Inhibition of TGF-β signaling, invasion, and growth of cutaneous squamous cell carcinoma by PLX8394

Abstract

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. The prognosis of patients with metastatic cSCC is poor emphasizing the need for new therapies. We have previously reported that the activation of Ras/MEK/ERK1/2 and transforming growth factor β (TGF-β)/Smad2 signaling in transformed keratinocytes and cSCC cells leads to increased accumulation of laminin-332 and accelerated invasion. Here, we show that the next-generation B-Raf inhibitor PLX8394 blocks TGF-β signaling in ras-transformed metastatic epidermal keratinocytes (RT3 cells) harboring wild-type B-Raf and hyperactive Ras. PLX8394 decreased phosphorylation of TGF-β receptor II and Smad2, as well as p38 activity, MMP-1 and MMP-13 synthesis, and laminin-332 accumulation. PLX8394 significantly inhibited the growth of human cSCC tumors and in vivo collagen degradation in xenograft model. In conclusion, our data indicate that PLX8394 inhibits several serine-threonine kinases in malignantly transformed human keratinocytes and cSCC cells and inhibits cSCC invasion and tumor growth in vitro and in vivo. We identify PLX8394 as a potential therapeutic compound for advanced human cSCC.

Fig. 1. PLX8394 decreases laminin-332 synthesis and inhibits Smad2 phosphorylation both in 3D spheroids and in 2D monolayer cultures in RT3 cells.

See also Supplementary Fig. S1. A HaCaT and RT3 cells were treated with a panel of inhibitors for 24 h in 2D monolayer conditions, followed by 3D spheroid formation. The inhibitors (LY294002 (PI3K inhibitor); PD98059, trametinib (MEK inhibitors); dabrafenib, PLX4720, PLX8394 (BRAF inhibitors) were added daily for 3 days. Laminin-332, phosphorylated ERK1/2 (p-ERK1/2), and total ERK1/2 (tot-ERK1/2) levels were analyzed by western blotting. β-actin was used as a loading control. Densitometric quantitation of laminin-332 and p-ERK1/2 levels corrected for β-actin or tot-ERK1/2 is shown below the blots. Values are relative to the levels (1.0) of DMSO control in HaCaT or RT3 cells. B RT3 cells were treated with dabrafenib (50 nM), PLX4720 (10 μM), or PLX8394 (10 μM) for 24 h in 2D monolayer conditions, followed by 3D spheroid formation with skin primary fibroblasts. The spheroids were grown for 24 h or 48 h before harvesting for western blotting. The levels of laminin-332, p-ERK1/2, tot-ERK1/2, p-Smad2, and tot-Smad2 were analyzed by western blotting. β-actin was used as a loading control. Densitometric quantitation of laminin-332, p-ERK1/2, and p-Smad2 levels corrected for β-actin, tot-ERK1/2, or tot-Smad2 is shown below the blots. Values are relative to the levels (1.0) of DMSO control in 24 h or 48 h samples. C A panel of primary (UT-SCC-12A, UT-SCC-91A, UT-SCC-105, UT-SCC-118) and metastatic (UT-SCC-7, UT-SCC-59A, UT-SCC-115) cSCC cell lines were treated with 10 μM PLX8394 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were grown 3 days. The levels of laminin-332, p-Smad2, and p-ERK1/2 were analyzed by western blotting. β-actin was used as a loading control. Densitometric quantitation of laminin-332, p-Smad2, and p-ERK1/2 levels corrected for β-actin, tot-Smad2, or tot-ERK1/2 is shown below the blots. Values are relative to the levels (1.0) of control samples (without PLX8394) of each cell line. RT3 cells were treated with increasing concentrations of PLX8394 for 4 h (D) or 18 h (E). The cells were then subjected to TGF-β (10 ng/ml, 30 min) and harvested for western blotting. p-Smad2 and tot-Smad2 levels were analyzed by western blotting. Densitometric quantitation of p-Smad2 levels corrected for tot-Smad2 is shown below the blots. Values are relative to the level (1.0) of DMSO plus TGF-β treated samples.

Fig. 2. PLX8394 inhibits TGF-β signaling by affecting TGF-β type II receptor kinase activity.

See also Supplementary Fig. S2. A RT3 cells were treated with increasing concentrations of PLX8394 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were grown for 3 days. The levels of laminin-332, p-TGFβRII, p-Smad2, tot-Smad2, and p-ERK1/2 were analyzed by western blotting. β-actin was used as a loading control. Densitometric quantitation of laminin-332, p-TGFβRII, p-Smad2, and p-ERK1/2 levels corrected for β-actin, tot-Smad2 or tot-ERK1/2 is shown below the blots. Values are relative to the levels (1.0) of DMSO treated control samples. B RT3 cells were treated with 10 μM PLX8394 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were grown for one to 5 days. The levels of laminin-332, p-Smad2, tot-Smad2 and p-TGFβRII were analyzed by western blotting. The graphs show relative protein expression to loading control from three independent biological replicates ±S.E.M. **p < 0.01, *<0.05; paired t-test. C RT3 cells were either left uninfected or infected (100 MOI) with control virus RAd66 or with adenovirus coding for constitutively active ALK5 (RAdCA-ALK5) for 48 h. The cells were then treated with 10 μM PLX8394 o/n. Phosphorylated Smad2 and tot-Smad2 levels were analyzed by western blotting. Box plots show data from four independent biological replicates (red dots), the second and third quartiles (the box), the median (blue line) and the mean (star) from all experiments ± S.D. *p < 0.05 (two-way ANOVA followed by Tukey post hoc test). D RT3 cells were treated with 10 µM PLX8394 and with TGF-β (10 ng/ml) in 2D condition for 1–4 days. The levels of Smad7, p-Smad2, and tot-Smad2 were analyzed by western blotting. β-actin was used as a loading control. Representative images from three independent biological replicates are shown. The graphs show relative Smad7 and p-Smad2 expression to loading control (β-actin and tot-Smad2, respectively) ± S.E.M.

Fig. 3. p38 MAPK regulates laminin-332 expression in RT3 cells.

See also Supplementary Fig. S3. A RT3 cells were treated in 2D condition with p38 signaling inhibitors SB203580 (10 μM) or BIRB796 (10 μM) for 24 h, followed by 3D spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days before harvesting for western blotting. Laminin-332 level was analyzed by western blotting and β-actin was used as a loading control. Representative images from three independent biological replicates are shown. B RT3 cells were treated with 10 μM PLX8394 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were grown for 3 days. The level of phosphorylated p38 (p-p38) was analyzed by western blotting and β-actin was used as a loading control. Representative images from three independent biological replicates are shown. C RT3 cells were treated with 10 μM PLX8394 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were grown for 1–5 days. The level of p-CREB was analyzed by western blotting. The graph shows relative p-CREB expression to β-actin from three independent biological replicates ± S.E.M. *<0.05; paired t-test. D RT3 cells were treated in 2D condition with 10 μM PLX8394 for 24 h, followed by treatments with p38 signaling activators TNF-α (10 ng/ml, 30 min), IL-1β (10 ng/ml, 30 min) and sorbitol (400 mM, 2 h). The level of p-CREB (arrow) was analyzed by western blotting and β-actin was used as a loading control. Densitometric quantitation of p-CREB levels corrected for β-actin is shown below the blots. Values are relative to the levels (1.0) of control samples (without PLX8394) of each activator.

Fig. 4. PLX8394 inhibits RT3 cell invasion through collagen I.

See also Supplementary Fig. S4. A RT3 cells were treated with 10 μM PLX8394 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days, after which they were transferred to a 96-well plate and embedded with a collagen I gel. The invasion was followed by a confocal microscope every 24 h during 5 days. RT3 cells, red. Scale bar, 500 μm. From each time point, 2–4 spheroids were imaged and analyzed. Three independent biological replicates were performed. B RT3 cell invasion from cocultured spheroids that were treated as in (A). Left, a representative graph from three biological replicates. Right, analysis of RT3 cell invasion from cocultured spheroids. The graph shows the difference in RT3 cell invasion between DMSO treated control samples and PLX8394 treated samples. The graph shows mean from three independent biological replicates (squares) ± SD. (Each replicate contained 3-4 spheroids). The p values are from paired t-test. C RT3 cells were treated with 10 μM SB203580 for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days, after which they were transferred to a 96-well plate and embedded with a collagen I gel. The invasion was followed by a confocal microscope every 24 h during 5 days. Left, a representative graph from three biological replicates. Right, analysis of RT3 cell invasion from cocultured spheroids. The graph shows difference in RT3 cell invasion between DMSO treated control samples and SB203580 treated samples. The graph shows mean from three independent biological replicates (squares) ± SD. (Each replicate contained 3-4 spheroids). The p value is from paired t-test. RT3 cells and cSCC cells were treated in 2D condition with PLX8394 (10 μM) for 24 h, followed by 3D spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days before harvesting for western blotting. MMP-1 (D and E) and MMP-13 (F) levels were analyzed by western blotting and β-actin was used as a loading control. Representative images from three independent biological replicates are shown. G RT3 cells were treated with 1 μM MMP Inhibitor III for 24 h in 2D condition, followed by spheroid formation with skin primary fibroblasts. The spheroids were allowed to grow for 3 days, after which they were transferred to a 96-well plate and embedded with a collagen I gel. The invasion was followed by a confocal microscope every 24 h during 5 days. Left, a representative graph from three biological replicates. Right, analysis of RT3 cell invasion from cocultured spheroids. The graph shows difference in RT3 cell invasion between DMSO treated control samples and MMP inhibitor treated samples. The graph shows mean from three independent biological replicates (squares) ± SD. (Each replicate contained 5–8 spheroids). The p values are from paired t-test.

Fig. 5. PLX8394 inhibits the growth of human cSCC xenografts and prevents the degradation of stromal collagen in cSCC tumors.

See also Supplementary Fig. S5. A UT-SCC-7 cells were either monocultured as 3D spheroids for 24 h, followed by TGF-β treatment (5 ng/ml) for additional 48 h, or treated with 10 μM PLX8394 for 24 h in 2D condition, followed by spheroid formation with primary human skin fibroblasts and the spheroids were allowed to grow for 3 days. Fibroblasts were monocultured as 3D spheroids for 3 days and harvested for western blotting. The levels of laminin-332, p-Smad2, and p-TGFβRII (arrow) were analyzed by western blotting. β-actin was used as a loading control. Representative images from three independent biological replicates are shown. B UT-SCC-7 cells (5 × 10⁶) were implanted subcutaneously into the back of SCID/SCID mice, and the mice were then fed by oral gavage with PLX8394 (150 mg/kg; n = 8 mice) and the control mice were fed with vehicle (n = 9 mice) once daily for 18 days. The tumor sizes were normalized to their initial sizes and log2-transformed. The p values are from Student’s t-test. C Control (n = 9) and PLX8394 (n = 8) xenograft tumors were stained with haematoxylin and eosin (HE). Histological analysis of the xenograft tumors revealed different growth pattern and viability of cSCC cells in PLX8394 xenograft tumors (right panel) compared to control tumors (left panel). D The cystic, necrotic and keratotic areas were determined using QuPath digital image analysis software version v0.2.3 and compared to tumor size. In PLX8394 tumors, there were more fibrinotic and keratotic areas, and cystic cavities were larger compared to control tumors. Arrows indicate fibrinotic, keratotic, and cystic areas. Scale bars, 200 μm. *p < 0.05. (Mann-Whitney U test). E Expression of laminin-332 γ2 chain was examined with immunohistochemistry in human cSCC xenograft tumors of untreated control (n = 9, left panel) and PLX8394 treated (n = 8, right panel) SCID mice. Scale bars, 100 µm. F, G Tissue sections of human cSCC tumor xenograft grown in mouse were dissected with laser capture microdissection (LCM). The tissue samples were decellularized with sonication followed by reduction and alkylation of proteins. The resultant proteins were digested using an s-trap column and the peptides were analyzed by liquid chromatography tandem mass spectrometry. The identified proteins were filtered for matrisome proteins and classified based on tumor/human origin and stromal/mouse origin. F Number of ECM proteins identified based on tissue source in the tumor microenvironment of cSCC xenograft. G Number of peptides of laminin-332 identified as tumor/human or stromal/mouse origin. H The area of strong staining intensity of laminin-332 γ2 chain was determined digitally using QuPath bioimage analysis software version v0.2.3 and compared to total tumor area excluding large necrotic and keratotic areas. I Control and PLX8394 tumors were stained with van Gieson to visualize total collagen and with collagen hybridizing peptide (CHP) to visualize degraded triple-helical collagen. The association of van Gieson and CHP staining was analyzed in adjacent sections of the tumors (black arrows). In control tumors, van Gieson and CHP co-staining was detected in wide areas of tumor margin indicating collagen degradation (left panels). In PLX8394 tumors there were less co-stained areas indicating reduced amount of degraded collagen (right panels). J The co-staining of total collagen (van Gieson) and degraded collagen (CHP) was scored negative (−) if the co-staining was detected in limited areas and positive (+) if both stainings were detected in wide areas of tumor margins. *p < 0.05 by Fisher’s exact test.

Fig. 6. A schematic representation of serine-threonine kinase inhibition by PLX8394.

PLX8394 inhibits TGF-β signaling by attenuating phosphorylation of TGFβRII and Smad2. Concurrently, PLX8394 inhibits TGF-β-induced Smad2 and p38 activation resulting in decreased MMP-1 and MMP-13 production. PLX8394 does not inhibit p38 activation induced by inflammatory cytokines or environmental stress. As a result, laminin-332 expression is downregulated leading to decreased cSCC cell invasion and tumor growth. The figure was created with BioRender.com.