Quantifying forms and functions of intestinal bile acid pools in mice.

Bile acids (BAs) are gastrointestinal metabolites that serve dual functions in lipid absorption and cell signaling. BAs circulate actively between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with intestinal cells in vivo remain ill-defined. Through multi-site sampling of nearly 100 BA species in individual wild type mice, as well as mice lacking the ileal BA transporter, Asbt/Slc10a2, we calculate the ileal BA pool in fasting C57BL/6J mice to be ~0.3 µmoles/g. Asbt-mediated transport accounts for ~80% of this pool and amplifies size, whereas passive absorption explains the remaining ~20%, and generates diversity. Accordingly, ileal BA pools in mice lacking Asbt are ~5-fold smaller than in wild type controls, enriched in secondary BA species normally found in the colon, and elicit unique transcriptional responses in cultured ileal explants. This work quantitatively defines ileal BA pools in mice and reveals how BA dysmetabolism can impinge on intestinal physiology.

Estrogen Therapy Induces Receptor-Dependent DNA Damage Enhanced by PARP Inhibition in ER+ Breast Cancer.

PURPOSE: Clinical evidence indicates that treatment with estrogens elicits anticancer effects in ∼30% of patients with advanced endocrine-resistant estrogen receptor α (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains underused. Mechanistic understanding may offer strategies to enhance therapeutic efficacy. EXPERIMENTAL DESIGN: We performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17ß-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDX), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models. RESULTS: Cells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacologic suppression of the DNA damage response via PARP inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence in BRCA1/2-mutant and BRCA1/2-wild-type cell line and PDX models. CONCLUSIONS: E2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.

Alternating 17ß-Estradiol and Aromatase Inhibitor Therapies Is Efficacious in Postmenopausal Women with Advanced Endocrine-Resistant ER+ Breast Cancer.

PURPOSE: Strategies to implement estrogen therapy for advanced estrogen receptor-positive (ER+) breast cancer are underdeveloped. Preclinical data suggest that cycling treatment with 17ß-estradiol followed by estrogen deprivation can control tumor growth long-term. PATIENTS AND METHODS: Postmenopausal women with advanced ER+/HER2- breast cancer with recurrence or progression on ≥ 1 antiestrogen or aromatase inhibitor (AI)-based therapy were eligible. Patients received 17ß-estradiol (2 mg orally, three times a day) for 8 weeks followed by AI (physician's choice) for 16 weeks, alternating treatments on an 8-week/16-week schedule until disease progression. Patients then optionally received continuous single-agent treatment until a second instance of disease progression. Endpoints included 24-week clinical benefit and objective response per RECIST, and tumor genetic alterations. RESULTS: Of 19 evaluable patients, clinical benefit rate was 42.1% [95% confidence interval (CI), 23.1%-63.9%] and objective response rate (ORR) was 15.8% (95% CI, 5.7%-37.9%). One patient experienced a grade 3 adverse event related to 17ß-estradiol. Among patients who received continuous single-agent treatment until a second instance of disease progression, clinical benefit was observed in 5 of 12 (41.7%) cases. Tumor ER (ESR1) mutations were found by whole-exome profiling in 4 of 7 (57.1%) versus 2 of 9 (22.2%) patients who did versus did not experience clinical benefit from alternating 17ß-estradiol/AI therapy. The only two patients to experience objective responses to initial 17ß-estradiol had tumor ESR1 mutations. CONCLUSIONS: Alternating 17ß-estradiol/AI therapy may be a promising treatment for endocrine-refractory ER+ breast cancer, including following progression on CDK4/6 inhibitors or everolimus. Further study is warranted to determine whether the antitumor activity of 17ß-estradiol differs according to ESR1 mutation status.

Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer.

Purpose: Clinical evidence indicates that treatment with estrogens elicits anti-cancer effects in ∼30% of patients with advanced endocrine-resistant estrogen receptor alpha (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains under-utilized. Mechanistic understanding may offer strategies to enhance therapeutic efficacy. Experimental Design: We performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived (LTED) ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17ß-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDXs), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models. Results: Cells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacological suppression of the DNA damage response via poly(ADP-ribose) polymerase (PARP) inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence in BRCA1/2 -mutant and BRCA1 /2-wild-type cell line and PDX models. Conclusions: E2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.

Natural Antibodies Alert the Adaptive Immune System of the Presence of Transformed Cells in Early Tumorigenesis.

Recent studies have revealed a critical role for natural Abs (NAbs) in antitumor immune responses. However, the role of NAbs in cancer immunosurveillance remains unexplored, mainly because of the lack of in vivo models that mimic the early recognition and elimination of transforming cells. In this article, we propose a role for NAbs in alerting the immune system against precancerous neoantigen-expressing cells immediately after they escape intrinsic tumor suppression mechanisms. We identify four distinct reproducible, trackable, MHC-matched neoantigen-expressing cell models that do not form tumors as the end point. This amplified readout in the critical window prior to tumor formation allows investigation of new mediators of cancer immunosurveillance. We found that neoantigen-expressing cells adoptively transferred in NAb-deficient mice persisted, whereas they were eliminated in wild-type mice, indicating that the circulating NAb repertoire alerts the immune system to the presence of transformed cells. Moreover, immunity is mounted against immunogenic and nonimmunogenic neoantigens contained in the NAb-tagged cells, regardless of whether the NAb directly recognizes the neoantigens. Beyond these neoantigen-expressing model systems, we observed a significantly greater tumor burden in chemically and virally induced tumor models in NAb-deficient mice compared with wild-type mice. Restoration of the NAb repertoire in NAb-deficient mice elicited the recognition and elimination of neoantigen-expressing cells and cancer. These data show that NAbs are required and sufficient for elimination of transformed cells early in tumorigenesis. These models can now be used to investigate how NAbs stimulate immunity via recognition receptors to eliminate precancerous cells.

An Efficient, Nonphylogenetic Method for Detecting Genes Sharing Evolutionary Signals in Phylogenomic Data Sets.

Assessing the compatibility between gene family phylogenies is a crucial and often computationally demanding step in many phylogenomic analyses. Here, we describe the Evolutionary Similarity Index (IES), a means to assess shared evolution between gene families using a weighted orthogonal distance regression model applied to sequence distances. The utilization of pairwise distance matrices circumvents comparisons between gene tree topologies, which are inherently uncertain and sensitive to evolutionary model choice, phylogenetic reconstruction artifacts, and other sources of error. Furthermore, IES enables the many-to-many pairing of multiple copies between similarly evolving gene families. This is done by selecting non-overlapping pairs of copies, one from each assessed family, and yielding the least sum of squared residuals. Analyses of simulated gene family data sets show that IES's accuracy is on par with popular tree-based methods while also less susceptible to noise introduced by sequence alignment and evolutionary model fitting. Applying IES to an empirical data set of 1,322 genes from 42 archaeal genomes identified eight major clusters of gene families with compatible evolutionary trends. The most cohesive cluster consisted of 62 genes with compatible evolutionary signal, which occur as both single-copy and multiple homologs per genome; phylogenetic analysis of concatenated alignments from this cluster produced a tree closely matching previously published species trees for Archaea. Four other clusters are mainly composed of accessory genes with limited distribution among Archaea and enriched toward specific metabolic functions. Pairwise evolutionary distances obtained from these accessory gene clusters suggest patterns of interphyla horizontal gene transfer. An IES implementation is available at https://github.com/lthiberiol/evolSimIndex.

Pervasive acquisition of CRISPR memory driven by inter-species mating of archaea can limit gene transfer and influence speciation.

CRISPR-Cas systems provide prokaryotes with sequence-specific immunity against viruses and plasmids based on DNA acquired from these invaders, known as spacers. Surprisingly, many archaea possess spacers that match chromosomal genes of related species, including those encoding core housekeeping genes. By sequencing genomes of environmental archaea isolated from a single site, we demonstrate that inter-species spacers are common. We show experimentally, by mating Haloferax volcanii and Haloferax mediterranei, that spacers are indeed acquired chromosome-wide, although a preference for integrated mobile elements and nearby regions of the chromosome exists. Inter-species mating induces increased spacer acquisition and may result in interactions between the acquisition machinery of the two species. Surprisingly, many of the spacers acquired following inter-species mating target self-replicons along with those originating from the mating partner, indicating that the acquisition machinery cannot distinguish self from non-self under these conditions. Engineering the chromosome of one species to be targeted by the other's CRISPR-Cas reduces gene exchange between them substantially. Thus, spacers acquired during inter-species mating could limit future gene transfer, resulting in a role for CRISPR-Cas systems in microbial speciation.

Evolution of Bacterial Gene Transfer Agents.

Bacterial gene transfer agents (GTAs) are small virus-like particles that package DNA fragments and inject them into cells. They are encoded by gene clusters resembling defective prophages, with genes for capsid head and tail components. These gene clusters are usually assumed to be maintained by selection for the benefits of GTA-mediated recombination, but this has never been tested. We rigorously examined the potential benefits of GTA-mediated recombination, considering separately transmission of GTA-encoding genes and recombination of all chromosomal genes. In principle GTA genes could be directly maintained if GTA particles spread them to GTA- cells often enough to compensate for the loss of GTA-producing cells. However, careful bookkeeping showed that losses inevitably exceed gains for two reasons. First, cells must lyse to release particles to the environment. Second, GTA genes are not preferentially replicated before DNA is packaged. A simulation model was then used to search for conditions where recombination of chromosomal genes makes GTA+ populations fitter than GTA- populations. Although the model showed that both synergistic epistasis and some modes of regulation could generate fitness benefits large enough to overcome the cost of lysis, these benefits neither allowed GTA+ cells to invade GTA- populations, nor allowed GTA+ populations to resist invasion by GTA- cells. Importantly, the benefits depended on highly improbable assumptions about the efficiencies of GTA production and recombination. Thus, the selective benefits that maintain GTA gene clusters over many millions of years must arise from consequences other than transfer of GTA genes or recombination of chromosomal genes.

Comparative Analysis of Surface Layer Glycoproteins and Genes Involved in Protein Glycosylation in the Genus Haloferax.

Within the Haloferax genus, both the surface (S)-layer protein, and the glycans that can decorate it, vary between species, which can potentially result in many different surface types, analogous to bacterial serotypes. This variation may mediate phenotypes, such as sensitivity to different viruses and mating preferences. Here, we describe S-layer glycoproteins found in multiple Haloferax strains and perform comparative genomics analyses of major and alternative glycosylation clusters of isolates from two coastal sites. We analyze the phylogeny of individual glycosylation genes and demonstrate that while the major glycosylation cluster tends to be conserved among closely related strains, the alternative cluster is highly variable. Thus, geographically- and genetically-related strains may exhibit diverse surface structures to such an extent that no two isolates present an identical surface profile.

Insights into origin and evolution of α-proteobacterial gene transfer agents.

Several bacterial and archaeal lineages produce nanostructures that morphologically resemble small tailed viruses, but, unlike most viruses, contain apparently random pieces of the host genome. Since these elements can deliver the packaged DNA to other cells, they were dubbed gene transfer agents (GTAs). Because many genes involved in GTA production have viral homologs, it has been hypothesized that the GTA ancestor was a virus. Whether GTAs represent an atypical virus, a defective virus, or a virus co-opted by the prokaryotes for some function, remains to be elucidated. To evaluate these possibilities, we examined the distribution and evolutionary histories of genes that encode a GTA in the α-proteobacterium Rhodobacter capsulatus (RcGTA). We report that although homologs of many individual RcGTA genes are abundant across bacteria and their viruses, RcGTA-like genomes are mainly found in one subclade of α-proteobacteria. When compared with the viral homologs, genes of the RcGTA-like genomes evolve significantly slower, and do not have higher %A+T nucleotides than their host chromosomes. Moreover, they appear to reside in stable regions of the bacterial chromosomes that are generally conserved across taxonomic orders. These findings argue against RcGTA being an atypical or a defective virus. Our phylogenetic analyses suggest that RcGTA ancestor likely originated in the lineage that gave rise to contemporary α-proteobacterial orders Rhizobiales, Rhodobacterales, Caulobacterales, Parvularculales, and Sphingomonadales, and since that time the RcGTA-like element has co-evolved with its host chromosomes. Such evolutionary history is compatible with maintenance of these elements by bacteria due to some selective advantage. As for many other prokaryotic traits, horizontal gene transfer played a substantial role in the evolution of RcGTA-like elements, not only in shaping its genome components within the orders, but also in occasional dissemination of RcGTA-like regions across the orders and even to different bacterial phyla.

Impact of a homing intein on recombination frequency and organismal fitness.

Inteins are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins. Many inteins contain a homing endonuclease (HEN) domain and rely on its activity for horizontal propagation. However, successful invasion of an entire population will make this activity redundant, and the HEN domain is expected to degenerate quickly under these conditions. Several theories have been proposed for the continued existence of the both active HEN and noninvaded alleles within a population. However, to date, these models were not directly tested experimentally. Using the natural cell fusion ability of the halophilic archaeon Haloferax volcanii we were able to examine this question in vivo, by mating polB intein-positive [insertion site c in the gene encoding DNA polymerase B (polB-c)] and intein-negative cells and examining the dispersal efficiency of this intein in a natural, polyploid population. Through competition between otherwise isogenic intein-positive and intein-negative strains we determined a surprisingly high fitness cost of over 7% for the polB-c intein. Our laboratory culture experiments and samples taken from Israel's Mediterranean coastline show that the polB-c inteins do not efficiently take over an inteinless population through mating, even under ideal conditions. The presence of the HEN/intein promoted recombination when intein-positive and intein-negative cells were mated. Increased recombination due to HEN activity contributes not only to intein dissemination but also to variation at the population level because recombination tracts during repair extend substantially from the homing site.

Horizontal gene transfer: building the web of life.

Horizontal gene transfer (HGT) is the sharing of genetic material between organisms that are not in a parent-offspring relationship. HGT is a widely recognized mechanism for adaptation in bacteria and archaea. Microbial antibiotic resistance and pathogenicity are often associated with HGT, but the scope of HGT extends far beyond disease-causing organisms. In this Review, we describe how HGT has shaped the web of life using examples of HGT among prokaryotes, between prokaryotes and eukaryotes, and even between multicellular eukaryotes. We discuss replacement and additive HGT, the proposed mechanisms of HGT, selective forces that influence HGT, and the evolutionary impact of HGT on ancestral populations and existing populations such as the human microbiome.

Inteins as indicators of gene flow in the halobacteria.

This research uses inteins, a type of mobile genetic element, to infer patterns of gene transfer within the Halobacteria. We surveyed 118 genomes representing 26 genera of Halobacteria for intein sequences. We then used the presence-absence profile, sequence similarity and phylogenies from the inteins recovered to explore how intein distribution can provide insight on the dynamics of gene flow between closely related and divergent organisms. We identified 24 proteins in the Halobacteria that have been invaded by inteins at some point in their evolutionary history, including two proteins not previously reported to contain an intein. Furthermore, the size of an intein is used as a heuristic for the phase of the intein's life cycle. Larger size inteins are assumed to be the canonical two domain inteins, consisting of self-splicing and homing endonuclease domains (HEN); smaller sizes are assumed to have lost the HEN domain. For many halobacterial groups the consensus phylogenetic signal derived from intein sequences is compatible with vertical inheritance or with a strong gene transfer bias creating these clusters. Regardless, the coexistence of intein-free and intein-containing alleles reveal ongoing transfer and loss of inteins within these groups. Inteins were frequently shared with other Euryarchaeota and among the Bacteria, with members of the Cyanobacteria (Cyanothece, Anabaena), Bacteriodetes (Salinibacter), Betaproteobacteria (Delftia, Acidovorax), Firmicutes (Halanaerobium), Actinobacteria (Longispora), and Deinococcus-Thermus-group.

Population and genomic analysis of the genus Halorubrum.

The Halobacteria are known to engage in frequent gene transfer and homologous recombination. For stably diverged lineages to persist some checks on the rate of between lineage recombination must exist. We surveyed a group of isolates from the Aran-Bidgol endorheic lake in Iran and sequenced a selection of them. Multilocus Sequence Analysis (MLSA) and Average Nucleotide Identity (ANI) revealed multiple clusters (phylogroups) of organisms present in the lake. Patterns of intein and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) presence/absence and their sequence similarity, GC usage along with the ANI and the identities of the genes used in the MLSA revealed that two of these clusters share an exchange bias toward others in their phylogroup while showing reduced rates of exchange with other organisms in the environment. However, a third cluster, composed in part of named species from other areas of central Asia, displayed many indications of variability in exchange partners, from within the lake as well as outside the lake. We conclude that barriers to gene exchange exist between the two purely Aran-Bidgol phylogroups, and that the third cluster with members from other regions is not a single population and likely reflects an amalgamation of several populations.

Distribution and evolution of the mobile vma-1b intein.

Inteins are self-splicing parasitic genetic elements found in all domains of life. These genetic elements are found in highly conserved positions in conserved proteins. One protein family that has been invaded by inteins is the vacuolar and archaeal catalytic ATPase subunits (vma-1). There are two intein insertion sites in this protein, "a" and "b." The b site was previously thought to be only invaded in archaeal lineages. Here we survey the distribution and evolutionary histories of the b site inteins and show that the intein is present in more lineages than previously annotated, including a bacterial lineage, Mahella australiensis 50-1 BON. We present evidence, through ancestral character state reconstruction and substitution ratios between host genes and inteins, for several transfers of this intein between divergent species, including an interdomain transfer between the archaea and bacteria. Although inteins may persist within a single population or species for long periods of time, transfer of the vma-1b intein between divergent species contributed to the distribution of this intein.

The role of reticulate evolution in creating innovation and complexity.

Reticulate evolution encompasses processes that conflict with traditional Tree of Life efforts. These processes, horizontal gene transfer (HGT), gene and whole-genome duplications through allopolyploidization, are some of the main driving forces for generating innovation and complexity. HGT has a profound impact on prokaryotic and eukaryotic evolution. HGTs can lead to the invention of new metabolic pathways and the expansion and enhancement of previously existing pathways. It allows for organismal adaptation into new ecological niches and new host ranges. Although many HGTs appear to be selected for because they provide some benefit to their recipient lineage, other HGTs may be maintained by chance through random genetic drift. Moreover, some HGTs that may initially seem parasitic in nature can cause complexity to arise through pathways of neutral evolution. Another mechanism for generating innovation and complexity, occurring more frequently in eukaryotes than in prokaryotes, is gene and genome duplications, which often occur through allopolyploidizations. We discuss how these different evolutionary processes contribute to generating innovation and complexity.