Studying the urine microbiome in superficial bladder cancer: samples obtained by midstream voiding versus cystoscopy.

BACKGROUND: Preliminary data suggest that the urinary microbiome may play a role in bladder cancer. Information regarding the most suitable method of collecting urine specimens is needed for the large population studies needed to address this. To compare microbiome metrics resulting from 16S ribosomal RNA gene sequencing between midstream, voided specimens and those obtained at cystoscopy. METHODS: Adults, with a history of superficial urothelial cell carcinoma (non-muscle invasive bladder cancer) being followed with periodic surveillance cystoscopy had a urine sample collected by a mid-stream, voided technique and then from the bladder at cystoscopy. Urine samples underwent 16S ribosomal RNA gene sequencing on the Illumina MiSeq platform. RESULTS: 22 subjects (8 female, 14 male) were included. There was no significant difference in beta diversity (diversity between samples) in all samples between collection methods. However, analysis by sex revealed a difference between voided and cystoscopy samples from the same individual in males (p = 0.006, Adonis test) but not in females (p = 0.317, Adonis test). No differences were seen by collection method in any alpha diversity (diversity within a sample) measurement or differential abundance of taxa. CONCLUSIONS: Beta diversity of the urine microbiome did differ by collection method for males only. This suggests that the urinary microbiomes of the two collection methods are not equivalent to each other, at least in males, which is the sex that bladder cancer occurs most frequently in. Therefore, the same collection method within a given study should be used.

Germline de novo mutation clusters arise during oocyte aging in genomic regions with high double-strand-break incidence.

Clustering of mutations has been observed in cancer genomes as well as for germline de novo mutations (DNMs). We identified 1,796 clustered DNMs (cDNMs) within whole-genome-sequencing data from 1,291 parent-offspring trios to investigate their patterns and infer a mutational mechanism. We found that the number of clusters on the maternal allele was positively correlated with maternal age and that these clusters consisted of more individual mutations with larger intermutational distances than those of paternal clusters. More than 50% of maternal clusters were located on chromosomes 8, 9 and 16, in previously identified regions with accelerated maternal mutation rates. Maternal clusters in these regions showed a distinct mutation signature characterized by C>G transversions. Finally, we found that maternal clusters were associated with processes involving double-strand-breaks (DSBs), such as meiotic gene conversions and de novo deletion events. This result suggested accumulation of DSB-induced mutations throughout oocyte aging as the mechanism underlying the formation of maternal mutation clusters.

A novel inborn error of the coenzyme Q10 biosynthesis pathway: cerebellar ataxia and static encephalomyopathy due to COQ5 C-methyltransferase deficiency.

Primary coenzyme Q10 (CoQ10 ; MIM# 607426) deficiencies are an emerging group of inherited mitochondrial disorders with heterogonous clinical phenotypes. Over a dozen genes are involved in the biosynthesis of CoQ10 , and mutations in several of these are associated with human disease. However, mutations in COQ5 (MIM# 616359), catalyzing the only C-methylation in the CoQ10 synthetic pathway, have not been implicated in human disease. Here, we report three female siblings of Iraqi-Jewish descent, who had varying degrees of cerebellar ataxia, encephalopathy, generalized tonic-clonic seizures, and cognitive disability. Whole-exome and subsequent whole-genome sequencing identified biallelic duplications in the COQ5 gene, leading to reduced levels of CoQ10 in peripheral white blood cells of all affected individuals and reduced CoQ10 levels in the only muscle tissue available from one affected proband. CoQ10 supplementation led to clinical improvement and increased the concentrations of CoQ10 in blood. This is the first report of primary CoQ10 deficiency caused by loss of function of COQ5, with delineation of the clinical, laboratory, histological, and molecular features, and insights regarding targeted treatment with CoQ10 supplementation.

Mutations in human homologue of chicken talpid3 gene (KIAA0586) cause a hybrid ciliopathy with overlapping features of Jeune and Joubert syndromes.

