<i>FOXM1</i> induces promoter hypermethylation of <i>p16^INK4A</i> gene in primary human oral keratinocytes.

<p>(<b>A</b>) Bisulfite modification and methylation specific absolute qPCR for the quantification of <i>p16^INK4A</i> promoter methylation status. Genomic DNA was first treated with sodium bisulfite prior to PCR pre-amplification of the promoter region of <i>p16^INK4A</i> (PCR^BS, 273 bp). Methylation specific (p16M-R/F) and methylation-independent (p16U-F/R) primers were then used to quantify the relative levels of methylated and unmethylated products within the PCR^BS sample using standard-curve based absolute qPCR method for each product, respectively. Melting analysis was performed to validate the qPCR specificity in detecting the two M and U products. (<b>B</b>) Bisulfite conversion and methylation specific qPCR were performed to measure the relative levels of unmethylated (U, melting temperature at 85.8°C) and methylated (M, 91.2°C) in either EGFP- or FOXM1-transduced primary NOK treated with either vehicle (DMSO) or 5Aza (1 µM, 3-day incubation with fresh drug replenishment daily). A total of n = 11 replicates from at least 4 independent experiments were performed. Statistical t-test significance notations *P<0.05 and ***P<0.001.</p

Upregulation of <i>FOXM1</i> (isoform B) induces a global shift in methylation pattern that mimics the cancer epigenome.

<p>(<b>A</b>) Genome-wide promoter microarray analysis of primary normal oral human keratinocytes expressing either <i>EGFP</i> (NOKG, black dots) or <i>FOXM1</i> (NOKF, yellow dots) and an established squamous cell carcinoma cell line (SCC15, red dots). Each dot represents a single gene. (<b>B</b>) A non-linear 2^nd order polynomial regression analyses were performed on the relative methylation patterns between NOKG vs NOKF (inverse correlation), NOKG vs SCC15 (inverse correlation) and NOKF vs SCC15 (positive correlation). (<b>C</b>) Gene selection criteria for differentially methylated genes between control (NOKG) and tests groups (NOKF and SCC15). 100-most hypermethylated and 100-most hypomethylated genes were inversely matched with differentially methylated genes from NOKF and SCC15. The adjacent gene lists show the shortlisted FOXM1-induced (also found in SCC15) differentially hypermethylated (red) and hypomethylated (green) genes compared to control NOKG cells. The CDKN2A (encodes <i>p16^INK4A</i>) gene, its promoter known to be hypermethylated in HNSCC, was included as a positive control for promoter hypermethylation. (<b>D</b>) Clinical tumour tissue sample correlation between the relative levels of methylation and gene expression of each shortlisted gene in a cohort of 10 patients with paired normal margin and HNSCC tumour tissue samples. Each dot represents mean ± SEM of each gene. Vertical error bars were derived from relative gene expression of 10 margin-tumour tissue pairs and horizontal error bars were derived from relative promoter methylation of 3 independent primary NOK (NOKG/NOKF) experiments. Correlation coefficient (R²) of a non-linear 2^nd order polynomial regression analyses were performed on all 30 candidate genes (left panel), 16 hypermethylated genes (middle panel) or 14 hypomethylated genes (right panel), respectively.</p

Upregulation of FOXM1 suppressed <i>p16^INK4A</i> expression in primary human oral keratinocytes.

