Abstract
E-Cadherin functions as a tumor suppressor in some invasive breast carcinomas and metastasis is promoted when its expression is lost. It has been observed, however, that in one of the most aggressive human breast cancers, inflammatory breast cancer (IBC), E-cadherin is overexpressed and this accounts for the formation of the lymphovascular embolus, a structure efficient at metastasis and resistant to chemotherapy through unknown cytoprotective mechanisms. Studies using a human xenograft model of IBC, MARY-X, indicate that the mechanism of E-cadherin overexpression is not transcriptional but related to altered protein trafficking. By real-time RT-PCR, E-cadherin transcript levels in MARY-X were 3- to 11-fold less than in other E-cadherin positive human breast carcinoma lines but the protein levels were 5- to 10-fold greater. In addition, several smaller E-cadherin protein fragments, e.g. 95 kDa, were present. To explain these observations, it was hypothesized that there may be altered protein trafficking. A real-time RT-PCR screen of candidate molecules generally known to regulate protein trafficking was conducted. The screen revealed 3.5- to 7-fold increased ExoC5 level and 10 to 20 fold decreased HRS and RAB7 levels, which was confirmed in human microdissected lymphovascular emboli. Since these alterations may only be correlative with E-cadherin overexpression, one of the molecules, Rab7, was selectively knocked down in MCF-7 cells. An increase in the full length 120 kDa E-cadherin and the de novo appearance of the 95 KD band were observed. These findings suggest that it is the altered E-cadherin trafficking that contributes to its oncogenic rather than suppressive role in IBC.
E-Cadherin, an adhesion protein present in normal epithelial cells within lateral junctions (zona adherens), is thought to function as a tumor suppressor in certain types of invasive breast carcinomas and metastasis is promoted when its expression is lost by gene mutation, promoter methylation or promoter repression by snail/slug and other mediators of epithelial-mesenchymal transition (EMT) (1-3). However, in the vast majority of human breast cancers, E-cadherin expression is not lost, but retained. In fact, the presence of E-cadherin immunoreactivity is routinely used to diagnose ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma where it is present, in contrast to lobular carcinoma in situ (LCIS) and infiltrating lobular carcinoma, where it is lost.
It has also been observed that in one of the most aggressive and metastatic human breast cancers, inflammatory breast cancer (IBC), E-cadherin is not only retained but overexpressed and distributed circumferentially 360° around the cancer cell membrane (4-6). This distribution accounts for the formation of the lymphovascular embolus. Lymphovascular emboli of both IBC as well as non-IBC tumors strongly express E-cadherin. The lymphovascular embolus is a structure efficient at metastatic dissemination and resistant to chemotherapy/radiotherapy through ill-defined cytoprotective mechanisms. E-cadherin seems to be a very important molecule for tumor cell survival. The mechanism of the overexpression of E-cadherin in IBC had not been previously investigated. This study used the human xenograft model of IBC, MARY-X (4-9), and other common, well established E-cadherin-positive and -negative human breast carcinoma cell lines to investigate the mechanism of E-cadherin overexpression in IBC and in the lymphovascular embolus.
