Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization.

Intracellular organelle organization is conserved in eukaryotic cells and is primarily achieved through active transport by motor proteins along the microtubule cytoskeleton. Microtubule post-translational modifications (PTMs) can contribute to microtubule diversity and differentially regulate motor-mediated transport. Here, we show that centrosome amplification, commonly observed in cancer and shown to promote aneuploidy and invasion, induces a global change in organelle positioning towards the cell periphery and facilitates nuclear migration through confined spaces. This reorganization requires kinesin-1 and is analogous to the loss of dynein. Cells with amplified centrosomes display increased levels of acetylated tubulin, a PTM that could enhance kinesin-1-mediated transport. Depletion of α-tubulin acetyltransferase 1 (αTAT1) to block tubulin acetylation rescues the displacement of centrosomes, mitochondria, and vimentin but not Golgi or endosomes. Analyses of the distribution of total and acetylated microtubules indicate that the polarized distribution of modified microtubules, rather than levels alone, plays an important role in the positioning of specific organelles, such as the centrosome. We propose that increased tubulin acetylation differentially impacts kinesin-1-mediated organelle displacement to regulate intracellular organization.

Centrosome amplification mediates small extracellular vesicle secretion via lysosome disruption.

Bidirectional communication between cells and their surrounding environment is critical in both normal and pathological settings. Extracellular vesicles (EVs), which facilitate the horizontal transfer of molecules between cells, are recognized as an important constituent of cell-cell communication. In cancer, alterations in EV secretion contribute to the growth and metastasis of tumor cells. However, the mechanisms underlying these changes remain largely unknown. Here, we show that centrosome amplification is associated with and sufficient to promote small extracellular vesicle (SEV) secretion in pancreatic cancer cells. This is a direct result of lysosomal dysfunction, caused by increased reactive oxygen species (ROS) downstream of extra centrosomes. We propose that defects in lysosome function could promote multivesicular body fusion with the plasma membrane, thereby enhancing SEV secretion. Furthermore, we find that SEVs secreted in response to amplified centrosomes are functionally distinct and activate pancreatic stellate cells (PSCs). These activated PSCs promote the invasion of pancreatic cancer cells in heterotypic 3D cultures. We propose that SEVs secreted by cancer cells with amplified centrosomes influence the bidirectional communication between the tumor cells and the surrounding stroma to promote malignancy.

Macrophages induce malignant traits in mammary epithelium via IKKε/TBK1 kinases and the serine biosynthesis pathway.

During obesity, macrophages infiltrate the breast tissue leading to low-grade chronic inflammation, a factor considered responsible for the higher risk of breast cancer associated with obesity. Here, we formally demonstrate that breast epithelial cells acquire malignant properties when exposed to medium conditioned by macrophages derived from human healthy donors. These effects were mediated by the breast cancer oncogene IKKε and its downstream target-the serine biosynthesis pathway as demonstrated by genetic or pharmacological tools. Furthermore, amlexanox, an FDA-approved drug targeting IKKε and its homologue TBK1, delayed in vivo tumour formation in a combined genetic mouse model of breast cancer and high-fat diet-induced obesity/inflammation. Finally, in human breast cancer tissues, we validated the link between inflammation-IKKε and alteration of cellular metabolism. Altogether, we identified a pathway connecting obesity-driven inflammation to breast cancer and a potential therapeutic strategy to reduce the risk of breast cancer associated with obesity.

Oxidative Stress in Cells with Extra Centrosomes Drives Non-Cell-Autonomous Invasion.

Centrosomal abnormalities, in particular centrosome amplification, are recurrent features of human tumors. Enforced centrosome amplification in vivo plays a role in tumor initiation and progression. However, centrosome amplification occurs only in a subset of cancer cells, and thus, partly due to this heterogeneity, the contribution of centrosome amplification to tumors is unknown. Here, we show that supernumerary centrosomes induce a paracrine-signaling axis via the secretion of proteins, including interleukin-8 (IL-8), which leads to non-cell-autonomous invasion in 3D mammary organoids and zebrafish models. This extra centrosomes-associated secretory phenotype (ECASP) promotes invasion of human mammary cells via HER2 signaling activation. Further, we demonstrate that centrosome amplification induces an early oxidative stress response via increased NOX-generated reactive oxygen species (ROS), which in turn mediates secretion of pro-invasive factors. The discovery that cells with extra centrosomes can manipulate the surrounding cells highlights unexpected and far-reaching consequences of these abnormalities in cancer.

Structural Centrosomal Abnormalities Push Cells toward Invasion.

