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Cancer Research 68, 4105, June 1, 2008. doi: 10.1158/0008-5472.CAN-07-6814
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

Cristina M. Karp1, Ting Ting Tan1, Robin Mathew2, Deidre Nelson1, Chandreyee Mukherjee1, Kurt Degenhardt1, Vassiliki Karantza-Wadsworth2,3 and Eileen White1,3

1 Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University; 2 Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey and 3 The Cancer Institute of New Jersey, New Brunswick, New Jersey

Requests for reprints: Eileen White, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. Phone: 732-235-5329; Fax: 732-235-5795; E-mail: ewhite{at}cabm.rutgers.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity, and contact inhibition while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila. [Cancer Res 2008;68(11):4105–15]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The vast majority of human cancers comprise epithelial-derived adult solid tumors that respond poorly to the currently available treatments. Epithelial cells have distinct properties necessitating that mechanisms of oncogenesis be addressed in this physiologically relevant cell type. We sought to develop models for epithelial tumor progression using primary epithelial cells, with the ability to apply genetics in a compound fashion, to identify genes that regulate tumor progression. Primary mouse kidney, prostate, mammary, and ovarian surface epithelial cells (C57B/6) immortalized by cooperating oncogenes become apparent as colonies and can be cloned and expanded (1–3). Specific inactivation or circumvention of the RB pathway (by expression of adenovirus E1A or c-myc) and inactivation of the p53 pathway are both necessary and sufficient for generation of immortal rat and mouse epithelial cells from multiple tissue types (1). For example, immortal baby mouse kidney (iBMK) and mammary epithelial cells are nontumorigenic and retain their normal epithelial character, including the ability to form tight junctions and establish polarity that is often lost in advanced cancers (1). Therefore, RB and p53 pathway inactivation is sufficient for immortalization in vitro while permitting retention of epithelial characteristics but is insufficient for tumorigenesis in vivo.

In addition to RB and p53 inactivation, the capacity for tumorigenesis additionally requires inactivation of p53-independent apoptosis either through the loss of proapoptotic bax and bak or bim, or through the gain of antiapoptotic bcl-2, events that can be selected for during tumorigenesis in vivo (2, 4, 5). Although inactivation of p53-independent apoptosis by modulation of Bcl-2 family members enables tumor growth, these cells retain epithelial characteristics. Activation of the Ras, mitogen-activated protein kinase (MAPK), or phosphatidylinositol 3-kinase (PI3K) pathways in iBMK cells also promotes tumorigenesis, but in contrast to defects in apoptosis, this is associated with the loss of epithelial characteristics through induction of an epithelial to mesenchymal transition (EMT; refs. 1, 5). Another mechanism to enable tumorigenesis is acquired through in vivo selection of nontumorigenic iBMK cells, which results in highly tumorigenic iBMK cells that have enabled tumor growth by an unknown mechanism (1). This selection for tumorigenesis in vivo also selects for loss of epithelial polarity and EMT, but the cells otherwise lack the phenotypically resemblance to cells with Ras, MAPK, or PI3K pathway activation. If or how polarity disruption and EMT contributes to tumorigenesis in this case is not yet understood. Thus, there seems to be at least four independent functions that promote tumorigenesis of immortal epithelial cells: apoptosis defect, MAPK pathway activation, PI3K pathway activation, and in vivo selection that may involve an unidentified activity associated with disruption of polarity.

EMT, the process through which cells transit from a polarized, epithelial phenotype to a highly motile mesenchymal phenotype, has emerged in recent years as an important factor in tumor progression (6). EMT causes pronounced morphologic and functional changes in cells, such as dissolution of epithelial tight junctions, reorganization of the actin cytoskeleton, loss of apicobasal polarity, and migration through basement membranes and tissues. The molecular mechanisms governing EMT involve several signaling pathways, including transforming growth factor-β (TGF-β), signal transducer and activator of transcription (STAT), Ras, Wnt, and Notch pathways (7, 8). EMTs during embryonic development are governed in part by STAT signaling pathway through the Snail family of transcription factors, first described in Drosophila (6). In some cases, EMT is partly due to direct repression of E-cadherin transcription and has been associated with Snail-induced repression of tight junction proteins, such as claudin-3, claudin-4, claudin-7, and occludin (9), both in early development and in cancer progression (10). E-cadherin repressor ZEB1 has been shown to repress cell polarity genes Crumbs3 (crb3), HUGHL2, and Plas1-associated tight junction protein in colon and invasive breast cancer, promoting EMT associated with tumor progression (11). Loss of epithelial polarity is a hallmark of EMT in normal development and cancer but the role of polarity determinants in this process in cancer in mammals is poorly understood. The proteins involved in polarity complexes are highly conserved between Drosophila and mammalian cells (12, 13). In Drosophila, disruption of polarity determinants causes overproliferation and disruption of tissue architecture, hence their designation as tumor suppressors. Although the genetic interactions between these Drosophila polarity determinants have been studied extensively, no links are established to analogous roles of their mammalian homologues in human cancer.

