HER2/ErbB2-induced breast cancer cell migration and invasion require p120 catenin activation of Rac1 and Cdc42

Abstract

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.

FIGURE 1.

ErbB2 induces expression of p120 in mammary epithelial cells. A, Affymetrix expression array (U74Av2) analysis of age-matched wild type glands compared with MMTV-c-Neu tumors revealed a ∼4-fold increase in p120 mRNA in tumors. The values represent the means ± S.D. *, p < 0.001. B, whole cell lysates of mouse mammary tumors or WT glands were generated, and samples were processed for Western blot analysis for p120 expression. C, freshly isolated epithelial cells from WT mammary glands and MMTV-c-Neu tumors were cultured on coverslips and processed for indirect immunofluorescence. The cells were stained with antibodies against E-cadherin, p120, and ZO-1. Note the mislocalization of p120 in tumor cells compared with WT mammary epithelial cells. D, the immortalized, nontransformed human mammary epithelial cell line MCF-10A was retrovirally transduced with a NeuN/ErbB2 expression cassette, and multiple populations were generated and analyzed for p120 expression (n = minimum of three replicates for each experiment). Scale bar, 20 μm.

FIGURE 2.

ErbB2-associated migration and invasion requires sustained expression of p120. A, species-specific oligodeoxynucleotides were used to generate lentiviral shRNA vectors (10). Stable p120 knockdown populations were generated from EN064, MCF-10A, 10ANeuN, and BT-474 cell lines. The mshRNA targets the mouse p120 mRNA, whereas hshRNA targets the human p120 mRNA. These sequences differ by three nucleotides. B, 10A or 10ANeuN cells that express either a nonsilencing shRNA (ns) or a p120-specific shRNA (p120kd) were cultured on coverslips and processed for indirect immunofluorescence. The cells were stained with antibodies against p120 and β-catenin and counterstained with DAPI. C, migration of ErbB2-overexpressing mammary epithelial cells requires sustained p120 expression. Wound healing was assessed in 10ANeuN cells expressing a nonsilencing p120 shRNA (ns) or a p120 species-specific shRNA (p120kd) over 14 h. D, ErbB2-dependent cells require p120 expression for migratory activity. EN064 and BT474 cell migration was analyzed with or without p120 suppression over a 12-h period of chemotactic migration in modified Boyden chambers toward 10% FBS. E, the ability of p120 to regulate cell invasion was examined with 10ANeuN ns and 10ANeuN kd cells in Martigel^TM-coated invasion chambers. The values represent the means ± S.D. **, p < 0.001; *, p < 0.05 compared with the same cell line not treated with inhibitor or transduced with the ns vector (n = minimum of three replicates for each experiment).

FIGURE 3.

p120 is required for ErbB2-induced activation of Rac1 and Cdc42. A, ErbB2 activation of Rac1 or Cdc42 requires p120 expression. Rac1 and Cdc42 activation levels were compared between parental MCF-10A, 10ANeuN, and 10ANeuN kd cells. The values represent the means ± S.D. *, p < 0.001 (n = 3). B, silencing p120 results in reduced phosphorylation of PAK1, an established target of Rac1 and Cdc42. C, add-back cell lines were generated by retroviral transduction with expression vectors for CA-Rac1 or CA-Cdc42 into 10ANeuN kd cells. The cells were serum-starved and subjected to chemotactic migration assays toward serum over a 16-h period. The values represent the means ± S.D. *, p < 0.001 (n = 3).

FIGURE 4.

Overexpression of p120 in MCF-10A cells mimics the ability of ErbB2 to induce migration but not invasion. A, cDNA clones for p120 isoforms 1 and 3 were generated from EN064 cell mRNA. Ectopic expression was achieved by retroviral transduction of both p120 isoforms into MCF-10A cells. Protein expression was compared with 10ANeuN and parental MCF-10A cells. pY1248 is a phosphorylated form of ErbB2, indicating active signaling from this receptor. B, cells were serum-starved overnight and subjected to Rac1/Cdc42 pulldown assays. MCF-10A cells expressing p120 (10Ap120) exhibited indistinguishable levels of activated Rac1 and Cdc42 compared with ErbB2 overexpression in MCF-10A (10ANeuN) cells. C and D, MCF-10A cells overexpressing ErbB2 (10ANeuN) or p120 (10Ap120) were serum-starved overnight, and chemotactic migration (C) and invasion (D) were assessed over a 14-h period. The values represent the means ± S.D. *, p < 0.001 (n = 3). E, 10A, 10ANeuN, and 10Ap120 cells were maintained in serum-free medium for 48 h, and conditioned medium was collected. The activity of MMP2/9 in conditioned medium was analyzed by gelatin zymography (n = 2).

