Abstract 354: A tissue microarray study to validate key immune-related proteins in melanoma
Authors: Chhabra Gagan , Thornton Jessica , Su Shengqin , Ndiaye Mary A. , Ahmad Nihal 
Published: June 2022

New immunomodulatory therapeutic targets and prognostic biomarkers hold promise in the management of melanoma. Using publicly available human melanoma datasets and bioinformatics analyses, we previously identified 21 immune-related genes that are differentially expressed in metastatic melanoma and have significant association with patient survival (Cancer Res 2021;81(13 Suppl):Abstract 873). Further, we shortlisted five candidate genes that have been implicated in pathogenesis of certain cancers; however, their role in melanoma is not known. These include, (i) Marginal Zone B and B1 cell specific protein (MZB1), an endoplasmic reticulum (ER)-localized protein that is shown to constitutively express in B cells as well as on cancer cells; (ii) SAM Domain, SH3 Domain and Nuclear Localization Signals 1 (SAMSN1), a negative regulator of B-cell activation that regulates cell spreading and cell polarization; (iii) NCK Associated Protein 1 Like (NCKAP1L) that controls lymphocyte development, activation, and proliferation, as well as phagocytosis and migration by neutrophils and macrophages; (iv) Kallikrein Related Peptidase 8 (KLK8), an epidermal protease involved in the skin barrier proteolytic cascade; and (v) Epstein Barr Virus Induced Gene 3 (EBI3) functions in innate immune responses. This study was aimed to validate the expression profile of these five proteins (MZB1, SAMSN1, NCKAP1L, KLK8 and EBI3) in clinical melanoma tissues along with S100, a melanoma biomarker. We performed fluorescent immunostaining of a human melanoma tissue microarray (ME2082c; US Biomax) consisting of 208 melanoma tissue cores (128 cases of primary malignant melanoma, 64 metastatic melanoma, 8 adjacent skin and 8 normal skin tissue) followed by multispectral Vectra Imaging coupled with inForm software analysis. Our results showed significantly increased expression of KLK8, SAMSN1 and EBI3 in S100-positive melanoma cells compared to S100-negative cells. Further, MZB1 and NCKAP1L protein levels were significantly increased in melanoma primary tumors and metastatic tissues compared to normal skin. Furthermore, an increasing trend for KLK8 and SAMSN1 proteins, though not significant, was observed in melanoma tissues. Interestingly, EBI3 expression was not found to be altered between the melanoma and skin tissues. Next, using in vitro cell culture analysis, we quantified MZB1 protein levels using Simple Western in various human melanoma cell lines and normal human melanocytes. We observed increased MZB1 protein levels in four human melanoma cell lines (A375, G361, HS294T, WM115) compared to normal melanocytes. Overall, our study suggests that MZB1 and NCKAP1L could be potential new targets for melanoma management. Additional validation and further evaluation of functional significance of these immune-related proteins using in vitro and in vivo models may lead to new prognostic and therapeutic strategies for melanoma management.

Citation Format: Gagan Chhabra, Jessica Thornton, Shengqin Su, Mary A. Ndiaye, Nihal Ahmad. A tissue microarray study to validate key immune-related proteins in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 354.