The collective migration of cells as a cohesive group is a hallmark of the tissue remodelling events that underlie embryonic morphogenesis, wound repair and cancer invasion. In such migration, cells move as sheets, strands, clusters or ducts rather than individually, and use similar actin- and myosin-mediated protrusions and guidance by extrinsic chemotactic and mechanical cues as used by single migratory cells. However, cadherin-based junctions between cells additionally maintain 'supracellular' properties, such as collective polarization, force generation, decision making and, eventually, complex tissue organization. Comparing different types of collective migration at the molecular and cellular level reveals a common mechanistic theme between developmental and cancer research.

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Collective cell migration in morphogenesis, regeneration and cancer

We present SCENIC, a computational method for simultaneous gene regulatory network reconstruction and cell-state identification from single-cell RNA-seq data (http://scenicaertslaborg) On a compendium of single-cell data from tumors and brain, we demonstrate that cis-regulatory analysis can be exploited to guide the identification of transcription factors and cell states SCENIC provides critical biological insights into the mechanisms driving cellular heterogeneity

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SCENIC: single-cell regulatory network inference and clustering.

PLoS BIOLOGY Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Jean-Philippe Coppe 1 , Christopher K. Patil 1[ , Francis Rodier 1,2[ , Yu Sun 3 , Denise P. Mun oz 1,2 , Joshua Goldstein 1¤ , Peter S. Nelson 3 , Pierre-Yves Desprez 1,4 , Judith Campisi 1,2* 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 2 Buck Institute for Age Research, Novato, California, United States of America, 3 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 California Pacific Medical Center Research Institute, San Francisco, California, United States of America Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA- damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment. Citation: Coppe JP, Patil CK, Rodier F, Sun Y, Mun oz DP, et al. (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): e301. doi:10.1371/journal.pbio.0060301 Introduction Cancer is a multistep disease in which cells acquire increasingly malignant phenotypes. These phenotypes are acquired in part by somatic mutations, which derange normal controls over cell proliferation (growth), survival, invasion, and other processes important for malignant tumorigenesis [1]. In addition, there is increasing evidence that the tissue microenvironment is an important determinant of whether and how malignancies develop [2,3]. Normal tissue environ- ments tend to suppress malignant phenotypes, whereas abnormal tissue environments such at those caused by inﬂammation can promote cancer progression. Cancer development is restrained by a variety of tumor suppressor genes. Some of these genes permanently arrest the growth of cells at risk for neoplastic transformation, a process termed cellular senescence [4–6]. Two tumor suppressor pathways, controlled by the p53 and p16INK4a/pRB proteins, regulate senescence responses. Both pathways integrate multiple aspects of cellular physiology and direct cell fate towards survival, death, proliferation, or growth arrest, depending on the context [7,8]. Several lines of evidence indicate that cellular senescence is a potent tumor-suppressive mechanism [4,9,10]. Many poten- tially oncogenic stimuli (e.g., dysfunctional telomeres, DNA PLoS Biology | www.plosbiology.org damage, and certain oncogenes) induce senescence [6,11]. Moreover, mutations that dampen the p53 or p16INK4a/pRB pathways confer resistance to senescence and greatly increase cancer risk [12,13]. Most cancers harbor mutations in one or both of these pathways [14,15]. Lastly, in mice and humans, a senescence response to strong mitogenic signals, such as those delivered by certain oncogenes, prevents premalignant lesions from progressing to malignant cancers [16–19]. Academic Editor: Julian Downward, Cancer Research UK, United Kingdom Received June 27, 2008; Accepted October 22, 2008; Published December 2, 2008 Copyright: O 2008 Coppe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: CM, conditioned medium; DDR, DNA damage response; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial–mesenchymal transition; GSE, genetic suppressor element; IL, interleukin; MIT, mitoxantrone; PRE, presenescent; PrEC, normal human prostate epithelial cell; REP, replicative exhaustion; SASP, senescence-associated secretory phenotype; SEN, senescent; shRNA, short hairpin RNA; XRA, X-irradiation * To whom correspondence should be addressed. E-mail: jcampisi@lbl.gov [ These authors contributed equally to this work. ¤ Current address: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America December 2008 | Volume 6 | Issue 12 | e301

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

We propose the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method for prioritizing single-guide RNAs, genes and pathways in genome-scale CRISPR/Cas9 knockout screens. MAGeCK demonstrates better performance compared with existing methods, identifies both positively and negatively selected genes simultaneously, and reports robust results across different experimental conditions. Using public datasets, MAGeCK identified novel essential genes and pathways, including EGFR in vemurafenib-treated A375 cells harboring a BRAF mutation. MAGeCK also detected cell type-specific essential genes, including BCR and ABL1, in KBM7 cells bearing a BCR-ABL fusion, and IGF1R in HL-60 cells, which depends on the insulin signaling pathway for proliferation.

