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Suppression of spindly delays mitotic exit and exacerbates cell death response of cancer cells treated with low doses of paclitaxel

Publication . Silva, Patrícia M. A.; Ribeiro, Nilza; Lima, Raquel T.; Andrade, Claudia; Diogo, Vania; Teixeira, Joana; Florindo, C.; Tavares, Alvaro; Vasconcelos, M. Helena; Bousbaa, Hassan

Microtubule-targeting agents (MTAs) are used extensively for the treatment of diverse types of cancer. They block cancer cells in mitosis through the activation of the spindle assembly checkpoint (SAC), the surveillance mechanism that ensures accurate chromosome segregation at the onset of anaphase. However, the cytotoxic activity of MTAs is limited by premature mitotic exit (mitotic slippage) due to SAC silencing. Here we have explored the dual role of the protein Spindly in chromosome attachments and SAC silencing to analyze the consequences of its depletion on the viability of tumor cells treated with clinically relevant doses of paclitaxel. As expected, siRNA-mediated Spindly suppression induced chromosome misalignment and accumulation of cells in mitosis. Remarkably, these cells were more sensitive to low-doses of paclitaxel. Sensitization was due to an increase in the length of mitotic arrest and high frequency of multinucleated cells, both correlated with an exacerbated post-mitotic cell death response as determined by cell fate profiling. Thus, by affecting both SAC silencing and chromosome attachment, Spindly targeting offers a double-edged sword that potentiates tumor cell killing by clinically relevant doses of paclitaxel, providing a rationale for combination chemotherapy against cancer. (C) 2017 Elsevier B.V. All rights reserved.

2017-05Journal article

Restricted

Dynein-dependent transport of spindle assembly checkpoint proteins off kinetochores toward spindle poles

Publication . Silva, PMA; Patrícia M.A. Silva; Reis, Rita M.; Bolanos-Garcia, Victor M.; Florindo, Claudia; Tavares, Alvaro; Bousbaa, Hassan

A predominant mechanism of spindle assembly checkpoint (SAC) silencing is dynein-mediated transport of certain kinetochore proteins along microtubules. There are still conflicting data as to which SAC proteins are dynein cargoes. Using two ATP reduction assays, we found that the core SAC proteins Mad1, Mad2, Bub1, BubR1, and Bub3 redistributed from attached kinetochores to spindle poles, in a dynein-dependent manner. This redistribution still occurred in metaphase-arrested cells, at a time when the SAC should be satisfied and silenced. Unexpectedly, we found that a pool of Hec1 and Mis12 also relocalizes to spindle poles, suggesting KMN components as additional dynein cargoes. The potential significance of these results for SAC silencing is discussed. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

2014-08Journal article

Open Access

Prenylated chalcone 2 ccts as an antimitotic agent and enhances the chemosensitivity of tumor cells to paclitaxel

Publication . Fonseca, Joana; Marques, Sandra; Silva, PMA; Patrícia M.A. Silva; Brandão, Pedro; Cidade, Honorina; Pinto, Madalena M.; Bousbaa, Hassan

We previously reported that prenylated chalcone 2 (PC2), the O-prenyl derivative (2) of 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone (1), induced cytotoxicity of tumor cells via disruption of p53-MDM2 interaction. However, the cellular changes through which PC2 exerts its cytotoxic activity and its antitumor potential, remain to be addressed. In the present work, we aimed to (i) characterize the effect of PC2 on mitotic progression and the underlying mechanism; and to (ii) explore this information to evaluate its ability to sensitize tumor cells to paclitaxel in a combination regimen. PC2 was able to arrest breast adenocarcinoma MCF-7 and non-small cell lung cancer NCI-H460 cells in mitosis. All mitosis-arrested cells showed collapsed mitotic spindles with randomly distributed chromosomes, and activated spindle assembly checkpoint. Live-cell imaging revealed that the compound induced a prolonged delay (up to 14 h) in mitosis, culminating in massive cell death by blebbing. Importantly, PC2 in combination with paclitaxel enhanced the effect on cell growth inhibition as determined by cell viability and proliferation assays. Our findings demonstrate that the cytotoxicity induced by PC2 is mediated through antimitotic activity as a result of mitotic spindle damage. The enhancement effects of PC2 on chemosensitivity of cancer cells to paclitaxel encourage further validation of the clinical potential of this combination.

