Tumor suppressor Alpha B-crystallin (CRYAB) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties

Article metrics

Abstract

Alpha B-crystallin (CRYAB) maps within the nasopharyngeal carcinoma (NPC) tumor-suppressive critical region 11q22-23 and its downregulation is significantly associated with the progression of NPC. However, little is known about the functional impact of CRYAB on NPC progression. In this study we evaluated the NPC tumor-suppressive and progression-associated functions of CRYAB. Activation of CRYAB suppressed NPC tumor formation in nude mice. Overexpression of CRYAB affected NPC progression-associated phenotypes such as loss of cell adhesion, invasion, interaction with the tumor microenvironment, invasive protrusion formation in three dimensional Matrigel culture, as well as expression of epithelial–mesenchymal transition-associated markers. CRYAB mediates this ability to suppress cancer progression by inhibition of E-cadherin cytoplasmic internalization and maintenance of β-catenin in the membrane that subsequently reduces the levels of expression of critical downstream targets such as cyclin-D1 and c-myc. Both ectopically expressed and recombinant CRYAB proteins were associated with endogenous E-cadherin and β-catenin, and, thus, the cadherin/catenin adherens junction. The CRYAB α-crystallin core domain is responsible for the interaction of CRYAB with both E-cadherin and β-catenin. Taken together, these results indicate that CRYAB functions to suppress NPC progression by associating with the cadherin/catenin adherens junction and modulating the β-catenin function.

Introduction

Nasopharyngeal carcinoma (NPC) is a malignancy associated with multiple genetic alterations. Loss of heterozygosity on chromosome 11q is frequently observed in NPC (Hui et al., 1996; Lo et al., 2000). Previously, through a monochromosome transfer approach and microsatellite typing, 11q13 and 11q22-23 were identified as tumor-suppressive critical regions (Cheng et al., 2000). Tumor-suppressor genes, THY1 and CADM1 (formerly called TSLC1) in 11q22-23, were identified; their loss is associated with invasive and metastatic properties characteristic of late- and advanced-stage NPC (Lung et al., 2005, 2006, 2010). These findings suggest that 11q22-23 harbors tumor-suppressor genes whose loss is associated with NPC progression. Alpha B-crystallin (CRYAB), located in the tumor-suppressive 11q22-23 region, is a small heat-shock protein and molecular chaperone. Downregulated CRYAB expression is associated with NPC progression (Lung et al., 2008), as well as testicular (Takashi et al., 1998) and breast (Lin et al., 2006) cancers, which indicate that CRYAB is a potential tumor-suppressor gene in these cancers.

Epithelial–mesenchymal transition (EMT) has an important role in cancer progression (Thiery et al., 2009). EMT is regulated by multiple signals during different stages in cancer progression. Imbalances in the cell polarity networks and disruption of cell–cell junctions initiate the EMT process (Lee et al., 2006). The adherens junction mainly functions to maintain the physical association between cells. Internalization of E-cadherin caused by aberrant cellular signals disrupts the adherens junction (Palacios et al., 2005). The consequent release of β-catenin from the membrane to the cytoplasm and nucleus activates its downstream transcriptional program and promotes EMT. EMT and β-catenin signaling are important in NPC development and progression (Chou et al., 2008). Loss of tumor-suppressor genes associated with NPC progression may facilitate EMT. To date, however, little is known about their association.

In this study, we aim to investigate the functional role of CRYAB in tumorigenesis and progression of NPC, as well as the molecular mechanisms responsible for CRYAB function. Re-expression of CRYAB in NPC cell lines reduced their tumorigenicity in vivo. NPC progression-associated phenotypes, as well as EMT-associated markers, were suppressed upon CRYAB re-expression. The effects of CRYAB on the internalization of E-cadherin and β-catenin functions, and its association with the cadherin/catenin adherens junction, were investigated in the present study.

Results

CRYAB suppresses NPC tumor formation in vivo

CRYAB was previously shown to be downregulated in NPC cell lines and tumors (Lung et al., 2008). We used a tetracycline-regulated gene expression system (Protopopov et al., 2002) to further study its tumor-suppressive function in vivo. pETE-Bsd-CRYAB was transfected into the tetracycline transcriptional activator (tTA)-expressing HONE1-2 NPC cell line. Stable transfectants, CRYAB42 and CRYAB46, were selected. As control, pETE-Bsd was transfected into HONE1-2 to generate the stable transfectant, BSD1. Both CRYAB mRNA and protein expression were induced in CRYAB42 and CRYAB46 in the absence of doxycycline (−dox) and verified by quantitative real-time PCR (Figure 1a) and western blot analyses (Figure 1b). In the presence of dox (+dox), the tTA transcriptional activator was inactivated, resulting in a reduction of CRYAB mRNA and protein expression.

Figure 1
figure1

CRYAB suppresses NPC tumorigenicity in vivo. (a) Real-time quantitative reverse transcription–PCR analysis of CRYAB gene expression in NP460, BSD1 vector-alone and CRYAB stable transfectants. NP460 was used as the positive control of CRYAB expression. The relative CRYAB expression fold differences of each cell line were compared with the BSD1 vector-alone transfectant control. ±dox: Treatment with and without dox. (b) Western blot analysis of CRYAB protein expression levels in NP460, BSD1 vector-alone and CRYAB stable transfectants (±dox). α-Tubulin was used for protein normalization. (c) Tumor growth kinetics of BSD1 vector-alone and CRYAB stable transfectants (±dox) in nude mice. Each data point represents the average tumor volume of all six sites inoculated for each cell line.

The BSD1 vector-alone and CRYAB stable transfectants were subcutaneously injected into athymic nude mice. The control BSD1 was highly tumorigenic and formed palpable tumors of 150 mm3 within 4 weeks in all six injection sites (Table 1). Overexpression of CRYAB in the absence of dox significantly suppressed tumorigenicity (Figure 1c). No tumors arising from CRYAB42 and only two tumors with CRYAB46 were observed at week 4 after injection. The difference was statistically significant (P<0.05). Addition of dox reduced the levels of CRYAB and partially reversed the tumor-suppressive effect. However, there was still a clear growth-inhibitory effect compared with BSD1. This may be attributed to some leakiness in the system as reported previously (Lung et al., 2010). Excised tumors (±dox) were examined by western blots and no longer express CRYAB (data not shown). These results are consistent with CRYAB being a potent suppressor of NPC tumor formation in vivo.

Table 1 Tumorigenicity assays of CRYAB transfectants

Overexpression of CRYAB affects NPC adhesion and invasion

Adhesion assays show that overexpression of CRYAB in CRYAB42 and CRYAB46 (−dox) significantly increases the cellular adhesive ability compared with BSD1 (P<0.05) (Figure 2a). Reduction of CRYAB expression in CRYAB42 and CRYAB46 (+dox) decreased the adhesive ability to similar levels observed in BSD1. In the transwell invasion assay, when CRYAB was overexpressed (−dox), the relative invasive ability was reduced to 62% and 74% (P<0.05), respectively, in CRYAB42 and CRYAB46 (Figure 2b), whereas the invasive ability in CRYAB42 and CRYAB46 (+dox) was restored. The migration potential of CRYAB42 and CRYAB46 was not changed significantly in the transwell migration assay under the same conditions as in the invasion assay (data not shown). Hence, the decreased cell number observed in the invasion assay was not due to change of migration ability. To further confirm inhibition of invasion by CRYAB, real-time quantitative cell migration and invasion assays were performed. In the invasion assay, 1:10 diluted Matrigel was coated in the chambers. Matrigel is a gelatinous protein mixture, which mimics the complex extracellular environment of the cancer cells. A rapid increase in cell index was observed after 30 h in BSD1 cells compared with CRYAB42 and CRYAB46, indicating their reduced invasion abilities (Figure 2c). At 50 h, the cell index of BSD1 was 1.5942, whereas the cell indices of CRYAB42 and CRYAB46 were 0.3606 and 0.2736, respectively. In the migration assay, the cell index did not show as much difference between BSD1 vector-alone and CRYAB stable transfectants. At 50 h, the cell index was 2.3856 for BSD1, and 1.8547 and 1.4072 for CRYAB42 and CRYAB46, respectively. Another Epstein–Barr virus-positive NPC cell line, C666, was also tested for its invasive ability; pCR3.1 and pCR3.1-CRYAB plasmids were transiently transfected into C666, followed by transwell invasion assay. The western blot shows that CRYAB is expressed in the transiently transfected C666 cells, which showed significantly reduced invasiveness when CRYAB is expressed (Figure 2d). These results suggest that overexpression of CRYAB potently affects both the adhesive and invasive properties of the NPC cells.

Figure 2
figure2

CRYAB affects NPC adhesion and invasion abilities. (a) Adhesion ability in BSD1 vector-alone and CRYAB stable transfectants. Significant increase of cell adherent ability was observed in CRYAB stable transfectants (−dox) as compared with BSD1 vector-alone and CRYAB stable transfectant (+dox) controls. Scale bar: 100 μm. The number of adherent cells in each cell line was counted. * and **Statistically significant difference from BSD1 vector-alone transfectants and CRYAB transfectants (+dox), respectively (P<0.05). (b) Transwell invasion assay of BSD1 vector-alone and CRYAB stable transfectants. Scale bar: 100 μm. Invading cells at the bottom surface of the transwell filter were stained and counted. Relative invasive ability in each cell line was calculated. * and **Statistically significant difference from BSD1 vector-alone transfectants and CRYAB transfectants (+dox) (P<0.05). (c) Quantitative assessment of migration and invasion abilities of BSD1 vector-alone and CRYAB stable transfectants. Real-time quantitative cell migration and invasion assays were performed using CIM-plate. Cell index represents the number of cells migrated or invaded through the chamber at different time points. (d) Transwell invasion assay of C666 with or without transient transfection of CRYAB. Scale bar 100 μm. *Statistically significant difference from C666 transient transfected with pCR3.1 (P<0.05). CRYAB protein expression in C666 with or without transient transfection of CRYAB was examined by western blotting.

CRYAB suppresses NPC progression-associated properties

To study CRYAB function in the tumor microenvironment, three dimensional (3D) Matrigel culture was used. When cultured in Matrigel for 14 days, BSD1 (±dox) formed large spheroids. Overexpression of CRYAB reduced the relative spheroid sizes to 54% and 61%, respectively, in CRYAB42 and CRYAB46 (−dox), compared with BSD1 (Figure 3a). Reduction of CRYAB expression (+dox) restored the relative spheroid sizes in CRYAB42 and CRYAB46. Significant reduction of viable cell numbers was observed by MTT assay for CRYAB42 and CRYAB46 after 2 weeks in 3D Matrigel culture (Figure 3b), indicating that reduction of spheroid sizes in CRYAB42 and CRYAB46 is due to decreased ability to proliferate in Matrigel culture.

