Stromal transcriptomics uncover LIF as a key effector in high tumor budding triple-negative breast cancer

Abstract

The tumor budding (TB) and fibroblast activation protein (FAP)-positive cancer-associated fibroblasts (CAFs) are associated with patient prognosis in triple-negative breast cancer (TNBC). However, the impact between TB and FAP-positive CAFs on TNBC progression remains poorly understood. This study seeks to investigate the transcriptomic profile of FAP-positive CAFs in high TB TNBC to identify CAFs-derived factors contributing to disease aggressiveness. The 170 formalin-fixed paraffin-embedded TNBC tissues were assessed for TB by pan-cytokeratin immunohistochemistry (IHC). Its clinicopathological correlations were examined through univariate and multivariate analyses, with overall survival (OS) evaluated using Kaplan-Meier analysis. CAFs whole transcriptomic profiles of 13 TNBCs was conducted using Templated Oligo-Sequencing and the differential expression was analyzed in R studio with the DESeq2 package. Pathways associated with differentially expressed genes were identified using the Enrichr package. The level of leukemia inhibitory factor (LIF) was explored by IHC in 141 TNBCs, and its prognostic significance was determined. The induction of TNBC cell migration and expression of programmed death-ligand 1 (PD-L1) by a recombinant LIF, along with attenuation by EC359, a LIF inhibitor, were investigated using Transwell assay and flow cytometry. High TB was observed in 46.5% of TNBC patients and significantly associated with shorter OS, in which the upregulated MAPK cascade, ERK1/ERK2 signaling, and protein kinase B regulation signaling pathways were involved. In FAP-positive CAFs, pathways related to tyrosine phosphorylation, STAT protein tyrosine phosphorylation, and G-protein-coupled receptor regulation were also elevated. The overexpressed genes in FAP-positive CAFs from high TB-TNBCs included ZNF235, ANKRD30B, SLC26A2, SPDYC, CDKN1C, LIF, and FAM83A. The secreted LIF was found in 41.1% of TNBC cases with high levels detected in CAFs, while 26.2% of cases showed high LIF production in cancer cells. Recombinant LIF significantly promoted breast cancer cell migration and enhanced PD-L1 expression. These effects were attenuated by EC359. This study highlights the correlation of high TB in TNBC with poor prognosis and upregulation of key signaling pathways in CAFs. FAP-positive CAFs overexpress genes like LIF, which promotes cell migration and PD-L1 expression. LIF inhibition reduces these effects, suggesting LIF as a potential therapeutic target in TB-associated TNBC progression and immunotherapy.