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

Abstract 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 ATAD2 HIGH 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.

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.

| INTRODUCTION
The bromodomain is an evolutionarily conserved, about 110 amino acid-long, protein-protein interaction domains that facilitates the recognition of acetylated lysine residues. This essential activity provides versatile functions to bromodomain-containing (BrD) proteins, primarily associated with the chromatin-templated gene transcription, recombination, replication and repair of the DNA. 1  all BrD members are epigenetic "readers," and numerous of them are known to be involved in the pathogenesis of distinct human diseases, including cancer. [2][3][4][5] Epigenetic dysregulation of gene expression contributes to tumorigenicity (at least partially) via facilitating the self-renewal of cancer cells. 6 Cancer cells that possess the ability to self-renew and to differentiate into the more specialized progeny are known as cancer stem cells (CSCs). This population exhibits highly metastatic potential and facilitates tumor relapse after treatment due to intrinsic resistance to standard therapies. 7,8 Moreover, the high plasticity of CSCs provides the transition between stem-like and non-stem-like states.
Therefore, it is difficult to unequivocally define the CSC population and to determine whether distinct tumor types are organized into a rigid hierarchy. 9 An increasing number of data demonstrates that molecular features characteristic for stem cells ("stemness") are indisputable for cancer progression and therapy resistance. 10,11 Although it is unclear whether the cancer stemness reflects the presence of bona fide CSCs, the molecular signatures sufficient for grading stem cell-like phenotype essentially contribute to the development of novel therapeutic approaches that may directly target the stem cell-like compartment of the tumor. 12 Recent reports suggest that several BrD members play a role in the regulation of the cancer stem cell population in distinct types of tumors. [13][14][15] Especially, the role of BRD4 protein, a member of BET transcriptional coactivators, has been well documented in several studies in vitro. BRD4 regulates the self-renewal of glioma, 16 medulloblastoma, 17 prostate, 18 breast 19 and stomach cancer stem cells, 20 and is essential for normal stem cell maintenance. 15,21 Similarly, TRIM28 (also known as TIF1-β or KAP1) facilitates stemness acquisition in distinct types of tumors, including breast, 22,23 lung 24 and melanoma, 25 and contributes to the stemness machinery of normal stem cells on several distinct levels. 26,27 However, little is known about other BrD family members and their association with cancer stemness. Here, we harnessed publicly available transcriptomic data from 27 distinct types of TCGA tumors to delineate the connection between specific BrD family proteins and cancer stemness measured with previously reported stemness indices or signatures. [10][11][12]28 Using the TIMER2.0 platform, 29 we reported that for most BrD family members, the differential expression in tumor tissues vs normal adjacent tissues could be observed. According to the GEPIA2 database, 30 for several BrD genes, we demonstrated a significant correlation with TCGA cancer patients' outcomes, mostly negative. These results were further validated with additional datasets from the Oncomine, 31 PrognoScan 32 and the GEO databases. Next, we used a transcriptome-based stemness index (mRNA-SI) and other stem cell-derived gene expression signatures [10][11][12]28 to analyze the relation between BrD proteins' expression and the level of tumor dedifferentiation. We observed that among 41 tested BrD family members, the correlation with cancer stemness across 27 tumor types was consistently positive or negative for only five and four BrD genes, respectively (namely ATAD2, BRD7, KAT2A, SMARCA4, TRIM28 [positive], and KAT2B, BAZ2B, SP100 and SMARCA2 [negative]). Using clinicopathologic data, we demonstrated that higher-grade tumors display significant upregulation of ATAD2 and SMARCA4 expression, and downregulation of SMARCA2 expression, which further confirms a universal relation of these BrD proteins' expression with cancer stemness. Moreover, the gene set enrichment analysis (GSEA) 33  for analyses (Table S1). All data is available online, and the access is unrestricted and does not require patients' consent or other permissions. The use of the data does not violate the rights of any person or any institution.

| The expression of BrD family members in distinct TCGA cohorts
The differential expression of BrD family members (Table S2)  The data regarding BrD family members' expression in other GEO datasets was retrieved from the online database, Oncomine (https:// www.oncomine.org/resource/login.html). 30 For further details, see Supporting Information Materials and Methods and Figure S1.

| The association between BrD family members expression and patients' outcome
The association between BrD family members expression and patients' overall survival (OS) across 27 solid TCGA tumor types was analyzed with the Survival_Map panel of the GEPIA2 database (http://gepia2.cancer-pku.cn/#index). 31 The hazard ratio was estimated using the Mantel-Cox test using the mean BrD family members' expression as a cut-off. As for additional GEO cohorts, the PrognoScan (http://dna00.bio.kyutech.ac.jp/PrognoScan/) 32

| TCGA genomic data
Genomic data for 27 solid TCGA tumors were directly downloaded from the cBioportal (www.cbioportal.org) database. 33

| Transcriptomic data
The RNA sequencing-based mRNA expression data were directly downloaded from the cBioportal. RNASeq V2 from TCGA is processed and normalized using RSEM. 34 Specifically, the RNASeq V2 data in cBioPortal corresponds to the rsem.genes.normalized_results file from TCGA. The Spearman's correlation was used for detection of co-expressed genes with P-value <.05 and FDR < 0.01 as cut-offs.

