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.

Linker for activation of T-cell family member2 (LAT2) a lipid raft adaptor protein for AKT signaling, is an early mediator of alkylphospholipid anti-leukemic activity.

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 µm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 µm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five µm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

Discrimination of Leishmania braziliensis variants by kDNA signatures produced by LSSP-PCR.

The conventional methods for identification and typing of Leishmania species depend on previous culture isolation of the parasites. Not infrequently, culture is unsuccessful and may result in misrepresentation of the heterogeneity of the original isolate. Thus, more reliable and precise identification of genotypes of Leishmania spp. is important for a better clinical and epidemiological understanding of the disease. We evaluated the potential of LSSP-PCR targeting kDNA minicircles in discriminating different variants of the parasite with the use of clinical samples directly or cultivated parasites. The 1st step of this procedure consists of the amplification of the minicircles by conventional PCR; the 2nd step is low-stringency amplification of the minicircles previously amplified, with the use of 1 of the primers. Although LSSP-PCR produced complex and distinct kDNA signatures for isolates representing different species, further experiments demonstrated that the approach had the potential for discriminating intraspecific variants of L. braziliensis. Thus, the generated profiles were too variable to be useful as markers for species identification. Moreover, we demonstrated that the approach can be directly applied to clinical samples. In conclusion, LSSP-PCR targeting kDNA minicircles produces profiles that reflect polymorphisms of the predominant classes of minicircles, and can be useful for studies aimed at discriminating Leishmania braziliensis genotypes without the need for previous cultivation of the parasite.