2016-08Journal article

Open Access

The spindle assembly checkpoint: new insights into its function, regulation and therapeutic implications

Publication . Silva, Patrícia Manuela Areias da; Tavares, Álvaro Augusto Marques; Bousbaa, Hassan

Genomic stability relies on the faithful chromosome segregation in mitosis, under the control of the Spindle Assembly Checkpoint (SAC). This surveillance mechanism monitors the nature of kinetochore-microtubule attachments (KT-MT) and prevents premature anaphase onset until all chromosomes achieve proper bipolar attachments and come under tension. To initiate anaphase and promote mitotic exit, the silencing of SAC is required. In this study, we proposed to shed light into the function and regulation of the SAC mechanism and to explore its potential as therapeutic target for cancer therapy. For that, two questions were asked that define the main objectives of the present thesis: i) how SAC proteins are functionally related to motor proteins to regulate KT-MT attachments and SAC silencing? and ii) to what extent could SAC proteins constitute relevant cancer biomarkers and/or relevant targets to kill cancer cells, either alone or in combination with currently used antimitotics? We found that RNAi-mediated co-depletion of the SAC protein Bub3 and the motor protein dynein partially restored functional KT-MT attachment, otherwise severely affect by individual depletion of each of the two proteins, leading to chromosome congression. An antagonistic relationship between Bub3 and dynein is suggested to ensure stable KT-MT attachments. In addition, we demonstrated that upon chromosome alignment at the metaphase plate, the core SAC proteins Mad1, Mad2, Bub1, BubR1, and Bub3 and the KMN components Hec1 and Mis12 are poleward transported by dynein, suggesting a role in SAC silencing. Second, and in a therapeutic perspective, we dissected the dual role of Spindly (kinetochore regulator of dynein), in chromosome attachment and SAC silencing, as a strategy to potentiate tumor cell killing. We found an upregulation of Spindly in human cancer cell lines and in patient samples which are correlated with tumor proliferation. Interestingly, Spindly inhibition enhanced the cytotoxic response of paclitaxel- and cisplatin-mediated chemotherapy, and sensitized human tumor cells to death. Overall, we provide new insight into the regulation of kinetochore-microtubule interactions by establishing a functional antagonistic relationship between Bub3 and dynein in regulating KT-MT attachment. Also, we provide new insight as to SAC components that are removed from the kinetochore by dynein during SAC silencing.

2017-09-15Doctoral thesis

Open Access

An overview of the spindle assembly checkpoint status in oral cancer

Publication . Teixeira, Jose Henrique; Silva, Patrícia; Reis, Rita Margarida; Moura, Ines Moranguinho; Marques, Sandra; Fonseca, Joana; Monteiro, Luis Silva; Bousbaa, Hassan

Abnormal chromosome number, or aneuploidy, is a common feature of human solid tumors, including oral cancer. Deregulated spindle assembly checkpoint (SAC) is thought as one of the mechanisms that drive aneuploidy. In normal cells, SAC prevents anaphase onset until all chromosomes are correctly aligned at the metaphase plate thereby ensuring genomic stability. Significantly, the activity of this checkpoint is compromised in many cancers. While mutations are rather rare, many tumors show altered expression levels of SAC components. Genomic alterations such as aneuploidy indicate a high risk of oral cancer and cancer-related mortality, and the molecular basis of these alterations is largely unknown. Yet, our knowledge on the status of SAC components in oral cancer remains sparse. In this review, we address the state of our knowledge regarding the SAC defects and the underlying molecular mechanisms in oral cancer, and discuss their therapeutic relevance, focusing our analysis on the core components of SAC and its target Cdc20.

2014Journal article

Open Access