Fig 1.
Metastatic SCNPC upregulates MET and RET.
Whole transcriptome RNA-Seq heatmaps of (A) UW rapid autopsy cohort (n = 98) and (B) SU2C cohort (n = 270) depicting AR-associated genes (AR, KLK3 and ACPP), NE-associated genes (SYP, CHGA, ASCL1 and LMO3), targets of cabozantinib (MET, RET, VEGFR2/KDR, AXL, TEK, KIT), and AR and NE activity scores. Results are expressed as log2 FPKM (transcripts) or as GSVA scores (activities) and are colored according to scale. Correlation analysis of AR activity scores (GSVA) and MET transcript expression (log2 FPKM) in (C) UW and (D) SU2C cohorts.
Fig 2.
The identification of MET and RET expressing SCNPC PDX models and responses to AMG-337 and cabozantinib in vitro.
(A) RNA-Seq heatmap of LuCaP PDX models and NCI-H660 cells showing AR-associated genes (AR, KLK3 and ACPP), NE-associated genes (SYP, CHGA, ASCL1 and LMO3), targets of cabozantinib (MET, RET, KDR, AXL, TEK, KIT). Results are expressed as log2 FPKM and are colored according to scale. (B) Cell viability assay of dissociated LuCaP PDX tumors and NCI-H660 cells. Cells were treated with vehicle control, AMG-337 (50 nM) or cabozantinib (2.5 μM) for 72h. Results are expressed as percent viable cells and are normalized to vehicle treated controls.
Fig 3.
Cabozantinib significantly decreases tumor volume and prolongs survival in SCNPC PDX models.
SCNPC PDX models were treated with 30 mg/kg of cabozantinib (LuCaP 93, n = 14; LuCaP 173.1 n = 14) or vehicle control (LuCaP 93, n = 14; LuCaP 173.1, n = 14) for up to 7.5 weeks. (A) LuCaP 93 and (B) LuCaP 173.1 normalized tumor volume measurements. LuCaP 93, p-value = 0.0175; LuCaP 173.1, p-value <0.0001. (C) LuCaP 93 and (D) LuCaP 173.1 overall survival curves using Kaplan-Meier analysis and the log-rank (Mantel-Cox) test. LuCaP 93, p-value < 0.0001; LuCaP 173.1, p-value < 0.0001. (E) LuCaP 93 and (F) LuCaP 173.1 normalized body weight measurements. LuCaP 93, p-value = 0.1677; LuCaP 173.1, p-value <0.0002. Black = control vs. red = cabozantinib.
Fig 4.
Cabozantinib treatment decreased microvessel density, but not proliferation and increased hypoxic stress in LuCaP 93 and LuCaP 173.1 patient-derived xenografts.
(A) Immunoblots of tumor lysates from LuCaP 93 and LuCaP 173.1 in vivo study tumors. Blots were probed with primary antibodies to MET, RET and ACTB. (B) Immunoblots of HUVEC cells and tumor lysates from LuCaP 93 and LuCaP 173.1 in vivo study tumors. HUVEC cell lysates were used as a positive control and blots were probed with primary antibodies to VEGFR2 and ACTB. (C) Graphs of Ki67 positive cells in control vehicle and cabozantinib-treated LuCaP 93 (p = 0.63, top graph) and 173.1 (p<0.001, bottom graph)* tumors. (D) Graphs of CD31 positive cells in control vehicle and cabozantinib-treated LuCaP 93 (p = 0.001; top graph) and LuCaP 173.1 (p = 0.01; bottom graph) tumors. (E) Graphs of Hexokinase II positive cells in control vehicle and cabozantinib-treated LuCaP 93 (p = 0.0184; top graph) and LuCaP 173.1 (p<0.001; bottom graph) tumors. Results are plotted as mean ± SD. *The cabozantinib-treated group in the LuCaP 173.1 study had only 3 tumors that could be assessed as the remaining tumors were too necrotic or too small to assess.
Fig 5.
RNA-Seq and pathway analysis of LuCaP 93 tumors from cabozantinib-treated animals compared to tumors from vehicle treated animals.
RNA was isolated for RNA-Seq from vehicle control (n = 3) and cabozantinib-treated (n = 3) LuCaP 93 tumor bearing animals. (A) Volcano plot representing up- and downregulated genes in response to cabozantinib treatment. Statistically significant changes in gene expression were based on limma and only included genes with p<0.05 and log2 Fold Change (FC)>1. Data points are colored according legend; NS = not significant. (B) Unsupervised clustering heatmap of significantly altered genes in response to cabozantinib treatment in LuCaP 93 PDX tumors. The top 20 upregulated genes are labeled. Data represents log2 FPKM values and are colored according to scale. (C) GSEA of significantly altered genes from (B) using the hallmark gene sets in MSigDB. Ctrl: vehicle control; Cabo: cabozantinib.