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Secretogranin II influences the assembly and function of MHC class I in melanoma
Experimental Hematology & Oncology volume 12, Article number: 29 (2023)
Melanoma is the deadliest form of skin cancer showing rising incidence over the past years. New insights into the mechanisms of melanoma progression contributed to the development of novel treatment options, such as immunotherapies. However, acquiring resistance to treatment poses a big problem to therapy success. Therefore, understanding the mechanisms underlying resistance could improve therapy efficacy. Correlating expression levels in tissue samples of primary melanoma and metastases revealed that secretogranin 2 (SCG2) is highly expressed in advanced melanoma patients with poor overall survival (OS) rates. By conducting transcriptional analysis between SCG2-overexpressing (OE) and control melanoma cells, we detected a downregulation of components of the antigen presenting machinery (APM), which is important for the assembly of the MHC class I complex. Flow cytometry analysis revealed a downregulation of surface MHC class I expression on melanoma cells that showed resistance towards the cytotoxic activity of melanoma-specific T cells. IFNγ treatment partially reversed these effects. Based on our findings, we suggest that SCG2 might stimulate mechanisms of immune evasion and therefore be associated with resistance to checkpoint blockade and adoptive immunotherapy.
To the Editor,
Melanoma is the deadliest skin cancer type and often associated with poor prognosis despite a variety of treatment options [1, 2]. The major histocompatibility complex class I (MHC-I) presents fragments of intracellular peptides on the cell surface to CD8 + T cells . MHC-I, the TAP complex (transporter associated with antigen processing) and chaperones located in the ER constitute the antigen presenting machinery (APM) [4,5,6]. Impairment of MHC-I assembly could affect the efficiency of immunotherapies relying on activation of CD8 + T cells. SCG2 belongs to the granin family and plays an essential role in secretory granule formation and biogenesis [7, 8]. We showed recently that high SCG2 expression correlates with low survival rate of melanoma patients with metastases . Here, we investigated the role of SCG2 in melanoma and its contribution to immunotherapy resistance.
Analysis of publicly available data of metastatic melanoma patients from DFCI, Nature Medicine 2019 (n = 121; Fig. 1A)  revealed that high intratumoral SCG2 expression (log2 SCG2 ≥ 1) correlated with a tendency towards lower OS compared to low intratumoral SCG2 expression (log2 SCG2 < 1; p = 0.0531). Data from a GSE database (GSE7553)  confirmed higher SCG2 levels in primary melanoma and metastases compared to normal skin (Fig. 1B) and higher levels of SCG2 in primary melanoma compared to nevi (Fig. 1C). By utilizing cell cycle analysis comparing empty vector (EV) control and ectopically SCG2 OE melanoma cells we ascertained no difference in cell cycle phases between both groups (Fig. 1E).
Next, microarray gene expression analysis followed by Reactome, KEGG, and gene ontology database analysis demonstrated that pathways involved in antigen presentation through MHC-I were impaired after SCG2 OE (Additional file 1). Additionally, SCG2 OE decreased the expression of several APM components (Fig. 1F–H).
Hereafter, we analyzed the expression of SCG2 and the HLA genes, which encode the heavy chain of the MHC-I complex, in melanoma patients (n = 87) from a GSE database (GSE7553) and found a highly significant negative correlation between SCG2 and HLA-A, HLA-B and HLA-C expression (Fig. 1I). Furthermore, flow cytometry revealed significantly reduced surface presentation of HLA-ABC on SCG2 OE melanoma cells (Fig. 1J). However, the percentage of HLA-ABC-positive cells was not altered (Additional file 2). We then performed a T cell cytotoxicity assay using SCG2 OE cells and cytotoxic T cells specific for melanoma antigen recognized by T cells (MART)-1. Our data indicate that SCG2 OE cells were more resistant to T cell-induced cytotoxicity compared to EV control cells (Fig. 1K, Additional file 3).
Next, we treated SCG2 OE cells with IFNγ, which enhances MHC-I expression through the activation of the Stat1-pathway12. We observed significant upregulation of HLA-ABC expression on SCG2 OE melanoma cells (Fig. 2A). The percentage of HLA-ABC-positive cells remained unchanged (Additional file 4). Quantification of STAT1 mRNA expression levels showed significant downregulation upon SCG2 OE. However, IFNγ treatment increased STAT1 mRNA expression in EV and SCG2 OE cell lines (Fig. 2B). Western blot analysis demonstrated increased total Stat1 and pStat1 levels after IFNγ treatment in EV and SCG2 OE cells (Fig. 2C). Moreover, we observed a decrease of total Stat1 and pStat1 in untreated SCG2 OE cells.
IFNγ treatment also increased TAP1, TAP2, and B2M mRNA expression in EV and SCG2 OE cells (Fig. 2D). Western blot analysis confirmed upregulation of TAP1, TAP2, and B2M (Fig. 2E).
Thereafter, we examined the effect of the IFNγ treatment on the sensitivity of SCG2 OE cells to T cell-mediated cytotoxicity. We detected a significantly higher sensitivity of IFNγ-treated EV and SCG2 OE cells compared to untreated cells (Fig. 2F, Additional files 5, 6). When comparing IFNγ-treated EV and SCG2 OE cells we found that SCG2 OE cells were less sensitive towards T cell-mediated cytotoxicity .
We demonstrate here that high intratumoral SCG2 levels correlated with worse prognosis for melanoma patients. SCG2 OE led to downregulation of APM components, which resulted in decreased MHC-I expression and reduced sensitivity of melanoma cells towards T cell-induced cytotoxicity. IFNγ treatment partially counteracted downregulation of APM components and MHC-I. However, IFNγ-treated SCG2 OE cells were still more resistant to T cell-induced cytotoxicity. Our results contribute to understanding melanoma immune evasion and the role of SCG2 in this process. Therefore, SCG2 could be a valuable prognostic factor, potentially influencing the success of checkpoint blockade and adoptive immunotherapy.
Availability of data and materials
The raw microarray data generated in this study are available in GEO under accession number GSE203179. Other data that support the findings of this study are available from the corresponding author upon request.
Antigen presenting machinery
Gene encoding calreticulin
Gene encoding calnexin
Human leukocyte antigen
Melanoma antigen recognized by T cells 1
Major histocompatibility complex
Standard error of the mean
Gene encoding tapasin
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We want to thank Sayran Arif-Said, Jennifer Dworacek and Marlene Pach for excellent technical assistance. We also thank Ni-Na Wang and Yiman Wang who provided scientific insights and expertise during the course of this research. We thank the Microarray Unit of the German Cancer Research Center (DKFZ, Heidelberg, Germany) Genomics and Proteomics Core Facility and Flow Cytometry Core Facility of the DKFZ for providing excellent technical assistance and equipment. We also want to thank the NCT-Gewebebank facility, Pathology Unit, University of Heidelberg, for the TMA slide-scanning service. This work is part of the doctoral thesis of Tamara Steinfass.
This project was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—project numbers 259332240/ RTG 2099 and 676288/ UT 112/1–1.
Ethics approval and consent to participate
Declaration of consent was performed based on the ethical votes 2010-318N-MA and 2014-835R-MA (ethics committee II of Heidelberg University, Germany) and was received from all patients included in the study. The study was performed in accordance with the Declaration of Helsinki.
The authors declare no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1: Table S1.
KEGG pathway analysis showing pathways predicted to be decreased in WM266-4 and C32 SCG2 OE melanoma cells compared to their control (EV). Table S2. Gene ontology pathway analysis showing pathways predicted to be decreased in WM266-4 and C32 SCG2 OE melanoma cells compared to their control (EV). Table S3. Reactome pathway analysis showing pathways predicted to be decreased in WM266-4 and C32 SCG2 OE melanoma cells compared to their control (EV).
Additional file 2: Figure S1.
SCG2 OE does not change the percentage of HLA-ABC-positive cells.
Additional file 3: Figure S2.
Correlation of high SCG2 expression with decreased MHC class I surface presentation on melanoma cells.
Additional file 4: Figure S3.
IFNγ treatment does not influence the percentage of HLA-ABC-positive cells or SCG2 expression.
Additional file 5: Figure S4.
SCG2 OE melanoma cells are more resistant to T cell-mediated cytotoxicity.
Additional file 6.
Additional materials and methods.
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Steinfass, T., Poelchen, J., Sun, Q. et al. Secretogranin II influences the assembly and function of MHC class I in melanoma. Exp Hematol Oncol 12, 29 (2023). https://doi.org/10.1186/s40164-023-00387-1
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