Targeting ROR1 inhibits epithelial-mesenchymal transition and metastasis

Abstract

Metastasis is responsible for 90% of cancer-related deaths. Strategies are needed that can inhibit the capacity of cancer cells to migrate across the anatomic barriers and colonize distant organs. Here, we show an association between metastasis and expression of a type I receptor tyrosine kinase-like orphan receptor, ROR1, which is expressed during embryogenesis and by various cancers, but not by normal postpartum tissues. We found that expression of ROR1 associates with the epithelial-mesenchymal transition (EMT), which occurs during embryogenesis and cancer metastasis. Breast adenocarcinomas expressing high levels of ROR1 were more likely to have gene expression signatures associated with EMT and had higher rates of relapse and metastasis than breast adenocarcinomas expressing low levels of ROR1. Suppressing expression of ROR1 in metastasis-prone breast cancer cell lines, MDA-MB-231, HS-578T, or BT549, attenuated expression of proteins associated with EMT (e.g., vimentin, SNAIL-1/2, and ZEB1), enhanced expression of E-cadherin, epithelial cytokeratins (e.g., CK-19), and tight junction proteins (e.g., ZO-1), and impaired their migration/invasion capacity in vitro and the metastatic potential of MDA-MB-231 cells in immunodeficient mice. Conversely, transfection of MCF-7 cells to express ROR1 reduced expression of E-cadherin and CK-19, but enhanced the expression of SNAIL-1/2 and vimentin. Treatment of MDA-MB-231 with a monoclonal antibody specific for ROR1 induced downmodulation of vimentin and inhibited cancer cell migration and invasion in vitro and tumor metastasis in vivo. Collectively, this study indicates that ROR1 may regulate EMT and metastasis and that antibodies targeting ROR1 can inhibit cancer progression and metastasis.

Figure 1. High-level Expression of ROR1 In Breast Cancer Is Associated With Shorter Metastasis-free Survival and EMT Gene Signature

(A) Graph was derived from published data available through the PubMed GEO database (GSE2603, GSE5327, GSE2034, and GSE12276). Kaplan-Meier curves depict the prognostic impact of ROR1 expression on overall metastasis-free survival. For each analysis, 582 cases were segregated into tertiles with group designated ROR1_H representing the one-third of the patients who had tumors with the highest levels of ROR1 mRNA, and the group designated ROR1_L representing the one-third of patients who had cancers with the lowest levels of ROR1 mRNA. The one-third of patients who had tumors with intermediate expression of ROR1 mRNA was designated as ^ROR1_M. Metastasis-free survival was determined by Kaplan-Meier analyses, and statistical differences were determined by log-rank test. The number of patients in each category, the total metastatic events, and the corresponding P values (chi-square test) are shown in the embedded tables. (B) Heat map showing the expression of ROR1 (top), EMT-related genes in primary breast cancer cells isolated from patients. (C) Heat map showing the expression of EMT-related genes isolated from cells treated with ROR1-siRNA or CTRL-siRNA. (D) Immunoblots of protein lysates of MDA-MB-231, HS-578T, or BT549 (as indicated on the bottom) transfected with CTRL-shRNA or ROR1-shRNA, as indicated at the top. (E) Immunoblots of protein lysates of MCF7 transfected with a control vector or a ROR1-expressing vector, as indicated at the top.

Figure 2

Expression Of ROR1 By Breast Cancer Cell Lines Is Associated With Features Of EMT And Higher Metastatic Potential. (A) Morphological changes (40x) of MDA-MB-231, HS-578T, or BT549 (as indicated on the left) transfected with CTRL-shRNA or ROR1-shRNA, as indicated at the top. (B) Expression of CK-19, E-cadherin, or vimentin were detected by immunofluorescence staining in MDA-MB-231 cells transfected with CTRL-shRNA or ROR1-shRNA under 63x magnification. (C) Morphological changes (40x) of MCF7 cells transfected with control vector or ROR1-expressing vector (as indicated at the top). (D) Expression of CK-19, E-cadherin, or vimentin was detected by immunofluorescence staining of MCF7 cells transfected with either control vector or ROR1-expression vector (63x magnification). (E) Assays for cell migration (left) or invasion (right) on MDA-MB-231, HS-578T, or BT549 transfected with either CTRL-shRNA (black) or ROR1-shRNA (white). All data were normalized to results of cells transfected with CTRL-shRNA, which did not differ from those noted for parental cell lines. Data are shown as mean±SEM (n=3); *P<0.05, **P<0.01, ***P<0.001, compared with CTRL-shRNA group. (F) Representative photomicrographs of CTRL-shRNA-transfected MDA-MB-231 (left) or ROR1-shRNA-transfected MDA-MB-231 (right) in assays for cell-migration (top) or invasion (bottom).

