The Shp2-induced epithelial disorganization defect is reversed by HDAC6 inhibition independent of Cdc42

Abstract

Regulation of Shp2, a tyrosine phosphatase, critically influences the development of various diseases. Its role in epithelial lumenogenesis is not clear. Here we show that oncogenic Shp2 dephosphorylates Tuba to decrease Cdc42 activation, leading to the abnormal multi-lumen formation of epithelial cells. HDAC6 suppression reverses oncogenic Shp2-induced multiple apical domains and spindle mis-orientation during division in cysts to acquire normal lumenogenesis. Intriguingly, Cdc42 activity is not restored in this rescued process. We present evidence that simultaneous reduction in myosin II and ERK1/2 activity by HDAC6 inhibition is responsible for the reversion. In HER2-positive breast cancer cells, Shp2 also mediates Cdc42 repression, and HDAC6 inhibition or co-suppression of ERK/myosin II promotes normal epithelial lumen phenotype without increasing Cdc42 activity. Our data suggest a mechanism of epithelial disorganization by Shp2 deregulation, and reveal the cellular context where HDAC6 suppression is capable of establishing normal epithelial lumenogenesis independent of Cdc42.

Figure 1. Expression of oncogenic Shp2 impairs normal epithelial lumen formation through reducing Cdc42 activity.

MDCK cells expressing GFP-Shp2-WT and GFP-Shp2-E76G were embedded in Matrigel. (a) After 2 days, cysts were extracted and fixed for immunofluorescence (IF) staining with antibodies as indicated. The percentage of cysts with single lumen is shown on the right (312–486 cysts were counted in each experiment, n=3). (b) After 7 days, cysts were extracted for IF staining, scale bar, 10 μm. Right panel shows the percentages of cysts with one single interior hollow lumen (584–767 cysts were counted in each experiment, n=3). (c) The 7-day cysts were harvested for western blot analysis with the indicated antibodies. (d) Cysts (2 days) were extracted for measuring the endogenous Cdc42 activity by G-LISA assay. (e) MDCK cells expressing Flag-Shp2-E76G were transfected with the expression vector of GFP or GFP-Cdc42-V12 and then embedded in Matrigel for 2 days. The formed cysts were fixed for IF stained. The percentage of cysts with single apical patch is shown below (203–241 green-positive cysts were counted in each experiment, n=3). Scale bars, 10 μm. All values are mean±s.d. from three independent experiments; statistical significance was analysed by two-tailed unpaired Student's t-test. *P<0.05, **P<0.01.

Figure 2. Regulation of Tuba-mediated Cdc42 activation by Shp2.

(a,b) HEK293T cells were transfected with HA-Tuba in the presence or absence of Flag-Shp2-E76G as indicated. Cells were harvested for (a) GST-PBD pull-down assay to determine endogenous Cdc42 activity, and (b) GEF activity of HA-tuba by GST-Cdc42-G15A pull-down assay. The representative Western blots for the assays are shown on the top. The relative Cdc42 and HA-Tuba GEF activity from three independent experiments are shown below. Data are presented as mean±s.d.; Two-tailed unpaired Student's t-test was performed, *P<0.05, **P<0.01. (c) HEK293T cells were transfected with or without Shp2 siRNA for 3 days, followed by transfection with the expression construct of HA-Tuba. HA-Tuba protein was immunoprecipitated with anti-HA beads for western blotting with anti-phosphotyrosine (4G10) and anti-HA antibodies. The total lysates were also subjected to Western blot analysis as indicated. (d) Cdc42 activity was measured by G-LISA assay in cells with and without Shp2 knockdown. Values are mean ±S.D from three independent experiments. **P<0.01 based on 2-tailed unpaired Student's t test. (e) Immunoprecipitated HA-Tuba protein from pervanadate-treated cells was incubated with increasing amounts of immunopurified Flag-Shp2-E76G protein at 30 °C for 30 min and analyzed by western blotting as indicated.

Figure 3. Dephosphorylation at Y430/Y515 by oncogenic Shp2 impairs Tuba-mediated Cdc42 activation and normal lumen formation.

(a) HEK293T cells were transiently transfected with HA-Tuba of WT and various mutants carrying Y→F mutation as indicated. Cells were treated with pervandate (50 μM) for 5 min before harvested for immunoprecipitation with anti-HA antibody. The immunoprecipitates were analysed by western blot with anti-phosphotyrosine (4G10) antibody followed by reporbing with anti-HA antibody. (b,c) HEK293T cells were transfected with control vector or HA-Tuba WT or YF (Y430/515F) mutant and harvested for (b) measuring GEF activity of HA-tuba by GST-Cdc42-G15A pull-down assay, and (c) endogenous Cdc42 activity by GST-PBD pull-down assay. The relative Cdc42 and HA-Tuba GEF activity from three independent experiments are shown. (d) MDCK cells were co-transfected with Tuba siRNA and WT or 430/515YF mutant of HA-Tuba and then cultured in Matrigel for 2 days. Cysts were extracted for IF staining with anti-GP135 (red) and anti-HA (green) antibodies and Hoechst (blue), scale bar,10 μm. The percentage of cysts with single apical patch is shown on the right (113–179 cysts were counted in each experiment, n=3). Data are presented as mean±s.d. from three independent experiments; two-tailed unpaired Student's t-test. *P<0.05, **P<0.01.

Figure 4. Role of HDAC6 in lumen formation and Microtubule acetylation in normal lumenogenesis.

MDCK cells expressing GFP-Shp2-WT and GFP-Shp2-E76G were transfected with HDAC6 siRNA. (a) Western blot analysis. RT-PCR shows the knockdown effect.(b) Cells grown in Matrigel for 2 days were fixed for immunostained with antibodies as indicated. The percentage of cysts with single apical domain is shown in the right (516–641 cysts were counted in each experiment, n=3). (c) Control MDCK cells were co-transfected with control vector or HDAC6-Flag with GFP-C1 (amount of DNA ratio at 3:1). These cells were embedded into Matrigel for 2 days. Cysts were fixed for IF staining as indicated. The percentage of GFP positive cysts with single apical domain is shown at the bottom (167–193 green cysts were counted in each experiment, n=3). (d) Control MDCK cells were transfected with GFP-Tubulin WT or K40R mutant and embedded in Matrigel for 2 days. Cysts were fixed for IF staining with anti-GP135 (red) and Hoechst (blue), scale bar, 10 μm. The percentage of cysts with single apical domain is shown below (104–121 green-positive cysts were counted in each experiment, n=3). Values are mean±s.d. from three independent experiments. **P<0.01 based on two-tailed unpaired Student's t-test.

Figure 5. HDAC6 inhibition allows normal apical domain formation independent of Cdc42 signal.

(a,b) MDCK expressing GFP-Shp2-WT or GFP-Shp2-E76G were embedded in Matrigel and treated with or without 1 μM of tubacin for 24 h. (a) Cyst were extracted for IF staining analysis, and the percentage of cysts with single apical domain is shown in the right (514–637 cysts were counted in each experiment, n=3.) (b) Cyst cultures were further treated with 10 μM of RO-3306 for 16 h to synchronize cells at the G2/M phase, and then released by replacement with normal medium for 40–50 min. Cysts were extracted for spindle analysis by α-tubulin IF and Hoechst staining. The angle between the spindle axis (thick line) and the thin line connecting the centre of cyst and the midpoint of spindle axis was measured. Scatter diagram of metaphase spindle angles are shown on the right. The data are presented as mean±s.e.m.; (n=20). (c) MDCK cells stably expressing GFP-Shp2-WT or E76G mutant were treated with tubacin (1 μM) for 2 days and harvested for Cdc42 activity by G-LISA assay. (d) GFP or GFP-Cdc42-N17-expressing MDCK cells in Matrigel and treated with tubacin (1 μM) for 2 days. Cysts were fixed and IF stained with antibodies as indicated. The percentage of cysts with single lumen is shown at the bottom (100 green-positive cysts were counted in each experiment, n=3). Values are mean±s.d. from three independent experiments.*P<0.05, **P<0.01 based on two-tailed unpaired Student's t-test. (e) MDCK cells in Matrigel were treated with 20 μM of aPKC inhibitor with or without tubacin (1 μM) for 24 h, followed by synchronization with RO-3306 for spindle observation. Right panel shows the spindle angle and data were presented as (b). ***P<0.001 based on two-tailed Student's t-test. Scale bars, 10 μm.

