Locally Advanced Rectal Cancer: POU2F3 as a prognostic biomarker and potential therapeutic target

Background: Rectal cancer (RC) represents about one-third of colorectal cancer (CRC), and most of them are Locally Advanced Rectal Cancer (LARC) at diagnosis. The gold standard approach for LARC patients is pre-operative chemoradiotherapy (pCRT) followed by surgery. However, response to pCRT varies considerably, with only 20% of patients achieving a pathological complete response (pCR). In responding patients, surgery might represent an over-treatment and a source of morbidity; on the other hand, non-responders are exposed to unnecessary toxicities and surgery delays. Nowadays, clinicopathological features have been proposed to predict pCRT response, however, their utility is currently limited due to low sensitivity and specificity. Therefore, novel predictors of response to pCRT are urgently needed. To date, several studies have been conducted for this purpose, but most of them are limited due to their single-omic approach, often insufficient to investigate the entire complexity of cancer. To fill this gap, the NanoInspired Biomedicine Laboratory of Padova has employed a multi-omic approach to identify a potential novel biomarker of pCRT response: a gene named POU2F3. Purposes of the study: • To evaluate the prognostic value of POU2F3 in LARC patients. • To determine in vitro whether POU2F3 is targeted by drugs used in clinical practice and whether they cause its up-regulation. • To evaluate in vitro if an enhancing activity exists between 5-Fluorouracil and a chemical compound that upregulates POU2F3. Materials and methods: 172 LARC patients’ data were obtained from The Cancer Genome Atlas (TCGA). HCT-15, HCT-116, and SW480 CRC cell lines were maintained in a humidified atmosphere in a Growth Medium, that was changed every three days. Cell lines were seeded in culture plates and after 24 h they were washed in PBS and incubated with nadolol and entinostat. Then, total RNA was extracted and concentration and purity were determined. cDNA was obtained through reverse transcription PCR and real-time PCR was performed for amplification and quantification. For cytotoxicity assay, cell lines were incubated with 5-FU, entinostat and a combination of both compounds. After 72 h resazurin dye was added to the wells, then cytotoxicity and IC50 were calculated through fluorescence assessment. Results: In the TCGA cohort, high POU2F3 expression significantly correlated with higher overall survival (OS) compared to low-expressing LARC patients. Therefore, we analysed three independent databases to identify chemical compounds targeting POU2F3 and its paralogs, POU2F1 and POU2F2. The histone deacetylase inhibitor entinostat and the b-blocker nadolol targeted POU2F3 and its paralogs respectively. We exposed CRC cell lines to both chemicals, and we studied POU2F3 expression: entinostat-treated and nadolol-treated cell lines showed a significant up-regulation and down-regulation respectively. To test the cytotoxicity effect of entinostat we chose 5-FU as a comparison, and we exposed CRC cell lines to both compounds at different concentrations. 5-FU demonstrated concentration-dependent cytotoxicity, whereas entinostat showed poor to no effect on cells. Then, we investigated the effects of a combined approach with both chemicals, and we observed significantly enhanced cytotoxicity. Conclusions: POU2F3 represents a potential prognostic biomarker for LARC patients and it is a promising therapeutic target. Dosing POU2F3 in clinical practice might identify patients that can truly benefit from pCRT, and spare others from unnecessary toxicity. The in vitro experiments suggested an empowering effect of entinostat on the classical 5-FU chemotherapy: this could lead to more efficient therapies and reduction of 5-FU dose, together with fewer side effects.