Reduction of Membrane Protein CRIM1 Decreases E-Cadherin and Increases Claudin-1 and MMPs, Enhancing the Migration and Invasion of Renal Carcinoma Cells

Abstract

CRIM1 is a membrane protein that has been reported to be related to cell proliferation. CRIM1 is expressed in renal carcinoma cells, but its involvement in proliferation and malignant transformation remains unclear. We analyzed whether alterations in the characteristics of cancer cells are observed following knockdown of CRIM1. Decreased expression of CRIM1 did not affect proliferation or anchorage-independent growth. The results of wound healing and invasion assays showed that reduced expression of CRIM1 increased cells’ migratory and invasive abilities. Expression analysis of factors involved in migration and invasion in CRIM1-knockdown cells revealed that expression of the cell adhesion factor E-cadherin declined and expression of claudin-1, which is upregulated in metastatic cancer cells, increased. In addition, increased expression of matrix metalloproteinase (MMP) 2 and MMP9, protease essential for cancer cell invasiveness, was observed. Furthermore, an increase in phosphorylated focal adhesion kinase (FAK), which increases cell migration, was observed. Increased expression of the E-cadherin transcription repressors Snail, Slug, and ZEB-1 were observed, and mRNA levels of E-cadherin were decreased. Therefore, expression of E-cadherin is thought to be decreased by both suppression of E-cadherin mRNA expression and promotion of degradation of the E-cadherin protein. In addition, expression of CRIM1 was decreased in renal cancer cells undergoing epithelial–mesenchymal transition (EMT) stimulated by tumor necrosis factor alpha (TNF-α). Thus, CRIM1 regulates the expression of several EMT-related factors and appears to play a role in suppressing migration and invasion through control of EMT.

CRIM1 was first identified as a membrane protein containing a cysteine-rich region that is involved in neural tissue development.^1,2) CRIM1 was subsequently found to be highly expressed in the kidney and placenta.^1–4) Analysis of CRIM1-deficient mice revealed that CRIM1 is necessary for normal kidney development.^5,6) Three-dimensional culture analysis revealed that CRIM1 is necessary for the viability of vascular endothelial cells and is indispensable for formation of tube structures by endothelial cells.^7,8) Furthermore, the extracellular domain of CRIM1 was reported to bind to factors such as vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-4.^9,10) Therefore, CRIM1 has been suggested to be involved in regulation of the activity of VEGF upon angiogenesis and control of the activity of BMP-4 during organogenesis. We recently found that CRIM1 is also highly expressed in kidney cancer cells (our unpublished observation); however, its role in these cells has not been elucidated.

Epithelial–mesenchymal transition (EMT) is required for the initiation of metastasis in cancer.¹¹⁾ Following EMT, epithelial cells lose their cell polarity and ability to adhere to surrounding cells, differentiating into mesenchymal cells and gaining migration and invasion abilities.^11,12) When EMT occurs, expression of several factors is altered.^11–13) The cell adhesion molecule E-cadherin decreases, and N-cadherin increases.^11–14) Expression of Snail, Slug and ZEB-1, which repress transcription of E-cadherin, is increased.^11–14) In addition, vimentin, an intermediate filament unique to mesenchymal cells, increases and β-catenin, responsible for cytoplasmic anchoring of cadherin, decreases.^11–14) Furthermore, the activity of focal adhesion kinase (FAK), which is involved in signal transduction from the adhesion point, mediating cell adhesion, is enhanced and expression of claudin-1, which is involved in the formation of tight junctions, is induced.^15–18) Expression of matrix metalloproteinase (MMP) 2 and 9 is also induced.^19,20) Changes in the expression of these proteins is used as a marker of EMT. In this study, we analyzed the motility and invasive potential of cells and the expression of EMT-related proteins using cells in which expression of CRIM1 was decreased using siCRIM1. Based on these results, we investigated whether CRIM1 is involved in progression to malignancy in renal cancer cells.

