GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation.

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5'-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis.

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.

Basal cell carcinoma of the umbilicus associated with sweat gland structures: Two case reports.

RATIONALE: Basal cell carcinoma (BCC) arising in the umbilicus is relatively rare, and in particular, there have been few reports mentioning peritumoral sweat gland structures histopathologically. We herein, report 2 cases of umbilical BCC with sweat gland structures within and around the tumor. PATIENT CONCERNS: A 61-year-old woman had a 2-year history of black exudative plaque in her umbilicus, and an 80-year-old woman had a 6-month history of dark brownish plaque in the umbilicus, with exudation 2 months prior to her first visit. DIAGNOSES: Based on the histopathological finding, both cases were confirmed as BCC. The results of immunohistochemical staining showed that the tumor cells were Ber-EP4 positive. In addition, EMA-positive glandular structures were seen within and around the tumor. INTERVENTIONS: Curative resection at the level of the linea alba on the bottom side was performed. OUTCOMES: No relapse has been observed since resection in either patient. LESSONS: We herein report 2 cases of umbilical BCC with sweat glands and ducts. Although whether peri- and/or intra-tumor sweat gland structures are the source of the tumor or arise by transdifferentiation from tumor cells remains unclear, these findings may provide clues to help understand the morphopathogenesis of umbilical BCC in the future.

Melanocyte-specific CD49a+ CD8+ T cells in vitiligo lesion potentiate to maintain activity during systemic steroid therapy.

Vitiligo is a common depigmenting skin disease that is often difficult to treat. Even if repigmentation is achieved by treatment, recurrence in the same lesion is often found within a year after stopping treatment. As a background of these issues, a subset of CD8+ T cells that recognize melanocyte-specific antigens or CD49a+ tissue-resident memory T cells that reside in the vitiligo lesion are thought to be involved. We investigated the MHC class I-restricted tyrosinase pentamer-positive CD8+ skin T cells in a progressive generalized vitiligo patient with HLA-A*02:01 who showed resistance to intravenous methylprednisolone pulse therapy. We found that HLA-A*02:01-restricted tyrosinase pentamer-positive CD8+ T cells remained in the lesions after the treatment and expressed IFN-γ and granzyme B. Interestingly, the expression of these cytokines in the pentamer-negative CD8+ T cells was decreased after intravenous methylprednisolone pulse therapy. These findings suggest that, in vitiligo patients, melanocyte-specific CD49a+ CD8+ T cells are in a potent activation state that is uncontrolled despite systemic immunosuppressive treatment, which may contribute to treatment resistance and local recurrence.

Characterization of human epithelial resident memory regulatory T cells.

Human resident memory regulatory T cells (Tregs) exist in the normal, noninflamed skin. Except one, all previous studies analyzed skin Tregs using full-thickness human skin. Considering that thick dermis contains more Tregs than thin epidermis, the current understanding of skin Tregs might be biased toward dermal Tregs. Therefore, we sought to determine the phenotype and function of human epidermal and epithelial Tregs. Human epidermis and epithelium were allowed to float on a medium without adding any exogenous cytokines and stimulations for two days and then emigrants from the explants were analyzed. Foxp3 was selectively expressed in CD4+CD103- T cells in the various human epithelia, as it is highly demethylated. CD4+CD103-Foxp3+ cells suppressed proliferation of other resident memory T cells. The generation and maintenance of epithelial Tregs were independent of hair density and Langerhans cells. Collectively, immune-suppressive CD4+CD103-Foxp3+ Tregs are present in the normal, noninflamed human epidermis and mucosal epithelia.

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.