Claudin-3 Overexpression Increases the Malignant Potential of Colorectal Cancer Cells: Roles of ERK1/2 and PI3K-Akt as Modulators of EGFR signaling

<div><p>The altered expressions of claudin proteins have been reported during the tumorigenesis of colorectal cancer. However, the molecular mechanisms that regulate these events in this cancer type are poorly understood. Here, we report that epidermal growth factor (EGF) increases the expression of claudin-3 in human colorectal adenocarcinoma HT-29 cells. This increase was related to increased cell migration and the formation of anchorage-dependent and anchorage-independent colonies. We further showed that the ERK1/2 and PI3K-Akt pathways were involved in the regulation of these effects because specific pharmacological inhibition blocked these events. Genetic manipulation of claudin-1 and claudin-3 in HT-29 cells showed that the overexpression of claudin-1 resulted in decreased cell migration; however, migration was not altered in cells that overexpressed claudin-3. Furthermore, the overexpression of claudin-3, but not that of claudin-1, increased the tight junction-related paracellular flux of macromolecules. Additionally, an increased formation of anchorage-dependent and anchorage-independent colonies were observed in cells that overexpressed claudin-3, while no such changes were observed when claudin-1 was overexpressed. Finally, claudin-3 silencing alone despite induce increase proliferation, and the formation of anchoragedependent and -independent colonies, it was able to prevent the EGF-induced increased malignant potential. In conclusion, our results show a novel role for claudin-3 overexpression in promoting the malignant potential of colorectal cancer cells, which is potentially regulated by the EGF-activated ERK1/2 and PI3K-Akt pathways.</p></div

NF-kappaB Is Involved in the Regulation of EMT Genes in Breast Cancer Cells

<div><p>The metastatic process in breast cancer is related to the expression of the epithelial-to-mesenchymal transition transcription factors (EMT-TFs) SNAIL, SLUG, SIP1 and TWIST1. EMT-TFs and nuclear factor-κB (NF-κB) activation have been associated with aggressiveness and metastatic potential in carcinomas. Here, we sought to examine the role of NF-κB in the aggressive properties and regulation of EMT-TFs in human breast cancer cells. Blocking NF-κB/p65 activity by reducing its transcript and protein levels (through siRNA-strategy and dehydroxymethylepoxyquinomicin [DHMEQ] treatment) in the aggressive MDA-MB-231 and HCC-1954 cell lines resulted in decreased invasiveness and migration, a downregulation of SLUG, SIP1, TWIST1, MMP11 and N-cadherin transcripts and an upregulation of E-cadherin transcripts. No significant changes were observed in the less aggressive cell line MCF-7. Bioinformatics tools identified several NF-κB binding sites along the promoters of SNAIL, SLUG, SIP1 and TWIST1 genes. Through chromatin immunoprecipitation and luciferase reporter assays, the NF-κB/p65 binding on TWIST1, SLUG and SIP1 promoter regions was confirmed. Thus, we suggest that NF-κB directly regulates the transcription of EMT-TF genes in breast cancer. Our findings may contribute to a greater understanding of the metastatic process of this neoplasia and highlight NF-κB as a potential target for breast cancer treatment.</p></div

The impact of claudin-3 silencing on EGF-induced effects in HT-29 cells.

<p>HT-29 cells were transfected with non-targenting control siRNA (scramble) or claudin-3 siRNA and treated with EGF as indicated. (<b>A</b>) Cells were grown for 24 h and the total cell lysates were obtained and analyzed for the expression of claudin-3 by immunoblotting; α-tubulin was used as a loading control. (<b>B</b>) Cells were seeded in 96-well plates for 48 h and proliferation was quantified using the crystal violet technique. The bar graph shows the ratio of the absorbance of transfected and/or EGF-treated cells to the control cells. (<b>C</b>) Representative photographs of anchorage-dependent colonies that were stained with crystal violet. The bar graph shows the proportion of fold increase in foci formation of the transfected and/or EGF-treated cells to control cells. (<b>D</b>) Representative images of anchorage-independent colonies. The bar graph shows the ratio of the fold increase in colony formation of transfected and/or EGF-treated cells to control cells. Error bars indicate the means ± SEM (n = 3); *p<0.05, **p<0.01, as determined by an ANOVA. <i>Claudin</i>; Cld. α<i>-tubulin</i>; α-tub.</p

Effects of the forced expression of claudins 1 and 3 on their subcellular distribution, TER and macromolecular permeability. HT-29 cells were transduced with retroviral vectors that contained claudin-1, claudin-3, or empty vector (pBABE).

<p>(<b>A</b>) The cells were grown and total cell lysates were harvested and analyzed by immunoblotting for the expression of claudin-1 and claudin-3. The numbers represent the ratio of the optical density of claudin-transduced to empty vector-transduced cells normalized by α-tubulin. (<b>B</b>) Cells were grown on glass coverslips and processed under permeabilizing conditions for the immunofluorescence analysis of claudin-1 and claudin-3 distribution; bar: 5 µm. (<b>C</b>) Cells were grown on Transwell inserts and the TER was measured using the Millicel-ERS system. The bar graphs show the TER values normalized for the area of the insert with the blank value subtracted. (<b>D</b>) The cells were grown on glass coverslips and processed for immunofluorescence under non-permeabilizing conditions to evaluate macromolecular permeability using the anti-uvomorulin/E-cadherin antibody; bar: 10 µm. (<b>E</b>) Cells were grown on glass coverslips and processed under permeabilizing conditions for immunofluorescence analysis of E-cadherin distribution. The stained cells were viewed with <b><i>FV10i-O</i></b> confocal microscope. Error bars indicate the means ± SEM (n = 3); **p<0.01 as determined by an ANOVA.</p

The impact of EGF treatment on the migration and proliferation of HT-29 cells.

<p>(<b>A, B</b>) HT-29 cells were grown in 6-well plates until confluent. Next, cell monolayers were wounded and treated with EGF, and cell migration in these regions was monitored for 24 h. (<b>C</b>) HT-29 cells were seeded in 96-well plates and treated with EGF at the indicated times, and proliferation was quantified using the crystal violet technique. The bar graph shows the ratio of the absorbance of EGF-treated to untreated cells (control). Bar: 100 µm. Error bars indicate the means ± SEM (n = 3); **p<0.01, ***p<0.001 as determined by t test.</p

The impact of claudin-1 and claudin-3 overexpression on anchorage-independent colony formation.

<p>HT-29^pBABE, HT-29^cld-1 and HT-29^cld-3 cells were seeded for an anchorage-independent colony formation assay as described in the Materials and Methods. The bar graph shows the ratio of the fold increase in colony formation of claudin-transduced cells to empty vector-transduced (pBABE) cells. Error bars indicate the means ± SEM (n = 3); ***p<0.001, as determined by an ANOVA.</p

Relative expression of EMT-phenotype markers after NF-κB/p65 signaling inhibition.

<p>The mRNA levels of <i>E-CADHERIN</i>, <i>N-CADHERIN</i> and <i>MMP11</i> were assessed in MDA-MB-231 (A) and HCC-1954 (B) cells at 8, 16 and 24 h of DHMEQ treatment. The data were expressed as the mean ± SD. * = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001.</p

Relative expression of the EMT-inducing factors after NF-κB/p65 signaling inhibition.

<p>The mRNA levels of <i>SNAIL1</i>, <i>SLUG</i>, <i>TWIST1</i>, and <i>SIP1</i> were assessed in MDA-MB-231 (A) and HCC-1954 (B) cells at 8, 16 and 24 h of DHMEQ treatment. NF-κB/p65 inhibition was evaluated at protein levels by western blot assay at 16 and 24 h of DHMEQ treatment. Ponceau staining was used as a loading control. Ctrl: control. The data were expressed as the mean ± SD. * = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001.</p

Migration assay.

<p>A representative wound healing assay evaluating cell migration at 24 h after DHMEQ treatment of MDA-MB-231 (A) HCC-1954 (B) and MCF-7 (C) cells is shown. The box plots represent migratory ability as indicated by the percent of wound closure. Magnification x100. The data were expressed as the mean ± SD. * = <i>p</i><0.05, ns = not statistically significant.</p

The impact of MEK1/2-ERK1/2 and PI3K-Akt inhibition on EGF-induced effects in HT-29 cells.

<p>(<b>A</b>) HT-29 cells were grown and treated with EGF for 5, 15, 30 and 60 min, after which the total cell lysates were harvested and analyzed by immunoblotting for p-ERK1/2, ERK1/2, p-Akt, and Akt. (<b>B</b>) Cells were grown and pretreated for 1 h with the indicated inhibitors before incubation with EGF for 48 h. Total cell lysates were harvested and analyzed by immunoblotting for claudin-3. α-Tubulin was used as a loading control. The numbers represent the ratio of the optical density of treated to untreated cells normalized by total protein or α-tubulin. Representative images of wound healing (<b>C</b>), anchorage-dependent colony formation (<b>D</b>) and anchorage-independent colony formation (<b>E</b>) assays. The cells were pretreated with the inhibitors as indicated, and the assays were performed as described in the Materials and Methods. For the anchorage-dependent assay, the bar graph shows the ratio of the fold increase in foci formation of treated to untreated cells (control). For the anchorage-independent assay, the bar graphs show the ratio of the fold increase in colony formation of treated to untreated cells (control). Bar: 200 µm. Error bars indicate the means ± SEM (n = 3); **p<0.01 as determined by an ANOVA.</p