Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF

Cancer‑associated fibroblasts (CAFs) play critical roles in the tumor microenvironment (TME); however, their characteristics under hypoxic conditions remain incompletely understood. The aim of the present study was to investigate the properties of hypoxic CAFs and identify their regulatory factors in colorectal cancer (CRC). CAFs cultured under normoxic and hypoxic conditions were analyzed using proliferation assays, co‑culture experiments, shotgun proteomics and single‑cell RNA sequencing. Clinical specimens were evaluated immunohistochemically using the desmoplastic reaction (DR) classification. In addition, the generalizability of the findings was validated by correlation analyses using The Cancer Genome Atlas database. Hypoxic CAFs showed enhanced proliferative capacity, and their conditioned medium promoted migration and chemoresistance in CRC cells. Shotgun proteomics revealed a significant increase in vitamin D‑binding protein in the conditioned media of hypoxic CAFs, while single‑cell RNA sequencing showed enrichment of genes related to bone metabolism and the phosphoinositide 3‑kinase‑AKT signaling pathway. Treatment of normal fibroblasts (NFs) with parathyroid hormone‑related protein (PTHrP) induced CAF‑like phenotypes, whereas treatment of CAFs with vitamin D led to morphological changes toward a NF‑like appearance. In clinical samples, the immature DR subtype, associated with poor prognosis, exhibited increased expression of the hypoxia marker hypoxia‑inducible factor‑1α, periostin and PTHrP, along with a significant association with Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. Furthermore, a strong positive correlation was observed between the copy numbers of PTHrP and KRAS across multiple cancer types, including CRC. These findings suggest that the PTHrP‑vitamin D‑rat sarcoma oncogene (RAS) axis functions as an important regulatory mechanism in hypoxic CAFs. PTHrP and vitamin D may influence each other's activity, and this axis may contribute to tumor progression within the TME. Therefore, the PTHrP‑vitamin D‑RAS axis could serve as a potential therapeutic target.