Prognostic utility of the ovarian cancer secretome: a systematic investigation.

BACKGROUND: Ovarian cancer is usually detected at an advanced stage with frequent recurrence. The recurrence-free survival and overall survival is influenced by the age at diagnosis, tumor stage and histological subtype. Nonetheless, quantifiable prognostic biomarkers are needed for early identification of the high-risk patients and for personalized medicine. Several studies link tumor-specific dysregulated expression of certain proteins with ovarian cancer prognosis. However, careful investigation of presence of these prognostically relevant proteins in ovarian cancer secretome is lacking. OBJECTIVE: To critically analyze the recent published data on prognostically relevant proteins for ovarian cancer and to carefully search how many of them are reported in the published ovarian cancer secretome datasets. DESIGN: A search for relevant studies in the past 2 years was conducted in PubMed and a comprehensive list of proteins associated with the ovarian cancer prognosis was prepared. These were cross-referred to the published ovarian cancer secretome profiles. The proteins identified in the secretome were further shortlisted based on a scoring strategy employing stringent criteria. RESULTS: A panel of seven promising secretory biomarkers associated with ovarian cancer prognosis is proposed. CONCLUSION: Scanning the ovarian cancer secretome datasets provides the opportunity to identify if tumor-specific biomarkers could be tested as secretory biomarkers. Detecting their levels in the body fluid would be more advantageous than evaluating the expression in the tissue, since it could be monitored multiple times over the course of the disease to have a better judgment of the prognosis and response to therapy.

N-glycosylation status of Trop2 impacts its surface density, interaction with claudin-7 and exosomal release.

Trophoblast antigen 2 (Trop2) is a type I transmembrane protein post-translationally modified by N-linked glycosylation. It was originally detected in trophoblasts but was later shown to be frequently overexpressed in many epithelial cancers. Recently, anti-Trop2 antibody-drug conjugate has been FDA approved for the treatment of metastatic triple-negative breast and urothelial carcinomas, making it an important tumor antigen. The current study explored the significance of N-glycosylation of Trop2 by substituting specific N-glycan addition sites by site-directed mutagenesis. The mutant proteins were characterized in transiently transfected HEK293 cells. The N-glycosylation mutants did not affect protein expression, stability, dimerization ability and matriptase mediated cleavage. However, N120A and N208A mutants showed decreased interaction with its binding partner claudin-7. Our earlier reported Trop2 mutant V194A, which shows aberrant glycosylation, also displayed hampered interaction with claudin-7. To further characterize the mutants, stable clones expressing wild type and mutant Trop2 were generated in OVCAR3 cell line. Interestingly, surface biotinylation assay showed significantly higher surface expression of N120A and N208A mutants whereas surface localization was drastically reduced for V194A Trop2 mutant. Though overexpression of wild type Trop2 did not cause any change in fibronectin-mediated FAK (Focal adhesion kinase) signaling; expression of N120A mutant, surprisingly downregulated FAK signaling. Furthermore, exosomal release of Trop2 was also decreased in N120A and N208A mutants. This data suggests that site-specific N-glycan addition determines Trop2 surface density, claudin-7 interaction and exosomal release.

Cysteine-rich secretory protein 3 plays a role in prostate cancer cell invasion and affects expression of PSA and ANXA1.

Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.

Mapping of the binding sites involved in PSP94-CRISP-3 interaction by molecular dissection of the complex.

BACKGROUND: Human Prostate Secretory Protein of 94 amino acids (PSP94) has been shown to bind human CRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known. METHODS: In order to identify the residues and/or regions involved in PSP94-CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP). RESULTS: For PSP94, amino acids Y(3), F(4), P(56) and the C-terminal ß-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C(37)A-C(73)A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with human PSP94. Based on our findings the most likely model of PSP94-CRISP-3 complex has been proposed. CONCLUSION: The terminal ß-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3. GENERAL SIGNIFICANCE: Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity.

Mammalian cysteine-rich secretory proteins interact with plasma membrane Ca2+ exporter PMCA4b.

BACKGROUND: Mammalian cysteine-rich secretory proteins (CRISPs) are predominantly expressed in the male reproductive tract. Knockout mice lacking two or more CRISPs show defects in sperm transport, sperm-egg interaction and Ca2+ homeostasis. CRISPs play redundant and specific roles via their binding partners. To understand this, a comprehensive analysis of CRISP interactome needs to be undertaken. OBJECTIVES: This study aimed to analyse CRISP4 binding partners on the plasma membrane of rat caudal spermatozoa. MATERIALS AND METHODS: Total proteins from rat caudal spermatozoa were subjected to immunoprecipitation using anti-CRISP4 antibody followed by liquid chromatography-mass spectrophotometry analysis. Plasma membrane localised proteins were shortlisted, and a key target was validated by co-immunoprecipitation and co-localisation. Co-transfection followed by co-immunoprecipitation was carried out for studying the interaction of full-length as well as deletion mutants of CRISPs with human plasma membrane calcium ATPase, isoform b (hPMCA4b). Calcium assays were performed using Fura-2-AM. The cholesterol binding ability of different CRISPs was evaluated in silico. RESULTS: The membrane-specific interactome of rat CRISP4 (rCRISP4) from caudal spermatozoa revealed PMCA4b as a novel binding partner, and their interaction was validated in rat spermatozoa. Human CRISP1 (hCRISP1) and hCRISP3 also interacted with PMCA4b via the N-terminal domain. Interestingly, hCRISP1 and rCRISP4 delayed PMCA4b-mediated calcium extrusion but hCRISP3 did not. In silico analysis demonstrated that hCRISP1 and rCRISP4 have higher binding affinity towards cholesterol than hCRISP3. The secretion profile of different CRISPs also showed that the ratio of secreted to cell-associated proteins was highest for hCRISP3. CONCLUSION: Our study identifies PMCA4b as a target of multiple mammalian CRISPs and unravels a new role of CRISPs in regulating calcium homeostasis. Differences in the interaction of different CRISPs with cholesterol may regulate their enrichment in the lipid rafts and redistribution in the membrane post-capacitation, thereby affecting their interaction with PMCA4b.

Comparison of Anti-Trop2 Extracellular Domain Antibodies Generated Against Peptide and Protein Immunogens for Targeting Trop2-Positive Tumour Cells.

Trophoblast antigen 2 (Trop2) is a transmembrane glycoprotein upregulated in multiple solid tumours. Trop2-based passive immunotherapies are in clinical trials, while Trop2 targeting CAR-T cell-based therapies are also reported. Information about its T- and B-cell epitopes is needed for it to be pursued as an active immunotherapeutic target. This study focused on identification of immunodominant epitopes in the Trop2 extracellular domain (ECD) that can mount an efficient anti-Trop2 antibody response. In silico analysis using various B-cell epitope prediction tools was carried out to identify linear and conformational B-cell epitopes in the ECD of Trop2. Three linear peptide immunogens were shortlisted and synthesized. Along with linear peptides, truncated Trop2 ECD that possesses combination of linear and conformational epitopes was also selected. Recombinant protein immunogen was produced in 293-F suspension culture system and affinity purified. Antisera against different immunogens were characterized by ELISA and Western blotting. Two anti-peptide antisera detected recombinant and ectopically expressed Trop2 protein; however, they were unable to recognize the endogenous Trop2 protein expressed by cancer cells. Antibodies against truncated Trop2 ECD could bind to the endogenous Trop2 expressed on the surface of cancer cells. In addition to their high avidity, these polyclonal anti-sera against truncated Trop2 protein also mediated antibody-dependent cell-mediated cytotoxicity (ADCC). In summary, our comparative analysis demonstrated the utility of truncated Trop2 ECD as a promising candidate to be pursued as an active immunotherapeutic molecule against Trop2-positive cancer cells.

Proteolytic cleavage of Trop2 at Arg87 is mediated by matriptase and regulated by Val194.

Proteolytic processing is an important post-translational modification affecting protein activity and stability. In the current study, we investigate the N-terminal cleavage of Trop2, a protein which is overexpressed in many cancers. We demonstrate that Trop2 is cleaved at Arg87 by a transmembrane serine protease, matriptase. Homology modeling and site-directed mutagenesis of amino acids in close proximity to the matriptase cleavage site reveal the importance of Val194 in regulating Trop2 cleavage. Co-immunoprecipitation studies confirm that amino acid substitutions at Arg87, Thr88, Lys189, Val194, and His195 do not affect Trop2 dimerization. However, cleavage of wild-type Trop2 by matriptase is inhibited when it is allowed to dimerize with a V194 A mutant monomer, further confirming the role of Val194 in matriptase-mediated N-terminal cleavage.

Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells.

Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR.

Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific.

The prostate secretory protein of 94 amino acids (PSP94) has been shown to interact with cysteine-rich secretory protein 3 (CRISP-3) in human seminal plasma. Interestingly, PSP94 expression is reduced or lost in the majority of the prostate tumours, whereas CRISP-3 expression is upregulated in prostate cancer compared with normal prostate tissue. To obtain a better understanding of the individual roles these proteins have in prostate tumourigenesis and the functional relevance of their interaction, we ectopically expressed either PSP94 or CRISP-3 alone or PSP94 along with CRISP-3 in three prostate cell lines (PC3, WPE1-NB26 and LNCaP) and performed growth inhibition assays. Reverse transcription-polymerase chain reaction and Western blot analysis were used to screen prostate cell lines for PSP94 and CRISP-3 expression. Mammalian expression constructs for human PSP94 and CRISP-3 were also generated and the expression, localization and secretion of recombinant protein were assayed by transfection followed by Western blot analysis and immunofluorescence assay. The effect that ectopic expression of PSP94 or CRISP-3 had on cell growth was studied by clonogenic survival assay following transfection. To evaluate the effects of co-expression of the two proteins, stable clones of PC3 that expressed PSP94 were generated. They were subsequently transfected with a CRISP-3 expression construct and subjected to clonogenic survival assay. Our results showed that PSP94 and CRISP-3 could each induce growth inhibition in a cell line specific manner. Although the growth of CRISP-3-positive cell lines was inhibited by PSP94, growth inhibition mediated by CRISP-3 was not affected by the presence or absence of PSP94. This suggests that CRISP-3 may participate in PSP94-independent activities during prostate tumourigenesis.