Sangkyou Lee

Phosphorylation on tyrosine-15 of p34Cdc2 by ErbB2 inhibits p34Cdc2 activation and is involved in resistance to taxol-induced apoptosis

ErbB2 overexpression confers resistance to taxol-induced apoptosis by inhibiting p34(Cdc2) activation. One mechanism is via ErbB2-mediated upregulation of p21(Cip1), which inhibits Cdc2. Here, we report that the inhibitory phosphorylation on Cdc2 tyrosine (Y)15 (Cdc2-Y15-p) is elevated in ErbB2-overexpressing breast cancer cells and primary tumors. ErbB2 binds to and colocalizes with cyclin B-Cdc2 complexes and phosphorylates Cdc2-Y15. The ErbB2 kinase domain is sufficient to directly phosphorylate Cdc2-Y15. Increased Cdc2-Y15-p in ErbB2-overexpressing cells corresponds with delayed M phase entry. Expressing a nonphosphorylatable mutant of Cdc2 renders cells more sensitive to taxol-induced apoptosis. Thus, ErbB2 membrane RTK can confer resistance to taxol-induced apoptosis by directly phosphorylating Cdc2.

Combined trastuzumab and paclitaxel treatment better inhibits ErbB‐2‐mediated angiogenesis in breast carcinoma through a more effective inhibition of Akt than either treatment alone

Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a humanized anti‐ErbB‐2 monoclonal antibody that has demonstrated antitumor function, especially in combination with other chemotherapies such as paclitaxel (Taxol; Bristol Myers‐Squibb, Princeton, NJ), in patients with tumors that overexpress ErbB‐2. Because the repeated administration of low‐dose chemotherapy, such as paclitaxel, endorsed an antiangiogenic effect in vitro, and because trastuzumab was shown to inhibit angiogenesis in tumor xenografts, the authors investigated whether ErbB‐2‐mediated angiogenic responses would be inhibited more effectively by the combined treatment of paclitaxel plus trastuzumab.

Quantitation of Aurora Kinase A Gene Copy Number in Urine Sediments and Bladder Cancer Detection

BACKGROUND Chromosome missegregation and the resulting aneuploidy is a common change in neoplasia. The Aurora kinase A (AURKA) gene, which encodes a key regulator of mitosis, is frequently amplified and/or overexpressed in cancer cells, and the level of AURKA amplification is associated with the level of aneuploidy. We examined whether AURKA gene amplification is a biomarker for the detection of bladder cancer. METHODS The effect of ectopic expression of Aurora kinase A (AURKA) using an adenoviral vector in simian virus 40-immortalized urothelial cells (SV-HUC) on centrosome multiplication and chromosome copy number was measured in vitro by immunofluorescence and fluorescence in situ hybridization (FISH), respectively. The FISH test was also used to examine AURKA gene copy number in exfoliated cells in voided urine samples from 23 patients with bladder cancer and 7 healthy control subjects (training set), generating a model for bladder cancer detection that was subsequently validated in an independent set of voided urine samples from 100 bladder cancer patients and 148 control subjects (92 healthy individuals and 56 patients with benign urologic disorders). An AURKA gene score (the proportion of cells with three or more AURKA signals) was used to produce receiver operating characteristic (ROC) curves and to calculate the specificity and sensitivity of the AURKA FISH test. Differences between mean AURKA scores in different pathogenetic groups of bladder cancer stratified according to histological grade and stage were tested by unpaired Mann-Whitney t tests or one-way Wilcoxon tests. All statistical tests were two-sided. RESULTS Forced overexpression of AURKA in urothelial cells induced amplification of centrosomes, chromosome missegregation, and aneuploidy, and natural overexpression was detectable in in situ lesions from patients with bladder cancer. The FISH test for the AURKA gene copy number performed on the validation set yielded a specificity of 96.6% (95% confidence interval [CI] = 92.3% to 98.5%) and sensitivity of 87% (95% CI = 79.0% to 92.2%) and an area under the ROC curve of 0.939 (95% CI = 0.906 to 0.971; P < .001). CONCLUSION Overexpression of AURKA can cause aneuploidy in urothelial cells, and the AURKA gene copy number is a promising biomarker for detection of bladder cancer.

Understanding the development of human bladder cancer by using a whole-organ genomic mapping strategy.

