KC inflammation and impaired KC viability and clonogenicity. Topically applying USP15 small interfering RNA significantly ameliorated imiquimod-induced psoriatic dermatitis and reduced the infiltration of T cells and neutrophils. In addition, we determined that IL-22 was a key cytokine that upregulated the expression of USP15. These findings provide insights regarding the mechanisms involved in the proliferation and inflammation of KCs mediated by IL-22, suggesting a potential IL-22-USP15-squamous cell carcinoma antigen 2 axis in the pathogenesis of psoriatic KCs.

The development of in vitro organotypic 3D vulvar models to study tumor-stroma interaction and drug efficacy.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) is a rare disease with a poor prognosis. To date, there's no proper in vitro modeling system for VSCC to study its pathogenesis or for drug evaluation. METHODS: We established healthy vulvar (HV)- and VSCC-like 3D full thickness models (FTMs) to observe the tumor-stroma interaction and their applicability for chemotherapeutic efficacy examination. VSCC-FTMs were developed by seeding VSCC tumor cell lines (A431 and HTB117) onto dermal matrices harboring two NF subtypes namely papillary fibroblasts (PFs) and reticular fibroblasts (RFs), or cancer-associated fibroblasts (CAFs) while HV-FTMs were constructed with primary keratinocytes and fibroblasts isolated from HV tissues. RESULTS: HV-FTMs highly resembled HV tissues in terms of epidermal morphogenesis, basement membrane formation and collagen deposition. When the dermal compartment shifted from PFs to RFs or CAFs in VSCC-FTMs, tumor cells demonstrated more proliferation, EMT induction and stemness. In contrast to PFs, RFs started to lose their phenotype and express robust CAF-markers α-SMA and COL11A1 under tumor cell signaling induction, indicating a favored 'RF-to-CAF' transition in VSCC tumor microenvironment (TME). Additionally, chemotherapeutic treatment with carboplatin and paclitaxel resulted in a significant reduction in tumor-load and invasion in VSCC-FTMs. CONCLUSION: We successfully developed in vitro 3D vulvar models mimicking both healthy and tumorous conditions which serve as a promising tool for vulvar drug screening programs. Moreover, healthy fibroblasts demonstrate heterogeneity in terms of CAF-activation in VSCC TME which brings insights in the future development of novel CAF-based therapeutic strategies in VSCC.

Exploring the molecular targets for Type 2 diabetes-induced Alzheimer's disease through bioinformatics analysis.

Aim: The purpose of this study was to elucidate the potential mechanisms of Alzheimer's disease (AD) induced by Type 2 diabetes mellitus (T2DM) through bioinformatics analysis, to provide new treatment targets for this disease. Methods: We used weighted gene coexpression network analysis and differentially expressed genes analysis to identify significantly differentially expressed genes shared by T2DM and AD. Molecular docking was used to predict possible protein targets for T2DM-induced AD. Results: The direct interaction of CD44 and STAT3 may play a significant role in the development of T2DM-induced AD. Conclusion: A new approach to treating T2DM-associated AD may be provided by these hub genes and their predicted molecular targets.

Human Papillary and Reticular Fibroblasts Show Distinct Functions on Tumor Behavior in 3D-Organotypic Cultures Mimicking Melanoma and HNSCC.

Human dermis can be morphologically divided into the upper papillary and lower reticular dermis. Previously, we demonstrated that papillary (PFs) and reticular (RFs) fibroblasts show distinct morphology and gene expression profiles. Moreover, they differently affect tumor invasion and epithelial-to-mesenchymal transition (EMT) in in vitro 3D-organotypic cultures of cutaneous squamous cell carcinoma (cSCC). In this study, we examined if these distinct effects of PFs and RFs can be extrapolated in other epithelial/non-epithelial tumors such as melanoma and head and neck squamous cell carcinoma (HNSCC). To this end, 3D-Full-Thickness Models (FTMs) were established from melanoma (AN and M14) or HNSCC cell lines (UM-SCC19 and UM-SCC47) together with either PFs or RFs in the dermis. The interplay between tumor cells and different fibroblasts was investigated. We observed that all the tested tumor cell lines showed significantly stronger invasion in RF-FTMs compared to PF-FTMs. In addition, RF-FTMs demonstrated more tumor cell proliferation, EMT induction and basement membrane disruption. Interestingly, RFs started to express the cancer-associated fibroblast (CAF) biomarker α-SMA, indicating reciprocal interactions eventuating in the transition of RFs to CAFs. Collectively, in the melanoma and HNSCC FTMs, interaction of RFs with tumor cells promoted EMT and invasion, which was accompanied by differentiation of RFs to CAFs.

