Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

Abstract

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.

Inhibition of proliferation and invasion in 2D and 3D models by 2-methoxyestradiol in human melanoma cells

“…These effects were further confirmed in the 3D artificial skin model with melanoma cells, as previously described (21, 22, 25). Untreated skin reconstructs were marked by melanoma invasion and/or migration through the basal membrane into the dermis, which is visible in both HE staining and fluorescence microscopy; invading melanoma cells nuclei are stained in blue by DAPI, below the basal membrane, and are shown in green by Laminin immunofluorescence.…”

“…Proteins were extracted and blotted (30 μg) as described by Faião-Flores and collaborators (2016) (21). After analysis, Western Blots were stripped once and reprobed for β-actin or GAPDH to show even protein loading.…”

FOXE1 supports the tumor promotion of Gli2 on papillary thyroid carcinoma by the Wnt/β‐catenin pathway

“…Transcription factors can promote and mediate gene expression, and tumor cell proliferation, apoptosis, and migration. As previously reported, Gli2 is a transcriptional activator of the Hh‐pathway target genes (Faião‐Flores et al, ; Lipinski, Gipp, Zhang, Doles, & Bushman, ) and plays important roles in carcinogenesis of several cancers (J. F. Li & Song, ; Narita et al, ). Overexpression of Gli2 in mice skin, driven by the Keratin 5 promoter, can produce BCCs (Grachtchouk et al, ).…”

Targeting insulin‐like growth factor 2 mRNA‐binding protein 1 (IGF2BP1) in metastatic melanoma to increase efficacy of BRAFV600E inhibitors

“…For example, IGF2BP family of proteins affects expression of multiple components of RAS‐RAF‐MAPK and PI3K‐AKT signaling pathways. Other well‐established IGF2BP1 targets such as CTNNB1, c‐myc, and GLI1 were shown to contribute to vemurafenib resistance. Overexpression of Gli1, the key transcription factor of Hedgehog (Hh) signaling pathway, is associated with the development of multiple tumor types.…”