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 3

Generation and screening of anti-human endotrophin.

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Generation and screening of anti-human endotrophin. (A) MCF-7 breast can...
(A) MCF-7 breast cancer cells (20,000 cells) were plated into a 96-well plate. Cells were then treated with 10 μm cisplatin, 10 μm cisplatin/100 ng/mL endotrophin, and 10 μm cisplatin/100 ng/mL endotrophin/10 μg/mL ETPmAb4 for 24 hours. Cell survival was measured using a CellTiter One Solution Cell Proliferation Assay. 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 = 3. (B) ETP transfected MCF-7 cells (100,000 cells) were plated into a 24-well plate with 500 μL media. Then 4 μL of supernatant was loaded for Western blotting. It is estimated that approximately 1 ng of ETP was loaded, compared with the standard ETP (2 ng band). So the concentration yields approximately 0.052 pg secreted ETP/hr/cell. (C) Total RNA was extracted from MCF-7, HCC1395, MDA-MB-231, ZR-75-1, and MDA-MB-453 cells. ETP expression was determined by qRT-PCR and normalized to GAPDH. Mean ± SEM, n = 3. (D) MCF-7 cells (5 × 105 cells) and ETP-MCF-7(5 × 105 cells) were harvested and total RNA was extracted. Expression levels of the EMT marker genes Twist, Snail, Cdh2, and Cdh1 were determined by qRT-PCR, then normalized to GAPDH. Mean ± SEM, n = 3. (E) ETP transfected MCF-7 cells (1 × 105 cells) were plated at the bottom chamber in a trans-well plate. Then 10 μg/mL, 2 μg/mL, 0.4 μg/mL, and 0.08 μg/mL ETPmAb4 was added to the bottom on day 2. SC macrophage cells (50,000 cells) were seeded at the top of the chamber on day 3. Migrated cells were counted after a 2 hour incubation. 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 = 3. 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.