Joao Silva

Scopus Publications

Mitotic Machinery Dysregulation in Lung Cancer: Biological Roles, Therapeutic Targeting, and Combination Strategies
Bárbara Pinto, João P. N. Silva, Patrícia M. A. Silva, Bruno Sarmento, Juliana Carvalho-Tavares, Hassan Bousbaa
Pharmaceutics, 2026
Lung cancer remains the leading cause of cancer-related mortality worldwide and is characterized by high aggressiveness and therapeutic resistance, partly driven by mitotic dysregulation. Key mitotic regulators, including kinases such as PLK1, AURKA, AURKB, and MPS1 and kinesins such as CENPE and Eg5, are frequently overexpressed in NSCLC and SCLC, contributing to chromosomal instability, aneuploidy, and highly proliferative tumor phenotypes. Although multiple inhibitors targeting these proteins have been developed, their clinical efficacy as monotherapies has been limited. This is largely due to insufficient target dependency, adaptive resistance mechanisms, mitotic slippage, activation of compensatory pathways, and dose-limiting toxicity. This review integrates current knowledge on the physiological roles of major mitotic regulators, their dysregulation in lung tumorigenesis, and the biological and pharmacological barriers that underlie the limited success of antimitotic drugs. We further highlight preclinical and clinical evidence supporting rational combination strategies designed to enhance the antitumor activity of mitotic inhibitors while minimizing toxicity. Together, these insights underscore the need for refined therapeutic approaches that better exploit vulnerabilities in mitotic control to improve outcomes for patients with lung cancer.
BCL-2 and BCL-xL in Cancer: Regulation, Function, and Therapeutic Targeting
João P. N. Silva, Bárbara Pinto, Patrícia M. A. Silva, Hassan Bousbaa
International Journal of Molecular Sciences, 2026
The BCL-2 family of proteins plays a central role in the regulation of apoptosis, with BCL-2 and BCL-xL representing two of its most prominent antiapoptotic members. This review explores the molecular regulation of BCL-2 and BCL-xL genes, emphasizing the structural domains that define the functions of the broader BCL-2 family. Beyond their canonical roles in preventing mitochondrial outer membrane permeabilization, both proteins contribute significantly to cancer development. Their overexpression enhances invasiveness and tumor progression, supports angiogenesis, and critically modulates cellular responses to chemotherapy, often conferring drug resistance. Additional non-apoptotic functions, including roles in metabolism, mitochondrial dynamics, and cellular homeostasis, further expand their biological relevance. Clinical trials exploring strategies to inhibit BCL-2 and BCL-xL, including selective BH3 mimetics and combination regimens, are discussed with emphasis on their potential and limitations in oncology. Overall, this review highlights the multifaceted contributions of BCL-2 and BCL-xL to cancer biology and underscores the importance of continued efforts to refine targeted therapeutic approaches.
Kinesin Spindle Protein (KIF11) in Mitosis and Cancer
João P. N. Silva, Patrícia M. A. Silva, Hassan Bousbaa
International Journal of Molecular Sciences, 2025
Kinesin spindle protein (KSP), also known as KIF11, is a member of the kinesin superfamily of motor proteins that plays a pivotal role in mitosis by regulating spindle assembly, chromosome alignment, and segregation. Its motor activity, which is essential for the proper organization of microtubules during mitosis, is crucial for maintaining genomic stability. KSP overexpression has been observed in several cancer types, where it promotes uncontrolled cell proliferation, making it a promising target for cancer therapy. This review provides a comprehensive analysis of the molecular mechanisms underlying KSP function, including its structural features, ATPase activity, and interactions with other mitotic proteins. Additionally, we review the regulation of KSP through post-translational modifications, such as phosphorylation, as well as the therapeutic strategies currently being explored to inhibit its activity in cancer treatment.
Time-Lapse Imaging to Analyze Cell Fate in Response to Antimitotics
Patrícia M. A. Silva, João P. N. Silva, Bárbara Pinto, Hassan Bousbaa
Methods in Molecular Biology, 2025
Limosilactobacillus reuteri AJCR4: A Potential Probiotic in the Fight Against Oral Candida spp. Biofilms
António Rajão, João P. N. Silva, Diana L. Almeida-Nunes, Paulo Rompante, Célia Fortuna Rodrigues, José Carlos Andrade
International Journal of Molecular Sciences, 2025
Oral candidiasis is one of the most common infections in the immunocompromised. Biofilms of Candida species can make treatments difficult, leading to oral infection recurrence. This research aimed to isolate a Lactobacillus with anti-Candida effects from the oral cavity. An oral Lactobacillus was isolated in caries-free individuals. The best isolate was evaluated against Candida spp. planktonic and biofilm forms. The bacterial impacts on Candida biofilms’ adhesion to acrylic discs were analyzed through an in vitro test. L. reuteri AJCR4 had the best anti-Candida activity in the preliminary screening. Results were promising in both planktonic and biofilms, particularly with C. albicans SC5314 and C. tropicalis ATCC750, where no viable cells were detected when using the cell-free supernatant (undiluted). In C. glabrata ATCC2001 and C. parapsilosis ATCC22019 biofilms, reductions of 3 Log10 and more than 2 Log10, respectively, were noted when using a cell suspension of L. reuteri ACJR4 (108 CFU/mL). On polymethyl methacrylate acrylic discs, the cell-free supernatant reduced Candida adhesion, resulting in no viable cell detection on the surface. In conclusion, L. reuteri AJCR4 demonstrated notable antifungal activity against Candida biofilms. This oral isolate and its postbiotic can be a potential alternative strategy to oral candidiasis, especially to treat recalcitrant infections.
Metformin Impairs Linsitinib Anti-Tumor Effect on Ovarian Cancer Cell Lines
Diana Luísa Almeida-Nunes, João P. N. Silva, Mariana Nunes, Patrícia M. A. Silva, Ricardo Silvestre, Ricardo Jorge Dinis-Oliveira, Hassan Bousbaa, Sara Ricardo
International Journal of Molecular Sciences, 2024
Ovarian cancer (OC) remains one of the leading causes of cancer-related mortality among women. Targeting the insulin-like growth factor 1 (IGF-1) signaling pathway has emerged as a promising therapeutic strategy. Linsitinib, an IGF-1 receptor (IGF-1R) inhibitor, has shown potential in disrupting this pathway. Additionally, metformin, commonly used in the treatment of type 2 diabetes, has been studied for its anti-cancer properties due to its ability to inhibit metabolic pathways that intersect with IGF-1 signaling, making it a candidate for combination therapy in cancer treatments. This study explores the anti-cancer effects of linsitinib and metformin on OVCAR3 cells by the suppression of the IGF-1 signaling pathway by siRNA-mediated IGF-1 gene silencing. The goal is to evaluate their efficacy as therapeutic agents and to emphasize the critical role of this pathway in OC cell proliferation. Cellular viability was evaluated by resazurin-based assay, and apoptosis was assessed by flux cytometry. The results of this study indicate that the combination of linsitinib and metformin exhibits an antagonistic effect (obtained by SynergyFinder 2.0 Software), reducing their anti-neoplastic efficacy in OC cell lines. Statistical analyses were performed using ordinary one-way or two-way ANOVA, followed by Tukey’s or Šídák’s multiple comparison tests. While linsitinib shows promise as a therapeutic option for OC, further research is needed to identify agents that could synergize with it to enhance its therapeutic efficacy, like the combination with standard chemotherapy in OC (carboplatin and paclitaxel).
Targeting the EGFR and Spindle Assembly Checkpoint Pathways in Oral Cancer: A Plausible Alliance to Enhance Cell Death
Mafalda Calheiros-Lobo, João P. N. Silva, Leonor Delgado, Bárbara Pinto, Luís Monteiro, Carlos Lopes, Patrícia M. A. Silva, Hassan Bousbaa
Cancers, 2024
Background/Objectives: Head and neck cancer (HNC) is among the most common cancer types globally, with its incidence expected to increase significantly in the coming years. Oral squamous cell carcinoma (OSCC), the predominant subtype, exhibits significant heterogeneity and resistance to treatment. Current therapies, including surgery, radiation, and chemotherapy, often result in poor outcomes for advanced stages. Cetuximab, an EGFR inhibitor, is widely used but faces limitations. This study explores the combined inhibition of EGFR and mitotic proteins to enhance treatment efficacy. Methods: We analyzed the effects of co-treating OSCC cells with small molecules targeting MPS-1 (BAY1217389), Aurora-B (Barasertib), or KSP (Ispinesib), alongside Cetuximab. The rationale is based on targeting EGFR-mediated survival pathways and the mitotic checkpoint, addressing multiple cell cycle phases and reducing resistance. Results: Our findings indicate that inhibiting MPS-1, Aurora-B, or KSP enhances Cetuximab’s therapeutic potential, promoting increased cancer cell death. Additionally, we examined EGFR, MPS-1, Aurora-B, and KSP expression in OSCC patient samples, revealing their clinicopathologic significance. Conclusions: This combinatorial approach suggests a promising strategy to improve treatment outcomes in OSCC.
Exploring the Therapeutic Implications of Co-Targeting the EGFR and Spindle Assembly Checkpoint Pathways in Oral Cancer
Mafalda Calheiros-Lobo, João P. N. Silva, Bárbara Pinto, Luís Monteiro, Patrícia M. A. Silva, Hassan Bousbaa
Pharmaceutics, 2024
Head and neck cancer (HNC), the sixth most common cancer worldwide, is increasing in incidence, with oral squamous cell carcinoma (OSCC) as the predominant subtype. OSCC mainly affects middle-aged to elderly males, often occurring on the posterior lateral border of the tongue, leading to significant disfigurement and functional impairments, such as swallowing and speech difficulties. Despite advancements in understanding OSCC’s genetic and epigenetic variations, survival rates for advanced stages remain low, highlighting the need for new treatment options. Primary treatment includes surgery, often combined with radiotherapy (RT) and chemotherapy (CT). Cetuximab-based chemotherapy, targeting the overexpressed epidermal growth factor receptor (EGFR) in 80–90% of HNCs, is commonly used but correlates with poor prognosis. Additionally, monopolar spindle 1 (MPS1), a spindle assembly checkpoint (SAC) component, is a significant target due to its role in genomic fidelity during mitosis and its overexpression in several cancers. This review explores EGFR and MPS1 as therapeutic targets in HNC, analyzing their molecular mechanisms and the effects of their inhibition on cancer cells. It also highlights the promise of combinatorial approaches, such as microtubule-targeting agents (MTAs) and antimitotic agents, in improving HNC therapies, patient outcomes, and survival rates.
Coupling Kinesin Spindle Protein and Aurora B Inhibition with Apoptosis Induction Enhances Oral Cancer Cell Killing
João P. N. Silva, Bárbara Pinto, Luís Monteiro, Patrícia M. A. Silva, Hassan Bousbaa
Cancers, 2024
Many proteins regulating mitosis have emerged as targets for cancer therapy, including the kinesin spindle protein (KSP) and Aurora kinase B (AurB). KSP is crucial for proper spindle pole separation during mitosis, while AurB plays roles in chromosome segregation and cytokinesis. Agents targeting KSP and AurB selectively affect dividing cells and have shown significant activity in vitro. However, these drugs, despite advancing to clinical trials, often yield unsatisfactory outcomes as monotherapy, likely due to variable responses driven by cyclin B degradation and apoptosis signal accumulation networks. Accumulated data suggest that combining emerging antimitotics with various cytostatic drugs can enhance tumor-killing effects compared to monotherapy. Here, we investigated the impact of inhibiting anti-apoptotic signals with the BH3-mimetic Navitoclax in oral cancer cells treated with the selective KSP inhibitor, Ispinesib, or AurB inhibitor, Barasertib, aiming to potentiate cell death. The combination of BH3-mimetics with both KSP and AurB inhibitors synergistically induced substantial cell death, primarily through apoptosis. A mechanistic analysis underlying this synergistic activity, undertaken by live-cell imaging, is presented. Our data underscore the importance of combining BH3-mimetics with antimitotics in clinical trials to maximize their effectiveness.
Maximizing Anticancer Response with MPS1 and CENPE Inhibition Alongside Apoptosis Induction
Bárbara Pinto, João P. N. Silva, Patrícia M. A. Silva, Daniel José Barbosa, Bruno Sarmento, Juliana Carvalho Tavares, Hassan Bousbaa
Pharmaceutics, 2024
Antimitotic compounds, targeting key spindle assembly checkpoint (SAC) components (e.g., MPS1, Aurora kinase B, PLK1, KLP1, CENPE), are potential alternatives to microtubule-targeting antimitotic agents (e.g., paclitaxel) to circumvent resistance and side effects associated with their use. They can be classified into mitotic blockers, causing SAC-induced mitotic arrest, or mitotic drivers, pushing cells through aberrant mitosis by overriding SAC. These drugs, although advancing to clinical trials, exhibit unsatisfactory cancer treatment outcomes as monotherapy, probably due to variable cell fate responses driven by cyclin B degradation and apoptosis signal accumulation networks. We investigated the impact of inhibiting anti-apoptotic signals with the BH3-mimetic navitoclax in lung cancer cells treated with the selective CENPE inhibitor GSK923295 (mitotic blocker) or the MPS1 inhibitor BAY1217389 (mitotic driver). Our aim was to steer treated cancer cells towards cell death. BH3-mimetics, in combination with both mitotic blockers and drivers, induced substantial cell death, mainly through apoptosis, in 2D and 3D cultures. Crucially, these synergistic concentrations were less toxic to non-tumor cells. This highlights the significance of combining BH3-mimetics with antimitotics, either blockers or drivers, which have reached the clinical trial phase, to enhance their effectiveness.
Combination Therapy as a Promising Way to Fight Oral Cancer
João P. N. Silva, Bárbara Pinto, Luís Monteiro, Patrícia M. A. Silva, Hassan Bousbaa
Pharmaceutics, 2023
BUBR1 as a Prognostic Biomarker in Canine Oral Squamous Cell Carcinoma
Leonor Delgado, Luís Monteiro, Patrícia Silva, Hassan Bousbaa, Fernanda Garcez, João Silva, Paula Brilhante-Simões, Isabel Pires, Justina Prada
Animals, 2022
RNA-Binding Proteins Driving the Regulatory Activity of Small Non-coding RNAs in Bacteria
Ana P. Quendera, André F. Seixas, Ricardo F. dos Santos, Inês Santos, João P. N. Silva, Cecília M. Arraiano, José M. Andrade
Frontiers in Molecular Biosciences, 2020
Plasmids for independently tunable, low-noise expression of two genes
João P. N. Silva, Soraia Vidigal Lopes, Diogo J. Grilo, Zach Hensel
Msphere, 2019

Joao Silva

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