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Article Open Access

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

  • Authors:
    • Shoichiro Nakajo
    • Mamoru Uemura
    • Hiroshi Kusafuka
    • Mao Osaki
    • Chikako Kusunoki
    • Nobuo Takiguchi
    • Mitsunobu Takeda
    • Yuki Sekido
    • Tsuyoshi Hata
    • Atsushi Hamabe
    • Takayuki Ogino
    • Norikatsu Miyoshi
    • Mitsuyoshi Tei
    • Yoshinori Kagawa
    • Hirofumi Yamamoto
    • Yuichiro Doki
    • Hidetoshi Eguchi
  • View Affiliations / Copyright

    Affiliations: Department of Gastroenterological Surgery, Graduate School of Medicine, University of Osaka, Suita, Osaka 565‑0871, Japan, Department of Surgery, Osaka Rosai Hospital, Sakai, Osaka 591‑8025, Japan, Department of Gastroenterological Surgery, Osaka General Medical Center, Osaka, Osaka 558‑8558, Japan
    Copyright: © Nakajo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 109
    |
    Published online on: April 7, 2026
       https://doi.org/10.3892/or.2026.9114
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Abstract

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.
View Figures

Figure 1

Primary culture and hypoxic culture
of fibroblasts. (A) CAF and NF morphology. (B) CAF and NF observed
by immunofluorescence staining. (C) Proliferation assay of CAF and
NF under both normoxic and hypoxic conditions. The data are shown
as the mean ± SD of three independent experiments. *P<0.05,
Student's t-test. CAF, cancer-associated fibroblast; NF, normal
fibroblast.

Figure 2

Investigation by co-culture of CAF
supernatant and HT29. (A) Proliferation assay using HT29 and HT29
co-cultured with supernatant of CAF cultured in both normoxia and
hypoxia. (B) Cell migration assay using both HT29 and HT29
co-cultured with supernatant of CAF cultured in both normoxia and
hypoxia. (C) Oxaliplatin and 5-FU sensitivity assay using both HT29
and HT29 co-cultured with supernatant of CAF cultured in both
normoxia and hypoxia. The data are shown as the mean ± SD of three
independent experiments. *P<0.05, Student's t-test. CAF,
cancer-associated fibroblast; 5-FU, 5-Fluorouracil.

Figure 3

Single-cell RNA sequencing of human
CAF and generation of data matrix. (A) CAF divided into 15
clusters. (B) CAF gene expression of α-SMA, HIF-1α and periostin.
(C) Selected clusters with high expression of α-SMA, HIF-1α and
periostin. (D) Volcano plots created from DEGs of cluster 2, 5, 6
and 7. (E) Gene list was generated from DEGs and KEGG pathway
analysis was performed. (F) Gene list was generated from DEGs and
GO analysis was performed. CAF, cancer-associated fibroblast;
α-SMA, α-smooth muscle actin; HIF-1α, hypoxia-inducible factor-1α;
DEG, differentially expressed gene; GO, Gene Ontology; KEGG, Kyoto
Encyclopedia of Genes and Genomes.

Figure 4

Selection of PTHrP. PTHrP effects on
NF and the influence of PTHrP and vitamin D on CAF. (A)
Immunofluorescence staining of NF with PTHrP. (B) Morphology of CAF
supplemented with PTHrP or vitamin D. (C) Proliferation assay using
both CAF and CAF supplemented with PTHrP or vitamin D. The data are
shown as the mean ± SD of three independent experiments.
*P<0.05, Student's t-test. PTHrP, parathyroid hormone-related
protein; NF, normal fibroblast; CAF, cancer-associated
fibroblast.

Figure 5

Examination of interrelationships
among PTHrP, vitamin D and RAS. (A and B) Representative image of
immunohistochemical staining of CRC tissue for HIF1-α, periostin,
PTHrP and VDR. (C) Correlation of copy numbers in CRC for PTHrP and
KRAS. (D) Correlation of copy numbers in various cancers for PTHrP
and KRAS. PTHrP, parathyroid hormone-related protein; RAS, rat
sarcoma oncogene; CRC, colorectal cancer; HIF-1α, hypoxia-inducible
factor-1α; VDR, vitamin D receptor; KRAS, Kirsten rat sarcoma viral
oncogene homolog.

Figure 6

Graphic abstract. Hypoxic CAFs
enhance tumor growth through PTHrP signaling and DBP secretion,
reducing local vitamin D levels in the peritumoral region. The
observed correlation between PTHrP expression in tumor cells and
RAS signaling suggests that the PTHrP-vitamin D-RAS axis may play a
key role in the tumor microenvironment. CAF, cancer-associated
fibroblast; PTHrP, parathyroid hormone-related protein; DBP,
vitamin D-binding protein; RAS, rat sarcoma oncogene.
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Copy and paste a formatted citation
Spandidos Publications style
Nakajo S, Uemura M, Kusafuka H, Osaki M, Kusunoki C, Takiguchi N, Takeda M, Sekido Y, Hata T, Hamabe A, Hamabe A, et al: Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF. Oncol Rep 55: 109, 2026.
APA
Nakajo, S., Uemura, M., Kusafuka, H., Osaki, M., Kusunoki, C., Takiguchi, N. ... Eguchi, H. (2026). Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF. Oncology Reports, 55, 109. https://doi.org/10.3892/or.2026.9114
MLA
Nakajo, S., Uemura, M., Kusafuka, H., Osaki, M., Kusunoki, C., Takiguchi, N., Takeda, M., Sekido, Y., Hata, T., Hamabe, A., Ogino, T., Miyoshi, N., Tei, M., Kagawa, Y., Yamamoto, H., Doki, Y., Eguchi, H."Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF". Oncology Reports 55.6 (2026): 109.
Chicago
Nakajo, S., Uemura, M., Kusafuka, H., Osaki, M., Kusunoki, C., Takiguchi, N., Takeda, M., Sekido, Y., Hata, T., Hamabe, A., Ogino, T., Miyoshi, N., Tei, M., Kagawa, Y., Yamamoto, H., Doki, Y., Eguchi, H."Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF". Oncology Reports 55, no. 6 (2026): 109. https://doi.org/10.3892/or.2026.9114
Copy and paste a formatted citation
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Spandidos Publications style
Nakajo S, Uemura M, Kusafuka H, Osaki M, Kusunoki C, Takiguchi N, Takeda M, Sekido Y, Hata T, Hamabe A, Hamabe A, et al: Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF. Oncol Rep 55: 109, 2026.
APA
Nakajo, S., Uemura, M., Kusafuka, H., Osaki, M., Kusunoki, C., Takiguchi, N. ... Eguchi, H. (2026). Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF. Oncology Reports, 55, 109. https://doi.org/10.3892/or.2026.9114
MLA
Nakajo, S., Uemura, M., Kusafuka, H., Osaki, M., Kusunoki, C., Takiguchi, N., Takeda, M., Sekido, Y., Hata, T., Hamabe, A., Ogino, T., Miyoshi, N., Tei, M., Kagawa, Y., Yamamoto, H., Doki, Y., Eguchi, H."Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF". Oncology Reports 55.6 (2026): 109.
Chicago
Nakajo, S., Uemura, M., Kusafuka, H., Osaki, M., Kusunoki, C., Takiguchi, N., Takeda, M., Sekido, Y., Hata, T., Hamabe, A., Ogino, T., Miyoshi, N., Tei, M., Kagawa, Y., Yamamoto, H., Doki, Y., Eguchi, H."Hypoxic CAF studies unveil PTHrP‑vitamin D‑RAS axis as pivotal in the CAF". Oncology Reports 55, no. 6 (2026): 109. https://doi.org/10.3892/or.2026.9114
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