Materials and Methods
Cell lines and xenograft studies. MARY-X was previously established from a patient with IBC and exhibited the phenotype of florid lymphovascular invasion with tumor emboli formation in nude/SCID mice (4-6). MARY-X gave rise to tight aggregates of tumor cells termed spheroids in vitro, which were propagated in suspension culture. These spheroids were maintained for periods up to 3 months by growing them in either keratinocyte serum-free medium with supplements (Life Technologies, Inc., Gaithersburg, MD, USA), or in minimal essential medium with 10% fetal calf serum (Life Technologies, Inc.). The spheroids exhibited high cell density (103 cells in a 120 μm diameter spheroid); remained viable in suspension culture for 12 weeks; never formed monolayers on either plastic, extracellular matrix-coated dishes or feeder layers of fibroblast, myoepithelial, or endothelial cells; and fully recapitulated the MARY-X phenotype of lymphovascular emboli when re-injected into nude/SCID mice. The crude minced fragments of MARY-X contained spheroids ranging in size from 10 μm to 600 μm. Spheroids of homogeneous size were obtained by filtration through variously sized filters (Becton Dickinson, Franklin Lakes, NJ, USA). A 100 μm filter was used to exclude spheroids greater than 100 μm; the filtrate was then subsequently passed through 40 μm and 20 μm filters, which isolated 20-40 μm spheroids and >40 μm spheroids. Within these spheroids, the individual tumor cells were held together by an overexpressed E-cadherin axis (4-9) which if neutralized with anti-E-cadherin or ethylenediaminetetraacetic acid, released individual single cells. It was assumed in the past that the basis of E-cadherin overexpression in MARY-X and the MARY-X spheroids was transcriptional in nature. However, because of the unique contribution of E-cadherin to the MARY-X phenotype of florid lymphovascular emboli in vivo and spheroids in vitro, this assumption was not made in the present study. The E-cadherin expression in MARY-X and MARY-X spheroids was compared with its expression in other E-cadherin-positive (MCF-7, HTB20, HTB27 and HCC202) and E-cadherin-negative (MDA-MB-231 and MDA-MB-468) breast carcinoma cell lines. All these latter cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin) at 37°C in a 5% CO2 atmosphere at constant humidity.
RNA isolation and cDNA synthesis. Total RNA was isolated from cultured cells using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Total RNA was eluted and dissolved in RNase-free water, and the concentration was determined using a NanoDrop® spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). For the first-strand cDNA synthesis, the SuperScript® III First-Strand Synthesis System (Invitrogen, Inc., Carlsbad, CA, USA), oligo(dT)20 and 2 μg of total RNA were used. The synthesized cDNA was used for real-time RT-PCR analysis of relative expression levels of target genes.
Real-time RT-PCR. Real-time RT-PCR was performed on an ABI 7500® real-time PCR system (Applied Biosystems, Foster City, CA, USA). Briefly, cDNA was combined with primer sets and the ABI Power SYBR Green PCR Master Mix. The following conditions were used: an initial denaturation at 95°C for 5 min followed by denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 68°C for 1 min for a total of 40 cycles. PCR products were analyzed on a 2.0% agarose gel. Gene expression levels were calculated relative to the housekeeping gene β-actin by using ABI 7500® System SDS software. All real time RT-PCR experiments were performed with a minimum of five replicates and results depicted as mean±standard deviation. The primers used were as follows:
Immunocytochemical studies and Western blot analysis. For the immunocytochemical studies on the MARY-X spheroids, primary murine monoclonal antibodies included anti-human E-cadherin (clone HECD-1, unconjugated; Zymed Laboratories, Inc., South San Francisco, CA, USA). Control antibodies included mouse IgG1 (clone ×40, unconjugated; BD Pharmingen, San Diego, CA, USA). Secondary antibodies included fluorescein isothiocyanate-conjugated goat antimouse IgG (BD Pharmingen). For the immunofluorescence studies, MARY-X spheroids were fixed in cold methanol:acetone (1:1) for 20 min. Cells were blocked with phosphate-buffered saline (PBS) plus 1% normal donkey serum for 30 min and followed by incubation with the respective antibodies for 45 min. For the unconjugated antibodies, incubation with secondary antibody occurred for 30 min. The nuclei were counterstained with Hoechst 33342 dye (5 g/ml) (Sigma-Aldrich, St. Louis, MO, USA). The stained cells were imaged on a Nikon E600 DIC Dual View microscope with an attached Roper CoolSnap CCD camera (Nikon Instruments, Melville, NY, USA) and MetaVue software (Universal Imaging Corporation, Downington, PA, USA). For the Western blot studies, antibodies against E-cadherin (H-108) and RAB7 (H-50), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies and Western blotting substrates were obtained from Pierce Technology Inc. (Rockford, IL, USA). To prepare protein lysates from cell lines, cells were lysed using ice-cold RIPA lysis buffer (Pierce Biotechnology, Inc.). Protein content was determined using BCA protein assay (Pierce Biotechnology, Inc.), and samples were stored at −80°C. For Western blot analysis, boiled protein was loaded onto a 4-12% Precast gradient gel, transferred to nitrocellulose membranes (Invitrogen Corporation, Inc.) and incubated with antibodies. Bound antibodies were detected by a chemiluminescent detection system (West Femto; Pierce Biotechnology, Inc.) according to the manufacturer's instructions.
