SUMMARY Clusters of circulating tumor cells (CTC-clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC-clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC-clusters have 23-50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC-clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC-cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC-clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC-clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and while rare, they greatly contribute to the metastatic spread of cancer.

For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of "the dose makes the poison," because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.

Proper coordination of cholesterol biosynthesis and trafficking is essential to human health. The sterol regulatory element binding proteins (SREBPs) are key transcription regulators of genes involved in cholesterol biosynthesis/uptake. We show here that microRNAs (miR-33a/b) embedded within introns of the SREBP genes target the ATP-binding cassette transporter A1 (ABCA1), an important regulator of high-density lipoprotein (HDL) synthesis and reverse cholesterol transport, for post-transcriptional repression. Antisense inhibition of miR-33 in cell lines causes upregulation of ABCA1 expression and increased cholesterol efflux, and injection of mice on a western-type diet with locked nucleic acid (LNA)-antisense oligonucleotides results in elevated plasma HDL. Collectively, our findings indicate that miR-33 acts in concert with the SREBP host genes to control cholesterol homeostasis, and suggest that miR-33 may represent a therapeutic target for ameliorating cardiometabolic diseases.

Summary Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem cells (ESCs). By comparing genetically identical mouse ESCs and iPSCs, we show here that the overall mRNA and miRNA expression patterns of these cell types are indistinguishable with the exception of a few transcripts and miRNAs encoded on chromosome 12qF1. Specifically, maternally expressed imprinted genes in the Dlk1-Dio3 cluster including Gtl2, Rian and Mirg as well as a larger number of miRNAs encoded within this region were aberrantly silenced in the majority of iPSC clones, irrespective of their cell type of origin. Consistent with a developmental role of the Dlk1-Dio3 gene cluster, iPSC clones with repressed Gtl2 contributed poorly to chimeras and failed to support the development of entirely iPSC-derived animals (“all-iPSC mice”). In contrast, iPSC clones with normal expression levels of these genes contributed to high-grade chimeras and generated viable all-iPSC mice. Importantly, treatment of an iPSC clone that had silenced Dlk1-Dio3 and failed to give rise to all-iPSC animals with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of exclusively iPSC-derived mice. Thus, the expression state of a single imprinted gene cluster distinguishes most murine iPSCs from ESCs and allows for the prospective identification of iPSC clones that have the full development potential of ESCs.

Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging, hence sampling circulating tumor cells (CTCs) may reveal drug resistance mechanisms. We established single cell RNA-sequencing profiles of 77 intact CTCs isolated from 13 patients (mean 6 CTCs/patient) using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing on AR inhibitor, compared with untreated cases indicates activation of noncanonical Wnt signaling (P=0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, while its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.

Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identify CTC-clusters in 30–40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis.

Tyrosine kinase inhibitors (TKIs) are effective treatments for non-small cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of one year of continuous treatment. A fundamental histological transformation from NSCLC to small cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumor samples and cell lines derived from resistant EGFR mutant patients revealed that RB is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared to resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.

Summary Sterol Regulatory Element-Binding Proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids, and therefore are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can result in excess stored fat and contribute to obesity, fatty liver disease and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls production of the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a mechanism where maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders.