Cancer Letters

Cancer Letters

Volume 515, 1 September 2021, Pages 86-95
Cancer Letters

PERK-eIF2α-ERK1/2 axis drives mesenchymal-endothelial transition of cancer-associated fibroblasts in pancreatic cancer

https://doi.org/10.1016/j.canlet.2021.05.021Get rights and content

Highlights

  • CAFs in PDAC can adopt an endothelial phenotype in vitro and in vivo.

  • UPR activation is essential for MEndoT induction in CAFs.

  • The PERK-eIF2α-ERK1/2 signaling branch regulates CAF MEndoT.

  • PERK inhibition suppresses tumor MEndoT and enhances the chemotherapeutic efficacy of gemcitabine.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by remarkable desmoplasia, usually driven by cancer-associated fibroblasts (CAFs), influencing patient prognosis. CAFs are a group of plastic cells responsible for tumor growth and metastasis. Fibroblasts have been reported to directly contribute to angiogenesis by undergoing mesenchymal-endothelial transition (MEndoT) after ischemic injury in the heart, brain, and hindlimbs. However, whether CAFs can undergo similar transdifferentiation in the hostile tumor microenvironment and directly contribute to tumor angiogenesis remains unclear. Herein, we provide evidence that CAFs can adopt an endothelial cell-like phenotype and directly contribute to tumor angiogenesis in vitro and in vivo. Furthermore, this program is regulated by the PERK-eIF2α-ERK1/2 axis. Pharmacological inhibition of PERK with GSK2606414 limited the phenotypic transition of CAFs. In conclusion, our results suggest that CAFs contribute to tumor angiogenesis by undergoing the MEndoT, thus representing therapeutic targets for improving PDAC prognosis.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers due to its devastating metastatic nature. It is characterized by remarkably dense and firm desmoplasia composed of cancer-associated fibroblasts (CAFs), extracellular matrix (ECM), leukocytes, and endothelial cells [1]. CAFs are the major contributors to desmoplasia, producing ECM and soluble factors that drive tumor progression [[1], [2], [3]].

Angiogenesis is indispensable for tumor growth and metastasis. Although PDAC is rather hypovascular, pro-angiogenic factors, such as VEGF and CXCL12, are overexpressed in PDAC. Further, blood vessel density is positively correlated with PDAC progression [4]. Previous studies have demonstrated that CAFs are involved in angiogenesis by releasing pro-angiogenic factors [5,6]. Genetic fate mapping revealed that fibroblasts in a murine myocardial infarction model were reprogrammed into endothelial cells, a process known as the mesenchymal-to-endothelial transition (MEndoT) [7,8]. Brumm et al. reported that astrocytes could also undergo MEndoT under serum deprivation in vitro [8]. Through lineage tracing and single-cell transcriptome sequencing, Meng et al. suggested that MEndoT contributes to peripheral circulation perfusion recovery in a mouse hindlimb ischemia model [9]. However, whether PDAC CAFs undergo a similar phenotype transition and directly contribute to tumor angiogenesis remains unclear.

Dense and firm desmoplasia creates a hypoxic, ischemic, and acidic tumor microenvironment (TME), compromising protein homeostasis and inducing endoplasmic reticulum (ER) stress within tumors [10]. Cell differentiation and transdifferentiation involve considerable protein synthesis, exerting enormous stress on the ER [11]. Consequently, cells initiate the unfolded protein response (UPR) in an attempt to maintain protein homeostasis. ER stress and UPR activation have been described in PDAC and contribute to tumorigenesis, angiogenesis, progression, cell transdifferentiation, metastasis, and treatment resistance [[9], [10], [11]]. However, the role of MEndoT in PDAC and its association with UPR remain unclear. Herein, we provide evidence that CAFs can act as a source of endothelial cells in PDAC by undergoing MEndoT regulated via PERK-eIF2α-ERK1/2 signaling.

Section snippets

Cell culture

With approval from the Ethics Committee, all human PDAC samples were provided by the pancreatic cancer department of Tianjin Medical University Cancer Institute & Hospital and collected from the donors with informed consent. The clinical features of patients whose tumors were used for CAFs isolation were provided in Table S1. We isolated human pancreatic CAFs from fresh PDAC surgery specimens using a culture outgrowth method [12,13].

In brief, fresh human PDAC surgical samples were cut into 1-mm3

CAFs transdifferentiate into the endothelial lineage in vitro

MEndoT induction was reported in fibroblasts of the heart, brain, and hindlimb after serum deprivation in vitro [[7], [8], [9]]. Thus, we seeded CAFs on Matrigel, subjecting them to different serum concentrations [15]. After 6–8 h of seeding, in contrast to 10% serum, low-concentration serum (≤2%) induced the formation of capillary-tube-like structures (Figs. S1D–F). CAFs exhibited pronounced tube formation, especially under 0% serum (Fig. 1A–C, S1D-F). We then subjected CAFs to serum

Discussion

Mounting evidence highlights CAFs as a heterogeneous and complex cell population [29]. In parallel to cancer progression, CAFs take on diverse functions that can support or suppress tumor growth [30]. Thus, a better understanding of their complex nature could help tailor therapy and improve patient prognosis [31]. The functional heterogeneity of CAFs may be ascribed to their diverse cellular sources [32]. Studies have shown that endothelial cells can alter their morphological and functional

Authors’ contributions

TianSuo Zhao, Wenrun Cai, and Xugang Sun designed, edited, and led out this study's experiments. Wenrun Cai and Xugang Sun performed most of the experiments. Fanjie jin performed some experiments, Di Xiao performed the statistical analysis. Hui Li,Huizhi Sun, Yifei Wang, and Yang Lu provided study material and technical support. Jihui Hao, Tiansuo Zhao, Chongbiao Huang, Xiuchao Wang, Song Gao, Hongwei Wang, and Chuntao Gao analyzed and discussed the data. All authors reviewed and approved the

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grants 81871968, 81525021, 81672431, 81672435, 81720108028, 81772633, 81702426, 81702427, 81572618, 81802432, 81802433 and 81871978), Key Program of Prevention and Treatment of Chronic Diseases of Tianjin (17ZXMFSY0010), the programs of Tianjin Prominent Talents, Tianjin Eminent Scholars and Tianjin Natural Science Fund for Distinguished Young Scholar.

References (38)

  • A.J. Brumm et al.

    Astrocytes can adopt endothelial cell fates in a p53-dependent manner

    Mol. Neurobiol.

    (2017)
  • S. Meng et al.

    Reservoir of fibroblasts promotes recovery from limb ischemia

    Circulation

    (2020)
  • E. Chevet et al.

    Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis

    Canc. Discov.

    (2015)
  • L. Tian et al.

    Primary cultures for pancreatic stellate cells (PSCs), methods in molecular biology

    Clifton, N.J.)

    (2019)
  • M. Waghray et al.

    GM-CSF mediates mesenchymal-epithelial cross-talk in pancreatic cancer

    Canc. Discov.

    (2016)
  • J.M. Axten et al.

    Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)

    J. Med. Chem.

    (2012)
  • I. Arnaoutova et al.

    In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract

    Nat. Protoc.

    (2010)
  • L. Kurian et al.

    Conversion of human fibroblasts to angioblast-like progenitor cells

    Nat. Methods

    (2013)
  • A.N. Hosein et al.

    Pancreatic cancer stroma: an update on therapeutic targeting strategies, Nature reviews

    Gastroenterol. Hepatol.

    (2020)
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