miR-378a regulates keratinocyte responsiveness to interleukin-17A in psoriasis.

BACKGROUND: Psoriasis is an immune-mediated inflammatory skin disease, in which an interplay between infiltrating immune cells and keratinocytes sustains chronic skin inflammation. Interleukin (IL)-17A is a key inflammatory cytokine in psoriasis and its main cellular targets are keratinocytes. OBJECTIVES: To explore the role of miR-378a in psoriasis. METHODS: Keratinocytes obtained from psoriatic skin and healthy epidermis were separated by magnetic sorting, and the expression of miR-378a was analysed by quantitative polymerase chain reaction. The regulation and function of miR-378a was studied using primary human keratinocytes. The expression of miR-378a was modulated by synthetic mimics, and nuclear factor kappa B (NF-κB) activity and transcriptomic changes were studied. Synthetic miR-378a was delivered to mouse skin in conjunction with induction of psoriasiform skin inflammation by imiquimod. RESULTS: We show that miR-378a is induced by IL-17A in keratinocytes through NF-κB, C/EBP-ß and IκBζ and that it is overexpressed in psoriatic epidermis. In cultured keratinocytes, ectopic expression of miR-378a resulted in the nuclear translocation of p65 and enhanced NF-κB-driven promoter activity even in the absence of inflammatory stimuli. Moreover, miR-378a potentiated the effect of IL-17A on NF-κB nuclear translocation and downstream activation of the NF-κB pathway. Finally, injection of miR-378a into mouse skin augmented psoriasis-like skin inflammation with increased epidermal proliferation and induction of inflammatory mediators. Mechanistically, miR-378a acts as a suppressor of NFKBIA/IκBζ, an important negative regulator of the NF-κB pathway in keratinocytes. CONCLUSIONS: Collectively, our findings identify miR-378a as an amplifier of IL-17A-induced NF-κB signalling in keratinocytes and suggest that increased miR-378a levels contribute to the amplification of IL-17A-driven skin inflammation in psoriasis.

BAFF Attenuates Immunosuppressive Monocytes in the Melanoma Tumor Microenvironment.

Emerging evidence indicates B-cell activating factor (BAFF, Tnfsf13b) to be an important cytokine for antitumor immunity. In this study, we generated a BAFF-overexpressing B16.F10 melanoma cell model and found that BAFF-expressing tumors grow more slowly in vivo than control tumors. The tumor microenvironment (TME) of BAFF-overexpressing tumors had decreased myeloid infiltrates with lower PD-L1 expression. Monocyte depletion and anti-PD-L1 antibody treatment confirmed the functional importance of monocytes for the phenotype of BAFF-mediated tumor growth delay. RNA sequencing analysis confirmed that monocytes isolated from BAFF-overexpressing tumors were characterized by a less exhaustive phenotype and were enriched for in genes involved in activating adaptive immune responses and NF-κB signaling. Evaluation of patients with late-stage metastatic melanoma treated with inhibitors of the PD-1/PD-L1 axis demonstrated a stratification of patients with high and low BAFF plasma levels. Patients with high BAFF levels experienced lower responses to anti-PD-1 immunotherapies. In summary, these results show that BAFF, through its effect on tumor-infiltrating monocytes, not only impacts primary tumor growth but can serve as a biomarker to predict response to anti-PD-1 immunotherapy in advanced disease. SIGNIFICANCE: The BAFF cytokine regulates monocytes in the melanoma microenvironment to suppress tumor growth, highlighting the importance of BAFF in antitumor immunity.

Alterations in the p53 pathway and their association with radio- and chemosensitivity in head and neck squamous cell carcinoma.

Chemotherapy and/or radiotherapy are established measures in treatment protocols of head and neck squamous cell carcinoma (HNSCC). However, we still lack reliable predictive markers for the response to radio- and chemotherapy. The p53 pathway is involved in stress response and thus might influence chemo-/radiosensitivity. Using 29 HNSCC cell lines previously characterized for p53 mutations, we simultaneously analyzed several key players in the p53 pathway by RT-PCR, transcript sequencing and immunohistochemistry, and investigated their association with chemosensitivity and radiosensitivity. Cell lines with p53 mutations were slightly more sensitive to cisplatin than those with wild-type p53. The type of mutation did not influence radio- or chemosensitivity. p14(ARF), an activator of p53, was lost or mutated in all cell lines. Three cell lines showed overexpression of HDM-2, a major negative regulator of p53; however, HDM-2 levels did not correlate with radio- or chemosensitivity. HPV-16 oncoproteins were detected in one highly chemoresistant cell line. Our findings suggest that molecular events resulting in the inactivation of the p53 pathway occur in all HNSCC cell lines. However, single alterations in the p53 pathway are not reliable predictors for the response to radio- or chemotherapy in HNSCC.

Tumor immune escape by the loss of homeostatic chemokine expression.

The novel keratinocyte-specific chemokine CCL27 plays a critical role in the organization of skin-associated immune responses by regulating T cell homing under homeostatic and inflammatory conditions. Here we demonstrate that human keratinocyte-derived skin tumors may evade T cell-mediated antitumor immune responses by down-regulating the expression of CCL27 through the activation of epidermal growth factor receptor (EGFR)-Ras-MAPK-signaling pathways. Compared with healthy skin, CCL27 mRNA and protein expression was progressively lost in transformed keratinocytes of actinic keratoses and basal and squamous cell carcinomas. In vivo, precancerous skin lesions as well as cutaneous carcinomas showed significantly elevated levels of phosphorylated ERK compared with normal skin, suggesting the activation of EGFR-Ras signaling pathways in keratinocyte-derived malignancies. In vitro, exogenous stimulation of the EGFR-Ras signaling pathway through EGF or transfection of the dominant-active form of the Ras oncogene (H-RasV12) suppressed whereas an EGFR tyrosine kinase inhibitor increased CCL27 mRNA and protein production in keratinocytes. In mice, neutralization of CCL27 led to decreased leukocyte recruitment to cutaneous tumor sites and significantly enhanced primary tumor growth. Collectively, our data identify a mechanism of skin tumors to evade host antitumor immune responses.

Reliable detection of p53 aberrations in squamous cell carcinomas of the head and neck requires transcript analysis of the entire coding region.

BACKGROUND: Aberrations of the p53 tumor suppressor gene are common events in squamous cell carcinomas of the head and neck (SCCHN). However, reported frequencies range considerably, and the predictive value of aberrant p53 is continuing to be an issue of controversy. These inconsistencies are possibly caused by methodical limitations. METHODS: In cell lines established from 23 SCCHN, we performed sequence analysis of p53 mRNA and genomic DNA, as well as protein detection using immunohistochemistry and Western blotting. RESULTS: Of the 23 SCCHN, 21 (91%) tumors have aberrant or no p53, including four aberrations (19%) located outside the usually examined exons 5 to 9. A second allele is present in 11, and the respective transcript in 4 tumors. Expression of protein could be detected in only 12 of the 21 p53 aberrant tumors (57%). CONCLUSIONS: Depending on the methodical approach applied, a considerable number of SCCHN may be misclassified regarding their p53 status.

Effects of tamoxifen on human squamous cell carcinoma lines of the head and neck.

Tamoxifen (TAM) is a well-tolerated compound in the treatment of breast cancer and is primarily considered to act by competition with estrogen receptors (ER). Here we investigated the in vitro efficacy and potentially underlying mechanisms of TAM in established cell lines of squamous cell carcinomas of the head and neck (SCCHN). Using proliferation and apoptosis assays the antitumor activity of TAM in five SCCHN and the breast carcinoma line MCF-7 (positive control) was determined. MCF-7 was more sensitive to low-dose TAM (below 1 microM), whereas SCCHN showed significant growth inhibition at higher TAM concentrations (5-10 microM). Growth curve analysis and apoptosis assays were indicative for a cytostatic effect of low-dose TAM and high-dose TAM led to cell loss by apoptosis in sensitive SCCHN. In order to further characterize the observed antitumor effects we determined the amount of steroid hormone receptors with the dextran-coated charcoal method and immunocytochemistry. In addition, production of transforming growth factor (TGF-)-alpha, -beta1 and -beta2 was measured by ELISA, and protein kinase C (PKC) activity was assessed with a radioligand assay. Except MCF-7, none of the SCCHN lines was positive for ER. TAM caused decreased TGF-alpha and increased TGF-beta levels in MCF-7, but not in SCCHN supernatants. Furthermore, the antiestrogen reduced PKC activity in MCF-7, but not in SCCHN. In the present in vitro system, the observed antitumor activity of high-dose TAM in SCCHN cannot be explained by estrogen antagonism, alterations of TGF-alpha/beta levels or decreased PKC activity.