Identification of a minority population of LMO2+ breast cancer cells that integrate into the vasculature and initiate metastasis.

Metastasis is responsible for most breast cancer-related deaths; however, identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage-traced cells integrate into the vasculature and have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by tumor necrosis factor-α and interleukin-6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.

Mesenchymal tumor cells drive adaptive resistance of Trp53-/- breast tumor cells to inactivated mutant Kras.

As precision medicine increases the response rate of treatment, tumors frequently bypass inhibition, and reoccur. In order for treatment to be effective long term, the mechanisms enabling treatment adaptation need to be understood. Here, we report a mouse model that, in the absence of p53 and the presence of oncogenic KrasG12D , develops breast tumors. Upon inactivation of KrasG12D , tumors initially regress and enter remission. Subsequently, the majority of tumors adapt to the withdrawal of KrasG12D expression and return. KrasG12D -independent tumor cells show a strong mesenchymal profile with active RAS-RAF-MEK-ERK (MAPK/ERK) signaling. Both KrasG12D -dependent and KrasG12D -independent tumors display a high level of genomic instability, and KrasG12D -independent tumors harbor numerous amplified genes that can activate the MAPK/ERK signaling pathway. Our study identifies both epithelial-mesenchymal transition (EMT) and active MAPK/ERK signaling in tumors that adapt to oncogenic KrasG12D withdrawal in a novel Trp53-/- breast cancer mouse model. To achieve long-lasting responses in the clinic to RAS-fueled cancer, treatment will need to focus in parallel on obstructing tumors from adapting to oncogene inhibition.

Upregulation of BMI1-suppressor miRNAs (miR-200c, miR-203) during terminal differentiation of colon epithelial cells.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of stem cell functions, including self-renewal and differentiation. In this study, we aimed to identify miRNAs that are upregulated during terminal differentiation in the human colon epithelium, and elucidate their role in the mechanistic control of stem cell properties. METHODS: "Bottom-of-the-crypt" (EPCAM+/CD44+/CD66alow) and "top-of-the-crypt" (EPCAM+/CD44neg/CD66ahigh) epithelial cells from 8 primary colon specimens (6 human, 2 murine) were purified by flow cytometry and analyzed for differential expression of 335 miRNAs. The miRNAs displaying the highest upregulation in "top-of-the-crypt" (terminally differentiated) epithelial cells were tested for positive correlation and association with survival outcomes in a colon cancer RNA-seq database (n = 439 patients). The two miRNAs with the strongest "top-of-the-crypt" expression profile were evaluated for capacity to downregulate self-renewal effectors and inhibit in vitro proliferation of colon cancer cells, in vitro organoid formation by normal colon epithelial cells and in vivo tumorigenicity by patient-derived xenografts (PDX). RESULTS: Six miRNAs (miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-345) were upregulated in "top-of-the-crypt" cells and positively correlated in expression among colon carcinomas. Overexpression of the three miRNAs with the highest inter-correlation coefficients (miR-200a, miR-200b, miR-200c) associated with improved survival. The top two over-expressed miRNAs (miR-200c, miR-203) cooperated synergistically in suppressing expression of BMI1, a key regulator of self-renewal in stem cell populations, and in inhibiting proliferation, organoid-formation and tumorigenicity of colon epithelial cells. CONCLUSION: In the colon epithelium, terminal differentiation associates with the coordinated upregulation of miR-200c and miR-203, which cooperate to suppress BMI1 and disable the expansion capacity of epithelial cells.

Inhibiting USP16 rescues stem cell aging and memory in an Alzheimer's model.

Alzheimer's disease (AD) is a progressive neurodegenerative disease observed with aging that represents the most common form of dementia. To date, therapies targeting end-stage disease plaques, tangles, or inflammation have limited efficacy. Therefore, we set out to identify a potential earlier targetable phenotype. Utilizing a mouse model of AD and human fetal cells harboring mutant amyloid precursor protein, we show cell intrinsic neural precursor cell (NPC) dysfunction precedes widespread inflammation and amyloid plaque pathology, making it the earliest defect in the evolution of the disease. We demonstrate that reversing impaired NPC self-renewal via genetic reduction of USP16, a histone modifier and critical physiological antagonist of the Polycomb Repressor Complex 1, can prevent downstream cognitive defects and decrease astrogliosis in vivo. Reduction of USP16 led to decreased expression of senescence gene Cdkn2a and mitigated aberrant regulation of the Bone Morphogenetic Signaling (BMP) pathway, a previously unknown function of USP16. Thus, we reveal USP16 as a novel target in an AD model that can both ameliorate the NPC defect and rescue memory and learning through its regulation of both Cdkn2a and BMP signaling.

Depletion of Trp53 and Cdkn2a Does Not Promote Self-Renewal in the Mammary Gland but Amplifies Proliferation Induced by TNF-α.

The mammary epithelium undergoes several rounds of extensive proliferation during the female reproductive cycle. Its expansion is a tightly regulated process, fueled by the mammary stem cells and these cells' unique property of self-renewal. Sufficient new cells have to be produced to maintain the integrity of a tissue, but excessive proliferation resulting in tumorigenesis needs to be prevented. Three well-known tumor suppressors, p53, p16INK4a, and p19ARF, have been connected to the limiting of stem cell self-renewal and proliferation. Here we investigate the roles of these three proteins in the regulation of self-renewal and proliferation of mammary epithelial cells. Using mammary epithelial-specific mouse models targeting Trp53 and Cdkn2a, the gene coding for p16INK4a and p19ARF, we demonstrate that p53, p16INK4a, and p19ARF do not play a significant role in the limitation of normal mammary epithelium self-renewal and proliferation, whereas in the presence of the inflammatory cytokine TNF-α, Trp53-/-Cdkn2a-/- mammary basal cells exhibit amplified proliferation.

LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

SMAD pathways govern epithelial proliferation, and transforming growth factor ß (TGF-ß and BMP signaling through SMAD members has distinct effects on mammary development and homeostasis. Here, we show that LEFTY1, a secreted inhibitor of NODAL/SMAD2 signaling, is produced by mammary progenitor cells and, concomitantly, suppresses SMAD2 and SMAD5 signaling to promote long-term proliferation of normal and malignant mammary epithelial cells. In contrast, BMP7, a NODAL antagonist with context-dependent functions, is produced by basal cells and restrains progenitor cell proliferation. In normal mouse epithelium, LEFTY1 expression in a subset of luminal cells and rare basal cells opposes BMP7 to promote ductal branching. LEFTY1 binds BMPR2 to suppress BMP7-induced activation of SMAD5, and this LEFTY1-BMPR2 interaction is specific to tumor-initiating cells in triple-negative breast cancer xenografts that rely on LEFTY1 for growth. These results suggest that LEFTY1 is an endogenous dual-SMAD inhibitor and that suppressing its function may represent a therapeutic vulnerability in breast cancer.

Single-cell transcriptional diversity is a hallmark of developmental potential.

Single-cell RNA sequencing (scRNA-seq) is a powerful approach for reconstructing cellular differentiation trajectories. However, inferring both the state and direction of differentiation is challenging. Here, we demonstrate a simple, yet robust, determinant of developmental potential-the number of expressed genes per cell-and leverage this measure of transcriptional diversity to develop a computational framework (CytoTRACE) for predicting differentiation states from scRNA-seq data. When applied to diverse tissue types and organisms, CytoTRACE outperformed previous methods and nearly 19,000 annotated gene sets for resolving 52 experimentally determined developmental trajectories. Additionally, it facilitated the identification of quiescent stem cells and revealed genes that contribute to breast tumorigenesis. This study thus establishes a key RNA-based feature of developmental potential and a platform for delineation of cellular hierarchies.

Serially transplantable mammary epithelial cells express the Thy-1 antigen.

BACKGROUND: Recent studies in murine mammary tissue have identified functionally distinct cell populations that may be isolated by surface phenotype or lineage tracing. Previous groups have shown that CD24medCD49fhigh cells enriched for long-lived mammary epithelial cells can be serially transplanted. METHODS: Flow cytometry-based enrichment of distinct phenotypic populations was assessed for their gene expression profiles and functional proliferative attributes in vitro and in vivo. RESULTS: Here, we show Thy-1 is differentially expressed in the CD24medCD49fhigh population, which allowed us to discern two functionally different populations. The Thy-1+CD24medCD49fhigh phenotype contained the majority of the serially transplantable epithelial cells. The Thy-1-CD24medCD49fhigh phenotype contains a rare progenitor population that is able to form primary mammary outgrowths with significantly decreased serial in vivo transplantation potential. CONCLUSIONS: Therefore, Thy-1 expression in the immature cell compartment is a useful tool to study the functional heterogeneity that drives mammary gland development and has implications for disease etiology.

Role of epithelial to mesenchymal transition associated genes in mammary gland regeneration and breast tumorigenesis.

Previous studies have proposed that epithelial to mesenchymal transition (EMT) in breast cancer cells regulates metastasis, stem cell properties and chemo-resistance; most studies were based on in vitro culture of cell lines and mouse transgenic cancer models. However, the identity and function of cells expressing EMT-associated genes in normal murine mammary gland homeostasis and human breast cancer still remains under debate. Using in vivo lineage tracing and triple negative breast cancer (TNBC) patient derived xenografts we demonstrate that the repopulating capacity in normal mammary epithelial cells and tumorigenic capacity in TNBC is independent of expression of EMT-associated genes. In breast cancer, while a subset of cells with epithelial and mesenchymal phenotypes have stem cell activity, in many cells that have lost epithelial characteristics with increased expression of mesenchymal genes, have decreased tumor-initiating capacity and plasticity. These findings have implications for the development of effective therapeutic agents targeting tumor-initiating cells.

A Quiescent Bcl11b High Stem Cell Population Is Required for Maintenance of the Mammary Gland.

Stem cells in many tissues sustain themselves by entering a quiescent state to avoid genomic insults and to prevent exhaustion caused by excessive proliferation. In the mammary gland, the identity and characteristics of quiescent epithelial stem cells are not clear. Here, we identify a quiescent mammary epithelial cell population expressing high levels of Bcl11b and located at the interface between luminal and basal cells. Bcl11bhigh cells are enriched for cells that can regenerate mammary glands in secondary transplants. Loss of Bcl11b leads to a Cdkn2a-dependent exhaustion of ductal epithelium and loss of epithelial cell regenerative capacity. Gain- and loss-of-function studies show that Bcl11b induces cells to enter the G0 phase of the cell cycle and become quiescent. Taken together, these results suggest that Bcl11b acts as a central intrinsic regulator of mammary epithelial stem cell quiescence and exhaustion and is necessary for long-term maintenance of the mammary gland.

CDX2 as a Prognostic Biomarker in Stage II and Stage III Colon Cancer.

Background The identification of high-risk stage II colon cancers is key to the selection of patients who require adjuvant treatment after surgery. Microarray-based multigene-expression signatures derived from stem cells and progenitor cells hold promise, but they are difficult to use in clinical practice. Methods We used a new bioinformatics approach to search for biomarkers of colon epithelial differentiation across gene-expression arrays and then ranked candidate genes according to the availability of clinical-grade diagnostic assays. With the use of subgroup analysis involving independent and retrospective cohorts of patients with stage II or stage III colon cancer, the top candidate gene was tested for its association with disease-free survival and a benefit from adjuvant chemotherapy. Results The transcription factor CDX2 ranked first in our screening test. A group of 87 of 2115 tumor samples (4.1%) lacked CDX2 expression. In the discovery data set, which included 466 patients, the rate of 5-year disease-free survival was lower among the 32 patients (6.9%) with CDX2-negative colon cancers than among the 434 (93.1%) with CDX2-positive colon cancers (hazard ratio for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). In the validation data set, which included 314 patients, the rate of 5-year disease-free survival was lower among the 38 patients (12.1%) with CDX2 protein-negative colon cancers than among the 276 (87.9%) with CDX2 protein-positive colon cancers (hazard ratio, 2.42; 95% CI, 1.36 to 4.29; P=0.003). In both these groups, these findings were independent of the patient's age, sex, and tumor stage and grade. Among patients with stage II cancer, the difference in 5-year disease-free survival was significant both in the discovery data set (49% among 15 patients with CDX2-negative tumors vs. 87% among 191 patients with CDX2-positive tumors, P=0.003) and in the validation data set (51% among 15 patients with CDX2-negative tumors vs. 80% among 106 patients with CDX2-positive tumors, P=0.004). In a pooled database of all patient cohorts, the rate of 5-year disease-free survival was higher among 23 patients with stage II CDX2-negative tumors who were treated with adjuvant chemotherapy than among 25 who were not treated with adjuvant chemotherapy (91% vs. 56%, P=0.006). Conclusions Lack of CDX2 expression identified a subgroup of patients with high-risk stage II colon cancer who appeared to benefit from adjuvant chemotherapy. (Funded by the National Comprehensive Cancer Network, the National Institutes of Health, and others.).

miR-142 regulates the tumorigenicity of human breast cancer stem cells through the canonical WNT signaling pathway.

MicroRNAs (miRNAs) are important regulators of stem and progenitor cell functions. We previously reported that miR-142 and miR-150 are upregulated in human breast cancer stem cells (BCSCs) as compared to the non-tumorigenic breast cancer cells. In this study, we report that miR-142 efficiently recruits the APC mRNA to an RNA-induced silencing complex, activates the canonical WNT signaling pathway in an APC-suppression dependent manner, and activates the expression of miR-150. Enforced expression of miR-142 or miR-150 in normal mouse mammary stem cells resulted in the regeneration of hyperproliferative mammary glands in vivo. Knockdown of endogenous miR-142 effectively suppressed organoid formation by BCSCs and slowed tumor growth initiated by human BCSCs in vivo. These results suggest that in some tumors, miR-142 regulates the properties of BCSCs at least in part by activating the WNT signaling pathway and miR-150 expression.

A cell-intrinsic role for TLR2-MYD88 in intestinal and breast epithelia and oncogenesis.

It has been postulated that there is a link between inflammation and cancer. Here we describe a role for cell-intrinsic toll-like receptor-2 (TLR2; which is involved in inflammatory response) signalling in normal intestinal and mammary epithelial cells and oncogenesis. The downstream effectors of TLR2 are expressed by normal intestinal and mammary epithelia, including the stem/progenitor cells. Deletion of MYD88 or TLR2 in the intestinal epithelium markedly reduces DSS-induced colitis regeneration and spontaneous tumour development in mice. Limiting dilution transplantations of breast epithelial cells devoid of TLR2 or MYD88 revealed a significant decrease in mammary repopulating unit frequency compared with the control. Inhibition of TLR2, its co-receptor CD14, or its downstream targets MYD88 and IRAK1 inhibits growth of human breast cancers in vitro and in vivo. These results suggest that inhibitors of the TLR2 pathway merit investigation as possible therapeutic and chemoprevention agents.

Usp16 contributes to somatic stem-cell defects in Down's syndrome.

Down's syndrome results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene-dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, which are trisomic for 132 genes homologous to genes on human chromosome 21, triplication of Usp16 reduces the self-renewal of haematopoietic stem cells and the expansion of mammary epithelial cells, neural progenitors and fibroblasts. In addition, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from histone H2A on lysine 119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal Usp16 allele or by short interfering RNAs, largely rescues all of these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and postnatal neural progenitors, whereas downregulation of USP16 partially rescues the proliferation defects of Down's syndrome fibroblasts. Taken together, these results suggest that USP16 has an important role in antagonizing the self-renewal and/or senescence pathways in Down's syndrome and could serve as an attractive target to ameliorate some of the associated pathologies.

Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice.

BACKGROUND & AIMS: Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells. METHODS: We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. RESULTS: Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased. CONCLUSIONS: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

Single-cell dissection of transcriptional heterogeneity in human colon tumors.

Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.

Removal of lactate dehydrogenase-elevating virus from human-in-mouse breast tumor xenografts by cell-sorting.

Lactate dehydrogenase-elevating virus (LDV) can infect transplantable mouse tumors or xenograft tumors in mice through LDV-contaminated mouse biological materials, such as Matrigel, or through mice infected with LDV. LDV infects specifically mouse macrophages and alters immune system and tumor phenotype. The traditional approaches to remove LDV from tumor cells, by transplanting tumors into rats or culturing tumor cells in vitro, are inefficient, labor-intensive and time-consuming. Furthermore, these approaches are not feasible for primary tumor cells that cannot survive tissue culture conditions or that may change phenotype in rats. This study reports that fluorescence-activated cell sorting (FACS) is a simple and efficient approach for purifying living primary human breast tumor cells from LDV(+) mouse stromal cells, which can be completed in a few hours. When purified from Matrigel contaminated LDV(+) tumors, sorted human breast tumor cells, as well as tumors grown from sorted cells, were shown to be LDV-free, as tested by PCR. The results demonstrate that cell sorting is effective, much faster and less likely to alter tumor cell phenotype than traditional methods for removing LDV from xenograft models. This approach may also be used to remove other rodent-specific viruses from models derived from distinct tissues or species with sortable markers, where virus does not replicate in the cells to be purified.

Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.

Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells.

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.

Association of reactive oxygen species levels and radioresistance in cancer stem cells.

The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.