UBE2N is essential for maintenance of skin homeostasis and suppression of inflammation.

UBE2N, a Lys63-ubiquitin conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n-knockout (KO) in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration, as well as signs of edema and blistering. Single cell transcriptomic analyses and RT-qPCR showed that Ube2n KO keratinocytes expressed elevated myeloid cell chemo-attractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemo-attractant Ccl27a. Consistently, the infiltrating immune cells were predominantly myeloid-derived cells including neutrophils and M1-like macrophages that expressed high levels of inflammatory cytokines such as Il1ß and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated inflammation, epidermal and dermal thickening, and immune infiltration of the Ube2n mutant skin. Together, these findings highlight a key role of keratinocyte-UBE2N in maintenance of epidermal homeostasis and skin immunity, and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

Corrigendum: Co-treatment of chloroquine and trametinib inhibits melanoma cell proliferation and decreases immune cell infiltration.

[This corrects the article DOI: 10.3389/fonc.2022.782877.].

UBE2N is essential for maintenance of skin homeostasis and suppression of inflammation.

UBE2N, a Lys63-ubiquitin conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n-knockout (KO) in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included eczematous inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration, as well as signs of edema and blistering. Single cell transcriptomic analyses and RT-qPCR showed that Ube2n KO keratinocytes expressed elevated myeloid cell chemo-attractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemo-attractant, Ccl27a. Consistently, the infiltrating immune cells of Ube2n-KO skin were predominantly myeloid-derived cells including neutrophils and M1-like macrophages that were highly inflammatory, as indicated by expression of Il1ß and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated eczema, epidermal and dermal thickening, and immune infiltration of the Ube2n mutant skin. Together, these findings highlight a key role of keratinocyte-UBE2N in maintenance of epidermal homeostasis and skin immunity and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

Co-Treatment of Chloroquine and Trametinib Inhibits Melanoma Cell Proliferation and Decreases Immune Cell Infiltration.

Autophagy is characterized as a cytoprotective process and inhibition of autophagy with medicinally active agents, such as chloroquine (CQ) is proposed as a prospective adjuvant therapy for cancer. Here, we examined the preclinical effects of CQ combined with the MEK inhibitor trametinib (TRA) on melanoma. We found that cotreatment of CQ and TRA markedly slowed melanoma growth induced in Tyr-CreER.BrafCa.Ptenfl/fl mice. Immunostaining showed that trametinib decreased Ki-67+ proliferating cells, and increased TUNEL+ apoptotic cells. The combo treatment induced a further decrease of Ki-67+ proliferating cells. Consistent with the in vivo findings, CQ and TRA inhibited melanoma cell proliferation in vitro, which was correlated by decreased cyclin D1 expression. In addition, we found that tissues treated with CQ and TRA had significantly decreased numbers of CD4+ and CD8+ T-lymphocytes and F4/80+ macrophages. Together, these results indicate that cotreatment of CQ and TRA decreases cancer cell proliferation, but also dampens immune cell infiltration. Further study is warranted to understand whether CQ-induced immune suppression inadvertently affects therapeutic benefits.

Potential Utility of Synthetic D-Lactate Polymers in Skin Cancer.

Increased breakdown of glucose through glycolysis in both aerobic and anaerobic conditions is a hallmark feature of mammalian cancer and leads to increased production of L-lactate. The high-level lactate present within the tumor microenvironment is reused as a crucial biofuel to support rapid cancer cell proliferation, survival, and immune evasion. Inhibitors that target the glycolysis process are being developed for cancer therapy. In this study, we report an approach of using synthetic D-lactate dimers to inhibit melanoma and squamous cell carcinoma cell proliferation and survival. We also provide in vivo evidence that intratumoral injection of D-lactate dimers induced an innate immune response and inhibited subcutaneous melanoma xenograft growth in immunodeficient mice. Our findings support a potential utility of D-lactate dimers in skin cancer treatment and therefore warrant further mechanistic studies.

Effect of FOCUS-PDCA procedure on improving self-care ability of patients undergoing colostomy for rectal câncer.

OBJECTIVE: To investigate the effect of the FOCUS-PDCA procedure on the self-care ability of patients undergoing colostomy for rectal cancer. METHOD: A nonrandomized controlled trial of 160 patients with rectal cancer undergoing colostomy. The control group received routine nursing intervention, and the observation group received the FOCUS-PDCA procedure. The self-care ability of the two groups was investigated 1 week and 1 month after surgery, and a comparative analysis was made between the groups. RESULTS: One week after surgery, the self-care ability of rectal cancer patients with colostomy increased from 39.09 points before implementation of the FOCUS-PDCA procedure to 60.15 points after implementation; an increase of 21.06%. One month after surgery, the self-care ability increased from 61.50 points to 83.13 points after implementation of the FOCUS-PDCA procedure; an increase of 21.63%. CONCLUSION: Application of the FOCUS-PDCA procedure improved the self-care ability of rectal cancer patients undergoing colostomy, improved their physical and mental health, reduced colostomy complications, and improved their quality of life. The results suggest that it is worth applying FOCUS-PDCA more widely.