RNA interference. Knockdown of RAB7 was achieved with an RNAi (RNA interference) approach, using small interfering RNA (siRNA) for transient, but effective knockdown. For the siRNA-knockdown experiments, RAB7 siRNA sequences included: 5′-GCUGCGUUCUGGUAUUUGAtt-3′ (sense) and 5′-UCAAAUACCAGAACGCAGCag-3′ (antisense). MCF-7 cells were transfected with 50 nM of either control siRNA (NCSI) or RAB7 siRNA. After 48 h, the cells were collected and analyzed for the expression of RAB7 by both real-time RT-PCR and Western blot.
Analysis of human material. Ten cases of primary human inflammatory breast cancer and five cases of non-IBC but with florid lymphovascular emboli were selected from the Information Warehouse patient database at Ohio State University Medical Center maintained in CoPath, Sunquest Information Systems, Inc., Tucson, USA and anonymized. All references to real patient identity were removed before study. These tissues were available as archival paraffin blocks and sectioned. Standard immunohistochemical protocols for E-cadherin were used. The antibody used was an anti-human E-cadherin (clone HECD-1, unconjugated; Zymed Laboratories, Inc., South San Francisco, CA, USA). The appropriate secondary antibody was used and the detection system applied was a peroxidase-conjugated streptavidin-biotin complex. Visualization occurred after the application of a DAB brown chromogen. Subsequent laser capture microdissection of the lymphovascular embolic areas and non-embolic areas were carried out using a Veritas™ microdissection instrument (Molecular Devices, Sunnyvale, CA, USA). 250 emboli from 10 cases of IBC and 5 cases of non-IBC consisting of a mean of 100 cells/embolus and control non-embolic areas were captured, pooled and subjected to RNA isolation, cDNA synthesis and real time RT-PCR analysis.
Institutional approvals. All animal and in vitro studies were approved by The Ohio State University's Animal Care and Use Committee, protocol 2007A0218 and by The Ohio State University's Institutional Biosafety Committee, protocol 2007R0057. Additional animal studies were approved by the University of Nevada, Reno's Institutional Animal Care and Use Committee, protocols 00439 and 00440.
Statistical analysis. Declarations of differences imply differences of statistical significance. For the comparative studies of mRNA and protein levels of E-cadherin and its trafficking-regulatory molecules among the different human breast carcinoma cell lines, significance was assessed by Student's t-test and ANOVA. p-Values <0.05 were judged to the statistically significant. SAS Analytics Software was used in the analysis (SAS Institute, Inc., CARY, USA).
Results
E-Cadherin overexpression produces the IBC phenotype. E-cadherin over-expression was responsible for the tight aggregates of the MARY-X spheroids in vitro (Figures 1A and 1B) as well as for the lymphovascular emboli in vivo (Figure 1C).
The mechanism of E-cadherin overexpression is not transcriptional. By real-time RT-PCR, E-cadherin mRNA levels were unexpectedly less in MARY-X and the MARY-X spheroids than in a number of different E-cadherin-positive human breast carcinoma cell lines including HTB20, HTB27, HCC202 and MCF-7, but expectedly more than in the E-cadherin-negative lines (MDA-MB-231 and MDA-MB-468) (Figure 2A). By Western blot analysis, however, the MARY-X and MARY-X spheroids exhibited 5- to 10-fold greater protein levels than any of the other E-cadherin-positive cell lines (Figure 2B). Increased levels of E-cadherin fragments were also observed (Figure 2B). Because MARY-X spontaneously forms spheroids in vitro whereas the other cell lines normally grow as monolayers, spheroid formation was induced in the other lines with previously established methods (9) in order to compare their spheroid state with the spheroid state of MARY-X. The protein levels of E-cadherin were not altered by the induction of spheroidgenesis in these other cell lines (data not shown) and the protein levels of the MARY-X spheroids remained unsurpassed.