| Stemness-associated scores
The mRNA-SI stemness score 12 and other stemness signatures (Ben-Porath_ES_core, Wong_ESC_core and Bhattacharya_ESC) used in this study were previously described. 10,11,28 Briefly, the mRNA-SI signature was calculated based on previously built predictive model

| Histologic tumor grades
The association between BrD family members' expression and the histologic tumor grade was assessed using the TISIDB portal (http://cis. hku.hk/TISIDB/index.php). 35 The correlation was calculated using Spearman's rank correlation coefficient (r).

| Gene set enrichment analysis
The GSEA (http://www.broad.mit.edu/gsea/index.html) 36 was used to detect the coordinated expression of a priori defined groups of genes within the tested samples. Gene sets are available from the Molecular Signatures Database (MSigDB, http://www.broad.mit.edu/gsea/. msigdb/msigdb_index.html). 37 All significantly DEGs were imported to GSEA. The GSEA was run according to the default parameters: each probe set was collapsed into a single gene vector (identified by its HUGO gene symbol), permutation number = 1000, and permutation type = "gene-sets." The FDR < 0.01 was used to correct for multiple comparisons and gene set sizes.

| The expression of BrD family members in lower and higher-grade tumors
As we have shown previously, higher-grade tumors clearly exhibit stemness characteristics mirrored by elevated mRNA-SI scores, especially in LIHC and UCEC ( Figure S5). 38 Therefore, we determined the association between the expression of BrD encoding genes and the tumor grade ( Figure 3A). We observed significantly higher expression of ATAD2 and significantly lower levels of SMARCA2 in de-differentiated tumors ( Figure 3B), while the level of other previously selected BrD family members (KAT2B, KAT2A and SMARCA4) was relatively unchanged. Although we did not detect statistical significance in the analyses of IHC staining from the Human Protein Atlas 37 ), we suggest that the level of ATAD2 protein is elevated while the level of SMARCA2 is depleted ( Figure S6) in the higher grade LGG tumors, further supporting our first observation.
To unequivocally confirm the enrichment of selected BrDassociated transcriptome profiles with the stemness signatures, we used additional GEO datasets in our studies (Table S4). The results presented in Figure S9 further validated our first observation of The transcriptome profiles associated with the expression of selected BrD family members are enriched with c-Myc and E2F transcription factor targets that significantly mirror the enrichment of mRNA-SI gene signature with the "Hallmarks of cancer" terms. (A) The GSEA using significantly DEGs (P < .05, FDR < 0.05) in TCGA patients divided into low-expressing or high-expressing ATAD2 cohorts (using the mean expression of ATAD2 as a cut-off) was performed with the MSigDB Hallmark (v7.4) collection as a reference. The heatmap presents the normalized enrichment score (NES). White-no statistical significance (P > .05) or no DEGs were detected. Only those Hallmark terms, that were previously determined as significantly enriched (violet) or depleted (yellow) in the mRNA-SI gene signature are presented in the heatmap. we excluded that the abovementioned association results strictly from shared localization. As presented in Figure S13, results obtained for FBXO32, ZHX1 and ANXA13 genes encoded within the same region as ATAD2 clearly emphasize the specificity of ATAD2 and MYC as well as ATAD2 and cancer stemness associations.
All the abovementioned results strongly support our claim that   10,11,28,39,40 The transcriptional program previously recognized in normal stem cells is commonly launched by different human epithelial cancers, which strongly suggests its' prevalence in gaining cancer stemness regardless of the tumor type, and stemness signatures were demonstrated as very efficient in quantifying cancer stemness (commonly reflected in the histopathological grade). 10,28 To date, only several members of BrD family were directly connected with cancer stemness. Especially, the engagement of BRD4, a member of BET transcriptional coactivators, as well as the role for TRIM28-a transcriptional co-repressor, known to mediate E3 SUMO/ubiquitin ligase activity, have been well established in mediating the self-renewal properties of cancer stem cells. [15][16][17][18][19][20][21][22][23][24][25][26][27] We have recently reported that TRIM28 overexpression closely associates with cancer stemness in breast cancer and melanomas and subsequently demonstrated that this phenomenon is very universal across diverse types of solid tumors. 22,25,38 Several potential modes of actions for TRIM28 in obtaining cancer stemness have been suggested including (a) transcriptional co-repression of differentiating genes 27 followed by (b) the enhancement of stem cell markers' expression. 41 Also, TRIM28 might act (c) by targeting for proteasomal degradation (through RING-mediated E3 ubiquitin ligase activity) various proteins, that is, AMPK, a "metabolic switch" that attenuates cancer stemness. 22 As for BRD4, Venkataraman et al 17 have demonstrated an indisputable role in mediating the self-renewal of cancer cells in c-Myc-driven medulloblastomas, which was further observed in gliomas, 16 stomach, 20 and liver tumors. 19 Similar to TRIM28, the exact mechanism of BRD4-associated cancer stemness-high phenotype is not clear, although unequivocally it depends on bromodomain activity. Surprisingly, using the TCGA transcriptomic data we did not observe a significant association of BRD4 expression with cancer stemness across 27 tested tumor types, in contrast to previously reported TRIM28 and newly discovered ATAD2 (and to some part also SMARCA4) and cancer de-differentiation status.
ATAD2, a chromatin modulator that possesses an AAA+ ATPase domain and a bromodomain, is normally overexpressed in nonspecialized cells, including embryonic stem cells, and in germ cells.
Recently, ATAD2 has been recognized as essential in supporting specific transcriptional programs in ESC cells, modulating their proliferation and differentiation. 42 Here, using the TCGA and GEO transcriptomic data, we report yet unrecognized association between ATAD2 overexpression and cancer stemness in solid tumors across distinct tumor types.
Several studies have previously reported significant upregulation of ATAD2 expression in solid tumors of distinct origins as well as its association with poor patients' outcome, especially in lung, breast, liver, ovarian and cervix cancers. 43 Our results demonstrate that a high ATAD2 level is significantly associated with a worse outcome in ACC, KIRP, LGG, LUAD, MESO, PAAD TCGA tumors, strongly suggesting that ATAD2 overexpression favors malignant transformation of unrelated cancer types. As previously reported, a high ATAD2 expression correlated with more aggressive tumor subgroups of cervical, 43 colorectal, 44 gastric 45 and liver cancer patients, 46 although a direct link with a cancer stem cell compartment was not tested.
Here, we demonstrate that ATAD2 upregulation positively correlates with a higher tumor grade of HNSC, KIRC, LGG, LIHC, OV, PAAD and UCEC tumors, and higher-grade tumors clearly display stem cell features, particularly stemness-related gene expression profiles.
Moreover, a significant correlation between the mean ATAD2 expression and the mean mRNA-SI score across tested tumor types suggests that strongly de-differentiated tumors overexpress ATAD2.
We report yet unrecognized correlation between ATAD2 upregulation and significant enrichment of stem cell-like phenotype in cancer and prove its' versatility across solid tumors. The transcriptome profiles of ATAD2 HIGH cancers are robustly overrepresented with predefined stemness gene signatures as well as with the targets for E2F and c-Myc transcription factors. Previously, ATAD2 has been identified as a transcriptional co-regulator modulating the expression of estrogen and androgen receptors or E2F and c-Myc transcription factors, all known as cancer/proliferation-promoting factors. 47 The pRB-E2F pathway tightly regulates ATAD2 expression, which is essential for the growth of normal and cancer cells. As a direct binding partner for both E2F and c-Myc, ATAD2 induces the expression of genes that facilitate cell cycle progression and inhibition of apoptosis in many different types of cancers, including breast, lung and prostate tumors. 48  Our results are in line with previously reported ATAD2 and MYC co-expression. c-Myc dysregulation accounts for most of the similarities between aggressive tumors and normal stem cell characteristics. Therefore, we suggest that the ATAD2-related cancer stem cell-like phenotype is mediated through both ATAD2 and c-Myc proteins and propose ATAD2 as a druggable target for de-differentiated tumors (especially those overexpressing MYC), which emerges achievable when considering its ATPase activity and its bromodomain. However, molecular studies are indispensable in order to determine the exact role for ATAD2 in cancer stem cell-like phenotype of solid tumors.

| CONCLUSIONS
To conclude, our results demonstrate that BrD family genes display diverse expression patterns in stem cell-like solid tumors. Among all tested BrD proteins, the newly discovered positive association between ATAD2 and cancer de-differentiation status emerges as universal regardless of the tumor type. Higher-grade tumors display significant upregulation of ATAD2 expression and high ATAD2 level corresponds to enhanced c-Myc transcriptional activity. Together, we suggest that ATAD2 might serve as a potential therapeutic target for de-differentiated solid tumors that strongly exhibit cancer stem cell-like characteristics.

ACKNOWLEDGMENTS
We would like to acknowledge Maciej Kaszubowski for his technical support in data acquisition and management.

CONFLICT OF INTEREST
The authors declare no conflict of interests.

DATA AVAILABILITY STATEMENT
Only publicly available data were used in this study, and data sources and handling of these data are described in the Materials and Methods and in the Supporting Information Material. Further information is available from the corresponding author upon request.