N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1–54 amino acids and the Armadillo repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1–54 amino acid sequence and Armadillo repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin

P120ctn isoform 1A restored the cytoplasmic E-cadherin levels and enhanced the invasiveness in SPC cells.

<p>SPC cells were transiently transfected with GFP-siRNA-p120ctn or with empty vector as control. 24 hours after transfection, an aliquot of cells was transfected again with p120ctn isoform 1A or 3A cDNA plasmids. (<b>A</b>) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal shown in the nucleus and cytoplasm indicates effective expression of GFP from GFP-siRNA-P120ctn, confirming the successful transfection. Depletion of p120ctn (I) reduced the E-cadherin levels (II). Transfection with empty vector (III) did not affect the E-cadherin levels (IV). Restitution of p120ctn isoform 1A (V) restored the cytoplasmic E-cadherin levels (VI), while restitution of p120ctn isoform 3A (VII) had no effects on the E-cadherin levels (VIII). (<b>B</b>) Levels of E-cadherin were then analyzed by Western blot assay. The results confirmed that depletion of p120ctn resulted in decreased E-cadherin levels. Restitution of p120ctn isoform 1A restored the E-cadherin levels, while restitution of p120ctn isoform 3A had no effects on E-cadherin expression. (<b>C</b>) The invasiveness of SPC cells were analyzed by Matrigel invasion assay. P120ctn ablation reduced the cell invasiveness in comparison with the control group transfected with vector alone (*, <i>P</i><0.01). Restitution of p120ctn isoform 1A and 3A both enhanced the invasiveness of SPC cells in comparison with the group with p120ctn ablation (Si-p120ctn) (**, <i>P</i><0.01, ***, <i>P</i><0.01).</p

Expression and localization of p120ctn and E-cadherin.

<p>(<b>A</b>) By the immunofluorescence method, the expression of E-cadherin and p120ctn were noted to be restricted to the cell membrane at cell-cell adherens junctions in HBE and H460cells, whereas in SPC and LTE cells, E-cadherin and P120ctn were confined to the cytoplasm. (<b>B</b>) Western blot analyses confirmed E-cadherin and p120ctn were expressed in all four cell lines. Of all p120ctn isoforms, only two isoforms, p120ctn isoforms 1 (120 kDa) and 3 (100 kDa), were detected.</p

P120ctn isoform 1A, isoform 3A and five p120ctn 1A deletion mutants M1 to M5.

<p>P120 isoform 1A contains the coiled-coil domain and a central armadillo domain. P120 isoform 3A lacks the coiled-coil domain. Five p120ctn 1A deletion mutants M1 to M5 fused to green fluorescent protein (GFP): M1 contains only N-terminal 1–101 amino acids; M2 has N-terminal 1–54 amino acids deleted; M3 contains only N-terminal 1–54 amino acids; M4 has N-terminal 55–101 amino acids deleted; M5 contains only N-terminal 55–101 amino acids.</p

Mutant 4 restored cytoplasmic E-cadherin and enhanced the invasiveness in SPC cells.

<p>SPC cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). (<b>A</b>) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn or M1–M5-GFP was expressed in the nucleus and cytoplasm. Expression (repletion) of M4 mutant (IX) restored cytoplasmic E-cadherin (X), while expression of the other mutants had no significant effects on the E-cadherin levels. (<b>B</b>) The levels of E-cadherin were analyzed by Western blot assay. Expression of M1–M5 mutants was detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated E-cadherin levels, whereas expression of the other mutants had no effects on the E-cadherin levels. (<b>C</b>) The invasiveness of SPC cells was analyzed by Matrigel invasion assay. Expression of M2 or M4 mutants enhanced the cell invasiveness in comparison with the group with ablated p120ctn (Si-p120ctn) (*, <i>P</i><0.01, **, <i>P</i><0.01), while expression of other three p120 1A mutants did not show significant effects.</p

Mutant 4 restored the E-cadherin to cell membrane and suppressed cell invasiveness in H460 cells.

<p>H460 cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). (<b>A</b>) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn, M1–M3, and M5-GFP was expressed in the nucleus and cytoplasm. GFP from GFP-si-P120ctn and M4-GFP was expressed in the nucleus, cytoplasm and on the cell membrane (IX). Expression or repletion of M4 mutant (IX) restored E-cadherin on the cell membrane (X), while expression of the other mutants had no significant effects on E-cadherin levels. (<b>B</b>) The levels of E-cadherin were then analyzed by Western blot assay. Expression of M1–M5 mutants were detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated the E-cadherin levels, whereas the other mutants had no effects on the E-cadherin levels. (<b>C</b>) The invasiveness of H460 cells was analyzed by Matrigel invasion assay. Repletion of M4 mutant reduced the cell invasiveness in comparison with the group of p120ctn ablation (Si-p120ctn) (**, <i>P</i><0.01), while repletion of M2 mutant enhanced the invasiveness (*, <i>P</i><0.01). Repletion of the other mutants (M1, M3 and M5) did not show significant effects on cell invasiveness in comparison with the group of 120ctn ablation.</p

Ascertaining an Appropriate Diagnostic Algorithm Using EGFR Mutation-Specific Antibodies to Detect EGFR Status in Non-Small-Cell Lung Cancer

<div><p>Background</p><p>Epidermal growth factor receptor (EGFR) mutation status is the most valuable indicator in the screening of non-small-cell lung cancer (NSCLC) patients for tyrosine kinase inhibitor (TKI) therapy. Accurate, rapid and economical methods of detecting EGFR mutations have become important. The use of two mutation-specific antibodies targeting the delE746-A750 mutation in exon 19 and L858R mutation in exon 21 makes this task possible, but the lack of consensually acceptable criteria for positive results limits the application of this antibody based mutation detection.</p> <p>Methods</p><p>We collected 399 specimens from NSCLC patients (145 resection specimens, 220 biopsy specimens, and 34 cytology specimens) whose EGFR mutation status had been detected by TaqMan PCR assay. Immunohistochemical (IHC) analyses using EGFR mutation-specific antibodies were employed for all samples. After staining and scoring, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated in accordance with different levels of positive grades in comparison with the results of PCR-based assay.</p> <p>Results</p><p>In IHC-based analyses, 144 cases were scored 0, 104 cases were scored 1+, 103 cases were scored 2+, and 48 cases were scored 3+. With the molecular-based results were set as the “gold standard”, the prevalence of mutation was 6.94% (10/144), 23.08% (24/104), 67.96% (70/103) and 100% (48/48), respectively, for samples with scores 0, 1+, 2+ and 3+. When score 3+ was considered positive, the specificity and PPV were 100%; if only score 0 was considered negative, 93.06% NPV was obtained.</p> <p>Conclusion</p><p>Patients with score 3+ have a perfect PPV (100%), and may accept TKI treatment directly without any molecular-based assays. Patients with score 0 had high NPV (93.06%), which could reach 97.22% when the detection of total EGFR was applied. However, samples with score 1+ or 2+ are unreliable and need further verification of EGFR mutation status by molecular-based assays.</p> </div

Promoter Methylation-Mediated Silencing of β-Catenin Enhances Invasiveness of Non-Small Cell Lung Cancer and Predicts Adverse Prognosis

<div><p>β-Catenin plays dual role in adhesion complex formation and the Wnt signaling pathway. Although β-catenin expression appears to be upregulated and Wnt signaling pathway is activated in the majority of cancers, its expression level seems to be lost in non-small cell lung cancer (NSCLC). We previously reported that the promoter of β-catenin was hypermethylated in two NSCLC cell lines. In the current study, we expanded our analysis for the methylation status of β-catenin promoter region and its protein expression in seven NSCLC cell lines and a series of 143 cases of primary human lung cancer with adjacent non-neoplastic tissues. Quantitative methylation specific PCR (qMSP) analysis showed methylation of β-catenin promoter region in five NSCLC cell lines, with increased β-catenin protein levels upon 5′-Aza-2′-deoxycytidine (5-aza-dC) treatment. The methylation status in SPC (methylated) and A549 (unmethylated) was confirmed by bisulfite sequencing PCR. 5-Aza-dC treatment inhibited invasiveness of SPC but not A549. Immunofluorescence analysis showed membranous β-catenin expression was lost in SPC and could be re-established by 5-aza-dC, while Wnt3a treatment led to nuclear translocation of β-catenin in both SPC and A549. Dual-luciferase assays indicated that 5-aza-dC treatment caused no significant increase in Wnt signaling activity compared with Wnt3a treatment. The effect of demethylation agent in SPC can be reversed by β-catenin depletion but not E-cadherin depletion which indicated that the methylation mediated β-catenin silencing might enhance NSCLC invasion and metastasis in an E-cadherin independent manner. Subsequent immunohistochemistry results further confirmed that β-catenin promoter hypermethylation correlated with loss of immunoreactive protein expression, positive lymph node metastasis, high TNM stage and poor prognosis. The present study implicates β-catenin promoter hypermethylation in the mechanism of epigenetic changes underlying NSCLC metastasis and progression, thus indicating the potential of β-catenin as a novel epigenetic target for the treatment of NSCLC patients.</p></div

Staining of biopsy specimens using EGFR mutation-specific antibodies.

<p>The total EGFR protein in biopsy samples could be stained with EGFR (D38B1) antibody (<b>A</b>, <b>D</b> and <b>G</b>, 40× and the upper left corner 200×). Samples without EGFR mutations were not stained with two mutation-specific antibodies (<b>B</b> and <b>C</b>, 40× and the upper left corner 200×). Samples with E746–A750 deletion mutation were stained with E746–A750 deletion (6B6) specific antibody (<b>E</b>, 40× and the upper left corner 200×) and samples with L858R mutation were stained with L858R mutant (43B2) specific antibody (<b>I</b>, 40× and the upper left corner 200×).</p

Staining of cytology specimens using EGFR mutation-specific antibodies.

<p>The total EGFR protein in cytology samples could be stained with EGFR (D38B1) antibody (A, D and G, 400×). Samples without EGFR mutations were not stained with two mutation-specific antibodies (B and C, 400×). Sample with E746–A750 deletion mutation were stained with E746–A750 deletion (6B6) specific antibody (E, 400×) and samples with L858R mutation were stained with L858R mutant (43B2) specific antibody (I, 400×).</p