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Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity

Abstract

E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here, we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas of cell–cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is PLEKHA7 dependent. The PLEKHA7–microprocessor complex co-precipitates with primary microRNAs (pri-miRNAs) and possesses pri-miRNA processing activity. PLEKHA7 regulates the levels of select miRNAs, in particular processing of miR-30b, to suppress expression of cell transforming markers promoted by the basolateral complex, including SNAI1, MYC and CCND1. Our work identifies a mechanism through which adhesion complexes regulate cellular behaviour and reveals their surprising association with the microprocessor.

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Figure 1: Polarized epithelial cells show distinct p120-associated populations at the junctions.
Figure 2: Biochemical separation of two distinct junctional complexes by proteomics.
Figure 3: PLEKHA7 suppresses anchorage-independent growth and expression of transformation-related markers.
Figure 4: The basolateral junctional complex promotes anchorage-independent growth and expression of transformation-related markers.
Figure 5: PLEKHA7 suppresses protein expression through miRNAs.
Figure 6: PLEKHA7 associates with DROSHA and DGCR8 at the ZA.
Figure 7: Localization of DROSHA and DGCR8 at the ZA is PLEKHA7 dependent.
Figure 8: PLEKHA7 regulates pri-miR-30b processing at the junctions.

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Acknowledgements

This work was supported by NIH R01 CA100467, R01 NS069753, P50 CA116201 (P.Z.A.); NIH R01 GM086435, Florida Department of Health, Bankhead-Coley 10BG11 (P.S.); NIH/NCI R01CA104505, R01CA136665 (J.A.C.); BCRF (E.A.P.); the Swiss Cancer League (S.C., Project KLS-2878-02-2012). A.K. is supported by the Jay and Deanie Stein Career Development Award for Cancer Research at Mayo Clinic. We thank Mayo Clinic’s Proteomics Core and B. Madden for assistance with mass spectrometry, B. Edenfield for immunohistochemistry, M. Takeichi, D. Radisky and M. Cichon for constructs, and D. Radisky, L. Lewis-Tuffin, J. C. Dachsel, B. Necela and the late G. Hayes for suggestions and comments.

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A.K. designed the study, conceived and designed experiments, carried out all experiments except those described below, analysed the data and wrote the manuscript. S.P.N. made constructs. P.P. and S.C. developed antibodies and constructs. R.W.F. provided technical support. L.R.C. assisted with IF. T.R.B., J.M.C. and E.A.T. carried out the NanoString experiment and assisted with qRT–PCRs. I.K.Y. and T.P. assisted with the ISH assay. E.A.P., P.S., S.B. and J.A.C. developed and provided tissue micro-arrays. P.Z.A. conceived and designed the study, conceived and designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Panos Z. Anastasiadis.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Distinct complexes exist at the junctions of epithelial cells.

Caco2 or PLEKHA7-GFP transfected MDCK cells were grown to polarize, subjected to IF for PLEKHA7 or GFP respectively and co-stained for (a) p120; (b,c) E-cadherin (Ecad); (d) Afadin, Actin (phalloidin), Myosin IIA; (e) phosphorylated p120: Y228; (f) phosphorylated p120: Y96; (g) phosphorylated p120: T310; (h,i) Rac1; (j) RhoA. Stained cells were imaged by confocal microscopy and image stacks were acquired, as in Fig. 1. Representative xy image stacks are shown and/or the merged composite xz images. Scale bars for xy images: 20 μM; for xz images: 5 μM.

Supplementary Figure 2 PLEKHA7 localization to the junctions is E-cadherin- and p120-dependent.

(a) Western blot of the lysates from the separated apical and basolateral fractions shown in Fig. 2 for E-cadherin (Ecad), α-catenin, and β-catenin. (b) Caco2 control (NT) or E-cadherin knockdown (shEcad) cells, stained by IF for E-cadherin and PLEKHA7. (c) p120 and PLEKHA7 IF stainings of Caco2 control (NT) cells, p120 knockdown (shp120) cells, and of p120 knockdown cells transfected either with the full length murine mp120-1A isoform (shp120 + 1A) or the murine mp120-4A isoform that lacks the N-terminal PLEKHA7-binding domain (shp120 + 4A). All scale bars: 20 μM.

Supplementary Figure 3 PLEKHA7 loss from the junctions results in increased anchorage-independent growth and related signalling.

(a) Demonstration of the PLEKHA7 mRNA knockdown by qRT–PCR after infection of Caco2 cells with two PLEKHA7 shRNAs (shPLEKHA7 no. 8 and no. 10) or non-target control shRNA (NT) (mean ± s.d. from n = 3 independent experiments; P < 0.0001, Student’s two-tailed t-test). Source data are provided in Supplementary Table 2. (b) Caco2 control (NT) and PLEKHA7 shRNA knockdown (shPLEKHA7) cells were stained and imaged for PLEKHA7 and p120. (c) Caco2 control (NT) or PLEKHA7 knockdown cells (shPLEKHA7 #8, #10) grown on soft agar and imaged for colony formation (images are in 2× magnification; see Fig. 3c for quantitation). (d) MDCK cells were infected with either control (NT) or PLEKHA7 shRNA (shPLEKHA7#8) and subjected to western blot for the markers shown. Phosphorylation sites are denoted by p-. Actin is the loading control. (e) Soft agar assay of Caco2 cells infected with either vector control (adGFP) or a SNAI1-expressing construct (adSNAI1) (images are in 2× magnification; see Fig. 3h for quantitation). (f) Western blot of control (NT) or NEZHA knockdown (shNEZHA#72 and #73 shRNAs) Caco2 cells for the markers shown; Actin is the loading control. (g) IF of control (NT) or NEZHA knockdown (shNEZHA) Caco2 cells for PLEKHA7 and NEZHA. (h) IF of Caco2 cells transfected with either wild type murine mp120-1A (1A) or the murine mp120-ΔARM1 (ΔARM1) construct that cannot bind E-cadherin, stained with the murine-specific p120 antibody 8D11 (mp120) and co-stained with PLEKHA7. (i) Western blot of pcDNA (vector control), mp120-ΔARM1, or mp120-1A transfected cells, for the markers shown; Actin is the loading control. Single and double stars on the p120 blot indicate the 1A and ΔARM1 bands, respectively, right above the endogenous p120 bands. Scale bars for xy images: 20 μM; for xz images: 2.5 μM; for panels c and e: 2 mm.

Supplementary Figure 4 PLEKHA7 is mis-localized or lost in breast and renal tumour tissues.

Representative immunohistochemistry images of (a) breast and (b) kidney (renal), normal and cancer tissues stained for PLEKHA7, p120 and E-cadherin (Ecad) (left panels) and the percentage of tissues that exhibit presence, absence, or mis-localization of the three markers examined (right panels). BC: breast cancer; RCC: renal cell carcinoma. Scale bars: 20 μM. Number of tissues examined per cancer type/stage; BC TMA: Benign, n = 8; DCIS, n = 12; ILC ER+ Her-, n = 10; IDC ER+, Her-, n = 16; IDC Her+, n = 16; IDC Triple Negative, n = 13; RCC TMA: Matched normal, n = 119; stage 1, n = 71; stage 2, n = 20; stage 3, n = 22; stage 4, n = 6. (c) Caco2 control (NT), PLEKHA7 knockdown (shPLEKHA7), and PLEKHA7, p120 (shPLEKHA7, shp120) double knockdown cells were grown on soft agar for colony formation assay (images are shown in 2× magnification; see Fig. 4a for quantitation) and (d) examined by western blot for E-cadherin (Ecad) levels; α-tubulin is the loading control. (e) Caco2 cells treated with either vehicle (DMSO) or the Src inhibitor PP2 (10 μM) were stained for p120 and phosphorylated p120: Y228. Scale bars 20 μM; for panel c: 2 mm.

Supplementary Figure 5 PLEKHA7 acts via miRNAs but not post-translational modification mechanisms.

(a) qRT–PCR analysis for the indicated mRNAs of Caco2 control (NT) or PLEKHA7 knockdown cells (shPLEKHA7) (mean ± s.d. from n = 3 independent experiments). (b) Western blot for the markers shown (Actin: loading control) of control (NT) or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells treated with 20 μg ml−1 cycloheximide (CHX) or (c) 10 μM MG-132, for the indicated time points. (d) Western blot of control (NT) or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells for the markers shown; Actin is the loading control. (e,f) miR-30b and miR-19a qRT–PCR analysis of PLEKHA7-knockdown (shPLEKHA7) Caco2 cells after ectopic re-expression of PLEKHA7 (LZRS-PLEKHA7) (mean ± s.d. from n = 3 independent experiments; P < 0.05, Student’s two-tailed t-test) (g) Caco2 cells transfected with the indicated anti-miRs (a-miR) were subjected to western blot for the markers shown. (h) Caco2 control (NT) and PLEKHA7 knockdown (shPLEKHA7) cells were transfected with either control or miR-30b mimic and blotted for the markers shown. (i) Caco2 cells infected with either vector control (adGFP) or a SNAI1-expressing construct (adSNAI1) were subjected to western blot for the markers shown; Actin is the loading control. Source data for panels a,e,f are provided in Supplementary Table 2.

