CD44 and RHAMM are essential for rapid growth of bladder cancer driven by loss of Glycogen Debranching Enzyme (AGL).

BACKGROUND: Loss of Amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL) drives rapid proliferation of bladder cancer cells by upregulating Hyaluronic acid(HA) Synthase (HAS2) mediated HA synthesis. However the role of HA receptors CD44 and Hyaluronan Mediated Motility Receptor (RHAMM) in regulating the growth of bladder cancer cells driven by loss of AGL has not been studied. METHODS: Western blot analysis and Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was carried out to study cellular apoptosis with HAS2, CD44 and RHAMM loss in bladder cancer cells with and without AGL expression. Proliferation and softagar assays were carried out to study cellular anchorage dependent and independent growth. Clinicopathologic analysis was carried out on bladder cancer patient datasets. RESULTS: Higher amounts of cleaved Cas3, Cas9 and PARP was observed in AGL low bladder cancer cell with loss of HAS2, CD44 or RHAMM. TUNEL staining showed more apoptotic cells with loss of HAS2, CD44 or RHAMM in AGL low bladder cancer cells. This revealed that bladder cancer cells whose aggressive growth is mediated by loss of AGL are susceptible to apoptosis with loss of HAS2, CD44 or RHAMM. Interestingly loss of either CD44 or RHAMM induces apoptosis in different low AGL expressing bladder cancer cell lines. Growth assays showed that loss of CD44 and RHAMM predominantly inhibit anchorage dependent and independent growth of AGL low bladder cancer cells. Clinicopathologic analysis revealed that high RHAMM mRNA expression is a marker of poor patient outcome in bladder cancer and patients with high RHAMM and low AGL tumor mRNA expression have poor survival. CONCLUSION: Our findings strongly point to the importance of the HAS2-HA-CD44/RHAMM pathway for rapid growth of bladder cancer cells with loss of AGL and provides rational for targeting this pathway at various steps for "personalized" treatment of bladder cancer patients based of their AGL expression status.

The multiMiR R package and database: integration of microRNA-target interactions along with their disease and drug associations.

microRNAs (miRNAs) regulate expression by promoting degradation or repressing translation of target transcripts. miRNA target sites have been catalogued in databases based on experimental validation and computational prediction using various algorithms. Several online resources provide collections of multiple databases but need to be imported into other software, such as R, for processing, tabulation, graphing and computation. Currently available miRNA target site packages in R are limited in the number of databases, types of databases and flexibility. We present multiMiR, a new miRNA-target interaction R package and database, which includes several novel features not available in existing R packages: (i) compilation of nearly 50 million records in human and mouse from 14 different databases, more than any other collection; (ii) expansion of databases to those based on disease annotation and drug microRNAresponse, in addition to many experimental and computational databases; and (iii) user-defined cutoffs for predicted binding strength to provide the most confident selection. Case studies are reported on various biomedical applications including mouse models of alcohol consumption, studies of chronic obstructive pulmonary disease in human subjects, and human cell line models of bladder cancer metastasis. We also demonstrate how multiMiR was used to generate testable hypotheses that were pursued experimentally.

A 20-gene model for molecular nodal staging of bladder cancer: development and prospective assessment.

BACKGROUND: Neoadjuvant chemotherapy before cystectomy confers a survival benefit in bladder cancer, but it has not been widely adopted since most patients do not benefit and we are at present unable to predict those that do. Since the most important predictor of recurrence after cystectomy is pathologically positive nodes, our aim was to assess techniques that define this stage for the selection of patients for neoadjuvant chemotherapy. METHODS: We developed a gene expression model (GEM) to predict the pathological node status in primary tumour tissue from three independent cohorts of patients who were clinically node negative. From a subset of transcripts detected faithfully by microarrays from both paired frozen and formalin-fixed tissues (32 pairs), we developed both the GEM and cutoffs that identified patient strata with raised risk of nodal involvement by use of two separate training cohorts (90 and 66 patients). We then assessed the GEM and cutoffs to predict node-positive disease in tissues from a phase 3 trial cohort (AUO-AB-05/95; 185 patients). FINDINGS: We developed a 20-gene GEM with an area under the curve of 0·67 (95% CI 0·60-0·75) for prediction of nodal disease at cystectomy in AUO-AB-05/95. The cutoff system identified patients with high relative risk (1·74, 95% CI 1·03-2·93) and low relative risk (0·70, 95% CI 0·51-0·96) of node-positive disease. Multivariate logistic regression showed the GEM predictor was independent of age, sex, pathological stage, and lymphovascular space invasion (coefficient 9·81, 95% CI 1·64-18·00; p=0·019). INTERPRETATION: Selecting patients for neoadjuvant chemotherapy on the basis of risk of node-positive disease has the potential to benefit high-risk patients while sparing other patients toxic effects and delay to cystectomy. FUNDING: US National Cancer Institute (R01CA143971).

Common introns within orthologous genes: software and application to plants.

The residence of spliceosomal introns within protein-coding genes can fluctuate over time, with genes gaining, losing or conserving introns in a complex process that is not entirely understood. One approach for studying intron evolution is to compare introns with respect to position and type within closely related genes. Here, we describe new, freely available software called Common Introns Within Orthologous Genes (CIWOG), available at http://ciwog.gdcb.iastate.edu/, which detects common introns in protein-coding genes based on position and sequence conservation in the corresponding protein alignments. CIWOG provides dynamic web displays that facilitate detailed intron studies within orthologous genes. User-supplied options control how introns are clustered into sets of common introns. CIWOG also identifies special classes of introns, in particular those with GC- or U12-type donor sites, which enables analyses of these introns in relation to their counterparts in the other genes in orthologous groups. The software is demonstrated with application to a comprehensive study of eight plant transcriptomes. Three specific examples are discussed: intron class conversion from GT- to GC-donor-type introns in monocots, plant U12-type intron conservation and a global analysis of intron evolution across the eight plant species.

Biomarkers for prognosis and treatment selection in advanced bladder cancer patients.

PURPOSE OF REVIEW: Patients with locally 'advanced' or muscle invasive bladder cancer have higher mortality rates than patients with nonmuscle invasive ('superficial') bladder cancer. Biomarkers can stratify clinical outcomes and thus promise to more accurately prognosticate and thus help assign patients to the appropriate treatments. The aim of this review is to summarize biomarker developments in the past year and to discuss their implications in prognosis and treatment selection in locally advanced bladder cancer. RECENT FINDINGS: Prognostic biomarkers for bladder cancer are identified at the DNA, RNA and/or protein levels. Some are new markers, whereas others are established markers with new roles in bladder cancer. Markers can report on the risk of disease recurrence or metastasis, or treatment responsiveness and thus are useful in determining 'who to treat' and 'what to treat with'. SUMMARY: The list of biomarkers for prognosis and treatment selection for advanced bladder cancer is growing. For most, their clinical relevance is unclear due to their lack of validation in external datasets. MicroRNAs and new techniques including next-generation sequencing offer additional opportunities for biomarker discovery, validation, and clinical applications.

Identifying therapeutic drug targets using bidirectional effect genes.

Prioritizing genes for translation to therapeutics for common diseases has been challenging. Here, we propose an approach to identify drug targets with high probability of success by focusing on genes with both gain of function (GoF) and loss of function (LoF) mutations associated with opposing effects on phenotype (Bidirectional Effect Selected Targets, BEST). We find 98 BEST genes for a variety of indications. Drugs targeting those genes are 3.8-fold more likely to be approved than non-BEST genes. We focus on five genes (IGF1R, NPPC, NPR2, FGFR3, and SHOX) with evidence for bidirectional effects on stature. Rare protein-altering variants in those genes result in significantly increased risk for idiopathic short stature (ISS) (OR = 2.75, p = 3.99 × 10-8). Finally, using functional experiments, we demonstrate that adding an exogenous CNP analog (encoded by NPPC) rescues the phenotype, thus validating its potential as a therapeutic treatment for ISS. Our results show the value of looking for bidirectional effects to identify and validate drug targets.

Induction of ER Stress by an AAV5 BDD FVIII Construct Is Dependent on the Strength of the Hepatic-Specific Promoter.

