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Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood-derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME). When patient tumors were implanted on aHM, mHM, and NOD/SCID/IL2rg-/- (NSG) cohorts, tumors appeared earlier and grew faster on NSG and mHM cohorts. We observed that immune cells differentiating in the aHM were relatively more capable of circulating peripherally, invading into tumors and interacting with the TME. A heterologous, human leukocyte antigen (HLA-A) matched cohort also yielded slower growing tumors than non-HLA-matched mHM, indicating that a less permissive immune environment inhibits tumor progression. When the aHM, mHM, and NSG cohorts were treated with immunotherapies mirroring what the originating patients received, tumor growth in the aHM accelerated, similar to the progression observed in the patients. This rapid growth was associated with decreased immune cell infiltration, reduced interferon gamma (IFNγ)-related gene expression, and a reduction in STAT3 phosphorylation, events that were replicated in vitro using tumor-derived cell lines. IMPLICATIONS: Engrafted adult HSPCs give rise to more tumor infiltrative immune cells, increased HLA matching leads to slower tumor initiation and growth, and continuing immunotherapy past progression can paradoxically lead to increased growth.

We have discovered that human vitiligo patients treated with narrow‐band UVB (NBUVB) demonstrated localized resistance to repigmentation in skin sites characterized by distinct cellular and molecular pathways. Using immunostaining studies, discovery‐stage RNA‐Seq analysis, and confirmatory in situ hybridization, we analyzed paired biopsies collected from vitiligo lesions that did not repigment after 6 months of NBUVB treatment (non‐responding) and compared them with repigmented (responding) lesions from the same patient. Non‐responding lesions exhibited acanthotic epidermis, had low number of total, proliferative, and differentiated melanocyte (MC) populations, and increased number of senescent keratinocytes (KCs) and of cytotoxic CD8+ T cells as compared with responding lesions. The abnormal response in the non‐responding lesions was driven by a dysregulated cAMP pathway and of upstream activator PDE4B, and of WNT/β‐catenin repigmentation pathway. Vitiligo‐responding lesions expressed high levels of WNT10B ligand, a molecule that may prevent epidermal senescence induced by NBUVB, and that in cultured melanoblasts prevented the pro‐melanogenic effect of α‐MSH. Understanding the pathways that govern lack of NBUVB‐induced vitiligo repigmentation has a great promise in guiding the development of new therapeutic strategies for vitiligo. [ABSTRACT FROM AUTHOR]

The crosslinked envelope of the mammalian epidermal corneocyte serves as a scaffold for assembly of the lipid barrier of the epidermis. Thus, deficient envelope crosslinking by keratinocyte transglutaminase (TGM1) is a major cause of the human autosomal recessive congenital ichthyoses characterized by barrier defects. Expectations that loss of some envelope protein components would also confer an ichthyosis phenotype have been difficult to demonstrate. To help rationalize this observation, the protein profile of epidermis from loricrin knockout mice has been compared to that of wild type. Despite the mild phenotype of the knockout, some 40 proteins were incorporated into envelope material to significantly different extents compared to those of wild type. Nearly half were also incorporated to similarly altered extents into the disulfide bonded keratin network of the corneocyte. The results suggest that loss of loricrin alters their incorporation into envelopes as a consequence of protein-protein interactions during cell maturation. Mass spectrometric protein profiling revealed that keratin 1, keratin 10, and loricrin are prominent envelope components and that dozens of other proteins are also components. This finding helps rationalize the potential formation of functional envelopes, despite loss of a single component, due to the availability of many alternative transglutaminase substrates.

