Targeting Ribosome Biogenesis - A Promising Therapeutic Approach to Overcome EMT-related Chemoresistance in Breast Cancer

In the field of cancer treatment, breast cancer treatment has always been a focus of research. In recent years, although there have been continuous advancements in breast cancer treatment methods, chemoresistance remains a major challenge. Epithelial - mesenchymal transition (EMT) plays a crucial role in chemoresistance of breast cancer. It endows cancer cells with enhanced invasiveness and metastatic potential, severely affecting patients' prognosis. Recently, a study published in eLife has brought new hope for solving this problem. Researchers found that targeting the ribosome biogenesis (RiBi) pathway may be an effective way to overcome EMT-related chemoresistance in breast cancer.

EMT: A Hurdle in Breast Cancer Treatment

EMT is a complex biological process. During this process, epithelial cells lose their polarity and intercellular junctions and acquire the characteristics of mesenchymal cells. This transformation gives cancer cells stronger migration and invasion abilities, enabling them to break through the basement membrane, enter the bloodstream, and colonize in distant organs, thus leading to tumor metastasis. In breast cancer, the occurrence of EMT is closely related to chemoresistance. Cancer cells that have undergone EMT become resistant to a variety of chemotherapeutic drugs, greatly reducing the effectiveness of chemotherapy, increasing the risk of recurrence, and lowering the survival rate of patients.

The molecular mechanisms involved in the EMT process are extremely complex, involving multiple growth factors, signaling pathways, and transcription factors. Growth factors such as transforming growth factor - β (TGF - β), epidermal growth factor (EGF), and Wnt can regulate the expression of EMT transcription factors such as Snail, Twist, and Zeb1/2 by activating signaling pathways like Smad2/3, PI3K/Akt, and ERK1/2, and further regulate hundreds of downstream EMT-related genes. This complexity leads to the co - existence of multiple EMT phenotypes at different stages of tumors, increasing the difficulty of treatment. Current treatment strategies targeting EMT mainly focus on blocking EMT stimuli, signal transduction, or mesenchymal features. However, these methods may promote the mesenchymal - epithelial transition (MET), which instead drives tumor progression. Therefore, it is urgent to find a treatment strategy that can effectively inhibit EMT while avoiding promoting MET.

Research Model: A Powerful Tool for Exploring EMT

To deeply study the EMT process, researchers constructed the Tri - PyMT EMT lineage - tracing model. This model combines MMTV - PyMT, Fsp1(S100a4)-Cre, and Rosa26 - mTmG transgenic mice, and traces EMT through a permanent RFP - to - GFP fluorescence switch induced by mesenchymal - specific Cre expression. Although this model has certain limitations in comprehensively tracing metastasis, it provides a unique perspective for studying the role of EMT in tumor progression and chemoresistance. Through this model, researchers found that, unlike cells that remain in the epithelial state (RFP+) in vivo, RFP + Tri PyMT cells actively transform into GFP + when cultured in vitro in a growth medium containing 10% FBS. During this process, researchers observed a group of cells that were double - positive for RFP and GFP (Doub+), accounting for approximately 2 - 5% of the total cells. Further studies showed that Doub+ cells represent tumor cells transitioning from an epithelial to a mesenchymal state. They express intermediate levels of both epithelial and mesenchymal markers, and have a higher proportion of cells in the S and G2/M phases of the cell cycle.

Ribosome Biogenesis: A Key Player in the EMT Process

Researchers used bulk RNA sequencing and single - cell RNA sequencing (scRNA - seq) technologies to conduct in - depth analyses of Tri - PyMT cells and found that the ribosome biogenesis (RiBi) pathway was significantly activated during the transition phase of EMT. In Doub+ cells, the KEGG_Ribosome pathway was significantly enriched. Compared with cells in the epithelial (Epi) or mesenchymal (Mes) phases, RiBi genes were significantly enriched in transition (Trans) cells. Further analysis revealed that RiBi activity showed an upward trend during the EMT transition phase, and this increase was transient. The RiBi activity was the lowest in cells with the latest EMT pseudotime.

Fig 1 Activation of ribosome biogenesis (RiBi) pathway in mesenchymal-to-epithelial transition (MET) during lung metastasis outgrowth.¹

Not only that, researchers also found that the RiBi pathway was equally activated during the MET process. By tracking the changes of GFP+/Epcam - Tri - PyMT cells in vivo, researchers found that during the outgrowth of lung metastases, tumor cells with epithelial phenotypes had elevated RiBi activity, and there was a significant positive correlation between RiBi gene upregulation and MET pseudotime. This indicates that the RiBi pathway plays an important role in both the EMT and MET processes.

