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. 2023 Jun 27;15(13):3360.
doi: 10.3390/cancers15133360.

Bioinformatics Screen Reveals Gli-Mediated Hedgehog Signaling as an Associated Pathway to Poor Immune Infiltration of Dedifferentiated Liposarcoma

Erik P Beadle  1 , Natalie E Bennett  1 , Julie A Rhoades  1   2   3   4   5
Affiliations

Bioinformatics Screen Reveals Gli-Mediated Hedgehog Signaling as an Associated Pathway to Poor Immune Infiltration of Dedifferentiated Liposarcoma

Erik P Beadle et al. Cancers (Basel). .

Abstract

Liposarcomas are the most diagnosed soft tissue sarcoma, with most cases consisting of well-differentiated (WDLPS) or dedifferentiated (DDLPS) histological subtypes. While both tumor subtypes can have clinical recurrence due to incomplete resections, DDLPS often has worse prognosis due to a higher likelihood of metastasis compared to its well-differentiated counterpart. Unfortunately, targeted therapeutic interventions have lagged in sarcoma oncology, making the need for molecular targeted therapies a promising future area of research for this family of malignancies. In this work, previously published data were analyzed to identify differential pathways that may contribute to the dedifferentiation process in liposarcoma. Interestingly, Gli-mediated Hedgehog signaling appeared to be enriched in dedifferentiated adipose progenitor cells and DDLPS tumors, and coincidentally Gli1 is often co-amplified with MDM2 and CDK4, given its genomic proximity along chromosome 12q13-12q15. However, we find that Gli2, but not Gli1, is differentially expressed between WDLPS and DDLPS, with a noticeable co-expression signature between Gli2 and genes involved in ECM remodeling. Additionally, Gli2 co-expression had a noticeable transcriptional signature that could suggest Gli-mediated Hedgehog signaling as an associated pathway contributing to poor immune infiltration in these tumors.

Keywords: Gli2; differentiation; hedgehog signaling; immune infiltration; liposarcoma; microenvironment.

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Conflict of interest statement

The authors declare no conflict of interest for this work and any subsequent publications that will be derived from these findings.

Figures

Figure 1
Figure 1
Comparative transcriptional analysis reveals enrichment of Hedgehog signaling in dedifferentiated adipose progenitors and dedifferentiated liposarcoma tumors. (A) Data accession and analysis pipeline. Tumor transcriptomics collected from GSE30929. Adipocyte differentiation transcriptomics collected from GSE20752. FDR p-value adjustment (0.05), no fold change threshold. (B) Differentially expressed genes between adipose progenitors and mature adipocytes. (C) Differentially expressed genes between WDLPS and DDLPS tumors. (D) Upregulated genes in DDLPS and Adipose progenitors. (E) Upregulated genes in WDLPS and adipocytes. (F) KEGG pathway analysis of overlapping upregulated genes in DDLPS and adipose progenitors. (G) KEGG pathway analysis of overlapping upregulated genes in WDLPS and adipocytes. Presented pathways were top 20 results based on fold enrichment, with an FDR cutoff of p < 0.05. (H) Hedgehog signaling canonically represses adipogenesis, but its overall role in the differentiation of liposarcoma is unknown.
Figure 2
Figure 2
Gli1 amplification can occur with MDM2 and CDK4 amplification. (A) Oncogenic transformation of adipocytes and adipose progenitor cells is often driven by chromosome 12q13-15, which includes MDM2 and CDK4. Gli1, a Hedgehog transcription factor, shares this chromosome segment. (B) Genomics data acquisition and analysis pipeline. (C) Amplification distribution of Gli1, MDM2, and CDK4 in the MSKCC patient cohort (n = 167). (D) Mutual exclusivity test of co-amplifications. * p < 0.05.
Figure 3
Figure 3
Gli2 has higher expression than Gli1 in DDLPS tumors but not as a result of Gli1 amplification. Gli1 and Gli2 mRNA expressions were evaluated in two separate cohorts (TCGA-SARC n = 58) (A) and GSE21124 (n = 50) of DDLPS tumors (B). Gli1 mRNA expression was compared to Gli1 genomic status in the respective cohorts (C,D), as was Gli2 mRNA expression (E,F). Pearson correlation analysis was performed to evaluate the strength of the relationship between Gli1 and Gli2 expression in DDLPS tumors in both cohorts (G,H). Statistical evaluation was performed following Rout’s outlier test with subsequent Student’s t-test or one-way ANOVA when appropriate. Outliers were removed as follows: 3B, Gli1 n = 6; 3C, Diploid n = 7, Gain n = 2, Amp. n = 1; 3D, Diploid, n = 1. Paired values from patient tumors were removed following outlier removal. (ns: not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Figure 4
Figure 4
Gli2 expression and downstream Hedgehog signaling are elevated in DDLPS tumors. Gli1 and Gli2 mRNA expressions were evaluated in two separate cohorts (GSE21122 n = 9 adipose tissue, n = 46 DDLPS) and GSE30929 (n = 52 WDLPS, n = 40 DDLPS) (A). Gli1 and Gli2 expression levels were profiled in 46 DDLPS tumors relative to normal fat samples (n = 9). mRNA z-scores were extracted from CBioportal and outlier corrected using Rout’s outlier test. Seven Gli1 expression outliers were identified, and the corresponding Gli2 expression values were removed from the analysis (B). Gli1 and Gli2 expression levels were evaluated to determine expression levels between WDLPS (n = 52) and DDLPS (n = 40) tumors. Six Gli1 outliers were removed as well as their corresponding Gli2 expression values. Significance was determined using two-way ANOVA with Sidak’s test for multiple comparisons (C). GSEA of expression files collected from GSE30929 cohort (D). (ns: not significant, ** p < 0.01, **** p < 0.0001).
Figure 5
Figure 5
Gene ontology and functional enrichment of Gli2 co-expressed genes. Using CBioPortal, two separate DDLPS cohorts were analyzed using co-expression analysis relative to Gli2. Genes were filtered to have a Spearman’s R of +/− 0.3 as well as q-value < 0.05. Gene lists were input and subsequently analyzed for enrichment across both cohorts. Genes with an R > +0.3 were preferentially enriched for cell cycle and signaling pathways (A,C). Genes with an R < −0.3 were preferentially enriched for inflammatory signaling (B,D). Top 10 pathways were selected based on fold enrichment and FDR cutoff (0.05).
Figure 6
Figure 6
Gli2 co-expression with ECM and immune cell markers. Gene clusters associated with adipose-derived fibroblasts and immune cell populations were identified using the Human Protein Atlas. Gli2 mRNA expression was compared across these markers within the TCGA-SARC DDLPS cohort (n = 58) to evaluate the relationship of Gli2 between stromal cells/fibroblasts (A) and immune cell populations (B). (C) Proposed Model of Gli-mediated Hedgehog (HH) signaling and potential influences over the DDLPS tumor microenvironment.
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