Prostate cancer (PCa) is a heterogeneous group of tumors with variable clinical courses. In order to improve patient outcomes, it is critical to clinically separate aggressive PCa (AG) from ...non-aggressive PCa (NAG). Although recent genomic studies have identified a spectrum of molecular abnormalities associated with aggressive PCa, it is still challenging to separate AG from NAG. To better understand the functional consequences of PCa progression and the unique features of the AG subtype, we studied the proteomic signatures of primary AG, NAG and metastatic PCa. 39 PCa and 10 benign prostate controls in a discovery cohort and 57 PCa in a validation cohort were analyzed using a data-independent acquisition (DIA) SWATH-MS platform. Proteins with the highest variances (top 500 proteins) were annotated for the pathway enrichment analysis. Functional analysis of differentially expressed proteins in NAG and AG was performed. Data was further validated using a validation cohort; and was also compared with a TCGA mRNA expression dataset and confirmed by immunohistochemistry (IHC) using PCa tissue microarray (TMA). 4,415 proteins were identified in the tumor and benign control tissues, including 158 up-regulated and 116 down-regulated proteins in AG tumors. A functional analysis of tumor-associated proteins revealed reduced expressions of several proteinases, including dipeptidyl peptidase 4 (DPP4), carboxypeptidase E (CPE) and prostate specific antigen (KLK3) in AG and metastatic PCa. A targeted analysis further identified that the reduced expression of DPP4 was associated with the accumulation of DPP4 substrates and the reduced ratio of DPP4 cleaved peptide to intact substrate peptide. Findings were further validated using an independently-collected tumor cohort, correlated with a TCGA mRNA dataset, and confirmed by immunohistochemical stains of PCa tumor microarray (TMA). Our study is the first large-scale proteomics analysis of PCa tissue using a DIA SWATH-MS platform. It provides not only an interrogative proteomic signature of PCa subtypes, but also indicates the critical roles played by certain proteinases during tumor progression. The spectrum map and protein profile generated in the study can be used to investigate potential biological mechanisms involved in PCa and for the development of a clinical assay to distinguish aggressive from indolent PCa.
PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is ...subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN’s C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN’s cellular stability.
High-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy in women. The low survival rate is largely due to drug resistance. Approximately 80% of patients who initially respond ...to treatment relapse and become drug-resistant. The lack of effective second-line therapeutics remains a substantial challenge for BRCA-1/2 wild-type HGSOC patients. Histone Deacetylases (HDACs) are promising targets in HGSOC treatment; however, the mechanism and efficacy of HDAC inhibitors are understudied in HGSOC. In order to consider HDACs as a treatment target, an improved understanding of their function within HGSOC is required. This includes elucidating HDAC6-specific protein-protein interactions. In this study, we carried out substrate trapping followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate HDAC6 catalytic domain (CD)-specific interactors in the context of BRCA-1/2 wild-type HGSOC. Overall, this study identified new HDAC6 substrates that may be unique to HGSOC. The HDAC6-CD1 mutant condition contained the largest number of significant proteins compared to the CD2 mutant and the CD1/2 mutant conditions, suggesting the HDAC6-CD1 domain has catalytic activity that is independent of CD2. Among the identified substrates were proteins involved in DNA damage repair including PARP proteins. These findings further justify the use of HDAC inhibitors as a combination treatment with platinum chemotherapy agents and PARP inhibitors in HGSOC.
Efficient recruitment of eligible participants is a significant challenge for clinical research studies. This challenge was exacerbated during the COVID-19 pandemic when in-person recruitment was not ...an option. In 2020, the University of Minnesota was tasked, as part of the National Cancer Institute's Serological Sciences Network for COVID-19 (SeroNet), to recruit participants for a longitudinal serosurveillance clinical research study with a goal of characterizing the COVID-19 vaccine-elicited immune response among immunocompromised individuals, which necessitated reliance on non-traditional strategies for participant recruitment. To meet our enrollment target of 300 transplant patients, 300 cancer patients, 100 persons living with HIV, and 200 immunocompetent individuals, we utilized targeted electronic health record (EHR)-based recruitment in addition to traditional recruitment tools, which was an effective combination of recruitment strategies. A significant advantage of patient portal messaging or other digital recruitment strategies such as email communication is timing. We reached 85 % (769 out of 900) of our enrollment target within one year with a 14.3 % response rate to invitations to participate in our study. This achievement is perhaps more salient given the COVID-19 pandemic-related constraints within which we were operating. We demonstrated that the EHR can be leveraged to quickly identify potentially eligible study participants either via EHR communication or mail. We also illustrate how the online portal MyChart can be used to efficiently send targeted recruitment messages.
High-grade serous ovarian cancer (HGSOC) is the most common form of ovarian cancer diagnosed in patients worldwide. Patients with BRCA1/2-mutated HGSOC have benefited from targeted treatments such as ...poly(ADP-ribose) polymerase inhibitors (PARPi). Despite the initial success of PARPi-based ovarian cancer treatment regimens, approximately 70% of patients with ovarian cancer relapse and the 5-year survival rate remains at 30%. PARPi exhibit variable treatment efficacy and toxicity profiles. Furthermore, the off-target effects of PARP inhibition have not yet been fully elucidated, warranting further study of these classes of molecules in the context of HGSOC treatment. Highly reproducible quantitative mass spectrometry-based proteomic workflows have been developed for the analysis of tumor tissues and cell lines. To detect the off-target effects of PARP inhibition, we conducted a quantitative mass spectrometry-based proteomic analysis of a BRCA1-mutated HGSOC cell line treated with low doses of two PARPi, niraparib and rucaparib. Our goal was to identify PARPi-induced protein signaling pathway alterations toward a more comprehensive elucidation of the mechanism of action of PARPi beyond the DNA damage response pathway. A significant enrichment of nuclear and nucleoplasm proteins that are involved in protein binding was observed in the rucaparib-treated cells. Shared upregulated proteins between niraparib and rucaparib treatment demonstrated RNA II pol promoter-associated pathway enrichment in transcription regulation. Pathway enrichment analyses also revealed off-target effects in the Golgi apparatus and the ER. The results from our mass spectrometry-based proteomic analysis highlights notable off-target effects produced by low-dose treatment of BRCA1-mutated HGSOC cells treated with rucaparib or niraparib.
The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) established a harmonized method for large-scale clinical proteomic studies. SWATH-MS, an instance of ...data-independent acquisition (DIA) proteomic methods, is an alternate proteomic approach. In this study, we used SWATH-MS to analyze remnant peptides from the original retrospective TCGA samples generated for the CPTAC ovarian cancer proteogenomic study. The SWATH-MS results recapitulated the confident identification of differentially expressed proteins in enriched pathways associated with the robust Mesenchymal high-grade serous ovarian cancer subtype and the homologous recombination deficient tumors. Hence, SWATH/DIA-MS presents a promising complementary or orthogonal alternative to the CPTAC proteomic workflow, with the advantages of simpler and faster workflows and lower sample consumption, albeit with shallower proteome coverage. In summary, both analytical methods are suitable to characterize clinical samples, providing proteomic workflow alternatives for cancer researchers depending on the context-specific goals of the studies.
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•SWATH-MS and iTRAQ-DDA are used to classify 103 high-grade serous ovarian cancer•SWATH-MS re-capitulates differentially expressed proteins in ovarian cancer subtypes•SWATH-MS is a robust proteomic approach for large-scale clinical proteomic studies
Biological Sciences; Proteomics; Cancer Systems Biology
Selected and multiple reaction monitoring involves monitoring a multiplexed assay of proteotypic peptides and associated transitions in mass spectrometry runs. To describe peptide and associated ...transitions as stable, quantifiable, and reproducible representatives of proteins of interest, experimental and analytical validation is required. However, inadequate and disparate analytical tools and validation methods predispose assay performance measures to errors and inconsistencies.
Implemented as a freely available, open-source tool in the platform independent Java programing language, MRMPlus computes analytical measures as recommended recently by the Clinical Proteomics Tumor Analysis Consortium Assay Development Working Group for "Tier 2" assays - that is, non-clinical assays sufficient enough to measure changes due to both biological and experimental perturbations. Computed measures include; limit of detection, lower limit of quantification, linearity, carry-over, partial validation of specificity, and upper limit of quantification.
MRMPlus streamlines assay development analytical workflow and therefore minimizes error predisposition. MRMPlus may also be used for performance estimation for targeted assays not described by the Assay Development Working Group. MRMPlus' source codes and compiled binaries can be freely downloaded from https://bitbucket.org/paiyetan/mrmplusgui and https://bitbucket.org/paiyetan/mrmplusgui/downloads respectively.
Approximately 20% of high-grade serous ovarian cancers are homologous-recombination (HR)-deficient due to genetic and epigenetic mutations of HR pathway genes including the tumor suppressor genes ...BRCA1 and 2. HR deficiency (HRD) compromises cells’ ability to efficiently repair DNA damage, but it also increases sensitivity to chemotherapeutic treatment strategies; however, not all ovarian cancer patients with HRD tumors exhibit positive responses to chemotherapy. Our previous iTRAQ-based comprehensive proteomic characterization of high-grade serous ovarian carcinomas found that lower levels of histone H4 acetylation at Lys12 and Lys16 (H4-K12acK16ac) were associated with HRD tumors compared with non-HRD tumors. In the current study, we developed and validated an H4-K12acK16ac parallel-reaction-monitoring (PRM)-targeted mass-spectrometry-based assay to analyze acetylation changes of histone H4 and to determine the association of these changes with total H4, histone acetyltransferase, and histone deacetylase (HDAC) levels. Whereas the levels of H4 and histone acetyltransferases were stable irrespective of HRD status, the levels of histone H4 acetylation and one HDAC, HDAC6, were elevated in the HRD tumors. Relative H4 acetylation levels were also analyzed by an antibody-based approach in additional ovarian tumors. It is possible that specific H4 acetylation at Lys12 and Lys16 associated with HRD could inform chemotherapeutic treatment modalities to improve ovarian cancer patients’ treatment response.
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