Monte M. Winslow

Targeted Deletion Reveals Essential and Overlapping Functions of the miR-17∼92 Family of miRNA Clusters

miR-17 approximately 92, miR-106b approximately 25, and miR-106a approximately 363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-1792 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17 approximately 92 cluster is also essential for B cell development. Absence of miR-17 approximately 92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b approximately 25 or miR-106a approximately 363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b approximately 25 and miR-17 approximately 92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 approximately 92 and its functions during B lymphopoiesis and lung development.

Treating metastatic cancer with nanotechnology

Metastasis accounts for the vast majority of cancer deaths. The unique challenges for treating metastases include their small size, high multiplicity and dispersion to diverse organ environments. Nanoparticles have many potential benefits for diagnosing and treating metastatic cancer, including the ability to transport complex molecular cargoes to the major sites of metastasis, such as the lungs, liver and lymph nodes, as well as targeting to specific cell populations within these organs. This Review highlights the research, opportunities and challenges for integrating engineering sciences with cancer biology and medicine to develop nanotechnology-based tools for treating metastatic disease.

NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21

Trisomy 21 results in Downs syndrome, but little is known about how a 1.5-fold increase in gene dosage produces the pleiotropic phenotypes of Downs syndrome. Here we report that two genes, DSCR1 and DYRK1A , lie within the critical region of human chromosome 21 and act synergistically to prevent nuclear occupancy of NFATc transcription factors, which are regulators of vertebrate development. We use mathematical modelling to predict that autoregulation within the pathway accentuates the effects of trisomy of DSCR1 and DYRK1A, leading to failure to activate NFATc target genes under specific conditions. Our observations of calcineurin-and Nfatc-deficient mice, Dscr1- and Dyrk1a–overexpressing mice, mouse models of Downs syndrome and human trisomy 21 are consistent with these predictions. We suggest that the 1.5-fold increase in dosage of DSCR1 and DYRK1A cooperatively destabilizes a regulatory circuit, leading to reduced NFATc activity and many of the features of Downs syndrome. More generally, these observations suggest that the destabilization of regulatory circuits can underlie human disease.

An Essential Switch in Subunit Composition of a Chromatin Remodeling Complex during Neural Development

Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, we show that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Krüppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits.

Calcineurin/NFAT signalling regulates pancreatic β-cell growth and function

The growth and function of organs such as pancreatic islets adapt to meet physiological challenges and maintain metabolic balance, but the mechanisms controlling these facultative responses are unclear. Diabetes in patients treated with calcineurin inhibitors such as cyclosporin A indicates that calcineurin/nuclear factor of activated T-cells (NFAT) signalling might control adaptive islet responses, but the roles of this pathway in β-cells in vivo are not understood. Here we show that mice with a β-cell-specific deletion of the calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1), develop age-dependent diabetes characterized by decreased β-cell proliferation and mass, reduced pancreatic insulin content and hypoinsulinaemia. Moreover, β-cells lacking Cnb1 have a reduced expression of established regulators of β-cell proliferation. Conditional expression of active NFATc1 in Cnb1-deficient β-cells rescues these defects and prevents diabetes. In normal adult β-cells, conditional NFAT activation promotes the expression of cell-cycle regulators and increases β-cell proliferation and mass, resulting in hyperinsulinaemia. Conditional NFAT activation also induces the expression of genes critical for β-cell endocrine function, including all six genes mutated in hereditary forms of monogenic type 2 diabetes. Thus, calcineurin/NFAT signalling regulates multiple factors that control growth and hallmark β-cell functions, revealing unique models for the pathogenesis and therapy of diabetes.

Suppression of lung adenocarcinoma progression by Nkx2-1

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of KrasLSL-G12D/+;p53flox/flox mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.

Calcineurin B1 Is Essential for Positive but Not Negative Selection during Thymocyte Development

During development, discrete cell fates often result from variation in the intensity of a particular signal. The mechanisms underlying these seemingly analog-to-digital switches are not understood. In developing T lymphocytes, low-intensity signals through the antigen receptor result in positive selection while more intense signals give rise to negative selection. By deleting the genetic locus encoding the regulatory B1 subunit of calcineurin specifically in thymocytes, we found an absolute requirement for calcineurin in positive selection. In contrast, calcineurin activity was dispensable in several models of negative selection. Unexpectedly, we found that removal of calcineurin activity from thymocytes results in inefficient ERK activation at the double-positive stage of thymocyte development, when selection occurs. These studies clarify the mechanism by which graded signals are converted to discrete outcomes in T cell development and further indicate that the developmental roles of calcineurin likely contribute to immunosuppression by calcineurin inhibitors.

Stage-specific sensitivity to p53 restoration during lung cancer progression

Tumorigenesis is a multistep process that results from the sequential accumulation of mutations in key oncogene and tumour suppressor pathways. Personalized cancer therapy that is based on targeting these underlying genetic abnormalities presupposes that sustained inactivation of tumour suppressors and activation of oncogenes is essential in advanced cancers. Mutations in the p53 tumour-suppressor pathway are common in human cancer and significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. Here we show that restoration of p53 in established murine lung tumours leads to significant but incomplete tumour cell loss specifically in malignant adenocarcinomas, but not in adenomas. We define amplification of MAPK signalling as a critical determinant of malignant progression and also a stimulator of Arf tumour-suppressor expression. The response to p53 restoration in this context is critically dependent on the expression of Arf. We propose that p53 not only limits malignant progression by suppressing the acquisition of alterations that lead to tumour progression, but also, in the context of p53 restoration, responds to increased oncogenic signalling to mediate tumour regression. Our observations also underscore that the p53 pathway is not engaged by low levels of oncogene activity that are sufficient for early stages of lung tumour development. These data suggest that restoration of pathways important in tumour progression, as opposed to initiation, may lead to incomplete tumour regression due to the stage-heterogeneity of tumour cell populations.

HMGA2 functions as a competing endogenous RNA to promote lung cancer progression

Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide. As the majority of patients present with invasive, metastatic disease, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA) for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-β co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-β signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.

Characterizing deformability and surface friction of cancer cells

Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell’s passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cells size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constrictions walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces.