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. 2012 Oct;14(10):974-85.
doi: 10.1593/neo.12942.

Tight junction proteins claudin-3 and claudin-4 control tumor growth and metastases

Xiying Shang  1 , Xinjian Lin, Edwin Alvarez, Gerald Manorek, Stephen B Howell
Affiliations

Tight junction proteins claudin-3 and claudin-4 control tumor growth and metastases

Xiying Shang et al. Neoplasia. 2012 Oct.

Abstract

The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), and activation of β-catenin pathway signaling as evidenced by increases in nuclear β-catenin, the dephosphorylated form of the protein, and transcriptional activity of β-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting β-catenin signaling.

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Figures

Figure 1
Figure 1
CLDN3 and CLDN4 control the growth of ovarian tumor xenografts in vivo. (A) Relative growth rate of 2008 (■), CLDN3KD.(◊), and CLDN4KD (○) tumors after SC inoculation of 1 x 106 cells in nu/nu mice (n = 16 for each tumor type). (B) Re-expression of siRNA-resistant CLDN3 or CLDN4 in the claudin knockdown cells, designated here as CLDN3KD-3 rescue and CLDN4KD-4 rescue, slowed the tumor growth in vivo. Six synonymous point mutations were introduced in shRNA target regions in full-length CLDN3 or CLDN4 cDNA and the siRNA-resistant cDNAs were cloned into pLVX-mCherry vector. Following infection with lentivirus produced from this vector in HEK298T cells, 2008 cells expressing high levels of the protein were isolated by fluorescence-activated cell sorting (FACS) and used to inoculate SC in nu/nu mice (n = 16 for each tumor type). (C) Quantification of Ki67-positive and TUNEL-positive cells and density of CD31-positive vessels in 2008, CLDN3KD, and CLDN4KD tumors.
Figure 2
Figure 2
Effect of CLDN3 and CLDN4 expression on TJ formation and cell adhesion. (A) TER in the monolayers of 2008, CLDN3KD, and CLDN4KD cells 3 days after seeding onto the transwell inserts (n = 6). (B) Paracellular permeability of 4-kDa FITC-dextran measured on day 3 after seeding the cells onto the transwell inserts. Vertical bars, ±SEM, n = 6. *P = 0.008 and **P = 0.001 (vs 2008 control). (C) Relative adhesion quantified at 6 hours after seeding on uncoated tissue culture dishes. Vertical bars, ±SEM, n = 4. *P = 0.001 and **P = 0.003 (vs 2008 control).
Figure 3
Figure 3
CLDN3 and CLDN4 control tumor cell migration and invasion. (A) Relative migration through an uncoated filter toward serum-containing medium in a Boyden chamber assay. The migratory ability of cells was compared by measuring the number of cells migrating through the uncoated filters. Vertical bars, ±SEM, n = 6. *P = 0.035 and **P = 0.005 (vs 2008 control). (B) Relativemotility as determined by the ability of 2008, CLDN3KD, and CLDN4KD cells to close a wound made by creating a scratch through a lawn of confluent cells. (C) Relative invasion of cells through a layer of Matrigel coated on the filter of a Boyden chamber measured at 24 hours after seeding. Vertical bars, ±SEM, n = 6. *P = 0.004 and **P = 0.002 (vs 2008 control).
Figure 4
Figure 4
CLDN3 and CLDN4 control metastatic potential of ovarian cells injected IV in nu/nu mice. (A) Fluorographs of mice 42 days after tail vein injection of 2 x 106 2008, CLDN3KD, or CLDN4KD cells molecularly engineered to express Ds-Red showing more extensive metastases when CLDN3 and CLDN4 are knocked down. (B) Representative fluorescent images of mouse lung lobes showing that knockdown of CLDN3 and CLDN4 increases the extent of metastasis to lung when imaged at 42 days after tail vein injection. Scale bar, 2 mm. (C) Quantification of total lung metastasis burden in the five mice of each group. The lungs were dissected, homogenized, and diluted for quantification of metastasis by red fluorescence. Vertical bars, ±SEM, n = 10. *P = 0.012 and **P = 0.001 (vs 2008 control). (D) Hematoxylin and eosin staining of fixed and paraffin-embedded tissue confirmed the presence of micrometastases in the lungs of mice injected with the CLDN4KD cells. Scale bar, 100 µm.
Figure 5
Figure 5
Effect of claudin expression on cell migration and invasion in vitro as well as growth and metastasis in vivo. (A) Relative motility as determined by the ability of HEY, HEY-CLDN3, and HEY-CLDN4 cells to close a wound made by creating a scratch through a lawn of confluent cells. (B) Relative invasion of HEY, HEY-CLDN3, and HEY-CLDN4 cells through a Matrigel-coated layer on the filter of a Boyden chamber measured at 12 hours after seeding. Vertical bars, ±SEM, n = 6. *P = 0.031 and **P = 0.014 (vs HEY control). (C) Relative growth rate of HEY (■), HEY-CLDN3 (▴), and CLDN4 (◆) tumors after SC inoculation of 1 x 106 cells in nu/nu mice (n = 16 for each tumor type). (D) Quantification of total lung metastatic burden as determined by measurement of extractable mCherry fluorescence at 43 days after tail vein injection of mCherry-labeled HEY, HEY-CLDN3, and HEY-CLDN4 cells. Vertical bars, ±SEM, n = 10. *P = 0.009 and **P = 0.005 (vs HEY-mCherry control).
Figure 6
Figure 6
CLDN3 and CLDN4 regulate the expression of E-cadherin. (A) Western blot analysis showing reduced expression of E-cadherin in CLDN3KD and CLDN4KD cells. (B) The relative levels of E-cadherin mRNA measured by real-time RT-PCR in CLDN3KD and CLDN4KD cells compared with 2008 cells. Vertical bars, ±SEM, n = 5. *P = 0.006 and **P = 0.002 (vs 2008 control).
Figure 7
Figure 7
Activation of β-catenin signaling on knockdown of CLDN3 and CLDN4. (A) Western blot analysis showing increased nuclear dephospho (active)-β-catenin levels in CLDN3KD and CLDN4KD cells. Relative levels of cytoplasmic and nuclear β-catenin were normalized against α-tubulin and histone H1, respectively, and expressed relative to the control from the mean of three separate analyses. (B) Immunocytochemical analysis of 2008, CLDN3KD, and CLDN4KD cells stained with antibody to β-catenin showing increased nuclear localization of β-catenin in the knockdown cells. (C) Western blot analysis showing increased GSK-3β phosphorylation in CLDN3KD and CLDN4KD cells. Relative levels of phospho-GSK-3β were normalized against total GSK-3β. The ratio in control 2008 cells is designated as 1. (D) Western blot analysis showing increased nuclear dephospho-β-catenin levels in CLDN3KD and CLDN4KD cells. Relative levels of active β-catenin (dephosphorylated form) were normalized against total β-catenin. The ratio in control 2008 cells is designated as 1. (E) Activation of β-catenin/TCF signaling in CLDN3KD and CLDN4KD cells. Cells were transiently co-transfected with the firefly luciferase TOPflash TCF reporter plasmid and the control plasmid pCMXβgal to normalize for transfection efficiency. The luciferase values were normalized for variations in transfection efficiency and are expressed as fold stimulation of luciferase activity compared with the 2008 control cultures. Vertical bars, ±SEM, n = 5. *P = 0.086 and **P = 0.016 (vs 2008 control). (F) Cyclin D1 protein levels in CLDN3KD and CLDN4KD cells relative to that in the 2008 cells were calculated densitometrically after normalization to the level of α-tubulin.
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