Epithelia-Sensory Neuron Cross Talk Underlies Cholestatic Itch Induced by Lysophosphatidylcholine.

BACKGROUND & AIMS: Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of antipruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus. METHODS: Pruritogenicity of lysophosphatidylcholine (LPC), LPA's precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC's pruritogenicity involving keratinocyte TRPV4 was studied using genetic and pharmacologic approaches, cultured keratinocytes, ion channel physiology, and structural computational modeling. Activation of pruriceptor sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in vitro and ex vivo Ca2+ imaging assays. Sera from patients with primary biliary cholangitis were used for measuring the levels of LPC and miR-146a. RESULTS: LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. Three-dimensional structural modeling, site-directed mutagenesis, and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4 activation by LPC induced extracellular release of miR-146a, which activated TRPV1+ sensory neurons to cause itch. LPC and miR-146a levels were both elevated in sera of patients with primary biliary cholangitis with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates. CONCLUSIONS: We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron cross talk, whereby it directly activates skin keratinocyte TRPV4, which rapidly releases miR-146a to activate skin-innervating TRPV1+ pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons, and central neural pathways supporting pruriception.

Effect of FOCUS-PDCA procedure on improving self-care ability of patients undergoing colostomy for rectal câncer / Efeito do procedimento FOCUS-PDCA na melhoria da capacidade de autocuidado de pacientes submetidos a colostomia por câncer retal / Efecto del procedimiento FOCUS-PDCA en la mejora de la capacidad de autocuidado de los pacientes sometidos a colostomía por cáncer de recto

ABSTRACT Objective: To investigate the effect of the FOCUS-PDCA procedure on the self-care ability of patients undergoing colostomy for rectal cancer. Method: A nonrandomized controlled trial of 160 patients with rectal cancer undergoing colostomy. The control group received routine nursing intervention, and the observation group received the FOCUS-PDCA procedure. The self-care ability of the two groups was investigated 1 week and 1 month after surgery, and a comparative analysis was made between the groups. Results: One week after surgery, the self-care ability of rectal cancer patients with colostomy increased from 39.09 points before implementation of the FOCUS-PDCA procedure to 60.15 points after implementation; an increase of 21.06%. One month after surgery, the self-care ability increased from 61.50 points to 83.13 points after implementation of the FOCUS-PDCA procedure; an increase of 21.63%. Conclusion: Application of the FOCUS-PDCA procedure improved the self-care ability of rectal cancer patients undergoing colostomy, improved their physical and mental health, reduced colostomy complications, and improved their quality of life. The results suggest that it is worth applying FOCUS-PDCA more widely.

RESUMO Objetivo: Investigar o efeito do procedimento FOCUS-PDCA na habilidade de autocuidado de pacientes submetidos a colostomia por câncer retal. Método: Um ensaio clínico não randomizado com 160 pacientes com câncer retal submetidos a colostomia. O grupo controle recebeu intervenção de enfermagem de rotina, e o grupo observação recebeu o procedimento FOCUS-PDCA. A capacidade de autocuidado dos dois grupos foi investigada por 1 semana e 1 mês após a cirurgia, e foi feita uma análise comparativa entre os grupos. Resultados: Em uma semana após a cirurgia a capacidade de autocuidado de pacientes com câncer retal com colostomia aumentou de 39,09 pontos antes da implementação do procedimento FOCUS-PDCA para 60,15 pontos após a implementação; um aumento de 21,06%. Em um mês após a cirurgia, a capacidade de autocuidado aumentou de 61,50 pontos para 83,13 pontos após a implantação do procedimento FOCUS-PDCA; um aumento de 21,63%. Conclusão: A aplicação do procedimento FOCUS-PDCA melhorou a capacidade de autocuidado de pacientes com câncer retal submetidos a colostomia, melhorou sua saúde física e mental, reduziu as complicações da colostomia e melhorou sua qualidade de vida. Os resultados sugerem que vale a pena aplicar o FOCUS-PDCA de forma mais ampla.