Adeno-associated virus 5 (AAV5)-human factor VIII-SQ (hFVIII-SQ; valoctocogene roxaparvovec) is an AAV-mediated product under evaluation for treatment of severe hemophilia A, which contains a B-domain-deleted hFVIII (hFVIII-SQ) transgene and a hybrid liver-specific promotor (HLP). To increase FVIII-SQ expression and reduce the vector dose required, a stronger promoter may be considered. However, because FVIII-SQ is a protein known to be difficult to fold and secrete, this could potentially induce endoplasmic reticulum (ER) stress. We evaluated the effect of two AAV5-hFVIII-SQ vectors with different liver-specific promoter strength (HLP << 100ATGB) on hepatic ER stress in mice. Five weeks after receiving vehicle or vector, the percentage of transduced hepatocytes and levels of liver hFVIII-SQ DNA and RNA increased dose dependently for both vectors. At lower doses, plasma hFVIII-SQ protein levels were higher for 100ATGB. This difference was attenuated at the highest dose. For 100ATGB, liver hFVIII-SQ protein accumulated dose dependently, with increased expression of ER stress markers at the highest dose, suggesting hepatocytes reached or exceeded their capacity to fold/secrete hFVIII-SQ. These data suggest that weaker promoters may require relatively higher doses to distribute expression load across a greater number of hepatocytes, whereas relatively stronger promoters may produce comparable levels of FVIII in fewer hepatocytes, with potential for ER stress.

Neurofilament light is a treatment-responsive biomarker in CLN2 disease.

OBJECTIVE: Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is a rare, progressive, fatal neurodegenerative pediatric disorder resulting from deficiencies of the lysosomal enzyme tripeptidyl peptidase 1 that are caused by mutations in TPP1. Identifying biomarkers of CLN2 disease progression will be important in assessing the efficacy of therapeutic interventions for this disorder. Neurofilament light is an intrinsic component of healthy neurons; elevated circulating extracellular neurofilament light is a biomarker of neuropathology in several adult-onset neurological diseases. Our objective was to assess whether circulating neurofilament light is a biomarker that is responsive to enzyme replacement therapy (ERT) in CLN2 disease. METHODS: Using an ultrasensitive immunoassay, we assessed plasma neurofilament light changes during disease progression in a canine model of CLN2 disease and in ERT clinical trial CLN2 disease patients. RESULTS: In tripeptidyl peptidase 1 (TPP1)-null dogs (N = 11), but not in control dogs [N = 6 (TPP1+/- ) and N = 27 (WT)], neurofilament light levels increased more than tenfold above initial low baseline levels during disease progression. Before treatment in 21 human subjects with CLN2 disease (age range: 1.72-6.85 years), neurofilament light levels were 48-fold higher (P < 0.001) than in 7 pediatric controls (age range: 8-11 years). Pretreatment neurofilament light did not significantly correlate with disease severity or age. In CLN2 disease subjects receiving ERT, neurofilament light levels decreased by 50% each year over more than 3 years of treatment. INTERPRETATION: Our data indicate that circulating neurofilament light is a treatment-responsive biomarker in CLN2 disease and could contribute to understanding of the pathophysiology of this devastating pediatric disorder.

Comparative Survival Analysis of Invasive Breast Cancer Patients Treated by a U.S. Military Medical Center and Matched Patients From the U.S. General Population.

OBJECTIVE: Many differences between U.S. military beneficiaries and the U.S. general population, including differences in health care access, are known factors affecting invasive breast cancer outcomes. Thus, comparing the two populations for any outcome differences and their contributing factors may provide insights to breast cancer prognosis. METHODS: Using a marginal Cox proportional hazards regression model, we compared disease-specific survival (DSS) and 5-year DSS rates between 418 patients from the Clinical Breast Care Project at the Walter Reed National Military Medical Center (CBCP-WR) and a set of 1:5 randomly matched patients from the Surveillance, Epidemiology, and End Results program. Patients were compared in the "demographic model" (adjusted by diagnosis year, age, and race) and the "overall model" (further adjusted by estrogen receptor, progesterone receptor, stage, and grade). RESULTS: In the "overall model," CBCP-WR patients were less likely overall to die from breast cancer (hazard ratio [HR] = 0.631, 95% confidence interval [CI] = 0.437-0.911; p = 0.014). This increase in survival was also significant in African American patients (HR = 0.524, 95% CI = 0.277-0.992; p = 0.047) and patients older than 50 (HR = 0.511, 95% CI = 0.306-0.854; p = 0.010). The advantage in 5-year DSS rate for CBCP-WR patients was 5.3% (93.1% vs. 87.8%; p < 0.001) in the "demographic model" and 3.4% (91.3% vs. 87.9%; p = 0.018) in the "overall model." CONCLUSION: CBCP-WR patients demonstrated significantly better DSS over matched SEER patients. Although a portion of the outcome disparity, i.e., 36% of the 5.3% DSS rate difference, could be explained by differences in tumor characteristics, the cause(s) behind the majority of the disparity has yet to be identified. Identification and further analysis of contributing factors to survival differences have the potential to improve clinical practice and outcomes for invasive breast cancer patients.