MATERIALS AND METHODS

Cell Culture and Treatment

ACHN human renal carcinoma cells were obtained from the American Type Culture Collection (ATC C, Manassas, VA, U.S.A.). Cells were cultured in RPMI1640 medium (Invitrogen, Carlsbad, CA, U.S.A.) containing 10% fetal bovine serum (FBS). Cells were maintained at 37°C with 5% CO₂. Treatments with recombinant human tumor necrosis factor alpha (TNF-α) (Wako, Osaka, Japan), and the MMP2 and MMP9 inhibitor G6001 (Calbiochem, San Diego, CA, U.S.A.) were performed for 48 h.

Knockdown of CRIM1 Expression

Stealth RNA interference oligos against CRIM1 (siCRIM1) were used for knockdown of CRIM1 gene expression. The siCRIM1 and control oligos (siNeg) were purchased from Invitrogen (Carlsbad, CA, U.S.A.). Either siCRIM1 or siNeg was transfected into cells using Lipofectamine RNAiMax reagent (Invitrogen). After 48 h of transfection, cells were harvested and identical numbers were seeded for each assay.

Cell Proliferation Assay

Cell proliferation was assessed using cell counting kit-8 (Dojindo, Kumamoto, Japan). Briefly, ACHN cells transfected with small interfering RNA (siRNA), were seeded in each well of 96-well plates at a density of 5×10³ cells/well. After 24, 48, or 72 h of culture, 10 µL of WST-8 was added to each well, and the plates were incubated for 2 h at 37°C. The absorbance of each well at 450 nm was measured using a microplate spectrophotometer. Experiments were carried out in triplicate.

Soft Agar Assay

For the soft agar assay, cells (2×10³) were suspended in 0.3% agar medium containing 2.5 or 10% FBS and layered on 0.5% agar-coated 35-mm dishes and cultivated for 3 weeks. The colonies formed were stained with 0.25% 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT, Sigma, St. Louis, MO, U.S.A.) for 24 h and the number of colonies (>0.07 mm in diameter) was counted.

Wound-Healing Assay

Wounds were created in confluent cells using a pipette tip. The cells were then rinsed with medium to remove floating cells and debris. The culture plates were incubated at 37°C. Wounds were measured at 0 and 24 h. The percentage of wound-healing was calculated using the equation: (percentage wound-healing)=average of ([gap length: 0 h]-[gap length: 24 h])/[gap length: 0 h]).

Invasion Assay

Cell culture inserts (8-µm pore size; Thermo Fisher Scientific, Waltham, MA, U.S.A.) coated with Matrigel (Corning, NY, U.S.A.) were used for cell invasion assays. ACHN cells transfected with siCRIM1 or siNeg were washed, and 5×10⁴ cells were suspended in serum-free RPMI1640 medium and placed on the Matrigel-coated cell culture inserts. Cell culture inserts were placed into 24-well plates containing RPMI1640 supplemented with 10% FBS. After 24 h of incubation, the non-invasive cells were removed with a cotton swab, and the invasive cells were fixed with 4% paraformaldehyde and stained with Giemsa staining solution. The invasive cells in 4 randomly-selected fields were imaged under a microscopy (×100 magnification) and counted.

Statistical Analysis

Data were presented as the mean±standard deviation (S.D.) Statistical significance was determined by Mann–Whitney U test.

Western Blotting

Cells were lysed in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, and 10% glycerol) without bromophenol blue and dithiothreitol, and sonicated for 10 s. The lysates were heated at 95°C for 5 min and then cleared by centrifugation. Protein concentration was determined by BCA kit (Pierce, Rockford, IL, U.S.A.), and then 1/20 vol of 1 mM dithiothreitol was added to the lysates. Aliquots of 30 µg of the lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon P Membrane (Merck Millipore; Billerica, MA, U.S.A.). The membranes were blocked with 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) (−), for 1 h and then incubated with primary antibodies CRIM-1 (Sigma), E-cadherin, N-cadherin, vimentin, β-catenin, claudin-1 (Cell Signaling Technology, Boston, MA, U.S.A.), phosphorylated FAK (pFAK), FAK (Upstate, Lake Placid, NY, U.S.A.) in PBS (−) containing 1% BSA and 0.05% Tween 20 for 12 h at 4°C. After washing, the blots were incubated with an HRP-conjugated secondary antibody and visualized with an enhanced chemiluminescence detection system (Western Lightning Plus-ECL, Parkin Elmer, Waltham, MA, U.S.A.).

RT Quantitative (q)PCR

Total RNA was isolated from ACHN cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized and PCR was carried out using THUNDERBIRD SYBR qPCR RT Set (TOYOBO Lifescience, Osaka, Japan). Primers with the following sequences were used: E-cadherin, 5′-CAG AAA GTT TTC CAC CAA AG-3′ (forward) and 5′-AAA TGT GAG CAA TTC TGC TT−3′ (reverse); Snail, 5′-CCC CAA TCG GAA GCC TAA CT-3′ (forward) and 5′-GCT GGA AGG TAA ACT CTG GAT TAG A-3′ (reverse) ; Slug, 5′-TGT TGC AGT GAG GGC AAG AA-3′ (forward) and 5′-GAC CCT GGT TGC TTC AAG GA-3′ (reverse); ZEB-1, 5′-GCC AAT AAG CAA ACG ATT CTG-3′ (forward) and 5′-TTT GGC TGG ATC ACT TTC AAG-3′ (reverse); MMP2, 5′-TTG ATG GCA TCG CTC AGA TC-3′ (forward) and 5′-TTG TCA CGT GGC GTC ACA GT-3′ (reverse); MMP9, 5′-GCA AGC TGG ACT CGG TCT TT-3′ (forward) and 5′-TGG CGC CCA GAG AAG AAG-3′ (reverse). The specificity of each assay was validated by dissociation curve analysis of the PCR product. PCR was carried out in triplicates for each gene being validated. The gene expression levels were normalized to the level of β-actin.

RESULTS

Effect of Reduction of CRIM1 Expression on Proliferation and Anchorage-Independent Growth of ACHN Cells

To determine whether CRIM1 is required for proliferation and anchorage-independent growth, we knocked down CRIM1 expression in ACHN cells using siRNA against CRIM1. Reduced expression of CRIM1 at 72 h after cell plating was confirmed by Western blotting (Fig. 1A). The effects of reduction of CRIM1 expression on proliferation and anchorage-independent growth were determined using cell proliferation and soft agar assays. No significant changes in cell proliferation or anchorage-independent growth were observed between siCRIM1-transfected and siNeg (control)-transfected ACHN cells (Figs. 1B, C). Thus, reduction of CRIM1 protein did not affected cell proliferation or anchorage-independent growth.

Fig. 1. Effect of CRIM1 Knockdown on Proliferation of ACHN Cells

(A) Western blot analysis of ACHN cells transfected with siCRIM1. ACHN cells were treated with siNeg (control) or siCRIM1 for 48 h. Cells were harvested and seeded at 5×10⁵ cells per 35-mm dish and cultured for the indicated time. Thirty micrograms of whole cell lysate was subjected to Western blotting with antibody to CRIM1. (B) Cellular proliferation was measured in siNeg- or siCRIM1-treated ACHN cells using cell counting kit-8 after reseeding equal numbers of cells. (C) Anchorage-independent growth was analyzed by soft agar assay. Cells were seeded in 0.3% agar medium containing 10% FBS at 2×10³ cells per 35-mm dish, and cultivated for 3 weeks. Colonies formed (>0.07 mm in diameter) were counted after staining with INT. Values are shown as the average±S.D. of triplicate culture dishes.

Effect of Reduction of CRIM1 Expression on Migration and Invasion of ACHN Cells

To determine whether CRIM1 is required for migration, we performed a wound-healing assay. The migration area of siCRIM1-transfected ACHN cells was approximately 72%, whereas that of control cells was approximately 32% (Fig. 2A). ACHN cells transfected with siCRIM1 thus showed greater migration ability than control cells (Figs. 2A, B). To determine whether CRIM1 is required for invasion, we performed a Matrigel-based Transwell invasion assay. The number of invasive siCRIM1-transfected cells was approximately three-fold higher than in control cells (Figs. 2C, D). These results suggest that CRIM1 suppresses both cell migration and invasion.

Fig. 2. Effect of CRIM1 Knockdown on Migration and Invasion of ACHN Cells

(A) Reduced expression of CRIM1 promoted cell migration in a wound healing assay. ACHN cells transfected with siNeg or siCRIM1 were replated and grown to confluence, followed by mechanical wounding. Representative photomicrographs of the wounded cell monolayer. (B) Percentage area of migrating cells at 24 h was calculated. Data are shown as the mean±S.D. * p<0.05. (C) Reduced expression of CRIM1 promoted invasive capability. Representative photomicrographs of ACHN cells transfected with siRNA that migrated across the Matrigel-coated membrane of the cell culture inserts. (D) Number of ACHN cells that migrated across the Matrigel within 24 h was counted in randomly-selected microscopic fields (×100 magnification). Data are shown as the mean±S.D. * p<0.05.

Effect of Reduction of CRIM1 Expression on the Expression of EMT-Related Factors

To determine whether CRIM1 is involved in the expression of EMT markers, we performed Western blot analysis. Western blots confirmed that CRIM1 reduction decreased E-cadherin expression and increased expression of both pFAK and claudin-1 (Figs. 3A, B). However, no significant differences in expression of vimentin, β-catenin, or FAK were observed between CRIM1-knockdown and control cells. The proportion of pFAK to FAK was higher in FAK proportion at 24 h, but the amount of pFAK increased with time. These results suggest that CRIM1 is involved in the expression of EMT-related factors.

Fig. 3. Effect of CRIM1 Knockdown on Expression of EMT-Related Molecules in ACHN Cells

Western blot analysis of ACHN cells transfected with siCRIM1. ACHN cells were treated with siNeg (control) or siCRIM1 for 48 h. Cells were harvested and seeded at 5×10⁵ cells per 35-mm dish and cultured for the indicated time. Thirty micrograms of whole cell lysate was loaded onto SDS-polyacrylamide gels. A, 10% and B, 14%. Antibodies against E-cadherin, N-cadherin, vimentin, β-catenin, pFAK, FAK, and claudin-1 were used. Actin was used as a loading control. The intensity of bands was calculated using Multi GAUGE version 3.0 software (FUJIFILM). Quantitative data for E-cadherin, pFAK/FAK and claudin-1 expression are shown in C–E, respectively.

Effect of Reduction of CRIM1 Expression on E-Cadherin mRNA Expression and on Transcriptional Regulators of E-cadherin

To investigate whether the decrease in E-cadherin protein observed in CRIM1-knockdown ACHN cells was due to changes in expression at the mRNA level, RT-qPCR was performed. The expression level of E-cadherin mRNA was lower in CRIM1-knockdown ACHN cells than in control cells (Fig. 4A). Furthermore, analysis of the expression of Snail, Slug, and ZEB-1, which suppress expression of the E-cadherin gene,^11–14) revealed that expression of all three factors increased as the expression of CRIM1 decreased (Figs. 4B–D).

Fig. 4. Effect of CRIM1 Knockdown on E-Cadherin mRNA Expression and on Transcriptional Regulators of E-Cadherin

ACHN cells were treated with siNeg (control) or siCRIM1 for 48 h. Cells were harvested and seeded at 5×10⁵ cells per 35-mm dish and cultured for 48 h. Expression of E-cadherin (A), and transcriptional repressors of E-cadherin, Snail (B), Slug (C), and ZEB-1 (D), in siCRIM1-transfected ACHN cells, as analyzed by RT-qPCR. Results are representatives of three independent experiments. Data are shown as the mean±S.D. * p<0.05.

MMPs Were Related to Decreased Expression of E-Cadherin Induced by Reduction of CRIM1 Expression

To investigate whether the decrease in protein levels of E-cadherin was due to enhancement of proteolysis, expression of MMP2 and MMP9, which are known to be involved in degradation of E-cadherin, was analyzed by RT-qPCR.²¹⁾ Expression levels of MMP2 and MMP9 mRNA were elevated in CRIM1-knockdown ACHN cells (Figs. 5A, B). Therefore, we used GM6001, a selective inhibitor of both MMP2 and MMP9, to investigate whether MMP2 and MMP9 are involved in the decrease in E-cadherin protein. Following CRIM1 knockdown and treatment with GM6001, levels of E-cadherin protein recovered, whereas levels of E-cadherin protein in control cells were not changed by GM6001 treatment (Fig. 5C). Thus, the decrease in E-cadherin protein following CRIM1 knockdown is mediated by both mRNA synthesis reduction and promotion of protein degradation.

Fig. 5. Matrix Metalloproteinases (MMPs) Were Related to Decreased Expression of E-Cadherin Induced by CRIM1 Knockdown

(A, B) Reduction of CRIM1 induces expression of MMPs. ACHN cells were treated with siNeg (control) or siCRIM1 for 48 h. Cells were harvested and seeded at 5×10⁵ cells per 35-mm dish and cultured for 48 h. Expression of MMP2 and MMP9 mRNA in siCRIM1-transfected ACHN cells was analyzed by RT-qPCR. Results are representatives of three independent experiments. Data are shown as the mean±S.D. * p<0.05. (C) Decreased expression of E-cadherin caused by CRIM1 knockdown was recovered by treatment with the MMP inhibitor GM6001. ACHN cells transfected with siRNA were harvested, re-plated and cultured for 24 h, and then GM6001 or dimethyl sulfoxide was added to the cells, followed by further culturing for 24 h. Thirty micrograms of whole cell lysate was subjected to Western blotting. Antibodies against E-cadherin were used. Actin was used as a loading control. The intensity of bands was calculated using Multi GAUGE version 3.0 software (FUJIFILM). Quantitative data for E-cadherin expression are shown.

Expression of CRIM1 Decreases by TNF-α

Stimulation with TNF-α has been reported to decrease E-cadherin expression and enhance the migratory ability and invasive ability of renal carcinoma cells.^20,22) Expression of claudin-1 has been reported to be increased by TNF-α in colorectal cancer cells. Deregulated expression of claudin-1 has been thought to involve in EMT.^17,18) Expression of CRIM1 under stimulation by TNF-α was analyzed by Western blotting. Expression of both E-cadherin and CRIM1 was decreased in ACHN cells stimulated with TNF-α (Fig. 6). Moreover, expression of claudin-1 was increased in TNF-α-treated ACHN cells (Fig. 6). The decrease in expression of CRIM1 was correlated with the expression change of both E-cadherin and claudin-1 which has been reported to be related to the enhancement of cell migration ability. This result suggests that CRIM1 plays a role in suppressing the migratory ability of cells under physiological conditions.

Fig. 6. Expression of CRIM1 Was Decreased by TNF-α

(A, B) Western blot analysis of ACHN cells treated with several concentrations of TNF-α for 48 h. Thirty micrograms of whole cell lysate was subjected to Western blotting. A, 10% and B, 14%. Antibodies against E-cadherin, CRIM1 and claudin-1 were used. Actin was used as a loading control. The intensity of bands was calculated using Multi GAUGE version 3.0 software (FUJIFILM). Quantitative data for E-cadherin, CRIM1 and claudin-1 expression are shown in C–E, respectively.

DISCUSSION

In this study, we demonstrated that decreasing the expression of CRIM1, a cell membrane protein in kidney cancer cells, has no effect on cell proliferation, but increases cell motility and invasive capacity. We also revealed that CRIM1 knockdown decreases the expression of E-cadherin, and increases pFAK and expression of claudin-1. These results strongly suggest that CRIM1 is involved in regulating expression of these factors and that it controls initiation of EMT, which is necessary for progression of cancer malignancy. CRIM1 is thought to contribute to cell–cell adhesion by maintaining the expression of E-cadherin, thereby suppressing cell mobility.

Expression of E-cadherin is known to decrease with EMT, and EMT plays an important role in cancer malignancy by promoting cancer invasion and metastasis.^11–14) Decreased expression of CRIM1 in cancer cells may promote EMT, followed by cancer invasion and metastasis. Claudin-1 is a protein involved in formation of tight junctions.^23–25) In colorectal cancer, deregulated expression of claudin-1 has been reported to cause tight junction failure and increase cell motility and metastatic potential.^23–25) In addition, it has been reported that expression of claudin-1 is increased by stimulation of the inflammatory cytokine TNF-α, promoting EMT.^17,18) Therefore, claudin-1 has been investigated for use as a marker of malignancy in cancer. We found that CRIM1 is involved in control of expression of claudin-1. Further study of CRIM1 may shed new light on promotion of malignancy in cancer cells. It has been reported that FAK is activated by phosphorylation, and that activated FAK increases cell motility.^15,16) We found that decreased expression of CRIM1 enhances FAK phosphorylation. Although the proportion of pFAK in CRIM1-knockdown ACHN cells was low in 24 h after reseeding, the proportion increased at 72 h after reseeding. Therefore, it appears that CRIM1 usually restricts the motility of cells by suppressing phosphorylation of FAK. There are many reports that FAK is involved in cancer proliferation, invasion and metastasis, and anti-cancer drugs targeting FAK are being developed.^15,16,26,27) Therefore, compounds that increase the activity of CRIM1, a membrane protein involved in FAK activity control, may also function as anti-cancer agents. Decreased expression of CRIM1 induced an increase in mRNA levels of Snail, Slug, and ZEB-1, which are transcriptional repressors of E-cadherin.^11–14) This suggests that CRIM1 is involved in suppression of Snail, Slug, and ZEB-1 gene expression, and that reduced CRIM1-mediated suppression promotes expression of these genes. Notch, a membrane protein, has been reported to be involved in transcriptional regulation of several genes.^28,29) The mechanism by which CRIM1 suppresses gene expression remains unclear, and is a promising subject for further research.

In addition, decreased expression of CRIM1 induced an increase in mRNA levels of MMP2 and 9; administration of inhibitors of MMP2 and 9 reversed the decrease in E-cadherin. MMP2 and 9 have been reported to be involved in degradation of E-cadherin.²¹⁾ These findings suggest that the decrease in E-cadherin mediated by CRIM1 knockdown is regulated at both the mRNA and protein levels. It is unknown whether CRIM1 and E-cadherin bind directly, but CRIM1 and E-cadherin have each been reported to be associated with β-catenin.³⁰⁾ CRIM1 may stabilize E-cadherin by forming a complex via β-catenin. We used a yeast two-hybrid system to search for intracellular binding factors of CRIM1 and obtained several candidate binding factors (Sugiyama et al., in preparation). Further study of these factors may reveal the mechanism by which E-cadherin stabilizes CRIM1.

CRIM1 has been reported to be involved in promotion of EMT in lung cancer and prostate cancer.^31,32) We demonstrated that loss of CRIM1 expression in renal carcinoma cells causes EMT. These conflicting results suggest that the function of CRIM1 may differ with tissue type or cellular origin. Analysis of the intracellular binding factors of CRIM1 may clarify the tissue-dependent modes of action of CRIM1.

TNF-α is known to induce EMT in cancer cells.^20,22) When ACHN cells were treated with TNF-α, expression of E-cadherin decreased, expression of claudin-1 increased and EMT occurred, but expression of CRIM1 simultaneously decreased. This result suggests that a decrease in expression of CRIM1 is required for EMT under physiological conditions. Methods to control the occurrence of EMT can contribute to development of cancer therapies. Therefore, investigation of compounds that decrease the expression of CRIM1 or molecular ligands that regulate CRIM1 activity may facilitate development of novel drugs for cancer treatment.

Conflict of Interest

The authors declare no conflict of interest.

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