Centrosome Amplification Research Articles

Hepatocyte Polyploidy: Driver or Gatekeeper of Chronic Liver Diseases.

Simple SummaryPolyploidy, a balanced amplification of the genome is a defining feature of the liver. Up to 90% of adult hepatocytes in rodents and around 40% of those in humans are polyploid. The polyploidy of these cells depends on both the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Remarkably, the liver is one of the few mammalian organs that display changes in ploidy content during normal homeostasis, regeneration and pathological conditions. Although polyploid hepatocytes were documented over a century ago, the significance of this original phenomenon in the pathophysiology of the liver remains unclear. In this review, we focused on the mechanisms regulating hepatocyte polyploidization both during liver development and under pathological conditions. We also detailed the effects of polyploidy on liver function and explored the fate and the role of the polyploid state during chronic liver diseases.Polyploidy, also known as whole-genome amplification, is a condition in which the organism has more than two basic sets of chromosomes. Polyploidy frequently arises during tissue development and repair, and in age-associated diseases, such as cancer. Its consequences are diverse and clearly different between systems. The liver is a particularly fascinating organ in that it can adapt its ploidy to the physiological and pathological context. Polyploid hepatocytes are characterized in terms of the number of nuclei per cell (cellular ploidy; mononucleate/binucleate hepatocytes) and the number of chromosome sets in each nucleus (nuclear ploidy; diploid, tetraploid, octoploid). The advantages and disadvantages of polyploidy in mammals are not fully understood. About 30% of the hepatocytes in the human liver are polyploid. In this review, we explore the mechanisms underlying the development of polyploid cells, our current understanding of the regulation of polyploidization during development and pathophysiology and its consequences for liver function. We will also provide data shedding light on the ways in which polyploid hepatocytes cope with centrosome amplification. Finally, we discuss recent discoveries highlighting the possible roles of liver polyploidy in protecting against tumor formation, or, conversely, contributing to liver tumorigenesis.

Centrosome, the Newly Identified Passenger through Tunneling Nanotubes, Increases Binucleation and Proliferation Marker in Receiving Cells

Type 1 tunneling nanotubes (TNTs-1) are long, cytoplasmic protrusions containing actin, microtubules and intermediate filaments that provide a bi-directional road for the transport of various components between distant cells. TNT-1 formation is accompanied by dramatic cytoskeletal reorganization offering mechanical support for intercellular communication. Although the centrosome is the major microtubule nucleating center and also a signaling hub, the relationship between the centrosome and TNTs-1 is still unexplored. We provide here the first evidence of centrosome localization and orientation towards the TNTs-1 protrusion site, which is implicated in TNT-1 formation. We also envision a model whereby synchronized reorientation of the Golgi apparatus along with the centrosome towards TNTs-1 ensures effective polarized trafficking through TNTs-1. Furthermore, using immunohistochemistry and live imaging, we observed for the first time the movement of an extra centrosome within TNTs-1. In this regard, we hypothesize a novel role for TNTs-1 as a critical pathway serving to displace extra centrosomes and potentially to either protect malignant cells against aberrant centrosome amplification or contribute to altering cells in the tumor environment. Indeed, we have observed the increase in binucleation and proliferation markers in receiving cells. The fact that the centrosome can be both as the base and the user of TNTs-1 offers new perspectives and new opportunities to follow in order to improve our knowledge of the pathophysiological mechanisms under TNT control.

E2F3 drives the epithelial-to-mesenchymal transition, cell invasion, and metastasis in breast cancer.

E2F3 is a transcription factor that may initiate tumorigenesis if overexpressed. Previously, we demonstrated that E2F3 mRNA is overexpressed in breast cancer and that E2F3 overexpression results in centrosome amplification and unregulated mitosis, which can promote aneuploidy and chromosome instability to initiate and sustain tumors. Further, we demonstrated that E2F3 leads to overexpression of the mitotic regulator Shugoshin-1, which until recently had unknown roles in cancer. This study aims to evaluate the roles of E2F3 and Shugoshin-1 in breast cancer metastatic potential. Here we demonstrated that E2F3 and Shugoshin-1 silencing leads to reduced cell invasion and migration in two mesenchymal triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and Hs578t). Moreover, E2F3 and Shugoshin-1 modulate the expression of epithelial-to-mesenchymal transition-associated genes such as Snail, E-Cadherin, and multiple matrix metalloproteinases. Furthermore, E2F3 depletion leads to reductions in tumor growth and metastasis in NOD-scid Gamma mice. Results from this study suggest a key role for E2F3 and a novel role for Shugoshin-1 in metastatic progression. These results can further help in the improvement of TNBC targeted therapies by interfering with pathways that intersect with the E2F3 and Shugoshin-1 signaling pathways.

