Overcoming radioresistance by targeting p21 in cancer

Radiation therapy is one of the most effective cytotoxic therapies to treat localized solid cancers, with external beam radiation and brachytherapy being the most used approach in clinical practice. Proton and charged particle therapy represents a promising advancement in the field, allowing for enhanced healthy tissue sparing and improved depth-dose distribution. Unfortunately, resistance to radiotherapy remains a major challenge in the eradication of human malignant tumors. Hence, understanding the molecular mechanisms at the base of radioresistance is crucial to improve effectiveness of therapy. Radiation induced tumor regression is driven by DNA damages production and activation of downstream cellular responses mainly regulated by p53. The cyclin-dependent kinase inhibitor p21 (CDKN1A), also known as p21WAF1/Cip1, is the main p53 transcriptional target and it is traditionally recognized as a tumor suppressor since it is involved in genomic stability preservation, cell cycle arrest or senescence induction, and DNA repair. Here we show that cytoplasmic p21 acts as an oncogene in colorectal cancer, uveal and cutaneous melanoma. Using HCT116 colorectal cancer cells and their counterpart knock-out for p21 – generated with CRISPR/Cas9 ribonucleofection approach – we demonstrated that p21 confers resistance to apoptosis promoting instead cell growth and senescence in 2D cells and 3D tumor spheroids, hence favoring radioresistance. Importantly, we observed a correlation between cytoplasmic accumulation of p21 after radiation treatment and diminished Caspases activation and apoptosis induction. Moreover, polysomes fractionation coupled with Next-Generation Sequencing analysis showed that loss of p21 induced substantial changes in the translatome, with reduced polysome-bound RNA, confirming altered gene expression post-radiation. In response to radiation, p21 recruits a multi-faced interaction network favoring survival after DNA-damage, cytoskeletal stability, vesicles trafficking and proteostasis and metabolic adaptation and rewiring. Finally, using the p21 inhibitor UC2288 or p21-targeting siRNAs treatment in combination with radiotherapy, we demonstrated that p21 inhibition also sensitizes other p53 wild type tumors to radiation. These findings support the hypothesis that targeting p21 could enhance the efficacy of radiotherapy, particularly in resistant cancers. This study provides valuable insights into the oncogenic role of p21 in tumor progression and radiotherapy response, proposing its potential as a biomarker and therapeutic target to overcome radioresistance in cancer.