Loss of Dab2 expression in breast cancer cells impairs their ability to deplete TGF-β and induce Tregs development via TGF-β

Abstract

Dab2 is a multifunctional adapter protein which is frequently under-expressed in a variety of cancers. It is implicated in many critical functions, including several signaling pathways, cell arrangement, differentiation of stem cells, and receptor endocytosis. Transforming growth factor-β (TGF-β) is a secreted multifunctional protein that controls several developmental processes and pathogenesis of many diseases. It has been documented that Dab2 played an important role in TGF-β receptors endocytosis. Here, we present evidence that re-expression of Dab2 in SK-BR-3 cell partially restored its ability to deplete TGF-β in surrounding medium by normalizing the trafficking of TGF-β receptors. We also demonstrate that the difference in TGF-β depletions produced by Dab2 expression was sufficient to impact on the conversion of naive CD4+ T cells to regulatory T cells (Tregs), and thus inhibited the proliferation of T cells. This work revealed a critical result that breast cancer cell was deficient in Dab2 expression and related receptor endocytosis-mediated TGF-β depletion, which may contribute to the accumulation of TGF-β in tumor microenvironment and the induction of immune tolerance.

Figure 1. Expression of Dab2 in breast cancer specimens.

(A) Ductal carcinoma in situ with microinvasion showing underexpression of Dab2 (original magnification ×200). (B) Invasive ductal carcinoma showing underexpression of Dab2 (×200). (C) Invasive lobular carcinoma showing underexpression of Dab2 (×200). (D) Medullary carcinoma cells showing partial Dab2 positivity (cytoplasm; 2+) (×200). (E) Normal breast epithelial cells showing strong Dab2 positivity in the cytoplasm (×200). (F) Epithelial cells in a galactocele served as positive control (arrows indicate positive cells) (×50).

Figure 2. Dab2 protein expression in three breast cancer cell lines demonstrated by western blots.

Western blot analysis of Dab2 protein expression in breast cancer cell lines. None of the 3 breast cancer cell lines showed detectable levels of Dab2 protein, while the nonmalignant mammary epithelial cell line MCF10A (positive control) showed the presence of Dab2 protein. Results were representative of 3 independent experiments.

Figure 3. The sensitivity of breast cancer cell lines to TGF-β.

(A) Induction of PAI-1 mRNA by TGF-β in breast cancer cell lines. MDA-MB-231 and SK-BR-3 cells were treated for 2 h with or without TGF-β (100 pM) before isolation of total RNA with Trizol. PAI-1 mRNA levels were determined with RT-Quantitative PCR. Data are means±SD of at least three independent experiments in each condition. * P<0.05. (B) TGF-β induced phosphorylation of Smad2 in SK-BR-3 cells. SK-BR-3 cells in complete medium was treated for 30 min with or without TGF-β(100pM) before being lysed with SDS sample buffer. The level of Phospho-Smad2 was analyzed by immunoblotting.

Figure 4. Time course curves of TGF-β depletion by SK-BR-3 and MLEC cells under different incubation conditions.

SK-BR-3 and MLEC cells were incubated in condition medium containing 35 or 60 pM of TGF-β for up to 8 h and aliquots of the incubation medium were collected at different time points. The remaining TGF-β content in those samples was quantified by a TGF-β reporter assay. (A) Curves of TGF-β depletion in SK-BR-3 cells in the presence of TGF-β at 35 and 60 pM. (B) Curves of TGF-β depletion in MLEC cells in the presence of TGF-β at 35 and 60 pM. (C) Curves of TGF-β depletion in SK-BR-3 cells at 4°C and 37°C (35 pM TGF-β). At 4°C, endocytosis was stopped. Data are Means±SD of at least three independent experiments in each condition. * P<0.05.(4°C vs 37°C)

Figure 5. Influence of Dab2 expression on TGF-β depletion in SK-BR-3 cells.

(A) Western blot analysis depicting Dab2 expression in SK-BR-3 cells after transfection with pcDNA3.1/Dab2. (B) TGF-β depletion by mock- and Dab2-transfected SK-BR-3 cells were measured with the reporter assay. Re-installation of Dab2 partially restored TGF-β depletion in the SK-BR-3 Dab2 cells, indicated by significant lower TGF-β content in its condition medium compared with the condition medium of SK-BR-3 V cells. (C) Influence of Dab2 on recycling of Tfn in breast cancer SK-BR-3 cells. Bar, 25 um. (D) After endocytosis, Tfn accumulates in the cytoplasm around the nucleus. The proportion of cells that showed diffued staining(indicative of Tfn recycling) within 15 min, out of a total of 50 cells examined, was calculated. *P<0.05.

Figure 6. Differentiation of CD4+CD25- naïve T cells from peripheral blood of healthy subjects.

(A) CD4+CD25- naïve T cells treated with negative control medium, condition medium from SK-BR-3 V cells (4 h), and condition medium from SK-BR-Dab2 cells (4 h). Cells were treated in the presence or absence of TGF-β antibody, harvested 96 h later, and analyzed by flow cytometry for CD4+CD25+Foxp3+ Tregs. Representative data from one determination are shown. (B) Proportion of CD4+CD25+Foxp3+ Tregs formed in different groups. Data are means ± SD of three independent experiment (*P<0.05).

Figure 7. The inhibitory effects of Tregs induced with condition mediums from mock- or Dab2-transfected SK-BR cells on T cell proliferation.

Condition medium from SK-BR-3 V cells or SK-BR-Dab2 cells were used to differentiate CD4+ CD25-naïve T cells to CD4+CD25+Foxp3+Tregs. The freshly differentiated Tregs were then co-cultured with CFSE-labeled CD4+CD25- naïve T cells at a ratio of 1:1 to examine the effects of Tregs on T cell proliferation. A lower mean fluorescence intensity (MFI) indicates more cells proliferation in this assay (CFSE label will be diluted 50% each time cell divides). (A) Representative data from 1 of 3 assays (medium-treated group, SK-BR-3 V medium group, and SK-BR-3 Dab2 medium group were treated with anti-CD3/CD28; negative control cells were untreated). (B) Means±SD of three independent experiments (MFI) in different groups (*P<0.05).

Figure 8. Proposed itinerary of TGF-β receptors in Dab2 absent cells or Dab2 re-expression cells.

TGF-β receptors undergoes endocytosis via clathrin-coated pits with involvement Dab2. Receptors enter the early endosomes and return to the cell membrane via Rab11-positive recycling endosomes. Dab2 can promote the transfer of receptors from early endosomes to recycling endosomes. When Dab2 expression is deficient, the TGF-β receptor may accumulate in the early endosomes after endocytosis, leading to the interruption of receptor movement to recycling endosomes, which hinders the return of receptors to cell membranes.

Figure 9. Model of how normal and cancer cells regulate the extracellular levels of TGF-β.

The concentration of extracellular TGF-β is maintained by the balance between cellular production of TGF-β and depletion by the endocytosis. The underexpression of Dab2 and impaired TGF-β depletion in cancer cells lead to increased extracellular TGF-β concentration and cancer progression.