BACKGROUND: In chicken, loss of TALPID3 results in non-functional cilia and short-rib polydactyly syndrome. This phenotype is caused by a frameshift mutation in the chicken ortholog of the human KIAA0586 gene, which encodes a novel coiled-coil domain protein essential for primary ciliogenesis, suggesting that KIAA0586 can be associated with ciliopathy in human beings. METHODS: In our patients with ciliopathy (http://www.clinicaltrials.gov: NCT00068224), we have collected extensive clinical and neuroimaging data from affected individuals, and performed whole exome sequencing on DNA from affected individuals and their parents. We analysed gene expression on fibroblast cell line, and determined the effect of gene mutation on ciliogenesis in cells derived from patients. RESULTS: We identified biallelic mutations in the human TALPID3 ortholog, KIAA0586, in six children with findings of overlapping Jeune and Joubert syndromes. Fibroblasts cultured from one of the patients with Jeune-Joubert syndrome exhibited more severe cilia defects than fibroblasts from patients with only Joubert syndrome; this difference was reflected in KIAA0586 RNA expression levels. Rescue of the cilia defect with full-length wild type KIAA0586 indicated a causal link between cilia formation and KIAA0586 function. CONCLUSIONS: Our results show that biallelic deleterious mutations in KIAA0586 lead to Joubert syndrome with or without Jeune asphyxiating thoracic dystrophy. Furthermore, our results confirm that KIAA0586/TALPID3 is essential in cilia formation in human beings, expand the KIAA0586 phenotype to include features of Jeune syndrome and provide a pathogenetic connection between Joubert and Jeune syndromes, based on aberrant ciliogenesis.

Diagnosis and treatment of cancer using genomics.

The field of cancer diagnostics is in constant flux as a result of the rapid discovery of new genes associated with cancer, improvements in laboratory techniques for identifying disease causing events, and novel analytic methods that enable the integration of many different types of data. These advances have helped in the identification of novel, informative biomarkers. As more whole genome sequence data are generated and analyzed, emerging information on the baseline variability of the human genome has shown the importance of the ancestral genomic background in patients with a potential disease causing variant. The recent discovery of many novel DNA sequence variants, advances in sequencing and genomic technology, and improved analytic methods enable the impact of germline and somatic genome variation on tumorigenesis and metastasis to be determined. New molecular targets and companion diagnostics are changing the way geneticists and oncologists think about the causes, diagnosis, and treatment of cancer.

Mutations in NOTCH1 cause Adams-Oliver syndrome.

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5' region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743-1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.

Germline variation in cancer-susceptibility genes in a healthy, ancestrally diverse cohort: implications for individual genome sequencing.

Technological advances coupled with decreasing costs are bringing whole genome and whole exome sequencing closer to routine clinical use. One of the hurdles to clinical implementation is the high number of variants of unknown significance. For cancer-susceptibility genes, the difficulty in interpreting the clinical relevance of the genomic variants is compounded by the fact that most of what is known about these variants comes from the study of highly selected populations, such as cancer patients or individuals with a family history of cancer. The genetic variation in known cancer-susceptibility genes in the general population has not been well characterized to date. To address this gap, we profiled the nonsynonymous genomic variation in 158 genes causally implicated in carcinogenesis using high-quality whole genome sequences from an ancestrally diverse cohort of 681 healthy individuals. We found that all individuals carry multiple variants that may impact cancer susceptibility, with an average of 68 variants per individual. Of the 2,688 allelic variants identified within the cohort, most are very rare, with 75% found in only 1 or 2 individuals in our population. Allele frequencies vary between ancestral groups, and there are 21 variants for which the minor allele in one population is the major allele in another. Detailed analysis of a selected subset of 5 clinically important cancer genes, BRCA1, BRCA2, KRAS, TP53, and PTEN, highlights differences between germline variants and reported somatic mutations. The dataset can serve a resource of genetic variation in cancer-susceptibility genes in 6 ancestry groups, an important foundation for the interpretation of cancer risk from personal genome sequences.

The Smad7-Skp2 complex orchestrates Myc stability, impacting on the cytostatic effect of TGF-ß.

