Off-tumor targets compromise antiangiogenic drug sensitivity by inducing kidney erythropoietin production

Abstract

Anti-VEGF drugs are commonly used for treatment of a variety of cancers in human patients, and they often develop resistance. The mechanisms underlying anti-VEGF resistance in human cancer patients are largely unknown. Here, we show that in mouse tumor models and in human cancer patients, the anti-VEGF drug-induced kidney hypoxia augments circulating levels of erythropoietin (EPO). Gain-of-function studies show that EPO protects tumor vessels from anti-VEGF treatment and compromises its antitumor effects. Loss of function by blocking EPO function using a pharmacological approach markedly increases antitumor activity of anti-VEGF drugs through inhibition of tumor angiogenesis. Similarly, genetic loss-of-function data shows that deletion of EpoR in nonerythroid cells significantly increases antiangiogenic and antitumor effects of anti-VEGF therapy. Finally, in a relatively large cohort study, we show that treatment of human colorectal cancer patients with bevacizumab augments circulating EPO levels. These findings uncover a mechanism of desensitizing antiangiogenic and anticancer effects by kidney-produced EPO. Our work presents conceptual advances of our understanding of mechanisms underlying antiangiogenic drug resistance.

Trial registration: ClinicalTrials.gov NCT00957125.

Keywords: angiogenesis; drug resistance; erythropoietin; hematopoiesis; tumor.

Conflict of interest statement

The authors declare no conflict of interest.

Published under the PNAS license.

Figures

Fig. 1.

Systemic anti-VEGF treatment augments kidney EPO production. (A and B) Kidney cortex CD31+ microvessels and hypoxia pimonidazole+ signals of NIIgG- and anti-VEGF–treated tumor-free and LLC tumor-bearing mice. White arrows point to CD31+ microvessels. CD31+ microvessels and pimonidazole+ signals were randomly quantified from 12 fields (n = 6 samples per group). (C) Western blotting analysis of CAIX in the NIIgG- and anti-VEGF–treated kidney cortex (n = 5 samples per group). (D) qPCR analysis of Hif1α mRNA and Hif2α mRNA expression levels of NIIgG- and anti-VEGF–treated kidney cortex of LLC tumor-bearing animals (n = 5 samples per group). (E) Western blotting analysis of HIF-1α and HIF-2α of the kidney cortex of tumor-free and LLC tumor-bearing animals (n = 3 samples per group). (F) ELISA measurements of EPO protein levels (n = 5 samples per group). (G) Epo mRNA levels of NIIgG- and anti-VEGF–treated tumors (n = 6 samples per group). (H) EPO protein staining of NIIgG- and anti-VEGF–treated kidney cortex of tumor-free and LLC tumor-bearing animals. White arrows point to EPO+ staining. Quantification of EPO positive signals (n = 6–8 random fields per group). (I, Left and Center) ELISA analysis of EPO protein levels and qPCR analysis of Epo mRNA levels of the kidney cortex of tumor-bearing animals (n = 6 tissue samples per group). (I, Right) Epo mRNA expression levels of livers of LLC tumor-bearing animals (n = 6 tissue samples per group). Data are means ± SEM. Animal experiments were repeated twice. *P < 0.05; **P < 0.01; ***P < 0.001. n.s., not significant. (Scale bars: 50 μm.)

Fig. 2.

EPO-EpoR–triggered endothelial cell signaling and functions. (A) RT-PCR analysis of Epor mRNA levels in different cell types. Bone marrow cell (BMC) served as a positive control. (B) Immunocytochemical staining of EpoR-positive signals in mEC cells (Upper) and LLC cells (Lower). (C) FACS analysis of EpoR expression on mEC cells. (D) Immunocytochemical staining of EpoR positive signals in LLC tumor tissue. (E and F) Western blotting analysis of phospho-ERK, total-ERK, and β-actin levels of each treated and nontreated samples. Mouse endothelial cells were stimulated with 1 U/mL human recombinant EPO for indicated time points. Densitometry of p-ERK–positive signals. Experiments were repeated twice. (Scale bars: 50 μm.)

Fig. 3.

Kidney-derived EPO confers anti-VEGF resistance. (A) Proliferation assay of sEpoR-Fc and control Fc-treated UT-7/EPO in the presence or absence of EPO protein (n = 6 samples per group). (B) Tumor growth rates (n = 6 animals per group). (C and D) Immunohistochemical analyses of CD31+ microvessels and vascular perfusion of 2,000-kDa dextran. Arrows in C, Upper point to CD31+ blood vessels and in Lower indicate perfused dextran+ signals. Quantifications of CD31+ vessel density and dextran blood perfusion (n = 10 samples per group). (E) Growth rates of various monotherapy- or combination therapy-treated LLC tumors (n = 6 animals per group). (F and G) Immunohistochemical analyses of CD31+ microvessels and vascular perfusion of 2,000-kDa dextran. Arrows in F, Upper point to CD31+ blood vessels and in Lower indicate perfused dextran+ signals (n = 10 samples for each group). Quantifications of CD31+ vessel density and dextran blood perfusion (n = 10 samples per group). *P < 0.05; **P < 0.01; ***P < 0.001. n.s., not significant. Data are means ± SEM. (Scale bars: 50 μm.)