Overexpression of the PLK4 Gene as a Novel Strategy for the Treatment of Autosomal Recessive Microcephaly by Improving Centrosomal Dysfunction.

Autosomal recessive microcephaly and chorioretinopathy (MCCRP) is a neurodevelopmental disorder characterized by delayed psychomotor development, growth retardation with dwarfism, and ocular abnormalities, and its occurrence has been found to be closely related to variants of the gene encoding centrosomes. However, the association between centrosomal duplication defects and the etiology of microcephaly syndromes is poorly understood. It is well known that polo-like kinase 4 (PLK4) is a key regulator of centriole duplication, and the abnormalities of centrosomal function caused by its protein variation need to be further explored in the pathogenesis of microcephaly. In our study, we found that a patient with microcephaly and chorioretinopathy harbored compound heterozygous missense variants NM_014264.4: c.2221C > T (p.Gln741*) and NM_014264.4: c.2062T > C (p.Tyr688His) in the PLK4 gene. Overexpression experiments of the variant PLK4 proteins then showed that the G741 variant rather than the T688H variant had lost centrosomal amplification ability, and the G741 variant but not the T688H variant induced centrosomal replication disorder, which further inhibited cell proliferation, cycle division and cytoskeleton morphology in HeLa cells. Moreover, the overexpression of the two variant proteins had inconsistent effects on the target protein PLK4 by western blot analysis, also indicating that T688H variant overexpression is not functionally equivalent to WT-PLK4 overexpression. Therefore, all data support the idea that the PLK4 mutation induces centriolar duplication disorder and reduces the efficiency of mitosis inducing cell death or cell proliferation in the etiology of microcephaly disorder.

Reciprocal interaction between SIRT6 and APC/C regulates genomic stability

SIRT6 is an NAD+-dependent deacetylase that plays an important role in mitosis fidelity and genome stability. In the present study, we found that SIRT6 overexpression leads to mitosis defects and aneuploidy. We identified SIRT6 as a novel substrate of anaphase-promoting complex/cyclosome (APC/C), which is a master regulator of mitosis. Both CDH1 and CDC20, co-activators of APC/C, mediated SIRT6 degradation via the ubiquitination-proteasome pathway. Reciprocally, SIRT6 also deacetylated CDH1 at lysine K135 and promoted its degradation, resulting in an increase in APC/C-CDH1-targeted substrates, dysfunction in centrosome amplification, and chromosome instability. Our findings demonstrate the importance of SIRT6 for genome integrity during mitotic progression and reveal how SIRT6 and APC/C cooperate to drive mitosis.

Sufu negatively regulates both initiations of centrosome duplication and DNA replication

Centrosome duplication and DNA replication are two pivotal events that higher eukaryotic cells use to initiate proliferation. While DNA replication is initiated through origin licensing, centrosome duplication starts with cartwheel assembly and is partly controlled by CP110. However, the upstream coordinator for both events has been, until now, a mystery. Here, we report that suppressor of fused protein (Sufu), a negative regulator of the Hedgehog (Hh) pathway playing a significant role in restricting the trafficking and function of glioma-related (Gli) proteins, acts as an upstream switch by facilitating CP110 phosphorylation by CDK2, promoting intranuclear Cdt1 degradation and excluding prereplication complex (pre-RC) components from chromosomes, independent of its canonical function in the Hh pathway. We found that Sufu localizes to both the centrosome and the nucleus and that knockout of Sufu induces abnormalities including centrosome amplification, increased nuclear size, multipolar spindle formation, and polyploidy. Serum stimulation promotes the elimination of Sufu from the centrosome by vesicle release at the ciliary tip and from the nucleus via protein degradation, which allows centrosome duplication and DNA replication to proceed. Collectively, this work reveals a mechanism through which Sufu negatively regulates the G1-S transition.

