Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Analysis of ovarian cancer cell secretome during epithelial to mesenchymal transition reveals a protein signature associated with advanced stages of ovarian tumors

Ovarian cancer (OvCA) is the most lethal neoplasia among gynecologic malignancies and faces high rates of new cases particularly in South America. In special, the High Grade Serous Ovarian Carcinoma (HGSC) presents very poor prognosis with deaths caused mainly by metastasis. Among several mechanisms involved in metastasis, the Epithelial to Mesenchymal Transition (EMT) molecular reprogramming represents a model for latest stages of cancer progression. EMT promotes important cellular changes in cellular adhesion and cell-cell communication, which particularly depends on the paracrine signaling from neighbor cells. Considering the importance of cellular communication during EMT and metastasis, here we analyzed the changes in the secretome of the ovarian cancer cell line Caov-3 induced to EMT by Epidermal Growth Factor (EGF). Using a combination of GEL-LC-MS/MS and stable isotopic metabolic labelling (SILAC), we identified up-regulated candidates during EMT as a starting point to identify relevant proteins for HGSC. Based on public databases, our candidate proteins were validated and prioritized for further analysis. Importantly, several of the protein candidates were associated with cellular vesicles, which are important to the cell-cell communication and metastasis. Furthermore, the association of candidate proteins with gene expression data uncovered a subset of proteins correlated with the mesenchymal subtype of ovarian cancer. Based on this relevant molecular signature for aggressive ovarian cancer, supported by protein and gene expression data, we developed a targeted proteomic method to evaluate individual OvCA clinical samples. The quantitative information obtained for 33 peptides, representative of 18 proteins, was able to segregate HGSC from other tumor types. Our study highlighted the richness of the secretome and EMT to reveal relevant proteins for HGSC, which could be used in further studies and larger patient cohorts as a potential stratification signature for ovarian cancer tumor that could guide clinical conduct for patient treatment.

LncRNA CERS6 - AS1 mediates the IGF2BP1/LIN28B axis to promote proliferation, migration and invasion in ovarian cancer

Ovarian cancer remains a significant clinical challenge for women's health, making it imperative to elucidate its underlying molecular mechanisms. LncRNA CERS6-AS1 and LIN28B-binding proteins play a vital role in the development of ovarian cancer. The expression levels of CERS6-AS1 and LIN28B in ovarian cancer tissues and cell lines were evaluated using qPCR. Protein levels were analyzed through IHC. The survival rate was assessed using the Kaplan-Meier curve. LIN28B or CERS6-AS1 was silenced via RNA interference (shRNA), and the effects on proliferation, migration, and invasion were examined through cell cloning, scratch assays, and Transwell assays. RNA pull-down and RIP experiments confirmed the binding of CERS6-AS1 to IGF2BP1 and enhances on the stability of LIN28B mRNA. Finally, LIN28B overexpression was performed for functional recovery experiments. Elevated expression of CERS6-AS1 and LIN28B was observed in both clinical ovarian cancer specimens and derived cell lines. The knockdown of LIN28B suppressed ovarian cancer cell proliferation, migration and invasion. Molecular investigations revealed that CERS6-AS1 stabilized LIN28B through IGF2BP1 binding. Furthermore, CERS6-AS1 depletion similarly attenuated malignant phenotypes, effects that were rescued by LIN28B overexpression. Collectively, the CERS6-AS1-regulated IGF2BP1/LIN28B signaling axis promoted ovarian cancer progression by enhancing tumor cell proliferation, migration and invasion, highlighting this pathway as a promising therapeutic target.

Phosphoproteome of signaling by ErbB2 in ovarian cancer cells

The gene for receptor tyrosine kinase ErbB2 is amplified in breast and ovarian tumours. The linear pathway by which signals are transduced through ErbB2 are well known. However, second generation questions that address spatial aspects of signaling remain. To address this, we have undertaken a mass spectrometry approach to identify phosphoproteins specific for ErbB2 using the inhibitors Lapatinib and CP724714 in ovarian cancer cells. The ErbB2 specific proteins identified in SKOV-3 cells were Myristoylated alanine-rich C-kinase substrate, Protein capicua homolog, Protein peptidyl isomerase G, Protein PRRC2C, Chromobox homolog1 and PRP4 homolog. We have evaluated three phosphoproteins PKM2, Aldose reductase and MARCKS in SKOV-3 cells. We observed that PKM2 was phosphorylated by EGF but was not inhibited by Lapatinib and CP724714. The activity of aldose reductase in reducing NADPH as a substrate was significantly higher in EGF stimulated cells which was inhibited by Lapatinib and CP724714 but not by Geftinib (EGFR inhibitor). MARCKS was phosphorylated on stimulation of SKOV-3 cells with EGF that was inhibited by Lapatinib and CP724714 which was dependent on the kinase activity of ErbB2. These results have identified phosphoproteins that are specific to ErbB2 which have not been previously reported and sets the basis for future experiments.