<div>Abstract<p>There continues to be interest in targeting epigenetic “readers, writers, and erasers” for the treatment of cancer and other pathologies. However, a mechanistic understanding is frequently lacking for the synergy observed when combining deacetylase and bromodomain inhibitors. Here we identify cell cycle and apoptosis regulator 2 (CCAR2) as an early target for acetylation in colon cancer cells treated with sulforaphane. N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 and β-catenin, interfering with Wnt coactivator functions of CCAR2, including in cells harboring genetically encoded CCAR2 acetylation. Protein domain arrays and pull-down assays identified acetyl “reader” proteins that recognized CCAR2 acetylation sites, including BRD9 and members of the bromodomain and extraterminal domain (BET) family. Treatment with the BET inhibitor JQ1 synergized with sulforaphane in colon cancer cells and suppressed tumor development effectively in a preclinical model of colorectal cancer. Studies with sulforaphane+JQ1 in combination implicated a BET/BRD9 acetyl switch and a shift in the pool of acetyl “reader” proteins in favor of BRD9-regulated target genes.</p>Significance:<p>These results highlight the competition that exists among the “readers” of acetylated histone and nonhistone proteins and provide a mechanistic basis for potential new therapeutic avenues involving epigenetic combination treatments.</p></div>
Abstract There continues to be interest in targeting epigenetic ‘readers, writers and erasers’ for the treatment of cancer and other pathologies. A mechanistic understanding is frequently lacking, however, for the synergy observed when combining deacetylase and bromodomain inhibitors. We identified cell cycle and apoptosis regulator 2 (CCAR2) as an early target for acetylation in colon cancer cells treated with sulforaphane (SFN). N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 (HDAC3) and β-catenin, interfering with Wnt coactivator functions of CCAR2, including in cells harboring genetically encoded CCAR2 acetylation. Protein domain arrays and pull-down assays identified acetyl ‘reader’ proteins that recognized CCAR2 acetylation sites, including BRD9 and members of the bromodomain and extraterminal domain (BET) family. The BET inhibitor JQ1 synergized with SFN in colon cancer cells, and suppressed tumor development effectively in a preclinical model of colorectal cancer. Studies with SFN+JQ1 in combination implicated a BET/BRD9 acetyl switch, and a shift in the pool of acetyl ‘reader’ proteins in favor of BRD9 regulated target genes. These results highlight the competition that exists among the ‘readers’ of acetylated histone and non-histone proteins, and provide a mechanistic basis for potential new therapeutic avenues involving epigenetic combination treatments. Citation Format: Gavin S. Johnson, Praveen Rajendran, Li Li, Ying-Shiuan Chen, W. Mohaiza Dashwood, Nhung Nguyen, Ahmet M. Ulusan, Furkan Ertem, Mutian Zhang, Deqiang Sun, Yun Huang, Shan Wang, Hon-Chiu E. Leung, David Lieberman, Laura M. Beaver, Emily Ho, Mark T. Bedford, Kyle Chang, Eduardo Vilar, Roderick H. Dashwood. CCAR2 acetylation establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2614.
Background Young adults and other working‐age adults with cancer are at risk for cancer‐related financial toxicity (FT), including material hardships, depletion of coping resources, and psychological burden. This study compares FT domains in young adults (18‐39 years old) (YAs), other working‐age adults (40‐64 years old), and older adults (≥65 years old) receiving cancer care. Methods A total of 311 adults were surveyed using the multi‐domain Economic Strain and Resilience in Cancer instrument measuring FT (0‐10 score indicating least to greatest FT; score ≥5 severe FT). Participants were receiving ambulatory care from March‐September 2019. Associations of age with overall FT and material hardship, coping resource depletion, and psychological burden FT domains were tested using Kruskal‐Wallis and χ 2 tests and multivariable generalized linear models with gamma distribution. Results YAs (median age, 31.5 years) comprised 9.6% of the sample; other working‐age adults comprised 56.9%. Overall, material, coping, and psychological FT scores were worse in younger age adults versus older adults ( P < .001 in all multivariable models). Compared with older adults, younger age adults demonstrated worse material hardship (median scores, 3.70 vs 4.80 vs 1.30 for YAs, other working‐age, and older adults, respectively; P < .001), coping resource depletion (4.50 vs 3.40 vs 0.80; P < .001), and psychological burden (6.50 vs 7.00 vs 1.00; P < .001). Fifty percent of YAs had severe overall FT versus 40.7% of other working‐age adults and 9.6% of older adults ( P < .001). Conclusions Younger age adults with cancer bore disproportionate FT. Interventions to address unmet needs are critical components for addressing FT in this population.