Supplementary Figure 6 The microprocessor complex localizes at the ZA.

(a) Northern blot analysis of control (NT) or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells using a miR-30b probe, indicating the pre-miR-30b and the mature miR-30b. U6 is the loading control. (b,c) IF of polarized Caco2 cells for p120 and DROSHA or DGCR8. Antibodies used here: DROSHA: Sigma and DGCR8: Sigma. The indication 2nd ab refers to the use here of a different antibody for DROSHA and DGCR8, compared to the antibodies used in Fig. 6d, e (for antibody details, see Supplementary Table 3). Enlarged details in boxes are shown on top of apical fields. The xz composite image of panel b is shown in Fig. 6f and of c in Fig. 6g. (d,e) IF of polarized MDCK cells for p120 and DROSHA (antibody: Abcam) or DGCR8 (antibody: Abcam). (f,g) siRNA-mediated knockdown of DROSHA (siDROSHA) and DGCR8 (siDGCR8) in Caco2 cells, shown both by IF (left side of each panel), and western blot (right side of each panel). Non-target (NT) siRNA is the control. p120 was used as a co-stain for the IF; Actin is the loading control for the blots; molecular weights (kDa) are indicated on the left side of each blot. (h) Western blots of PLEKHA7, p120, DROSHA, and DGCR8 IPs for PLEKHA7, p120 and E-cadherin (Ecad). IgG is the negative IP control. Scale bars: 20 μM; for enlarged parts of panels b and c: 3 μM.

Supplementary Figure 7 Regulation of the microprocessor at the ZA is PLEKHA7-depended.

(ac) IF of Caco2 cells transfected with either the wild type murine mp120-1A (1A) construct or the murine mp120-ΔARM1 (ΔARM1) construct that cannot bind E-cadherin, both stained with the murine-specific p120 antibody 8D11 (mp120) and co-stained with PLEKHA7, DROSHA, or DGCR8. Arrows indicate affected junctional staining of the indicated markers in transfected cells. (d) IF of control (NT) or p120 knockdown (shp120) Caco2 cells for DROSHA and p120. (e) qRT–PCR of control (NT) and p120 knockdown (shp120) Caco2 cells for the miRNAs shown (mean ± s.d. from n = 3 independent experiments; P < 0.05, Student’s two-tailed t-test). (f) IF of control (DMSO) or Nocodazole (10 μM for 8 h) treated Caco2 cells for PLEKHA7, DROSHA, or DGCR8. (g,h) IF of control (NT) or NEZHA knockdown (shNEZHA) Caco2 cells for DROSHA and DGCR8, co-stained with NEZHA. (i) qRT–PCR of control (NT), PLEKHA7 knockdown (shPLEKHA7), PLEKHA7 + p120 (shPLEKHA7 + shp120), and PLEKHA7 + Cadherin-11 (shPLEKHA7 + shCad11) double knockdown Caco2 cells for the indicated miRNAs (individual data points and mean from n = 2 independent experiments are shown). (j,k) IF of control (NT), PLEKHA7 knockdown (shPLEKHA7), and PLEKHA7 + p120 double knockdown (shPLEKHA7 + shp120) Caco2 cells for DROSHA and DGCR8, co-stained with p120. (l,m) IF of control (NT), PLEKHA7 knockdown (shPLEKHA7), and PLEKHA7 + Cadherin-11 double knockdown (shPLEKHA7 + Cad11) Caco2 cells for DROSHA and DGCR8, co-stained with Cadherin 11 (Cad11). Non-specific cytoplasmic or nuclear background appears respectively for the two Cadherin-11 antibodies used in the IF (see Methods for antibody details). All scale bars: 20 μM. Source data for panels e and i are provided in Supplementary Table 2.

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Kourtidis, A., Ngok, S., Pulimeno, P. et al. Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity. Nat Cell Biol 17, 1145–1157 (2015). https://doi.org/10.1038/ncb3227

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