Here, we identify crb3, the mammalian orthologue of Drosophila and polarity determinant crumbs, as a gene whose expression is lost in iBMK cells rendered tumorigenic through in vivo selection. We further show that Crb3 functions to maintain epithelial junction formation and apicobasal polarity required for contact-inhibited growth and to suppress invasion and metastasis. These findings suggest the existence of a mammalian pathway for tumor suppression analogous to that long known to control tumor growth in Drosophila.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture and transfection. Derivation of iBMK cell lines was previously described (4). Stable iBMK cells expressing human Bcl-2 or vector-only controls were derived by electroporation with pCDNA3.1-hBcl-2 or selection vector only (Invitrogen), respectively, followed by selection (W2, gentamicin; D3, zeocin) and ring cloning. Multiple clones from each condition were expanded and characterized. For red fluorescent protein (RFP) expression, cells were cotransfected with the pDsRed2-C1 (Clontech) and pCDNA3.1zeo (W2.3.1-5, Invitrogen) or pPUR (D3.zeo-2, BD Biosciences) vectors followed by selection with zeocin or puromycin, respectively, and ring cloning. For each cell line, three individual clones were characterized in animals for fluorescence and tumor growth, and one individual clone was selected for further analysis. All cell lines were maintained in DMEM (Life Technologies/Invitrogen) containing 10% fetal bovine serum (FBS). For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, cells were plated in 96-well plates at a density of 8,000 per well and treated after 24 h with 3 µmol/L etoposide for 48 h. MTT was added to the wells and incubated at 37°C for 3 h followed by the aspiration of the medium and addition of DMSO. The plates were read using SpectraMax 250 (Molecular Devices).

Microarray analysis. Gene expression analysis was carried out using the Affymetrix Mouse 430A 2.0 chips (accession number GSE10682). Initial scaling was done using the Affymetrix Microarray Suite Expression Software version 5.0 and subsequent analysis was done using GeneSpring Software version 6.1. Tumor-derived cell lines (TDCL) were compared with duplicate samples of the parental cell line from which they were derived. Three different filtering conditions were applied: expression percentage restriction, retaining only those genes that had a raw expression value of 80.0 or more in at least one of the five samples followed by a filtering selecting for genes with a Flag value of present in all the samples being compared, and a pairwise fold change analysis comparing each TDCL to the parental cell line. Genes that underwent at least 3-fold or more up-regulation or down-regulation in the TDCLs, compared with the parental cell line, were retained.

Retrovirus infection. The png retroviral vector and mycCrb3-png, mycCrb3{Delta}ERLI-png, and mycCrb3mutFERM{Delta}ERLI-png were generously provided by Dr. Benjamin Margolis (University of Michigan Medical School, Ann Arbor, MI). The production of amphotropic retroviruses and cell infection was performed as described previously (14). Selection of cells stably expressing the retroviral vector was done using selection medium containing 500 µg/mL puromycin 48 h after infection. Cells were selected for 6 d before use or until all noninfected cells had died.

Immunoblotting, immunofluorescence, immunohistochemistry, and confocal microscopy. Immunoblotting was performed using the rabbit polyclonal antibody against Crb3 (15), a generous gift from Dr. Benjamin Margolis. For immunofluorescence analysis, cells were grown on glass coverslips and fixed in ice-cold acetone for 10 min and indirect immunofluorescence was performed as described previously (16). The following antibodies were used: rabbit anti-occludin (Abcam), mouse monoclonal anti-pan-cadherin clone CH-19 (Sigma-Aldrich), mouse monoclonal anti-β-catenin, rabbit polyclonal anti-ZO-1 (Zymed), mouse anti-bromodeoxyuridine (BrdUrd)FITC (BD Biosciences), and polyclonal rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology, Inc.). Immunohistochemistry was performed on paraffin-embedded sections of tumors using polyclonal rabbit anti-cleaved caspase-3 (Asp175). Confocal microscopy was performed using a Zeiss LSM510-META confocal microscope system at the Neuroscience Imaging Facility (W.M. Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ).

Three-dimensional morphogenesis assay and imaging. Three-dimensional culture of TDCL 5D on a reconstituted basement membrane was performed according to the protocol previously described for the immortalized human mammary epithelial cell line MCF-10A (17). Time-lapse microscopy was performed as previously described (1). In brief, cells were cultured in the time-lapse chamber equipped with controlled environmental conditions. The time-lapse microscopy system consisted of an Olympus IX71 inverted microscope fitted with 37°C temperature and 5% CO2 controlled environmental chamber (Solent Scientific) and a CoolSnap ES charge-coupled device camera. Image capturing and analysis were performed using Image-Pro Plus software (Media Cybernetics). Phase-contrast images (100x) at multiple fields were obtained for the indicated time period.

Tumor growth assay. Tumor formation in nude mice by s.c. injection using 107 cells in 0.1 mL PBS was performed essentially as described previously (4). Briefly, 107 cells were injected in each of five mice for each cell line and tumors were measured regularly. Tumor volumes were calculated using the formula length (mm) x width2 (mm)2 / 2. Each point represents the mean value for all five mice in each group. To visualize RFP-expressing cells in mice, animals were imaged using the Illumatool imaging and camera system (Lightools Research).

