Overexpression of IGF-1 During Early Development Expands the Number of Mammary Stem Cells and Primes them for Transformation.

Insulin-like growth factor I (IGF-1) has been implicated in breast cancer due to its mitogenic and anti-apoptotic effects. Despite substantial research on the role of IGF-1 in tumor progression, the relationship of IGF-1 to tissue stem cells, particularly in mammary tissue, and the resulting tumor susceptibility has not been elucidated. Previous studies with the BK5.IGF-1 transgenic (Tg) mouse model reveals that IGF-1 does not act as a classical, post-carcinogen tumor promoter in the mammary gland. Pre-pubertal Tg mammary glands display increased numbers and enlarged sizes of terminal end buds, a niche for mammary stem cells (MaSCs). Here we show that MaSCs from both wild-type (WT) and Tg mice expressed IGF-1R and that overexpression of Tg IGF-1 increased numbers of MaSCs by undergoing symmetric division, resulting in an expansion of the MaSC and luminal progenitor (LP) compartments in pre-pubertal female mice. This expansion was maintained post-pubertally and validated by mammosphere assays in vitro and transplantation assays in vivo. The addition of recombinant IGF-1 promoted, and IGF-1R downstream inhibitors decreased mammosphere formation. Single-cell transcriptomic profiles generated from 2 related platforms reveal that IGF-1 stimulated quiescent MaSCs to enter the cell cycle and increased their expression of genes involved in proliferation, plasticity, tumorigenesis, invasion, and metastasis. This study identifies a novel, pro-tumorigenic mechanism, where IGF-1 increases the number of transformation-susceptible carcinogen targets during the early stages of mammary tissue development, and "primes" their gene expression profiles for transformation.

TGF-ß-induced DACT1 biomolecular condensates repress Wnt signalling to promote bone metastasis.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFß and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2-a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

Dynamic role of the codon 72 p53 single-nucleotide polymorphism in mammary tumorigenesis in a humanized mouse model.

Female breast cancer (BrCa) is the most common noncutaneous cancer among women in the United States. Human epidemiological studies reveal that a p53 single-nucleotide polymorphism (SNP) at codon 72, encoding proline (P72) or arginine (R72), is associated with differential risk of several cancers, including BrCa. However, the molecular mechanisms by which these variants affect mammary tumorigenesis remain unresolved. To investigate the effects of this polymorphism on susceptibility to mammary cancer, we used a humanized p53 mouse model, homozygous for either P72 or R72. Our studies revealed that R72 mice had a significantly higher mammary tumor incidence and reduced latency in both DMBA-induced and MMTV-Erbb2/Neu mouse mammary tumor models compared to P72 mice. Analyses showed that susceptible mammary glands from E-R72 (R72 x MMTV-Erbb2/Neu) mice developed a senescence-associated secretory phenotype (SASP) with influx of proinflammatory macrophages, ultimately resulting in chronic, protumorigenic inflammation. Mammary tumors arising in E-R72 mice also had an increased influx of tumor-associated macrophages, contributing to angiogenesis and elevated tumor growth rates. These results demonstrate that the p53 R72 variant increased susceptibility to mammary tumorigenesis through chronic inflammation.

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

Phylogenetic analysis and expression profiling of the pattern recognition receptors: Insights into molecular recognition of invading pathogens in Manduca sexta.

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 ß-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect.

A comprehensive analysis of the Manduca sexta immunotranscriptome.

As a biochemical model, Manduca sexta has substantially contributed to our knowledge on insect innate immunity. The RNA-Seq approach was implemented in three studies to examine tissue immunotranscriptomes of this species. With the latest and largest focusing on highly regulated process- and tissue-specific genes, we further analyzed the same set of data using BLAST2GO to explore functional aspects of the larval fat body (F) and hemocyte (H) transcriptomes with (I) or without (C) immune challenge. Using immunity-related sequences from other insects, we found 383 homologous contigs and compared them with those discovered based on relative abundance changes. The major overlap of the two lists validated our previous research designed for gene discovery and transcript profiling in organisms lacking sequenced genomes. By concatenating the contigs, we established a repertoire of 232 immunity-related genes encoding proteins for pathogen recognition (16%), signal transduction (53%), microbe killing (13%) and others (18%). We examined their transcript levels along with attribute classifications and detected prominent differences in nine of the 30 level 2 gene ontology (GO) categories. The increase in extracellular proteins (155%) was consistent with the highly induced synthesis of defense molecules (e.g., antimicrobial peptides) in fat body after the immune challenge. We identified most members of the putative Toll, IMD, MAPK-JNK-p38 and JAK-STAT pathways and small changes in their mRNA levels. Together, these findings set the stage for on-going analysis of the M. sexta immunogenome.

Pyrosequencing-based expression profiling and identification of differentially regulated genes from Manduca sexta, a lepidopteran model insect.

Although Manduca sexta has significantly contributed to our knowledge on a variety of insect physiological processes, the lack of its genome sequence hampers the large-scale gene discovery, transcript profiling, and proteomic analysis in this biochemical model species. Here we report our implementation of the RNA-Seq cDNA sequencing approach based on massively parallel pyrosequencing, which allows us to categorize transcripts based on their relative abundances and to discover process- or tissue-specifically regulated genes simultaneously. We obtained 1,821,652 reads with an average length of 289 bp per read from fat body and hemocytes of naïve and microbe-injected M. sexta larvae. After almost all (92.1%) of these reads were assembled into 19,020 contigs, we identified 528 contigs whose relative abundances increased at least 5- and 8-fold in fat body and hemocytes, respectively, after the microbial challenge. Polypeptides encoded by these contigs include pathogen recognition receptors, extracellular and intracellular signal mediators and regulators, antimicrobial peptides, and proteins with no known sequence but likely participating in defense in novel ways. We also found 250 and 161 contigs that were preferentially expressed in fat body and hemocytes, respectively. Furthermore, we integrated data from our previous study and generated a sequence database to support future gene annotation and proteomic analysis in M. sexta. In summary, we have successfully established a combined approach for gene discovery and expression profiling in organisms lacking known genome sequences.