Effects of vascular-endothelial protein tyrosine phosphatase inhibition on breast cancer vasculature and metastatic progression - PubMed (original) (raw)

. 2013 Aug 21;105(16):1188-201.

doi: 10.1093/jnci/djt164. Epub 2013 Jul 30.

Nisha Gupta, Brian P Walcott, Matija Snuderl, Cristina T Kesler, Nathaniel D Kirkpatrick, Takahiro Heishi, Yuhui Huang, John D Martin, Eleanor Ager, Rekha Samuel, Shuhan Wang, John Yazbek, Benjamin J Vakoc, Randall T Peterson, Timothy P Padera, Dan G Duda, Dai Fukumura, Rakesh K Jain

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Effects of vascular-endothelial protein tyrosine phosphatase inhibition on breast cancer vasculature and metastatic progression

Shom Goel et al. J Natl Cancer Inst. 2013.

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Abstract

Background: The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels.

Methods: AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)-deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided.

Results: AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ(2) test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel-Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation.

Conclusions: Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments.

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Figures

Figure 1.

Figure 1.

Effect of AKB-9778, a vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor, on Tie-2 signaling in endothelial cells. A) Tie-2 tyrosine phosphorylation in human umbilical vein endothelial cell (HUVEC) lysates after AKB-9778 treatment. B) Levels of phosphorylated AKT (Ser473), total AKT, phosphorylated endothelial nitric oxide synthase (eNOS) (Ser1177), and total eNOS in HUVEC lysates after treatment with AKB-9778 (numbers below blot indicate densitometric ratios as labeled). C) Phosphorylated AKT (Ser473 residue), total AKT, phosphorylated eNOS (Ser1177 residue), and total eNOS levels in HUVEC lysates or TIE2-shRNA HUVEC lysates after exposure in vitro to AKB-9778 (numbers below blot indicate densitometric ratios as labeled). D) Tie-2 tyrosine phosphorylation in murine whole lung lysates 1 hour after intravenous injection of AKB-9778 or control vehicle. E) Tie-2 tyrosine phosphorylation in murine orthotopic 4T1 mammary carcinoma lysates 1 hour after intravenous injection of AKB-9778 or control vehicle. F) Levels of phosphorylated AKT (Ser473), total AKT, phosphorylated eNOS (Ser1177), and total eNOS in HUVEC lysates after 30 minutes of treatment with angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2), and/or AKB-9778.

Figure 2.

Figure 2.

Effect of vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibition on embryonic and tumor angiogenesis in zebrafish. A) Representative epifluorescence micrographs of Flk-1:GFP zebrafish embryos treated with control vehicle (DMSO) (top panel) or AKB-9778 (middle panel) at 48 hours postfertilization. Lower panels (left and right) show high-powered images of AKB-9778–treated embryos at 48 hours postfertilization. DLAV = dorsal longitudinal anastomotic vessel; DA = dosal aorta; ISV = intersegmental vessel. Scale bars: top panel = 100 µm; middle panel = 75 µm; bottom panels = 50 µm. B) Percentage of vessels that sprouted from the dorsal aorta to reach the dorsal longitudinal anastomotic vessel in zebrafish embryos treated with control vehicle (DMSO) or AKB-9778 (*P < .01 by χ2 test; n = 5–6 embryos per group). Table shows total vessels counted with data as means and 95% confidence intervals (CIs). C) 4T1-dsRed mammary carcinomas growing in Flk-1:GFP zebrafish embryos treated with control vehicle (left panel) or AKB-9778 (right panel). D) Vessel density (determined as green fluorescent protein [GFP]-positive area/tumor area) from experiment in (C) (*P < .01 by two-sided t test; n = 6 embryos per group). Value for each embryo is determined as the mean of five individual micrographs taken throughout each tumor. Bars show mean ± standard deviation (control mean = 0.08, 95% CI = 0.04 to .11; AKB-9778 mean = 0.02, 95% CI = 0.01 to 0.03).

Figure 3.

Figure 3.

Impact of adjuvant therapy with AKB-9778 on the growth of spontaneous micrometastases. A) Schema demonstrating model for adjuvant AKB-9778 therapy. Orthotopically grown 4T1 tumors were resected at 5mm in diameter. B) Number of macroscopic lung metastases in control vs AKB-9778–treated mice (*P = .03 by two-sided t test; n = 10 per group) after 3 weeks of treatment. C) Distribution of the size of macrometastatic lung nodules (*P < 0.01 by two-sided χ2 test). D) Representative images of lungs visualized after resection in control (upper panels) and AKB-9778 treated (lower panels) mice (scale bars = 10mm). E) Total number of macroscopically detected metastases (including lung, liver, bone, lymph node, and soft tissue) (*P = .03 by two-sided t test; n = 10 per group). For all panels, error bars represent standard deviation.

Figure 4.

Figure 4.

Effect of vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibition on extravasation of disseminated tumor cells into distant organ parenchyma. A–C) 4T1 mammary carcinoma cells were injected intravenously, and therapy with control or AKB-9778 commenced 12 hours later. Lungs were examined for micrometastases after 8 days. A) Representative hematoxylin and eosin–stained sections of micrometastases. Control lungs (upper panels) show micrometastatic cells that have breached vascular boundaries and entered the alveolar airspace. AKB-9778–treated lungs (lower panels) show cells tracking within vessels yet to extravasate (scale bars = 100 µm). B) Number of intra- vs extravascular micrometastases (*P < .01 by two-sided Fisher exact test; n = 6 mice per group; data show total number of metastases combined for all mice in each group). C) Representative images of micrometastases. Images show hematoxylin and eosin–stained sections of micrometastases and serial sections (separated by 5 microns) showing the same metastasis stained for collagen IV (brown). Control mice show extravasation of tumor cells through the vascular basement membrane, but AKB-9778–treated mice show tumor cells retained within vessels (scale bars = 100 µm). D) Representative images of experimental MMTV-PyVT tumor cell micrometastases treated with control (left panel) or AKB-9778 (right panel) (scale bars = 100 µm). E) Mouse survival after adjuvant therapy with doxorubicin ± AKB-9778. (*P = .05 using log-rank [Mantel–Cox] test; n = 8–9 mice per group) using a model of 4T1 spontaneous metastasis.

Figure 5.

Figure 5.

Structural changes within established primary tumor vessels invoked by vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibition. A) Colocalization of CD31-positive area (endothelial cells) and NG2-positive area (perivascular cells) (*P = .05 by two-sided t test; n = 6 per group) in 4T1 tumors. B) Colocalization of CD31-positive area (endothelial cells) and desmin-positive area (perivascular cells) (*P = .01 by two-sided t test; n = 6 per group) in 4T1 tumors. C) Mean distance between desmin-positive pericytes and CD31=positive endothelial cells in microns (*P < .01 by two-sided t test; n = 6 per group) in 4T1 tumors. D) Colocalization of CD31+ area (endothelial cells) and NG2+ area (perivascular cells) (*P = .05 by two-sided t test; n = 6 per group) in E0771 tumors (*P < .01 by two-sided t test; n = 6 per group). E) Enhanced vascular pericyte coverage in AKB-9778–treated tumors (right panel) compared with control-treated tumors (left panel) (red: CD31; green: NG2; blue: DAPI). F–G) Vessel diameter in control vs AKB-9778–treated 4T1 (F) and E0771 (G) tumors (F: *P < .01 by two-sided t test, n = 6 per group; G: *P < .01 by two-sided t test, n = 6 per group). H–I) Vessel density in control vs AKB-9778–treated 4T1 (H) and E0771 (I) tumors (H: *P = .05 by two-sided t test, n = 6 per group; I: *P = .05 by two-sided t test, n = 6 per group). Error bars represent standard deviation for all panels.

Figure 6.

Figure 6.

Effect of vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibition on tumor vessel function, hypoxia, and radiosensitivity. A) Percentage change in permeability from baseline to after 72 hours of treatment in control vs AKB-9778–treated 4T1 tumors (*P = .03 by two-sided t test; n = 3 per group). B) Intravital micrographs taken 0 minutes and 1 hour after intravenous injection of fluorescent bovine serum albumin after treatment with control (top panels) or AKB-9778 (lower panels) (scale bars = 200 µm). C and D) Lectin positive area in control vs AKB-9778–treated 4T1 (C) and E0771 (D) tumors (C: *P = .03 by two-sided t test, n = 6 per group; D: *P = .05 by two-sided t test, n = 5 per group). E and F) Enhanced vessel perfusion by lectin in AKB-9778–treated tumors (lower panel) compared with control (upper panel) (E: 4T1l; F: E0771). G and H) Relative hypoxic fraction assessed by pimonidazole staining in control vs AKB-9778–treated 4T1 (G) and E0771 (H) tumors (G: *P = .05 by two-sided t test, n = 10–13 per group; H: *P = .04 by two-sided t test, n = 9–10 per group). For (A) and (B), data were obtained after 72 hours of treatment, and for (C–H), treatment was commenced at tumor diameter of 3mm and continued for 10 days. I) Intravital optical frequency domain imaging of 4T1 mammary carcinomas at baseline (day 0, diameter 2–3mm) and after 4 days of treatment with AKB-9778 or control vehicle. Images show perfused vessels only. Scale bars represent 4mm. J) 4T1 tumor growth curves from combined AKB-9778 and radiotherapy. Treatment was begun at diameter 3mm, and radiotherapy given 1 week later (*P < .01 comparing green and pink lines by analysis of variance; n = 7 per group). For all panels, error bars represent standard deviation.

Figure 7.

Figure 7.

Role of endothelial nitric oxide synthase in vascular endothelial protein tyrosine phosphatase (VE-PTP)–induced tumor vascular alterations. A) Human umbilical vein endothelial cell (HUVEC) nitric oxide production in vitro after treatment with control, AKB-9778, or NG-Monomethyl-L-arginine (L-NMMA). Nitric oxide production was determined by quantification of the fluorescent signal from DAF-2T. B) Representative images of DAF-2T fluorescence (green) in HUVECs after treatment with control (left panel), AKB-9778 (center panel), or AKB-9778 and L-NMMA (right panel) (scale bars = 20 μM). C) E0771 mammary carcinoma was implanted orthotopically into either wild-type (WT) C57Bl/6 mice or NOS3 -/- mice (KO). At tumor diameter of 3mm, mice were treated with either AKB-9778 or control vehicle (n = 5 per group). Data show tumor growth curves. eNOS = endothelial nitric oxide synthase. D) Vessel diameter in E0771 orthotopic mammary carcinomas treated with AKB-9778 or control vehicle in NOS3 -/- mice (P = .40 by two-sided t test; n = 5 per group). E) Vessel density in E0771 orthotopic mammary carcinomas treated with AKB-9778 or control vehicle in NOS3 -/- mice (P = .77 by two-sided t test; n = 5 per group). For all panels, error bars represent standard deviation.

Figure 8.

Figure 8.

The effects of vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibition in mouse models of breast cancer. Inhibition of the VE-PTP domain in endothelial cells activates Tie-2 signaling, in turn promoting a more stable vessel phenotype. In early tumor growth, this prevents early destabilization and hence slows the initial phase of tumor progression. In established tumors, vessels show structural and functional changes of normalization, resulting in improved tumor perfusion (which occurs because of an increased production of nitric oxide [NO] by endothelial nitric oxide synthase [eNOS]). In the setting of micrometastasis, stabilization of distant organ vessels prevents extravasation of tumor cells, delaying micrometastatic progression and prolonging survival.

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