Background Colorectal cancer (CRC) is among the deadliest cancers, wherein early dissemination of tumor cells, and consequently, metastasis formation, are the main causes of mortality and poor prognosis. Cofilin-1 (CFL-1) and its modulators, LIMK1/SSH1, play key roles in mediating the invasiveness by driving actin cytoskeleton reorganization in various cancer types. However, their clinical significance and prognostic value in CRC has not been fully explored. Here, we evaluated the clinical contribution of these actin regulators according to TNM and consensus molecular subtypes (CMSs) classification. Methods CFL-1, LIMK1 and SSH1 mRNA/protein levels were assessed by real-time PCR and immunohistochemical analyses using normal adjacent and tumor tissues obtained from a clinical cohort of CRC patients. The expression levels of these proteins were associated with clinicopathological features by using the chi square test. In addition, using RNA-Seq data of CRC patients from The Cancer Genome Atlas (TCGA) database, we determine how these actin regulators are expressed and distributed according to TNM and CMSs classification. Based on gene expression profiling, Kaplan–Meier survival analysis was used to evaluated overall survival. Results Bioinformatic analysis revealed that LIMK1 expression was upregulated in all tumor stages. Patients with high levels of LIMK1 demonstrated significantly lower overall survival rates and exhibited greater lymph node metastatic potential in a clinical cohort. In contrast, CFL-1 and SSH1 have expression downregulated in all tumor stages. However, immunohistochemical analyses showed that patients with high protein levels of CFL-1 and SSH1 exhibited greater lymph node metastatic potential and greater depth of local invasion. In addition, using the CMSs classification to evaluate different biological phenotypes of CRC, we observed that LIMK1 and SSH1 genes are upregulated in immune (CMS1) and mesenchymal (CMS4) subtypes. However, patients with high levels of LIMK1 also demonstrated significantly lower overall survival rates in canonical (CMS2), and metabolic (CMS3) subtypes. Conclusions We demonstrated that CFL-1 and its modulators, LIMK1/SSH1, are differentially expressed and associated with lymph node metastasis in CRC. Finally, this expression profile may be useful to predict patients with aggressive signatures, particularly, the immune and mesenchymal subtypes of CRC.

Radiotherapy (RT) is an effective non-surgical treatment used in colorectal cancer (CRC), which is one of the major causes of cancer mortality. In patients with advanced rectal cancer, this approach is used as neoadjuvant therapy to reduce local recurrence risk. However, RT has a small impact on metastasis formation, which remains an obstacle in clinical practice, being responsible for the high mortality rates. Also, local tumor recurrence after RT tends to be more aggressive, with shorter survival expectancy. This suggests that RT surviving cells develop resistance mechanisms that allow their survival and increase metastatic potential. Despite efforts in this field, the knowledge concerning the post-irradiation behavior and the radioresistant cancer cells phenotype is still limited. Therefore, it is important to elucidate the mechanisms that contribute to the acquisition of this resistant phenotype to improve therapeutic success. In this study, we investigated the characteristics of surviving cells that were submitted to a clinically relevant treatment protocol of hypofractionated irradiation. The cell lines were chosen due to the different levels of genomic instability and genetic mutations and were exposed to 25 Gy delivery in 5 daily fractions. Irradiation surviving cells (ISC) from both cell lines, SW-480 and HCT-116, gain resistance to re-irradiation, increasing their autophagic process and assuming a polyploid giant cancer cell phenotype. In addition, HCT-116 ISC became senescent, while SW-480 ISC show an increase in mesenchymal markers and a decrease in epithelial markers. Long-term evaluation of the ISCs confirmed senescence in HCT-116, while SW-480 exhibited a transient dormancy state, resuming its growth after 14 days. SW-480 ISC also generates a heterogeneous progeny with three distinct phenotypes: rounded and highly proliferative cells, elongated mesenchymal cells, and polyploid giant cancer cells. Furthermore, both ISC enhance the secretion of extracellular vesicles and cytokines (like IL-8, MCP-1, VEGF, and GM-CSF) in a cell-specific manner. Based on these results and the identification of proteins and pathways differentially regulated in ISCs we can contribute to the identification of therapeutic targets for neoadjuvant therapies, in order to improve the prognosis of CRC patients. Citation Format: Josiane Weber Tessmann, Murilo Ramos Rocha, Renata Ivo Vasconcelos, Danielle Lazarin-Bidoia, Raphael Rodrigues Corrêa, Rafael Soares Lindoso, Paulo Emílio Corrêa Leite, Celso Vataru Nakamura, Jose Andres Morgado-Diaz. Hypofractionated radiation therapy promotes distinct resistant cellular fates as a survival mechanism in two colorectal cell lines: senescence or transient dormancy followed by heterogeneous repopulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 211.