Pathogen Analysis of Superficial Mucocutaneous Mycosis in a Tertiary A-level Hospital from 2007 to 2018.

OBJECTIVE: This study aimed to analyze the fungal species of pathogens isolated from patients with superficial mucocutaneous mycosis from May 2007 to December 2018. METHODS: A retrospective analysis was carried out to determine the pathogenic fungi isolated from patients with superficial fungal infections in the Medical Mycology Clinical Laboratory, Department of Dermatology and Venereology, Union Hospital, from May 2007 to December 2018. RESULTS: A total of 7639 strains were obtained, belonging to 21 genera and 36 species. They mainly consisted of Candida (3707/7639, 48.53%) and dermatophytes (3594/7639, 47.05%). The specimens were skin scales, nail shavings, secretions on the nail grooves, broken or diseased hair and dandruff, secretions or pseudomembrane of the external genitalia, and the oral mucosa. A total of 7300 patients were enrolled in this study, including 3301 males and 3999 females aged 2 months to 92 years old with a median age of 46.04 years old except for 633 patients whose ages were unknown. Two strains of different species were isolated from each of 339 patients at different body sites. The most frequent species were Trichophyton rubrum complex (2906/7639, 38.04%), Candida albicans (2619/7639, 34.28%), and unclassified Candida spp. Dermatophytes were mostly isolated from glabrous skin (2138/3594, 59.49%), with T. rubrum complex being the predominant species. Candida strains were most commonly isolated from mucosal sites (1979/3707, 53.39%), and C. albicans was the most prevalent causative agent. CONCLUSION: The main distribution of pathogenic fungal species isolated from patients with superficial mycosis from 2007 to 2018 in Wuhan, Hubei province and the surrounding areas was that Candida slightly outnumbered dermatophytes. Among all of the isolated strains, T. rubrum complex was the most abundant.

Multicentre clinicopathological study of adenoid cystic carcinoma: A report of 296 cases.

AIMS: Adenoid cystic carcinoma (ACC) is a distinctive tumour. Limited studies involving a large population have reported multicentre systematic analyses of the clinical, pathological and immunohistochemical (IHC) features of ACC as well as the potential role of IHC markers in the prognosis of ACC. METHODS AND RESULTS: The clinical, histopathological and IHC data of 296 cases obtained from two tertiary hospitals were analysed. The age at onset ranged from 12 to 87 years with a median age of 52 years. The male-to-female ratio was 1:1.3. Patients with ACC arising from the lacrimal gland were younger than those with tumours arising from other sites. Patients with tumours in the extra auditory canal and nasopharynx were older than those with tumours in other locations. Histopathologically, solid type ACC was the most frequent in the nasal cavity and paranasal sinus (6/51) group. Tumours arising from the oral cavity most commonly showed perineural invasion (10/60) and margin positivity (11/60). IHC analyses showed that CK8/18, CK7, CK14, epithelial membrane antigen and CD117 were expressed in 35/35 (100%), 87/88 (98.8%), 26/27 (96.2%), 42/43 (97.6%) and 113/120 (94.1%) patients, respectively. CK5/6, P63, smooth muscle actin, calponin and S100 were positively expressed in 73/73 (100%), 111/124 (89.5%), 38/43 (88.3%), 41/50 (82.0%) and 61/92 (66.3%) cases, respectively. S100 proteins were expressed in 54 (54/77) primary cases and two (2/9) metastatic cases (p = 0.013). CONCLUSIONS: ACC is a distinctive tumour that mainly affects middle-aged and elderly individuals, with a mild female predominance. Loss of expression of S100 proteins may be a poor prognostic factor associated with metastasis.

Feasibility Study for the Long-Term Management of Refractory Hyperkeratotic Eczema with Calcipotriol and Betamethasone Dipropionate (Daivobet®), Viaminate and Concomitant Conventional Therapies: A Retrospective Study.