Abstract Heterocyclic amines (HCAs) produced during high-temperature cooking have been studied extensively in terms of their genetic effects, but recent work has implicated epigenetic mechanisms involving non-coding RNAs. We reported that colon tumors induced in the rat by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have altered microRNA (miRNA) signatures linked to dysregulated pluripotency factors, such as c-Myc and Krüppel-like factor 4 (Klf4). The corresponding miRNAs prioritized from PhIP-induced colon tumors were examined in other target organs from a one-year carcinogenicity bioassay, and compared with miRNAs dysregulated in the Apc-mutant polyposis in rat colon (Pirc) genetic model. Multiple let-7 family members were downregulated in colon, skin, lung, small intestine, and Zymbal’s gland tumors, and were associated with Myc and Hmga2 upregulation. A “PhIP miRNA signature” with the profile mir-21high | mir-126low | mir-29low | mir-215low | mir-145low was linked to reduced expression of Klf4 in multiple target organs of the rat, and was predictive of poor prognosis in human pan-cancer and colorectal cancer datasets (https://cancergenome.nih.gov/). Findings from The Cancer Genome Atlas suggested that PhIP signature miRNAs might serve as an arbiter of HCA exposure, defining a subset of human tumors linked to environmental carcinogen exposure. We conclude that future studies should examine the miRNA signatures of other HCAs, and determine their possible predictive value for human risk assessment. This work was supported in part by NIH grants CA090890, CA122959, ES00210, and ES023512, the John S. Dunn Foundation, and a Chancellor’s Research Initiative. We are most thankful to Robert Hawk for providing a graduate student fellowship in aid of Y-S Chen. Citation Format: Ying-Shiuan Chen, Rong Wang, Wan-Mohaiza Dashwood, Christiane Löhr, David E. Williams, Emily Ho, Susanne Mertens-Talcott, Roderick H. Dashwood. A miRNA signature linked to human environmental risk defined from carcinogen-induced and genetically-driven colon carcinogenesis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4428. doi:10.1158/1538-7445.AM2017-4428
There is growing evidence that DNA repair factors have clinical value for cancer treatment. Nucleotide excision repair (NER) proteins, including excision repair cross-complementation group 2 (ERCC2), play a critical role in maintaining genome integrity. Here, we examined ERCC2 expression following epigenetic combination drug treatment. Attention was drawn to ERCC2 for three reasons. First, from online databases, colorectal cancer (CRC) patients exhibited significantly reduced survival when ERCC2 was overexpressed in colon tumors. Second, ERCC2 was the most highly downregulated RNA transcript in human colon cancer cells, plus Ercc2 in rat tumors, after treatment with the histone deacetylase 3 (HDAC3) inhibitor sulforaphane (SFN) plus JQ1, which is an inhibitor of the bromodomain and extraterminal domain (BET) family. Third, as reported here, RNA-sequencing of polyposis in rat colon (Pirc) polyps following treatment of rats with JQ1 plus 6-methylsulfinylhexyl isothiocyanate (6-SFN) identified Ercc2 as the most highly downregulated gene. The current work also defined promising second-generation epigenetic drug combinations with enhanced synergy and efficacy, especially in metastasis-lineage colon cancer cells cultured as 3D spheroids and xenografts. This investigation adds to the growing interest in combination approaches that target epigenetic 'readers', 'writers', and 'erasers' that are deregulated in cancer and other pathologies, providing new avenues for precision oncology and cancer interception.
Abstract Background: Complex interrelationships govern the dynamic interactions between gut microbes, the host, and exogenous drivers of disease outcome. A multi-omic approach to cancer prevention by spinach was pursued in the polyposis in rat colon (Pirc) model of colorectal cancer. Results: Spinach fed for 26 weeks (10% w/w, freeze-dried in the diet) exhibited significant antitumor efficacy in the Pirc model and, despite the Apc-mutant genetic background, no changes were detected in b-catenin protein expression. To provide mechanistic leads, we performed 16S rRNA sequencing of the gut microbiome and unbiased transcriptomic analyses of colon polyps and matched normal-looking tissues. For dietary spinach intervention groups, increased microbiome diversity coincided with reversal of taxonomic composition in both wild type and Apc-mutant rats. Metagenomic prediction implicated linoleate and butanoate metabolism, tricarboxylic acid cycle, and pathways in cancer, which was supported by mechanistic leads from metabolomic analyses. Specifically, anticancer outcomes were linked to several spinach-derived linoleate bioactives with known anti-inflammatory and proapoptotic mechanisms, as well as N -aceto-2-hydroxybutanoate changes that were consistent with altered butanoate metabolism stemming from the increased a-diversity of the gut microbiome. On the other hand, L-glutamate and N -acetylneuraminate were reduced markedly in colon tumors from spinach-fed rats, implicating deregulated mitochondrial energetics and altered cell surface glycans involved in oncogenic signaling networks, immune evasion, and other pathways in cancer. Conclusions: Tumor suppression by dietary spinach was observed for the first time in the Apc-mutant Pirc model, and was independent of the mechanistic targeting of b-catenin. Marked reshaping of the gut microbiome in spinach-fed rats occurred along with changes in host transcriptomics and RNA-miRNA networks. Metabolomics provided mechanistic support for linoleate and butanoate metabolism, which are both strongly linked to anticancer outcomes in the colon. Tumor-associated changes in L-glutamate and N -acetylneuraminate warrant further investigation in the context of synthetic lethality and circumventing deregulated signaling networks in colorectal cancer patients.
Abstract There is growing interest in the crosstalk between the gut microbiome, metabolomic features, and disease pathogenesis. Colorectal cancer is a major health burden worldwide, linked in part to modifiable risk factors associated with diet and lifestyle (1). The differential roles of the metabolites in targeting Wnt/b-catenin signaling were recently reported (2). The current investigation compared long-term (26 week) and acute (3 day) dietary spinach intake in a genetic model of colorectal cancer. Metabolomic analyses in the polyposis in rat colon (Pirc) model and in wildtype animals corroborated key contributions to anticancer outcomes by spinach-derived linoleate bioactives and a butanoate metabolite linked to increased a-diversity of the gut microbiome (3). Combining linoleate and butanoate metabolites in human colon cancer cells revealed enhanced apoptosis and reduced cell viability, paralleling the apoptosis induction observed in colon tumors from rats given long-term spinach treatment. Mechanistic studies in cell-based assays and in vivo implicated the linoleate and butanoate metabolites in targeting histone deacetylase (HDAC) activity and the interferon-g (IFN-g) signaling axis. Clinical translation of the findings from this investigation to at-risk patients might provide valuable quality-of-life benefits by delaying surgical interventions and drug therapies with adverse side effects (4,5). References 1. Xi, Y., Xu, P. Global colorectal cancer burden in 2020 and projections to 2040. Transl Oncol 2021;14: 101174 2. Liu, F. et al. Suppression of membranous LPR5 recycling, Wnt/β-catenin signaling, and colon carcinogenesis by 15-LOX-1 peroxidation of linoleic acid in PI3P. Cell Rep 2020;32:108049. 3. Chen, Y.-S. et al. Dietary spinach reshapes the gut microbiome in an Apc-mutant genetic background: mechanistic insights from integrated multi-omics. Gut Microbes 2021;13:1972756. 4. Ulusan, A.M. et al. Optimization of erlotinib plus sulindac dosing regimens for intestinal cancer prevention in an Apc-mutant model of Familial Adenomatous Polyposis (FAP). Cancer Prev Res (Phila) 2021;14:325-336. 5. Samadder, N.J. et al. Association of sulindac and erlotinib vs placebo with colorectal neoplasia in Familial Adenomatous Polyposis: secondary analysis of a randomized clinical trial. JAMA Oncol 2018:4:671-677. Citation Format: Ying-Shiuan Chen, Jia Li, Sultan Neja, Rani Menon, Arul Jayaraman, Kyongbum Lee, Wan Mohaiza Dashwood, Shan Wang, Sabeeta Kapoor, Praveen Rajendran, Ke Zhang, Roderick Dashwood. Metabolomics of acute vs. chronic spinach intake in an Apc-mutant genetic background: linoleate and butanoate metabolites targeting HDAC activity and IFN-γ signaling [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A013.
Abstract DNA repair genes have potential clinical value in predicting cancer prognosis and treatment outcomes. Nucleotide excision repair (NER) proteins like ERCC2 play a critical role in maintaining genome integrity by recognizing and unwinding DNA at sites of damage1.Aberrant expression of ERCC2 alters NER capacity, influencing treatment outcomes2. The current investigation examined the expression of ERCC2 following epigenetic combination treatment in colorectal cancer (CRC) cells and preclinical models. Attention was drawn to ERCC2 based on three observations. First, from online databases, when ERCC2 was overexpressed in colon tumors the corresponding CRC patients had reduced overall survival3. Second, ERCC2 was the most highly downregulated gene when dietary histone deacetylase 3 (HDAC3) inhibitor sulforaphane (SFN) was combined withJQ1, an inhibitor of the bromodomain and extra terminal domain family, in human colon cancer cells and in colon polyps from the polyposis in rat colon (Pirc) model4. Third, as reported here for the first time, RNA-seq analyses of Pirc colon polyps from rats treated withJQ1 and the SFN analog 6-SFN5 identified Ercc2 as the most highly downregulated gene. RNA-seq data were corroborated by RT-qPCR and immunoblotting experiments. There is much interest in combination approaches that target epigenetic ‘readers, writers, and erasers' that are deregulated in cancer and other pathologies. The current work identified promising second-generation inhibitors with enhanced synergy and antitumor efficacy, especially in metastatic cells cultured in three-dimensions. Drug combinations decreased HDAC3, BRD4 and ERCC2 expression, while DNA damage and apoptosis markers were increased both in spheroids and in a mouse xenograft model. This investigation has potential clinical relevance for the identification of robust biomarkers that predict enhanced antitumor outcomes in CRC patients.1. Gillet LC, Schärer OD. Molecular mechanisms of mammalian global genome nucleotide excision repair. Chem Rev 2006;106:253-76.2. Shuck SC, Short EA, Turchi JJ. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology. Cell Res 2008;18:64-72.3. Liu J et al., The Differential Expression of Core Genes in Nucleotide Excision Repair Pathway Indicates Colorectal Carcinogenesis and Prognosis. Biomed Res Int 2018;2018:9651320.4. Rajendran P et al., Acetylation of CCAR2 establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition. Cancer Res 2019;79:918-27.5. Rajendran Pet al., HDAC turnover, CtIP acetylation and dysregulated DNA damage signaling in colon cancer cell treated with sulforaphane and related dietary isothiocyanates. Epigenetics 2013;8:612-23. Citation Format: Sabeeta Kapoor, Trace Gustafson, Mutian Zhang, Ying-Shiuan Chen, Jia Li, Nhung Nguyen, Jorge Enrique Perez, Wan Mohaiza Dashwood, Praveen Rajendran, Roderick Dashwood. Combined deacetylase and bromodomain inhibition downregulates ERCC2 and suppresses growth of metastatic colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2106.
There continues to be interest in targeting epigenetic 'readers, writers and erasers' for the treatment of cancer and other pathologies. A mechanistic understanding is frequently lacking, however, for the synergy observed when combining deacetylase and bromodomain inhibitors. We identified cell cycle and apoptosis regulator 2 (CCAR2) as an early target for acetylation in colon cancer cells treated with sulforaphane (SFN). N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 (HDAC3) and β-catenin, interfering with Wnt coactivator functions of CCAR2, including in cells harboring genetically encoded CCAR2 acetylation. Protein domain arrays and pull-down assays identified acetyl 'reader' proteins that recognized CCAR2 acetylation sites, including BRD9 and members of the bromodomain and extraterminal domain (BET) family. The BET inhibitor JQ1 synergized with SFN in colon cancer cells, and suppressed tumor development effectively in a preclinical model of colorectal cancer. Studies with SFN+JQ1 in combination implicated a BET/BRD9 acetyl switch, and a shift in the pool of acetyl 'reader' proteins in favor of BRD9 regulated target genes. These results highlight the competition that exists among the 'readers' of acetylated histone and non-histone proteins, and provide a mechanistic basis for potential new therapeutic avenues involving epigenetic combination treatments.Citation Format: Gavin S. Johnson, Praveen Rajendran, Li Li, Ying-Shiuan Chen, W. Mohaiza Dashwood, Nhung Nguyen, Ahmet M. Ulusan, Furkan Ertem, Mutian Zhang, Deqiang Sun, Yun Huang, Shan Wang, Hon-Chiu E. Leung, David Lieberman, Laura M. Beaver, Emily Ho, Mark T. Bedford, Kyle Chang, Eduardo Vilar, Roderick H. Dashwood. CCAR2 acetylation establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2614.
