Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy

Abstract

Tumor vessels are characterized by abnormal morphology and hyperpermeability that together cause inefficient delivery of chemotherapeutic agents. Although vascular endothelial growth factor has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here we show that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TECs exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards extracellular matrix stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 knockout mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anticancer drug cisplatin, significantly reduced tumor growth in wild-type mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

Fig.1. TRPV4 channel expression and function in normal and tumor endothelial cells

A) Western blot analysis of TRPV4 expression in normal (NEC) and tumor-derived endothelial cells (TEC). B) Quantitative analysis of the Western blots showing significant (p≦0.05) reduction in TRPV4 expression in TEC. C) Representative traces showing relative changes in cytosolic calcium in response to a selective TRPV4 agonist, GSK1016790A (100 nM) in Fluo-4 loaded normal and tumor endothelial cells (n=300). Arrow denotes the time when the cells were stimulated with the TRPV4 agonist D) Quantitative analysis of cytosolic calcium influx induced by GSK1016790A in NEC and TEC. (F/F0 = ratio of normalized Fluo-4 fluorescence intensity relative to time 0). The results shown are mean ± SEM from 3 independent experiments. The significance was set at p≦ 0.05.

Fig.2. Vessel malformations and tumor growth are enhanced in TRPV4 knockout mice

A) Time-dependent growth of the tumors in WT and TRPV4KO mice. Mouse Lewis lung carcinoma (LLC) cells (2 × 10⁶) were subcutaneously injected in to wild type C57BL/6 mice (WT) or TRPV4 knockout mice in C57BL/6 background (TRPV4KO) and tumor growth was measured using calipers at indicated days. The data shown are ± SEM of three independent experiments (n=8-10 mice for each group). B) Immunohistochemical analysis showing increased vessel diameter (feret) in tumors (21 days) from TRPV4 knockout mice (TRPV4KO) compared to wild type mice (WT). C) Quantitative analysis of microvessel diameter in tumors from WT and TRPV4KO mice. D) Frozen sections of tumors (10 μm thickness) were stained with CD31 (green) and α-SMA (red) to measure pericyte coverage (matured vessels). E) Quantitative analysis of pericyte covered microvessels in tumors from WT and TRPV4KO mice. The results shown are mean ± SEM from 3 independent experiments. The significance was set at p≦ 0.05.

Fig.3. TRPV4 overexpression restores mechanosensitivity towards ECM stiffness, and reduces migration in TEC

A) Representative images showing normal (NEC) and tumor (TEC) endothelial cell spreading (indicative of mechanosensitivity) on intermediate (370 Pa) and high (2280 Pa) stiffness ECM gels. Cells were transfected with either EGFP alone or TRPV4-EGFP B) Quantitative analysis of projected cell areas of NEC and TEC (expressing EGFP alone) and TEC+TRPV4 (expressing TRPV4-EGFP) on ECM gels of intermediate and high stiffness (370 and 2280 Pa). The results shown are mean ± SEM from 3 independent experiments. The significance was set at p≦ 0.05. NS= non-significant. C) Time lapse phase contrast micrographs showing migration of TEC-EGFP (TEC) and TEC+TRPV4-EGFP (TEC+TRPV4) cells plated on the surface of ECM gels of 370 Pa stiffness. Cells were allowed to spread for 4 h at 37°C and later shifted on to a microscope stage and random cell migration was recorded every 10 min using time lapse microscopy. Dashed line denotes the border of the leading edge at time 0. D) Quantification of cell migration as measured by marking the centroid of migrating cells overtime. Note: NEC migrated with a speed of 10 μm/h (Supplementary Fig.S5) suggesting that overexpression of TRPV4 normalized TEC migration. The results shown are mean ± SEM from 3 independent experiments. The significance was set at p≦ 0.05.

Fig.4. TRPV4 overexpression normalizes abnormal angiogenesis by tumor EC through the inhibition of abnormal Rho activity

A) Phase contrast micrographs showing the normalizing effects of TRPV4 overexpression on TEC angiogenic behavior when plated on 2D Matrigels (at high densities; 8 × 10⁴ well that cause collapse of tubular networks) and 3D Matrigels. NEC-EGFP (NEC), TEC-EGFP (TEC) and TEC-TRPV4-EGFP (TEC+TRPV4) cells were plated and cultured on the surface of Matrigel for 18 h (2D) or mixed in Matrigel and cultured for 14 days (3D). Note that the overexpression of TRPV4 in TEC cells restored tube formation on 2D Matrigel and normalized abnormal tubes in 3D Matrigel. Scale bar= 10 μm. B) Phase contrast micrographs showing the normalizing effects of pharmacological activation of TRPV4 with GSK1016790A (100 nM) on TEC angiogenic behavior when plated on 2D (at high densities; 8 × 10⁴/ well that cause collapse of tubular networks). Scale bar= 10 μm. C) Representative Western blot showing TRPV4 expression in TEC untreated or treated with GSK1016790A (100 nM) for 24 h. Densitometry analysis of relative changes in TRPV4 expression measured by normalizing the levels of TRPV4 with that of tubulin. D) Representative Western blot showing the levels of active-Rho and total Rho for TEC and TEC+TRPV4 cells. Rho activity was analyzed in TEC (EGFP) and TEC+TRPV4 cells cultured under regular growth conditions using the Rhotekin-RBD binding assay. Densitometry analysis of relative changes in Rho activity. Rho activity levels were measured by normalizing the levels of active Rho with that of total Rho.

Fig.5. TRPV4 activation with a small molecule activator together with Cisplatin reduces tumor growth in WT mice

A) Syngeneic tumors (LLC) were injected in the back of WT (C57BL/6) mice and tumor growth was monitored for 21 days. TRPV4 activator, GSK1016790A (GSK) was injected i.p. everyday starting from day 7 (after palpable tumors were observed) until 21 days. Cisplatin was injected i.p. (once/week) 3 days after the injection of GSK1016790A. Frozen sections of tumors (10 μm thickness; from 21 day) were stained with CD31 (green) and α-SMA (red) to measure pericyte coverage (matured vessels). B) Quantitative analysis of pericyte covered microvessels in tumors from control, GSK, Cisplatin, and GSK + Cisplatin treated mice. The results shown are mean ± SEM from 3 independent experiments. The significance was set at p≦ 0.05. C) Tumor volumes among mice groups. Note that tumor growth was reduced in GSK + Cisplatin (*) treated mice. However, treatment with either of the drug alone did not inhibit tumor growth, indicating that GSK treatment improved Cisplatin delivery through the normalization of the abnormal tumor vasculature.