Comparison of PC-PLC protein expression, activity and cellular localization in A431-AD and in a model of cancer-initiating A431-SPH.

<p><b>A)</b> Western blot analysis of PC-PLC relative levels in total cell lysates (left panels) and in the cytoplasmic and nuclear fractions (right panels). Nucleoporin and actin were used to ensure the quality of fractions separation and protein quantitative loading control, respectively. Densitometric analyses were performed and results (mean ± SD of three independent experiments) are shown as relative fold change of PC-PLC protein levels normalized to the actin (for total lysates and cytoplasmic fractions) or nucleoporin (for nuclear fractions) levels. Data are presented relative to the A431-AD cell line. <b>B)</b> PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total cell lysates. P = 0.002. <b>C, D)</b> CSLM analyses of the single central optical section (top panels) and Z-projection of 25 optical sections taken from the bottom to the edge (3-D reconstruction, bottom panels) of the A431 sphere cells cultured for 72 hours, then fixed, permeabilized and stained for PC-PLC (green), EGFR (red) and nuclei (blue) detection. Scale bar, 40 μm.</p

ALCAM expression in thyroid cell lines.

<p>Analysis of TT and MZ-CRC1 cell lysates showing ALCAM protein expression in these thyroid cell lines. SKOV-3 and TPC-1 cell lysates were included as controls. Total lysates from each cell line were resolved by 4–12% SDS-PAGE and immunoblotted with anti-ALCAM antibodies. Arrow indicates the fully glycosylated ALCAM isoform. Beta-actin was used as a loading control.</p

ALCAM expression in normal and tumor human thyroid tissue samples.

<p>(A) Total protein extracts (30 µg) from 11 papillary thyroid carcinomas (Ca Pap), 5 medullary thyroid carcinomas (Ca Mid), and 5 normal thyroid tissues (Thyroid) were resolved by 4–12% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with anti-ALCAM antibody. (*) indicates a non-specific background band. Beta-actin was used as a loading control. (B) Western blot analysis of ALCAM expression of total protein extracts (30 µg) from three PTCs, two MTCs, and three CTRLs, treated (+) or not (−) with N-glycosidase F. Beta-actin was used as a loading control.</p

PC-PLC protein expression and activity in the non-tumoral keratinocyte HaCaT and in the A431-AD squamous carcinoma cell lines.

<p>Subcellular localization of PC-PLC (grey) on the plasma membrane of unfixed cells (<b>A</b>) and in different cellular compartments of fixed and permeabilized cells (<b>B)</b> detected by CLSM analyses. Single central optical sections are shown. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. <b>C</b>) Western Blot analysis of the relative PC-PLC protein expression in HaCaT and A431-AD total cell lysates (left) and in their cytoplasmic and nuclear fractions (right). Nucleoporin and β-actin were used to ensure the quality of fractions’ separation and protein quantitative loading, respectively. <b>D)</b> Absolute PC-PLC activity (pmoles/μg protein x min; mean ± SD, n = 6) measured by Amplex Red assay in total cell lysates. P = 0.0001.</p

Comparison of PC-PLC protein expression in A431-AD and CasKi-AD cells and effects of D609 on cell proliferation and stemness potential of CaSki cells.

<p><b>A)</b> Western blot analyses of the relative PC-PLC protein expression in A431-AD and CaSki-AD total cell lysates. β-actin was used as quantitative loading control. Densitometric analyses were performed and results are shown as relative fold change of PC-PLC protein levels normalized to the actin level. Data represent the mean (± SD) of three independent experiments, and are presented relative to the A431-AD cell line. <b>B</b>) Proliferation assays performed on CaSki-AD cells seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (left panel) and of live (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. <b>C</b>) CaSki-SPH cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counts (mean ± SD, n = 3) of total present cells (left panel) and of alive (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test. <b>D</b>) Sphere Formation Efficiency (SFE) of CaSki-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 2). Statistical analyses were performed using one-way ANOVA, P = 0.064. <b>E)</b> Real Time PCR analyses of the stemness markers OCT4, NANOG, NESTIN (left panel) and ALDH1 (right panel) expressed by the CaSki-AD cell line either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test. (OCT4, P = 0.301; NANOG, P = 0.413; NESTIN, P = 0.173; ALDH1, P = 0.019).</p

MOESM2 of The genomics of desmoplastic small round cell tumor reveals the deregulation of genes related to DNA damage response, epithelial–mesenchymal transition, and immune response

Additional file 2. Table S1. List of somatic mutations identified in each patient. Table S2. List, information and literature supply for genes mutated or copy number altered described in the main text as belonging to DDR or MErT/EMT categories. Table S3. List of somatic copy number aberrations identified by EXCAVATOR2. Table S4. List of gains with at least two copies and losses with homozygous deletions. Table S5. List of recurrent amplified genes on chromosome 1. Table S6. List of recurrently amplified genes of chromosome 1, belonging to the two significant biological functions identified by Ingenuity Pathway Analysis (IPA®, Qiagen; Bioinformatics, Redwood City, CA, USA; http://www.qiagen.com/ingenuity ). For the entire name of the genes, reported as gene ID, see Table S5. Table S7. List of recurrent deleted genes on chromosome 6

Changes in ALCAM expression induced by ionomycin or phorbol myristate acetate (PMA).

<p>Total lysates (Lys) and conditioned medium (CM) from TPC-1 cells, treated or not with 1 µM ionomycin (iono) or 50 ng/mL PMA for 2 h, were resolved by 3–8% SDS-PAGE and immunoblotted with ALCAM antibody. Both treatments led to an increase of secreted ALCAM isoforms; especially PMA induced a gain of 60-kDa isoform expression. Arrow indicates the membrane-localized ALCAM isoform in lysate, and arrowheads indicate shed-ALCAM isoforms in CM, respectively. Beta-actin was used as a loading control.</p

Effects of PC-PLC inhibition on EGFR, ERK and AKT phosphorylation in the HaCat and A431-AD cells.

<p>Representative Western blot analyses of total cell lysates from HaCaT (left panels) and A431-AD (right panels) cells cultured in the presence or absence of 50 μg/ml of D609. Cell lysates were immunoblotted with the following antibodies: pEGFR (Tyr1068), EGFR, pERK1/2 (Thr202/Tyr204), ERK 1/2, pAKT (Ser473), AKT and β-actin. β-actin was used as a quantitative loading control. Histograms below each panel represent the relative optical densities of phospho-protein levels normalized to the total protein level (mean values ± SD of three independent experiments) and are presented relative to the untreated sample at 24h. Statistical analyses were performed between treated samples (24h and 48 h) and untreated control sample at 24h, using the t-test.</p

Release of ALCAM is sensitive to ADAM17/TACE silencing.

<p>(A) Analysis of lysates from TPC-1 and A2774 cells, resolved on 4–12% SDS-PAGE and immunoblotted with anti-ADAM17/TACE antibodies. Arrows indicate inactive (130 kDa) and active (80 kDa) forms of ADAM17/TACE enzyme. Normalization of results was obtained with immunoblotting analysis of beta-actin. (B) Expression of ADAM17/TACE protein by TPC-1 cells transfected with an ADAM17/TACE-specific small interfering RNA (siRNA) (OTP17), or with non-targeting siRNA (NT) as detected by western blot. The amount of protein was calculated by comparative densitometric scanning with beta-actin. (C) ELISA detection of ALCAM release by TPC-1 cells after transfection with siRNA specifically inhibiting ADAM-17/TACE (OTP17, black column) or with non-targeting siRNA (NT, white column). (D) Conditioned medium (CM) from TPC-1 cells, cultured with pervanadate (PV) (60 min), epidermal growth factor (EGF) (24 h), or medium alone (ctr, 24 h) in the presence (black columns) or in absence (white columns) of CGS27023A (CGS) was assessed by ELISA for ALCAM. Columns, means of three experiments (cells cultured in presence of 10, 1, or 0.1 µM CGS); bars, SD. *, P<0.05. Grey bar: in absence of CGS, but in presence of orthovanadate (OV).</p

Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma

<div><p>Purpose</p><p>The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them.</p><p>Results</p><p>Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).</p><p>Conclusions</p><p>These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.</p></div