Dynamic variations in epithelial-to-mesenchymal transition (EMT), ATM, and SLFN11 govern response to PARP inhibitors and cisplatin in small cell lung cancer.

Small cell lung cancer (SCLC) is one of the most aggressive forms of cancer, with a 5-year survival <7%. A major barrier to progress is the absence of predictive biomarkers for chemotherapy and novel targeted agents such as PARP inhibitors. Using a high-throughput, integrated proteomic, transcriptomic, and genomic analysis of SCLC patient-derived xenografts (PDXs) and profiled cell lines, we identified biomarkers of drug sensitivity and determined their prevalence in patient tumors. In contrast to breast and ovarian cancer, PARP inhibitor response was not associated with mutations in homologous recombination (HR) genes (e.g., BRCA1/2) or HRD scores. Instead, we found several proteomic markers that predicted PDX response, including high levels of SLFN11 and E-cadherin and low ATM. SLFN11 and E-cadherin were also significantly associated with in vitro sensitivity to cisplatin and topoisomerase1/2 inhibitors (all commonly used in SCLC). Treatment with cisplatin or PARP inhibitors downregulated SLFN11 and E-cadherin, possibly explaining the rapid development of therapeutic resistance in SCLC. Supporting their functional role, silencing SLFN11 reduced in vitro sensitivity and drug-induced DNA damage; whereas ATM knockdown or pharmacologic inhibition enhanced sensitivity. Notably, SCLC with mesenchymal phenotypes (i.e., loss of E-cadherin and high epithelial-to-mesenchymal transition (EMT) signature scores) displayed striking alterations in expression of miR200 family and key SCLC genes (e.g., NEUROD1, ASCL1, ALDH1A1, MYCL1). Thus, SLFN11, EMT, and ATM mediate therapeutic response in SCLC and warrant further clinical investigation as predictive biomarkers.

Comparison of Breast Cancer Molecular Features and Survival by African and European Ancestry in The Cancer Genome Atlas.

Importance: African Americans have the highest breast cancer mortality rate. Although racial difference in the distribution of intrinsic subtypes of breast cancer is known, it is unclear if there are other inherent genomic differences that contribute to the survival disparities. Objectives: To investigate racial differences in breast cancer molecular features and survival and to estimate the heritability of breast cancer subtypes. Design, Setting, and Participants: Among a convenience cohort of patients with invasive breast cancer, breast tumor and matched normal tissue sample data (as of September 18, 2015) were obtained from The Cancer Genome Atlas. Main Outcomes and Measures: Breast cancer­free interval, tumor molecular features, and genetic variants. Results: Participants were 930 patients with breast cancer, including 154 black patients of African ancestry (mean [SD] age at diagnosis, 55.66 [13.01] years; 98.1% [n = 151] female) and 776 white patients of European ancestry (mean [SD] age at diagnosis, 59.51 [13.11] years; 99.0% [n = 768] female). Compared with white patients, black patients had a worse breast cancer-free interval (hazard ratio, HR=1.67; 95% CI, 1.02-2.74; P = .043). They had a higher likelihood of basal-like (odds ratio, 3.80; 95% CI, 2.46-5.87; P < .001) and human epidermal growth factor receptor 2 (ERBB2 [formerly HER2])­enriched (odds ratio, 2.22; 95% CI, 1.10-4.47; P = .027) breast cancer subtypes, with the Luminal A subtype as the reference. Blacks had more TP53 mutations and fewer PIK3CA mutations than whites. While most molecular differences were eliminated after adjusting for intrinsic subtype, the study found 16 DNA methylation probes, 4 DNA copy number segments, 1 protein, and 142 genes that were differentially expressed, with the gene-based signature having an excellent capacity for distinguishing breast tumors from black vs white patients (cross-validation C index, 0.878). Using germline genotypes, the heritability of breast cancer subtypes (basal vs nonbasal) was estimated to be 0.436 (P = 1.5 × 10−14). The estrogen receptor­positive polygenic risk score built from 89 known susceptibility variants was higher in blacks than in whites (difference, 0.24; P = 2.3 × 10−5), while the estrogen receptor­negative polygenic risk score was much higher in blacks than in whites (difference, 0.48; P = 2.8 × 10−11). Conclusions and Relevance: On the molecular level, after adjusting for intrinsic subtype frequency differences, this study found a modest number of genomic differences but a significant clinical survival outcome difference between blacks and whites in The Cancer Genome Atlas data set. Moreover, more than 40% of breast cancer subtype frequency differences could be explained by genetic variants. These data could form the basis for the development of molecular targeted therapies to improve clinical outcomes for the specific subtypes of breast cancers that disproportionately affect black women. Findings also indicate that personalized risk assessment and optimal treatment could reduce deaths from aggressive breast cancers for black women.

Loss of Glycogen Debranching Enzyme AGL Drives Bladder Tumor Growth via Induction of Hyaluronic Acid Synthesis.

PURPOSE: We demonstrated that amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL) is a tumor growth suppressor and prognostic marker in human bladder cancer. Here we determine how AGL loss enhances tumor growth, hoping to find therapeutically tractable targets/pathways that could be used in patients with low AGL-expressing tumors. EXPERIMENTAL DESIGN: We transcriptionally profiled bladder cell lines with different AGL expression. By focusing on transcripts overexpressed as a function of low AGL and associated with adverse clinicopathologic variables in human bladder tumors, we sought to increase the chances of discovering novel therapeutic opportunities. RESULTS: One such transcript was hyaluronic acid synthase 2 (HAS2), an enzyme responsible for hyaluronic acid (HA) synthesis. HAS2 expression was inversely proportional to that of AGL in bladder cancer cells and immortalized and normal urothelium. HAS2-driven HA synthesis was enhanced in bladder cancer cells with low AGL, and this drove anchorage-dependent and independent growth. siRNA-mediated depletion of HAS2 or inhibition of HA synthesis by 4-methylumbelliferone (4MU) abrogated in vitro and xenograft growth of bladder cancer cells with low AGL. AGL and HAS2 mRNA expression in human tumors was inversely correlated in patient datasets. Patients with high HAS2 and low AGL tumor mRNA expression had poor survival, lending clinical support to xenograft findings that HAS2 drives growth of tumors with low AGL. CONCLUSIONS: Our study establishes HAS2-mediated HA synthesis as a driver of growth of bladder cancer with low AGL and provides preclinical rationale for personalized targeting of HAS2/HA signaling in patients with low AGL-expressing tumors.

Resistance to PARP inhibitors by SLFN11 inactivation can be overcome by ATR inhibition.

Poly(ADP-ribose) polymerase inhibitors (PARPIs) kill cancer cells by trapping PARP1 and PARP2. Talazoparib, the most potent PARPI inhibitor (PARPI), exhibits remarkable selectivity among the NCI-60 cancer cell lines beyond BRCA inactivation. Our genomic analyses reveal high correlation between response to talazoparib and Schlafen 11 (SLFN11) expression. Causality was established in four isogenic SLFN11-positive and -negative cell lines and extended to olaparib. Response to the talazoparib-temozolomide combination was also driven by SLFN11 and validated in 36 small cell lung cancer cell lines, and in xenograft models. Resistance in SLFN11-deficient cells was caused neither by impaired drug penetration nor by activation of homologous recombination. Rather, SLFN11 induced irreversible and lethal replication inhibition, which was independent of ATR-mediated S-phase checkpoint. The resistance to PARPIs by SLFN11 inactivation was overcome by ATR inhibition, mechanistically because SLFN11-deficient cells solely rely on ATR activation for their survival under PARPI treatment. Our study reveals that SLFN11 inactivation, which is common (~45%) in cancer cells, is a novel and dominant resistance determinant to PARPIs.

SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html.

Role in tumor growth of a glycogen debranching enzyme lost in glycogen storage disease.

BACKGROUND: Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances. METHODS: Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis. RESULTS: We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2. CONCLUSIONS: Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth.

Translation initiation factor eIF3b expression in human cancer and its role in tumor growth and lung colonization.