Structural centrosomal aberrations have long been described in cancer, but their impact on cell physiology and tumorigenesis remains unclear. Ganier et al. (2018) show that centrosome structural abnormalities facilitate cell dissemination by promoting budding of epithelial mitotic cells.

Over-elongation of centrioles in cancer promotes centriole amplification and chromosome missegregation.

Centrosomes are the major microtubule organising centres of animal cells. Deregulation in their number occurs in cancer and was shown to trigger tumorigenesis in mice. However, the incidence, consequence and origins of this abnormality are poorly understood. Here, we screened the NCI-60 panel of human cancer cell lines to systematically analyse centriole number and structure. Our screen shows that centriole amplification is widespread in cancer cell lines and highly prevalent in aggressive breast carcinomas. Moreover, we identify another recurrent feature of cancer cells: centriole size deregulation. Further experiments demonstrate that severe centriole over-elongation can promote amplification through both centriole fragmentation and ectopic procentriole formation. Furthermore, we show that overly long centrioles form over-active centrosomes that nucleate more microtubules, a known cause of invasiveness, and perturb chromosome segregation. Our screen establishes centriole amplification and size deregulation as recurrent features of cancer cells and identifies novel causes and consequences of those abnormalities.

Loss of E-cadherin provides tolerance to centrosome amplification in epithelial cancer cells.

Centrosome amplification is a common feature of human tumors. To survive, cancer cells cluster extra centrosomes during mitosis, avoiding the detrimental effects of multipolar divisions. However, it is unclear whether clustering requires adaptation or is inherent to all cells. Here, we show that cells have varied abilities to cluster extra centrosomes. Epithelial cells are innately inefficient at clustering even in the presence of HSET/KIFC1, which is essential but not sufficient to promote clustering. The presence of E-cadherin decreases cortical contractility during mitosis through a signaling cascade leading to multipolar divisions, and its knockout promotes clustering and survival of cells with multiple centrosomes. Cortical contractility restricts centrosome movement at a minimal distance required for HSET/KIFC1 to exert its function, highlighting a biphasic model for centrosome clustering. In breast cancer cell lines, increased levels of centrosome amplification are accompanied by efficient clustering and loss of E-cadherin, indicating that this is an important adaptation mechanism to centrosome amplification in cancer.

Dividing with Extra Centrosomes: A Double Edged Sword for Cancer Cells.

The presence of supernumerary centrosomes is a hallmark of human tumours. Recent work in animal models suggests that extra centrosomes are not just bystanders in cancer but can accelerate tumourigenesis in the absence of the tumour suppressor p53. Centrosome amplification could indeed actively participate in tumour progression through the induction of chromosome instability, disruption of tissue architecture and promoting cell invasion. Paradoxically, however, centrosome amplification is rather poorly tolerated in normal cells and there are several hurdles cells need to overcome in order to efficiently proliferate in the presence of extra centrosomes. Here, we review the adaptation mechanisms that allow cells to efficiently divide in the presence of extra centrosomes and how these could be exploited to develop selective cancer therapies.

Centrosome amplification and cancer: Branching out.

Despite being a common feature of human cancer, the role of supernumerary centrosomes in tumourigenesis is still poorly understood. We have recently described a novel role for centrosome amplification in promoting cell invasion that could impact tumor progression.

Oncogene-like induction of cellular invasion from centrosome amplification.

Centrosome amplification has long been recognized as a feature of human tumours; however, its role in tumorigenesis remains unclear. Centrosome amplification is poorly tolerated by non-transformed cells and, in the absence of selection, extra centrosomes are spontaneously lost. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumours, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumour progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behaviour is similar to that induced by overexpression of the breast cancer oncogene ERBB2 (ref. 4) and indeed enhances invasiveness triggered by ERBB2. Our data indicate that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell-cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation.

The cell-cycle regulator c-Myc is essential for the formation and maintenance of germinal centers.

Germinal centers (GCs) are sites of intense B cell proliferation and are central for T cell-dependent antibody responses. However, the role of c-Myc, a key cell-cycle regulator, in this process has been questioned. Here we identified c-Myc(+) B cell subpopulations in immature and mature GCs and found, by genetic ablation of Myc, that they had indispensable roles in the formation and maintenance of GCs. The identification of these functionally critical cellular subsets has implications for human B cell lymphomagenesis, which originates mostly from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the c-Myc(+) GC subpopulations may be at a particularly high risk for malignant transformation.

Centrosomes and cilia in human disease.