Loricrin is a major component of the cornified cell envelope, a highly insoluble structure composed of covalently cross-linked proteins. Although loricrin knockout mice only exhibit a mild transient phenotype at birth, they show a marked delay in the formation of an epidermal barrier in utero. We recently discovered that induction of a compensatory response to repair the defective barrier is initiated by amniotic fluid via activation of NF-E2-related factor 2 and identified Sprr2d and Sprr2h as direct transcriptional targets. Proteomic analysis suggested that other proteins were also incorporated into the loricrin knockout cell envelope, in addition to the small proline rich proteins. Here we present evidence suggesting that the late cornified envelope 1 proteins are also compensatory components as determined by their localization within the loricrin knockout cell envelope via immunoelectron microscopy. We also demonstrate that late cornified envelope 1 genes are upregulated at the transcriptional level in loricrin knockout mouse skin and confirm that late cornified envelope 1 genes are transcriptional targets of NRF2. Our present study further highlights the complexity and importance of a compensatory mechanism that evolved in terrestrial animals to ensure the formation of a functional epidermal barrier.

Keratin intermediate filaments (KIFs) protect the epidermis against mechanical force, support strong adhesion, help barrier formation, and regulate growth. The mechanisms by which type I and II keratins contribute to these functions remain incompletely understood. Here, we report that mice lacking all type I or type II keratins display severe barrier defects and fragile skin, leading to perinatal mortality with full penetrance. Comparative proteomics of cornified envelopes (CEs) from prenatal KtyI(-/-) and KtyII(-/-)(K8) mice demonstrates that absence of KIF causes dysregulation of many CE constituents, including downregulation of desmoglein 1. Despite persistence of loricrin expression and upregulation of many Nrf2 targets, including CE components Sprr2d and Sprr2h, extensive barrier defects persist, identifying keratins as essential CE scaffolds. Furthermore, we show that KIFs control mitochondrial lipid composition and activity in a cell-intrinsic manner. Therefore, our study explains the complexity of keratinopathies accompanied by barrier disorders by linking keratin scaffolds to mitochondria, adhesion, and CE formation.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.02.017 Byline: Jillian Guttormsen (a), Maranke I. Koster (c), John R. Stevens (b), Dennis R. Roop (c), Trevor Williams (d), Quinton A. Winger (a) Keywords: AP-2; Tcfap2c; Skin; Epidermis; Keratinocyte; Filaggrin; Keratin; Differentiation; Embryo development Abstract: Summary Sentence: Conditional ablation of AP-2[gamma] results in a delay in skin development and abnormal expression of p63, K14, K1, filaggrin, repetin and secreted Ly6/Plaur domain containing 1, key genes required for epidermal development and differentiation. The development of the epidermis, a stratified squamous epithelium, is dependent on the regulated differentiation of keratinocytes. Differentiation begins with the initiation of stratification, a process tightly controlled through proper gene expression. AP-2[gamma] is expressed in skin and previous research suggested a pathway where p63 gene induction results in increased expression of AP-2[gamma], which in turn is responsible for induction of K14. This study uses a conditional gene ablation model to further explore the role of AP-2[gamma] in skin development. Mice deficient for AP-2[gamma] exhibited delayed expression of p63, K14, and K1, key genes required for development and differentiation of the epidermis. In addition, microarray analysis of E16.5 skin revealed delayed expression of additional late epidermal differentiation genes: filaggrin, repetin and secreted Ly6/Plaur domain containing 1, in mutant mice. The genetic delay in skin development was further confirmed by a functional delay in the formation of an epidermal barrier. These results document an important role for AP-2[gamma] in skin development, and reveal the existence of regulatory factors that can compensate for AP-2[gamma] in its absence. Author Affiliation: (a) Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT, USA (b) Mathematics and Statistics, Utah State University, Logan, UT, USA (c) Department of Dermatology and Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, Aurora, CO, USA (d) Departments of Craniofacial Biology and Cell and Developmental Biology, University of Colorado Denver, Aurora, CO, USA Article History: Received 13 September 2007; Revised 7 February 2008; Accepted 8 February 2008

Mutations in ras and p53 are the most prevalent mutations found in human nonmelanoma skin cancers. Although some p53 mutations cause a loss of function, most result in expression of [...]