The functional activity of E-cadherin controls tumor cell metastasis at multiple steps

Abstract

E-cadherin is a tumor suppressor protein, and the loss of its expression in association with the epithelial mesenchymal transition (EMT) occurs frequently during tumor metastasis. However, many metastases continue to express E-cadherin, and a full EMT is not always necessary for metastasis; also, positive roles for E-cadherin expression in metastasis have been reported. We hypothesize instead that changes in the functional activity of E-cadherin expressed on tumor cells in response to environmental factors is an important determinant of the ability of the tumor cells to metastasize. We find that E-cadherin expression persists in metastatic lung nodules and circulating tumor cells (CTCs) in two mouse models of mammary cancer: genetically modified MMTV-PyMT mice and orthotopically grafted 4T1 tumor cells. Importantly, monoclonal antibodies that bind to and activate E-cadherin at the cell surface reduce lung metastasis from endogenous genetically driven tumors and from tumor cell grafts. E-cadherin activation inhibits metastasis at multiple stages, including the accumulation of CTCs from the primary tumor and the extravasation of tumor cells from the vasculature. These activating mAbs increase cell adhesion and reduce cell invasion and migration in both cell culture and three-dimensional spheroids grown from primary tumors. Moreover, activating mAbs increased the frequency of apoptotic cells without affecting proliferation. Although the growth of the primary tumors was unaffected by activating mAbs, CTCs and tumor cells in metastatic nodules exhibited increased apoptosis. Thus, the functional state of E-cadherin is an important determinant of metastatic potential beyond whether the gene is expressed.

Keywords: E-cadherin; E-cadherin-positive tumors; MMTV-PyMT breast cancer model; breast cancer metastasis; circulating tumor cells.

Fig. 1.

E-cadherin activation inhibits tumor metastasis in the MMTV-PyMT mouse model of breast cancer. (A) Schema of MMTV-PyMT breast cancer mouse model study. The MMTV-PyMT or FVB control female mice (Fig. 2) received i.p. injections of either 19.1-10 neutral E-cadherin-specific mAb or 56-4 E-cadherin activating mAb twice weekly. (B) All palpable masses were measured weekly, using external calipers. (C) Metastatic nodules counted in Bouin’s fluid fixation (Top) and H&E staining (Middle) of the lung from 14‐wk‐old MMTV-PyMT mice. (B and C, n = 14–16) ***P < 0.001 compared with neutral Ab-treated control. (D) Representative microscopic images of immunofluorescence staining for E-cadherin in metastases of MMTV-PyMT mice. (Scale bars, 50 μm.)

Fig. 2.

Circulating tumor cells are reduced by E-cadherin activation in the breast cancer models. mRNA levels for several tumor markers were analyzed by qRT-PCR, and the estimated number of CTCs based on levels of mRNA expression in cultured Py2T or 4T1 cells. (A) MMTV-PyMT model; CTCs in the peripheral blood were detected by mRNA levels (FVB wild-type, n = 3; MMTV-PyMT, n = 5 to 7) *P < 0.05; **P < 0.01; ***P < 0.001 compared with neutral Ab-treated control. (B–D) 4T1 tumor cell grafted metastatic mouse model study. Mouse epithelial 4T1 Luc2 cells expressing human E-cadherin (4T1 Luc-hE) were injected into mammary fat pads of BALB/c mice. After 3 d, the mice were given i.p. injections of either 46H7 neutral or 19A11 activating mAb twice weekly until the end of the experiments. (B) Schema. (C) Quantification of metastatic tumor nodules (n = 10) ***P < 0.001 compared with neutral Ab-treated control. (D) CTCs were detected by mRNA levels of Luc2 and hE-cadherin expression in 4T1 Luc-hE orthotopic grafted mouse model based on levels of expression in cultured cells (no graft group, n = 3; tumor cell graft group, n = 8 to 9). **P < 0.01; ***P < 0.001 compared with neutral Ab-treated control in tumor cell grafted group.

Fig. 3.