Centrioles are microtubule-derived structures that are essential for the formation of centrosomes, cilia and flagella. The centrosome is the major microtubule organiser in animal cells, participating in a variety of processes, from cell polarisation to cell division, whereas cilia and flagella contribute to several mechanisms in eukaryotic cells, from motility to sensing. Although it was suggested more than a century ago that these microtubule-derived structures are involved in human disease, the molecular bases of this association have only recently been discovered. Surprisingly, there is very little overlap between the genes affected in the different diseases, suggesting that there are tissue-specific requirements for these microtubule-derived structures. Knowledge of these requirements and disease mechanisms has opened new avenues for therapeutical strategies. Here, we give an overview of recent developments in this field, focusing on cancer, diseases of brain development and ciliopathies.

Cytokinesis failure occurs in Fanconi anemia pathway-deficient murine and human bone marrow hematopoietic cells.

Fanconi anemia (FA) is a genomic instability disorder characterized by bone marrow failure and cancer predisposition. FA is caused by mutations in any one of several genes that encode proteins cooperating in a repair pathway and is required for cellular resistance to DNA crosslinking agents. Recent studies suggest that the FA pathway may also play a role in mitosis, since FANCD2 and FANCI, the 2 key FA proteins, are localized to the extremities of ultrafine DNA bridges (UFBs), which link sister chromatids during cell division. However, whether FA proteins regulate cell division remains unclear. Here we have shown that FA pathway-deficient cells display an increased number of UFBs compared with FA pathway-proficient cells. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein FANCM was recruited to the UFBs at a later stage. The increased number of bridges in FA pathway-deficient cells correlated with a higher rate of cytokinesis failure resulting in binucleated cells. Binucleated cells were also detectable in primary murine FA pathway-deficient hematopoietic stem cells (HSCs) and bone marrow stromal cells from human patients with FA. Based on these observations, we suggest that cytokinesis failure followed by apoptosis may contribute to bone marrow failure in patients with FA.

A mechanism linking extra centrosomes to chromosomal instability.

Chromosomal instability (CIN) is a hallmark of many tumours and correlates with the presence of extra centrosomes. However, a direct mechanistic link between extra centrosomes and CIN has not been established. It has been proposed that extra centrosomes generate CIN by promoting multipolar anaphase, a highly abnormal division that produces three or more aneuploid daughter cells. Here we use long-term live-cell imaging to demonstrate that cells with multiple centrosomes rarely undergo multipolar cell divisions, and the progeny of these divisions are typically inviable. Thus, multipolar divisions cannot explain observed rates of CIN. In contrast, we observe that CIN cells with extra centrosomes routinely undergo bipolar cell divisions, but display a significantly increased frequency of lagging chromosomes during anaphase. To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number. We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division. These segregation errors are a consequence of cells passing through a transient 'multipolar spindle intermediate' in which merotelic kinetochore-microtubule attachment errors accumulate before centrosome clustering and anaphase. These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumours. We propose that this mechanism may be a common underlying cause of CIN in human cancer.

Centrosomes and cancer: how cancer cells divide with too many centrosomes.

Precise control of centrosome number is crucial for bipolar spindle assembly and accurate transmission of genetic material to daughter cells. Failure to properly control centrosome number results in supernumerary centrosomes, which are frequently found in cancer cells. This presents a paradox: during mitosis, cells with more than two centrosomes are prone to multipolar mitoses and cell death, however, cancer cells possessing extra centrosomes usually divide successfully. One mechanism frequently utilized by cancer cells to escape death caused by multipolar mitoses is the clustering of supernumerary centrosomes into bipolar arrays. An understanding of the molecular mechanisms by which cancer cells can suppress multipolar mitoses is beginning to emerge. Here, we review what's currently known about centrosome clustering mechanisms and discuss potential strategies to target these mechanisms for the selective killing of cancer cells.

Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes.

Multiple centrosomes in tumor cells create the potential for multipolar divisions that can lead to aneuploidy and cell death. Nevertheless, many cancer cells successfully divide because of mechanisms that suppress multipolar mitoses. A genome-wide RNAi screen in Drosophila S2 cells and a secondary analysis in cancer cells defined mechanisms that suppress multipolar mitoses. In addition to proteins that organize microtubules at the spindle poles, we identified novel roles for the spindle assembly checkpoint, cortical actin cytoskeleton, and cell adhesion. Using live cell imaging and fibronectin micropatterns, we found that interphase cell shape and adhesion pattern can determine the success of the subsequent mitosis in cells with extra centrosomes. These findings may identify cancer-selective therapeutic targets: HSET, a normally nonessential kinesin motor, was essential for the viability of certain extra centrosome-containing cancer cells. Thus, morphological features of cancer cells can be linked to unique genetic requirements for survival.

Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis.

Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells.