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CDX2 is expressed in most colon cancers, but approximately 4% do not express this transcription factor. Lack of CDX2 expression marks a subset of cancers with a more aggressive natural history. Adjuvant chemotherapy primarily benefits patients with stage II CDX2-negative tumors. During the past decade, disease-free survival among patients with stage III colon cancer has increased significantly owing to the introduction of new adjuvant chemotherapy regimens. 1 – 3 This therapeutic success, however, has not translated into longer disease-free survival among patients with earlier-stage (stage I or II) cancer. 4 The lack of simple, reliable criteria for the identification of patients with early-stage disease who are at high risk for relapse has made it difficult to identify patients in whom the hazards of multiagent chemotherapy may be offset by benefits with respect to disease-specific survival. 4 – 9 To address this problem, researchers have explored the . . .

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.

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. Messenger RNA molecules take the information encoded in a gene's DNA sequence and turn it into instructions for building a protein. However, if certain smaller molecules of RNA—called microRNAs—bind to a messenger RNA molecule, they ‘silence’ it, which prevents the information in the messenger RNA from being translated to make a protein. Despite their small size, microRNAs are very powerful. These molecules are able to simultaneously inhibit the translation of hundreds of messenger RNAs and perform many roles, including controlling cell growth and maintaining populations of stem cells. Furthermore, microRNAs have been linked to different aspects of the growth of cancerous cells. Certain microRNAs appear to suppress tumors by regulating the growth of the stem cells found there, while others appear to be ‘hyperactive’ in cancers—including breast cancer, colon cancer, and blood cancer. In 2009, researchers compared the amount of microRNA in breast cancer stem cells that are highly capable of forming tumors with the amount in other cancer cells within the same tumor. Amongst other differences, two microRNAs (called miR-142 and miR-150) were found to be hyperactive in human breast cancer stem cells. One of them, miR-142, is known to target a gene called APC that inhibits the renewal of normal stem cells. Mutations in the APC gene have been linked to colon cancer, and scientists have suggested that the mutations inactivate APC in cancer cells to promote unregulated cell growth. Breast tumors rarely have mutations in the APC gene, but Isobe et al. wondered whether microRNAs that target this gene might also promote the growth of these tumor cells. Isobe et al.—including several of the researchers involved in the 2009 work—show that miR-142 does target the APC gene in human breast cancer stem cells, and silences it. With the gene silenced, a cancer-promoting pathway turns on and more miR-150 is made. Increasing the amount of either miR-142 or miR-150 causes excessive cell growth in breast tissue and can form abnormal breast tissue in mice. Reducing the amount of miR-142 in human breast cancer stem cells slows the growth of breast tumors. Although they only make up a small population of human breast cancer cells, focusing on breast cancer stem cells could uncover the cancer-promoting pathways that are activated in human breast cancers.

Not all cells in a tumor are alike, but our ability to characterize cancer heterogeneity in detail has been limited. Dalerba et al . use high-throughput single-cell expression analysis to define clinically relevant subpopulations in normal and cancerous colon tissue. 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.

Reactive oxygen: role in tumour radiation resistance Michael Clarke and colleagues find that cancer stem cell in breast tumours have lower levels of reactive oxygen species (ROS) than the rest of the tumour cells. This property renders cancer stem cells less sensitive to radiation therapy, which may cause radio resistance in breast cancer. This study shows that cancer stem cell in breast tumours have lower levels of reactive oxygen species (ROS) than the rest of the tumour cells. This property renders cancer stem cells less sensitive to radiation therapy, which may cause radioresistance in breast cancer. 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 1 , 2 and haematopoietic stem cells and early progenitors 3 , 4 , 5 , 6 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 7 , 8 , 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.

BackgroundMicroRNAs (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-derivedxenografts (PDX). ResultsSix 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.ConclusionIn 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.

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.

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.

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. The contribution of EMT in mammary gland homeostasis and human breast cancer is still unclear. Here, using in vivo lineage tracing and breast cancer PDXs the authors demonstrate that the repopulating capacity in normal mammary epithelial cells and tumorigenic capacity in breast cancer is independent of expression of EMT-associated genes.

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. An analysis of somatic tissues derived from mouse models of Down’s syndrome shows reduced self-renewal capacities in various cell types, with these defects partially dependent on triplication of the Usp16 gene; overexpression and knockout studies in human cells shows that USP16 has a role in Down’s syndrome-related proliferation defects, making this gene an attractive option for further study. Excess Usp16 linked to Down's syndrome People with Down's syndrome have abnormalities in multiple tissues including mental retardation and early ageing. The disease is often the result of full or partial trisomy of chromosome 21, but the molecular mechanisms underlying the observed cellular defects remain largely unknown. An analysis of haematopoietic stem cells in the Down's syndrome mouse model Ts65Dn has revealed a reduced self-renewal associated with the proliferation of cells expressing three copies of the Usp16 gene, which encodes a deubiquitination enzyme involved in chromatin remodelling and cell cycle progression. In a second Down's syndrome mouse model, Ts1Cje, haematopoietic stem cells were not defective. Downregulation of USP16 rescued the functional defects of affected Ts65Dn cells. Overexpression of USP16 in normal human fibroblasts reduced their proliferative capacity and USP16 downregulation partially rescued human Down's syndrome fibroblast proliferation defects. The authors propose that USP16 is a potential target for therapeutics designed to ameliorate the pathologies associated with this syndrome.