Brignola, Chiara (2024) Combined treatment with β-carotene and 5-Fluorouracil to overcome uL3-mediated chemoresistance in colorectal cancer. [Tesi di dottorato]

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Abstract

Despite recent therapeutic advancements, the development of multidrug resistance (MDR) and metastasis remains a serious concern for the efficacy of chemotherapy against colorectal cancer (CRC). The identification of novel relevant biomarkers as well as therapeutic targets is a key issue for CRC management. Our previous data demonstrated that ribosomal protein uL3 is a stress sensing molecule essential for cellular response to certain chemotherapeutics in CRC cells lacking p53. Specifically, uL3 downregulation is associated with drug resistance, alteration in cell motility, epithelial-mesenchymal transition and autophagy. Plant-derived nutraceuticals have shown the ability to improve cancer cell response to cytotoxic drugs providing a safer choice to defeat MDR. Here, we report the transcriptomic analysis of a large cohort of CRC patients revealing that lower uL3 levels positively correlated with shorter progression-free survival as well as worse response to treatment, suggesting uL3 as a novel prognostic molecular biomarker for CRC. uL3 low transcriptional state was found to be associated with increased expression of specific ATP-binding cassette (ABC) transporters, involved in the export of a wide range of chemotherapeutics outside the cells. Moreover, metabolomic and transcriptomic studies demonstrated that uL3 downregulation triggered redox metabolic reprogramming and dysregulated the gene expression of several markers of ferroptosis, a form of programmed cell death involving iron-dependent accumulation of lipid reactive oxygen species. We specifically identified ribosomal protein uL3 as a negative regulator of Solute Carrier Family 7 Member 11 (SLC7A11), which plays a major role in regulating cell fate via its key function in the uptake of cystine for the synthesis of glutathione. Of note, a poor outcome was linked to uL3 downregulation concurrent with high expression of SLC7A11 in CRC patients. By using a model of multidrug-resistant CRC cells devoid of p53 and stably silenced of uL3, we investigated the combination of erastin (a pharmacological inhibitor of SLC7A11) plus uL3 to trigger ferroptosis. The effects of the combined treatment on tumor growth and invasiveness were analysed in uL3-silenced CRC cell-derived chorioallantoic membrane (CAM) xenografts, which showed a high propensity to metastasize to the liver and lungs of chick embryos. The discovery of a targetable vulnerability of multidrug-resistant uL3-silenced CRC cells generated by high SLC7A11 levels will prompt us to investigate the activity of natural compounds specifically targeting this transporter in order to induce ferroptosis in both in vitro and CAM-based in vivo models of p53-null CRCs. Among the plant-derived nutraceuticals that have been studied as reversal agents of MDR, β-carotene has emerged as an adjuvant of conventional chemotherapeutics affecting the expression of ABC transporters. Hence, we investigated the effect of a combined treatment based on β-carotene plus 5-FU, the gold-standard of CRC chemotherapy. Our results indicated that this treatment may overcome 5-FU chemoresistance by significantly reducing the expression levels of analysed ABC transporters and consequently inducing a G2/M cell cycle arrest associated with the induction of apoptosis in 2D and 3D cell culture models. In conclusion, this PhD thesis shed light on novel molecular signatures associated with uL3-based chemoresistance that can be relevant in identifying vulnerability to specific programmed cell deaths triggered by natural products, revealing a potential role of these compounds as adjuvants of conventional chemotherapeutics for the design of individualized therapy for CRCs expressing low levels of p53 and uL3.

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