Migration assay. The invasive capacity of TDCLs was measured using an in vitro Transwell assay (18). Cells, in DMEM containing 0.1% bovine serum albumin, were added to the upper well of Transwell chambers (Corning, Inc.) containing an 8-µm porous membrane. Lower chambers contained 10% FBS in DMEM. Cells were cultured for 2 to 4 h before invading cells were fixed, stained with crystal violet, and examined under a bright-field microscope. Cells were counted in five random fields per membrane and results were presented as mean ± SE of three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Selection in vivo for acquisition of tumorigenesis. Ten million wild-type nontumorigenic W2.3.1-5 iBMK cells were introduced s.c. into a series of nude mice and thereby subjected to selection in vivo for tumor growth (Fig. 1A ). Following 2 months in vivo, 1 to 10 nontumorigenic wild-type iBMK cells acquired the capacity for tumor growth, appearing initially as small clonal nodules (Fig. 1A). These clonal tumors that arise following a long latency were excised and returned to cell culture as TDCLs. Seven of these TDCLs were then injected s.c. into nude mice along with the original parental cell line (W2.3.1) to monitor tumor growth kinetics. Despite 3 months of selection in vivo, TDCLs returned to cell culture readily and were profoundly tumorigenic relative to the parental, nontumorigenic W2.3.1 cells (Fig. 1A and B). TDCLs were also more tumorigenic than iBMK cells rendered defective for apoptosis by deficiency in both bax and bak (D3; ref. 4) or by overexpression of bcl-2 (W2.Bcl2-3; Fig. 1B; ref. 5). As defects in apoptosis enable tumorigenesis, we determined if the TDCLs incurred mutations, rendering them resistant to apoptosis by testing TDCLs for sensitivity to apoptosis inducer staurosporine or etoposide. All TDCLs retain sensitivity to staurosporine or etoposide and readily undergo apoptosis similarly to the parental W2.3.1-5 cell line (Fig. 1C). Moreover, the apoptotic sensitivity of TDCLs was assessed in vivo by examining the presence of cleaved, activated caspase-3 in tumor sections. Tumors from TDCL 5D displayed frequent cells positive for activated caspase-3, whereas sections from tumors derived from Bax/Bak-deficient, apoptosis-defective D3 cells did not (Fig. 1D). Thus, although the TDCLs acquire a stable tumor-promoting function through in vivo selection, they did not acquire resistance to apoptosis. This suggests that TDCLs acquired stable genetic or epigenetic change(s) through selection in vivo that confers functional tumorigenic capability distinct from blockade of apoptosis.


Figure 1
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  		Figure 1. Selection scheme for TDCLs. A, schematic representation of mouse model used for obtaining TDCLs. Primary mouse epithelial kidney cells were immortalized by inactivation of the RB and p53 pathways (1). Wild-type, nontumorigenic W2.3.1 iBMK cells were injected s.c. in mice and tumor growth occurred with long latency (3–4 mo). TDCLs were returned to in vitro cell culture and reinjected into animals where tumor growth occurred within 2 wk. Clonal emergence of W2.3.1 RFP-expressing tumors by whole animal optical imaging after 3 mo and H&E staining of emerging clonal nodules following selection for growth for 3 mo in vivo. B, tumorigenic capacity of TDCLs. Parental W2.3.1 cells (red) and TDCLs (orange) were injected s.c. into nude mice, and tumor volumes were measured over time. W2.Bcl2-3 (green) and D3.zeo-2 (black) cells were included as controls. C, TDCLs retain apoptotic sensitivity. W2.3.1 and TDCLs were cultured for 24 h with 0.4 µmol/L staurosporine or for 48 h with 3 µmol/L etoposide, and viability was assessed by trypan blue exclusion for staurosporine and MTT assay for etoposide. White columns, untreated; black columns, treated. Apoptotic-defective Bcl-2–expressing (W2.Bcl2-3) and Bax- and Bak-deficient D3.zeo-2 cells were included as controls, as these cells are resistant to staurosporine-induced apoptosis. D, TDCL 5D cells retain apoptotic sensitivity in vivo. Paraffin-embedded sections from Bax/Bak-deficient, apoptosis-defective D3 tumors and TDCL 5D tumors were subjected to immunohistochemistry for active caspase-3, showing apoptosis induction (brown staining) of TDCL 5D tumors in vivo that is absent in D3 tumors.

 
Acquisition of tumorigenesis results in an altered pattern of gene expression. Comparison of the gene expression pattern of four independently derived TDCLs (Fig. 2A ) cultured in vitro revealed a strikingly similar pattern of gene expression changes relative to the parental W2.3.1-5 cells (Fig. 2A). In contrast, TDCLs produced from W2.3.1-5 cells expressing bcl-2 did not display a significant change in the pattern of gene expression, consistent with the lack of selection pressure given that these cells readily form tumors due to defective apoptosis. Most of the up-regulated genes in the TDCLs were IFN-regulated genes often referred to as the IFN cluster (Fig. 2A; refs. 19–23); the up-regulation of many was validated at the mRNA and protein levels (data not shown) and the significance of which is currently under investigation.


Figure 2
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  		Figure 2. TDCLs acquire loss of crb3 expression and defects in epithelial tight junction formation. A, gene tree analysis of parental W2.3.1-5 and TDCL 5A to TDCL 5D. All samples were analyzed in duplicate (Affymetrix 430; accession number GSE10682). B, Crb3 expression is repressed in TDCLs. List of the 15 most repressed genes in TDCLs from the gene expression analysis. C, Western blot showing reduced expression of Crb3 protein in TDCL 5A to TDCL 5D relative to parental W2.3.1-5 and expression of vimentin and E-cadherin in TDCL 5A to TDCL 5D relative to parental W2.3.1-5. D, localization of junction proteins occludin, ZO-1, pan-cadherin, E-cadherin, and β-catenin to intercellular junction in parental W2.3.1-5 and aberrant localization in TDCL 5D. The localization of junction proteins in TDCLs is either fragmented or localized to the cytoplasm.