Figure 3

Silencing ROR1 Inhibits The Metastatic Potential Of Mammary Fat Pad Xenografts. (A) Diagram depicting Stage I or II of the study. Mouse cartoon is modified from reference (29). (B) Tumor volumes over time (days) during stage I. (C) Weight of the tumors excised from each group. (D) Ex vivo photon flux of primary tumors of each group. (E–F) The in vivo (e) lung photon flux or (f) liver photon flux of each mouse during stage II was normalized with primary-tumor photon flux for each mouse. Histograms depict the normalized lung and liver photon flux of each group. (G) The in vivo lung photon flux during stage II of each group. (H) Horizontal bars indicate mean ex vivo lung photon flux of mice on d21 for each group (left). To the right are representative bioluminescence images of the extirpated lungs from each group. (I) Histograms represent lung-weight-index for each group. (J) Representative H&E-stained lung sections. (K) Horizontal bars indicate mean ex vivo liver photon flux of mice on d21 for each group (left). To the right are representative bioluminescence-images of extirpated livers on d21 of each group. (l) Representative H&E-stained sections of the liver on d21 of mice injected for each group. Data are shown as mean±SEM; P>0.05 is considered not significant (N.S.), *P<0.05, **P<0.01, ***P<0.001, compared with CTRL-shRNA group.

Figure 4

Silencing ROR1 Inhibits Experimental Pulmonary Metastasis Of MDA-MB-231. (A) Kaplan-Meier survival curves of mice injected i.v. with 5×10⁵ CTRL-shRNA-transfected or ROR1-shRNA-transfected cells (P<0.001 by log-rank test). (B) The in vivo lung photon flux of each group over time following injection (left). Representative bioluminescence images of mice from each group are depicted to the right. (C–E) Representative H&E-stained sections of the lung on (c) d3, (d) d21, and (e) d28. (F) Bottom histograms provide ex vivo lung GFP photon flux on d28 for each group. Representative bioluminescence images of the lungs (top) extirpated on d28. (G) The lung-weight-index from each group on d28 (bottom). Representative photographs of the lungs (top) of each group. (H) Kaplan-Meier survival curves of mice injected i.c. with 1×10⁵ CTRL-shRNA-transfected or ROR1-shRNA-transfected cells (P=0.0017 by log-rank test). (I) Representative bioluminescence images of mice following i.c. tumor injection. The white boxes define the area from which we acquired the bioluminescence data presented in (j). (J) The histograms provide the normalized in vivo bone photon flux of each group. (K) The ex vivo bone photon flux of the extracted pelvic bones of each group on d21. Representative bioluminescence images of the extracted pelvic bones are depicted to the right. (L) Representative H&E-stained histological bone sections of mice from each. Mouse cartoon is modified from reference (29). Mouse cartoon is modified from reference (29). Data are shown as the mean±SEM *P<0.05, **P<0.01, ***P<0.001, compared with CTRL-shRNA group.

Figure 5

An Anti-ROR1 Monoclonal Antibody D10 Inhibits Cancer-cell Migration And Metastasis. (A) D10 mAb causes internalization of ROR1. MDA-MB-231 cells were stained with control-IgG-Alexa647 (red), or D10-Alexa647 for 30 min on ice, and then either kept on ice (blue) or transferred to 37° C for 1h (orange) prior to flow cytometry. (B) Confocal microscopy of D10-stained (green) MDA-MB-231 cells before and after 1h incubation at 37 °C. (C) MDA-MB-231 cells were treated with or without (−) control IgG (IgG) or D10 for 24h prior to staining with a fluorochrome-labeled, non-cross-blocking anti-ROR1, without loss in viability. Mean fluorescence intensity (MFI) of treated cells is shown (***P<0.001 by One-way ANOVA). (D) Representative Immunoblots probed for vimentin (top) or β-actin (bottom) of lysates prepared from MDA-MB-231 before (0h) or after 1, 4, or 24 h treatment with D10 or control IgG. The ratios of vimentin to β-actin band-intensity are provided below. (E) Immunoprecipitates of MDA-MB-231 cell-lysate using control IgG (IgG) or anti-ROR1 (ROR1) were used for immunoblot analyses probed with antibodies specific for vimentin (top) or ROR1 (bottom). (F) Histograms provide the number of migrated MDA-MB-231 cells that were pre-treated for 1 h with D10 or control IgG. (G) Representative photomicrographs of migration (left) or invasion (right) of MDA-MB-231 following treatment with control IgG or D10. (H) Left, histograms depicting the in vivo lung photon flux. Right, Representative H&E-stained sections of the lungs. (I) Graph depicts the normalized in vivo lung photon flux. (G) Representative bioluminescence images of tumor-injected mice treated with IgG (top) or D10 (bottom). (K) Histogram depicts the lung-weight-index. (L) Representative H&E-stained sections of the lungs. Data are shown as mean±SEM; *P<0.05, **P<0.01, ***P<0.001, compared with IgG group.