The proteins of extracellular vesicles (EVs) provide proteomic signatures that reflect molecular features of EV-producing cells, including cancer cells. Detection of cancer cell EV proteins is of great interest due to the development of novel predictive diagnostic approaches. Using targeted mass spectrometry with stable-isotope-labeled peptide standards (SIS), we measured in this study the levels of 34 EV-associated proteins in vesicles and whole lysate derived from the colorectal cancer (CRC) cell lines Caco-2, HT29 and HCT116. We also evaluated the abundance of 13 EV-associated proteins (FN1, TLN1, ITGB3, HSPA8, TUBA4A, CD9, CD63, HSPG2, ITGB1, GNAI2, TSG101, PACSIN2, and CDC42) in EVs isolated from blood plasma samples from 11 CRC patients and 20 healthy volunteers. Downregulation of TLN1, ITGB3, and TUBA4A with simultaneous upregulation of HSPG2 protein were observed in cancer samples compared to healthy controls. The proteomic cargo of the EVs associated with CRC represents a promising source of potential prognostic markers.

Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all-trans-retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3–12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear proteome in DNA damage recognition in the RUNX1-triggered pathway, and in the p53-regulation pathway. By applying scheduled multiple reaction monitoring using stable isotopically labeled peptide standards (MRM/SIS), we found a persistent increase in the content of the following proteins: PRAM1, CEPBP, RBPJ, and HIC1 in the HL-60 cell nuclear fraction during ATRA-induced granulocytic differentiation. In the case of STAT1, CASP3, PARP1, and PRKDC proteins, a transient increase in their content was observed at early time points (3–12 h) after the ATRA treatment. Obtained data on nuclear proteome composition and dynamics during granulocytic differentiation could be beneficial for the development of new treatment approaches for leukemias with the mutated p53 gene.

Colorectal cancer (CRC) and lung cancer (LC) are leading causes of cancer-related mortality, highlighting the need for minimally invasive diagnostic, prognostic, and predictive markers for these cancers. Proteins secreted by a tumor into the extracellular space directly, known as the tumor secretome, as well as proteins in the extra-cellular vesicles (EVs), represent an attractive source of biomarkers for CRC and LC. We performed proteomic analyses on secretome and EV samples from LC (A549, NCI-H23, NCI-H460) and CRC (Caco2, HCT116, HT-29) cell lines and targeted mass spectrometry on EVs from plasma samples of 20 patients with CRC and 19 healthy controls. A total of 782 proteins were identified across the CRC and LC secretome and EV samples. Of these, 22 and 44 protein markers were significantly elevated in the CRC and LC samples, respectively. Functional annotation revealed enrichment in proteins linked to metastasis and tumor progression for both cancer types. In EVs isolated from the plasma of patients with CRC, ITGB3, HSPA8, TUBA4A, and TLN1 were reduced, whereas FN1, SERPINA1, and CST3 were elevated, compared to healthy controls. These findings support the development of minimally invasive liquid biopsy methods for the detection, prognosis, and treatment monitoring of LC and CRC.

Vestibular schwannomas are relatively rare intracranial tumors compared to other brain tumors. Data on the molecular features, especially on schwannoma proteome, are scarce. The 41 cerebrospinal fluid (liquor) samples were obtained during the surgical removal of vestibular schwannoma. Obtained peptide samples were analyzed by shotgun LC-MS/MS high-resolution mass spectrometry. The same peptide samples were spiked with 148 stable isotopically labeled peptide standards (SIS) followed by alkaline fractionation and scheduled multiple reaction monitoring (MRM) for quantitative analysis. The natural counterparts of SIS peptides were mapped onto 111 proteins that were Food and Drug Administration (FDA)-approved for diagnostic use. As a result, 525 proteins were identified by shotgun LC-MS/MS with high confidence (at least two peptides per protein, FDR < 1%) in liquor samples. Absolute quantitative concentrations were obtained for 54 FDA-approved proteins detected in at least five experimental samples. Since there is lack of data on the molecular landscape of vestibular schwannoma, the obtained datasets are unique and one of the first in its field.

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3–72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).