Abstract 1294: Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma

Abstract Background. PDK1, is a master activator of multiple prosurvival and oncogenic protein kinases such as AKT, PKC, RSK and SGK families in oncology[1]. On the other hand, overexpression and aberrant activation of Aurora-A kinase (AurA) have been functionally linked to oncogenic transformation mainly through development of centrosome amplification and chromosomal instability[2]. In this study, the molecular mechanisms at the simultaneous PDK1/AurA inhibition using OXID-pyridonyl[3] compounds were explored in panel of tumor cell lines carrying different oncogenic mutations (PI3KCA, K-RAS, PTEN, RB-1, EGFR and TP53). Materials and Methods. Established human cancer cell lines from breast, glioblastoma, renal prostate, colon, lung and Ewing sarcoma were studied. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72h-exposure with increasing concentrations (from 0.014 to 30 µM). Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds to investigate the functional crosstalk between the two pathways in sensitive cancer cell lines. Results. We assessed anti-proliferative activity of single-agents SA16, IB35, VI8 and VI18 in a panel of 15 cancer cell lines and observed that three Ewing sarcoma and one colon carcinoma cell lines were sensitive (6647,TC71, SKNMC and HCT116), with IC50s values between 2.6 and 25 µM after 72h-exposure, whereas all the studied cell lines were resistant to IB35 and VI18. These results were confirmed by soft agar clonogenic formation studies. AurA, PDK-1, p-PDK1 ser241, AKT, p-AKT ser473 protein levels were characterized but no differences in their basal levels were observed between OXID-pyridonyl compounds -sensitive or -resistant cell lines. SA16 effect at the protein level was evaluated after 5 to 120 min in sensitive cell lines. In SKNMC, TC71 and HCT116 cells, we didn't observe a modulation of AurA or p-PDK1 ser241 while a decrease of p-AKT was observed in SKNMC and HCT116 cells. In addition, a decrease of the p-p44/42 MAPK was observed after 30 min-exposure in SKNMC cells. On the other hand, p-RB1, p-Cdc2, P21 and C-MYC were not regulated at the protein level with SA16 treatment up to 120 min-exposure. In resistant cell lines, U87MG and PC3, both p-PDK1 ser241 and AurA levels were not modulated up to 2h of treatment with SA16. Conclusion. Here we report the characterization of two potent first-in-class dual PDK1-AurA inhibitors, SA16 and VI8. Our findings suggest that innovative PDK1/AurA dual-target molecules which could represent an attractive lead scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma. [1] Nagashima K et al. J Biol Chem. 2011. [2] Yan M et al. Med Res Rev. 2016. [3] Sestito, S et al. Proceedings 2019. Citation Format: Maria Eugenia Riveiro, Simona Rapposelli, Samuel Huguet, Ramiro Vazquez, Mohamed Bekradda, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai. Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1294.

Deletion of a pseudogene within a fragile site triggers the oncogenic expression of the mitotic CCSER1 gene.

The oncogenic role of common fragile sites (CFS), focal and pervasive gaps in the cancer genome arising from replicative stress, remains controversial. Exploiting the TCGA dataset, we found that in most CFS the genes residing within the associated focal deletions are down-regulated, including proteins involved in tumour immune recognition. In a subset of CFS, however, the residing genes are surprisingly overexpressed. Within the most frequent CFS in this group, FRA4F, which is deleted in up to 18% of cancer cases and harbours the CCSER1 gene, we identified a region which includes an intronic, antisense pseudogene, TMSB4XP8. TMSB4XP8 focal ablation or transcriptional silencing elicits the overexpression of CCSER1, through a cis-acting mechanism. CCSER1 overexpression increases proliferation and triggers centrosome amplifications, multinuclearity, and aberrant mitoses. Accordingly, FRA4F is associated in patient samples to mitotic genes deregulation and genomic instability. As a result, cells overexpressing CCSER1 become sensitive to the treatment with aurora kinase inhibitors. Our findings point to a novel tumourigenic mechanism where focal deletions increase the expression of a new class of "dormant" oncogenes.

BAP1-inactivated melanocytic tumors show prominent centrosome amplification and associated loss of primary cilia.

BRCA1-associated protein (BAP1) is a tumor suppressor whose loss is associated with various malignancies. The primary cilium is an organelle involved in signal transduction and cell cycle progression. Primary cilia have been shown to be absent in melanoma but retained to some extent in melanocytic nevi, and the severity of dysplasia influences the degree of cilia loss. Additionally, studies have revealed roles for BAP1 in centrosome and mitotic spindle formation. Because the primary cilium is nucleated on the mother centriole, we examined the connection between the presence of primary cilia and the formation of centrosomes in BAP1-inactivated melanocytic tumors (BIMTs). We evaluated the cilia and centrosomes in 11 BIMTs and five conventional melanocytic nevi using immunofluorescence staining of acetylated alpha-tubulin and gamma-tubulin. We found that, compared to nevi, BIMTs show loss of primary cilia and amplification of centrosomes. Occasional nevi also showed increased centrioles; however, these foci of amplification were more likely to be ciliated than those in BIMTs. Although centrosome amplification does not absolutely correlate with loss of primary cilia in melanocytic neoplasms, absence of BAP1 exacerbates the phenotype. Moreover, aberrant centrosome and cilia formation are likely critical in the pathogenesis of other BAP1-inactivated tumors.

Binding between ROCK1 and DCTN2 triggers diabetes‑associated centrosome amplification in colon cancer cells.

Type 2 diabetes increases the risk various types of cancer and is associated with a poor prognosis therein. There is also evidence that the disease is associated with cancer metastasis. Centrosome amplification can initiate tumorigenesis with metastasis in vivo and increase the invasiveness of cancer cells in vitro. Our previous study reported that type 2 diabetes promotes centrosome amplification via the upregulation and centrosomal translocation of Rho-associated protein kinase 1 (ROCK1), which suggests that centrosome amplification is a candidate biological link between type 2 diabetes and cancer development. In the present study, functional proteomics analysis was used to further investigate the molecular pathways underlying centrosome amplification by targeting ROCK1 binding partners. High glucose, insulin and palmitic acid were used to induce centrosome amplification, and immunofluorescent staining was employed to visualize centrosomal alterations. Combined with immunoprecipitation, mass spectrometry-based proteomics analysis was used to identify ROCK1 binding proteins, and protein complex disruption was achieved by siRNA-knockdown. In total, 1,148 ROCK1 binding proteins were identified, among which 106 proteins were exclusively associated with the treated samples, 193 were only associated with the control samples, and 849 were found in both the control and treated samples. Of the proteins with evidence of centrosomal localization, Dynactin subunit 2 (DCTN2) was confirmed to be localized to the centrosomes. Treating the cells with high glucose, insulin and palmitic acid increased the protein levels of ROCK1 and DCTN2, promoted their binding with each other, and triggered centrosome amplification. Disruption of the protein complex by knocking down ROCK1 or DCTN2 expression partially attenuated centrosome amplification, while simultaneous knockdown of both proteins completely inhibited centrosome amplification. These results suggested ROCK1-DCTN2 binding as a signal for the regulation of centrosome homeostasis, which is key for diabetes-associated centrosome amplification, and enriches our knowledge of centrosome biology. Therefore, the ROCK1-DCTN2 complex may serve as a target for inhibiting centrosome amplification both in research or future therapeutic development.

Patterns of selection against centrosome amplification in human cell lines.

The presence of extra centrioles, termed centrosome amplification, is a hallmark of cancer. The distribution of centriole numbers within a cancer cell population appears to be at an equilibrium maintained by centriole overproduction and selection, reminiscent of mutation-selection balance. It is unknown to date if the interaction between centriole overproduction and selection can quantitatively explain the intra- and inter-population heterogeneity in centriole numbers. Here, we define mutation-selection-like models and employ a model selection approach to infer patterns of centriole overproduction and selection in a diverse panel of human cell lines. Surprisingly, we infer strong and uniform selection against any number of extra centrioles in most cell lines. Finally we assess the accuracy and precision of our inference method and find that it increases non-linearly as a function of the number of sampled cells. We discuss the biological implications of our results and how our methodology can inform future experiments.

Hexavalent chromium induces amplification via ROS‐TF6‐PLK4 pathway

Hexavalent chromium is classified as one of the group one carcinogens in 2012 by the IARC. Accumulated evidence indicates that centrosome amplification is sufficient to initiation tumorigenesis. Since chromium can cause centrosome amplification, it is logical to suspect that chromium causes tumorigenesis via centrosome amplification. The present study investigated the molecular mechanisms underlying chromium-elicited centrosome amplification using non-cancerous lung cells as an experimental model. We found that hexavalent chromium K2Cr2O7 was sub-toxic the cells at the concentration of 0.5 µM or below. At the sub-toxic concentrations, it induced centrosome amplification significantly. Treatment of the cells by chromium increased the intracellular level of reactive oxygen species (ROS), activated ATF-6, and increased the protein level of PLK4. Antioxidant N-acetylcysteine was able to inhibit the chromium-triggered ROS, ATF-6 activation, increment in PLK expression and centrosome amplification. Knockdown of ATF-6 inhibited the production of AFT6 active fragment, the PLK4 expression and centrosome amplification by chromium, without affecting the chromium-produced ROS. Knockdown of PLK4 inhibited the centrosome amplification, but did not modify the ROS production and AFT6 activation. In conclusion, our results suggest that the hexavalent chromium evokes centrosome amplification via ROS-ATF-6-PLK4 signaling pathways. Understanding the molecular mechanisms underlying the chromium-induced centrosome amplification is helpful for the assessment of centrosome amplification in the chromium-caused tumorigenesis.

PCID2 Impacts Centrosome Duplication by Regulating the Nuclear Export of BARD1 mRNA in Breast Cancer Cells

The nuclear export of proteins and other macromolecules out of the nucleus to different intracellular locations is a significant and crucial function of the cell that affects cellular processes such as DNA synthesis, RNA transcription, protein processing, the cell cycle, and apoptosis. Chromosome region maintenance (CRM1) is the major export receptor within the cell, regulating the localization of most proteins. One nuclear export cargo of CRM1 is BARD1, which dimerizes with BRCA1 to regulate DNA repair in the nucleus, while the pair also function in the cytoplasm as negative regulators of centrosome amplification during cell cycle. Despite cooperation at both of these locations BARD1 and BRCA1 are independently exported from the nucleus to the cytoplasm. Our laboratory is interested characterizing the protein PCID2, which is involved in CRM1-mediated protein export and localizes to the centrosome, where it potentially regulates centrosome duplication. We previously demonstrated that PCID2 helps mediate CRM1 dependent export of BRCA1 from the nucleus to the centrosome in Hs578T Breast Cancer cells. The present work aims to determine the role of PCID2 in BARD1 nuclear export and localization in these same cells. PCID2 siRNA knockdowns were used and confirmed via western blot, and BARD1 localization was viewed via immunofluorescence. PCID2 knockdown caused a twenty percent decrease in BARD1 protein in the nucleus and a thirty percent decrease in BARD1 protein at the centrosome. The loss of BARD1 at centrosomes was tied to a fifteen percent increase in Hs578T cells demonstrating excess centrosomes duplication. The loss of BARD1 at both locations in the absence of PCID2 suggests this protein is not involved in the CRM1 mediated export of BARD1 protein from nucleus to centrosomes, and instead changes in cellular localization of BARD1 may be due to an observed 50% decrease in total BARD1 protein with PCID2 knockdown. Interestingly, PCID2 is also a part of the TREX-2 mRNA nuclear export complex; therefore loss of PCID2 may have instead impacted the nuclear export, and translation of BARD1 mRNA. Indeed, PCID2 siRNA knockdown led to a two-fold increase in nuclear BARD1 mRNA as observed by in situ hybridization. Nuclear accumulation of BARD1 mRNA likely contributed the overall decrease in BARD1 expression. The correlation between loss of PCID2 and centrosome amplification suggests PCID2 plays impacts centrosome duplication by regulating both BARD1 mRNA export and BRCA1 protein export; with the loss of PCID2 creating an overall decrease in these negative regulators at the centrosome. Understanding PCID2’s effect on centrosome duplication helps to determine it's impact on cell cycle regulation and define it's potential role as tumor suppressor in Breast Cancer.

The Nek2 centrosome-mitotic kinase contributes to the mesenchymal state, cell invasion, and migration of triple-negative breast cancer cells

Nek2 (NIMA‐related kinase 2) is a serine/threonine-protein kinase that localizes to centrosomes and kinetochores, controlling centrosome separation, chromosome attachments to kinetochores, and the spindle assembly checkpoint. These processes prevent centrosome amplification (CA), mitotic dysfunction, and chromosome instability (CIN). Our group and others have suggested that Nek2 maintains high levels of CA/CIN, tumor growth, and drug resistance. We identified that Nek2 overexpression correlates with poor survival of breast cancer. However, the mechanisms driving these phenotypes are unknown. We now report that overexpression of Nek2 in MCF10A cells drives CA/CIN and aneuploidy. Besides, enhanced levels of Nek2 results in larger 3D acinar structures, but could not initiate tumors in a p53+/+ or a p53−/− xenograft model. Nek2 overexpression induced the epithelial-to-mesenchymal transition (EMT) while its downregulation reduced the expression of the mesenchymal marker vimentin. Furthermore, either siRNA-mediated downregulation or INH6’s chemical inhibition of Nek2 in MDA-MB-231 and Hs578t cells showed important EMT changes and decreased invasion and migration. We also showed that Slug and Zeb1 are involved in Nek2 mediated EMT, invasion, and migration. Besides its role in CA/CIN, Nek2 contributes to breast cancer progression through a novel EMT mediated mechanism.

