Glycoproteomics Identifies Plexin-B3 as a Targetable Cell Surface Protein Required for the Growth and Invasion of Triple-Negative Breast Cancer Cells.

Driven by the lack of targeted therapies, triple-negative breast cancers (TNBCs) have the worst overall survival of all breast cancer subtypes. Considering that cell surface proteins are favorable drug targets and are predominantly glycosylated, glycoproteome profiling has significant potential to facilitate the identification of much-needed drug targets for TNBCs. Here, we performed N-glycoproteomics on six TNBCs and five normal control (NC) cell lines using hydrazide-based enrichment. Quantitative proteomics and integrative data mining led to the discovery of Plexin-B3 (PLXNB3), a previously undescribed TNBC-enriched cell surface protein. Furthermore, siRNA knockdown and CRISPR-Cas9 editing of in vitro and in vivo models show that PLXNB3 is required for TNBC cell line growth, invasion, and migration. Altogether, we provide insights into N-glycoproteome remodeling associated with TNBCs and functional evaluation of an extracted target, which indicate the surface protein PLXNB3 as a potential therapeutic target for TNBCs.

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma.

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies.

In-depth proteomic analyses of ovarian cancer cell line exosomes reveals differential enrichment of functional categories compared to the NCI 60 proteome.

Molecular communication between cancer cells and its stromal microenvironment is a key factor for cancer progression. Alongside classic secretory pathways, it has recently been proposed that small membranous vesicles are alternative mediators of intercellular communication. Exosomes carry an effector-rich proteome with the ability to modulate various functional properties of the recipient cell. In this study, exosomes isolated from four epithelial ovarian cancer cell lines (OVCAR3, OVCAR433, OVCAR5 and SKOV3) were characterized using mass spectrometry-based proteomics. Using an optimized workflow consisting of efficient exosome solubilization and the latest generation of proteomic instrumentation, we demonstrate improved detection depth. Systematic comparison of our cancer cell line exosome proteome against public data (Exocarta) and the recently published NCI 60 proteome revealed enrichment of functional categories related to signaling biology and biomarker discovery.

Organelle resolved proteomics uncovers PLA2R1 as a novel cell surface marker required for chordoma growth.

Chordomas are clinically aggressive tumors with a high rate of disease progression despite maximal therapy. Given the limited therapeutic options available, there remains an urgent need for the development of novel therapies to improve clinical outcomes. Cell surface proteins are attractive therapeutic targets yet are challenging to profile with common methods. Four chordoma cell lines were analyzed by quantitative proteomics using a differential ultracentrifugation organellar fractionation approach. A subtractive proteomics strategy was applied to select proteins that are plasma membrane enriched. Systematic data integration prioritized PLA2R1 (secretory phospholipase A2 receptor-PLA2R1) as a chordoma-enriched surface protein. The expression profile of PLA2R1 was validated across chordoma cell lines, patient surgical tissue samples, and normal tissue lysates via immunoblotting. PLA2R1 expression was further validated by immunohistochemical analysis in a richly annotated cohort of 25-patient tissues. Immunohistochemistry analysis revealed that elevated expression of PLA2R1 is correlated with poor prognosis. Using siRNA- and CRISPR/Cas9-mediated knockdown of PLA2R1, we demonstrated significant inhibition of 2D, 3D and in vivo chordoma growth. PLA2R1 depletion resulted in cell cycle defects and metabolic rewiring via the MAPK signaling pathway, suggesting that PLA2R1 plays an essential role in chordoma biology. We have characterized the proteome of four chordoma cell lines and uncovered PLA2R1 as a novel cell-surface protein required for chordoma cell survival and association with patient outcome.

Pleiotropic action of renal cell carcinoma-dysregulated miRNAs on hypoxia-related signaling pathways.

The von Hippel-Lindau (VHL) gene is lost in ≈ 70% of all renal cell carcinomas (RCCs); however, increasing evidence supports the involvement of alternative mechanisms in the regulation of VHL expression, including suppression by microRNAs (miRNAs). miRNAs are small, noncoding RNA molecules that regulate gene expression through binding to target mRNAs. In this study, we found that miRNAs, which are dysregulated in cases of RCC, can target multiple members of RCC-related signaling pathways. Importantly, both VHL and the hypoxia-inducible factor 1-α gene are experimentally validated and are likely direct targets of miR-17-5p and miR-224, as shown by both luciferase assay and Western blot analysis. We found a negative correlation between miR-17-5p and its two predicted targets, VEGF-A and EGLN3, and between miR-224 and its targets SMAD4 and SMAD5 in RCC specimens, suggesting that downstream signaling pathways are also modulated by clear cell RCC-dysregulated miRs. Results from our bioinformatics analysis show that a single miRNA molecule can target multiple components of the same pathway and that multiple miRNAs can target the same molecule. Our results also indicate that miRNAs represent a mechanism for the inactivation of VHL in cases of RCC and can elucidate a new dimension in cancer pathogenesis. As such, miRNAs exemplify new potential therapeutic targets with a significant effect on both tumor growth and metastatic potential.

Characterization of the 12q15 MDM2 and 12q13-14 CDK4 amplicons and clinical correlations in osteosarcoma.

The chromosomal region 12q13-15 is recurrently amplified in osteosarcoma (OS), but its importance in bone tumor development remains unknown. Although there are two major candidate genes (MDM2, a TP53 downregulator, and CDK4, involved in cell cycle progression) considered to be the driving genes in this region, the size of the amplicon and number of genes involved have not been determined. In this study, we used 130 classical OS and 15 parosteal OS to determine MDM2 and CDK4 amplification frequency in OS. Tumors in which these genes were amplified were used to map the 12q13-15 amplified region and to determine its correlation with clinical prognosis. The 12q13-15 amplification was more prevalent in parosteal OS (67% of cases) than in high-grade classical OS (12%). Quantitative real-time PCR of MDM2, CDK4, and 25 other genes showed that this region contains two different amplicons: one at 12q15 centered on MDM2 and one at 12q13-14 centered on CDK4. Both regions were frequently co-amplified in both types of OS, and MDM2 and CDK4 amplification was correlated with higher expression levels for both genes. Univariate and multivariate analyses of clinical data indicated that classical OS patients whose tumors exhibited MDM2 amplification were more likely to be older at diagnosis (median age 32.6 vs. 17.8 years) and female (66.7 vs. 33.3%) than those without gene amplification. There was no association with other clinical parameters. In conclusion, co-amplification of MDM2 and CDK4 in two separate amplicons occurs frequently in parosteal OS and less so in classical high-grade OS.

Dysregulation of kallikrein-related peptidases in renal cell carcinoma: potential targets of miRNAs.

Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies and currently no diagnostic marker exists. Kallikrein-related peptidases (KLKs) have been implicated in numerous cancers including ovarian, prostate, and breast carcinoma. KLKs 5, 6, 10, and 11 have decreased expression in RCC when compared to normal kidney tissue. Our bioinformatic analysis indicated that the KLK 1, 6, and 7 genes have decreased expression in RCC. We experimentally verified these results and found that decreased expression of KLKs 1 and 3 were significantly associated with the clear cell RCC subtype (p<0.001). An analysis of miRNAs differentially expressed in RCC showed that 61 of the 117 miRNAs that were reported to be dysregulated in RCC were predicted to target KLKs. We experimentally validated two targets using two independent approaches. Transfection of miR-224 into HEK-293 cells resulted in decreased KLK1 protein levels. A luciferase assay demonstrated that hsa-let-7f can target KLK10 in the RCC cell line ACHN. Our results, showing differential expression of KLKs in RCC, suggest that KLKs could be novel diagnostic markers for RCC and that their dysregulation could be under miRNA control. The observation that KLKs could represent targets for miRNAs suggests a post-transcriptional regulatory mechanism with possible future therapeutic applications.

Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors.

PDX1+/NKX6-1+ pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of ß cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2+ cells generate ß-like cells (C-PEPTIDE+/NKX6-1+) more efficiently compared to GP2- and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.

Neonatal transfer of membrane-bound stem cell factor improves survival and heart function in aged mice after myocardial ischemia.

Stem cell mobilization to injured tissue contributes to neovascularization, resulting in regeneration after myocardial infarction (MI). We previously showed that direct cardiac injection of a recombinant lentivirus (LV) that engineers expression of membrane-bound stem cell factor (mSCF) improves outcomes immediately after MI. In this study, we evaluated the effect of neonatal LV/mSCF transduction on MI outcomes in aged mice. We constructed a recombinant LV harboring an α-myosin heavy chain promoter that drives mSCF expression and injected it into the temporal vein of neonatal mice. One year later, sustained expression of mSCF in the adult mouse hearts was detected by genomic and quantitative RT-PCR and immunohistochemistry. To evaluate the contribution of neonatal LV/mSCF delivery to recovery from MI, we induced an MI in adult LV/mSCF-transduced, LV only-transduced, and nontransduced control mice. Strikingly, LV/mSCF transduction reduced infarct scar size, enhanced angiogenesis, improved ventricular function, and significantly increased survival of the mice. Regional overexpression of CD11b, a marker of monocytes and proangiogenic cells, was observed on monocytes isolated from the infarcted hearts of LV/mSCF-transduced mice. Our data suggest a model of neonatal gene delivery that leads to sustained mSCF expression during adulthood to aid recovery from MI and prevent heart failure.

The clinical utility of miR-21 as a diagnostic and prognostic marker for renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney. Increasing evidence suggests that microRNAs are dysregulated in RCC and are important factors in RCC pathogenesis. miR-21 is a known oncogene with tumor-promoting effects in many types of cancer. In this study, we analyzed miR-21 in 121 cases of healthy kidney and different RCC subtypes, including clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC), and oncocytoma. Total RNA was extracted, and the expression of miR-21 was measured with real-time quantitative RT-PCR using miR-21-specific probes. The expression of miR-21 was significantly up-regulated in RCC compared with healthy kidney. There was a significant difference in the expression levels between RCC subtypes, with the highest levels of expression in ccRCC and pRCC subtypes. miR-21 expression distinguished ccRCC and pRCC from chRCC and oncocytoma with 90% specificity (95% CI, 63.9% to 98.1%) and 83% sensitivity (95% CI, 53.5% to 97.6%). Significantly higher miR-21 levels were associated with higher stage and grade. Patients who were miR-21 positive had statistically significant shorter disease-free and overall survival rates. Thus, miR-21 is up-regulated in RCC, and its expression levels can be used as a diagnostic marker to distinguish ccRCC and pRCC from chRCC and oncocytoma. Moreover, it has potential as a prognostic marker in RCC, although it is not independent of tumor stage and grade.

Accurate molecular classification of kidney cancer subtypes using microRNA signature.

BACKGROUND: Renal cell carcinoma (RCC) encompasses different histologic subtypes. Distinguishing between the subtypes is usually made by morphologic assessment, which is not always accurate. OBJECTIVE: Our aim was to identify microRNA (miRNA) signatures that can distinguish the different RCC subtypes accurately. DESIGN, SETTING, AND PARTICIPANTS: A total of 94 different subtype cases were analysed. miRNA microarray analysis was performed on fresh frozen tissues of three common RCC subtypes (clear cell, chromophobe, and papillary) and on oncocytoma. Results were validated on the original as well as on an independent set of tumours, using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis with miRNA-specific primers. MEASUREMENTS: Microarray data were analysed by standard approaches. Relative expression for qRT-PCR was determined using the ΔΔC(T) method, and expression values were normalised to small nucleolar RNA, C/D box 44 (SNORD44, formerly RNU44). Experiments were done in triplicate, and an average was calculated. Fold change was expressed as a log(2) value. The top-scoring pairs classifier identified operational decision rules for distinguishing between different RCC subtypes and was robust under cross-validation. RESULTS AND LIMITATIONS: We developed a classification system that can distinguish the different RCC subtypes using unique miRNA signatures in a maximum of four steps. The system has a sensitivity of 97% in distinguishing normal from RCC, 100% for clear cell RCC (ccRCC) subtype, 97% for papillary RCC (pRCC) subtype, and 100% accuracy in distinguishing oncocytoma from chromophobe RCC (chRCC) subtype. This system was cross-validated and showed an accuracy of about 90%. The oncogenesis of ccRCC is more closely related to pRCC, whereas chRCC is comparable with oncocytoma. We also developed a binary classification system that can distinguish between two individual subtypes. CONCLUSIONS: MiRNA expression patterns can distinguish between RCC subtypes.