ADAPT Identifies an ESCRT Complex Composition That Discriminates VCaP From LNCaP Prostate Cancer Cell Exosomes

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC-MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes

Studies on the actin-binding protein HS1 in platelets

<p>Abstract</p> <p>Background</p> <p>The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1^-/-mice.</p> <p>Results</p> <p>The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1^-/-mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay.</p> <p>Conclusion</p> <p>This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin.</p

Comparative genome analysis of cortactin and HSI:the significance of the F-actin binding repeat domain

Background: In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein I (HSI) although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution.Results: Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HSI exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HSI is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HSI.Conclusions: Comparative analysis of the genomic organization and amino acid sequences of cortactin and HSI provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HSI lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which is of importance for the major role of HSI in apoptosis and for cortactin in cell migration.</p

Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis

Adaptive dynamic artificial poly-ligand targeting: Aptamer-based profiling of liquid biopsies to improve the accuracy of breast cancer diagnoses in women with dense breast tissue

Introduction: Breast cancer screening relies upon mammography, but for women with dense breast tissue this method is often uninformative. Routine screening identifies suspicious breast lesions in some women, but the pain and risk associated with follow-up biopsies along with the poor accuracy of traditional histopathology urgently call for improved approaches to breast cancer screening. This is especially important for those high-risk patients for whom mammography is of limited value. We describe a non-invasive liquid biopsy method of profiling plasma exosome preps designed to improve the accuracy and safety of breast cancer screening for women with dense breast tissue. Results: We incubated plasma samples (300 microliters per sample) from breast cancer patients (n=60) and a control cohort (n=60) with a high-complexity DNA aptamer library using a modified SELEX scheme, termed “adaptive dynamic artificial poly-ligand targeting (ADAPTTM)”. Differentially bound (cancer vs. non-cancer) aptamers were recovered from precipitated exosomes and were identified by deep sequencing. Two thousand aptamer sequences were resynthesized and used to probe a larger set of 500 plasma samples from a patient cohort (n=206) and a control cohort comprised of self-reported healthy volunteers (n=117) and patients whose biopsies led to a diagnosis of non-cancer (n=177). We employed several statistical models to build a cancer/non-cancer predictor, including a Random Generalized Linear Model (RGLM) and a Random Forest Model (RFM). Both models yielded an equivalent classification performance with areas under the receiver-operator characteristic curve (ROC AUC) of 0.7. Testing the prediction performance by 100 Out-of-Bag permutations or by pre-filtered (read cutoff and estimated sample size) cross-validation (CV) resulted in ROC AUC values of 0.66 and 0.62, respectively. When samples were randomly assigned to groups, the aptamers were no longer able to distinguish the groups (ROC AUC = 0.54), indicating that the underlying information driving the model is truly specific to cancer. Importantly, incorporation of BIRAD results as a clinical covariate did not influence model performance, signifying that predictions by ADAPTTM were independent of breast tissue density. Conclusions: We have identified a set of 2000 DNA aptamers that distinguish women with breast cancer from women without breast cancer. Our liquid biopsy approach requires only 300 microliters of plasma and is amenable to high-throughput processing. By employing a number of statistical approaches including rigorous cross-validation, we consistently achieve cross validation ROC AUC values approaching 0.7. The performance of the predictor was not affected by BIRAD scores, supporting its potential utility in difficult cases where imaging is insufficient, such as in women with dense breast tissue. Further optimization of the aptamer library and testing on additional samples should improve performance. Upon complete validation, an ADAPTTM – derived breast cancer test may serve as a vital diagnostic adjunct that can be easily incorporated into standard clinical practice. Citation Format: Domenyuk V, Zhong Z, Wang J, Stark A, Chen W, Xiao N, Miglarese MR, Famulok M, Mayer G, Spetzler DB. Adaptive dynamic artificial poly-ligand targeting: Aptamer-based profiling of liquid biopsies to improve the accuracy of breast cancer diagnoses in women with dense breast tissue. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-01-08