Enhancing gemcitabine uptake by promoting dimerization of ENT1 transporter improves response rates of patients with sarcoma
Aditya Ganju, Shyam Rao, Mark A. Dickson, Robert A. Lefkowitz, Chris Thompson, Jin Cheng, Katia Manova, Adriana Haimovitz-Friedman, Gary Schwartz, Zhigang Zhang, Zvi Fuks, William D. Tap, Richard Kolesnick
Aditya Ganju, Shyam Rao, Mark A. Dickson, Robert A. Lefkowitz, Chris Thompson, Jin Cheng, Katia Manova, Adriana Haimovitz-Friedman, Gary Schwartz, Zhigang Zhang, Zvi Fuks, William D. Tap, Richard Kolesnick
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Research Article Cell biology Oncology

Enhancing gemcitabine uptake by promoting dimerization of ENT1 transporter improves response rates of patients with sarcoma

Abstract

Transport of nucleoside chemotherapeutic drugs into tumor cells is primarily accomplished through equilibrative nucleoside transporter 1 (ENT1), which is considered constitutively active. Drugs are redistributed via ENT1 across lipid bilayers via facilitated diffusion. Here we discover that ENT1 is not constitutively active, but rather requires activation of acid sphingomyelinase (ASMase) by gemcitabine, generating ceramide-rich platforms (CRPs) on external plasma membranes of endothelial and tumor cells into which ENT1 inserts, dimerizing therein to functionalize transmembrane gemcitabine transport. Whereas sarcoma cells synthesize minimal ASMase, they take up gemcitabine poorly in vitro and in murine xenografts. A strategy designed to augment gemcitabine-induced ASMase secretion into the extravascular space by ASMase-rich neo-angiogenic cells, which then targets tumor cell plasma membranes, yields “bystander” CRPs on sarcoma cells and ENT1 insertion therein, conferring markedly enhanced gemcitabine uptake and xenograft response. Engaging this biology in a prospective phase II clinical trial in advanced sarcoma yielded robust volumetric changes in evaluated tumors that developed early and were often durable.

Authors

Aditya Ganju, Shyam Rao, Mark A. Dickson, Robert A. Lefkowitz, Chris Thompson, Jin Cheng, Katia Manova, Adriana Haimovitz-Friedman, Gary Schwartz, Zhigang Zhang, Zvi Fuks, William D. Tap, Richard Kolesnick

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Figure 1

Nucleoside transporter ENT1 requires CRPs for functionalization of gemcitabine uptake.

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Nucleoside transporter ENT1 requires CRPs for functionalization of gemci...
(A) Uptake of gemcitabine (10 μM gemcitabine plus tracer [14C] gemcitabine) in proliferative and growth-arrested BAECs was performed in the presence and absence of ENT1 inhibitors (100 nM NBMPR and 10 μM dilazep). Proliferative cells were 50%–60% confluent; growth-arrested cells were used 3 days post-confluence. Inhibitors were delivered 1 hour before gemcitabine. Cell-associated radioactivity was quantified by scintillation counting. Shown is 1 representative of 2 independent experiments performed in triplicate. (B) Confocal images showing colocalization of ENT1 in a CRP 1 minute after 1 µM gemcitabine. Nuclei were stained with DAPI (blue), ENT1 with rabbit anti-ENT1 followed by Alexa Fluor 488–conjugated anti-rabbit IgG (green), and ceramide with mouse anti-ceramide MID 15B4 followed by Cy3-conjugated anti-mouse IgM (red). Merged images indicate ENT1 localization in CRPs. Data are representative of 10 cells/group from 1 of 2 independent experiments. (C) Time course of gemcitabine-induced CRP formation (1 μM) in BAECs. Data represent the mean ± SEM. n = 2. **P < 0.01, *P < 0.05 vs. 0-second point by 1-way ANOVA. (D) Dose response of gemcitabine-induced CRP formation in BAECs at 1 minute (gemcitabine dose 0.1–30 μM). Data represent the mean ± SEM. n = 2. ***P < 0.001, **P < 0.01, *P < 0.05 vs. 0 μM by 1-way ANOVA. (E) 3D still image of CRPs in control and gemcitabine-treated cells at 1 minute after 1 μM gemcitabine treatment in proliferative BAECs. The 3D still image was acquired using Imaris software by generating Z-stacks during the image acquisition as described in Methods. (F) CRP volumes from E. Data represent mean ± SEM from 2 independent experiments using 10 cells/sample. Two-tailed Student’s t test. *P < 0.05. (G) Confocal images showing CRP formation and ENT1 localization in growth-arrested BAECs treated with gemcitabine and/or bSMase (Bacillus cereus; 5 units/well). Cells were stained as in B. Data are representative of 10 cells/group from 1 of 2 independent experiments. (H) Gemcitabine uptake in growth-arrested BAECs pretreated with bSMase (5 units/well). Cells were processed as in A. Data are representative of 3 independent experiments.