14-3-3γ prevents centrosome duplication by inhibiting NPM1 function.

14-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication, whereas E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3γ led to an increase in centrosome duplication and over-rode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3γ in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.

Cancer biology: Messages in extracellular vesicles depend on centrosome number

Extra centrosomes are linked to cancer-associated errors in cell division, metastasis and signaling. A new study reveals that centrosome amplification disrupts lysosome function, leading to the release of small extracellular vesicles and to invasive activity in pancreatic cells.

20. Increased Polo-like kinase 1 (Plk1) expression promotes centrosome amplification, chromosomal instability, and tumor formation in a mouse model

Polo-like kinase 1 (PLK1) functions as a regulator of mitotic events. By completing a meta-analysis of The Cancer Genome Atlas, we determined that PLK1 overexpression is positively associated with genome-wide copy number alterations and a poorer prognosis in multiple human cancers. However, it is uncertain whether elevated PLK1 expression is a downstream marker for aggressive tumors or represents a driving force behind tumor progression. To address this question, we established a mouse model that ubiquitously express exogenous Plk1 in a graded manner [Plk1TA/+ (heterozygous) and Plk1TA/TA (homozygous)].

Mitotic kinases as drivers of the epithelial-to-mesenchymal transition and as therapeutic targets against breast cancers.

Biological therapies against breast cancer patients with tumors positive for the estrogen and progesterone hormone receptors and Her2 amplification have greatly improved their survival. However, to date, there are no effective biological therapies against breast cancers that lack these three receptors or triple-negative breast cancers (TNBC). TNBC correlates with poor survival, in part because they relapse following chemo- and radio-therapies. TNBC is intrinsically aggressive since they have high mitotic indexes and tend to metastasize to the central nervous system. TNBCs are more likely to display centrosome amplification, an abnormal phenotype that results in defective mitotic spindles and abnormal cytokinesis, which culminate in aneuploidy and chromosome instability (known causes of tumor initiation and chemo-resistance). Besides their known role in cell cycle control, mitotic kinases have been also studied in different types of cancer including breast, especially in the context of epithelial-to-mesenchymal transition (EMT). EMT is a cellular process characterized by the loss of cell polarity, reorganization of the cytoskeleton, and signaling reprogramming (upregulation of mesenchymal genes and downregulation of epithelial genes). Previously, we and others have shown the effects of mitotic kinases like Nek2 and Mps1 (TTK) on EMT. In this review, we focus on Aurora A, Aurora B, Bub1, and highly expressed in cancer (Hec1) as novel targets for therapeutic interventions in breast cancer and their effects on EMT. We highlight the established relationships and interactions of these and other mitotic kinases, clinical trial studies involving mitotic kinases, and the importance that represents to develop drugs against these proteins as potential targets in the primary care therapy for TNBC.

Centrosome amplification mediates small extracellular vesicle secretion via lysosome disruption

SummaryBidirectional 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.

Genotoxic stress-activated DNA-PK-p53 cascade and autophagy cooperatively induce ciliogenesis to maintain the DNA damage response

The DNA-PK maintains cell survival when DNA damage occurs. In addition, aberrant activation of the DNA-PK induces centrosome amplification, suggesting additional roles for this kinase. Here, we showed that the DNA-PK-p53 cascade induced primary cilia formation (ciliogenesis), thus maintaining the DNA damage response under genotoxic stress. Treatment with genotoxic drugs (etoposide, neocarzinostatin, hydroxyurea, or cisplatin) led to ciliogenesis in human retina (RPE1), trophoblast (HTR8), lung (A459), and mouse Leydig progenitor (TM3) cell lines. Upon genotoxic stress, several DNA damage signaling were activated, but only the DNA-PK-p53 cascade contributed to ciliogenesis, as pharmacological inhibition or genetic depletion of this pathway decreased genotoxic stress-induced ciliogenesis. Interestingly, in addition to localizing to the nucleus, activated DNA-PK localized to the base of the primary cilium (mother centriole) and daughter centriole. Genotoxic stress also induced autophagy. Inhibition of autophagy initiation or lysosomal degradation or depletion of ATG7 decreased genotoxic stress-induced ciliogenesis. Besides, inhibition of ciliogenesis by depletion of IFT88 or CEP164 attenuated the genotoxic stress-induced DNA damage response. Thus, our study uncovered the interplay among genotoxic stress, the primary cilium, and the DNA damage response.