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MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.

Integrin-regulated FAK-Src signaling in normal and cancer cells.

Cell adhesion and migration are essential for embryonic development, tissue regeneration, but also for tumor development. The physical link between the extracellular matrix (ECM) and the actin cytoskeleton is mainly mediated by receptors of the integrin family. Through signals transduced upon integrin ligation to ECM proteins, this family of proteins plays key roles in regulating tumor growth and metastasis as well as tumor angiogenesis. During melanoma development, changes in integrin expression, intracellular control of integrin functions and signals perceived from integrin ligand binding impact upon the ability of tumor cells to interact with their environment and enable melanoma cells to convert from a sessile, stationary to a migratory and invasive phenotype. Antagonists of several integrins are now under evaluation in clinical trials to determine their potential as therapeutics for malignant melanoma and other kinds of cancer.

Integrin signaling in malignant melanoma.

The Dickkopf (DKK) genes were originally identified as factors inducing head formation in Xenopus. The genes code for inhibitors that are involved in Wnt signaling. We speculate that loss of DKK expression plays a role in development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of DKK, especially DKK-3 transcription. We found that DKK-1, -2 and -3 were downregulated or lost in all cell lines and in most of the tumor samples analysed. Reduced DKK-3 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription-polymerase chain reaction RT-PCR analysis. Functional assays with stable DKK-3 transfected cell lines revealed that DKK-3 expression increased cell-cell adhesion and decreased cell migration. Further, downregulation of fibronectin, snail-1 and re-expression of E-cadherin was found in the DKK-3 expressing cell clones supporting a role of DKK-3 in tumor progression. Our studies thus indicate that loss of DKK-3 expression may contribute to melanoma progression.

Expression of Dickkopf genes is strongly reduced in malignant melanoma.

High malignancy and early metastasis are hallmarks of melanoma. Here, we report that the transcription factor Snail1 inhibits expression of the tumor suppressor CYLD in melanoma. As a direct consequence of CYLD repression, the protooncogene BCL-3 translocates into the nucleus and activates Cyclin D1 and N-cadherin promoters, resulting in proliferation and invasion of melanoma cells. Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo. Analysis of a tissue microarray with primary melanomas from patients revealed an inverse correlation of Snail1 induction and loss of CYLD expression. Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression. Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

https://rupress.org/jem/article-pdf/206/1/221/1199209/20082044.pdf

Down-regulation of CYLD expression by Snail promotes tumor progression in malignant melanoma

A fundamental event in the development and progression of malignant melanoma is the deregulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of tumor progression in melanoma and thus the most important member of the AP-1 transcription factor family for this disease. Interestingly, we revealed that c-Jun expression was regulated on the post-transcriptional level and therefore speculated that miRNAs could be involved in c-Jun regulation. We determined seed sequences for miR-125b and miR-527 in the coding region of c-Jun mRNA that hints at the direct involvement of miRNA-dependent regulation on the protein level. We found that the expression of miR-125b was significantly reduced in malignant melanoma cell lines and tissue samples compared with melanocytes, whereas miR-527 remained unchanged. In further functional experiments, treatment of melanoma cells with pre-miR-125b resulted in strong suppression of cellular proliferation and migration, supporting the role of miR-125b in melanoma. In addition, transfection of pre-miR-125b led to strong downregulation of c-Jun protein but not mRNA expression in melanoma cells. Luciferase assays using reporter plasmids containing the miR-125b seed sequence in the luciferase coding region confirmed the direct interaction with miR-125b. Furthermore, immunoprecipitation of Ago-2 revealed that c-Jun mRNA accumulated in the RNA-induced silencing complex after pre-miR-125b transfection in melanoma cells. In summary, we identified an important role for miR-125b in malignant melanoma. Moreover, we demonstrated post-transcriptional regulation of c-Jun by this miRNA and showed that c-Jun is a main mediator of the effects of miR-125b on melanoma cells.

Silke Kuphal

Papers

Integrin signaling in malignant melanoma.

Expression of Dickkopf genes is strongly reduced in malignant melanoma.

Down-regulation of CYLD expression by Snail promotes tumor progression in malignant melanoma

MicroRNA miR-125b controls melanoma progression by direct regulation of c-Jun protein expression

AP-1/c-Jun transcription factors: regulation and function in malignant melanoma.