The mechanism of E-cadherin overexpression involves its redistribution. Not only were the levels of E-cadherin protein substantially increased in MARY-X and its derived spheroids but also the distribution of E-cadherin was strikingly altered compared to normal epithelial cells within ducts and acini. In normal epithelial cells, E-cadherin localized to the lateral junctions which were present focally along the lateral border between epithelial cells (data not shown). But in MARY-X and its derived spheroids, E-cadherin escaped the confines of the lateral junctions and was present circumferentially 360° around the plasma membrane of the cancer cell (Figure 1B). E-Cadherin similarly escaped the confines of the lateral junctions and was present circumferentially 360° around the plasma membranes of the cells contained within the lymphovascular emboli of IBC (Figure 1C).
The mechanism of E-cadherin overexpression and redistribution involves altered trafficking. To explain these observations, it was hypothesized that E-cadherin trafficking may be altered in MARY-X and its derived spheroids. A real-time RT-PCR screen of molecules thought to regulate protein trafficking was conducted (Figure 3). The results of this screen demonstrated that EXOC5 was significantly increased 3.5- to 7-fold (p=0.01) (Figure 3D), and both (HGS) and Rab7 were significantly decreased 10 to 20 fold (p=0.01) (Figures 3G and 3J) in MARY-X spheroids. These proteins are thought to play key roles in regulating lysosomal degradation and endosomal recycling. The other molecules tested, which included ARF6, NM23, RAB5, RAB11, RALA and SRC, showed no significant decreases compared to RAB7 and HRS in the MARY-X spheroids (Figures 3A, 3H, 3I, 3K, 3L and 3M). The other molecules tested, which included EXOC2, EXOC4, EXOC6 and EXOC7, showed no significant increases compared to EXOC5 in the MARY-X spheroids (Figures 3B, 3C, 3E and 3F). Since these molecular alterations which were present in the MARY-X spheroids may only be correlative with E-cadherin overexpression and redistribution, the expression of one of these molecules, RAB7, was modulated by knocking it down in MCF-7 cells to see the effects on E-cadherin expression. This knockdown was achieved effectively (Figure 4) and resulted not only in an increase in full length E-cadherin but also the de novo appearance of its 95 kDa fragment (Figure 4), a fragment which was also observed in MARY-X (Figure 2B).
The mechanism of E-cadherin overexpression, redistribution and altered trafficking is applicable to lymphovascular emboli in IBC and non-IBC cases. Real-time RT-PCR studies of pooled microdissected lymphovascular emboli from both IBC as well as non-IBC cases revealed a 3-fold increase in EXOC5 (p=0.01) and a 5- to 10-fold decrease in RAB7 and HRS, respectively, compared to non-embolic areas of the non-IBC cancers (p=0.01) data not shown. With respect to these trafficking molecules, the findings in the actual lymphovascular emboli from the human cases resembled the findings in the MARY-X spheroids.
