The association between bromodomain proteins and cancer stemness in different solid tumor types.

Cancer stemness, which covers the stem cell-like molecular traits of cancer cells, is essential for tumor development, progression and relapse. Both transcriptional and epigenetic aberrations are essentially connected with cancer stemness. The engagement of bromodomain (BrD) proteins-a family of epigenetic factors-has been presented in the pathogenesis of several tumor types, although their association with cancer stemness remains largely unknown. Here, we harnessed TCGA and GEO databases and used several bioinformatic tools (ie, Oncomine, PrognoScan, GEPIA2, TIMER2.0, TISIDB, GSEA, R2 platform) to characterize the association between the BrD family members' expression and cancer stemness in solid tumors. Our results demonstrate that significant upregulation of ATAD2 and SMARCA4, and downregulation of SMARCA2 is consistently associated with enriched cancer stem cell-like phenotype, respectively. Especially, higher-grade tumors that display stem cell-like properties overexpress ATAD2. In contrast to most BrD members, the gene expression profiles of ATAD2HIGH expressing tumors are strongly enriched with known markers of stem cells and with specific targets for c-Myc transcription factor. For other BrD proteins, the association with cancer de-differentiation status is rather tumor-specific. Our results demonstrate for the first time the relation between distinct BrD family proteins and cancer stemness across 27 solid tumor types. Specifically, our approach allowed us to discover a robust association of high ATAD2 expression with cancer stemness and reveal its' versatility in tumors. As bromodomains are attractive targets from a chemical and structural perspective, we propose ATAD2 as a novel druggable target for de-differentiated tumors, especially those overexpressing MYC.

The Association between TIF1 Family Members and Cancer Stemness in Solid Tumors.

Cancer progression entails a gradual loss of a differentiated phenotype in parallel with the acquisition of stem cell-like features. Cancer de-differentiation and the acquisition of stemness features are mediated by the transcriptional and epigenetic dysregulation of cancer cells. Here, using publicly available data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and harnessing several bioinformatic tools, we characterized the association between Transcriptional Intermediary Factor 1 (TIF1) family members and cancer stemness in 27 distinct types of solid tumors. We aimed to define the prognostic value for TIF1 members in predicting a stem cell-like cancer phenotype and patient outcome. Our results demonstrate that high expression of only one member of the TIF1 family, namely TIF1ß (also known as Tripartite Motif protein 28, TRIM28) is consequently associated with enriched cancer stemness across the tested solid tumor types, resulting in a worse prognosis for cancer patients. TRIM28 is highly expressed in higher grade tumors that exhibit stem cell-like traits. In contrast to other TIF1 members, only TIF1ß/TRIM28-associated gene expression profiles were robustly enriched with stemness markers regardless of the tumor type. Our work demonstrates that TIF1 family members exhibit distinct expression patterns in stem cell-like tumors, despite their structural and functional similarity. Among other TIF1 members, only TRIM28 might serve as a marker of cancer stemness features.

Melanoma Stem Cell-Like Phenotype and Significant Suppression of Immune Response within a Tumor Are Regulated by TRIM28 Protein.

TRIM28 emerged as a guard of the intrinsic "state of cell differentiation", facilitating self-renewal of pluripotent stem cells. Recent reports imply TRIM28 engagement in cancer stem cell (CSC) maintenance, although the exact mechanism remains unresolved. TRIM28 high expression is associated with worse melanoma patient outcomes. Here, we investigated the association between TRIM28 level and melanoma stemness, and aligned it with the antitumor immune response to find the mechanism of "stemness high/immune low" melanoma phenotype acquisition. Based on the SKCM TCGA data, the TRIM28 expression profile, clinicopathological features, expression of correlated genes, and the level of stemness and immune scores were analyzed in patient samples. The biological function for differentially expressed genes was annotated with GSEA. Results were validated with additional datasets from R2: Genomics Analysis and Visualization Platform and in vitro with a panel of seven melanoma cell lines. All statistical analyses were accomplished using GraphPad Prism 8. TRIM28HIGH-expressing melanoma patients are characterized by worse outcomes and significantly different gene expression profiles than the TRIM28NORM cohort. TRIM28 high level related to higher melanoma stemness as measured with several distinct scores and TRIM28HIGH-expressing melanoma cell lines possess the greater potential of melanosphere formation. Moreover, TRIM28HIGH melanoma tumors were significantly depleted with infiltrating immune cells, especially cytotoxic T cells, helper T cells, and B cells. Furthermore, TRIM28 emerged as a good predictor of "stemness high/immune low" melanoma phenotype. Our data indicate that TRIM28 might facilitate this phenotype by direct repression of interferon signaling. TRIM28 emerged as a direct link between stem cell-like phenotype and attenuated antitumor immune response in melanoma, although further studies are needed to evaluate the direct mechanism of TRIM28-mediated stem-like phenotype acquisition.

Therapeutic melanoma vaccine with cancer stem cell phenotype represses exhaustion and maintains antigen-specific T cell stemness by up-regulating BCL6.

We developed a therapeutic, gene-modified, allogeneic melanoma vaccine (AGI-101H), which, upon genetic modification, acquired melanoma stem cell-like phenotype. Since its initial clinical trial in 1997, the vaccine has resulted in the long-term survival of a substantial fraction of immunized patients (up to 20 years). Here, we investigated the potential molecular mechanisms underlying the long-lasting effect of AGI-101H using transcriptome profiling of patients' peripheral T lymphocytes. Magnetically-separated T lymphocytes from AGI-101H-immunized long-term survivors, untreated melanoma patients, and healthy controls were subjected to transcriptome profiling using the microarray analyses. Data were analyzed with a multitude of bioinformatics tools (WebGestalt, DAVID, GSEA) and the results were validated with RT-qPCR. We found substantial differences in the transcriptomes of healthy controls and melanoma patients (both untreated and AGI-101H-vaccinated). AGI-101H immunization induced similar profiles of peripheral T cells as tumor residing in untreated patients. This suggests that whole stem cells immunization mobilizes analogous peripheral T cells to the natural adaptive anti-melanoma response. Moreover, AGI-101H treatment activated the TNF-α and TGF-ß signaling pathways and dampened IL2-STAT5 signaling in T cells, which finally resulted in the significant up-regulation of a BCL6 transcriptional repressor, a known amplifier of the proliferative capacity of central memory T cells and mediator of a progenitor fate in antigen-specific T cells. In the present study, high levels of BCL6 transcripts negatively correlated with the expression of several exhaustion markers (CTLA4, KLRG1, PTGER2, IKZF2, TIGIT). Therefore, Bcl6 seems to promote a progenitor fate for cancer-experienced T cells from AGI-101H-vaccinated patients by repressing the exhaustion markers.

The role of TRIM family proteins in the regulation of cancer stem cell self-renewal.

The tripartite-motif (TRIM) family of proteins represents one of the largest classes of putative single protein RING-finger E3 ubiquitin ligases. The members of this family are characterized by an N-terminal TRIM motif containing one RING-finger domain, one or two zinc-finger domains called B boxes (B1 box and B2 box), and a coiled-coil region. The TRIM motif can be found in isolation or in combination with a variety of C-terminal domains, and based on C-terminus, TRIM proteins are classified into 11 distinct groups. Because of the complex nature of TRIM proteins, they are implicated in a variety of cellular functions and biological processes, including regulation of cell proliferation, cell division and developmental processes, cancer transformation, regulation of cell metabolism, autophagocytosis, modification of chromatin status, regulation of gene transcription, post-translational modifications, and interactions with pathogens. Here, we demonstrate the specific activities of TRIM family proteins that contribute to the cancer stem cell phenotype. A growing body of evidence demonstrates that several TRIM members guarantee the acquisition of stem cell properties and the ability to sustain stem-like phenotype by cancer cells using distinct mechanisms. For other members, further work is needed to understand their full contribution to stem cell self-renewal. Identification of TRIM proteins that possess the potential to serve as therapeutic targets may result in the development of new therapeutic strategies. Finally, these strategies may result in the disruption of the machinery of stemness acquisition, which may prevent tumor growth, progression, and overcome the resistance to anticancer therapies.