RESUMEN Objetivo: Investigar el efecto del procedimiento FOCUS-PDCA sobre la capacidad de autocuidado de pacientes sometidos a colostomia por cáncer de recto. Método: Un ensayo controlado no aleatorizado de 160 pacientes con cáncer de recto sometidos a colostomia. El grupo de control recibió una intervención de enfermería de rutina y el grupo de observación recibió el procedimiento FOCUS-PDCA. La capacidad de autocuidado de los dos grupos se investigó 1 semana y 1 mes después de la cirugía, y se realizó un análisis comparativo entre los grupos. Resultados: En una semana después de la cirugía la capacidad de autocuidado de los pacientes con cáncer de recto con colostomía aumentó de 39,09 puntos antes de la implementación del procedimiento FOCUS-PDCA a 60,15 puntos después de la implementación; un aumento del 21,06%. En un mes después de la cirugía, la capacidad de autocuidado aumentó de 61,50 puntos a 83,13 puntos después de la implementación del procedimiento FOCUS-PDCA; un aumento del 21,63%. Conclusión: La aplicación del procedimiento FOCUS-PDCA mejoró la capacidad de autocuidado de los pacientes con cáncer de recto sometidos a colostomía, mejoró su salud física y mental, redujo las complicaciones de la colostomía y mejoró su calidad de vida. Los resultados sugieren que vale la pena aplicar FOCUS-PDCA de manera más amplia.

The Ubiquitin-Modifying Enzyme A20 Terminates C-Type Lectin Receptor Signals and Is a Suppressor of Host Defense against Systemic Fungal Infection.

C-type lectin receptors (CLRs) play key roles in antifungal defense. CLR-induced NF-κB is central to CLR functions in immunity, and thus, molecules that control the amplitude of CLR-induced NF-κB could profoundly influence host defense against fungal pathogens. However, little is known about the mechanisms that negatively regulate CLR-induced NF-κB, and molecules which act on the CLR family broadly and which directly regulate acute CLR-signaling cascades remain unidentified. Here, we identify the ubiquitin-editing enzyme A20 as a negative regulator of acute NF-κB activation downstream of multiple CLR pathways. Absence of A20 suppression results in exaggerated CLR responses in cells which are A20 deficient and also cells which are A20 haplosufficient, including multiple primary immune cells. Loss of a single allele of A20 results in enhanced defense against systemic Candida albicans infection and prolonged host survival. Thus, A20 restricts CLR-induced innate immune responses in vivo and is a suppressor of host defense against systemic fungal infection.

UBE2N Promotes Melanoma Growth via MEK/FRA1/SOX10 Signaling.

UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry-based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma.Significance: These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. Cancer Res; 78(22); 6462-72. ©2018 AACR.

Epidermal CYLD inactivation sensitizes mice to the development of sebaceous and basaloid skin tumors.

The deubiquitinase-encoding gene Cyld displays a dominant genetic linkage to a wide spectrum of skin-appendage tumors, which could be collectively designated as CYLD mutant-syndrome (CYLDm-syndrome). Despite recent advances, little is understood about the molecular mechanisms responsible for this painful and difficult-to-treat skin disease. Here, we generated a conditional mouse model with epidermis-targeted expression of a catalytically deficient CYLDm through K14-Cre-mediated deletion of exon 9 (hereafter refer to CyldEΔ9/Δ9 ). CyldEΔ9/Δ9 mice were born alive but developed hair and sebaceous gland abnormalities and dental defects at 100% and 60% penetrance, respectively. Upon topical challenge with DMBA/TPA, these animals primarily developed sebaceous and basaloid tumors resembling human CYLDm-syndrome as opposed to papilloma, which is most commonly induced in WT mice by this treatment. Molecular analysis revealed that TRAF6-K63-Ubiquitination (K63-Ub), c-Myc-K63-Ub, and phospho-c-Myc (S62) were markedly elevated in CyldEΔ9/Δ9 skin. Topical treatment with a pharmacological c-Myc inhibitor induced sebaceous and basal cell apoptosis in CyldEΔ9/Δ9 skin. Consistently, c-Myc activation was readily detected in human cylindroma and sebaceous adenoma. Taken together, our findings demonstrate that CyldEΔ9/Δ9 mice represent a disease-relevant animal model and identify TRAF6 and c-Myc as potential therapeutic targets for CYLDm-syndrome.

Reactive oxygen species, but not Ca2+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion.

We investigated the role of pH, reactive oxygen species (ROS), Ca2+, and the mitochondrial permeability transition (MPT) in pH-dependent ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 3 h to simulate ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (cyclosporin A and N-methyl-4-isoleucine cyclosporin) and antioxidants (desferal, diphenylphenylene diamine, and 2-mercaptopropionyl glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with tetramethylrhodamine methyl ester, calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca2+ imaging, myocytes were coloaded with rhod-2 and fluo-4 to evaluate mitochondrial and cytosolic Ca2+, respectively. After 10 min of reperfusion at pH 7.4, calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion, antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing. Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca2+ increased during ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca2+ overloading again occurred late after reperfusion. This late Ca2+ overloading was blocked by MPT inhibition. Intramitochondrial Ca2+ chelation by cold loading/warm incubation of BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca2+ overloading appears to be the consequence of bioenergetic failure after the MPT and is not a factor promoting MPT onset.