FIGURE 5.

Ectopic expression of p120 accelerates experimental metastasis. A, 474L/vec and 474L/p120–2 cells were maintained in complete medium, and whole cell lysates were prepared in radioimmune precipitation assay buffer. Protein expression was compared by Western blot. B, evaluation of p120 overexpression on ErbB2-dependent cell migration in Transwell® assays. Migration in response to 10% serum for 16 h was examined for cells containing the empty vector (474L/vec) or 474L/p120–1 and 474L/p120–2 mixed population cell lines that overexpress isoforms 1 and 3 of p120,. *, p < 0.001 (n = 3). C, representative photographs of an 8-week-old female NCR nu/nu mice following intracardiac injections with 474L/vec or 474L/p120–2 cells and noninvasively imaged utilizing luciferase-mediated bioluminescence. The images were captured at 0, 15, and 45 days after tumor cell injection. D, extent of metastases was assessed for individual animals in the experimental metastasis model. The bioluminescence intensity above a negative tissue threshold was calculated, and any mice that had intensities higher than this threshold were considered positive for metastases (n = 7).

FIGURE 6.

Src activation is required for ErbB2 induction of p120 expression. A, EN518- and ErbB2-expressing mouse mammary tumor cells require Src activation for migratory activity. EN518 cell migration was analyzed with or without the following inhibitors: Iressa (Ir; 1 μm), PD168393 (PD16; 1 μm), PD98059 (PD98, 20 μm), and PP2 (10 μm) over an 8-h period of chemotactic migration in modified Boyden chambers toward 10% FBS. Con, control. B, blockade of ErbB2, EGFR, and Src prevents induction of p120 expression. Heregulin (5 μg/ml) induction of p120 expression in SKBR3 cells can be attenuated by pretreatment with various inhibitors. SKBR3 cells were stimulated with heregulin (5 μg/ml) over a 2-h time course, and lysates were probed for alterations in p120 expression. Total and phosphorylated forms of each target of the inhibited proteins were assessed to confirm inhibitor efficacy (supplemental Fig. 3B). C and D, SkBr3, 10A, and 10ANeuN cells were transiently transfected with a Dharmacon siRNA SMART pool against human Src. 48 h post-transfection the cells were harvested and assessed for p120 expression. (n = minimum of three replicates for each experiment).

FIGURE 7.

Expression of p120 is maintained in most, if not all, primary human breast cancers and distal metastasis regardless of receptor status. A, representative paraffin-embedded samples of 60 primary human breast cancers were selected based on receptor composition and were immunohistochemically stained for p120 catenin. Expression of p120 was retained in all samples and differed only by intracellular location. B, the distinct patterns of subcellular localization of p120 in lobular and ductal primary tumors were preserved in unpaired samples of distal metastases. C, p120 expression is maintained in distal metastases. Representative samples of nine matched pairs of primary breast cancers and their corresponding metastases were stained for p120 catenin and evaluated for expression levels and subcellular localization. Two representative pairs are shown. Bars, 20 μm.

FIGURE 8.

A schematic depiction of p120 in the context of ErbB2 regulation of migration/invasion and metastasis. Oncogenic amplification and overexpression of ErbB2 initiates metastatic signals mediated by Src. Our model shows p120 to be a critical intermediary that transduces the pro-migratory signals originating from ErbB2 through Src. Other studies have shown that ErbB2-dependent cells modulate Src activity, which stabilizes functional ErbB2/ErbB3 heterodimers (5). Elevated Src activity may directly contribute to the cytoplasmic pool of p120. It is not clear whether this fraction of p120 requires phosphorylation by Src to propagate ErbB2 signaling. However, it is evident that p120 is necessary to induce Rac1 and Cdc42 activity in highly migratory, ErbB2-dependent cells.