Discussion
The hypothesis suggested by the present study is that E-cadherin is overexpressed in MARY-X and its derived spheroids because key proteins which regulate E-cadherin trafficking are altered (Figure 5). The mechanism is clearly not transcriptional and probably not translational but rather post-translational. Although the full length E-cadherin cDNA was sequenced in MARY-X and MARY-X spheroids with no mutations found, alternate splicing or rearrangements (data not shown), a translational component cannot be completely excluded to explain overexpression of E-cadherin. Certainly, altered translational regulation by ribosomal initiation factors or microRNAs could explain the overexpression but, they alone, would not explain the redistribution of E-cadherin observed 360° circumferentially around the cancer cell. This latter observation suggests the post-translational mechanism of altered trafficking. The present results showed that mRNAs encoding the endocytic trafficking regulators RAB7 and HRS are substantially decreased in the MARY-X spheroids compared to other E-cadherin-positive breast carcinoma cell lines, while EXOC5 mRNA is significantly increased compared to other E-cadherin-positive breast carcinoma cell lines. RAB7, a member of the Rab family, functions in the endocytic pathway of mammalian cells by regulating traffic from early to late endosomes. RAB7, specifically, is a monomeric GTPase, localized to multivesicular bodies and late endosomes, enhancing lysosome biogenesis (10). HRS, also referred to as hepatocyte growth factor-regulated tyrosine kinase substrate or HGS, is a ubiquitin-binding trafficking gatekeeper on sorting endosomes that controls the lysosomal degradation of ubiquitinated cargo such as endothelial growth factor receptors; its activity is regulated in turn by HRS tyrosine phosphorylation, ubiquitination, and degradation (11). Decreased RAB7 and HRS not only decreases lysosomal degradation but shuttles E-cadherin to the recycling endosomes (Figure 5). EXOC5 is a key component of the exocyst complex which also promotes endosomal recycling (Figure 5).
Lysosomal protein degradation may also involve ubiquitination. Ubiquitination is the conjugation of a 76 amino acid ubiquitin moiety to a target protein. This process has been reported to mark cellular proteins for proteasomal degradation (12, 13) and plasma membrane proteins for internalization, endocytic trafficking and lysosomal degradation (14-16). E-Cadherin is specifically thought to be ubiquitinated by the E3 ligase Hakai (17-19). Ubiquitinated E-cadherin is thought to be degraded in lysosomes after trafficking through RAB5- and RAB7-positive endosomal compartments (17). In these studies, the proteasome appeared to play an indirect role in E-cadherin degradation downstream of SRC activation (20). Interestingly, increased lysosomal targeting of E-cadherin has been postulated to represent a unique mechanism for the down-regulation of cell-cell adhesion during EMT (21). The present study is dealing with the opposite phenomenon, decreased lysosomal targeting for the up-regulation of cell-cell adhesion to foster the epithelial state.
Because it could be argued that both the increased EXOC5 and the decreased HRS and RAB7 were only correlative with E-cadherin overexpression, one of these molecules, RAB7, was arbitrarily chosen and targeted for knockdown. Similarly, HRS or overexpressed EXOC5 in MCF-7 could also have been chosen for knockdown in order to examine the effects on E-cadherin. RAB7 knockdown not only increased full-length E-cadherin but also induced the de novo appearance of the 95 KD E-cadherin fragment in MCF-7 cells, making the MCF-7 cells resemble the MARY-X spheroids more. This was additional proof that the molecules thought to regulate E-cadherin trafficking actually account for the unique E-cadherin post-translational processing and overexpression in the MARY-X spheroids. The 95 kDa fragment was not the only E-cadherin fragment observed in MARY-X and its derived spheroids. Understanding the nature and genesis of these other fragments may further elucidate the mechanisms of altered E-cadherin trafficking in IBC.
The present findings of decreased HRS and RAB7 and increased EXOC5 in the MARY-X spheroids were also observed in the lymphovascular tumoral emboli from human cases, suggesting that these observations have relevance in vivo.
The altered trafficking of E-cadherin in MARY-X spheroids and the lymphovascular emboli of both IBC as well as non-IBC cases, therefore, accounts for its overexpression and accumulation. E-cadherin accumulation and, subsequently, overexpression is responsible for both the formation of the MARY-X spheroids as well as the lymphovascular emboli and confers upon both structures a resistance to apoptosis and a survival advantage. It can be argued that, in the setting of the lymphovascular tumoral embolus, E-cadherin is functioning not as a suppressor gene but rather as an oncogene.
Acknowledgements
This study was supported by the Department of Defense Breast Cancer Research Program Grants BC990959, BC024258 and BC053405, the American Airlines–Susan Komen Promise Grant KG08128702 and the University of Nevada Vasco A. Salvadorini Endowment.
- Received July 29, 2010.
- Revision received September 22, 2010.
- Accepted September 26, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved