Secretome processing for proteomics: A methods comparison.

The cancer cell secretome comprises a treasure-trove for biomarkers since it reflects cross-talk between tumor cells and their surrounding environment with high detectability in biofluids. In this study, we evaluated six secretome sample processing workflows coupled to single-shot mass spectrometry: (1) Protein concentration by ultrafiltration with a molecular weight cut-off (MWCO) filter and sample preparation through in-gel digestion (IGD); (2) Acetone protein precipitation coupled to IGD; (3) MWCO filter-based protein concentration followed by to in-solution digestion (ISD); (4) Acetone protein precipitation coupled to ISD; (5) Direct ISD; (6) Secretome lyophilization and ISD. To this end, we assessed workflow triplicates in terms of total number of protein identifications, unique identifications, reproducibility of protein identification and quantification and detectability of small proteins with important functions in cancer biology such as cytokines, chemokines, and growth factors. Our findings revealed that acetone protein precipitation coupled to ISD outperformed the other methods in terms of the number of identified proteins (2246) and method reproducibility (correlation coefficient between replicates (r = 0.94, CV = 19%). Overall, especially small proteins such as those from the classes mentioned above were better identified using ISD workflows. Concluding, herein we report that secretome protein precipitation coupled to ISD is the method of choice for high-throughput secretome proteomics via single shot nanoLC-MS/MS.

VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

Protein homeostasis maintained by HOOK1 levels promotes the tumorigenic and stemness properties of ovarian cancer cells through reticulum stress and autophagy.

BACKGROUND: Ovarian cancer has a high mortality rate mainly due to its resistance to currently used therapies. This resistance has been associated with the presence of cancer stem cells (CSCs), interactions with the microenvironment, and intratumoral heterogeneity. Therefore, the search for new therapeutic targets, particularly those targeting CSCs, is important for improving patient prognosis. HOOK1 has been found to be transcriptionally altered in a substantial percentage of ovarian tumors, but its role in tumor initiation and development is still not fully understood. METHODS: The downregulation of HOOK1 was performed in ovarian cancer cell lines using CRISPR/Cas9 technology, followed by growth in vitro and in vivo assays. Subsequently, migration (Boyden chamber), cell death (Western-Blot and flow cytometry) and stemness properties (clonal heterogeneity analysis, tumorspheres assay and flow cytometry) of the downregulated cell lines were analysed. To gain insights into the specific mechanisms of action of HOOK1 in ovarian cancer, a proteomic analysis was performed, followed by Western-blot and cytotoxicity assays to confirm the results found within the mass spectrometry. Immunofluorescence staining, Western-blotting and flow cytometry were also employed to finish uncovering the role of HOOK1 in ovarian cancer. RESULTS: In this study, we observed that reducing the levels of HOOK1 in ovarian cancer cells reduced in vitro growth and migration and prevented tumor formation in vivo. Furthermore, HOOK1 reduction led to a decrease in stem-like capabilities in these cells, which, however, did not seem related to the expression of genes traditionally associated with this phenotype. A proteome study, along with other analysis, showed that the downregulation of HOOK1 also induced an increase in endoplasmic reticulum stress levels in these cells. Finally, the decrease in stem-like properties observed in cells with downregulated HOOK1 could be explained by an increase in cell death in the CSC population within the culture due to endoplasmic reticulum stress by the unfolded protein response. CONCLUSION: HOOK1 contributes to maintaining the tumorigenic and stemness properties of ovarian cancer cells by preserving protein homeostasis and could be considered an alternative therapeutic target, especially in combination with inducers of endoplasmic reticulum or proteotoxic stress such as proteasome inhibitors.

Loss of VRK1 alters the nuclear phosphoproteome in the DNA damage response to doxorubicin.

Dynamic chromatin remodeling requires regulatory mechanisms for its adaptation to different nuclear function, which are likely to be mediated by signaling proteins. In this context, VRK1 is a nuclear Ser-Thr kinase that regulates pathways associated with transcription, replication, recombination, and DNA repair. Therefore, VRK1 is a potential regulatory, or coordinator, molecule in these processes. In this work we studied the effect that VRK1 depletion has on the basal nuclear and chromatin phosphoproteome, and their associated pathways. VRK1 depletion caused an alteration in the pattern of the nuclear phosphoproteome, which is mainly associated with nucleoproteins, ribonucleoproteins, RNA splicing and processing. Next, it was determined the changes in proteins associated with DNA damage that was induced by doxorubicin treatment. Doxorubicin alters the nuclear phosphoproteome affecting proteins implicated in DDR, including DSB repair proteins NBN and 53BP1, cellular response to stress and chromatin organization proteins. In VRK1-depleted cells, the effect of doxorubicin on protein phosphorylation was reverted to basal levels. The nuclear phosphoproteome patterns induced by doxorubicin are altered by VRK1 depletion, and is enriched in histone modification proteins and chromatin associated proteins. These results indicate that VRK1 plays a major role in processes requiring chromatin remodeling in its adaptation to different biological contexts.

Kinase activities in pancreatic ductal adenocarcinoma with prognostic and therapeutic avenues.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho-subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho-subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype-associated activity profiles can guide therapeutic combination approaches in tumor and stroma-enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance.

Protein kinase D drives the secretion of invasion mediators in triple-negative breast cancer cell lines.

The protein kinase D (PKD) family members regulate the fission of cargo vesicles at the Golgi complex and play a pro-oncogenic role in triple-negative breast cancer (TNBC). Whether PKD facilitates the secretion of tumor-promoting factors in TNBC, however, is still unknown. Using the pharmacological inhibition of PKD activity and siRNA-mediated depletion of PKD2 and PKD3, we identified the PKD-dependent secretome of the TNBC cell lines MDA-MB-231 and MDA-MB-468. Mass spectrometry-based proteomics and antibody-based assays revealed a significant downregulation of extracellular matrix related proteins and pro-invasive factors such as LIF, MMP-1, MMP-13, IL-11, M-CSF and GM-CSF in PKD-perturbed cells. Notably, secretion of these proteins in MDA-MB-231 cells was predominantly controlled by PKD2 and enhanced spheroid invasion. Consistently, PKD-dependent secretion of pro-invasive factors was more pronounced in metastatic TNBC cell lines. Our study thus uncovers a novel role of PKD2 in releasing a pro-invasive secretome.

IGF1R signaling induces epithelial-mesenchymal plasticity via ITGAV in cutaneous carcinoma.

BACKGROUND: Early cutaneous squamous cell carcinomas (cSCCs) generally show epithelial differentiation features and good prognosis, whereas advanced cSCCs present mesenchymal traits associated with tumor relapse, metastasis, and poor survival. Currently, the mechanisms involved in cSCC progression are unclear, and the established markers are suboptimal for accurately predicting the clinical course of the disease. METHODS: Using a mouse model of cSCC progression, expression microarray analysis, immunofluorescence and flow cytometry assays, we have identified a prognostic biomarker of tumor relapse, which has been evaluated in a cohort of cSCC patient samples. Phosphoproteomic analysis have revealed signaling pathways induced in epithelial plastic cancer cells that promote epithelial-mesenchymal plasticity (EMP) and tumor progression. These pathways have been validated by genetic and pharmacological inhibition assays. RESULTS: We show that the emergence of epithelial cancer cells expressing integrin αV (ITGAV) promotes cSCC progression to a mesenchymal state. Consistently, ITGAV expression allows the identification of patients at risk of cSCC relapse above the currently employed clinical histopathological parameters. We also demonstrate that activation of insulin-like growth factor-1 receptor (IGF1R) pathway in epithelial cancer cells is necessary to induce EMP and mesenchymal state acquisition in response to tumor microenvironment-derived factors, while promoting ITGAV expression. Likewise, ITGAV knockdown in epithelial plastic cancer cells also blocks EMP acquisition, generating epithelial tumors. CONCLUSIONS: Our results demonstrate that ITGAV is a prognostic biomarker of relapse in cSCCs that would allow improved patient stratification. ITGAV also collaborates with IGF1R to induce EMP in epithelial cancer cells and promotes cSCC progression, revealing a potential therapeutic strategy to block the generation of advanced mesenchymal cSCCs.