Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Boston University School of Medicine

Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ∼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation. 

Cytokinesis failure triggers hippo tumor suppressor pathway activation.

Tetraploidy and tumor development.

Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin-remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric noncoding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. The cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.

Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors.

Genomic instability leads to a wide spectrum of genetic changes, including single nucleotide mutations, structural chromosome alterations, and numerical chromosome changes. The accepted view on how these events are generated predicts that separate cellular mechanisms and genetic events explain the occurrence of these types of genetic variation. Recently, new findings have shed light on the complexity of the mechanisms leading to structural and numerical chromosome aberrations, their intertwining pathways, and their dynamic evolution, in somatic as well as in germ cells. In this review, we present a critical analysis of these recent discoveries in this area, with the aim to contribute to a deeper knowledge of the molecular networks leading to adverse outcomes in humans following exposure to environmental factors. The review illustrates how several technological advances, including DNA sequencing methods, bioinformatics, and live-cell imaging approaches, have contributed to produce a renewed concept of the mechanisms causing genomic instability. Special attention is also given to the specific pathways causing genomic instability in mammalian germ cells. Remarkably, the same scenario emerged from some pioneering studies published in the 1980s to 1990s, when the evolution of polyploidy, the chromosomal effects of spindle poisons, the fate of micronuclei, were intuitively proposed to share mechanisms and pathways. Thus, an old working hypothesis has eventually found proper validation.

Genomic instability: Crossing pathways at the origin of structural and numerical chromosome changes.

Centrosome amplification is a common feature of both solid and hematological human malignancies. Extra centrosomes are not merely innocent bystanders in cancer cells, but rather promote tumor progression by disrupting normal cellular architecture and generating chromosome instability. Consequently, centrosome amplification correlates with advanced tumor grade and overall poor clinical prognosis. By contrast, extra centrosomes are adversely tolerated in non-transformed cells and hinder cell proliferation. This suggests that in addition to acquiring extra centrosomes, cancer cells must also adapt to overcome the deleterious consequences associated with them. Here, we review evidence that implicates core components of the Hippo tumor suppressor pathway as having key roles in both the direct and indirect regulation of centrosome number. Intriguingly, functional inactivation of the Hippo pathway, which is common across broad spectrum of human cancers, likely represents one key adaptation that enables cancer cells to tolerate extra centrosomes. 

The interplay between centrosomes and the Hippo tumor suppressor pathway.

Tetraploid cells are genetically unstable and have the capacity to promote the development and/or progression of human malignancies. It is now estimated that ~40 % of all solid tumors have passed through a tetraploid intermediate stage at some point during their development. Understanding the biological characteristics of tetraploid cells that impart oncogenic properties is therefore a highly relevant and fundamentally important aspect of cancer biology. Here, we describe strategies to efficiently generate and purify tetraploid cells for use in cell biological studies.

Generation and Purification of Tetraploid Cells.

The nuclear envelope, composed of two lipid bilayers and numerous accessory proteins, has evolved to house the genetic material of all eukaryotic cells. In so doing, the nuclear envelope provides a physical barrier between chromosomes and the cytoplasm. Once believed to be highly stable, recent studies demonstrate that the nuclear envelope is prone to rupture. These rupture events expose chromosomal DNA to the cytoplasmic environment and have the capacity to promote DNA damage. Thus nuclear rupture may be an unappreciated mechanism of mutagenesis.

Nuclear envelope rupture drives genome instability in cancer.

Despite intense efforts, the cure rates of childhood and adult solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. We have found that the majority of childhood solid tumors, including rhabdoid tumors, neuroblastoma, medulloblastoma, and Ewing sarcoma, express an active DNA transposase, , that can promote site-specific genomic rearrangements in human cells. Using functional genetic approaches, we discovered that mouse and human cells deficient in nonhomologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA damage signaling inhibitors, we identified AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. We found that expression of PGBD5, but not its nuclease activity-deficient mutant, was sufficient to induce sensitivity to AZD6738. Depletion of endogenous PGBD5 conferred resistance to AZD6738 in human tumor cells. PGBD5-expressing tumor cells accumulated unrepaired DNA damage in response to AZD6738 treatment and underwent apoptosis in both dividing and G-phase cells in the absence of immediate DNA replication stress. Accordingly, AZD6738 exhibited nanomolar potency against most neuroblastoma, medulloblastoma, Ewing sarcoma, and rhabdoid tumor cells tested while sparing nontransformed human and mouse embryonic fibroblasts in vitro. Finally, treatment with AZD6738 induced apoptosis and regression of human neuroblastoma and medulloblastoma tumors engrafted in immunodeficient mice in vivo. This effect was potentiated by combined treatment with cisplatin, including substantial antitumor activity against patient-derived primary neuroblastoma xenografts. These findings delineate a therapeutically actionable synthetic dependency induced in PGBD5-expressing solid tumors.

Neil J. Ganem

Department of Pharmacology & Experimental Therapeutics,
Department of Medicine,
Section of Hematology and Oncology,
Department of Medicine, Section of Hematology and Oncology

Boston University School of Medicine

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Neil J. Ganem

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pharmacology & Experimental Therapeutics,Department of Medicine,Section of Hematology and Oncology,Department of Medicine, Section of Hematology and Oncology

Boston University School of Medicine

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Department of Pharmacology & Experimental Therapeutics,
Department of Medicine,
Section of Hematology and Oncology,
Department of Medicine, Section of Hematology and Oncology