In most human cancers the Myc proto-oncogene is highly activated. Dysregulation of Myc oncoprotein contributes to tumorigenesis in numerous tissues and organs. Thus, targeting Myc stability could be a crucial step for cancer therapy. Here we report Smad7 as a key molecule regulating Myc stability and activity by recruiting the F-box protein, Skp2. Ectopic expression of Smad7 downregulated the protein level of Myc without affecting the transcription level, and significantly repressed its transcriptional activity, leading to inhibition of cell proliferation and tumorigenic activity. Furthermore, Smad7 enhanced ubiquitylation of Myc through direct interaction with Myc and recruitment of Skp2. Ablation of Smad7 resulted in less sensitivity to the growth inhibitory effect of TGF-ß by inducing stable Myc expression. In conclusion, these findings that Smad7 functions in Myc oncoprotein degradation and enhances the cytostatic effect of TGF-ß signaling provide a possible new therapeutic approach for cancer treatment.

SDF-1α mediates wound-promoted tumor growth in a syngeneic orthotopic mouse model of breast cancer.

Increased growth of residual tumors in the proximity of acute surgical wounds has been reported; however, the mechanisms of wound-promoted tumor growth remain unknown. Here, we used a syngeneic, orthotopic mouse model of breast cancer to study mechanisms of wound-promoted tumor growth. Our results demonstrate that exposure of metastatic mouse breast cancer cells (4T1) to SDF-1α, which is increased in wound fluid, results in increased tumor growth. Both, wounding and exposure of 4T1 cells to SDF-1α not only increased tumor growth, but also tumor cell proliferation rate and stromal collagen deposition. Conversely, systemic inhibition of SDF-1α signaling with the small molecule AMD 3100 abolished the effect of wounding, and decreased cell proliferation, collagen deposition, and neoangiogenesis to the levels observed in control animals. Furthermore, using different mouse strains we could demonstrate that the effect of wounding on tumor growth and SDF-1α levels is host dependent and varies between mouse strains. Our results show that wound-promoted tumor growth is mediated by elevated SDF-1α levels and indicate that the effect of acute wounds on tumor growth depends on the predetermined wound response of the host background and its predetermined wound response.

A small molecule (pluripotin) as a tool for studying cancer stem cell biology: proof of concept.

BACKGROUND: Cancer stem cells (CSC) are thought to be responsible for tumor maintenance and heterogeneity. Bona fide CSC purified from tumor biopsies are limited in supply and this hampers study of CSC biology. Furthermore, purified stem-like CSC subpopulations from existing tumor lines are unstable in culture. Finding a means to overcome these technical challenges would be a useful goal. In a first effort towards this, we examined whether a chemical probe that promotes survival of murine embryonic stem cells without added exogenous factors can alter functional characteristics in extant tumor lines in a fashion consistent with a CSC phenotype. METHODOLOGY/PRINCIPAL FINDINGS: The seven tumor lines of the NCI60 colon subpanel were exposed to SC-1 (pluripotin), a dual kinase and GTPase inhibitor that promotes self-renewal, and then examined for tumorigenicity under limiting dilution conditions and clonogenic activity in soft agar. A statistically significant increase in tumor formation following SC-1 treatment was observed (p<0.04). Cloning efficiencies and expression of putative CSC surface antigens (CD133 and CD44) were also increased. SC-1 treatment led to sphere formation in some colon tumor lines. Finally, SC-1 inhibited in vitro kinase activity of RSK2, and another RSK2 inhibitor increased colony formation implicating a role for this kinase in eliciting a CSC phenotype. CONCLUSIONS/SIGNIFICANCE: These findings validate a proof of concept study exposure of extant tumor lines to a small molecule may provide a tractable in vitro model for understanding CSC biology.

Transient tumor-fibroblast interactions increase tumor cell malignancy by a TGF-Beta mediated mechanism in a mouse xenograft model of breast cancer.

Carcinoma are complex societies of mutually interacting cells in which there is a progressive failure of normal homeostatic mechanisms, causing the parenchymal component to expand inappropriately and ultimately to disseminate to distant sites. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumor microenvironment and then to normal fibroblasts as it traverses the underlying connective tissue towards the bloodstream. The interaction of tumor cells with stromal fibroblasts influences tumor biology by mechanisms that are not yet fully understood. Here, we report a role for normal stroma fibroblasts in the progression of invasive tumors to metastatic tumors. Using a coculture system of human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts, we found that medium conditioned by cocultures of the two cell types (CoCM) increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. Transient treatment of MCF10CA1a cells with CoCM in vitro accelerated tumor growth at orthotopic sites in vivo, and resulted in an expanded pattern of metastatic engraftment. The effects of CoCM on MCF10CA1a cells were dependent on small amounts of active TGF-beta1 secreted by fibroblasts under the influence of the tumor cells, and required intact ALK5-, p38-, and JNK signaling in the tumor cells. In conclusion, these results demonstrate that transient interactions between tumor cells and normal fibroblasts can modify the acellular component of the local microenvironment such that it induces long-lasting increases in tumorigenicity and alters the metastatic pattern of the cancer cells in vivo. TGF-beta appears to be a key player in this process, providing further rationale for the development of anti-cancer therapeutics that target the TGF-beta pathway.

Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel.

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six-color fluorescence-activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co-expression patterns correlated with the colony-formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account.

Five strategies for accelerating the war on cancer in an era of budget deficits.

In recent years, the National Institutes of Health's largest institute, the National Cancer Institute (NCI), has adapted to difficult economic conditions by leveraging its robust infrastructure -- which includes risk factor surveillance and population monitoring, research centers (focused on basic, translation, clinical, and behavioral sciences), clinical trials and health care research networks, and rigorously validated statistical models -- to maximize the impact of scientific progress on the public health. To continue advancement and realize the opportunity of significant, population-level changes in cancer mortality, the NCI recommends that five national-level actions be taken: (1) significantly increase enrollment of Medicare patients into cancer clinical trials through adequate physician reimbursement, (2) increase NCI/Centers for Medicare and Medicaid Services collaboration on clinical trials research to evaluate the therapeutic efficacy of anticancer drugs, (3) establish a national outcomes research demonstration project to test strategies for measuring and improving health care quality and provide an evidence base for public policy, (4) leverage existing tobacco-control collaborations and possible new authorities at the U.S. Food and Drug Administration to realize the outstanding health gains possible from a reduction in tobacco use, and (5) increase colorectal cancer screening rates though intensified collaboration between federal agencies working to address barriers to access and use of screening. These cost-effective strategies provide the opportunity for extraordinary results in an era of budget deficits. Of the chronic diseases, cancer has the strongest national research infrastructure that can be leveraged to produce rapid results to inform budget prioritization and public policy, as well as mobilize new projects to answer critical public health questions.

Acute wounds accelerate tumorigenesis by a T cell-dependent mechanism.

We investigated the influence of acute wounding on tumor growth in a syngeneic mouse breast cancer model. Metastatic mouse breast cancer cells (4T1) were orthotopically injected into the mammary fat pads of BALB/c mice, and animals were wounded locally by full thickness dermal incisions above the mammary fat pads or remotely above the scapula 9 days later. Local, but not remote, wounding increased tumor size when compared with sham treatment. Injection of wound fluid close to the tumor site increased tumor growth, whereas in vitro wound fluid compared with serum increased the proliferation rate of 4T1 cells. Our results show that wound stroma can unfavorably influence growth of nearby tumors. This effect is T cell-dependent, as local wounding had no effect on tumor growth in nu/nu mice. The effect of wounding on tumor growth can be mimicked by acellular wound fluid, suggesting that T cells secrete or mediate secretion of cytokines or growth factors that then accelerate tumor growth. Here, we define an experimental model of wound-promoted tumor growth that will enable us to identify mechanisms and therapeutic targets to reduce the negative effect of tissue repair on residual tumors.