Figure 3
figure3

CRYAB suppresses NPC progression-associated properties. (a) 3D Matrigel culture assay of BSD1 vector-alone and CRYAB stable transfectants. Scale bar: 100 μm. The relative spheroid sizes of each cell line compared with BSD1 vector-alone were counted and calculated 2 weeks after inoculation. * and **Statistically significant difference from BSD1 vector-alone transfectants and CRYAB transfectants (+dox) (P<0.05). (b) Cell viability in 3D Matrigel culture was measured by MTT assay. *Statistically significant difference from BSD1 vector-alone transfectants and CRYAB transfectants (+dox) (P<0.05). *Statistically significant difference from BSD1 vector-alone transfectants and CRYAB transfectants (+dox) (P<0.05). (c) Invasive protrusion structure formation in 3D Matrigel culture of BSD1 vector-alone and CRYAB stable transfectants. The BSD1 vector-alone transfectant and CRYAB transfectants (+dox) showed a disorganized morphology, with invasive protrusion structures in 3D Matrigel culture 4 weeks after inoculation, whereas CRYAB transfectants (−dox) formed polarized cell spheroids. Higher magnification ( × 10) images of invasive protrusion structures and polarized cell spheroids are shown. Phalloidin was used to stain the actin filaments. Scale bar: 50 μm. (d) Real-time quantitative PCR analysis of E-cadherin and Vimentin in BSD1 vector-alone and CRYAB stable transfectants (±dox). The relative fold differences of E-cadherin and Vimentin expression of each cell line were compared with the BSD1 vector-alone transfectant control. *Statistically significant differences (P<0.05). (e) Western blot analysis of vimentin and E-cadherin in the parental NPC cell line HONE1-2, BSD1 vector-alone and CRYAB stable transfectants (±dox). α-Tubulin was used for normalization.

EMT is a central process occurring during cancer progression (Thiery et al., 2009). Loss of epithelial polarity and formation of disorganized, non-cohesive and irregular invasive protrusions in 3D culture are hallmarks of the morphological phenotypes indicative of EMT (Grunert et al., 2003). In 3D Matrigel culture, BSD1 (±dox) and CRYAB42 and CRYAB46 (+dox) showed typical EMT phenotypes with disorganized, non-cohesive and irregular invasive protrusions. By contrast, CRYAB42 and CRYAB46 (−dox) showed polarized and well-organized spheroids (Figure 3c). In addition to morphological changes, we examined the expression levels of the EMT-associated markers E-cadherin and vimentin. Upregulation of E-cadherin and downregulation of vimentin were observed in CRYAB42 and CRYAB46 (−dox) at both the mRNA (P=0.02 and 0.026 for E-cadherin; P=0.034 and 0.037 for vimentin) (Figure 3d) and protein (Figure 3e) levels, as compared with the parental cell line HONE1-2 and the BSD1 vector-alone transfectant. The +dox treatment restored the altered expression of E-cadherin and vimentin in CRYAB42 and CRYAB46 clones. CRYAB, thus, has a potential role in suppressing NPC progression-associated properties.

CRYAB affects E-cadherin localization

Disruption of cell–cell junctions such as the cadherin/catenin adherens junction is associated with EMT (Thomson et al., 2011). In BSD1 vector-alone transfectants, E-cadherin internalized from the cell membrane to the cytoplasm, indicating disruption of the cadherin/catenin adherens junction in these cells. By contrast, E-cadherin remained in the cell membrane in CRYAB42 and CRYAB46, indicating maintenance of the cadherin/catenin adherens junction in the cell membrane (Figure 4a), which was also observed by immunohistochemical staining in clinical samples (Supplementary Figure 1). This difference was statistically significant (P<0.05) and was confirmed after subcellular fractionation and western blot analysis. In the membrane fractions, E-cadherin accumulated to higher levels in the CRYAB42 and CRYAB46 clones. The E-cadherin levels were similar in the nuclear fractions and were weak in the cytoplasmic fractions (Figure 4b). Subcellular fractionation and western blot analysis of CRYAB showed that the majority of CRYAB protein in the CRYAB42 and CRYAB46 clones was located in the cytoplasm. CRYAB was moderately expressed in the cell membrane and was very weak in the nucleus (Figure 4c).

Figure 4
figure4

CRYAB function associated with inhibition of cytoplasmic internalization of E-cadherin. (a) Immunofluorescence staining analysis of E-cadherin. CRYAB inhibits the internalization of E-cadherin. The arrows indicate membrane-bound E-cadherin. Scale bar: 1 μm. E-cadherin localization from different fields in a minimum of 150 cells was counted and the percentage of cells showing E-cadherin localization at different sites was calculated. *Statistically significant differences (P<0.05). (b) Subcellular localization of E-cadherin in BSD1 vector-alone and CRYAB stable transfectants. Subcellular fractionation followed by immunoblotting was performed with BSD1 vector-alone and CRYAB stable transfectants. α-Tubulin was used as a marker for normalization of the cytoplasmic fraction. Histone3 (H3) was used as a marker for normalization of the nuclear fraction. The membrane fraction was normalized to the Coomassie Blue staining of the proteins in that fraction. (c) Subcellular localization of CRYAB in BSD1 vector-alone and CRYAB stable transfectants. Subcellular fractionation followed by immunoblotting was performed with BSD1 vector-alone and CRYAB stable transfectants. α-Tubulin was used as a marker for normalization of the cytoplasmic fraction. Histone3 (H3) was used as a marker for normalization of the nuclear fraction. The membrane fraction was normalized to the Coomassie Blue staining of the proteins in that fraction.

CRYAB affects β-catenin function

β-Catenin, when released from the cadherin/catenin adherens junction, translocates to the cytoplasm and nucleus, and acts as a transcription factor to promote tumor progression and EMT (Heuberger and Birchmeier, 2010). As CRYAB suppressed the internalization of E-cadherin, we further investigated whether CRYAB affected β-catenin function. Immunofluorescence staining showed that β-catenin was located throughout the cytoplasm and nucleus in the control BSD1, whereas it localized mainly to the cell membrane in the CRYAB42 and CRYAB46 transfectants (Figure 5a). This difference was statistically significant (P<0.05). Membrane localization of β-catenin was also observed by immunohistochemical staining in clinical samples (Supplementary Figure 1). This observation was further confirmed after subcellular fractionation and subsequent western blot analysis. In both nuclear and cytoplasmic fractions, the β-catenin levels were reduced in CRYAB42 and CRYAB46 as compared with BSD1, whereas in the membrane fractions, there were increased levels of β-catenin in the CRYAB42 and CRYAB46 clones (Figure 5b).

Figure 5
figure5

CRYAB affects β-catenin function. (a) Immunostaining of β-catenin in BSD1 vector-alone and CRYAB stable transfectants are shown. The arrow indicates typical membrane staining. Scale bar 1 μm. β-Catenin localization from different fields in a minimum of 500 cells was counted and the percentage of cells showing β-catenin localization at different sites was calculated. *Statistically significant differences (P<0.05). (b) Subcellular localization of β-catenin in BSD1 vector-alone and CRYAB stable transfectants. Subcellular fractionation followed by immunoblotting was performed with BSD1 vector-alone and CRYAB stable transfectants. α-Tubulin was used as a marker for normalization of the cytoplasmic fraction. Histone3 (H3) was used as a marker for normalization of the nuclear fraction. The membrane fraction was normalized to the Coomassie Blue staining of the proteins in that fraction. (c) Western blot analysis of β-catenin and its downstream targets c-myc and cyclin-D1 in the parental NPC cell line HONE1-2, BSD1 vector-alone and CRYAB stable transfectants. α-Tubulin was used for normalization. (d) Western blot analysis of β-catenin and its downstream targets c-myc and cyclin-D1 in the NPC cell line CNE2, with or without transient transfection of CRYAB. α-Tubulin was used for normalization.

The NPC cell line HONE1-2 and the BSD1 vector-alone stable transfectant strongly express β-catenin and its downstream targets c-myc and cyclin-D1. By contrast, in CRYAB42 and CRYAB46 (−dox), the protein expression levels of β-catenin, c-myc and cyclin-D1 were downregulated; reduction of CRYAB expression in CRYAB42 and CRYAB46 (+dox) restored these expression levels (Figure 5c). Downregulation of β-catenin, c-myc and cyclin-D1 was also observed in another NPC cell line, CNE2, when CRYAB was transiently expressed (Figure 5d). These results indicate that overexpression of CRYAB maintains β-catenin in the cell membrane and downregulates target genes such as c-myc and cyclin-D1.

The CRYAB protein is associated with the cadherin/catenin adherens junction

Previous studies showed that CRYAB interacts (Ghosh et al., 2007b) and colocalizes (Maddala and Rao, 2005) with β-catenin, as well as interacts with the kidney-specific cadherin (Thedieck et al., 2008). CRYAB is, therefore, potentially associated with the cadherin/catenin adherens junction. In co-immunoprecipitation assays using CRYAB (Figure 6a) and β-catenin (Figure 6b) antibodies, the association between CRYAB and endogenous β-catenin and E-cadherin was detected in CRYAB42 and CRYAB46. Moreover, the level of β-catenin associating with E-cadherin was dramatically increased in the CRYAB42 and CRYAB46 clones compared, indicating a strong association of β-catenin and E-cadherin with CRYAB.

Figure 6
figure6

CRYAB protein is associated with the cadherin/catenin complex. Ectopically expressed CRYAB protein is associated with endogenous cadherin/catenin complex in vivo as shown by co-immunoprecipitation assay using CRYAB or β-catenin antibodies. Cell lysates prepared from BSD1 vector-alone and CRYAB stable transfectants were precipitated with antibodies against CRYAB (a) and β-catenin (b), or purified IgG controls, followed by immunoblotting with the indicated antibodies. Input: Cell lysate without immunoprecipitation. (c) A schematic diagram showing the CRYAB full-length and truncated variants. (d) The purified recombinant CRYAB protein interacts with endogenous E-cadherin and β-catenin. The total cell lysate of the BSD1 vector-alone transfectant was incubated with a GST control, GST-CRYAB full-length or GST-CRYAB truncated variants. Immunoblotting with anti-GST antibodies served as loading controls.

This association was further confirmed in glutathione S-transferase (GST) pull-down assays. GST-tagged CRYAB full-length and truncated proteins (Figure 6c), namely CRYAB-ΔN (deleted N-terminus), CRYAB-ΔN+core (deleted N-terminus and α-crystallin core domain) and CRYAB-ΔC (deleted C-terminus), were expressed, purified and incubated with total cell lysates of the vector-alone BSD1. Endogenous E-cadherin was pulled down by the CRYAB full-length, CRYAB-ΔN and CRYAB-ΔC, but not by the CRYAB-ΔN+core proteins. Endogenous β-catenin was pulled down by CRYAB full-length, moderately pulled down by CRYAB-ΔN and CRYAB-ΔC, but not pulled-down by the CRYAB-ΔN+core proteins (Figure 6d). This indicates that the α-crystallin core domain is responsible for the interaction of CRYAB with both E-cadherin and β-catenin. The N-terminus and C-terminus of CRYAB are partially responsible for the interaction with β-catenin. Taken together, these results indicate that CRYAB is likely to be associated with the cadherin/catenin adherens junction in NPC.

Discussion

Our earlier study showed that CRYAB is a candidate tumor-suppressor gene in NPC (Lung et al., 2008). In the current study, we show that overexpression of CRYAB significantly suppresses NPC tumorigenicity in nude mice. However, there is still a clear growth inhibition in the CRYAB stable transfectants as compared with the BSD1 vector-alone transfectant control, even when expression of CRYAB is repressed by dox. This may be attributed to some leakiness of CRYAB expression in vivo, which was also reported in earlier studies using this dox-regulated system (Li et al., 2004; Lung et al., 2006). The expression of CRYAB is lost from all excised tumors of both CRYAB42 and CRYAB46 (±dox), indicating CRYAB is clearly inactivated in the tumorigenic revertants, and further supports the tumor-suppressive function of CRYAB in vivo. The nude mouse assay provides key functional evidence that CRYAB suppresses NPC growth in vivo and is a potent tumor-suppressor gene in NPC.

CRYAB is correlated with lymph node metastasis in NPC (Lung et al., 2008); reduced in peri-neural invasion of head and neck cutaneous squamous cell carcinoma (Solares et al., 2010); and is significantly associated with adverse ovarian cancer patient survival (Stronach et al., 2003). In this current study, overexpression of CRYAB functionally suppresses tumor progression-associated properties such as loss of cell adhesion and invasion, as well as viability and appearance of irregular invasive protrusion formation in 3D Matrigel culture in NPC, providing functional evidence of the impact of CRYAB in NPC progression.

EMT has a crucial role in cancer progression. In NPC, EMT was shown to affect cancer invasion in both Epstein–Barr virus-dependent and independent manners (Lin et al., 2009; Kong et al., 2010). E-cadherin and vimentin are two well-studied markers associated with EMT that are aberrantly expressed in NPC (Zheng et al., 1999; Lo et al., 2003). The observed expression changes of E-cadherin and vimentin in the CRYAB stable transfectants further suggest association of CRYAB with EMT. Loss of cell polarity and disruption of cell–cell junctions such as the adherens junction is involved in the initiation of EMT (Lee et al., 2006). E-cadherin is one of the major components in the adherens junctions and it is internalized from the cell membrane to the cytoplasm during disruption of the adherens junction (Lu et al., 2003). Inhibition of cytoplasmic internalization of E-cadherin in the CRYAB stable transfectants indicates that CRYAB may be involved in the disruption of the cadherin/catenin adherens junction and EMT initiation. Recently, nuclear localization of E-cadherin was reported in Merkel cell carcinomas, solid pseudopapillary tumors of the pancreas and renal cell carcinomas (Chetty and Serra, 2008). The detailed mechanisms of E-cadherin nuclear localization are still poorly understood. p120, which regulates the trafficking of E-cadherin, reportedly may have a role (Reynolds and Roczniak-Ferguson, 2004). The nuclear localization of E-cadherin in BSD1 and CRYAB stable transfectants remains to be investigated further.

Disruption of the cadherin/catenin adherens junction releases the membrane-bound β-catenin into the cytoplasm. The β-catenin then further accumulates and interacts with the T-cell factor/lymphoid enhancer factor (TCF/LEF) complex to enhance the transcription of genes promoting tumor progression (Nelson and Nusse, 2004). In the current study, overexpression of CRYAB in NPC suppresses the β-catenin oncogenic function by maintaining the membrane-bound β-catenin. c-myc and cyclin-D1 are well-studied β-catenin/TCF/LEF target genes (Heuberger and Birchmeier, 2010). Downregulation of c-myc and cyclin-D1 in the CRYAB stable and transient transfectants further indicates that suppression of β-catenin is associated with overexpression of CRYAB.

In the cadherin/catenin adherens junctions, E-cadherin interacts with p120–catenin, γ-catenin and β-catenin. β-Catenin interacts with cortical actin-associated α-catenin, which links the adherens junctions to the actin cytoskeleton and mediates its mechanical stability (Green et al., 2010). In our study, we demonstrated the interaction among CRYAB, E-cadherin and β-catenin in vitro and in vivo, which suggests the involvement of CRYAB in the cadherin/catenin adherens junction. Previous studies found that the CRYAB protein interacts with β-catenin at the N-terminus, the α-crystallin core domain and the C-terminus (Ghosh et al., 2007b), and interacts with Ksp–cadherin, another member of the cadherin family, in the N-terminus (Thedieck et al., 2008). Our GST pull-down assay shows that the α-crystallin core domain is mainly responsible for the interaction with β-catenin. The moderate effects seen with the N-terminal and C-terminal fragments are consistent with the previous finding of Ghosh et al. (2007b). The α-crystallin core domain, but not the N-terminus or the C-terminus, is required for the interaction with E-cadherin. The α-crystallin core domain is the major interaction site of CRYAB with cytoskeleton proteins and also facilitates actin polymerization (Ghosh et al., 2007a; Ohto-Fujita et al., 2007). The interaction of CRYAB with E-cadherin and β-catenin in the α-crystallin core domain may facilitate the interaction between the adherens junction and the actin cytoskeleton, which is required for adhesion strengthening, and junctional plaque assembly and maintenance (Green et al., 2010).

Growing evidence suggests the impact of chromosome 11q tumor-suppressor genes in NPC progression (Lung et al., 2006, 2010). Our current study now provides clear evidence that CRYAB suppresses tumorigenesis, as well as progression-associated phenotypes and EMT-associated markers in NPC. CRYAB inhibits E-cadherin cytoplasmic internalization and also affects the oncogenic function of β-catenin by maintaining membrane-bound β-catenin. In addition, we demonstrate that the CRYAB protein is also associated with the cadherin/catenin adherens complex through the α-crystallin core domain. Thus, CRYAB is a potent tumor-suppressor gene, which is associated with tumor progression in NPC. Additional studies are needed to unravel the detailed mechanisms of CRYAB function in the cadherin/catenin adherens complex.

Materials and methods

Cell lines and culture conditions

The NPC cell lines, CNE2 (Sizhong et al., 1983) and C666 (Cheung et al., 1999), the immortalized nasopharyngeal epithelial cell line, NP460 (Li et al., 2006), and the engineered NPC HONE1-2 cells (Protopopov et al., 2002) were cultured as described previously (Lung et al., 2008). Stable transfectants with the CRYAB transgene or the pETE-Bsd vector alone were maintained in culture medium containing 5 μg/ml blasticidin.

Quantitative reverse transcription–PCR analysis

Quantitative reverse transcription–PCR analysis was performed as described previously (Cheung et al., 2009). CRYAB and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) Taqman probes and the SYBR Green PCR master mix were purchased from Applied Biosystems (ABI, Carlsbad, CA, USA). The sequences of primers are shown in Supplementary Table 1.

Western blot analysis

Preparation of cell lysates, sodium dodecyl sulfate–PAGE and transfer were performed as described previously (Lung et al., 2005). CRYAB (SPA-222; Stressgen, Victoria, BC, Canada), E-cadherin (36/E-cadherin; BD Biosciences Labware, MA, USA), vimentin (RV202; BD Biosciences), β-catenin (L54E2; Cell Signaling Technology, Danvers, MA, USA), cyclin-D1 (sc-753; Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-myc (sc-764; Santa Cruz Biotechnology), α-tubulin (Ab-1; Calbiochem, Darmstadt, Germany) and histone H3 (sc-10809; Santa Cruz Biotechnology) antibodies were used for western blots.

Transient and stable transfection of CRYAB

The 528 bp CRYAB cDNA fragment was subcloned from pcDNA3-CRYAB (Simon et al., 2007) into the pCR3.1 vector (Invitrogen, Carlsbad, CA, USA) at the HindIII and EcoRI sites, and into the pETE-Bsd vector (Protopopov et al., 2002) at the BglII and NotI sites. Transient and stable transfection of CRYAB was performed with the Lipofectamine 2000 reagent (Invitrogen), as described previously (Lung et al., 2006).

In vivo tumorigenicity assay

The tumorigenicity of cell lines was examined by subcutaneous injection as described previously (Lung et al., 2006). In brief, 1 × 107 cells were injected into six sites on three 6 to 8-week-old female immunodeficient athymic BALB/C nu/nu nude mice and tumor growth was measured weekly. To inhibit tetracycline-regulated expression of CRYAB, 200 μg/ml dox was added to the drinking water (Lung et al., 2010).

Adhesion assay

A total of 1 × 104 cells was seeded in a 96-well plate and incubated in culture medium for 8 h, followed by washing in phosphate-buffered saline twice and fixing with 70% ethanol for 15 min at room temperature. The remaining adherent cells were washed by phosphate-buffered saline and stained with 0.5% crystal violet for 10 min. Adherent cells in each well were counted.

Invasion assay

A total of 2 × 105 cells was seeded into the chamber of a 24-well Matrigel-coated membrane filter (Becton Dickinson Labware, Franklin Lakes, NJ, USA). The cell invasion assay was performed as described previously (Lung et al., 2010).

Real-time quantitative cell migration and invasion assays

The assays were performed using the xCELLigence System (Roche, Penzberg, Germany) for real-time cell analysis. In brief, cells are seeded in a 16-well CIM-plate containing electronic sensors for direct study of cell migration and invasion on the xCELLigence RTCA DP Instrument (Roche). For the migration assay, 40 000 cells were seeded directly on the upper chamber of the CIM-plate. For the invasion assay, 40 000 cells were seeded on the upper chamber coated with 1:10 diluted Matrigel (BD Biosciences). The bottom chambers were filled with Dulbecco's modified Eagle's medium plus 5% serum as chemoattractants. Migration and invasion were monitored for 70 h. Cell index represents the number of cells inside the wells based on the measured electrical impedance.

Matrigel culture spheroid and invasive protrusion formation assays

A Matrigel basement membrane matrix (BD Biosciences) was coated onto 24-well plates and then 2000 cells were seeded on top. For the spheroid formation assay, the Matrigel culture was incubated at 37 °C for 14 days and spheroids formed in the culture were counted and spheroid sizes were measured under an inverted light microscope (Nikon Instruments, Melville, NY, USA). For protrusion formation, the Matrigel culture was incubated at 37 °C for 30 days. Fixing and staining were performed as described previously (Wong et al., 2012).

MTT assay

A Matrigel basement membrane matrix (BD Biosciences) was coated onto 24-well plates and then 2000 cells were seeded on top. The MTT assay was performed as described previously (Lung et al., 2006).

Immunofluorescent staining

Immunofluorescence staining was performed as described previously (Leung et al., 2008) using the following primary antibodies as mentioned previously: β-catenin (1:200) and E-cadherin (1:100). Cells with different β-catenin and E-cadherin localization were counted using the ImageJ software (NIH, Bethesda, MD, USA). The percentage of cells with membrane or cytoplasmic and nuclear localization of β-catenin or E-cadherin was calculated.

Subcellular fractionation

Subcellular fractionation was performed following the protocol by Abcam (http://www.abcam.com/ps/pdf/protocols/subcellular_fractionation.pdf). In brief, cells were lysed with a subcellular fractionation buffer and cell lysates were centrifuged. The pellet was washed in fractionation buffer and resuspended in nuclear buffer; this is the nuclear fraction. The supernatant was re-centrifuged at a higher speed (40 000 r.p.m.); the supernatant, after ultra-centrifugation, is the cytoplasmic fraction. The pellet was washed with fractionation buffer and resuspended in nuclear buffer; this is the membrane fraction.

Co-immunoprecipitation

The co-immunoprecipitation assay was performed as described previously (Zhang et al., 2010). In brief, cells were lysed and then pre-cleared with rProtein-G-agarose (Invitrogen) and incubated with CRYAB (Stressgen) (1:250), β-catenin (Cell Signaling Technology) (1:250) or a control purified rabbit IgG (Invitrogen), at 4 °C overnight. Lysates were then immunoprecipitated with rProtein-G-agarose, washed, and then subjected to sodium dodecyl sulfat-PAGE and western blot analysis.

GST recombinant protein expression and purification

GST recombinant protein expression and purification were performed as described previously (Wong et al., 2011). The cloning and purification of the truncated CRYAB proteins are described in the Supplementary information and Supplementary Table 2.

Statistical analysis

Student's t-test was performed for adhesion, invasion and 3D Matrigel culture assays; quantitative real-time reverse transcription–PCR; and immunofluorescence staining for statistical comparison between the BSD1 vector-alone and CRYAB stable transfectants. A P-value of <0.05 was considered statistically significant.

References

  1. Cheng Y, Stanbridge EJ, Kong H, Bengtsson U, Lerman MI, Lung ML . (2000). A functional investigation of tumor suppressor gene activities in a nasopharyngeal carcinoma cell line HONE1 using a monochromosome transfer approach. Genes Chromosomes Cancer 28: 82–91.

  2. Chetty R, Serra S . (2008). Nuclear E-cadherin immunoexpression: from biology to potential applications in diagnostic pathology. Adv Anat Pathol 15: 234–240.

  3. Cheung AK, Lung HL, Ko JM, Cheng Y, Stanbridge EJ, Zabarovsky ER et al. (2009). Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma. Proc Natl Acad Sci USA 106: 14478–14483.

  4. Cheung ST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS et al. (1999). Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein–Barr virus. Int J Cancer 83: 121–126.

  5. Chou J, Lin YC, Kim J, You L, Xu Z, He B et al. (2008). Nasopharyngeal carcinoma—review of the molecular mechanisms of tumorigenesis. Head Neck 30: 946–963.

  6. Ghosh JG, Houck SA, Clark JI . (2007a). Interactive sequences in the stress protein and molecular chaperone human alphaB crystallin recognize and modulate the assembly of filaments. Int J Biochem Cell Biol 39: 1804–1815.

  7. Ghosh JG, Shenoy Jr AK, Clark JI . (2007b). Interactions between important regulatory proteins and human alphaB crystallin. Biochemistry 46: 6308–6317.

  8. Green KJ, Getsios S, Troyanovsky S, Godsel LM . (2010). Intercellular junction assembly, dynamics, and homeostasis. Cold Spring Harb Perspect Biol 2: a000125.

  9. Grunert S, Jechlinger M, Beug H . (2003). Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. Nat Rev Mol Cell Biol 4: 657–665.

  10. Heuberger J, Birchmeier W . (2010). Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling. Cold Spring Harb Perspect Biol 2: a002915.

  11. Hui AB, Lo KW, Leung SF, Choi PH, Fong Y, Lee JC et al. (1996). Loss of heterozygosity on the long arm of chromosome 11 in nasopharyngeal carcinoma. Cancer Res 56: 3225–3229.

  12. Kong QL, Hu LJ, Cao JY, Huang YJ, Xu LH, Liang Y et al. (2010). Epstein–Barr virus-encoded LMP2A induces an epithelial–mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma. PLoS Pathog 6: e1000940.

  13. Lee JM, Dedhar S, Kalluri R, Thompson EW . (2006). The epithelial–mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 172: 973–981.

  14. Leung AC, Wong VC, Yang LC, Chan PL, Daigo Y, Nakamura Y et al. (2008). Frequent decreased expression of candidate tumor suppressor gene, DEC1, and its anchorage-independent growth properties and impact on global gene expression in esophageal carcinoma. Int J Cancer 122: 587–594.

  15. Li HM, Man C, Jin Y, Deng W, Yip YL, Feng HC et al. (2006). Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase. Int J Cancer 119: 1567–1576.

  16. Li J, Wang F, Protopopov A, Malyukova A, Kashuba V, Minna JD et al. (2004). Inactivation of RASSF1C during in vivo tumor growth identifies it as a tumor suppressor gene. Oncogene 23: 5941–5949.

  17. Lin DI, Barbash O, Kumar KG, Weber JD, Harper JW, Klein-Szanto AJ et al. (2006). Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF (FBX4–alphaB crystallin) complex. Mol Cell 24: 355–366.

  18. Lin JC, Liao SK, Lee EH, Hung MS, Sayion Y, Chen HC et al. (2009). Molecular events associated with epithelial to mesenchymal transition of nasopharyngeal carcinoma cells in the absence of Epstein–Barr virus genome. J Biomed Sci 16: 105.

  19. Lo AK, Liu Y, Wang XH, Huang DP, Yuen PW, Wong YC et al. (2003). Alterations of biologic properties and gene expression in nasopharyngeal epithelial cells by the Epstein–Barr virus-encoded latent membrane protein 1. Lab Invest 83: 697–709.

  20. Lo KW, Teo PM, Hui AB, To KF, Tsang YS, Chan SY et al. (2000). High resolution allelotype of microdissected primary nasopharyngeal carcinoma. Cancer Res 60: 3348–3353.

  21. Lu Z, Ghosh S, Wang Z, Hunter T . (2003). Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of beta-catenin, and enhanced tumor cell invasion. Cancer Cell 4: 499–515.

  22. Lung HL, Bangarusamy DK, Xie D, Cheung AK, Cheng Y, Kumaran MK et al. (2005). THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma. Oncogene 24: 6525–6532.

  23. Lung HL, Cheung AK, Cheng Y, Kwong FM, Lo PH, Law EW et al. (2010). Functional characterization of THY1 as a tumor suppressor gene with antiinvasive activity in nasopharyngeal carcinoma. Int J Cancer 127: 304–312.

  24. Lung HL, Cheung AK, Xie D, Cheng Y, Kwong FM, Murakami Y et al. (2006). TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma. Cancer Res 66: 9385–9392.

  25. Lung HL, Lo CC, Wong CC, Cheung AK, Cheong KF, Wong N et al. (2008). Identification of tumor suppressive activity by irradiation microcell-mediated chromosome transfer and involvement of alpha B-crystallin in nasopharyngeal carcinoma. Int J Cancer 122: 1288–1296.

  26. Maddala R, Rao VP . (2005). alpha-Crystallin localizes to the leading edges of migrating lens epithelial cells. Exp Cell Res 306: 203–215.

  27. Nelson WJ, Nusse R . (2004). Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303: 1483–1487.

  28. Ohto-Fujita E, Fujita Y, Atomi Y . (2007). Analysis of the alphaB-crystallin domain responsible for inhibiting tubulin aggregation. Cell Stress Chaperones 12: 163–171.

  29. Palacios F, Tushir JS, Fujita Y, D'Souza-Schorey C . (2005). Lysosomal targeting of E-cadherin: a unique mechanism for the downregulation of cell–cell adhesion during epithelial to mesenchymal transitions. Mol Cell Biol 25: 389–402.

  30. Protopopov AI, Li J, Winberg G, Gizatullin RZ, Kashuba VI, Klein G et al. (2002). Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21. J Gene Med 4: 397–406.

  31. Reynolds AB, Roczniak-Ferguson A . (2004). Emerging roles for p120–catenin in cell adhesion and cancer. Oncogene 23: 7947–7956.

  32. Simon S, Fontaine JM, Martin JL, Sun X, Hoppe AD, Welsh MJ et al. (2007). Myopathy-associated alphaB-crystallin mutants: abnormal phosphorylation, intracellular location, and interactions with other small heat shock proteins. J Biol Chem 282: 34276–34287.

  33. Sizhong Z, Xiukung G, Yi Z . (1983). Cytogenetic studies on an epithelial cell line derived from poorly differentiated nasopharyngeal carcinoma. Int J Cancer 31: 587–590.

  34. Solares CA, Boyle GM, Brown I, Parsons PG, Panizza B . (2010). Reduced alphaB-crystallin staining in perineural invasion of head and neck cutaneous squamous cell carcinoma. Otolaryngol Head Neck Surg 142: S15–S19.

  35. Stronach EA, Sellar GC, Blenkiron C, Rabiasz GJ, Taylor KJ, Miller EP et al. (2003). Identification of clinically relevant genes on chromosome 11 in a functional model of ovarian cancer tumor suppression. Cancer Res 63: 8648–8655.

  36. Takashi M, Katsuno S, Sakata T, Ohshima S, Kato K . (1998). Different concentrations of two small stress proteins, alphaB crystallin and HSP27 in human urological tumor tissues. Urol Res 26: 395–399.

  37. Thedieck C, Kalbacher H, Kratzer U, Lammers R, Stevanovic S, Klein G . (2008). alpha B-Crystallin is a cytoplasmic interaction partner of the kidney-specific cadherin-16. J Mol Biol 378: 145–153.

  38. Thiery JP, Acloque H, Huang RY, Nieto MA . (2009). Epithelial–mesenchymal transitions in development and disease. Cell 139: 871–890.

  39. Thomson S, Petti F, Sujka-Kwok I, Mercado P, Bean J, Monaghan M et al. (2011). A systems view of epithelial–mesenchymal transition signaling states. Clin Exp Metastasis 28: 137–155.

  40. Wong VC, Chen H, Ko JM, Chan KW, Chan YP, Law S et al. (2012). Tumor suppressor Dual-specificity Phosphatase 6 (DUSP6) impairs cell invasion and epithelial–mesenchymal transition (EMT)-associated phenotype. Int J Cancer 130: 83–95.

  41. Wong VC, Ko JM, Qi RZ, Li PJ, Wang LD, Li JL et al. (2011). Abrogated expression of DEC1 during oesophageal squamous cell carcinoma progression is age- and family history-related and significantly associated with lymph node metastasis. Br J Cancer 104: 841–849.

  42. Zhang J, Yu L, Wu X, Zou L, Sou KK, Wei Z et al. (2010). The interacting domains of hCdt1 and hMcm6 involved in the chromatin loading of the MCM complex in human cells. Cell Cycle 9: 4848–4857.

  43. Zheng Z, Pan J, Chu B, Wong YC, Cheung AL, Tsao SW . (1999). Downregulation and abnormal expression of E-cadherin and beta-catenin in nasopharyngeal carcinoma: close association with advanced disease stage and lymph node metastasis. Hum Pathol 30: 458–466.

Download references

Acknowledgements

We thank Patrick Vicart for the pcDNA3-CRYAB construct. We acknowledge the funding support from the Research Grants Council grants, and the University Grants Council of Hong Kong Special Administrative Region, People's Republic of China, for AoE/M-06/08 to MLL; The University of Hong Kong Seed Funding Programme for Basic Research to HLL; and the Swedish Cancer Society, the Swedish Research Council, the Swedish Institute and Karolinska Institute to ERZ. We acknowledge the Area of Excellence Hong Kong NPC Research Tissue Bank for the NPC specimens and tissue blocks.

Author information

Correspondence to H L Lung or M L Lung.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Supplementary Figure (PDF 1315 kb)

Supplementary Information (DOC 47 kb)

Rights and permissions

Reprints and Permissions

About this article

Keywords

  • Alpha B-crystallin
  • nasopharyngeal carcinoma
  • E-cadherin
  • β-catenin

Further reading