Patrícia M.A. Silva

Monitoring the fidelity of mitotic chromosome segregation by the spindle assembly checkpoint

Accurate chromosome segregation relies on activity of the spindle assembly checkpoint, a surveillance mechanism that prevents premature anaphase onset until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Defects in this mechanism contribute to chromosome instability and aneuploidy, a hallmark of malignant cells. Here, we review the molecular mechanisms of activation and silencing of the spindle assembly checkpoint and its relationship to tumourigenesis.

High CDC20 expression is associated with poor prognosis in oral squamous cell carcinoma

OBJECTIVES Human Cell Division Cycle 20 (CDC20) homolog is a crucial target of the spindle assembly checkpoint. It is an activator of the Anaphase-Promoting Complex/Cyclosome (APC/C) which promotes anaphase onset and mitotic exit through the ubiquitination of securin and cyclin B1. Overexpression of CDC20 was previously reported in oral squamous cell carcinoma (OSCC). Here, we propose to explore the clinicopathological significance of CDC20 overexpression and its potential use as a prognostic marker in OSCC. METHODS Using tissue microarray technology, we analyzed CDC20 expression in 65 primary OSCC tissues by immunohistochemistry. Statistical analysis was performed to evaluate the clinicopathological and prognostic significance of CDC20 expression in OSCC. RESULTS Of the 65 cases of patients with OSCC studied, 37 (56.9%) showed high CDC20 protein expression. No clinicopathological features were correlated with CDC20 expression. Importantly, in univariable analysis, OSCC patients with higher CDC20 protein expression showed significantly shorter cancer-specific survival rate (P = 0.018). Multivariable analysis identified high CDC20 expression as an independent prognostic factor (P = 0.032). CONCLUSION High CDC20 expression is associated with poor prognosis in OSCC and may be used to identify high-risk OSCC patients and may serve as a therapeutic target.

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

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.

The spindle assembly checkpoint: perspectives in tumorigenesis and cancer therapy

Loss or gain of chromosomes, a condition known as aneuploidy, is a common feature of tumor cells and has therefore been proposed as the driving force for tumorigenesis. Such chromosomal instability can arise during mitosis as a result of mis-segregation of the duplicated sister chromatids to the two daughter cells. In normal cells, missegregation is usually prevented by the spindle assembly checkpoint (SAC), a sophisticated surveillance mechanism that inhibits mitotic exit until all chromosomes have successfully achieved bipolar attachment to spindle microtubules. Complete abrogation of SAC activity is lethal to normal as well as to tumor cells, as a consequence of massive chromosome mis-segregation. Importantly, many human aneuploid tumor cells exhibit a weakened SAC activity that allows them to tolerate gains or losses of a small number of chromosomes; and interfering with this SAC residual activity may constitute a suitable strategy to kill cancer cells. This review focuses on the potential link between SAC and tumorigenesis, and the therapeutic strategy to target the SAC for cancer treatment.

An Overview of the Spindle Assembly Checkpoint Status in Oral Cancer

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.

Prenylated Chalcone 2 Acts as an Antimitotic Agent and Enhances the Chemosensitivity of Tumor Cells to Paclitaxel

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.

Spindle Assembly Checkpoint as a Potential Target in Colorectal Cancer: Current Status and Future Perspectives

Abstract Colorectal cancer (CRC), one of the most common malignancies worldwide, is often diagnosed at an advanced stage, and resistance to chemotherapeutic and existing targeted therapy is a major obstacle to its successful treatment. New targets that offer alternative clinical options are therefore urgently needed. Recently, perturbation of the spindle assembly checkpoint (SAC), the surveillance mechanism that maintains anaphase inhibition until all chromosomes reach the metaphase plate, has been regarded as a promising target to fight cancer cells, either alone or in combination regimens. Consistent with this strategy, many cancers, including CRC, exhibit altered expression of SAC genes. In this article, we review our current knowledge on SAC activity status in CRC, and on current anti‐CRC strategies and future therapeutic perspectives on the basis of SAC targeting experiments in vitro and in animal models.

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

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.

Co‐silencing of human Bub3 and dynein highlights an antagonistic relationship in regulating kinetochore–microtubule attachments

We previously reported that the spindle assembly checkpoint protein Bub3 is involved in regulating kinetochore–microtubule (KT‐MT) attachments. Also, Bub3 was reported to interact with the microtubule motor protein dynein. Here we examined how this interaction contributes to KT‐MT attachments. Depletion of Bub3 or dynein induced misaligned chromosomes, consistent with their role in KT‐MT attachments. Unexpectedly, co‐silencing of both proteins partially suppressed the misalignment phenotype and restored chromosome congression. Consistent with these observations, KT‐MT attachments in co‐depleted cells were stable, able to drive chromosome congression, and produce inter‐ and intra‐kinetochore stretch, indicating they are functional. We suggest that a mutual antagonism exists between Bub3 and dynein to ensure optimal KT‐MT attachments.

Mitosis inhibitors in anticancer therapy: When blocking the exit becomes a solution

Current microtubule-targeting agents (MTAs) remain amongst the most important antimitotic drugs used against a broad range of malignancies. By perturbing spindle assembly, MTAs activate the spindle assembly checkpoint (SAC), which induces mitotic arrest and subsequent apoptosis. However, besides toxic side effects and resistance, mitotic slippage and failure in triggering apoptosis in various cancer cells are limiting factors of MTAs efficacy. Alternative strategies to target mitosis without affecting microtubules have, thus, led to the identification of small molecules, such as those that target spindle Kinesins, Aurora and Polo-like kinases. Unfortunately, these so-called second-generation of antimitotics, encompassing mitotic blockers and mitotic drivers, have failed in clinical trials. Our recent understanding regarding the mechanisms of cell death during a mitotic arrest pointed out apoptosis as the main variable, providing an opportunity to control the cell fates and influence the effectiveness of antimitotics. Here, we provide an overview on the second-generation of antimitotics, and discuss possible strategies that exploit SAC activity, mitotic slippage/exit and apoptosis induction, in order to improve the efficacy of anticancer strategies that target mitosis.