Identification of FBXO25-interacting proteins using an integrated proteomics approach

FBXO25 is one of 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of Skp1, Rbx1, Cullin1 and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FANDs). Combining two-step affinity purification followed by mass spectrometry with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, -actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FANDs, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions

Agrin and Perlecan Mediate Tumorigenic Processes in Oral Squamous Cell Carcinoma

<div><p>Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.</p></div

Treatment with chondroitinase ABC decreased SCC-9 LN-1 cell adhesion to extracellular matrix, but not SCC-9 LN-1 cell migration and increased sensibility of SCC-9 LN-1, SCC-9 and A431 cells to cisplatin.

<p>(A) SCC-9 LN-1 had a lower ability to adhere to extracellular matrix proteins (Matrigel) after treatment with 0.1 U/ml of chondroitinase for 4 h/37°C in serum free media (n = 3, triplicate, Student’s <i>t</i>-test, * indicates p<0.05). (B) SCC-9 LN-1 was treated with 0.1 U/ml for 4 h/37°C in serum free media were seeded in the upper chamber of 96-well transwell plates (n = 3, triplicate). RPMI media, which was supplemented with 1% FBS, was added in the lower chamber. (C) SCC-9 LN-1, SCC-9 and A431 cells treated with increasing concentrations of cisplatin (0–100 µM) for 48 h in the presence of 0.1 U/ml of chondroitinase showed increased sensibility to cisplatin, calculated by a non-linear regression of a dose-response curves (log[µM cisplatin] vs normalized response). Data are expressed as means ± SD from one independent experiment.</p

The role of agrin and perlecan in cell viability.

<p>The viability of SCC-9 (n = 2, A) and SCC-9 LN-1 (n = 3, B) was significantly reduced after siRNA-knockdown of agrin, but no difference in viability was observed in perlecan knockdown. The viability of A431 was not altered neither by agrin knockdown nor by perlecan knockdown (n = 3, C) (One-way ANOVA followed by Tukey’s test, different letters indicate statistically difference at p<0.05).</p

mRNA expression levels of agrin and perlecan.

<p>(A) Validation of higher expression of agrin and perlecan by qRT-PCR in OSCC tumor tissues<b>.</b> Agrin and Perlecan showed higher mRNA expression levels in human OSCC tumor tissues compared to control tissues by qRT-PCR (Kruskal-Wallis followed by Dunn’s test, n = 16, *p<0.05). (B) Agrin showed higher mRNA expression levels in SCC-9 compared to A431 and SCC-9 LN-1 cell lines. (C) Perlecan showed higher mRNA expression levels in SCC-9 LN-1 compared with A431 and SCC-9 cell lines. The data were normalized with glyceraldehyde-3-phosphate dehydrogenase gene, used as internal reference). Each bar represents means ± SD of at least two independent experiments in triplicates (one-way ANOVA followed by Tukey’s test. Different letters indicate statistically difference at p<0.05).</p

The role of agrin and perlecan in cell adhesion.

<p>Knockdown of agrin decreased adhesion to Matrigel in SCC-9 (n = 2, A) and SCC-9 LN-1 (n = 3, B), while no difference was observed in A431 (n = 3, C). When perlecan was silenced by siRNA no difference was observed in SCC-9 adhesion to Matrigel (n = 2, D) but a significant reduction was observed in SCC-9 LN-1 (n = 3, E) and A431 (n = 3, F) adhesion to Matrigel (Student’s <i>t</i>-test, * indicates p<0.05).</p

Loss of membrane-bound syndecan-1 localization after PMA treatment in SCC-9 cells.

<p>SCC-9 cells were treated with PMA for 5 min, 30 min and 24 h, fixed, and labeled for syndecan-1 with goat anti-syndecan-1 antibody. (A) Immunofluorescence images revealed diminished syndecan-1 in membrane localization after 30 min and 24 h of PMA treatment. Green: anti-goat antibody conjugated with Alexa Fluor 488. Blue: DAPI. (B) Example of masks used for quantification of syndecan-1 in the cell surface membrane. (C) Quantification performed by the Operetta high content image system showed loss of cell membrane of syndecan-1 after 30 min and 24 h of PMA treatment. Three independent experiments were performed. Columns represent mean ± SD (n = 3) and * and ** indicate p<0.05 and p<0.01, respectively.</p

Synthetic syndecan-1-derived peptide promoted migration in SCC-9 cells by transwell migration assay.

<p>The migration of HaCaT and SCC-9 cells treated with synthetic syndecan-1-derived peptide (SYN-1) and its scrambled in a concentration of 10 µM in the absence of FBS was evaluated by transwell assay. The measurements were normalized with the control (scrambled peptide). SYN-1 did not induce migration in HaCaT cells (A). SCC-9 cell migration was statistically significant when compared to scrambled peptide (B). Three experiments were performed with triplicates. Columns represent mean ± SD (n = 3) and * indicate p<0.05.</p

Identification of proteins in the conditioned media by LC-MS/MS according to the ratio of quantitative value.

<p>The HaCaT and SCC-9 cells were treated with PMA for 24 h, the media were collected, the proteins were digested with trypsin and analyzed by LC-MS/MS. The data were submitted to Mascot search engine and the .dat files from Mascot output were analyzed in Scaffold Q+, which calculates the quantitative value by normalizing spectral counts across the experiments. The ratio of quantitative value of the PMA-stimulated cells/DMSO-treated cells for each protein is shown in the table. The proteins exclusively found in one condition are indicated as “only” and the proteins that are not present are indicated as “absent”.</p

Identification of proteins in the conditioned media by LC-MS/MS according to the ratio of quantitative value (normalized spectral counts), as indicated in <b>Table 1</b>.

<p>Identification of proteins in the conditioned media by LC-MS/MS according to the ratio of quantitative value (normalized spectral counts), as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043521#pone-0043521-t001" target="_blank"><b>Table 1</b></a>.</p