Epithelial-to-Mesenchymal Transition Is Not a Major Modulating Factor in the Cytotoxic Response to Natural Products in Cancer Cell Lines

Numerous natural products exhibit antiproliferative activity against cancer cells by modulating various biological pathways. In this study, we investigated the potential use of eight natural compounds (apigenin, curcumin, epigallocatechin gallate, fisetin, forskolin, procyanidin B2, resveratrol, urolithin A) and two repurposed agents (fulvestrant and metformin) as chemotherapy enhancers and mesenchymal-to-epithelial (MET) inducers of cancer cells. Screening of these compounds in various colon, breast, and pancreatic cancer cell lines revealed anti-cancer activity for all compounds, with curcumin being the most effective among these in all cell lines. Although some of the natural products were able to induce MET in some cancer cell lines, the MET induction was not related to increased synergy with either 5-FU, irinotecan, gemcitabine, or gefitinib. When synergy was observed, for example with curcumin and irinotecan, this was unrelated to MET induction, as assessed by changes in E-cadherin and vimentin expression. Our results show that MET induction is compound and cell line specific, and that MET is not necessarily related to enhanced chemosensitivity

Epigenetic Mechanisms Underlying the Dynamic Expression of Cancer-Testis Genes, PAGE2, -2B and SPANX-B, during Mesenchymal-to-Epithelial Transition

Cancer-testis (CT) genes are expressed in various cancers but not in normal tissues other than in cells of the germline. Although DNA demethylation of promoter-proximal CpGs of CT genes is linked to their expression in cancer, the mechanisms leading to demethylation are unknown. To elucidate such mechanisms we chose to study the Caco-2 colorectal cancer cell line during the course of its spontaneous differentiation in vitro, as we found CT genes, in particular PAGE2, -2B and SPANX-B, to be up-regulated during this process. Differentiation of these cells resulted in a mesenchymal-to-epithelial transition (MET) as evidenced by the gain of epithelial markers CDX2, Claudin-4 and E-cadherin, and a concomitant loss of mesenchymal markers Vimentin, Fibronectin-1 and Transgelin. PAGE2 and SPAN-X up-regulation was accompanied by an increase in Ten-eleven translocation-2 (TET2) expression and cytosine 5-hydroxymethylation as well as the disassociation of heterochromatin protein 1 and the polycomb repressive complex 2 protein EZH2 from promoter-proximal regions of these genes. Reversal of differentiation resulted in down-regulation of PAGE2, -2B and SPANX-B, and induction of epithelial-to-mesenchymal transition (EMT) markers, demonstrating the dynamic nature of CT gene regulation in this model

Nuclear co-localization of CDX2 and SPANX-B in differentiating Caco-2 cells.

<p>Immunofluorescent staining of differentiating Caco-2 cells with DAPI counterstaining reveals overlapping SPANX-B (Alexa Fluor 488: green) and CDX2 (Alexa Fluor 568: red) expression; (20× magnification) (<b>A</b>). More than 60 to 80% of the cells show double-labeling when analyzed quantitatively (<b>B</b>).</p

Predictive Gene Signature for Pyrazolopyrimidine Derivative c-Src Inhibitor 10a Sensitivity in Melanoma Cells.

Melanoma is a highly aggressive cancer with poor prognosis. Although more than 80% of melanomas harbor an activating mutation in genes within the MAPK pathway, which are mutually exclusive, usefulness of therapies targeting MAPK pathway are impeded by innate and/or acquired resistance in most patients. In this study, using melanoma cells, we report the efficacy of a recently developed pyrazolo[3,4-]pyrimidine derived c-Src inhibitor 10a and identify a molecular signature which is predictive of 10a chemosensitivity. We show that the expression of TMED7, PLOD2, XRCC5, and NSUN5 are candidate biomarkers for 10a sensitivity. Although an undifferentiated/mesenchymal/invasive status of melanoma cells is associated with resistance to 10a, we show here for the first time that melanoma cells can be sensitized to 10a via treatment with valproic acid, a histone deacetylase inhibitor

<i>TET</i> expression during Caco-2 SD.

<p>mRNA of all three <i>TET</i> genes increase gradually during Caco-2 SD (<b>A</b>). An increase in only a 25 kD version (<b>B</b>), but not the full-length TET2 protein (<b>C</b>) occurs simultaneously with the increase in mRNA. BAPTA-AM treatment results in a modest decrease in the 25 kD TET2 protein, with the generation of a larger mw version (<b>D</b>). P values, as determined by one-way ANOVA, are 0.03, 0.01, and 0.07, for Tet1, Tet2, and Tet3, respectively.</p

Increased hydroxymethylation of <i>PAGE2</i> and <i>SPANX-B</i> during Caco-2 spontaneous differentiation.

<p>CHIP experiments using an anti-hmC antibody and primers corresponding to +31 to +182 and +68 to +184 bp from the transcription start site of the <i>PAGE2</i> and <i>SPANX-B</i> genes, respectively. P values (one-way ANOVA) are 0.001 and 0.07 for PAGE2 and SPANX-B, respectively.</p

SPANX-B and vimentin expression are mutually exclusive in differentiating Caco-2 cells.

<p>Immunofluorescent staining of differentiating Caco-2 cells with DAPI counterstaining reveals a gradual increase in nuclear SPANX-B (green) with a concomitant decrease in cytoplasmic vimentin expression (red); (20× magnification) (<b>A</b>). Less than 1% of SPANX-B positive cells showed staining for vimentin on day 0 (<b>B</b>).</p

Bisulfide sequencing of <i>PAGE2, -2B</i> and <i>SPANX-B</i> promoter-proximal regions.

<p>Filled and empty circles represent methylated and unmethylated cytosines, respectively. % methylation within each analysed region, based on the 10 clones sequenced is indicated. CpG residues proximal to <i>PAGE2, -2B</i> and <i>SPANX-B</i> promoters during Caco2 differentiation at days 0, 10 and 30 reveals no statistically significant change (by one-way ANOVA).</p

Up-regulation of CT genes in parallel to MET in the Caco-2 SD model.

<p>Relative mRNA expression of CT genes (<i>PAGE2</i>, <i>-2B</i> and <i>SPANXB</i>) (<b>A</b>), epithelial genes (<i>E-cadherin (CDH1)</i>, <i>claudin 4(CLDN4)</i>, <i>CDX2</i>) (<b>B</b>), and mesenchymal genes (<i>fibronectin 1 (FN1), vimentin (VIM)</i>, <i>transgelin (TAGLN)</i>) (<b>C</b>) as determined by quantitative PCR at days 0, 10, 20 and 30 post-confluence. Data represent average of two experiments. Change in expression levels for all genes between days 0 and 30 is statistically significant (P<0.0001, by two way ANOVA with Tukey's post hoc test).</p

Overlapping TET2 and CT gene expression in differentiating Caco-2 cells.

<p>Double immunofluorescence staining for TET2 (Alexafluor 568: red) and SPANX-B or PAGE2 (Alexafluor 488: green) with DAPI counterstaining 20 days post-confluence show overlapping nuclear expression Magnification: 40× (<b>A</b>). More than 95% of cells expressing PAGE2 or SPANX-B were also positive for TET2 staining (<b>B</b>).</p