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Enhancer of zeste homolog 2 (EZH2) and SET domain bifurcated 1 (SETDB1, also known as ESET) are oncogenic methyltransferases implicated in a number of human cancers. These enzymes typically function as epigenetic repressors of target genes by methylating histone H3 K27 and H3-K9 residues, respectively. Here, we show that EZH2 and SETDB1 are essential to proliferation in 3 SCC cell lines, HSC-5, FaDu, and Cal33. Additionally, we find both of these proteins highly expressed in an aggressive stem-like SCC sub-population. Depletion of either EZH2 or SETDB1 disrupts these stem-like cells and their associated phenotypes of spheroid formation, invasion, and tumor growth. We show that SETDB1 regulates this SCC stem cell phenotype through cooperation with ΔNp63α, an oncogenic isoform of the p53-related transcription factor p63. Furthermore, EZH2 is upstream of both SETDB1 and ΔNp63α, activating these targets via repression of the tumor suppressor RUNX3. We show that targeting this pathway with inhibitors of EZH2 results in activation of RUNX3 and repression of both SETDB1 and ΔNp63α, antagonizing the SCC cancer stem cell phenotype. This work highlights a novel pathway that drives an aggressive cancer stem cell phenotype and demonstrates a means of pharmacological intervention.

Our understanding of cancer and the key pathways that drive cancer survival has expanded rapidly over the past several decades. However, there are still important challenges that continue to impair patient survival, including our inability to target cancer stem cells (CSCs), metastasis, and drug resistance. The transcription factor p63 is a p53 family member with multiple isoforms that carry out a wide array of functions. Here, we discuss the critical importance of the ΔNp63α isoform in cancer and potential therapeutic strategies to target ΔNp63α expression to impair the CSC population, as well as to prevent metastasis and drug resistance to improve patient survival.

Bromodomain containing protein 4 (BRD4) plays a critical role in controlling the expression of genes involved in development and cancer. Inactivation of BRD4 inhibits cancer growth, making it a promising anticancer drug target. The cancer stem cell (CSC) population is a key driver of recurrence and metastasis in patients with cancer. Here we show that cancer stem-like cells can be enriched from squamous cell carcinomas (SCC), and that these cells display an aggressive phenotype with enhanced stem cell marker expression, migration, invasion, and tumor growth. BRD4 is highly elevated in this aggressive subpopulation of cells, and its function is critical for these CSC-like properties. Moreover, BRD4 regulates ΔNp63α, a key transcription factor that is essential for epithelial stem cell function that is often overexpressed in cancers. BRD4 regulates an EZH2/STAT3 complex that leads to increased ΔNp63α-mediated transcription. Targeting BRD4 in human SCC reduces ΔNp63α, leading to inhibition of spheroid formation, migration, invasion, and tumor growth. These studies identify a novel BRD4-regulated signaling network in a subpopulation of cancer stem-like cells, elucidating a possible avenue for effective therapeutic intervention. Significance: This study identifies a signaling cascade driven by BRD4 that upregulates ΔNp63α to promote cancer stem-like properties, which has potential therapeutic implications for the treatment of squamous cell carcinomas.

p63 (also known as TP63) is a transcription factor of the p53 family, along with p73. Multiple isoforms of p63 have been discovered and these have diverse functions encompassing a wide array of cell biology. p63 isoforms are implicated in lineage specification, proliferative potential, differentiation, cell death and survival, DNA damage response and metabolism. Furthermore, p63 is linked to human disease states including cancer. p63 is critical to many aspects of cell signaling, and in this Cell science at a glance article and the accompanying poster, we focus on the signaling cascades regulating TAp63 and ΔNp63 isoforms and those that are regulated by TAp63 and ΔNp63, as well the role of p63 in disease.

Human breast tumors with abundant hyaluronan (HA) and high expression of the HA synthesizing enzyme HAS2 are aggressive and exhibit poorer survival than tumors with low HA/HAS2. The vitamin D receptor (VDR) and its ligand 1,25(OH)2D3 (1,25D) exert anti-tumor effects in cancer cells and inhibit breast cancer growth in animals, but considerable heterogeneity has been reported in different model systems. Genomic profiling has demonstrated that, while a few genes are commonly regulated by 1,25D in all VDR positive cell lines, the majority of VDR regulated genes are cell line specific. In general, cell lines derived from the more aggressive tumor subtypes such as Triple Negative Breast Cancer (TNBC) express low levels of VDR and are less sensitive to 1,25D than cell lines derived from less aggressive subtypes. Through genomic profiling of TNBC cells derived from wild-type (WT) and VDR knockout (VDRKO) mice, we identified HAS2 as a 1,25D repressed gene. To understand the mechanistic impact of vitamin D and Has2 on HA synthesis and tumorigenic potential in TNBC cells, we utilized the human TNBC cell line Hs578T, which is representative of the mesenchymal/stem-like (MSL) subtype of TNBC. 1,25D inhibited HAS2 expression and HA synthesis in Hs578T cells, and also down-regulated CD44, a breast cancer stem cell marker which acts as the receptor for HA, the product of HAS2. Further studies determined that Hs578T cells are heterogeneous with respect to morphology and HAS2 expression. We therefore sorted parental Hs578T cells into HAhigh and HAlow populations. HAhigh populations exhibited elevated HA production, smaller size, increased proliferation and higher motility than HAlow populations. Through genomic profiling, HAhigh populations demonstrated higher levels of expression of genes involved in the enzymatic production and degradation of HA (HAS2, HAS2-AS, HYAL1), expression of extracellular matrix (ECM) proteins involved in HA binding and motility (PTX3, CEMIP, ACAN) and epithelial-mesenchymal transition (SNAI1) compared to HAlow populations. Despite their more aggressive phenotype, HAhigh populations retained expression of VDR protein at levels comparable to that of parental Hs578T cells and HAlow subclones. Treatment with 1,25D decreased production of HA in both HAhigh and HAlow populations, but significantly reduced expression of genes involved in the production of HA (HAS2, HAS2-AS) and HA binding and motility (ACAN, CEMIP) only in the HAhigh populations. This study suggests that 1,25D/VDR can suppress HAS2 and HA production and signaling in the ECM, and thus may reduce the aggressive phenotype of TNBC. Citation Format: Carmen J. Narvaez, Seamus Balinth, JoEllen Welsh. Vitamin D inhibits hyaluronan synthesis and extracellular matrix gene expression in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3742.