PURPOSE: Discovery transcriptomic analyses suggest eukaryotic initiation factor 3b (eIF3b) is elevated in human bladder and prostate cancer, yet its role as a prognostic factor or its requirement in the maintenance or progression of human cancer is not established. Here, we determine the therapeutic potential of eIF3b by examining the clinical relevance of its expression in human cancer tissues and its role in experimental tumor models. EXPERIMENTAL DESIGN: We examined mRNA expression of eIF3b in bladder (N = 317) and prostate (N = 566) tissue samples and protein expression by immunohistochemistry in 143 bladder tumor samples as a function of clinicopathologic features. The impact of eIF3b depletion by siRNA in human cancer lines was evaluated in regard to in vitro cell growth, cell cycle, migration, in vivo subcutaneous tumor growth, and lung colonization. RESULTS: eIF3b mRNA expression correlated to tumor grade, stage, and survival in human bladder and prostate cancer. eIF3b protein expression stratified survival in human bladder cancer. eIF3b depletion reduced in vitro cancer cell growth; inhibited G1-S cell-cycle transition by changing protein but not RNA expression of cyclin A, E, Rb, and p27Kip1; inhibited migration; and disrupted actin cytoskeleton and focal adhesions. These changes were associated with decreased protein expression of integrin α5. Integrin α5 depletion phenocopied effects observed with eIF3b. eIF3b-depleted bladder cancer cells formed fewer subcutaneous tumors that grew more slowly and had reduced lung colonization. CONCLUSION: eIF3b expression relates to human bladder and prostate cancer prognosis, is required for tumor growth, and thus a candidate therapeutic target.

Contributions of KRAS and RAL in non-small-cell lung cancer growth and progression.

INTRODUCTION: KRAS mutations are poor prognostic markers for patients with non-small-cell lung cancer (NSCLC). RALA and RALB GTPases lie downstream of RAS and are implicated in RAS-mediated tumorigenesis. However, their biological or prognostic role in the context of KRAS mutation in NSCLC is unclear. METHODS: Using expression analysis of human tumors and a panel of cell lines coupled with functional in vivo and in vitro experiments, we evaluated the prognostic and functional importance of RAL in NSCLC and their relationship to KRAS expression and mutation. RESULTS: Immunohistochemical (N = 189) and transcriptomic (N = 337) analyses of NSCLC patients revealed high RALA and RALB expression was associated with poor survival. In a panel of 14 human NSCLC cell lines, RALA and RALB had higher expression in KRAS mutant cell lines whereas RALA but not RALB activity was higher in KRAS mutant cell lines. Depletion of RAL paralogs identified cell lines that are dependent on RAL expression for proliferation and anchorage independent growth. Overall, growth of NSCLC cell lines that carry a glycine to cystine KRAS mutation were more sensitive to RAL depletion than those with wild-type KRAS. The use of gene expression and outcome data from 337 human tumors in RAL-KRAS interaction analysis revealed that KRAS and RAL paralog expression jointly impact patient prognosis. CONCLUSION: RAL GTPase expression carries important additional prognostic information to KRAS status in NSCLC patients. Simultaneously targeting RAL may provide a novel therapeutic approach in NSCLC patients harboring glycine to cystine KRAS mutations.

A framework to select clinically relevant cancer cell lines for investigation by establishing their molecular similarity with primary human cancers.

Experimental work on human cancer cell lines often does not translate to the clinic. We posit that this is because some cells undergo changes in vitro that no longer make them representative of human tumors. Here, we describe a novel alignment method named Spearman's rank correlation classification method (SRCCM) that measures similarity between cancer cell lines and human tumors via gene expression profiles, for the purpose of selecting lines that are biologically relevant. To show utility, we used SRCCM to assess similarity of 36 bladder cancer lines with 10 epithelial human tumor types (N = 1,630 samples) and with bladder tumors of different stages and grades (N = 144 samples). Although 34 of 36 lines aligned to bladder tumors rather than other histologies, only 16 of 28 had SRCCM assigned grades identical to that of their original source tumors. To evaluate the clinical relevance of this approach, we show that gene expression profiles of aligned cell lines stratify survival in an independent cohort of 87 bladder patients (HR = 3.41, log-rank P = 0.0077) whereas unaligned cell lines using original tumor grades did not. We repeated this process on 22 colorectal cell lines and found that gene expression profiles of 17 lines aligning to colorectal tumors and selected based on their similarity with 55 human tumors stratified survival in an independent cohort of 177 colorectal cancer patients (HR = 2.35, log-rank P = 0.0019). By selecting cell lines that reflect human tumors, our technique promises to improve the clinical translation of laboratory investigations in cancer.