The search for the genomic sequences involved in human cancers can be greatly facilitated by maps of genomic imbalances identifying the involved chromosomal regions, particularly those that participate in the development of occult preneoplastic conditions that progress to clinically aggressive invasive cancer. The integration of such regions with human genome sequence variation may provide valuable clues about their overall structure and gene content. By extension, such knowledge may help us understand the underlying genetic components involved in the initiation and progression of these cancers. We describe the development of a genome-wide map of human bladder cancer that tracks its progression from in situ precursor conditions to invasive disease. Testing for allelic losses using a genome-wide panel of 787 microsatellite markers was performed on multiple DNA samples, extracted from the entire mucosal surface of the bladder and corresponding to normal urothelium, in situ preneoplastic lesions, and invasive carcinoma. Using this approach, we matched the clonal allelic losses in distinct chromosomal regions to specific phases of bladder neoplasia and produced a detailed genetic map of bladder cancer development. These analyses revealed three major waves of genetic changes associated with growth advantages of successive clones and reflecting a stepwise conversion of normal urothelial cells into cancer cells. The genetic changes map to six regions at 3q22–q24, 5q22–q31, 9q21–q22, 10q26, 13q14, and 17p13, which may represent critical hits driving the development of bladder cancer. Finally, we performed high-resolution mapping using single nucleotide polymorphism markers within one region on chromosome 13q14, containing the model tumor suppressor gene RB1, and defined a minimal deleted region associated with clonal expansion of in situ neoplasia. These analyses provided new insights on the involvement of several non-coding sequences mapping to the region and identified novel target genes, termed forerunner (FR) genes, involved in early phases of cancer development.

Meta-Analysis of the Luminal and Basal Subtypes of Bladder Cancer and the Identification of Signature Immunohistochemical Markers for Clinical Use

Background It has been suggested that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to chemotherapy. We aimed to validate these subtypes in several clinical cohorts and identify signature immunohistochemical markers that would permit simple and cost-effective classification of the disease in primary care centers. Methods We analyzed genomic expression profiles of bladder cancer in three cohorts of fresh frozen tumor samples: MD Anderson (n = 132), Lund (n = 308), and The Cancer Genome Atlas (TCGA) (n = 408) to validate the expression signatures of luminal and basal subtypes and relate them to clinical follow-up data. We also used an MD Anderson cohort of archival bladder tumor samples (n = 89) and a parallel tissue microarray to identify immunohistochemical markers that permitted the molecular classification of bladder cancer. Findings Bladder cancers could be assigned to two candidate intrinsic molecular subtypes referred to here as luminal and basal in all of the datasets analyzed. Luminal tumors were characterized by the expression signature similar to the intermediate/superficial layers of normal urothelium. They showed the upregulation of PPARγ target genes and the enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. In addition, luminal tumors were characterized by the overexpression of E-Cadherin, HER2/3, Rab-25, and Src. Basal tumors showed the expression signature similar to the basal layer of normal urothelium. They showed the upregulation of p63 target genes, the enrichment for TP53 and RB1 mutations, and overexpression of CD49, Cyclin B1, and EGFR. Survival analyses showed that the muscle-invasive basal bladder cancers were more aggressive when compared to luminal cancers. The immunohistochemical expressions of only two markers, luminal (GATA3) and basal (KRT5/6), were sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy. Interpretation The molecular subtypes of bladder cancer have distinct clinical behaviors and sensitivities to chemotherapy, and a simple two-marker immunohistochemical classifier can be used for prognostic and therapeutic stratification. Funding U.S. National Cancer Institute and National Institute of Health.

Forerunner genes contiguous to RB1 contribute to the development of in situ neoplasia

We used human bladder cancer as a model system and the whole-organ histologic and genetic mapping strategy to identify clonal genetic hits associated with growth advantage, tracking the evolution of bladder cancer from intraurothelial precursor lesions. Six putative chromosomal regions critical for clonal expansion of intraurothelial neoplasia and development of bladder cancer were identified by using this approach. Focusing on one of the regions, which includes the model tumor suppressor RB1, we performed allelotyping of single-nucleotide polymorphic sites and identified a 1.34-Mb segment around RB1 characterized by a loss of polymorphism associated with the initial expansion of in situ neoplasia. This segment contains several positional candidate genes referred to by us as forerunner genes that may contribute to such expansion. We subsequently concentrated our efforts on the two neighbor genes flanking RB1, namely ITM2B and CHC1L, as well as P2RY5, which is located inside RB1. Here, we report that ITM2B and P2RY5 modulated cell survival and were silenced by methylation or point mutations, respectively, and thus by functional loss may contribute to the growth advantage of neoplasia. We also show that homozygous inactivation of P2RY5 was antecedent to the loss of RB1 during tumor development, and that nucleotide substitutions in P2RY5 represent a cancer predisposing factor.

WP760, a melanoma selective drug

PurposeOur goal was to perform studies on the specificity and antimelanoma mechanism of a novel bis-anthracycline, WP760. WP760 initially identified in the NCI 160 screen as anti-melanoma.MethodsThe methyl thiazolyl tetrazolium reduction (MTT) assay was used to test tumor cell growth inhibition; confocal microscopy to view WP760 intracellular distribution; flow cytometry for cell-cycle arrest and apoptosis; and Western blotting was employed to identify and compare quantities and kinetics of cell growth related molecule levels.ResultsWP760 induced G2/M-phase cell-cycle arrest and apoptosis in melanoma cell lines and short-term melanoma explants established from clinical specimens in a time and concentration dependent manner at nM concentrations. In contrast, effects on fibroblasts and A549 lung cancer cells required higher concentrations, suggesting that WP760 possesses selectivity for melanoma. Molecular studies indicated that WP760 induced p53 stabilization, checkpoint kinase 2 and p27Kip1 protein upregulation, and activation of caspase-3. Endogenous nitric oxide (NO) production has been implicated in the chemoresistance of melanoma; WP760 caused inhibition of the inducible nitric oxide synthase (iNOS) protein as well as inhibition of phosphorylation of ERK, known to drive the iNOS pathway. Based on WP760 localization into mitochondria, and caspase-3 inhibitor block the killing of WP760, the intrinsic pathway of apoptosis appears to have been activated.ConclusionsOur results indicate that WP760 affects a critical and unique set of growth regulatory effects in melanoma, and is a promising candidate for further preclinical studies.

Aurora Kinase A is a Biomarker for Bladder Cancer Detection and Contributes to its Aggressive Behavior

The effects of AURKA overexpression associated with poor clinical outcomes have been attributed to increased cell cycle progression and the development of genomic instability with aneuploidy. We used RNA interference to examine the effects of AURKA overexpression in human bladder cancer cells. Knockdown had minimal effects on cell proliferation but blocked tumor cell invasion. Whole genome mRNA expression profiling identified nicotinamide N-methyltransferase (NNMT) as a downstream target that was repressed by AURKA. Chromatin immunoprecipitation and NNMT promoter luciferase assays revealed that AURKA’s effects on NNMT were caused by PAX3-mediated transcriptional repression and overexpression of NNMT blocked tumor cell invasion in vitro. Overexpression of AURKA and activation of its downstream pathway was enriched in the basal subtype in primary human tumors and was associated with poor clinical outcomes. We also show that the FISH test for the AURKA gene copy number in urine yielded a specificity of 79.7% (95% confidence interval [CI] = 74.2% to 84.1%), and a sensitivity of 79.6% (95% CI = 74.2% to 84.1%) with an AUC of 0.901 (95% CI = 0.872 to 0.928; P < 0.001). These results implicate AURKA as an effective biomarker for bladder cancer detection as well as therapeutic target especially for its basal type.

In-frame cDNA library combined with protein complementation assay identifies ARL11-binding partners.

The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5′-untranslated regions (UTRs) that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5′-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an “in-frame cDNA library.” We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11), cellular retinoic acid binding protein 2 (CRABP2), and phosphoglycerate mutase 1 (PGAM1). Thus, the in-frame cDNA library without 5′-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.

Dysregulation of EMT Drives the Progression to Clinically Aggressive Sarcomatoid Bladder Cancer

The sarcomatoid variant of urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARCs and 84 cases of conventional urothelial carcinomas (UCs), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs showed a distinct mutational landscape with enrichment of TP53, RB1, and PIK3CA mutations. They were related to the basal molecular subtype of conventional UCs and could be divided into epithelial/basal and more clinically aggressive mesenchymal subsets based on TP63 and its target genes expression levels. Other analyses revealed that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of cell cycle and EMT networks, and nearly half exhibited a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer.