Exploring the fibronectin/p21 (CDKN1A) functional axis in breast cancer as a marker of an aggressive disease subgroup.

Abstract

e12557 Background: Breast cancer is a highly heterogeneous disease concerning both cancer cells and the tumor microenvironment (TME). Fibronectin ( FN1 ) is a protein identified as an extracellular matrix (ECM) component in breast cancer TME and its expression correlates with the epithelial to mesenchymal transition (EMT) and the triple-negative phenotype. Cyclin-dependent kinase inhibitor 1A ( CDKN1A/p21 ) is a multifaced protein with roles in cell cycle regulation, DNA damage response, and cellular senescence induction, that can function either as an oncogene or as a tumor suppressor. To study a possible interaction pathway between cancer cells and the TME we assessed p21 expression in tumor cells and FN1, in conjunction to clinicopathological parameters. Methods: We employed Immunohistochemistry (IHC) for p21 (CDKN1A) and Fibronectin (FN1) protein expression on FFPE breast cancer samples and the Breast Invasive Carcinoma, TCGA, Firehose legacy data set through cBioPortal for analysis of patient clinicopathological parameters. Correlations were tested through the SPSS package and gene set enrichment analysis was done through g.Profiler online tool. Results: Here, by p21 and Fibronectin IHC in breast cancer FFPE tissue samples, we identified a statistically significant positive correlation (p=0.02) between p21 nuclear expression and a complex Fibronectin expression pattern in both cancer cells and TME. Next, we confirmed a positive correlation of p21 and FN1 through analysis of the Breast Invasive Carcinoma, TCGA data set for both proteins, in both ER-positive (p=1.41e -24 ) and ER-negative populations (p=3.73e -7 ). In addition, we identified in the same data set a patient subgroup overexpressing simultaneously both proteins (p21 high, FN1 high) with statistically significant worse overall survival (OS) (p=0.0193, HR 0.479). According to gene set enrichment analysis of the p21/FN1 double positive subgroup of patients we identified a gene signatures that encompassed gene ontology terms related to system development (p=1.755x10 -65 ), extracellular matrix organization (p=1.315x10 -59 ), cell adhesion (p=4.012x10 -50 ) and cell migration (p=7.471x10 -34 ). Finally, in order to test a possible link between DNA damage-induced p21 expression and the simultaneous upregulation of fibronectin, we subjected breast cancer cell lines to Etoposide treatment in vitro and tested through immunofluorescence for protein expression. Concurrent upregulation of p21 and fibronectin in vitro by cancer cells, further supported the notion of a complex interplay between tumor cells and TME in breast cancer. Conclusions: Collectively, our preliminary results indicate that ECM fibronectin deposition and cancer cell fibronectin expression in relation to p21 in breast cancer is a complex phenomenon, regulated by diverse molecular pathways, with a possible impact on disease outcomes.