<p>(<b>A</b>) FOXM1 significantly supresses <i>p16^INK4A</i> mRNA and protein expression (inset figure) in primary normal human keratinocytes. GAPDH was used as a control for protein loading. Control cells (mock-transduced with empty retroviral particles or EGFP-transduced) did not show significant suppression of p16^INK4A expression. (<b>B</b>) Knockdown of a FOXM1-target gene <i>HELLS</i>, which regulates genome-wide methylation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034329#pone.0034329-Dennis1" target="_blank">[14]</a>, induced <i>p16^INK4A</i> and simultaneously suppressed <i>DNMT1</i> and <i>DNMT3B</i>, but not <i>DNMT3A</i> mRNA expression in a FOXM1-transformed malignant cell line (SVFN5) expressing constitutive levels of endogenous <i>HELLS </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034329#pone.0034329-Gemenetzidis1" target="_blank">[8]</a>. Each bar represents a mean ± SEM of triplicate transfection (48 h) with either siCTRL or siHELLS. *P<0.05, **P<0.01 and ***P<0.001 indicate the level of statistical significance compared to controls. (<b>C</b>) Endogenous <i>FOXM1</i> (isoform B) mRNA expression levels in 8 strains of primary human normal oral keratinocytes, 5 dysplastic and 11 HNSCC cell lines. Total <i>FOXM1</i> mRNA expression levels were measured in the EGFP and FOXM1-transduced NOK (NOKG and NOKF), respectively. (<b>D</b>–<b>J</b>) Third-order polynomial regression analyses were performed to obtain the R² coefficient of determination values which indicate the significance of co-expression between each gene with <i>FOXM1</i> across the 24 cell strains/lines indicated in panel C.</p

RhoC knockdown decrease cellular migration and is rescued by ectopic RhoC overexpression.

<div><p>Initial screen of PDAC cell lines revealed high expression levels of RhoC protein in HPAF and Panc0403 pancreatic cancer cells. Endogenous RhoC was silenced using shRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">Figures S1, S2</a>). </p> <p>shRhoC (stable RhoC knockdown: two different constructs were used: shRhoC1 and shRhoC2) resulted in a significant reduction of cell migration compared with the parental or pSilencer (empty vector control) cell lines towards 10% FBS (A) or through Fibronectin (B). Transient over-expression of RhoC in shRhoC cells (pTag2BRhoC) restored cell migration significantly and comparable to parental cell line. pTag2B is the empty vector control cell line. Similarly, another cell line (HPAF) demonstrated RhoC-dependent migration (C). Here the summary data of both ShRhoC are presented for migration towards 10% FBS. ** p<0.001, * p<0.01, ANOVA. Individual data points represent technical/biological repeats with summary statistics represented by mean ± SEM.</p></div

Src activation is downstream of RhoC-enhanced integrin α5β1 trafficking, partially contributing to increased cell migration.

<div><p>(A)Total RhoA and RhoB were unaltered upon transfection with various RhoC constructs in Capan1 cells. Rotekin immunoprecipitation binding assays revealed an increase of active RhoC-GTP in nRhoC cells (RhoA-GTP, RhoB-GTP levels were unaltered). Densitometric analysis for various RhoC-harboring constructs’ cell lines are normalized to parental Capan1 levels from triplicate experiments. </p> <p>(B)Upon plating on fibronectin, there was an increased level of phospho-Src (Tyr416) in nRhoC cells only. This increase was abrogated upon treatment with integrin α5β1-neutralizing antibody. Both cRhoC and nDCT cells showed significantly lower phospho-Src than parental Capan1 cells after treatment (IgG controls: no difference from the non-treated cells). </p> <p>(C)Functionally, both nRhoC and cRhoC cells showed enhanced migration in the IgG control conditions as compared with the parental, nEV, or nDCT cells. Blocking cells with 8μg/ml of the integrin α5β1-neutralizing antibody significantly diminished cell migration of both the nRhoC and cRhoC cells but did not alter basal migratory capacity of the nDCT cells. However, in nRhoC cells this reduction in migration capacity did not return to the level of parental or nEV cells. </p> <p>(D)Similarly in the endogenously high-expressing Panc0403 cells (also for HPAF (data not shown)) and the vector control transfected (pSilencer) cell line there was an abrogation of the enhanced migratory capacity upon blockade with integrin α5β1-neutralizing antibody compared with the lack of effect on the shRhoC cell line. </p> <p>*p<0.05, **p<0.001, Student’s t-test, error bars: SEM. </p></div

RhoC co-localized with integrin α5β1 at cell protrusions and in the peri-nuclear region.

<div><p>(A) In parental Capan1 cells there was minimal diffuse expression of RhoC (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">Figures S1, S2</a>) and, similarly, diffuse expression of integrin α5β1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s008" target="_blank">Figures S3, S4</a>). </p> <p>(B) In nRhoC cells, the exogenous RhoC co-localized with integrin α5β1 at a cell protrusion (white arrows and boxes) and in the peri-nuclear region (red arrows and boxes). </p> <p>(C) This was in contrast to cRhoC cells which showed co-localization of the V5 tag (cRhoC) with integrin α5β1 at the periphery of cells but not in the peri-nuclear region. (D) nDCT cells showed no co-localization of the V5 tag with integrin α5β1 at the plasma membrane (white box). In a few cells, diffuse cytoplasmic staining could be seen (red box). Scale bar: 10µm.</p> <p>This co-localization (confirmed by co-localization software in the confocal microscope) in distinct cellular compartments could be seen in 3D during Transwell migration (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s008" target="_blank">Figures S3, S4</a>). Further explanation of the likely cellular compartment of this co-localization is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s011" target="_blank">Figure S6</a>. </p> <p>(E) Immuno-precipitation (IP) using anti-RhoC antibody (against C-terminal 100-193 amino acids) confirmed the interaction of RhoC and integrin α5β1. Densitometric quantification, when normalization was carried out for the immuno-precipitation reaction (IgG, F) or for the input (total RhoC, G), of the IP-bound integrin α5β1 showed significant increase of integrin α5β1 interaction with RhoC in both nRhoC and cRhoC cell lines. A significant reduction in this interaction with integrin α5β1 was observed in the nDCT cells as compared with parental cell lines. Appropriate IgG controls and reverse IP are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s012" target="_blank">Figure S7</a>. B:IP-bound fraction; UB: IP-unbound fraction; total: total lysate. *p<0.05, **p<0.01, Student’s t-test, error bars: SEM. </p></div

RhoC enhanced integrin α5β1 internalization and recycling upon fibronectin adherence.

<div><p>The well-established Biotin-labeling assay (labeling integrin α5β1 with Biotin and allowing internalization and recycling (separate assays: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s001" target="_blank">Methods S1</a>) followed by cleavage of Biotin and measurement of integrin by ELISA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#B18" target="_blank">18</a>]) to compare the internalization and recycling rates of integrin α5β1. Graphs represent summary data from three representative individual experiments. The trend-line shown is second-order polynomial regression fit for the data, as previously used [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#B46" target="_blank">46</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#B47" target="_blank">47</a>]. Thus, compared with parental Capan1 cells, nRhoC cells demonstrated significantly increased internalization (A) and recycling (B) of integrin α5β1 on fibronectin-coated surface. The masking of RhoC CAAX in cRhoC cells resulted in a significant increase in internalization, but not recycling, of integrin α5β1 (compared with parental Capan1 cells). However, deletion of the C-terminal of RhoC in nDCT cells resulted in significant reduction in internalization and recycling of integrin α5β1. </p> <p>Compared with the parental HPAF and pSilencer (empty vector) cells, shRhoC (stable RhoC knockdown) cells showed a significant decline of integrin α5β1 internalization (C) and recycling (D) on a fibronectin-coated surface. The dramatic reduction in the HPAF-shRhoC cells was not due to a vector artifact, since HPAF-pSilencer cells actually showed a significant enhancement of the recycling rate. Similar data were obtained after knock-down of endogenous RhoC in Panc0403 (not shown). In addition, there was no change in Transferrin receptor recycling after manipulation of RhoC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s013" target="_blank">Figure S8</a>). *** p<0.0001, ** p<0.001, * p<0.01, ANOVA. Error bars: SEM. </p></div

RhoC over-expression altered paxillin distribution and accelerated focal adhesion turnover.

<div><p>(A-B) Z-stacked confocal images showed differential distribution of paxillin staining (green) in ‘non-spreading’ (i) and ‘spreading’ (ii), Capan1 (A) and nRhoC (B) cells, 60 minutes after plating on the fibronectin-coated surface. Main images presented are in the XY plane with Z axis at the basal (ventral) aspects of cells as shown in the XZ and YZ sections at the top and right of composite images respectively (the cross-section of images in X, Y and Z axes are denoted by green, red and blue lines respectively). The obvious focal staining at the cell protrusion end of ‘spread’ Capan1 cells (Aii) was not present in the ‘spread’ nRhoC cells (Bii, arrowheads: cell protrusion ends). In the few ‘non-spreading’ cells, there were greater amounts of paxillin staining along the basal membrane of Capan1 cells (Ai) than that of nRhoC (Aii) cells. Scale bar: 5μm.</p> <p>(C-E) Focal adhesion ‘disassembly assay’ (NOC: nocodazole) showed that, compared to Capan 1, nRhoC cells had accelerated disassembly and re-assembly of mature focal adhesions (FA: large 4-5 μm) and small focal adhesion (1μm). Mean Intensity of peripheral paxillin staining showed similar levels 30 minutes after washout of the nocodazole treatment in Capan1 and nRhoC cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s001" target="_blank">Methods S1</a>). However, while nRhoC cells had decreased the paxillin intensity levels significantly by 15 minutes after recovery from nocodazole, Capan1 cells took 30 minutes to achieve similar reduction, suggesting a quicker turnover of new focal adhesions in nRhoC cells. The size of focal adhesions after nocodazole treatment also demonstrates a quicker recovery/ re-assembly of stable large focal adhesions in nRhoC cells as compared to Capan1 cells. This could well explain the rapid movement and the increased cell spreading and decreased adhesions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone-0081575-g001" target="_blank">Figures 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone-0081575-g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">Videos S1, S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s008" target="_blank">S3</a>), as a result of introduction of RhoC in nRhoC cells. *p<0.05, Mann-Whitney test. Summary statistics in the box-whisker plot represented median ± inter-quartile range. </p></div

RhoC enhanced cell invasion into three-dimensional (3D) organotypic culture models.

<div><p>(A) Capan1 cells formed ductal structures on the surface of the gel in organotypic cultures, whereas the invading cells remained as individual cells within the gel. In contrast, invading nRhoC cells re-formed large ductal structures within the gel (red arrowheads within the lumen). Pronounced co-localization of RhoC and integrin α5β1 at the peri-nuclear region and cell protrusion end was observed particularly in invading nRhoC cells (inset).</p> <p>(B-C) Quantification of the total invading cell numbers (B), and average invading distance (C) at various time points confirmed a significant increase of invading nRhoC cell numbers compared with Capan1 cell numbers (though the invading distance was comparable). **p<0.001, Wilcoxon signed-rank test. </p> <p>(D)In human PDAC, co-localization of RhoC and integrin α5β1 peri-nuclear and cellular processes was seen in cancer cells (but not in stromal cells) where it was particularly prominent in those areas with increased peri-tumoral fibronectin (see merge picture and insets (E) and (F)). Scale bar 50 μm (20 μm for E and F).</p></div

RhoC overexpression causes rapid spreading and movement of Capan1 cells.

<div><p>Initial screen of PDAC cell lines revealed low expression levels of RhoC protein in Capan1 pancreatic cancer cells which were then transfected with RhoC constructs (nRhoC, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">Figures S1, S2</a>). </p> <p>(A)Cells were plated on the fibronectin-coated surface and incubated at 37°C till the indicated time-point, then fixed and stained with Crystal Violet before imaging with Zeiss Axiovert 200M microscope. nRhoC cells (Capan1 cells with N-terminus tagged RhoC overexpression) spread more within the first two hours as compared to the rounded parental Capan1 cells. </p> <p>(B)A selection of images from time-lapse videos (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">videos S1, S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s008" target="_blank">S3</a> for detailed information) of cell movement during two hours after plating on fibronectin-coated surface. These images demonstrate that nRhoC transfected cells had accelerated cell spreading and contraction, associated with rapid movement, as compared to Capan1 cells (arrows). Time of capture is indicated at the top-left corner of each image (m= minutes). </p> <p>Scale bar: 20µm.</p></div