DCLK1 and Lgr5 have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. Epithelial–mesenchymal transition (EMT) is a key development program that is often activated during cancer invasion and metastasis, and also imparts a self-renewal capability to disseminating cancer cells. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we observed a relative decrease in DCLK1 expression in the colonic crypts, with significant shift towards stromal staining at peak (12 days post infection) hyperplasia, whereas staining for Lgr5 and Msi-1 increased several fold. When hyperplasia was regressing (days 20–34), an expansion of DCLK1 +ve cells in the CR-infected crypts compared with that seen in uninfected control was recorded. Purified colonic crypt cells exhibiting epigenetic modulation of the transforming growth factor-β (TGFβ), Wnt and Notch pathways on 12 or 34 days post infection formed monolayers in vitro, and underwent trans-differentiation into fibroblast-like cells that stained positive for vimentin, fibronectin and DCLK1. These cells when trypsinized and regrown in soft agar, formed colonospheres/organoids that developed into crypt-like structures (colonoids) in Matrigel and stained positive for DCLK1. Mice exhibiting 12 or 34 days of TMCH were given azoxymethane once for 8 h (Gp1) or weekly for 3 weeks (Gp2), and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as colonospheres in soft agar and nodules/tumors in nude mice. Crypt cells isolated from Gp2 animals failed to form the monolayers, but developed into colonospheres in soft agar and nodules/tumors in nude mice. Thus, both hyperplasia and increased presence of DCLK1 +ve cells promote cellular transformation in response to a second hit. The TMCH model, therefore, provides an excellent template to study how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection.

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4−/− (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc Min/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.

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The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., Myc and BCL-2. Here we determined the effects of the BET protein inhibitor JQ1 and/or histone deacetylase (HDAC) inhibitor panobinostat (PS) on cultured (JeKo1 and MO2058) and primary human MCL cells harvested from the excised MCL involved lymph nodes. Treatment with JQ1 (100 to 2000 nM), but not its inactive enantiomer (R-JQ1), dose-dependently increased the % of cells in the G1 phase while reducing the % of S phase cells, while concomitantly inducing apoptosis in the cultured (MO2058 > JeKo1) MCL cells. Treatment with JQ1 was also dose-dependently lethal against primary MCL cells. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of MYC, BCL2 and CDK6 promoter in JeKo1 and MO2058 cells. Total RNA from the untreated and JQ1-treated cells was used for the quantitative PCR analysis, which showed depletion of the mRNA of c-MYC, BCL2 and CDK6 genes in JQ1-treated cells. While it had no effect on acetylated histone H3 and BRD4, JQ1 treatment dose-dependently depleted the protein levels of MYC, BCL2, CDK6 and pSer2 RNA POL II, but induced the levels of p21, p27 and cleaved PARP in MCL cells. As compared to each agent alone, co-treatment with JQ1 (but not its inactive enantiomer, R-JQ1) and panobinostat (PS) synergistically induced apoptosis of the cultured and primary MCL cells (combination indices< 1.0 by isobologram analyses), but not of normal CD34+ hematopoietic progenitor cells. This was associated with greater attenuation of MYC, BCL2, MCL1 and cyclin D1, but increase in the levels of p21 and cleaved PARP in cultured MCL cells. Co-treatment with JQ1 and PS was also synergistically lethal against carfilzomib-resistant JeKo1 cells (> 10 fold resistant), which were isolated following selection under a continuous exposure to increasing levels of carfilzomib. Following the tail vein infusion and engraftment of JeKo1 cells (5 million cells/mouse) in the bone marrow and spleen of NOD/SCID mice, co-treatment with JQ1 (50 mg/kg/day, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, administered IP) and PS (5 mg/kg, IP) versus treatment with vehicle control, or JQ1 or PS alone, resulted in significant in vivo attenuation of c-MYC, BCL-2 and cyclin D1 levels in the harvested MCL cells from the mice (p < 0.01). Collectively, these pre-clinical findings demonstrate that the combined treatment with BRD4 antagonist and pan-HDAC inhibitor is a synergistically effective epigenetic therapy targeted against human MCL cells, regardless of their sensitivity to proteasome inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A124. Citation Format: Bhavin Shah, Laxmi Jakkula, Warren Fiskus, Sunil Sharma, Jun Qi, John A. Valenta, Leasha J. Schaub, Melissa Rodriguez, Santhana G.T. Devaraj, James E. Bradner, Kapil N. Bhalla. Co-treatment with BRD4 antagonist and histone deacetylase inhibitor is synergistically lethal against Mantle Cell Lymphoma (MCL) cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A124.