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. 2008 Jun 1;68(11):4105-15.
doi: 10.1158/0008-5472.CAN-07-6814.

Role of the polarity determinant crumbs in suppressing mammalian epithelial tumor progression

Cristina M Karp  1 , Ting Ting Tan, Robin Mathew, Deidre Nelson, Chandreyee Mukherjee, Kurt Degenhardt, Vassiliki Karantza-Wadsworth, Eileen White
Affiliations

Role of the polarity determinant crumbs in suppressing mammalian epithelial tumor progression

Cristina M Karp et al. Cancer Res. .

Abstract

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.

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Figures

Figure 1
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 pathway (1). Wild type, non-tumorigenic W2.3.1 iBMK were injected subcutaneously in mice and tumor growth occurred with long latency (three to four months). TDCLs were returned to in vitro cell culture and reinjected into animals where tumor growth occurred within two weeks. Clonal emergence of W2.3.1-RFP expressing tumors by whole animal optical imaging after three months and hematoxylin and eosin staining of emerging clonal nodules following selection for growth for three months in vivo. (B) Tumorigenic capacity of TDCLs. Parental W2.3.1 cells (red) and TDCLs (orange) were injected subcutaneously 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 hours with 0.4mM staurosporine or for 48 hrs with 3 μM etoposide, and viability was assessed by trypan blue exclusion for staurosporine and MTT assay for etoposide (white bars: untreated; black bars: 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 known to be resistant to staurosporine-induced apoptosis. (D) TDCL 5D cells retain apoptotitc 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.
Figure 2
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 TDCLs 5A-D. All samples were analyzed in duplicate (Affymetrix 430), accession no. GPLxxxx. (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 A-D relative to parental W2.3.1-5 and expression of vimentin and E-cadherin in TDCL A-D relative to parental W2.3.1-5. (D) Localization of junction proteins occludin, ZO-1, cadherin, and β-catenin to intracellular 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.
Figure 3
Figure 3
Role of Crb3 in controlling tight junction formation. (A) Graphic representation of wild type Crumbs 3 protein domain structure and schematic illustrating the Myc-tagged Crb3 and Crb3 mutants22. The Crb3 mutants contain a deletion of the C-terminal domain (ΔERLI) 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-Crb3ΔERLI in W2.3.1-5 cells. (C) Crb3 expression restores tight junction formation in TDCL 5D. TDCL 5D png vector infected or wild type Crb3 expressing vector infected cells and stained with for tight junction markers occludin, and ZO-1. Immunofluorescence showin 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 to png vector control. Mutant crb3 expression impairs tight junction formation in parental W2.3.1-5 cells. W2.3.1-5 parental cells png vector infected or Crb3ΔERLI 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 co-localization of Crb3 with occludin and ZO-1 in TDCL 5DCrb3 cells.
Figure 4
Figure 4
Crumbs3 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 as the cells migrated in and filled the void using computerized video multifield time-lapse microscopy for three days. 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 uninfected or infected with the empty retroviral vector png, or the wild type Crbs3 expressing vector were cultured in Matrigel and monitored by time-lapse microscopy. (C) Spheroids were co-stained with for β-catenin (green) and active caspase-3 (red) and imaged by confocal microscopy at 14 days. Crb3 expression confers single layer cell growth and apoptotic lumen formation in three-dimensional culture. 20–30 spheroids/microscopic field were counted in five different fields for each cell line and the experiment was repeated three times (p<0.05). Greater than 90% of TDCL 5D cells expressing Crbs3 formed spheroids with hollow lumens compared to less than 10% of TDCL 5D infected with the png vector.
Figure 5
Figure 5
Role of Crb3 in regulation of growth arrest and in vitro migration. (A) Immunofluorescence staining showing BrdU incorporation, as a measure of proliferation in TDCL 5D png and 5D Crb3 cells or W2.3.1-5 png and W2.3.1-5 Crb3ΔERLI with increasing cell density. Crb3 deficient TDCL 5D png with impaired tight junction formation continued to incorporate BrdU even after the cells reached confluency, whereas the TDCL 5D Crb3 suppressed proliferation in response to tight junction formation, contact inhibition. (B) Graphs summarizing the number of positive BrdU cells with increasing time and cell density. 50–100 cells/microscopic field were counted in four different fields for each cell line and the experiment was repeated three times. Results are presented as means ± SD of the number of cells/field, p<0.05 (p= 0.0348). (C) TDCL 5D png and 5D Crb3 and (B) W2.3.1-5 png and W2.3.1-5 Crb3ΔERLI cells were cultured to confluency in the upper well of transwell chambers. After 24 hrs 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/experiment and the experiment was replicated three times. Representative microscopic fields of crystal violet stained cells are shown. (D) Graphs summarizing the results of in vitro migration assay. Results are presented as means ± SD of the number of cells/field, evaluated in five-fields/membrane, p<0.05 (p=0.037).
Figure 6
Figure 6
Crb3 expression suppresses metastasis. (A) Tumorigenic capacity of TDCL 5D and png and Crb3 transfectants compared to parental W2.3.1-5 cells. Animals were injected subcutaneously with 107 cells and tumor formation was monitored over time. The parental W2.3.1-5 cells failed to form tumors by 20 days post injection while the TDCL 5D formed subcutaneous tumors as expected. Expression of Crb3 in TDCL 5D did not suppress subcutaneous tumor growth. (B) Subcutaneous tumor growth of TDCL 5D Crb3 is associated with the loss of Crb3 expression. TDCL 5D Crb3 tumors that formed 3 weeks post 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 5×105 cells TDCL 5D png (black, n=6) and TDCL 5D Crb3 (red, n=6). Most (5/6) of the TDCL 5D png injected animals succumbed to multiple bone and kidney metastases by 10 weeks 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.
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