Skp2 Deletion Unmasks a p27 Safeguard that Blocks Tumorigenesis in the Absence of pRb and p53 Tumor Suppressors

SummarypRb and p53 are two major tumor suppressors. Here, we found that p53 activates expression of Pirh2 and KPC1, two of the three ubiquitin ligases for p27. Loss of p53 in the absence of Skp2, the third ubiquitin ligase for p27, shrinks the cellular pool of p27 ubiquitin ligases to accumulate p27 protein. In the absence of pRb and p53, p27 was unable to inhibit DNA synthesis in spite of its abundance, but could inhibit division of cells that maintain DNA replication with rereplication. This mechanism blocked pRb/p53 doubly deficient pituitary and prostate tumorigenesis lastingly coexistent with bromodeoxyuridine-labeling neoplastic lesions, revealing an unconventional cancer cell vulnerability when pRb and p53 are inactivated

RHOA Is a Modulator of the Cholesterol-Lowering Effects of Statin

<div><p>Although statin drugs are generally efficacious for lowering plasma LDL-cholesterol levels, there is considerable variability in response. To identify candidate genes that may contribute to this variation, we used an unbiased genome-wide filter approach that was applied to 10,149 genes expressed in immortalized lymphoblastoid cell lines (LCLs) derived from 480 participants of the Cholesterol and Pharmacogenomics (CAP) clinical trial of simvastatin. The criteria for identification of candidates included genes whose statin-induced changes in expression were correlated with change in expression of <em>HMGCR</em>, a key regulator of cellular cholesterol metabolism and the target of statin inhibition. This analysis yielded 45 genes, from which <em>RHOA</em> was selected for follow-up because it has been found to participate in mediating the pleiotropic but not the lipid-lowering effects of statin treatment. <em>RHOA</em> knock-down in hepatoma cell lines reduced <em>HMGCR, LDLR</em>, and <em>SREBF2</em> mRNA expression and increased intracellular cholesterol ester content as well as apolipoprotein B (APOB) concentrations in the conditioned media. Furthermore, inter-individual variation in statin-induced <em>RHOA</em> mRNA expression measured <em>in vitro</em> in CAP LCLs was correlated with the changes in plasma total cholesterol, LDL-cholesterol, and APOB induced by simvastatin treatment (40 mg/d for 6 wk) of the individuals from whom these cell lines were derived. Moreover, the minor allele of rs11716445, a SNP located in a novel cryptic <em>RHOA</em> exon, dramatically increased inclusion of the exon in <em>RHOA</em> transcripts during splicing and was associated with a smaller LDL-cholesterol reduction in response to statin treatment in 1,886 participants from the CAP and Pravastatin Inflamation and CRP Evaluation (PRINCE; pravastatin 40 mg/d) statin clinical trials. Thus, an unbiased filter approach based on transcriptome-wide profiling identified <em>RHOA</em> as a gene contributing to variation in LDL-cholesterol response to statin, illustrating the power of this approach for identifying candidate genes involved in drug response phenotypes.</p> </div

Transmembrane protein 55B is a novel regulator of cellular cholesterol metabolism.

ObjectiveInterindividual variation in pathways affecting cellular cholesterol metabolism can influence levels of plasma cholesterol, a well-established risk factor for cardiovascular disease. Inherent variation among immortalized lymphoblastoid cell lines from different donors can be leveraged to discover novel genes that modulate cellular cholesterol metabolism. The objective of this study was to identify novel genes that regulate cholesterol metabolism by testing for evidence of correlated gene expression with cellular levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) mRNA, a marker for cellular cholesterol homeostasis, in a large panel of lymphoblastoid cell lines.Approach and resultsExpression array profiling was performed on 480 lymphoblastoid cell lines established from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, and transcripts were tested for evidence of correlated expression with HMGCR as a marker of intracellular cholesterol homeostasis. Of these, transmembrane protein 55b (TMEM55B) showed the strongest correlation (r=0.29; P=4.0E-08) of all genes not previously implicated in cholesterol metabolism and was found to be sterol regulated. TMEM55B knockdown in human hepatoma cell lines promoted the decay rate of the low-density lipoprotein receptor, reduced cell surface low-density lipoprotein receptor protein, impaired low-density lipoprotein uptake, and reduced intracellular cholesterol.ConclusionsHere, we report identification of TMEM55B as a novel regulator of cellular cholesterol metabolism through the combination of gene expression profiling and functional studies. The findings highlight the value of an integrated genomic approach for identifying genes that influence cholesterol homeostasis

Transmembrane Protein 55B Is a Novel Regulator of Cellular Cholesterol Metabolism

ObjectiveInterindividual variation in pathways affecting cellular cholesterol metabolism can influence levels of plasma cholesterol, a well-established risk factor for cardiovascular disease. Inherent variation among immortalized lymphoblastoid cell lines from different donors can be leveraged to discover novel genes that modulate cellular cholesterol metabolism. The objective of this study was to identify novel genes that regulate cholesterol metabolism by testing for evidence of correlated gene expression with cellular levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) mRNA, a marker for cellular cholesterol homeostasis, in a large panel of lymphoblastoid cell lines.Approach and resultsExpression array profiling was performed on 480 lymphoblastoid cell lines established from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, and transcripts were tested for evidence of correlated expression with HMGCR as a marker of intracellular cholesterol homeostasis. Of these, transmembrane protein 55b (TMEM55B) showed the strongest correlation (r=0.29; P=4.0E-08) of all genes not previously implicated in cholesterol metabolism and was found to be sterol regulated. TMEM55B knockdown in human hepatoma cell lines promoted the decay rate of the low-density lipoprotein receptor, reduced cell surface low-density lipoprotein receptor protein, impaired low-density lipoprotein uptake, and reduced intracellular cholesterol.ConclusionsHere, we report identification of TMEM55B as a novel regulator of cellular cholesterol metabolism through the combination of gene expression profiling and functional studies. The findings highlight the value of an integrated genomic approach for identifying genes that influence cholesterol homeostasis

Transmembrane protein 55B is a novel regulator of cellular cholesterol metabolism.

ObjectiveInterindividual variation in pathways affecting cellular cholesterol metabolism can influence levels of plasma cholesterol, a well-established risk factor for cardiovascular disease. Inherent variation among immortalized lymphoblastoid cell lines from different donors can be leveraged to discover novel genes that modulate cellular cholesterol metabolism. The objective of this study was to identify novel genes that regulate cholesterol metabolism by testing for evidence of correlated gene expression with cellular levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) mRNA, a marker for cellular cholesterol homeostasis, in a large panel of lymphoblastoid cell lines.Approach and resultsExpression array profiling was performed on 480 lymphoblastoid cell lines established from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, and transcripts were tested for evidence of correlated expression with HMGCR as a marker of intracellular cholesterol homeostasis. Of these, transmembrane protein 55b (TMEM55B) showed the strongest correlation (r=0.29; P=4.0E-08) of all genes not previously implicated in cholesterol metabolism and was found to be sterol regulated. TMEM55B knockdown in human hepatoma cell lines promoted the decay rate of the low-density lipoprotein receptor, reduced cell surface low-density lipoprotein receptor protein, impaired low-density lipoprotein uptake, and reduced intracellular cholesterol.ConclusionsHere, we report identification of TMEM55B as a novel regulator of cellular cholesterol metabolism through the combination of gene expression profiling and functional studies. The findings highlight the value of an integrated genomic approach for identifying genes that influence cholesterol homeostasis

Identification of candidate genes associated with statin.

<p>HepG2 (n = 4), Hep3B (n = 3) and 480 LCLs derived from participants of the Cholesterol and Pharmacogenetics (CAP) clinical trial were exposed to either 2 µM activated simvastatin or sham buffer for 24 hours, and gene expression was quantified on the Illumina HTref8v3 bead chip. Genes expressed in both LCLs and normal human liver (n = 60, GEO dataset GSE28893) were identified and tested for evidence of statin-induced changes in gene expression.</p

Relationship between <i>RHOA</i> haplotypes H3B and H2 and statin-induced changes in LDL-cholesterol response in the CAP (N = 580) and PRINCE (N = 1306) clinical trials.

<p>Linear regression of statin-induced changes (delta log) in LDL-cholesterol with adjustment age, sex, BMI, smoking status, and clinical trial was used to test for association with <i>RHOA</i> haplotypes. Graph depicts mean percent change in LDL-cholesterol +/− SE.</p

Expression characteristics of identified candidate genes.

<p>Expression characteristics of identified candidate genes.</p