Residual OXPHOS is required to drive primary and metastatic lung tumours in an orthotopic breast cancer model.

Background: Fast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells. Methods: 4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice. Results: OXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways. Conclusion: In summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.

Bioenergetic and Metabolic Adaptation in Tumor Progression and Metastasis.

The ability of cancer cells to adjust their metabolism in response to environmental changes is a well-recognized hallmark of cancer. Diverse cancer and non-cancer cells within tumors compete for metabolic resources. Metabolic demands change frequently during tumor initiation, progression and metastasis, challenging our quest to better understand tumor biology and develop novel therapeutics. Vascularization, physical constraints, immune responses and genetic instability promote tumor evolution resulting in immune evasion, opportunities to breach basement membrane barriers and spread through the circulation and lymphatics. In addition, the unfolded protein response linked to the ubiquitin proteasome system is a key player in addressing stoichiometric imbalances between nuclear and mitochondrially-encoded protein subunits of respiratory complexes, and nuclear-encoded mitochondrial ribosomal protein subunits. While progressive genetic changes, some of which affect metabolic adaptability, contribute to tumorigenesis and metastasis through clonal expansion, epigenetic changes are also important and more dynamic in nature. Understanding the role of stromal and immune cells in the tumor microenvironment in remodeling cancer cell energy metabolism has become an increasingly important area of research. In this perspective, we discuss the adaptations made by cancer cells to balance mitochondrial and glycolytic energy metabolism. We discuss how hypoxia and nutrient limitations affect reductive and oxidative stress through changes in mitochondrial electron transport activity. We propose that integrated responses to cellular stress in cancer cells are central to metabolic flexibility in general and bioenergetic adaptability in particular and are paramount in tumor progression and metastasis.

Mitochondrial DNA Affects the Expression of Nuclear Genes Involved in Immune and Stress Responses in a Breast Cancer Model.

Tumor cells without mitochondrial (mt) DNA (ρ0 cells) are auxotrophic for uridine, and their growth is supported by pyruvate. While ATP synthesis in ρ0 cells relies on glycolysis, they fail to form tumors unless they acquire mitochondria from stromal cells. Mitochondrial acquisition restores respiration that is essential for de novo pyrimidine biosynthesis and for mitochondrial ATP production. The physiological processes that underpin intercellular mitochondrial transfer to tumor cells lacking mtDNA and the metabolic remodeling and restored tumorigenic properties of cells that acquire mitochondria are not well understood. Here, we investigated the changes in mitochondrial and nuclear gene expression that accompany mtDNA deletion and acquisition in metastatic murine 4T1 breast cancer cells. Loss of mitochondrial gene expression in 4T1ρ0 cells was restored in cells recovered from subcutaneous tumors that grew from 4T1ρ0 cells following acquisition of mtDNA from host cells. In contrast, the expression of most nuclear genes that encode respiratory complex subunits and mitochondrial ribosomal subunits was not greatly affected by loss of mtDNA, indicating ineffective mitochondria-to-nucleus communication systems for these nuclear genes. Further, analysis of nuclear genes whose expression was compromised in 4T1ρ0 cells showed that immune- and stress-related genes were the most highly differentially expressed, representing over 70% of those with greater than 16-fold higher expression in 4T1 compared with 4T1ρ0 cells. The monocyte recruiting chemokine, Ccl2, and Psmb8, a subunit of the immunoproteasome that generates MHCI-binding peptides, were the most highly differentially expressed. Early monocyte/macrophage recruitment into the tumor mass was compromised in 4T1ρ0 cells but recovered before mtDNA could be detected. Taken together, our results show that mitochondrial acquisition by tumor cells without mtDNA results in bioenergetic remodeling and re-expression of genes involved in immune function and stress adaptation.

A semi-automated technique for adenoma quantification in the ApcMin mouse using FeatureCounter.

Colorectal cancer is a major contributor to death and disease worldwide. The ApcMin mouse is a widely used model of intestinal neoplasia, as it carries a mutation also found in human colorectal cancers. However, the method most commonly used to quantify tumour burden in these mice is manual adenoma counting, which is time consuming and poorly suited to standardization across different laboratories. We describe a method to produce suitable photographs of the small intestine of ApcMin mice, process them with an ImageJ macro, FeatureCounter, which automatically locates image features potentially corresponding to adenomas, and a machine learning pipeline to identify and quantify them. Compared to a manual method, the specificity (or True Negative Rate, TNR) and sensitivity (or True Positive Rate, TPR) of this method in detecting adenomas are similarly high at about 80% and 87%, respectively. Importantly, total adenoma area measures derived from the automatically-called tumours were just as capable of distinguishing high-burden from low-burden mice as those established manually. Overall, our strategy is quicker, helps control experimenter bias, and yields a greater wealth of information about each tumour, thus providing a convenient route to getting consistent and reliable results from a study.

Exome Sequencing Diagnoses X-Linked Moesin-Associated Immunodeficiency in a Primary Immunodeficiency Case.

Background: We investigated the molecular etiology of a young male proband with confirmed immunodeficiency of unknown cause, presenting with recurrent bacterial and Varicella zoster viral infections in childhood and persistent lymphopenia into early adulthood. Aim: To identify causative functional genetic variants related to an undiagnosed primary immunodeficiency. Method: Whole genome microarray copy number variant (CNV) analysis was performed on the proband followed by whole exome sequencing (WES) and trio analysis of the proband and family members. A >4 kbp deletion identified by repeated CNV analysis of exome sequencing data along with three damaging missense single nucleotide variants were validated by Sanger sequencing in all family members. Confirmation of the causative role of the candidate gene was performed by qPCR and Western Blot analyses on the proband, family members and a healthy control. Results: CNV identified our previously reported interleukin 25 amplification in the proband; however, the variant was not validated to be a candidate gene for immunodeficiency. WES trio analysis, data filtering and in silico prediction identified a novel, damaging (SIFT: 0; Polyphen 1; Grantham score: 101) and disease-causing (MutationTaster) single base mutation in the X chromosome (c.511C > T p.Arg171Trp) MSN gene not identified in the UCSC Genome Browser database. The mutation was validated by Sanger sequencing, confirming the proband was hemizygous X-linked recessive (-/T) at this locus and inherited the affected T allele from his non-symptomatic carrier mother (C/T), with other family members (father, sister) confirmed to be wild type (C/C). Western Blot analysis demonstrated an absence of moesin protein in lymphocytes derived from the proband, compared with normal expression in lymphocytes derived from the healthy control, father and mother. qPCR identified significantly lower MSN mRNA transcript expression in the proband compared to an age- and sex-matched healthy control subject in whole blood (p = 0.02), and lymphocytes (p = 0.01). These results confirmed moesin deficiency in the proband, directly causative of his immunodeficient phenotype. Conclusion: These findings confirm X-linked moesin-associated immunodeficiency in a proband previously undiagnosed up to 24 years of age. This study also highlights the utility of WES for the diagnosis of rare or novel forms of primary immunodeficiency disease.

Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts.

Neisseria meningitidis (meningococcus) can cause meningococcal disease, a rapidly progressing and often fatal disease that can occur in previously healthy children. Meningococci are found in healthy carriers, where they reside in the nasopharynx as commensals. While carriage is relatively common, invasive disease, associated with hypervirulent strains, is a comparatively rare event. The basis of increased virulence in some strains is not well understood. New Zealand suffered a protracted meningococcal disease epidemic, from 1991 to 2008. During this time, a household carriage study was carried out in Auckland: household contacts of index meningococcal disease patients were swabbed for isolation of carriage strains. In many households, healthy carriers harbored strains identical, as determined by laboratory typing, to the ones infecting the associated patient. We carried out more-detailed analyses of carriage and disease isolates from a select number of households. We found that isolates, although indistinguishable by laboratory typing methods and likely closely related, had many differences. We identified multiple genome variants and transcriptional differences between isolates. These studies enabled the identification of two new phase-variable genes. We also found that several carriage strains had lost their type IV pili and that this loss correlated with reduced tumor necrosis factor alpha (TNF-α) expression when cultured with epithelial cells. While nonpiliated meningococcal isolates have been previously found in carriage strains, this is the first evidence of an association between type IV pili from meningococci and a proinflammatory epithelial response. We also identified potentially important metabolic differences between carriage and disease isolates, including the sulfate assimilation pathway. IMPORTANCENeisseria meningitidis causes meningococcal disease but is frequently carried in the throats of healthy individuals; the factors that determine whether invasive disease develops are not completely understood. We carried out detailed studies of isolates, collected from patients and their household contacts, to identify differences between commensal throat isolates and those that caused invasive disease. Though isolates were identical by laboratory typing methods, we uncovered many differences in their genomes, in gene expression, and in their interactions with host cells. In particular, we found that several carriage isolates had lost their type IV pili, a surprising finding since pili are often described as essential for colonization. However, loss of type IV pili correlated with reduced secretion of a proinflammatory cytokine, TNF-α, when meningococci were cocultured with human bronchial epithelial cells; hence, the loss of pili could provide an advantage to meningococci, by resulting in a dampened localized host immune response.

Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA.

We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory (super)complexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest pathophysiological processes for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.

De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.