Suppression of MAPK signaling in BRAF‐activated PTEN‐deficient melanoma by blocking β‐catenin signaling in cancer‐associated fibroblasts

Abstract

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β-catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF-activated PTEN-deficient mouse melanoma was significantly suppressed in vivo after blocking β-catenin signaling in CAFs. Further analysis revealed that expression of phospho-Erk1/2 and phospho-Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial-mesenchymal transition (EMT)-like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF-MEK-ERK signaling cascade in BRAF-activated melanoma and may offer a new approach to abrogate host-dependent drug resistance in targeted therapy.

The β-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts

“…10 Further studies showed that targeted ablation of β-catenin in murine stromal fibroblasts had opposite biological effects on melanoma development depending on the timing of β-catenin ablation. 10,14 Despite these interesting results, the mechanisms by which β-catenin regulates the biological properties of human stromal fibroblasts and their interactions with melanoma cells and the ECM remain largely unknown. To address this question, we used inducible lentiviral shRNAs (Fig.…”

“…To understand whether β-catenin is related to YAP nuclear localization in melanoma-associated fibroblasts, we collected tumor tissues from mice bearing Braf V600E ; Pten lox5 - Fb or Braf V600E ; Pten lox5 -bcat/Fb melanoma 14 for double staining using an anti-YAP antibody and an anti-α-SMA antibody. A majority of α-SMA-positive fibroblasts in control Braf V600E ; Pten lox5 - Fb melanomas showed red fluorescent staining of YAP (Fig.…”

“…Mouse Braf V600E ; Pten lox5/lox5 melanoma sections were obtained, as previously reported. 14 Human melanoma cell lines, A375 [ BRAF(V600E) ; PTEN(mut) ] and SK-MEL-24 [ BRAF(V600E); PTEN(mut) ; CDKN2A ], were purchased from American Type Culture Collection (ATCC, Manassas, VA) and were maintained in Dulbecco's modified Eagle medium (DMEM) in a humidified incubator at 37 °C with 5% CO 2 . All culture media were supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) 10,000 U/ml penicillin and 10,000 U/ml streptomycin (PS) unless otherwise stated.…”

“…Western blotting was performed as previously described. 14 The following antibodies were used: anti-β-catenin (ThermoFisher, MA1–301, 1:2000), anti-F-actin (Abcam, ab205, 1:500), anti-paxillin (BD Biosciences, 610051, 1:2000), anti-vimentin (Abcam, ab92547, 1:1000), anti-fibronectin (Sigma-Aldrich, F3648, 1:2000), anti-YAP (Novus, NB110–58358, 1:200), anti-actin (1:2000, Abcam, ab3280) and anti-GAPDH (Invitrogen, AM4300, 1:1000).…”

“…10 Surprisingly, in an in vivo study using a melanoma-CAF mouse model combining Braf V600E ; Pten fl/fl mouse melanoma cells 11,12 with stromal fibroblasts of the genotype Ctnnb1 fl/fl ; Col1α2-CreER , 13 we found that the growth of the Braf V600E ; Pten fl/fl melanoma was significantly suppressed upon β-catenin ablation in stromal fibroblasts following tumor formation, and this occurred through the downregulation of Erk/Mapk signaling. 14 Despite the abundance of experimental evidence demonstrating the significance of β-catenin activity in CAFs, the molecular mechanisms underlying the functional association between β-catenin and the tumor-promoting and ECM remodeling abilities of CAFs have not been fully described.…”