Cadherin-11 in poor prognosis malignancies and rheumatoid arthritis: common target, common therapies

Abstract

Cadherin-11 (CDH11), associated with epithelial to mesenchymal transformation in development, poor prognosis malignancies and cancer stem cells, is also a major therapeutic target in rheumatoid arthritis (RA). CDH11 expressing basal-like breast carcinomas and other CDH11 expressing malignancies exhibit poor prognosis. We show that CDH11 is increased early in breast cancer and ductal carcinoma in-situ. CDH11 knockdown and antibodies effective in RA slowed the growth of basal-like breast tumors and decreased proliferation and colony formation of breast, glioblastoma and prostate cancer cells. The repurposed arthritis drug celecoxib, which binds to CDH11, and other small molecules designed to bind CDH11 without inhibiting COX-2 preferentially affect the growth of CDH11 positive cancer cells in vitro and in animals. These data suggest that CDH11 is important for malignant progression, and is a therapeutic target in arthritis and cancer with the potential for rapid clinical translation.

Figure 1. CDH11 mRNA and protein are expressed in ductal carcinoma in situ and invasive breast carcinoma and regulates growth of MDA-MB-231 breast cancer transplanted cells

(A) CDH11 transcript is significantly increased in invasive carcinoma compared to normal breast (http://cancergenome.nih.gov), (B) and in invasive cancer stroma compared to normal stroma [11]. (C) Significant CDH11 up-regulation is also seen as an early event in ductal carcinoma in-situ [12]. The fold changes (FC) in the expression level of CDH11 and statistical analysis p values (Welch's t-test) were as follows: (A) Invasive ductal carcinoma, invasive lobular carcinoma and invasive mixed carcinoma vs. normal breast: p<0.0001 for all, FC: 1.75, 2.27, 2.44 respectively, (B) Invasive breast carcinoma stroma vs. normal breast stroma: p<0.0001, FC: 14.239. (C) Ductal carcinoma in-situ vs. normal breast: p=0.0194, FC: 2.160, invasive ductal carcinoma vs. normal breast: p=0.0034, FC: 2.896, invasive lobular carcinoma vs. normal breast: p=0.0077, FC: 2.603, invasive mixed carcinoma vs. normal breast: p=0.0015, FC: 3.413. Data presented as bars and whiskers (D) CDH11 immunohistochemical staining of human DCIS (left to right: comedo-type, cribriform and another comedo-type) and invasive ductal carcinoma as well as adjacent normal epithelium. (E) Growth inhibition of CDH11 positive MDA-MB-231 xenografts (>70%) upon treatment with 13C2 or control IgG with 0.5 mg initial I.P. injection followed by 0.1 mg subsequent injections (x3/week) for a month. (p=0.0365 compared to control (IgG), 28 d, two-tailed student's t-test). (F) Growth inhibition of established MDA-MB-231 tumors (>40%) upon I.P. treatment with 13C2 antibody or control (IgG) at 20 mg per kg body weight (x2/week) beginning when subcutaneous tumors were palpable. (p=0.0394 for 13C2 compared to control (IgG), 17 d post treatment start, two-tailed student's t-test). (G,H): Inhibitory effect of CDH11 knockdown on tumor growth in mice. Athymic nude mice were s. c. injected with 1-2×10⁶ MDA-MB-231 cells stably expressing (G) CDH11 siRNA, or (H) shRNA or controls into 1 of 4 mammary fat pads, such that all cell lines were represented in a minimum 10 total injections each (minimum of 2x per specific locus). For ILC images, please see Supplementary Fig. S2. The red arrows indicate CDH11 membranous staining. Black arrows point to a cell within the region that is magnified in the small insets. Scale bar: 45 μM. Small insets are 2.5x magnification of large images. Data are presented as means ± SEM. IDC, ILC and IMC: Invasive ductal, lobular and mixed carcinomas respectively, Invas: invasive.

Figure 2. CDH11 regulates colony growth and proliferation of MDA-MB-231 breast cancer cells and PC-3 prostate cancer cells

(A) Western blot analysis of CDH11 in MDA-MB-231 cells stably expressing CDH11 shRNA (33 or 34 clonal cells) or siRNA (4A or 6A pooled cell lines). (B) Effect of CDH11 depletion on proliferation of MDA-MB-231 cells measured using crystal violet staining after 5 days. (C) Effect of CDH11 depletion on anchorage-independent colony formation in soft agar. (D) Western blot analysis of CDH11 in PC3 cells CDH11 shRNA, (E) CDH11 knockdown fails to significantly reduce the proliferation of PC3 cells but (F) colony formation is significantly reduced upon CDH11 depletion. (EV, 1B): Empty vector. (Luc) scrambled control. GAPDH was used as a loading control for western blot. Phase image using a 4x objective on a Zeiss inverted microscope. Data are presented as means ± SEM (Student's t-test).

Figure 3. CDH11 knockdown significantly inhibits migration and mediates colony formation of MDA-MB-231 breast and PC-3 prostate cancer cells and is required for the growth and invasion of LN229 glioblastoma cells

(A) Effect of CDH11 depletion on the ability of MDA-MB-231 cells and (B) PC3 cells to migrate (x3 separate fields in each well from triplicate wells) 16hr after wounding. (C) Effect of CDH11 depletion on the formation of branched networks on Matrigel™. (D-F) CDH11 or N-cadherin was knocked down in LN229 cells using shRNA. (D) Western blot showing significant reduction in CDH11 and N-cadherin protein, 48 hours post infection with lentivirus containing shRNA. (E) CDH11 knockdown reduces migration and (F) growth of LN229 cells. Note that CDH11 depletion was more effective than N-cadherin knockdown in growth inhibition of LN229 cells. P values determined at day 5: ***p=0.0001, **p=0.0016, *p=0.0425, Ncad#41 vs. pLKO: not significantly different. Columns and bars show the mean and SEM respectively (two-tailed student's t-test).

