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. 2020 Apr 3;12(4):868.
doi: 10.3390/cancers12040868.

TIE2 Induces Breast Cancer Cell Dormancy and Inhibits the Development of Osteolytic Bone Metastases

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Free PMC article

TIE2 Induces Breast Cancer Cell Dormancy and Inhibits the Development of Osteolytic Bone Metastases

Florian Drescher et al. Cancers (Basel). .
Free PMC article

Abstract

Breast cancer (BCa) cells disseminating to the bone can remain dormant and resistant to treatments for many years until relapsing as bone metastases. The tyrosine kinase receptor TIE2 induces the dormancy of hematopoietic stem cells, and could also induce the dormancy of BCa cells. However, TIE2 is also a target for anti-angiogenic treatments in ongoing clinical trials, and its inhibition could then restart the proliferation of dormant BCa cells in bone. In this study, we used a combination of patient data, in vitro, and in vivo models to investigate the effect of TIE2 in the dormancy of bone metastases. In BCa patients, we found that a higher TIE2 expression is associated with an increased time to metastases and survival. In vitro, TIE2 decreased cell proliferation as it increased the expression of cyclin-dependent kinase inhibitors CDKN1A and CDKN1B and arrested cells in the G0/G1 phase. Expression of TIE2 also increased the resistance to the chemotherapeutic 5-Fluorouracil. In mice, TIE2 expression reduced tumor growth and the formation of osteolytic bone metastasis. Together, these results show that TIE2 is sufficient to induce dormancy in vitro and in vivo, and could be a useful prognostic marker for patients. Our data also suggest being cautious when using TIE2 inhibitors in the clinic, as they could awaken dormant disseminated tumor cells.

Keywords: TIE2; bone metastasis; breast cancer; cancer relapse; chemotherapy resistance; dormancy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
High TIE2 expression in the primary tumor is associated with an increased time to the detection of metastases in breast cancer patients. Analysis of metastasis-free survival using the PROGgene database. The median TIE2 mRNA level in the primary tumor was taken as a bifurcation point. Results are presented as (A) Kaplan–Meier plots for the Sotiriou (GSE2990) and Loi datasets (GSE6532), or as (B) a forest plot, indicating the overall hazard ratio (HR) for metastasis-occurrence and 95% confidence interval (CI). Survival analysis was performed using a log-rank test, p < 0.078, and * p < 0.05.
Figure 2
Figure 2
High expression of TIE2 in the primary tumor of breast cancer patients is associated with increased overall survival. Analysis of overall survival using the PROGgene database. The median TIE2 mRNA level in the primary tumor was taken as a bifurcation point. Results are presented as (A) Kaplan–Meyer plots for the Pawitan (GSE1456) and Miller datasets (GSE3494), or as (B) a forest plot, indicating the overall hazard ratio (HR) for metastasis-occurrence and 95% confidence interval (CI). The horizontal line divides datasets with a HR higher or lower than 1, the dotted lines separate the datasets with a HR lower than 0.85 and higher than 1.15. Survival analysis was performed using a log-rank test, p < 0.078, and ** p < 0.01.
Figure 3
Figure 3
TIE2 expression is low in cancer cell lines in culture. (A) Evaluation of TIE2 protein expression in breast cancer (BCa) and prostate cancer (PCa) cell lines using flow cytometry. (B) Evaluation of TIE2 mRNA expression using RT-qPCR. (C) MDA-MB-231 and PC-3 cells were transduced with lentiviral particles to over-express TIE2 or GFP. Expression was measured by flow cytometry five days after transduction (before selection) and three weeks after transduction (after selection). (D) MDA-MB-231 and PC-3 cells were transduced to express TIE2 using a Tet-On inducible system. Transduced cells were selected with puromycin, and TIE2 expression was measured by flow cytometry (after selection). MDA-MB-231 cells were then cultured for three weeks, and PC-3 cells were cultured for one week, and TIE2 expression was measured again (later passages). Flow cytometry results are represented as contour plots overlaid with outliers. Values indicate the percentage of TIE2+ cells. TIE2 mRNA expression is represented as the average ± SEM vs. MDA-MB-468 cells.
Figure 4
Figure 4
TIE2 expression is decreased in the primary tumor of breast cancer patients. Relative TIE2 expression was compared between the normal breast tissue and the primary tumor of BCa patients diagnosed with invasive ductal breast carcinoma [33,34], invasive breast carcinoma [35], or ductal breast carcinoma [36,37]. Relative TIE2 expressions are represented as box-plots and were compared using an unpaired Student’s t-test.
Figure 5
Figure 5
TIE2 expression reduces the proliferation of MCF-7 cells. (A) MCF-7 TIE2tet and MCF-7 eGFPtet cells were cultured in the presence or absence of doxycycline (Dox, 0.5-1 µg/mL). Proliferation was assessed by MTT. Values are represented as the average ± SEM. *** p < 0.001 vs. vehicle-treated cells using a two-way ANOVA with Bonferroni post-test. (B) Representative histograms of a cell cycle analysis of MCF-7 eGFPtet and MCF-7 TIE2tet cells cultured in the presence or absence of Dox (0.5–1 µg/mL) for 9 days. Percentages of non-proliferating (G0/G1) and proliferating (S + G2/M) cells are indicated. Average percentages of cells in G0/G1, S, and G2/M ± SEM of three independent cell cycle analyses. (C) Expression of MKI67, PCNA, CDKN1A, CDKN1B, and cyclin D1 (CCND1) in MCF-7 TIE2tet or eGFPtet cells cultured ±Dox (1 µg/mL) for 9 days. Results are represented as the average gene expression ±SEM vs. MCF-7 TIE2tet vehicle-treated cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 using a two-way ANOVA with Bonferroni post-test.
Figure 6
Figure 6
TIE2 expression increases the resistance to 5-Fluorouracil in MCF-7 cells. MCF-7 TIE2tet and MCF-7 eGFPtet cells were cultured in the presence or absence of doxycycline (1 µg/mL) during 10 days of the experiment. 5-Fluorouracil (5-FU) was added to the cells for 4 days. Proliferation was assessed by MTT, and values are represented as the average ± SEM. *** p < 0.001 vs. vehicle-treated cells and analyzed using a two-way ANOVA with Bonferroni post-test.
Figure 7
Figure 7
TIE2 reduces the growth of orthotopic 4T1 TIE2tet tumors. (A) 4T1 TIE2tet cells were cultured in the presence or absence of doxycycline for 2 days before being inoculated bilaterally, in the 4th mammary fat pad of mice receiving or not doxycycline in their drinking water (n = 7 per group). (B) Water consumption and (C) tumor volume were measured throughout the experiment, and (D) the weight of excised tumors was measured at the time of the euthanasia. Results are represented as the average ± SEM. ** p < 0.01 and *** p < 0.001 vs. control mice using a two-way ANOVA with Bonferroni post-test (panels B and C). †† p < 0.01 using an unpaired Student’s t-test (panel D).
Figure 8
Figure 8
TIE2 reduces osteolysis of 4T1 TIE2tet bone metastasis. (A) 4T1 TIE2tet cells were cultured in the presence or absence of doxycycline for 2 days before being inoculated in the left cardiac ventricle of mice receiving or not doxycycline in their drinking water (n = 7 per group). (B) Water consumption was measured throughout the experiment. Osteolytic lesions were assessed using (C) radiographs of the hind limbs of mice (white arrows indicate osteolysis area), and (D) the osteolysis area was measured. * p < 0.05 using a Mann–Whitney test. (E) Summary of the occurrence of osteolytic lesions on endpoint radiographs and of skeletal tumor burden on tissue sections. (F) The presence of skeletal tumor burden was assessed using tissue sections stained with hematoxylin, eosin, and orange G (representative sections). The yellow dotted lines contour and indicate the skeletal tumor burden.

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