Human melanomas and ovarian cancers overexpressing mechanical barrier molecule genes lack immune signatures and have increased patient mortality risk

Abstract

We have identified eight genes whose expression in human melanoma metastases and ovarian cancers is associated with a lack of Th1 immune signatures. They encode molecules with mechanical barrier function in the skin and other normal tissues and include filaggrin (FLG), tumor-associated calcium signal transducer 2 (TACSTD2), and six desmosomal proteins (DST, DSC3, DSP, PPL, PKP3, and JUP). This association has been validated in an independent series of 114 melanoma metastases. In these, DST expression alone is sufficient to identify melanomas without immune signatures, while FLG and the other six putative barrier molecules are overexpressed in a different subset of melanomas lacking immune signatures. Similar associations have been identified in a set of 186 ovarian cancers. RNA-seq data from 471 melanomas and 307 ovarian cancers in the TCGA database further support these findings and also reveal that overexpression of barrier molecules is strongly associated with early patient mortality for melanoma (p = 0.0002) and for ovarian cancer (p < 0.01). Interestingly, this association persists for FLG for melanoma (p = 0.012) and ovarian cancer (p = 0.006), whereas DST overexpression is negatively associated with CD8⁺ gene expression, but not with patient survival. Thus, overexpression of FLG or DST identifies two distinct patient populations with low immune cell infiltration in these cancers, but with different prognostic implications for each. These data raise the possibility that molecules with mechanical barrier function in skin and other tissues may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction.

Keywords: Adherens junction; TIL; cancer immunology; desmosome; filaggrin; immune privilege; immunosuppression; melanoma; ovarian cancer; tumor microenvironment.

Figure 1.

Filaggrin and desmosome-associated gene transcripts in a subset of melanomas lacking immune signature genes.

Figure 2.

Transcriptional profiling of 113 melanoma metastases and 180 ovarian adenocarcinomas and of melanoma cell lines. Self-organizing heat maps display selected immune signature transcripts together with the expression of mechanical barrier molecules. Genes highlighted in yellow are associated with mechanical barrier function, and in green constitute a Th1 immune signature. For melanoma (A), categories of gene expression are grouped within yellow boxes, from the right, into four subclusters: (z) robust immune signatures, and lacking barrier molecule expression, (y) low or mixed immune signatures and sporadic barrier molecule expression, (x) absent immune signatures with DST overexpression, and (w) overexpression of at least three barrier molecule genes and low level of immune genes. For ovarian cancer (B), yellow boxes mark 3 of the same categories (z, w, x). For both graphs, orange rectangles identify a small subset of tumors in subcluster (w) with high expression of some barrier molecules (but not DST), and limited expression of chemokines and IRF1. (C) Gene–gene matrix correlation between barrier molecule genes in melanoma metastases and their matched cell lines. Gene expression data are from a subset of melanoma metastases (n = 15) for which matched cell lines (n = 15) were available. Each square represents the Pearson product-moment correlation (R), obtained by correlating: (i) genes within tumor metastases, (ii) genes within tumor cell lines or (iii) genes between tumor cell lines and matched tumor metastases. Pink indicates direct correlation, while green inverse correlation. Genes in tumor metastases are labeled with their gene symbol, whereas genes in tumor cell lines are labeled by “cl,” followed by their gene symbol.

Figure 3.

A Filaggrin staining in human tissues: (from top to bottom) normal skin, spleen, placenta, metastatic melanoma, metastatic melanoma and ovarian carcinoma. Specimens are double-stained with filaggrin (blue) and CD45 (purple for spleen, placenta and melanoma, and brown for ovarian cancer); a methyl green counterstain identifies nuclei. (B) Visual analog scale grading filaggrin expression in metastatic melanoma. Scores are based on the number of positively stained cells per 40X field. White arrowheads indicate positively staining cells in levels 1 and 2. (C) Filaggrin staining in melanoma TMA cores with low and high CD8⁺ infiltration. Levels of staining: 0 (white), 1 (dotted gray), 2 (dark diagonal hash lines), 3 (black), n = number of tumor cores examined.

Figure 4.

(A) Periplakin (blue) and (B) desmoplakin (blue) staining in melanoma and ovarian carcinoma specimens. Melanoma and ovarian carcinoma specimens are double-stained with CD45 (purple for melanoma, and brown for ovarian CA), and a methyl green counterstain identify nuclei. Tissues controls for each panel are spleen (top left, negative control), and normal skin (bottom left, positive control).

Figure 5.

Overexpression of barrier molecules identifies melanomas that lack immune signatures and are largely distinct from those that overexpress endothelin receptor B or WNT/β-catenin genes. (A) Overexpression of genes in each tumor is shown by a red bar (z > 1.5), and reduced expression is shown by a blue bar (z< −1.5). Th1 immune genes (CD8A, CD8B, IFN-gamma, CXCL10) are grouped at the top (Immune); then ETNBR (E) ; then barrier molecule genes (FLG, TACSTD2, DST, DSP, DSC3, PPL, PKP3, JUP) are grouped in the middle (Barrier molecules), and genes in the β-catenin/WNT pathway (EFNB3, APC2, MYC, TCF12, VEGFA, WNT7B, SOX2, SOX11, FZD3) are grouped at the bottom. This image was obtained from the TCGA bioportal (cbioportal.org); (B) The proportion of 437 “cold” melanomas lacking overexpression of Th1 immune genes in the TCGA database are shown, with overexpression of barrier molecule genes (OBM), β-catenin/WNT genes (BCAT), the endothelin B receptor gene (ETBNR) alone or in combination, as indicated; (C) Tumors were organized into three groups based on the number of Th1-immune genes overexpressed (0 = Th1-none; 1–2 = Th1-low; 3–4 = Th1-high); For each tumor, the number of the eight barrier molecule genes that are overexpressed is represented by a circle, the number of the nine β-catenin/WNT1 genes that are overexpressed is identified by an x, and overexpression of ETNBR is indicated by a plus sign.

Figure 6.

Associations between overall survival and overexpression of barrier molecule genes or β-catenin/WNT1 genes in melanoma or ovarian cancer, in The Cancer Genoma Atlas. Overall survival of patients with melanoma is significantly decreased with overexpression of genes for barrier molecules filaggrin (A), TACSTD2 (B), but not dystonin (C). Survival is significantly decreased for all melanomas overexpressing any of the eight barrier molecules (D). Survival is not associated with overexpression of endothelin receptor B (E) or any of the β-catenin/WNT genes (EFNB3, APC2, MYC, TCF12, VEGFA, WNT7B, SOX2, SOX11, FZD3) in melanoma (F). In ovarian cancer, overexpression of FLG is associated with worse survival (G), whereas DST overexpression is not associated with lower survival (H). On the other hand, expression of one or more of the barrier molecules FLG, TACSTD2, DST, DSC3, JUP was associated with decreased patient survival (I). In each graph, the red line represents the tumors with overexpression of one or more of the selected genes. Overexpression was based on a z score of 1.5 or greater.