In vivo selection for metastasis promoting genes in the mouse

Abstract

Here, we report the identification of a metastasis promoting factor by a forward genetic screen in mice. A retroviral cDNA library was introduced into the nonmetastatic cancer cell line 168FARN, which was then orthotopically transplanted into mouse mammary fat pads, followed by selection for cells that metastasize to the lung. The genes encoding the disulfide isomerase ERp5 and beta-catenin were found to promote breast cancer invasion and metastasis. Disulfide isomerases (thiol isomerases), which catalyze disulfide bond formation, reduction, and isomerization, have not previously been implicated in cancer cell signaling and tumor metastasis. Overexpression of ERp5 promotes both in vitro migration and invasion and in vivo metastasis of breast cancer cells. These effects were shown to involve activation of ErbB2 and phosphoinositide 3-kinase (PI3K) pathways through dimerization of ErbB2. Activation of ErbB2 and PI3K subsequently stimulates RhoA and beta-catenin, which mediate the migration and invasion of tumor cells. Inhibition of ErbB2 and PI3K reverses the phenotypes induced by ERp5. Finally, ERp5 was shown to be up-regulated in human surgical samples of invasive breast cancers. These data identify a link between disulfide isomerases and tumor development, and provide a mechanism that modulates ErbB2 and PI3K signaling in the promotion of cancer progression.

Fig. 1.

Identification of ERp5 as a metastasis-promoting gene. (A) The scheme for the forward genetic screen. (B) Model establishment: 168FARN tumor cell growth in the primary site after transplantation (1 × 10⁶ cells) to mammary fat pad in BALB/cJ mice (B Upper Left). Dissection of mice 7 weeks posttransplantation shows no metastasis in secondary organs (B Lower). 168FARN cells are able to form lung metastasis after tail vein injection (1 × 10⁶ cells) (B Upper Right). (C and D) Screening and validation: Tumor cell growth in the primary site after injection of 168FARN cells (1 × 10⁶ cells) containing a cDNA library in mammary fat pad. (C) Two of five mice developed lung metastasis. Transplantation of 168FARN cells stably overexpressing ERp5 in mammary fat pad leads to lung metastasis. (D) Mice were imaged 6–8 weeks after transplantation.

Fig. 2.

ERp5 promotes tumor cell migration and invasion in vitro. MDA-MB-436 and MCF7 cells were serum-starved for 24 h before the transwell assay. Complete medium was added to the bottom wells of the transwell chambers. Cells that migrated to the lower surface of the filters were fixed and counted 12 h after they were added to the upper chamber. Stable ERp5 overexpression in human breast cancer MDA-MB-436 cells causes 100% more migrated cells (A) and 60% more invaded cells (B) in a transwell assay compared with these cells with a control vector. The expression of ERp5 in human breast cancer MCF7 cells causes a phenotype change from nonmigratory and noninvasive to significant migration (A) and invasion (B). Suppression of cell surface disulfide exchange blocks cell migration and invasion. Treatment of cells with 1 mM DTNB reduces migration of MDA-MB-436 cells by 40%, reduces migration of MDA-MB-436 cells overexpressing ERp5 by >80%, and reduces migration of MCF7 cells overexpressing ERp5 by >60% compared with DMSO treatment as a control (C). DTNB (1 mM) has a similar effect on cell invasion: MDA-MB-436 cell invasion is inhibited by 30%; invasion by MDA-MB-436 cells overexpressing ERp5 is inhibited by 80%; and invasion by MCF7 cells overexpressing ERp5 is inhibited by >80% (D). DTNB (1 mM) has no effect on cell growth (E).

Fig. 3.

ERp5 activates ErbB2 pathway. (A) The expression level of total ErbB2 does not change after ERp5 overexpression in human breast cancer MDA-MB-436 and MCF7 cells. (B) Immunoprecipitation of phosphorylated ErbB2 demonstrates ErbB2 is activated by ERp5 overexpression in MDA-MB-436 and MCF7 cells. (C) Immunoblots under nonreducing condition show that ERP5 promotes dimerization of ErbB2 in MDA-MB-436 and MCF7 cells. (D) Knockdown of ErbB2 by a specific shRNA reduces the effect of ERp5 on migration and invasion in MDA-MB-436 and MCF7 cells.

Fig. 4.

ERp5 activates PI3K pathway. (A) Immunoprecipitation of phosphorylated PI3K in MDA-MB-436 and MCF7 cells demonstrates that ERp5 overexpression activates PI3K, whereas it has no effect on the expression level of total PI3K. (B) PI3K inhibitors Ly294002 (10 μM) and wortmannin (0.1 μM) reverse the effect of ERp5 in the migration and invasion assay in MDA-MB-436 and MCF7 cells. (C) Stable ERp5 overexpression in MDA-MB-436 and MCF7 cells leads to the activation of RhoA and Akt but has no effect on the total protein expression level of these two proteins. The activation of Akt subsequently up-regulates β-catenin.

Scheme 1.

The molecular mechanisms of ERp5 in promoting tumor invasion and metastasis.

Fig. 5.

ERp5 expression is up-regulated in the clinical tumor samples of invasive ductal carcinoma. (A) Immunostaining with an ERp5 antibody of the tumor tissue array containing normal breast tissues and breast ductal carcinoma at various stages was scored on a scale of 0 to 3: 0 represents no staining, and 3+ represents the strongest staining. ERp5 expression is elevated in T2N0 and T2N1 compared with normal breast duct and T1N0 tumors. (B) The percentage of tumor samples on the array with different scores was tabulated. The percentage of tumor samples with 2+ scores is higher in invasive breast cancer: 21.7 and 37.5% of T2N0 and T2N1 stage, respectively, compared with 0% in normal breast ductal structure and 4.3% in T1N0 tumors.