Human endotrophin as a driver of malignant tumor growth
Dawei Bu, Clair Crewe, Christine M. Kusminski, Ruth Gordillo, Alexandra L. Ghaben, Min Kim, Jiyoung Park, Hui Deng, Wei Xiong, Xiao-Zheng Liu, Per Eystein Lønning, Nils Halberg, Adan Rios, Yujun Chang, Anneliese Gonzalez, Ningyan Zhang, Zhiqiang An, Philipp E. Scherer
Dawei Bu, Clair Crewe, Christine M. Kusminski, Ruth Gordillo, Alexandra L. Ghaben, Min Kim, Jiyoung Park, Hui Deng, Wei Xiong, Xiao-Zheng Liu, Per Eystein Lønning, Nils Halberg, Adan Rios, Yujun Chang, Anneliese Gonzalez, Ningyan Zhang, Zhiqiang An, Philipp E. Scherer
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Research Article Endocrinology Oncology

Human endotrophin as a driver of malignant tumor growth

Abstract

We have previously reported that the carboxy-terminal proteolytic cleavage product of the COL6α3 chain that we refer to as “endotrophin” has potent effects on transformed mammary ductal epithelial cells in rodents. Endotrophin (ETP) is abundantly expressed in adipose tissue. It is a chemoattractant for macrophages, exerts effects on endothelial cells and through epithelial-mesenchymal transition (EMT) enhances progression of tumor cells. In a recombinant form, human endotrophin exerts similar effects on human macrophages and endothelial cells as its rodent counterpart. It enhances EMT in human breast cancer cells and upon overexpression in tumor cells, the cells become chemoresistant. Here, we report the identification of endotrophin from human plasma. It is circulating at higher levels in breast cancer patients. We have developed neutralizing monoclonal antibodies against human endotrophin and provide evidence for the effectiveness of these antibodies to curb tumor growth and enhance chemosensitivity in a nude mouse model carrying human tumor cell lesions. Combined, the data validate endotrophin as a viable target for anti-tumor therapy for human breast cancer and opens the possibility for further use of these new reagents for anti-fibrotic approaches in liver, kidney, bone marrow and adipose tissue.

Authors

Dawei Bu, Clair Crewe, Christine M. Kusminski, Ruth Gordillo, Alexandra L. Ghaben, Min Kim, Jiyoung Park, Hui Deng, Wei Xiong, Xiao-Zheng Liu, Per Eystein Lønning, Nils Halberg, Adan Rios, Yujun Chang, Anneliese Gonzalez, Ningyan Zhang, Zhiqiang An, Philipp E. Scherer

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Figure 2

Effects of human endotrophin on human cells in vitro.

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Effects of human endotrophin on human cells in vitro. (A) T47D breast ca...
(A) T47D breast cancer cells (5 × 105 cells) were plated into 6-well plates and treated with endotrophin (0.1 μg/mL) 3 times (every other day). Total RNA was then extracted from each well. The EMT marker genes Twist, Snail, Cdh2, and Cdh1 were determined by qRT-PCR, then normalized to GAPDH. Mean ± SEM, n = 3. (B) Primary human mesothelial cells (5 × 105 cells) were plated into a 6-well plate and were treated with endotrophin (0.1 μg/mL and 1 μg/mL) 3 times (every other day). Total RNA was extracted from each well and EMT genes Twist, Snail, Cdh2, and Cdh1 were determined by qRT-PCR and normalized to GAPDH. Mean ± SEM, n = 3. (C) HUVEC cells (40,000 cells) were plated into 24-well plate. When the cells reached 90% confluence, the monolayer was scratched with a 1 mL pipette tip to create 2 perpendicular straight lines across the center of the well. Cells were then treated with increasing concentrations of endotrophin (10 pg/mL, 100 pg/mL, 1 ng/mL, 10 ng/mL, 0.1 μg/mL, and 1 μg/mL). Images were obtained using a Nikon Cool Scope microscope (Nikon) after a 20 hour incubation. Migrating cell numbers were evaluated using ImageJ software. Statistical significance of the curve fit parameters was tested using the extra sum of squares F test with P < 0.05 considered significant. Goodness of curve fit is described using r2. Mean ± SEM, n = 4. Scale bars: 100 μm. (D) HUVEC cells (5 × 105 cells) were plated at the top chamber in a trans-well plate. Endotrophin (0.1 μg/mL) was then added with or without 1% FBS in the lower chamber, then incubated for 16 hours. Images were then obtained on a Nikon Cool Scope microscope (Nikon) after a 16 hours incubation. Mean ± SEM, n = 4. Scale bars: 100μm (E) HUVEC cells (40,000 cells) were plated into a gel-coated 24-well plate. Cells were then treated with endotrophin (10 pg/mL, 100 pg/mL, 1 ng/mL, 10 ng/mL, 0.1 μg/mL, and 1 μg/mL), with or without 1% FBS for 16 hours. A cell-permeable dye, Calcein, was added for fluorescent monitoring of tube formation. Images were then obtained using a Nikon Cool Scope microscope (Nikon) after 16 hours. The statistical significance of the effect of FBS treatment on node number was tested by comparing the curve fits using a sum of squares F test. Goodness of fit of each individual regression is described using r2. Mean ± SEM, n = 3. Scale bars: 100μm (F) SC macrophage cells (50,000 cells) were seeded at the top of the chamber. Endotrophin (0.1 μg/mL) was added with 1% FBS in the lower chamber and incubated for 2 hours. Migrated cells were then counted after 2 hours. Mean ± SEM, n = 4. In all cases, data was represented as mean ± SEM and statistical significance (***P < 0.0001) was calculated using unpaired, 2-tailed t-test w/Holm-Sidak correction for multiple comparisons.