Ablation of p120-catenin enhances invasion and metastasis of human lung cancer cells

Abstract

p120-catenin, a member of the Armadillo gene family, has emerged as both a master regulator of cadherin stability and an important modulator of small GTPase activities. Therefore, it plays novel roles in tumor malignant phenotype, such as invasion and metastasis. We have reported previously that abnormal expression of p120-catenin is associated with lymph node metastasis in lung squamous cell carcinomas (SCC) and adenocarcinomas. To investigate the role and possible mechanism of p120-catenin in lung cancer, we knocked down p120-catenin using small interfering RNA (siRNA). We found that ablation of p120-catenin reduced the levels of E-cadherin and beta-catenin proteins, as well as the mRNA of beta-catenin. Furthermore, p120-catenin depletion inactivated RhoA, but increased the activity of Cdc42 and Rac1, and promoted proliferation and the invasive ability of lung cancer cells both in vitro and in vivo. Our data reveal that p120-catenin gene knockdown enhances the metastasis of lung cancer cells, probably by either depressing cell-cell adhesion due to lower levels of E-cadherin and beta-catenin, or altering the activity of small GTPase, such as inactivation of RhoA and activation of Cdc42/Rac1.

Figure 1

Expression of p120‐catenin (p120ctn) after transfection of p120ctn‐small invasive (si)RNA.Following transfection with siRNAs as described in the Materials and Methods section, p120ctn expression was analyzed by Western blot and reverse transcription – polymerase reaction (RT‐PCR). To test the specificity of our siRNAs, we re‐introduced p120ctn cDNA to three clonal cell lines. (A) Protein bands representing p120ctn isoforms were detected at approximately 120 kDa (isoform 1) and 100 kDa (isoform 3) in BE1 and control cells. The clonal cell lines designated siRNA1‐3 revealed more than 90% reduction in p120ctn. (B) RT‐PCR analysis indicates that mRNA expression of p120ctn produced several specific bands, in BE1 and control cells. After transfection with p120ctn‐siRNA, the BE1 cells showed a reduction or the complete absence of all p120ctn isoforms. (C) Lanes 1–3 were siRNA‐transfected cells transfected with mouse p120ctn 1A, and lanes 4–6 were the siRNA‐transfected cells transfected with p120ctn 3A. At approximately 120 kDa and 100 kDa, protein bands representing p120ctn isoform 1 or isoform 3, respectively, were detected. SPC (D–F) and LTE (G–I) cells showed similar results.

Figure 2

Down‐regulation of endogenous p120‐catenin (p120ctn) expression reduces the expression of both E‐cadherin and β‐catenin.
Expression of E‐cadherin and β‐catenin was analyzed by Western blot (A) and reverse transcription – polymerase reaction (RT‐PCR) (B). (A) E‐cadherin and β‐catenin protein levels were severely reduced in BE1/SPC/LTE‐p120ctn‐small invasive (si)RNA cells, compared with untransfected cells and control cells, which had specific bands at 120 kDa and 92 kDa, respectively. (B) There was no significant change in mRNA levels of E‐cadherin, and a specific band at 585 bp was detected in all cells. The expression of β‐catenin was reduced in the p120ctn knockdown cells. The band positions are indicated on the left with molecular weight markers.

Figure 3

Restoration of p120‐catenin (p120ctn) rescued the expression of E‐cadherin and β‐catenin, and inhibited the invasion of SPC cell line.Re‐expression of E‐cadherin and β‐catenin was analyzed by Western blot (A–C). (A) E‐cadherin and β‐catenin protein levels were significantly increased in BE1 small invasive (si)RNA+ p120ctn isoform 1A cells or +p120ctn isoform 3A cells, compared with BE1siRNA+ control cells, which had specific bands at 120 kDa and 92 kDa, respectively. There was a similar change in LTE‐siRNA (B) and SPC‐siRNA (C) cell lines. (D) Serum‐stimulated Matrigel invasion assay of SPC cell line. The average number of migrating cells that had migrated through the pores was counted after 18 h. Statistical analysis showed that fewer of the SPC siRNA+ p120ctn‐1A‐expressing cells (13 ± 5.57) appeared to have migrated compared to SPC siRNA+ control (31 ± 3.61) and SPC siRNA+ 3A‐expressing cells (18 ± 7.02). The average number of cells that had migrated in each cell shown in bars was counted.

Figure 4

Knockdown of p120‐catenin (p120ctn) decreased the activity of RhoA and increased the activity of Cdc42 and Rac1.Expression of total Cdc42, and Rac1 was analyzed by Western blot. Activity of these proteins was measured as described in the Materials and Methods section. (A) Cdc42 and Rac1 activities increased in the p120ctn‐knockdown BE1 cells, which were determined using pull‐down assay. Total Cdc42 and Rac1 protein did not change. (B) The activity of RhoA was obviously decreased in (si)RNA‐transfected BE1 cells. Results of RhoA G‐lisa in p120 knockdown cells and control cells. Columns with error bars indicate the activities of RhoA. SPC (C, D) and LTE (E, F) cells showed similar results.

Figure 5

p120‐catenin (p120ctn) knockdown promotes lung cancer cell growth and migration.We added the result of BE1, which has representative results, to Fig. 5. (A, B) FCM analysis indicated that cells transfected with small invasive (si)RNA showed a significant increase in number at the S stage and a significant reduction in number at the G1 stage, compared with the untransfected cells and control group. (C, D) The number of cells that migrated through the pores was counted after 16 h and 24 h. No migrated cells were detected in the untransfected and control groups, while migratory p120ctn knockdown cells were detected after 16 h of culture. Although there were migratory cells in untransfected cells and control cell groups after 24 h, more migratory cells were detected in the p120ctn‐siRNA cell culture.

Figure 6

The decreased expression of p120‐catenin (p120ctn) promotes BE1 cell growth, and tumors lacking p120ctn have malignant features in vivo.The tumors on the back of the mice, which were injected with untransfected cells (A) or control cells (B), were smaller than the tumors in the mice with transfected cells (C). The mean tumor weights in the BE1‐p120ctn^(–) group was significantly higher than in either the BE1 or control groups (D). Hematoxylin and eosin staining reveals that tumors in the BE1 group and control groups had clear boundaries (E). The p120ctn knockdown tumors showed characteristics of invasion (F). Similar observations were seen in the metastatic tumors. Liver metastatic tumor in BE1 cell‐injected mice had clear boundaries with noninvasive growth (G). Tumors in mice injected with BE1‐p120ctn^(–) cells showed characteristics of invasion (H), and nude mice injected with BE1‐p120ctn^(–) cells also developed leg (I) and heart (J) metastases, and pleural fluid formation (K), in which we can see the tumor cells by Papanicolaou staining (arrow). (E, F × 100 magnification, G–J × 200, and K ×400).