Figure 4. Structural modeling of celecoxib, DMC and other small molecule inhibitors binding to CDH11 and inhibiting the growth of MDA-MB-231 cells. Sd-133 binding capability to CDH11 was validated by SPR

(A) EC1 homodimer interface of CDH11 (PDB: 2A4C); one monomer is represented by the Van der Waals molecular surface (green) and the other by a ribbon. P1 is a hydrophobic, concave surface binding to two W residues from the partner EC1 monomer. P2 is a small pocket defined by the EC1 domain itself. Virtual screening was carried out with the residues lining P1 and P2. (B) The EC1 interface with the A strand motif ‘SGWVW’ of the partner EC1 domain (C-atoms-green) contains two W residues. Only residues (black) that make favorable hydrophobic, van der Waals and hydrogen bond contact with the motif (red) are highlighted (H-bonds-dashed lines). (C) 3D structural model of celecoxib and (D) DMC with interactive residue side chains at the tryptophan W-binding pocket (F7, L24, S26, Y37, A75, A77, E87, S90, F92 and W4) are shown in stick rendering, with the carbon atoms of CDH sidechains colored white and the carbon atoms of the inhibitors colored green. The polypeptide backbones are rendered as ribbons. The red broken lines indicate potential intermolecular hydrogen bonds. Oxygen atoms are shown in red, flourine in pale green, nitrogen in blue, and sulfur in yellow. (E) Blocking CDH11 with celecoxib and (F) DMC significantly reduced the proliferation of CDH11 positive MDA-MB-231 as measured using MTS assay. (G) Native gel comparison of cadherin-11 EC1-2 in the absence (left) and presence (right) of celecoxib. Celecoxib was solubilized in DMSO and mixed with purified CDH11 EC1-2 in a 1:1 molar ratio. Note that celecoxib reduces the dimer fraction. (H) Recombinant modified CDH11 protein was immobilized on a Biacore® CM5 Surface by thiol coupling method. Wild type cadherin-11 was injected at various concentrations using Biacore T-200 instrument. Each concentration was injected twice, which showed good binding reproducibility. Colored lines represent real data-points and black lines represent curve fits. (I) 2D structure of the active compounds. (J)Sd-133 competed with CDH11 (ligand) in binding to immobilized CDH11 protein on the surface of the chip.

Figure 5. Development of small molecule inhibitors and their effect on CDH11 function-inhibition

(A) Blocking CDH11 with sd-133 significantly reduced the proliferation of CDH11 positive MDA-MB-231 as measured by MTS assay. (B) Sd-133 did not inhibit the growth of CDH11-negative MDA-MB-435 melanoma or MCF7 breast cancer cell lines. (C) Sd-037 and Sd-133 significantly impaired MDA-MB-231 outgrowth on Matrigel™. (D) Sd-133 fails to change Matrigel™ morphology of CDH11 negative MDA-MB-435 and MCF7 cells. (E) Effect of sd-133 on anchorage independent colony growth in soft agar. (F) Colony growth at various sizes when MDA-MB-231 cells were treated with Sd-133. (G) Likely binding model of Sd-133. W-binding pocket residues are highlighted (C-atoms-white; H-bonds-red dotted lines). Residues F7, L24, S26, Y37, A75, A77, E87, S90, and F92 contribute hydrophobic interactions and a water mediated interaction with Sd-133. The hydrophobic and H-bond interaction between Sd-133 and CDH11 is similar to that of the two W as seen in (Fig. 5F). (H) Diagram of the concave surface of P1 and P2. W-binding pocket residues are highlighted (C-atoms-white; H-bonds-red dotted lines). Sd-133 is locked into the cavity with H-bond networks on the outside of the concave surface. (I) The H-bond and hydrophobic interactions of Sd-037 and (J) Sd-073 are similar to Sd-133. (K) Superimposition of Cadherin-11 inhibitors Sd-133, Sd-037 and Sd-073 (C-atoms-white) with the W of a partner EC1 monomer motif (C-atoms-green). C: control. Columns and bars show the mean and ESM respectively.

Figure 6. 4-[5-(2,5-Dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzene sulfonamide (DMC) significantly reduced the proliferation of MDA-231 cells but had no effect on CDH11-negative MCF7 cells in vivo

Nude mice were implanted with 6x10⁶ MDA231 (N=4/cohort) or MCF7 cells (N=3/cohort) in the mammary fat pad. Once the tumors became palpable, animals were treated with (150 mg/kg/day) of DMC or vehicle for 48 hours and euthanized 2 hours post final treatment. (A) Representative ki67 immunostaining (proliferation marker), TUNEL (apoptosis marker) and corresponding H&E sections from xenografts. (B) DMC significantly lowered the proliferation rate of MDA231 transplanted cells but not MCF7 cells. (C) DMC did not have any significant effect on the apoptosis rate of cancer cells. Arrows indicate representative proliferative cells. Black arrow-heads demonstrate apoptotic cells and red arrow-heads indicate mitotic-figures. Scale bar= 20 micrometers. Columns and bars show the mean and ESM respectively (student's t-test). ***p=0.0006