Further exploration revealed that the activation of the ERK and mTOR signaling pathways is closely related to the upregulation of the RiBi pathway. Doub+ cells showed significantly higher levels of phosphorylated extracellular signal - regulated kinase (p - ERK) and phosphorylated mammalian target of rapamycin complex 1 (p - mTORC1) than RFP+ or GFP+ cells in response to serum stimulation. Meanwhile, the phosphorylation level of Rps6, an essential ribosome protein of the 40 S subunit, was also significantly increased. These results suggest that the elevation of the RiBi pathway in cells during the EMT transition phase is related to the abnormal activation of ERK and mTOR signals, and this activation may endow tumor cells with the ability to synthesize nascent proteins, thus helping them complete phenotypic changes.

Targeting the RiBi Pathway: A New Direction for Inhibiting EMT/MET

Since the RiBi pathway is so crucial in the EMT/MET process, does inhibiting the RiBi pathway affect the EMT/MET ability of tumor cells? Researchers verified this. RNA polymerase I (Pol I) is a key enzyme that mediates the transcription of ribosomal RNA (rRNA). Small molecules BMH21 and CX5461 are specific Pol I inhibitors that can inhibit rRNA transcription and disrupt ribosome assembly. The study found that after treating RFP+/Epcam + cells with BMH21 or CX5461, the fluorescence switch of the cells was inhibited. Most cells remained in the RFP+ state, and the expression of mesenchymal markers was significantly blocked, while the epithelial marker was retained. During the MET process, BMH21 also significantly prevented the re - acquisition of Epcam by GFP+/Epcam - Tri - PyMT cells, indicating impaired MET ability.

In addition to drug inhibition, researchers also verified the importance of RiBi activity by genetically regulating ribosomal proteins. Using Lenti - shRNAs to target two key genes of the 40S subunit, Rps24 and Rps28, after effectively knocking down these two genes, the number of nucleoli in cells decreased, RiBi activity declined, and both the EMT (RFP to GFP switch) and MET (re - acquisition of Epcam) processes were inhibited. These results fully demonstrate that elevated RiBi activity is crucial for tumor cells to maintain their ability to switch between epithelial and mesenchymal states.

Combination Therapy: A New Strategy to Enhance Chemotherapy Efficacy

Researchers also evaluated the effect of combining the inhibition of the RiBi pathway with chemotherapeutic drugs. In vitro experiments showed that when unsorted Tri - PyMT cells were treated with BMH21, the accumulation of GFP + cells decreased, while treatment with the chemotherapeutic drug cyclophosphamide (CTX) led to an increase in GFP + cells, which was consistent with the previously discovered EMT - mediated resistance to CTX. Further studies found that when BMH21 was combined with CTX, the sensitivity of tumor cells to treatment was significantly enhanced, and at low concentrations (100 - 200 nM) of BMH21, it showed the best synergy with CTX. This synergistic effect was not limited to CTX. When BMH21 was combined with other commonly used chemotherapeutic drugs in breast cancer treatment (such as 5FU, cisplatin, doxorubicin, gemcitabine, and paclitaxel), synergistic trends were also observed.

Fig 2 RNA Pol I inhibitor synergizes with chemo drug in vitro.¹

In in vivo experiments, researchers established a competitive metastasis assay and an experimental lung metastasis model of human breast cancer cells (MDA - MB231 - LM2). The results showed that the combination treatment of BMH21 and CTX had the strongest inhibitory effect on the growth of metastatic tumors, significantly reducing the number and size of lung metastasis nodules, and had obvious inhibitory effects on both epithelial and mesenchymal tumor cells. Analysis of scRNA - seq data from human breast cancer patients found that the RiBi activity in tumor cells was related to the EMT state, and patients with high RiBi activity had a worse prognosis.

Conclusion: Promising Prospects and Future Steps

This study reveals the important role of the RiBi pathway in the EMT and MET processes of breast cancer, providing a new treatment strategy to overcome EMT-related chemoresistance in breast cancer. Targeting the RiBi pathway can not only inhibit the EMT/MET ability of tumor cells but also synergize with chemotherapeutic drugs to enhance the efficacy of chemotherapy, bringing new hope to patients with advanced breast cancer. However, the current research is still in the basic and animal experiment stage. In the future, further clinical trials are needed to verify the effectiveness and safety of this treatment strategy in human patients. It is expected that this achievement can be applied to clinical practice as soon as possible to benefit more breast cancer patients.

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Reference

1. Ban, Yi, et al. "Targeting ribosome biogenesis as a novel therapeutic approach to overcome EMT-related chemoresistance in breast cancer." Elife 12 (2024): RP89486. https://doi.org/10.7554/eLife.89486.3. Distributed under the Open Access license CC BY 4.0, without modification.