CCAR1 and CCAR2 as gene chameleons with antagonistic duality: Preclinical, human translational, and mechanistic basis.

Cell Cycle and Apoptosis Regulator 1 (CCAR1) and Cell Cycle and Apoptosis Regulator 2 (CCAR2) have emerged as key players in physiology and pathophysiology, with critical roles in the DNA damage response, nuclear receptor function, and Wnt signaling, among other activities. Contradictory reports exist on the functional duality of CCAR1 and CCAR2 as either tumor promoters or suppressors, suggesting that CCAR1 and CCAR2 have the hallmarks of gene chameleons. We review herein the mechanistic, preclinical, and human translational findings for CCAR1 and CCAR2, based on available RNA and protein expression data from human studies, The Cancer Genome Atlas (TCGA) data mining, gene knockout mouse models, and cell-based assays. Multiple factors contribute to the divergent activities of CCAR1 and CCAR2, including tissue type, mutation/genetic background, protein-protein interactions, dynamic regulation via posttranslational modifications, and alternative RNA splicing. An array of protein partners interact with CCAR1 and CCAR2 in the context of tumor promotion and suppression, including ß-catenin, androgen receptor, p21Cip1/Waf1, tumor protein p53 (p53), sirtuin 1, and histone deacetylase 3. Genetic changes frequently found in cancer, such as TP53 mutation, also serve as critical determinants of survival outcomes in cancer patients. This review seeks to provide the impetus for further investigation into CCAR1 and CCAR2 as potential master regulators of metabolism, aging, and cancer.

Heterocyclic Analogs of Sulforaphane Trigger DNA Damage and Impede DNA Repair in Colon Cancer Cells: Interplay of HATs and HDACs.

SCOPE: DNA repair inhibitors have broad clinical applications in tumor types with DNA repair defects, including colorectal cancer (CRC). Structural analogs of the anticancer agent sulforaphane (SFN) were investigated as modifiers of histone deacetylase (HDAC) and histone acetyltransferase (HAT) activity, and for effects on DNA damage/repair pertinent to human CRC. METHODS AND RESULTS: In the polyposis in rat colon (Pirc) model, single oral administration of SFN and structurally related long-chain isothiocyanates (ITCs) decreased histone deacetylase 3 (HDAC3) expression and increased pH2AX levels markedly in adenomatous colon polyps, extending prior observations on HDAC3 inhibition/turnover in cell-based assays. Colon cancer cells at a high initial plating density had diminished cytotoxicity from SFN, whereas novel tetrazole-containing heterocyclic analogs of SFN retained their efficacy. The potent SFN analogs triggered DNA damage, cell cycle arrest, apoptosis, and loss of a key DNA repair regulator, C-terminal binding protein (CtBP) interacting protein (CtIP). These SFN analogs also altered HAT/HDAC activities and histone acetylation status, lowered the expression of HDAC3, P300/CBP-associated factor (PCAF) and lysine acetyltransferase 2A (KAT2A/GCN5), and attenuated homologous recombination (HR)/non-homologous end joining (NHEJ) repair activities in colon cancer cells. CONCLUSION: Novel tetrazole-containing heterocyclic analogs of SFN provide a new avenue for chemosensitization in colon cancer cells via modulation of HAT/HDAC activities and associated DNA damage/repair signaling pathways.

Long noncoding RNAs and sulforaphane: a target for chemoprevention and suppression of prostate cancer.

Long noncoding RNAs (lncRNAs) have emerged as important in cancer development and progression. The impact of diet on lncRNA expression is largely unknown. Sulforaphane (SFN), obtained from vegetables like broccoli, can prevent and suppress cancer formation. Here we tested the hypothesis that SFN attenuates the expression of cancer-associated lncRNAs. We analyzed whole-genome RNA-sequencing data of normal human prostate epithelial cells and prostate cancer cells treated with 15 µM SFN or dimethylsulfoxide. SFN significantly altered expression of ~100 lncRNAs in each cell type and normalized the expression of some lncRNAs that were differentially expressed in cancer cells. SFN-mediated alterations in lncRNA expression correlated with genes that regulate cell cycle, signal transduction and metabolism. LINC01116 was functionally investigated because it was overexpressed in several cancers, and was transcriptionally repressed after SFN treatment. Knockdown of LINC01116 with siRNA decreased proliferation of prostate cancer cells and significantly up-regulated several genes including GAPDH (regulates glycolysis), MAP1LC3B2 (autophagy) and H2AFY (chromatin structure). A four-fold decrease in the ability of the cancer cells to form colonies was found when the LINC01116 gene was disrupted through a CRISPR/CAS9 method, further supporting an oncogenic function for LINC01116 in PC-3 cells. We identified a novel isoform of LINC01116 and bioinformatically investigated the possibility that LINC01116 could interact with target genes via ssRNA:dsDNA triplexes. Our data reveal that chemicals from the diet can influence the expression of functionally important lncRNAs, and suggest a novel mechanism by which SFN may prevent and suppress prostate cancer.

A functional pseudogene, NMRAL2P, is regulated by Nrf2 and serves as a coactivator of NQO1 in sulforaphane-treated colon cancer cells.

SCOPE: The anticancer agent sulforaphane (SFN) acts via multiple mechanisms to modulate gene expression, including the induction of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent signaling and the inhibition of histone deacetylase activity. Transcriptomics studies were performed in SFN-treated human colon cancer cells and in nontransformed colonic epithelial cells in order to pursue new mechanistic leads. METHODS AND RESULTS: RNA-sequencing corroborated the expected changes in cancer-related pathways after SFN treatment. In addition to NAD(P)H quinone dehydrogenase 1 (NQO1) and other well-known Nrf2-dependent targets, SFN strongly induced the expression of Loc344887. This noncoding RNA was confirmed as a novel functional pseudogene for NmrA-like redox sensor 1, and was given the name NmrA-like redox sensor 2 pseudogene (NMRAL2P). Chromatin immunoprecipitation experiments corroborated the presence of Nrf2 interactions on the NMRAL2P genomic region, and interestingly, NMRAL2P also served as a coregulator of NQO1 in human colon cancer cells. Silencing of NMRAL2P via CRISPR/Cas9 genome-editing protected against SFN-mediated inhibition of cancer cell growth, colony formation, and migration. CONCLUSION: NMRAL2P is the first functional pseudogene to be identified both as a direct transcriptional target of Nrf2, and as a downstream regulator of Nrf2-dependent NQO1 induction. Further studies are warranted on NMRAL2P-Nrf2 crosstalk and the associated mechanisms of gene regulation.