Rap1 integrates tissue polarity, lumen formation, and tumorigenic potential in human breast epithelial cells

Abstract

Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here, we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their nonmalignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to the formation of acinar structures with correct polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities and baseline growth. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

Figure 1

Down-modulation of Rap1 activity in malignant T4-2 cells restores tissue polarity in three-dimensional lrECM culture. A, phase-contrast images and Rap1 expression and activity levels in S1 and T4-2 cells cultured in three-dimensional lrECM. Bar, 10 μm. B, Western blot of endogenous Rap1 and exogenous DN-Rap1 in T4-vector and T4-DN-Rap1 cells. C, morphologies of T4-vector treated with and without AG1478 and T4-DN-Rap1 colonies in three-dimensional lrECM. Cell polarity was examined by staining markers for basal (α6-integrin), basolateral (β-catenin), and apical (GM130) membrane domains. Bar, 5 μm. D, cell proliferation in three-dimensional lrECM was determined by measuring colony size (left) and Ki-67–positive colonies (right). Columns, averages across three independent experiments; bars, SD. *, P < 0.05.

Figure 2

Bim-mediated lumina are formed in T4-DN-Rap1 cells but not in AG1478-treated T4-2 cells. A, phase-contrast images after 15 d of culture in three-dimensional lrECM, showing prominent lumina in acini of T4-DN-Rap1, but not in those of AG1478-treated T4-vector cells. The localization of actin filaments in apical membrane domains confirmed lumen formation and establishment of apical polarity in T4-DN-Rap1 acini. Apoptotic cell death was examined by staining for active caspase-3 at day 10 of culture. B, expression level of Bim in T4-vector, AG1478-treated T4-vector, and T4-DN-Rap1 cells at day 10 of culture in three-dimensional lrECM. Bar, 5 μm.

Figure 3

T4-DA-Rap1 cells show dose-dependent resistance to reversion by inhibitors of EGFR and MAPK. A, phase-contrast images and immunofluorescence localization of α6-integrin in T4-DA-Rap1 cells with or without 70 nmol/L AG1478 in three-dimensional lrECM. B, levels of β1-integrin, EGFR, and phospho-EGFR in three-dimensional lrECM–cultured T4-vector cells, and T4-DA-Rap1 cells in the presence and absence of 70 nmol/L AG1478. C, dose-dependent effect of reverting agents on T4-DA-Rap1 cells in three-dimensional lrECM. Cells were treated with different doses of inhibitors against EGFR (AG1478 and mAb225), MEK (PD98059), or PI3K (LY294002). Bar, 5 μm.

Figure 4

Level of Rap1 activity affects invasiveness and tumorigenic potential of T4-2 cells. A, invasiveness of T4-vector, T4-DA-Rap1, and T4-DN-Rap1 cells was examined using Matrigel-coated transwell filters. Quantification at 48 h after plating. B, the transfectants were s.c. injected into nude mice to examine tumorigenic potential. Tumor size measured at 8 wks after injection and mice bearing tumors larger than 10 mm³ were scored. C, histology of tumors derived from T4-vector and T4-DA-Rap1 cells.

Figure 5

Rap1 reverts the T4-2 malignant phenotype through different signaling pathways from those targeted by EGFR or β1-integrin. Activity and expression of the components of (A) EGFR-MAPK and (B) PTEN/Akt pathways were examined by Western blotting. C, the levels of FOXO1 protein and mRNA were determined by Western and reverse transcription-PCR analyses, respectively.