BACKGROUND: The available treatments for refractory hyperkeratotic eczema are inadequate with frustrating results. We, therefore, incorporated Calcipotriol and Betamethasone Dipropionate (Daivobet®), and Viaminate into the mainstay treatment to improve the clinical symptoms. The study aimed to evaluate the efficacy of Daivobet ® and Viaminate as a potential treatment alternative for refractory hyperkeratotic eczema. PATIENTS AND METHODS: Between 2013 and 2015, 61 patients diagnosed with refractory hyperkeratotic eczema (RHE) who had shown inadequate response to conventional therapies were pooled from a single center. Besides, they were all treated with Daivobet ® , Viaminate, and an occlusive dressing mixture containing 5% salicylic acid ointment and 25% zinc oxide paste following inadequate response to conventional therapies (corticosteroids plus 25% zinc oxide paste and 5% salicylic acid ointment). Investigators Global Assessment (IGA) and Patient-Oriented Eczema Measure (POEM) assessed baseline and outcome measures for the degree of hyperkeratinization (0-clear; 3-moderate; 4-severe). RESULTS: Of the 61 patients, 49 (80.3%) patients presented with moderate RHE and 12 (19.7%) with severe RHE. After 24 weeks of treatment, the period for loss of keratinization was significantly lower in patients with moderate RHE (3.9±1.9 weeks) than those with severe RHE (10.8±1.0 weeks) with a P-value <0.01. Furthermore, they required a significantly shorter total treatment duration (10.6 ± 4.3 weeks) than those with severe RHE (20.3±3.6 weeks) with a P-value of <0.01. However, there were no significant differences in post hoc analysis at week 36 with P-values of 0.46 and 1.00 for IGA and POEM, respectively. CONCLUSION: Our results showed that the incorporation of Viaminate and Daivobet® into mainstay treatment was effective and safe for the long-term management of RHE.

Hyaluronidase-Functionalized Silica Nanocarrier for Enhanced Chemo-Immunotherapy through Inducing Immunogenic Cell Death.

The chemo-immunotherapy has become a highly prospective method for cancer treatment, and it has been known that chemotherapeutic drugs [e.g., doxorubicin (DOX)] could trigger antitumor immune responses. Yet, insufficient tumor penetrability and weak immunogenic cell death (ICD) severely limits the therapeutic effect of chemo-immunotherapy against cancer. Herein, we report the design of DOX-loaded silica nanocarriers (DOX@HMSPHs) with hyaluronidase functionalization, which could increase the permeability of drug and induce enhanced ICD effect through the degradation of hyaluronic acid (HA) in the extracellular matrix (ECM). Interestingly, the controlled release of DOX from DOX@HMSPHs in the acidic microenvironment induced ICD of tumor cells to release tumor antigens and damage-associated molecular patterns, promoting the antigen-presentation of dendritic cells (DCs) and the activation of specific tumor immunity. Moreover, HMSPHs could be used as an immune adjuvant to promote maturation of DCs, thereby promoting the activation of tumor infiltrating cytotoxic T lymphocytes. This strategy presents a concept to improve the efficacy of chemo-immunotherapy through degradation of HA in the ECM.

The potential of Diosgenin in treating psoriasis: Studies from HaCaT keratinocytes and imiquimod-induced murine model.

AIMS: Psoriasis is a cutaneous disease mainly characterized by keratinocyte hyperproliferation, abnormal epidermal differentiation, inflammation and angiogenesis. In this study, we aimed to report the therapeutic potential of Diosgenin on psoriasis-like models and explore the underlying mechanisms. MAIN METHODS: For in vitro studies, we initially evaluated the bioeffects of Diosgenin on keratinocytes by detecting the cell viability, cell cycle and apoptosis in HaCaT cells. To mimic psoriatic conditions, we established hyperproliferative model by stimulating HaCaT cells with LPS/IL-22 and inflammatory model by LPS/TNF-α stimulation. Meanwhile, differentiation in HaCaT cells and angiogenesis in HUVECs/HMEC-1 were observed. The influence of Diosgenin on above-mentioned conditions was examined. For in vivo studies, we dosed imiquimod (IMQ) -induced mice with Diosgenin and conducted hematoxylin-eosin (HE), TUNEL assay and immunohistochemistry (IHC) to evaluate histological changes, apoptosis and the status of keratinocyte proliferation, epidermal differentiation, vascularity and cutaneous inflammatory cell infiltration respectively. KEY FINDINGS: Results showed that Diosgenin inhibited HaCaT cell growth through cell cycle arrest and NFκB inhibition while induced apoptosis by regulating Caspase3, Bax and Bcl-2 protein expression. After Diosgenin treatment, NFκB nuclear translocation and IL-22 receptor dependent pathways were suppressed in LPS/IL-22 induced HaCaT cells respectively. Furthermore, Diosgenin downregulated proinflammatory cytokines through TLR4/Myd88 inhibition and upregulated several differentiation markers' expression in HaCaT cells. Additionally, Diosgenin inhibited vascular formation partially by reducing the VEGF-α expression in keratinocytes. In animal studies, Diosgenin attenuated psoriatic lesions on mice accordingly. SIGNIFICANCE: This study suggests that Diosgenin might be a promising candidate for developing anti-psoriatic agents.

Identification and Interaction Analysis of Key Genes and MicroRNAs in Systemic Sclerosis by Bioinformatics Approaches.

Systemic sclerosis (SSc) is a highly heterogeneous autoimmune disease with a high mortality rate. However, the cellular and molecular mechanisms of SSc remain unclear. Here, we identified the key hub genes and microRNAs (miRNAs) that modulate the occurrence and development of SSc. We downloaded the microarray dataset GSE95065 from the Gene Expression Omnibus (GEO) database and then analyzed the data by using GEO2R. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for functional pathway enrichment analyses of differentially expressed genes (DEGs), and Cytoscape software was used to generate the protein-protein interaction (PPI) network. In addition, OmicsNet was used to predict the miRNAs for the hub genes of SSc. As a result, 783 DEGs were identified, of which 770 genes (142 up-regulated genes and 628 down-regulated genes) were matched to the genes in SSc skin samples. Gene Ontology (GO) analyses by DAVID indicated that the up-regulated genes were mainly involved in immune response, and the down-regulated genes were greatly enriched in glycinergic synaptic transmission. In the PPI network, 22 nodes were selected as key genes, including several members of the chemokine family. Furthermore, after uploading these key genes to the OmicsNet tool, we found that hsa-miR-26b-5p might target CXCL9 and CXCL13. Moreover, we demonstrated that the hsa-miR-26b-5p inhibitor might inhibit fibrosis in TGF-ß-activated fibroblasts, which would be a promising target for SSc therapy.

Increased Cthrc1 Activates Normal Fibroblasts and Suppresses Keloid Fibroblasts by Inhibiting TGF-ß/Smad Signal Pathway and Modulating YAP Subcellular Location.

Keloid may induce severe impairment of life quality for the patients, although keloid is a cutaneous benign tumor. Collagen triple helix repeat containing protein 1 (Cthrc1) was identified as a novel gene that was originally found in adventitial fibroblasts after arterial injury. To address the role of Cthrc1 in keloid, the expression level of Cthrc1 was assessed in normal skin and keloid tissue, as well as in normal fibroblasts (NFs) and keloid fibroblasts (KFs) by using quantitative PCR, Western blotting and immunohistochemical analysis. The results showed that Cthrc1 was increased in keloid tissue and KFs as compared with normal skin and NFs. Meanwhile, CCK8 and Transwell assays found the cellular proliferation and migration of KFs were increased as compared with NFs. Further, to verify the function of Cthrc1 in NFs and KFs, we increased Cthrc1 expression by transfecting lentivirus (LV) vectors LV-Cthrc1. The cellular proliferation and migration, collagen synthesis and the influence on TGF-ß and YAP signaling were tested. The cellular proliferation and migration were increased in NFs-Cthrc1 as compared with NFs-control. Meanwhile, YAP expression and nuclear-location was increased in NFs-Cthrc1. On the contrary, when Cthrc1 was overexpressed in KFs, the cellular migration was suppressed and YAP expression was reduced and transferred to cytoplasm in KFs-Cthrc1 as compared with KFs-control. But the expression level of collagen I was decreased and pSmad2/3 nucleus transfer was suppressed in both NFs-Cthrc1 and KFs-Cthrc1 by using Western blotting and immunofluorescence. Increased Cthrc1 activated NFs by promoting YAP nucleus translocation, whereas suppressed KFs by inhibiting YAP nucleus translocation. Enhanced Cthrc1 decreased collagen I in both NFs and KFs by inhibiting TGF-ß/Smad pathway. In conclusion, Cthrc1 may play a role in the pathogenesis of keloid by inhibiting collagen synthesis and fibroblasts migration via suppressing TGF-ß/Smad pathway and YAP nucleus translocation.