Tuberin regulates E-cadherin localization: implications in epithelial-mesenchymal transition

Abstract

The tuberous sclerosis complex 2 (TSC2) gene encodes the protein tuberin, which functions as a key negative regulator of both mammalian target of rapamycin (mTOR) C1-dependent cell growth and proliferation. Loss-of-function mutations of TSC2 result in mTORC1 hyperactivity and predispose individuals to both tuberous sclerosis and lymphangioleiomyomatosis. These overlapping diseases have in common the abnormal proliferation of smooth muscle-like cells. Although the origin of these cells is unknown, accumulating evidence suggests that a metastatic mechanism may be involved, but the means by which the mTOR pathway contributes to this disease process remain poorly understood. In this study, we show that tuberin regulates the localization of E-cadherin via an Akt/mTORC1/CLIP170-dependent, rapamycin-sensitive pathway. Consequently, Tsc2(-/-) epithelial cells display a loss of plasma membrane E-cadherin that leads to reduced cell-cell adhesion. Under confluent conditions, these cells detach, grow in suspension, and undergo epithelial-mesenchymal transition (EMT) that is marked by reduced expression levels of both E-cadherin and occludin and increased expression levels of both Snail and smooth muscle actin. Functionally, the Tsc2(-/-) cells demonstrate anchorage-independent growth, cell scattering, and anoikis resistance. Human renal angiomyolipomas and lymphangioleiomyomatosis also express markers of EMT and exhibit an invasive phenotype that can be interpreted as consistent with EMT. Together, these results suggest a novel relationship between TSC2/mTORC1 and the E-cadherin pathways and implicate EMT in the pathogenesis of tuberous sclerosis complex-related diseases.

Figure 1

Tsc2 regulates E-cadherin localization. Immunofluorescence analysis of wild-type rat kidney epithelial NRK52E cells (A) and Tsc2(−/−) rat kidney tumor epithelial LEF2 cells (B) using anti-E-cadherin antibodies (green). C: In loss-of-function experiments, NRK52E cells were transiently transfected with Cy3-labeled (red) Tsc2 siRNA or control siRNA (siCntrl) and analyzed for E-cadherin expression (green). Western blot shows effects of siRNA on Tsc2 and E-cadherin expression. α-Tubulin serves as the loading control. C, control siRNA. D: In gain-of-function analyses, LEF2 cells were transiently transfected with Tsc2 expression plasmid (red) and stained for E-cadherin (green). Two examples are shown. Nuclei were stained with DAPI (blue).

Figure 2

Membrane E-cadherin localization is dependent on mTORC1 activity. A: Effects of rapamycin (RAP, 200 nmol/L, 24 hours), wortmannin (1 μmol/L, 24 hours), or both inhibitors on E-cadherin distribution in LEF2 cells. Green, E-cadherin; blue, nuclei. B: Western blot analysis shows expression of phospho-S6(Ser235/236), phospho-AKT(Ser473), and E-cadherin for the conditions shown in A after a short exposure to inhibitors: rapamycin (RAP) for one hour and wortmannin for ten minutes. β-Actin serves as a loading control.

Figure 3

Influence of Akt-mTORC1-CLIP170 on E-cadherin localization. A: Immunofluorescence analysis of E-cadherin (green) in LEF2 cells transfected with Myr-Akt (red). B: NRK52E cells were transfected with mLST8 (left) or Rheb(Q64L) expression plasmids (right). Small insets show transfected cells (asterisk) in red. E-cadherin is shown in green. C: Examples of LEF2 cells that were transiently transfected with CLIP170 or control (Cy3) siRNA, followed by immunofluorescence analysis of E-cadherin (green). Nuclei were stained with DAPI (blue). Asterisks marks the transfected cells.

Figure 4

Tsc2 regulates cell-cell adhesion. A: Hanging drop assays were performed in HEK293T cells transfected with indicated vectors. Arrows highlight examples of aggregates of more than four cells 2 hours after plating. B: The number of cell aggregates per 100 cells counted after transfection of the indicated vectors was tabulated. The Western blot shows the expression of E-cadherin and the transgenes used in this experiment. C: Cell aggregation of wild-type (NRK52E) and mutant (LEF2 and ERC18M) cells in hanging drop assays. Graph indicates average number of aggregates per 100 cells counted. Arrows indicate cell aggregates. **P < 0.01.

Figure 5

Rapamycin affects E-cadherin-mediated cell-cell adhesion. A: NRK52E cells form aggregates that are dependent on E-cadherin function. Cells were pretreated with EGTA (2 mmol/L) or functional blocking anti-E-cadherin antibody (1:100) (Ab) or untreated followed by cell agitation assay. The number of aggregates per high-power field was tabulated. B: Aggregation of ERC18M [Tsc2(−/−)] cells with or without rapamycin (RAP) in a cell agitation assay. C: Response of the LEF2 [Tsc2(−/−)] cells to rapamycin alone or in combination with EGTA or anti-E-cadherin antibody to form aggregates relative to vehicle (DMSO) control in a cell agitation assay. Each experiment was repeated in triplicate. Arrows highlight cell aggregates. *P < 0.05; **P < 0.01.

Figure 6

Tsc2(−/−) cells detach and grow independent of anchorage. A: Growth pattern of wild-type NRK52E cells compared with that of the two independently derived Tsc2(−/−) cell lines (LEF2 and ERC18M) at confluence. Arrowheads indicate refractile cells that have detached from the plates. B: Cell viability assessment by trypan blue exclusion of the detached cells under different growth conditions. White bars, 10% FBS; black bars, 0.1% FBS. C: Colony formation in soft agar of wild-type and mutant cells assessed 2 weeks after plating and stained with 0.4% iodonitrotetrazolium solution. Graph indicates number of colonies for each cell type. *P < 0.05, compared with NRK52E. D: Effects of Tsc2 siRNA on postconfluent NRK52E cells. Left: Photomicrographs depicting the relative abundance of detached, refractile cells in control or Tsc2 siRNA-transfected cells. Right: The number of viable (based on trypan blue exclusion), nonadherent cells was counted. *P < 0.05 compared with control (siNC).

Figure 7

Nonadherent Tsc2(−/−) epithelial cells express markers of EMT. A: Western blot analysis of E-cadherin, occludin, and SMA in adherent (Adh) and nonadherent (N-Adh) cells derived from two Tsc2(−/−) epithelial tumor cell lines, LEF2 and ERC18M. β-Actin served as a loading control. B: RT-PCR analysis of LEF2 cells showing expression of E-cadherin, Snail, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control in adherent and nonadherent states. C: SMA expression in adherent and nonadherent LEF2 cells analyzed by immunofluorescence analysis (left) and Western blotting analysis (right). DAPI-stained nuclei show corresponding cells in the same fields as the SMA immunofluorescence. D: Expression of SMAD proteins in adherent and nonadherent cells.

Figure 8

Nonadherent Tsc2(−/−) cells exhibit functional evidence of EMT. A: Anoikis assay of wild-type NRK52E and Tsc2(−/−) LEF2 cells shows the proportion of viable cells when cultured on poly-(2-hydroxyethyl methacrylate) plates over 3 days under high- and low-serum conditions. B: Comparison of adherent (Adh) and nonadherent (N-Adh) Tsc2(−/−) LEF2 and ERC18M cells in the cell scattering assay. Top panels illustrate the appearance of compact and scattered colonies seen in the adherent and nonadherent mutant cells, respectively. The proportion of compact and scattered colonies is tabulated as a percentage of the total number of colonies counted. At least 100 colonies were assessed for each category. C: Effects of rapamycin (RAP, 200 nmol/L) on the morphology of LEF2 cells at confluence. Detaching refractile cells are indicated by arrowheads. DMSO, vehicle control. D: Effects of rapamycin (RAP) on the expression of E-cadherin and SMA in nonadherent LEF2 cells. A change in phospho-S6 indicates the rapamycin effect. β-Actin served as a loading control. E: Effects of rapamycin on anoikis in LEF2 cells.

Figure 9

AML and LAM show evidence of EMT. A: Western blot analysis shows the expression of EMT markers (smooth muscle actin [α-sm-actin] and fibronectin), total and phospho (p)-Smad2, total and phospho-S6, and actin (loading control) in tissue lysates from human normal kidney (NK), AML, normal lung, and LAM samples. B: Immunohistochemical analyses of Snail in LAM and AML. Note nuclear staining in the abnormal cells. Right panels: high-power views of the boxed areas. C: High-magnification view of a LAM nodule showing heterogeneous expression of Snail, suggesting distinct populations of LAM cells. D: Immunohistochemical analysis of TGF-β in LAM tissue. Note the absence of TGF-β expression in normal epithelium (arrow in right panel [high-magnification]). E: Immunofluorescence analysis of E-cadherin in LAM compared with normal bronchial epithelium.

Figure 10

LAM and AML cells exhibit an invasive growth pattern. Immunohistochemical analysis of LAM (top panels) using anti-smooth muscle actin antibody to highlight the LAM cells. Left panel: infiltration of LAM cells (brown) within lung parenchyma. Right panel: tumor cells (arrowheads) surrounding a blood vessel (arrow) in perivascular lymphatic spaces. Bottom panels: phospho-S6 expressing AML cells invading the normal kidney parenchyma. Arrow in left panel indicates a normal collecting duct with phospho-S6 immunoreactivity. Arrows in right panel indicate normal renal tubules surrounded by AML cells.