 
Similar to the up-regulated genes, there was a unifying pattern of repressed genes in all TDCLs, and these genes had the common theme of plasma membrane proteins known or predicted to play a role in epithelial adhesion and junction formation (Fig. 2B, blue). Among the 15 most repressed genes in TDCL 5D (0.36-fold or lower expression relative to parental W2.3.1-5), the most repressed gene (AU015319) is crb3 (Fig. 2B), a murine homologue of the Drosophila apicobasal polarity determent Crumbs. crb3 is the mammalian isoform of Drosophila crb that is exclusively expressed in epithelial tissues, and Western blotting for Crb3 protein expression revealed high expression in parental W2.3.1-5 cells and dramatically reduced expression in all four of the TDCLs, with undetectable expression in TDCL 5D (Fig. 2C).

In addition to crb3, another highly repressed gene in TDCLs is Tm4sf10/Bcmp1/Vab-9 (Fig. 2B), a poorly characterized adherent junction and claudin-related protein that regulates adhesion through discs large (Dlg) in Drosophila Crumbs pathway (24). Claudin-3 and claudin-4 were also repressed in TDCLs (Fig. 2B) and are transmembrane proteins that are the major structural elements in epithelial tight junctions (25). Another repressed gene, Tacstd1/EpCam (Fig. 2B) is a homophilic adhesion regulator and claudin-interacting protein (26). It is possible that acquisition of a single defect in epithelial junction formation resulted in the coordinate down-regulation of expression of multiple cell junction components.

Acquisition of tumorigenesis is associated with EMT and deficient tight junction formation. Commonly used molecular markers for EMT include increased expression of vimentin, a mesenchymal marker, and decreased E-cadherin expression, a hallmark of metastatic carcinoma (27). To determine if TDCLs underwent an EMT, we assayed vimentin and E-cadherin protein expression levels in comparison with the parental cell line. Western blot analysis revealed an increase in vimentin in all TDCLs compared with W2.3.1-5 and a concomitant down-regulation of the E-cadherin levels in TDCL 5C and TDCL 5D (Fig. 2C). These results suggest that selection for tumorigenesis in TDCLs also selects for acquisition of properties associated with an EMT.

The repression of crb3 expression and that of other junction/adhesion regulators suggested that selection for tumorigenicity in vivo resulted in loss of epithelial junction formation. Therefore, junction formation was examined in parental W2.3.1-5 and TDCLs by immunofluorescence for the tight junction proteins occludin and ZO-1 and for the adherens junction proteins cadherins and β-catenin in culture in vitro.

Staining for the tight junction markers occludin and ZO-1 in W2.3.1-5 cells (Fig. 2D) revealed a smooth, continuous, and well-defined staining at the membranes between cells that was initiated by cell-cell contact as cells began to form a monolayer. In contrast, TDCL 5D (Fig. 2D) and the other TDCLs (data not shown) displayed a diffuse staining pattern for occludin and a faint, discontinuous, saw-tooth–like staining pattern of ZO-1, even after monolayer formation, suggesting impaired tight junction formation. Staining for β-catenin, pan-cadherin, and E-cadherin revealed smooth, continuous staining at cell-cell contacts in parental W2.3.1-5 that was reduced in TDCL 5D (Fig. 2D) and the other TDCLs (data not shown). These results suggest that tight junction formation was severely impaired in TDCLs and adherens junctions are affected to a lesser degree in TDCLs compared with the parental cells. Thus, essential characteristics of epithelial cell junction formation were lost in TDCLs coincident with acquisition of tumorigenicity.

Restoration of Crb3 expression in TDCL 5D corrects defective epithelial junction formation. To test the hypothesis that repressed crb3 expression was responsible for defective tight junction formation in TDCLs, crb3 expression was restored in TDCL 5D where crb3 endogenous levels were undetectable. TDCL 5D was infected with either a retroviral vector alone (png) or a vector driving expression of myc-tagged Crb3 (15) and cells stably expressing Crb3 were generated (Fig. 3A and B ). Importantly, Crb3 localized to the plasma membrane and substantially rescued tight junction formation in TDCL 5D as indicated by increased occludin and ZO-1 at the junctions between cells compared with the png vector control (Fig. 3C). Thus, the loss of crb3 expression in TDCL 5D is in part responsible for impaired junction formation.


