The expression of the Nectin complex in human breast cancer and the role of Nectin-3 in the control of tight junctions during metastasis

Abstract

Introduction: Nectins are a family of integral protein molecules involved in the formation of functioning Adherens and Tight Junctions (TJ). Aberrant expression is associated with cancer progression but little is known how this effects changes in cell behaviour. This study aimed to ascertain the distribution of Nectins-1 to -4 in human breast cancer and the effect on junctional integrity of Nectin-3 modulation in human endothelial and breast cancer cells.

Methods: A human breast tissue cohort was processed for Q-PCR and immunohistochemistry for analysis of Nectin-1/-2/-3/-4. Nectin-3 over-expression was induced in the human breast cancer cell line MDA-MB-231 and the human endothelial cell line HECV. Functional testing was carried out to ascertain changes in cell behaviour.

Results: Q-PCR revealed a distinct reduction in node positive tumours and in patients with poor outcome. There was increased expression of Nectin-1/-2 in patients with metastatic disease, Nectin-3/-4 was reduced. IHC revealed that Nectin-3 expression showed clear changes in distribution between normal and cancerous cells. Nectin-3 over-expression in MDA-MB-231 cells showed reduced invasion and migration even when treated with HGF. Changes in barrier function resulted in MDAN3 cells showing less change in resistance after 2h treatment with HGF (p<0.001). Nectin-3 transformed endothelial cells were significantly more adhesive, irrespective of treatment with HGF (p<0.05) and had reduced growth. Barrier function revealed that transformed HECV cells had significantly tighter junctions that wildtype cells when treated with HGF (p<0.0001). HGF-induced changes in permeability were also reduced. Overexpression of Nectin-3 produced endothelial cells with significantly reduced ability to form tubules (p<0.0001). Immunoprecipitation studies discovered hitherto novel associations for Nectin-3. Moreover, HGF appeared to exert an effect on Nectin-3 via tyrosine and threonine phosphorylation.

Conclusions: Nectin-3 may be a key component in the formation of cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells.

Figure 1. Expression of the Nectin family in human breast cancer.

(A) and (B) Expression of Nectin-1 to -4 and nodal status of breast cancer patients at the transcript level. (C) and (D) Transcript levels and patient outcome. (E) and (F) Nectin transcript expression and metastatic disease in human breast cancer. (G) Nectins and patient survival. (H) Immunohistochemistry of Nectin-1 (left), Nectin-2 (middle) and Nectin-3 of representative patients tissues.

Figure 2. Immunohistochemical staining of Nectin-3 in human breast cancer and background tissue to show concentrated Nectin-3 inclusions in tumour sections.

Figure 3. Evaluation of Nectin expression in human breast cancer cell lines using RT-PCR (A).

Nectin-3 expression in human breast cancer cell lines showing sequential RT-PCR (B). Effect of cell confluency and Nectin-3 transcript and protein expression as assessed using Western Blotting (C). Confirmation of over-expression of Nectin-3 in human breast cancer and endothelial (HECV) cell lines (D).

Figure 4. Effect of Nectin-3 over-expression and knockdown in the human breast cancer cell line MDA-MB-231.

(A) Nectin-3 caused reduced motility, (B) decreased invasion, (C) and (D) reduced in vitro growth (even after treatment with HGF at 40 ng/ml), (E) increased trans-epithelial resistance (TER) and (F) reduced in vivo tumour growth. Knockdown of Nectin-3 resulted in increased motility in MDA-MB-231 (G) and MCF-7 (H) cells.

Figure 5. Effect of Nectin-3 over-expression in the human endothelial cell line, HECV.

(A) and (B) Nectin-3 caused increased resistance and (C) and (D) reduced paracellular permeability. No change was seen in in vitro growth assays (E and F). Nectin-3 over-expression caused reduced adhesion (G), but increased tubule formation (H).

Figure 6. Immunoprecipitation study of Nectin-3 (A, an antibody to the C-terminal region; B, an antibody to an internal region) and proteins that are involved in cell to cell adhesion (A).

Immunoprecipitation of relevant proteins probed with Nectin-3 (B). Ezrin precipitation and confirmation of Nectin-3 interaction (C). Phosphorylation study of Nectin-3 after treatment with HGF (40 ng/ml) and/or the ROCK inhibitor Y-27632 (D). Effect of Nectin-3 over-expression on the protein expression of Nectin-1, -2 and -4 (E). Effect of Nectin-3 over-expression on the protein expression of potential binding partners (F).