Figure 6. The involvement of ERK/Myosin II in restoring normal lumenogenesis by HDAC6 inhibition.

Shp2-WT and E76G cells were embedded in Matrigel for 2 days. (a) Western blot analysis of 2-day cysts that had been daily treated with tubacin (1 μM). (b) Cyst cultures were treated with Y27632 (10 μM), U0126 (10 μM) or their combination for 2 days. Cysts were extracted for IF staining with antibodies as indicated, scale bar, 10 μm. The percentage of cysts with single apical domain is shown below (323–381 cysts were counted in each experiment, n=3). Data are presented as mean±s.d. from three independent experiments; two-tailed unpaired Student's t-test. **P<0.01. (c) The cysts were harvested for Cdc42 activity by G-LISA assay. (d) Control MDCK cells were transfected with the vector GFP-C1 in the presence of control vector or HDAC6-Flag as indicated (amount of DNA ratio at 1:3). These cells were embedded in Matrigel and treated with Y27632/U0126. Cysts were extracted and fixed for IF staining with GP135. Percentage of cysts with single apical domain (158–163 green-positive cysts were counted in each experiment, n=3) is shown below and analysed by two-tailed unpaired Student's t-test. **P<0.01. (e) Control MDCK cells transfected with the vector GFP-C1 or GFP-Cdc42-N17 were embedded in Matrigel and treated without and with Y27632/ U0126. Cysts were extracted and stained with GP135 (red) and Hoechst (blue). The bottom panel shows the percentage of cysts with single apical domain (104–117 green positive were counted in each experiment, n=3). Statistical significance was analysed by two-tailed unpaired Student's t-test. **P<0.01. (f) Control MDCK cells were treated with of aPKC inhibitor (20 μM) without and with Y27632/U0126. The percentage of cysts with single apical domain is shown below (346–393 cysts were counted in each experiment, n=3). Values are mean±s.d. from three independent experiments. **P<0.01 based on two-tailed unpaired Student's t-test.

Figure 7. Shp2 and HDAC6 influence epithelial organization of HER2-positive breast cancer cells.

(a) Various breast cancer cell lines were lysed and subjected for Cdc42 activity assay by GST-PBD pull-down. Relative endogenous Cdc42 activity from three independent experiments is shown. Statistical significance was analysed by two-tailed unpaired Student's t-test. *P<0.05, **P<0.01. (b) BT474 cells were transfected with Shp2 siRNA, and then harvested for Cdc42 activity assay. Data are presented as mean±s.d.; statistical significance was analysed by 2-tailed unpaired Student's t-test. *P<0.05, (c) BT474 cells were daily treated with Tubacin at the indicated concentration for 2 days, and harvested for western blot analysis. (d) Cells were cultured in Matrigel with daily treatment of tubacin (5 μM) for 2 weeks. These cysts were extracted for IF staining with anti-β-catenin (red) and Hoechst (blue). Percentage of cysts with single lumen (300 cysts were counted, n=3)Were analysed by two-tailed unpaired Student's t-test. **P<0.01. (e) Cdc42 activity was analysed in 2 days of cysts. (f) BT474 cells treated with or without Y27632 (10 μM) and U0126 (10 μM) for 2 weeks were extracted for IF staining. Statistical significance was analysed by two-tailed unpaired Student's t-test. **P<0.01. (g) Cdc42 activity in 2-day cysts by G-LISA assay.