Fig. 4.

EPO inhibition increases antitumor activity by anti-VEGF drugs. (A–C) Immunohistochemical analyses of PCNA+ proliferating tumor cells, TUNEL+ apoptotic tumor cells, and tumor hypoxia. Arrows in A indicate PCNA+ proliferating tumor cells and arrows in B point to TUNEL+ apoptotic tumor cells. CAIX positive hypoxic signals are indicated with green signals in C. DAPI in blue was used for counterstaining of cell nuclei. (D–G) Quantification of PCNA+ proliferating tumor cells, TUNEL+ apoptotic tumor cells, apoptotic index, and CAIX positive hypoxic signals (n = 10 random fields per group; six animals per group). (H–J) Immunohistochemical analyses of PCNA+ proliferating tumor cells, TUNEL+ apoptotic tumor cells, and tumor hypoxia. Arrows in H indicate PCNA+ proliferating tumor cells and arrows in I point to TUNEL+ apoptotic tumor cells. CAIX-positive hypoxic signals are indicated with green signals in J. DAPI in blue was used for counterstaining of cell nuclei. (K–N) Quantification of PCNA+ proliferating tumor cells, TUNEL+ apoptotic tumor cells, apoptosis index, and CAIX-positive hypoxic signals (n = 10 random fields per group; six animals per group). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Data are means ± SEM. (Scale bars: A, B, H, and I, 50 μm; C and J, 100 μm.)

Fig. 5.

Nonhematopoietic ablation of EpoR increases anti-VEGF sensitivity. (A) Tumor growth rates (n = 6 animals per group). (B) Analyses of red blood cells, hemoglobin levels, and hematocrits (n = 6 animals per group). (C–E) Immunohistochemical analyses of CD31+ microvessels and vascular perfusion of 2,000-kDa dextran (n = 6 animals per group). Arrows in C, Upper point to CD31+ blood vessels and in Lower indicate perfused dextran+ signals (n = 10 samples per group). Quantifications of CD31+ vessel density and dextran blood perfusion (n = 10 samples per group). (F–K) Immunohistochemical analyses of PCNA+ proliferating tumor cells, Ki67+ proliferating tumor cells, cleaved caspase-3+ apoptotic tumor cells, TUNEL+ apoptotic tumor cells, and tumor hypoxia (n = 10 random fields per group; six animals per group). Epor (−/−) indicates EpoR(−/−)::HG1-EpoR mice. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Data are means ± SEM. (Scale bars: 50 μm.)

Fig. 6.

Chemotherapy in combination of anti-VEGF therapy elevated EPO expression. (A) Treatment schedule with anti-VEGF drug in combination of cisplatin and vinorelbine. (B) Tumor growth rates (n = 5 animals per group). (C) Immunohistochemical analyses of CD31+ microvessels Arrows point to CD31+ blood vessels. (Scale bars: 50 μm.) (D–F) Quantifications of CD31+ vessel density in tumor, kidney, and liver (n = 8 random fields per group). (G) ELISA analysis of plasma EPO protein levels (n = 5 samples per group). (H) ELISA analysis of kidney EPO protein levels (n = 4–5 samples per group). (I) ELISA analysis of plasma EPO protein levels of bevacizumab plus chemotherapeutics (9.31 ± 1.17 mIU) and chemotherapeutics alone-treated (18.32 ± 3.44 mIU) colorectal cancer patients (n = 34 patients in chemotherapy alone group; n = 32 patients in bevacizumab plus chemotherapy group). (J) ELISA analysis of plasma EPO protein levels of control (12.5 ± 0.107 mIU) and bevacizumab plus chemotherapeutic docetaxel-epirubicin–treated (16.8 ± 0.124 mIU) breast cancer patients (n = 61 patients per group). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Data are means ± SEM.

Fig. 7.

Schematic diagram of systemic anti-VEGF treatment-induced drug resistance. Systemic administration of anti-VEGF drugs such as bevacizumab enters into the circulation to target both tumor and healthy vasculatures such as kidney blood vessels. Anti-VEGF–induced vessel regression in the kidney cortex leads to tissue hypoxia, which induces HIF-1α and HIF-2α expressions. HIF-1α and HIF-2α at transcription level target the Epo promoter for high production of EPO in peritubular interstitial cells in kidney cortex. Kidney-derived EPO enters the circulation to stimulate angiogenesis and eventually contributes to antiangiogenic drug resistance. These findings provide mechanistic insights of off-tumor targets of antiangiogenic drugs in the cancer patients for development of drug resistance.