Figure 3
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  		Figure 3. Role of Crb3 in controlling tight junction formation. A, graphic representation of wild-type Crb3 protein domain structure and schematic illustrating the myc-tagged Crb3 and Crb3 mutants (22). The Crb3 mutants contain a deletion of the COOH-terminal domain ({Delta}ERLI) and three point mutations in the FERM domain (Crb3mutFERM). B, Western blots illustrating the stable expression of myc-Crb3 in TDCL 5D and the mutant myc-Crb3{Delta}ERLI in W2.3.1-5 cells. C, Crb3 expression restores tight junction formation in TDCL 5D. Left, TDCL 5D png vector–infected or wild-type Crb3-expressing vector-infected cells stained for tight junction markers occludin and ZO-1. Immunofluorescence showing restoration of tight junctions indicated by alterations in ZO-1 and occludin. Expressed Crb3 localized to the plasma membrane and rescued tight junction formation in TDCL 5D as indicated by increased occludin and ZO-1 at cell junctions compared with png vector control. Mutant crb3 expression impairs tight junction formation in parental W2.3.1-5 cells. Right, W2.3.1-5 parental cells, png vector–infected or Crb3{Delta}ERLI-expressing vector-infected, were immunostained for tight junction markers occludin and ZO-1. Immunofluorescence showing disruption of tight junctions indicated by alterations in ZO-1 and occludin. D, immunofluorescence showing colocalization of Crb3 with occludin and ZO-1 in TDCL 5D Crb3 cells.

 
Inhibition of Crb3 function prevents junction formation in W2.3.1-5 cells. Because interfering with Crb3 function disrupts junction formation (28), in a complimentary approach, we tested if expression of dominant-negative mutant of crb3 disrupted polarity in parental, nontumorigenic W2.3.1-5 cells. The ERLI motif on Crb3 binds to the PDZ domain on PALS1 and PALS1 binds PATJ to form the Crb3/PALS1/PATJ functional protein complex. Deletion of the ERLI motif in Crb3 disrupts the Crb3/PALS1/PATJ complex, and thereby polarity and junction formation in mammalian cells (15, 28), and produces multilayer cell growth in Drosophila (29). Mutation of the FERM-binding motif in Crb3 has similar effects but the interacting protein is not known (15).

Infection with retroviral vectors driving expression of dominant-negative, myc-tagged mutant forms of crb3, Crb3{Delta}ERLI, and Crb3{Delta}ERLImutFERM was used to assess the effect on junction formation in W2.3.1-5 cells (Fig. 3A and B). Expression of either Crb3{Delta}ERLI (Fig. 3C) or Crb3{Delta}ERLImutFERM (data not shown) efficiently disrupted tight junction formation in W2.3.1-5 cells (Fig. 3C). Thus, as in other epithelial cell types (30, 31), Crb3 is an essential regulator of tight junction formation in iBMK cells.

Requirement for Crb3 for establishing apicobasal polarity. To test if loss of crb3 and junction formation caused defect polarity and possibly altered migration in TDCLs, we used multifield time-lapse microscopy to examine a wound-healing response as a means to study directional cell migration in vitro. Parental W2.3.1-5 cells responded to the wound in vitro with a high mitotic rate and a wave of coordinated and directional migration into the wound area followed by tight junction and fusion sheet formation as indicated by the disappearance of the distinction between individual cells (Fig. 4A, below the red line ; Supplementary Video 1). In contrast, wounded TDCL 5D cell monolayers displayed random, uncoordinated cell movements and the cell population failed to efficiently migrate to fill the void (Fig. 4A; Supplementary Video 2). There was also failure of tight junction formation (no fusion sheet), and cells piled on top of one another (multilayer growth) and succumbed to apoptosis indicated by the accumulation of highly refractile apoptotic corpses (Fig. 4A; Supplementary Video 2). TDCL 5D infected with the png vector behaved similarly (Fig. 4A; Supplementary Video 3). TDCL 5D expressing crb3, however, displayed a marked improvement in coordinated directional migration, junction formation (fusion sheet formation, below the red line), contact-inhibited growth, and suppression of apoptosis (Fig. 4A; Supplementary Video 4). These findings support a substantial role for deficiency in crb3 expression in the loss of epithelial characteristics acquired during selection for the capacity for tumor growth in vivo.


Figure 4
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  		Figure 4. crb3 rescues migration, polarity, and tight junction formation. A, iBMK parental, TDCLs, or derivatives infected with the retroviral vector (5D/png) or the Crb3-expressing retroviral vector (5D/Crb3) were cultured to a confluent monolayer. Scratching a line through the monolayer with a pipette tip then disrupted a small area. The open gap was then monitored using computerized video multifield time-lapse microscopy for 3 d as the cells migrated in and filled the void. Epithelial fusion sheet resulting from tight junction formation is indicated below the red line. B, restoration of Crb3 expression establishes single-layer cell growth, apicobasal polarity, and lumen formation in TDCL 5D spheroids. TDCL 5D cells uninfected or infected with the empty retroviral vector png or the wild-type Crb3-expressing vector were cultured in Matrigel and monitored by time-lapse microscopy. C, spheroids were costained for β-catenin (green) and active caspase-3 (red) and imaged by confocal microscopy at 14 d. Crb3 expression confers single-layer cell growth and apoptotic lumen formation in three-dimensional culture. Twenty to 30 spheroids per microscopic field were counted in five different fields for each cell line and the experiment was repeated thrice (P < 0.05). Greater than 90% of TDCL 5D cells expressing Crb3 formed spheroids with hollow lumens compared with <10% of TDCL 5D infected with the png vector.

