Acute degradation of nucleolin reveals its novel functions in cell cycle progression and cell division in triple negative breast cancer

The Abstract

This research delves into the critical functions of nucleoli, which are pivotal nuclear sub-compartments integral to numerous vital cellular processes, most notably the assembly of ribosomes. Given that the majority of nucleolar proteins are indispensable for cell viability, their complete elimination or functional suppression through conventional experimental approaches presents a significant technical hurdle. This inherent difficulty has historically constrained a comprehensive understanding of the precise biological roles these nucleolar proteins play in maintaining cellular homeostasis and their involvement in the complex progression of cancer.

Introduction

Nucleoli represent prominent and highly dynamic organelles within the nucleus of eukaryotic cells, serving as the primary sites for ribosome biogenesis, a fundamental process for all living cells. Beyond their well-established role in synthesizing ribosomal RNA and assembling ribosomal subunits, nucleoli are increasingly recognized as multi-functional hubs involved in a myriad of other cellular activities, including stress responses, cell cycle regulation, and even viral replication. The proteins residing within these nucleolar structures are often essential, meaning their complete absence or severe impairment would be lethal to the cell. This essentiality poses a considerable challenge for researchers attempting to decipher their specific contributions to cellular processes and disease states using traditional gene knockout or knockdown methodologies. Such techniques often lead to cell death before researchers can fully observe the nuanced impacts of protein abrogation. Consequently, our understanding of how these vital proteins contribute to normal cellular function and, critically, how their dysregulation might drive pathologies like cancer, has been limited. Triple Negative Breast Cancer (TNBC) stands out as a particularly aggressive and challenging subtype of breast cancer, characterized by the absence of estrogen receptor, progesterone receptor, and HER2 expression. This lack of conventional therapeutic targets makes TNBC notoriously difficult to treat, often resulting in poorer prognoses compared to other breast cancer types. Therefore, identifying novel therapeutic targets and understanding their underlying biological mechanisms in TNBC is of paramount importance.

Methods

To circumvent the aforementioned technical obstacles and achieve an unprecedented level of characterization regarding the biological activities of a key nucleolar protein, we strategically employed the Auxin Inducible Degron (AID) proteolytic system. This innovative system allows for rapid and inducible degradation of specific proteins, thereby enabling researchers to study the acute consequences of protein loss without the confounding effects of long-term compensatory mechanisms or cellular lethality often associated with constitutive knockouts. The AID system was meticulously paired with CRISPR/Cas9 knock-in gene-editing technology, ensuring precise and efficient tagging of endogenous proteins for targeted degradation. Our focus was directed towards Nucleolin (NCL), a remarkably abundant and highly conserved nucleolar protein known to participate in diverse cellular processes, including ribosomal RNA processing, mRNA stability, and DNA replication. By applying this sophisticated molecular toolkit to Triple Negative Breast Cancer (TNBC) cells, we aimed to dissect NCL’s multifaceted roles in a clinically relevant context. Following the acute abrogation of NCL, we implemented a multifaceted analytical approach to comprehensively characterize its impact on TNBC cell behavior. This integrated strategy included live-cell imaging, which provided dynamic insights into cellular morphology and progression through the cell cycle in real-time. Complementing this, RNA-sequencing was performed to unveil global transcriptomic changes, allowing us to identify genes whose expression levels were altered upon NCL depletion. Simultaneously, quantitative proteomics was employed to map changes in protein abundance across the entire proteome, offering a direct view of how NCL abrogation affects the cellular protein landscape. The combination of these powerful techniques provided a holistic understanding of the immediate and downstream consequences of NCL loss. Finally, to validate the molecular role of NCL and translate our in vitro findings into a clinical context, we utilized extensive in silico analyses, leveraging publicly available genomic and proteomic datasets from actual TNBC patients. This computational approach allowed us to correlate NCL expression levels with the abundance of other critical cellular regulators, thereby strengthening the clinical relevance of our experimental observations.

Results

The acute abrogation of endogenous Nucleolin (NCL) in Triple Negative Breast Cancer (TNBC) cells elicited profound and widespread effects, significantly impacting both the overall transcriptome and the proteome of these cells. Our comprehensive analyses revealed that the most critically affected cellular components were those directly involved in fundamental processes such as ribosome biogenesis and, notably, the precise progression through the cell cycle. This suggests a central role for NCL in orchestrating these essential cellular machinery components. Unexpectedly, one of the most striking and novel findings was that the depletion of NCL severely compromised the ability of cancer cells to effectively complete cytokinesis, the final stage of cell division where the cytoplasm divides to form two distinct daughter cells. This impairment in cytokinesis ultimately led to a pronounced accumulation of bi-nucleated cells, indicating a failure in proper cellular segregation following nuclear division. Furthermore, our robust in silico analyses, conducted on extensive datasets derived from human TNBC patients, provided compelling validation for our experimental findings. These analyses confirmed a significant correlation between NCL abundance and the levels of various key regulators of both cell cycle progression and chromosome segregation in clinical TNBC samples. This correlation strongly supports the idea that NCL’s role in supporting cell division, as observed in our experimental models, is highly relevant in the human disease context. Critically, we also discovered that the targeted degradation of NCL significantly enhanced the activity of existing pharmaceutical inhibitors of cellular mitosis. Specifically, NCL degradation markedly improved the efficacy of compounds such as APCin, an inhibitor of the Anaphase Promoting Complex (APC), a crucial ubiquitin ligase that orchestrates cell cycle progression by targeting specific proteins for degradation during mitosis. This synergistic effect highlights a promising therapeutic avenue, suggesting that NCL inhibition could sensitize TNBC cells to mitotic stress and improve the effectiveness of current chemotherapeutic strategies.

Conclusions

Our comprehensive investigation has uncovered a compelling and previously unrecognized role for Nucleolin (NCL) in actively supporting the successful completion of cell division within Triple Negative Breast Cancer models. This discovery positions NCL not merely as a general facilitator of cell growth, but as a critical player in the precise and often vulnerable process of cytokinesis. Furthermore, our findings strongly indicate that the targeted abrogation of NCL has the remarkable potential to enhance the therapeutic activity of existing and future inhibitors designed to disrupt mitotic progression. This suggests a novel strategy for improving the efficacy of cancer treatments, particularly in challenging cases like TNBC, by sensitizing cancer cells to division-disrupting agents and potentially overcoming resistance mechanisms.

Key Concepts And Disclosures

The key concepts central to this investigation include the innovative application of the Auxin-inducible degron system for rapid protein depletion, the intricate process of the cell cycle and its regulation, and the specific functional roles of Nucleolin (NCL) within the nucleolus and broader cellular context.

Additionally, it is important to note certain disclosures relevant to this research. There are no declarations concerning ethics approval or consent to participate, nor is consent for publication applicable in this context. Regarding competing interests, it is disclosed that DP and CMC are inventors named on patent application WO2017011411 A1, which pertains to methods and compositions relating to anti-NCL recombinant immunoagents. Furthermore, DP and TJM serve as consultants and hold equity in Koru Biopharma, a company actively engaged in the development of anti-NCL agents.