Susanne Klein-Scory

Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma

Improved non‐invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non‐coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non‐invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR‐1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR‐1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2‐1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2‐1f) and not from miR‐1246. In addition, we observed a remarkable stability of RNU2‐1f in serum and provide experimental evidence that hsa‐miR‐1246 is likely a pseudo microRNA. In a next step, RNU2‐1f was measured by qRT‐PCR and normalized to cel‐54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI = (87.7, 99.9)] and 90.6% [95% CI = (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies.

Characterization of soluble and exosomal forms of the EGFR released from pancreatic cancer cells

AIMS Members of the epidermal growth factor receptor (EGFR) family represent validated targets for anti-cancer therapy and EGFR inhibitors have also shown efficacy in pancreatic carcinoma. We here described in detail molecular forms of the EGF receptor released by pancreatic cancer cells. We found peptides specific for the EGFR in the secretomes of five pancreatic cancer cell lines. Secretomes from cultured cancer cells are widely used as sources for serum biomarker discovery. MAIN METHODS The detailed analysis of EGFR forms in secretomes of human pancreatic cancer cells is a compilation of results from mass spectrometry (MS) and Western blotting with intracellular and extracellular domain specific antibodies. KEY FINDINGS Pancreatic cancer cells secrete a 110 kDa soluble form of the EGFR (sEGFR) representing the ligand binding extracellular EGFR domains and presumably released by ectodomain shedding. At the same time, as constituents of exosomes, the EGFR is released as full-length intact receptor (170 kDa) and as a 65 kDa processed form, the C-terminal remnant fragment that corresponds to the intracellular kinase domain. SIGNIFICANCE The detailed characterization of diverse EGFR forms released by pancreatic cancer cells in vitro and presumably in vivo bears important implications for functional studies, for the validation of soluble EGFR as a serum biomarker and for the design of targeted therapies.

A catalogue of proteins released by colorectal cancer cells in vitro as an alternative source for biomarker discovery.

Improved methods for the early diagnosis of colorectal cancer by way of sensitive and specific tumour markers are highly desirable. Therefore, efficient strategies for biomarker discovery are urgently needed. Here we present an approach that is based on the direct experimental access to proteins released by SW620 human colorectal cancer cells in vitro. A 2‐D map and a catalogue of this subproteome – here termed the secretome – were established comprising more than 320 identified proteins which translate into approximately 220 distinct genes. As the majority of the secretome constituents were nominally cellular proteins, we directly compared the secretome and the total proteome by 2‐D‐DIGE analysis. We provide evidence that unspecific release through cell death, classical secretion, ectodomain shedding, and exosomal release contribute to the secretome in vitro, presumably reflecting the mechanisms in vivo which lead to the occurrence of tumour‐specific proteins in the circulation. These data together with the fact that the SW620 secretome catalogue, as presented here, does comprise a large number of known and novel biomarker candidates, validates our approach to isolate and characterize the tumour cell secretome in vitro as a rich source for tumour biomarkers.

Soluble E-cadherin as a serum biomarker candidate: elevated levels in patients with late-stage colorectal carcinoma and FAP.

Various strategies have been tested to identify serum biomarkers in patients with cancer. Recently, the entire of proteins released by cultured tumor cells into the media, the so‐called secretome, has been suggested as a promising source for biomarker discovery. Ectodomains of membrane proteins cleaved from the cell surface represent a surprisingly abundant and apparently stable subset of this subproteome. Aiming for the detection of serum biomarkers for patients with colorectal cancer (CRC), we have previously detected significant amounts of the soluble form of E‐cadherin in the secretomes of CRC cells. Here, we report a comprehensive analysis of sE‐cadherin levels in sera from patients with CRC, colorectal adenoma, inflammatory bowel disease and familial adenomatous polyposis (FAP). Whereas mean sE‐cadherin levels in patients with inflammatory bowel disease (mean: 4.7 μg/ml, SD: 1.5 μg/ml), with adenomas (mean: 4.6 μg/ml, SD: 3.0 μg/ml) and early stage cancers (mean: 4.9 μg/ml, SD: 4.7 μg/ml) do not significantly differ from healthy controls (mean: 4.8 μg/ml, SD: 1.9 μg/ml), patients with Stage III and Stage IV carcinomas display a significant increase (mean: 6.1 μg/ml, SD: 2.6 μg/ml). In individual patients with late‐stage CRC, sE‐cadherin serum levels directly reflect their disease status over time. These findings suggest a potential application of sE‐cadherin as an alternative diagnostic biomarker for monitoring disease particularly in patients with carcinoembryonic antigen negative tumors. In patients with FAP, on the other hand, we also detected a significant increase of serum sE‐cadherin levels (mean: 5.8 μg/ml, SD: 2.8 μg/ml), but this was regardless of their tumor load and colectomy status.

New insights in the composition of extracellular vesicles from pancreatic cancer cells: implications for biomarkers and functions

BackgroundPancreatic cancer development is associated with characteristic alterations like desmoplastic reaction and immune escape which are mediated by the cell-cell communication mechanism and by the microenvironment of the cells. The whole of released components are important determinants in these processes. Especially the extracellular vesicles released by pancreatic cancer cells play a role in cell communication and modulate cell growth and immune responses.ResultsHere, we present the proteomic description of affinity purified extracellular vesicles from pancreatic tumour cells, compared to the secretome, defined as the whole of the proteins released by pancreatic cancer cells. The proteomic data provide comprehensive catalogues of hundreds of proteins, and the comparison reveals a special proteomic composition of pancreatic cancer cell derived extracellular vesicles. The functional analysis of the protein composition displayed that membrane proteins, glycoproteins, small GTP binding proteins and a further, heterogeneous group of proteins are enriched in vesicles, whereas proteins derived from proteasomes and ribosomes, as well as metabolic enzymes, are not components of the vesicles. Furthermore proteins playing a role in carcinogenesis and modulators of the extracellular matrix (ECM) or cell-cell interactions are components of affinity purified extracellular vesicles.ConclusionThe data deepen the knowledge of extracellular vesicle composition by hundreds of proteins that have not been previously described as vesicle components released by pancreatic cancer cells. Extracellular vesicles derived from pancreatic cancer cells show common proteins shared with other vesicles as well as cell type specific proteins indicating biomarker candidates and suggesting functional roles in cancer cell stroma interactions.

Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε

BackgroundInactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells.MethodsHigh resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction.ResultsWe identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization.ConclusionBased on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry.

A soluble form of the giant cadherin Fat1 is released from pancreatic cancer cells by ADAM10 mediated ectodomain shedding

In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.

Uncoupled responses of Smad4-deficient cancer cells to TNFα result in secretion of monomeric laminin-γ2

BackgroundFunctional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-γ2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFα on tumor cells.ResultsSmad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFα with an increased expression of all three chains encoding laminin-332; coincubation with TGFβ and TNFα leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFα can induce expression of the γ2 and β3 but not the α3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of γ2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble γ2 is associated with increased cell migration.ConclusionsLoss of Smad4 may lead to uncoupled induction of laminin-γ2 in response to TNFα and may therefore represent one of the mechanisms which underlie accumulation of laminin-γ2 at the invasive margin of a tumor. The finding, that γ2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.

High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition

BackgroundSmad4 is a tumour suppressor frequently inactivated in pancreatic and colorectal cancers. We have recently reported loss of Smad4 in every fourth carcinoma of the uterine cervix. Smad4 transmits signals from the TGF-β superfamily of cytokines and functions as a versatile transcriptional co-modulator. The prevailing view suggests that the tumour suppressor function of Smad4 primarily resides in its capability to mediate TGF-β growth inhibitory responses. However, accumulating evidence indicates, that the acquisition of TGF-β resistance and loss of Smad4 may be independent events in the carcinogenic process. Through inducible reexpression of Smad4 in cervical cancer cells we wished to shed more light on this issue and to identify target genes implicated in Smad4 dependent tumor suppression.MethodsSmad4-deficient human C4-II cervical carcinoma cells were used to establish inducible Smad4 reexpression using the commercial Tet-on™ system (Clontech). The impact of Smad4 reexpression on cell growth was analysed in vitro and in vivo. Transcriptional responses were assessed through profiling on cDNA macroarrays (Clontech) and validated through Northern blotting.ResultsClones were obtained that express Smad4 at widely varying levels from approximately physiological to 50-fold overexpression. Smad4-mediated tumour suppression in vivo was apparent at physiological expression levels as well as in Smad4 overexpressing clones. Smad4 reexpression in a dose-dependent manner was associated with transcriptional induction of the extracellular matrix-associated genes, BigH3, fibronectin and PAI-1, in response to TGF-β. Smad4-dependent regulation of these secreted Smad4 targets is not restricted to cervical carcinoma cells and was confirmed in pancreatic carcinoma cells reexpressing Smad4 after retroviral transduction and in a stable Smad4 knockdown model. On the other hand, the classical cell cycle-associated TGF-β target genes, c-myc, p21 and p15, remained unaltered.ConclusionOur results show that Smad4-mediated tumour suppression in cervical cancer cells is not due to restoration of TGF-β growth inhibitory responses. Rather, tumour cell-ECM interactions may be more relevant for Smad4-mediated tumour suppression. C4-II cells with a high level inducible Smad4 expression may serve as a model to indicate further Smad4 targets responsive to diverse environmental stimuli operative in vivo.

Significance of Liquid Biopsy for Monitoring and Therapy Decision of Colorectal Cancer

PURPOSE: Despite therapeutic improvements, all patients with nonresectable metastatic colorectal cancer (mCRC) acquire resistance to treatment probably due to the growth of mutated clones. In contrast to tissue-based studies, liquid biopsies have enabled the opportunity to reveal emerging resistance to treatment by detecting mutated clones and noninvasively monitoring clonal dynamics during therapy. METHODS: The courses of three patients with mCRC who were initially RAS wild-type were monitored longitudinally using liquid biopsy with long-term follow-up of up to 20 sequential samples. Detection of fragmented RAS mutated circulating cell-free DNA (cf)DNA in plasma was performed by BEAMing. In addition, plasma digital droplet PCR was used to detect and quantify BRAF and PIK3CA mutated cfDNA. Changes of mutational load were correlated with imaging data. RESULTS: A combination of liquid biopsy and radiological imaging enabled visualization of the occurrence of clonal redistribution after discontinuation of anti-EGFR mAb therapy, as well as emerging RAS mutations during therapy with anti-EGFR mAb indicating resistance. Furthermore, we found that growth of RAS mutated clones is independent of direct selective pressure by anti-EGFR therapy, which is a significant and new finding of this study. CONCLUSIONS: Our findings demonstrated the whole spectrum of clonal selection and redistribution of mutated cell clones leading to acquired resistance. Given our observation that the growth of RAS mutated clones can evolve even in the absence of anti-EGFR mAb therapy, there is a clear imperative to monitor RAS mutations in serial blood draws in all RAS wild-type patients in general and independent of the therapy.