E-cadherin activation reduces the metastatic colonization from circulation. (A) Schema. Mouse epithelial 4T1 Luc-hE cells were injected into the tail-vein of BALB/c mice. One day after inoculation, the mice were treated with i.p. injections of either 46H7 neutral or 19A11 activating mAb twice weekly until the end of the experiments. (B) Representative microscopic images of H&E stained sections (Top) or lungs fixed with Bouin’s (Bottom). (C) Quantification of metastatic tumor nodules (n = 15). (Scale bar, 500 μm.) **P < 0.01 compared with neutral Ab-treated control group.

Fig. 4.

E-cadherin activating mAbs inhibit invasiveness and migration and enhance cell adhesion of PyMT primary spheroids and in vitro cell cultures. (A) MMTV-PyMT tumor-derived spheroids were mixed with a suspension of 1–2 spheroids/µl with a 3D extracellular matrix and incubated for 5 d in the presence of mAbs. Representative image of organoid spheroids and enlarged views (Bottom). (Scale bars, 100 µm.) (B) Quantification of A. Calculation of invasion as a function of the longest invasive distance emanating from the spheroid. Average of the longest invasive distance (µm) per spheroid (n = 30 spheroids per group). ***P < 0.001 compared with neutral Ab treatment. (C–F) 4T1 cells. (C) For cell adhesion assay, activating mAbs and Fab fragments stimulated adhesion of cells to pure E-cadherin substrate. 4T1 cells were untreated, pretreated with 3 µg/mL neutral mAb, 19.1-10, or activating mAbs 18-5 or 56-4 for 2 h, and cell adhesion strength to E-cadherin-coated capillary tubes was evaluated using increasing laminar flow to determine the force required to detach cells. (D) Migration. (E) Invasion assay of cells invading through a basement membrane. The cells were treated with of 3 µg/mL neutral mAb, 19.1-10, or activating mAbs 18-5 or 56-4 for 24 h. (F) Epithelial morphology. 4T1 cells were treated with of 3 µg/mL neutral mAb, 19.1-10, or activating mAb 56-4 for 48 h. ***P < 0.001 compared with neutral Ab treatment.

Fig. 5.

E-cadherin activation increases cancer cell-specific apoptosis. Sections of lung from neutral or activating mAb-treated mice described in Fig. 2B were examined for cleaved caspase-3 by immunofluorescence (A). Cells were measured in at least 10,000 cells, and each percentage represents the average of three randomly chosen fields of 1 sample (×10; n = 4 per group; no graft, n = 3). (B and C) 4T1 Luc-hE and MCF10a cells in culture were treated with 46H7 neutral or 19A11 activating mAb for 24 h. (B) Immunofluorescence staining for cleaved caspase-3 was tested in the 4T1 Luc-hE and MCF10a cells. Each percentage represents the average of three randomly chosen fields of 1 sample (×10; n = 3 per group). (C) Total RNA was prepared and analyzed for expression of the indicated transcripts by qRT-PCR, using specific primers. ***P < 0.001 compared with neutral Ab treatment. (D–F) Schema (D). Mouse was treated with i.p. injections of either 46H7 neutral or 19A11 activating mAb twice weekly until the end of the experiments. One day after beginning the treatment, 4T1 Luc-hE cells were i.v. injected into the tail vein and whole-blood was collected at the indicated times. CTCs expressing hE-cadherin were sorted by FACS. (E) Percentage of CTCs expressing hE-cadherin in blood. Population of cells expressing hE-cadherin per 10,000 cells was measured in peripheral blood mononuclear cells (PBMC) of each mouse by flow cytometry (n = 4). (F) mRNA expression of Bcl-xL (Left) and Bax (Right) in CTCs isolated by hE-cadherin. The levels were normalized by mRNA expression of hE-cadherin, and the fold change was assessed with the control group treated with neutral mAb 46H7 at each point. *P < 0.05; **P < 0.01; ***P < 0.001 compared with neutral Ab treatment. ns, not significant.