 
Crb3 is required for epithelial apicobasal polarity in three-dimensional morphogenesis. Another means to examine proper junction formation and polarity characteristics of epithelial cells is through three-dimensional growth in extracellular matrix (Matrigel) where cells form spheroids that organize into an epithelial layer with apicobasal polarity forming a lumen through apoptosis (3, 17). To test the role of crb3 in epithelial organization in three-dimensional cultures, TDCL 5D, TDL 5D png, and TDCL 5D Crb3 were grown in Matrigel and evaluated morphologically by time-lapse microscopy, and for junctions, organization and spatial localization of apoptosis by β-catenin and active caspase-3 staining by confocal microscopy.

TDCL 5D and vector png–transduced derivatives form solid spheroids in Matrigel with high efficiency typified by random cellular organization and spatial localization of apoptosis and multilayer cell growth (Fig. 4B; Supplementary Video 5). In contrast, TDCL 5D expressing crb3 formed spheroids composed of a single epithelial cell layer with hollow lumens (Fig. 4B; Supplementary Video 6). This suggested that lack of Crb3 and tight junction formation disrupted the three-dimensional epithelial organization by preventing tight junction formation. The spheroids were stained for β-catenin (green) and active caspase-3 (red). Interestingly, in some spheroids, β-catenin, a basolateral marker, was also observed at the apical surface, a fact reported by other studies on loss of apicobasal polarity of cancer cells (32, 33). Confocal microscopy showed that 90% of TDCL 5D cells overexpressing crb3 formed spheroids with hollow lumens compared with 10% TDCL 5D transduced with the png vector, and these lumens were coincident with detection of active caspase-3 (Fig. 4C). In contrast, TDCL 5D and TDCL 5D png generated solid spheroids with random cellular organization and caspase-3 activation (Fig. 4C). Thus, restoring crb3 expression in TDCL 5D cells rescued loss of specialized cell-cell contacts, polarized morphology, and proper epithelial organization and function while suppressing multilayer cell growth that may affect tumorigenesis.

Role of Crb3 in regulating growth arrest through contact inhibition. Striking evidence from the time-lapse microscopy in particular, also supported by the findings from three-dimensional culture, suggested that subconfluent crb3-expressing epithelial cells (either W2.3.1-5 or TDCL 5D Crb3) divide rapidly, but once confluence was reached and tight junctions form, mitosis was suppressed due to contact inhibition. This tight junction formation-associated suppression of cell division was absent in Crb3-deficient cells, which may contribute to multilayer cell growth. To test the role of Crb3 in mediation of contact-inhibited growth, TDCL 5D png and TDCL 5D Crb3 were followed over time and during increasing cell density for occurrence of DNA synthesis by BrdUrd labeling (Fig. 5B ). The results indicated that TDCL 5D png continued to incorporate BrdUrd, even when cells were at high density and reached confluency, at which point TDCL 5D Crb3 failed to incorporate BrdUrd. Conversely, we tested if disruption of Crb3 function in W2.3.1-5 cells relieved contact inhibition. W2.3.1-5 cells displayed decreased BrdUrd incorporation and with increasing cell density (Fig. 5), as expected. Furthermore, coincident with tight junction formation, W2.3.1-5 cells expressing the dominant-negative Crb3{Delta}ERLI continued DNA synthesis even when at high cell density (Fig. 5B). These results indicate that impairing tight junction formation in iBMK cells leads to failure of contact inhibition leading to unrestrained proliferation. This is remarkably similar to the cellular overgrowth phenotype of Drosophila imaginal disc tissue with defects in junction formation (13).


Figure 5
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  		Figure 5. Role of Crb3 in regulation of growth arrest and in vitro migration. A, immunofluorescence staining showing BrdUrd incorporation as a measure of proliferation in TDCL 5D png and TDCL 5D Crb3 cells or W2.3.1-5 png and W2.3.1-5 Crb3{Delta}ERLI with increasing cell density. Crb3-deficient TDCL 5D png with impaired tight junction formation continued to incorporate BrdUrd even after the cells reached confluency, whereas the TDCL 5D Crb3 suppressed proliferation in response to tight junction formation and contact inhibition. B, graphs summarizing the number of positive BrdUrd cells with increasing time and cell density. The percentage of positive cells per microscopic field was counted in four different fields for each cell line and the experiment was repeated thrice. Columns, mean of the number of cells per field; bars, SD. P < 0.0001 for TDCL 5D Crb3 versus TDCL 5D png at day 8 and P < 0.05 (P = 0.028) for W2.3.1-5 png versus W2.3.1-5 Crb3{Delta}ERLI at 11 d. C, TDCL 5D png and TDCL 5D Crb3 cells and W2.3.1-5 png and W2.3.1-5 Crb3{Delta}ERLI cells were cultured to confluency in the upper well of Transwell chambers. After 24 h, the medium was changed in the upper chamber to serum-depleted and cells passing through the filter were fixed, stained, and counted. Cells were cultured in triplicate wells per experiment and the experiment was replicated thrice. Representative microscopic fields of crystal violet–stained cells are shown. D, graphs summarizing the results of in vitro migration assay. Columns, mean of the number of cells per field, evaluated in five fields per membrane; bars, SD. P < 0.05 (P = 0.037).

 
Role of Crb3 in suppressing in vitro migration. As TDCL 5D displayed a profound defect in migration as a sheet in the wound-healing assay caused by defective tight junction formation, we also assessed the migratory ability of individual cells. In the process of migration as a sheet, epithelial cells must form biological barriers in which individual cells associate with each other through tight junctions and migrate as a cohesive unit. This process is distinct from migration of independent, single, unattached cells that measures the ability of individual cells to move through porous membranes by a process that may be linked to increased intravasation, invasion, and capacity for metastasis. The invasive behavior of TDCL 5D png and TDCL 5D Crb3 was assessed in parallel with W2.3.1-5 png and W2.3.1-5 Crb3{Delta}ERLI using an in vitro migration assay. As illustrated in Fig. 5C and D, png vector–transduced TDCL 5D efficiently migrated as individual cells across a porous membrane, whereas restoring crb3 expression in TDCL 5D cells suppressed migration capabilities. Parental W2.3.1-5 cells displayed reduced migration likely due to proper tight junction formation often appearing to transverse the pores as continuous sheets of cells (Fig. 5C and D). Expressing mutant Crb3{Delta}ERLI in W2.3.1-5 cells greatly increased the migratory capacity, although the cells still retained some capacity to form cell-cell contacts (Fig. 5C and D). Thus, restoring Crb3 expression in TDCLs repressed migration through a porous membrane, which may have been impaired by tight junction formation, whereas disrupting Crb3 function in W2.3.1-5 cells repressed junction formation and stimulated migration. This suggested that tight junction formation dependent on Crb3 reduced the potential for cell migration most likely by sustaining intercellular interactions.

Crb3 expression suppresses metastatic potential. To examine the role of Crb3 in suppressing tumor growth, the tumorigenicity of TDCL 5D png and TDCL 5D Crb3 was evaluated following s.c. injection. Surprisingly, both vector- and Crb3-transduced TDCLs formed tumors at similar rates (Fig. 6A ). However, immunohistochemistry for myc-Crb3 expression revealed a striking loss of Crb3 staining in the expanding growth regions at the perimeter of TDCL 5D Crb3 tumors (Fig. 6B). Although all the cells were positive for Crb3 at the start of tumor formation, 3 weeks later, most were negative for Crb3 expression in tumors in vivo. Furthermore, loss of transgene expression is not observed under conditions where expression is required for tumor growth (data not shown; ref. 5). These findings indicate selection for loss of Crb3 expression during tumor expansion, suggesting that its expression imparts a growth disadvantage in tumors in vivo. Moreover, expression of Crb3{Delta}ERLI in W2.3.1 cells and disruption of Crb3 function were not sufficient to confer tumorigenic capacity (data not shown), indicating that impairing tight junction formation may not be sufficient for tumorigenesis. Thus, other genetic or epigenetic changes in TDCLs are responsible for enabling tumor growth independent of the role of the loss of Crb3 in disrupting junctions and polarity and enabling proliferation at high cell density.


Figure 6
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  		Figure 6. Crb3 expression suppresses metastasis. A, tumorigenic capacity of TDCL 5D png and Crb3 transfectants compared with parental W2.3.1-5 cells. Animals were injected s.c. with 107 cells and tumor formation was monitored over time. The parental W2.3.1-5 cells failed to form tumors by 20 d after injection, whereas the TDCL 5D formed s.c. tumors as expected. Expression of Crb3 in TDCL 5D did not suppress s.c. tumor growth. B, s.c. tumor growth of TDCL 5D Crb3 is associated with the loss of Crb3 expression. TDCL 5D Crb3 tumors that formed 3 wk after injection were examined by immunohistochemistry for myc-Crb3 using the myc tag. Crb3-positive cells with Crb3 localized to cellular junctions (left) were found in central (top) but not expanding, peripheral regions (right) of the tumor. C, Crb3 expression suppresses metastasis. Kaplan-Meier survival curve of mice injected (tail vein) with 5 x 105 cells, TDCL 5D png (black; n = 6), and TDCL 5D Crb3 (red; n = 6). Most (five of six) of the TDCL 5D png–injected animals succumbed to multiple bone and kidney metastases by 10 wk, whereas all of the TDCL 5D Crb3–injected animals remained tumor-free. D, immunohistochemistry showing metastasis to the spinal cord, illustrated by purple cells invading the gray matter (pink area) in animals injected with TDCL 5D png cells.

 
Because loss of Crb3 expression was associated with tumor expansion, we also evaluated the requirement for Crb3 loss in a model for metastatic growth. TDCL 5D png and TDCL 5D Crb3 cells (5 x 105) were injected into the tail vein of nude mice that were monitored over time for tumor growth and for intravasation and colonization of organs. Multiple tumors from TDCL 5D–injected cells were found extensively colonizing the kidney and bone, especially the legs and spinal cord, in five of six injected mice (Fig. 6C and D). In contrast, all of the TDCL 5D Crb3–injected animals remained tumor-free (Fig. 6C). This suggested that restoring epithelial junction formation by expressing crb3 limits invasiveness and metastatic potential. Increased intercellular adhesion and attachment among tumor cells may be expected to diminish migration, intravasation, and invasiveness, in addition to its role in mediating contact-inhibited growth. Thus, loss of crb3 expression in tumors in vivo may be one means to overcome a barrier to tumor progression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
One of the primary diagnostic features in malignant tumors of epithelial origin is a profound disruption of cell architecture and organization. Although the link between loss of epithelial organization and tumor progression has been previously made, an important unanswered question remains: is it correlative or does the loss of architecture contribute to progression toward malignancy in mammalian tumors? In Drosophila, however, most of the genes associated with tumor suppression function to establish and maintain epithelial cell junctions and polarity, the loss of which disrupts tissue organization and produces an overgrowth phenotype. If this process is evolutionarily conserved, there may be a role for the mammalian homologues of neoplastic tumor suppressor genes in mammalian cancer that has yet to be elucidated.

The tumor suppressors identified in Drosophila are prominently composed of genes that regulate epithelial junctions and polarity determination. In the Drosophila Crumbs pathway, the polarity regulators Crumbs, Scribble (Scrib), Lethal giant larvae (Lgl), and Dlg function to suppress overgrowth and tumorigenesis. Perturbation in the function of Drosophila crumbs, scrib, lgl, or dlg leads to disruption of polarity and to cell overgrowth in the epithelial tissue of imaginal discs (12–14, 34, 35). There are three junction complexes conserved in invertebrates and mammals that act together to establish apicobasal polarity and junction formation (12, 14): mammalian Crb3/PALS1/PATJ or Drosophila Crumbs/Stardust/dPATJ, mammalian Par-3/Par-6/aPKC or Drosophila Baz/Par-6/aPKC, and mammalian Scrib/Dlg/Lgl or Drosophila Scrib/Dlg/Lgl. Direct interaction between these proteins and protein complexes and their localization to the cellular membranes establishes junction formation and apicobasal polarity.

It is not surprising that a tumor suppression mechanism identified in Drosophila, such as polarity regulation, is conserved in mammals. Nonetheless, establishing that this is the case is important and supported by the location of crb3 at 19p13.3, which is frequently deleted in carcinomas and where multiple tumor suppressor genes reside (36). Advanced cancers often have undergone a partial or full EMT that disrupts epithelial junction formation that may contribute to invasion and metastasis. Previous studies have shown that mutations in polarity genes in Drosophila lead to uncontrolled proliferation and abnormal tissue architecture (12). An EMT can be achieved by epithelial tumor cells reactivating one of the developmental EMT programs controlled by TGF-β, receptor tyrosine kinase/RAS, Wnt, Notch, Hedgehog, or nuclear factor-{kappa}B (8). Alternatively, in mammalian cells, a partial or full EMT can be achieved by oncogenic activation of signal transduction pathways, such as the MAPK and PI3K pathways. Increased levels of PI3K contribute to loss of polarity and increased cell proliferation through its downstream effectors Akt and Rac1 in human mammary tumors (37). The ability of STAT pathways to disrupt polarity by direct repression of E-cadherin by Snail or Twist is also documented (8). STAT3 activation by β4integrin in conjunction with Erb2 signaling contributes to disruption of epithelial polarity and overproliferation in aggressive breast tumors (38). Moreover, activation of Erb2 signaling disrupts apicobasal polarity by associating with Par-6-aPKC components of the Par polarity complex (39). Our data suggest that direct perturbation of the downstream effectors of polarity, such as the polarity determinant Crb3, can also lead to phenotypic changes that facilitate tumor progression.

Comparing the global expression profiles of four independent TDCLs with the nontumorigenic parental cell line from which they were derived showed striking down-regulation of Crb3, an important determinant of tight junction formation (15) along with coordinate down-regulation of other known or putative cell junction/adhesion proteins. Previous studies have shown a correlation between tumor progression and the reduction in tight junctions, suggesting their importance as key aspect that cancer cells must overcome to metastasize (40). We established that TDCLs have lost the capacity for contact-inhibited growth and expressing Crb3 substantially restored proper tight junction formation leading to inhibition of cell proliferation. Thus, the loss of Crb3 expression in TDCLs plays a significant role in altering epithelial three-dimensional organization by conferring multilayer cell growth associated with absent contact inhibition that may contribute to tumorigenesis.

Previous studies have shown a correlation between tumor metastasis and a reduction in tight junctions (40). Our studies showed that restoring crb3 expression in TDCL cells reduced the migratory capacity and limited the invasiveness and metastatic potential of TDCLs. The dramatic loss of Crb3 expression in tumors in vivo also suggests that it is a growth disadvantage. Taken together, these results establish that Crb3 plays an important role in maintaining the epithelial phenotype, and down-regulation or loss of function of Crb3 contributes to tumorigenic progression. Our data suggest that Crb3 is required for tight junction formation, establishment of polarity and contact-inhibited growth, and suppression of migration, which suppresses metastasis. Thus, in mammalian epithelial tumors, as in Drosophila, similar mechanisms may control tumor growth. It will be of great interest to examine the status of Crb3 in human tumors and the possible contribution of Crb3 deregulation to the tumor-promoting functions of developmental and oncogenic pathways that modulate polarity in human cancers.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: The Howard Hughes Medical Institute and NIH grant R37CA53370 (E. White).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Benjamin Margolis for the generous gift of Crb3 and Crb3 mutant retroviral expression vectors and Crb3 antibody and Tissue Analytic Services and the Gene Expression Core Facilities at the Cancer Institute of New Jersey for tissue preparation and microarray analysis, respectively.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 12/21/07. Revised 3/26/08. Accepted 4/ 1/08.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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