SUMMARY Basal p53 levels are tightly suppressed under normal conditions. Disrupting this regulation results in elevated p53 levels to induce cell cycle arrest, apoptosis and tumor suppression. Here, we report the suppression of basal p53 levels by a nuclear, p53-regulated long noncoding RNA that we termed PURPL (p53 upregulated regulator of p53 levels). Targeted depletion of PURPL in colorectal cancer cells results in elevated basal p53 levels and induces growth defects in cell culture and in mouse xenografts. PURPL associates with MYBBP1A, a protein that binds to and stabilizes p53, and inhibits the formation of the p53-MYBBP1A complex. In the absence of PURPL, MYBBP1A interacts with and stabilizes p53. Silencing MYBBP1A significantly rescues basal p53 levels and proliferation in PURPL-deficient cells, suggesting that MYBBP1A mediates the effect of PURPL in regulating p53. These results reveal a p53-PURPL autoregulatory feedback loop and demonstrate a role for PURPL in maintaining basal p53 levels.

Key Points NGS-based prognostic panels may identify individuals at risk for HHMs despite not being designed for this purpose. Variant allele frequency >0.4 and gene of interest may be predictive of germ line origin.

A series of pyridine-based derivatives of the antimetastatic Ru(III) complex imidazolium [trans-RuCl(4)(1H-imidazole)(DMSO-S)] (NAMI-A) have been synthesized along with their sodium-ion compensated analogues. These compounds have been characterized by X-ray crystallography, electron paramagnetic resonance (EPR), NMR, and electrochemistry, with the goal of probing their noncovalent interactions with human serum albumin (hsA). EPR studies show that the choice of imidazolium ligands and compensating ions does not strongly influence the rates of ligand exchange processes in aqueous buffer solutions. By contrast, the rate of formation and persistence of interactions of the complexes with hsA is found to be strongly dependent on the properties of the axial ligands. The stability of noncovalent binding is shown to correlate with the anticipated ability of the various pyridine ligands to interact with the hydrophobic binding domains of hsA. These interactions prevent the oligomerization of the complexes in solution and limit the rate of covalent binding to albumin amino acid side chains. Electrochemical studies demonstrate relatively high reduction potentials for these complexes, leading to the formation of Ru(II) species in aqueous solutions containing biological reducing agents, such as ascorbate. However, EPR measurements indicate that while noncovalent interactions with hsA do not prevent reduction, covalent binding produces persistent mononuclear Ru(III) species under these conditions.

The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using highthroughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.T he caudal-type homeobox 1 (CDX1) transcription factor controls enterocyte differentiation in the colon, where its expression is excluded from the crypt-base stem cell compartment. CDX1 is also central to the capacity of a colorectal cancer (CRC) cell line to differentiate, and it is a negative marker of CRC stem cells (1-3). In 19% of CRC cell lines assayed in a large study, CDX1 expression was completely lost due to promoter methylation and was down-regulated in a further 13% as a result of hemimethylation of the promoter (4). Comparison of CDX1 expression in colorectal adenocarcinoma versus matched normal tissue showed downregulation of CDX1 in 73% of tumors, which was also attributable to promoter methylation (5). Expression of CDX1 also correlates inversely with that of the polycomb complex protein BMI1, which is necessary for the maintenance of quiescent injury-inducible stem cells in the normal crypt and is expressed in cancer stem cells (2, 6-8). The mechanism underlying this inverse correlation has not yet been elucidated.In addition to these correlative data, CDX1 has also been shown to promote directly the expression of structural proteins important for epithelial differentiation including cytokeratin 20 (KRT20) (1), villin (VIL) (9), and FABP1 (10). Introduction...