Protein-protein interactions of the nuclear receprot NR4A2

The Gastroenterology and Molecular cell biology research group examines biological mechanisms that are important in the development of gastric cancer. The peptide hormone gastrin is known to play a role in organization and maintenance of the gastric epithelium, however, its role in carcinogenesis is debated. Gastrin induces the expression of the nuclear transcription factor NR4A2. NR4A2 expression has been suggested as prognostic factors and potential therapeutic targets for patients with different cancer types, including gastric cancer. The biological functions of NR4A2 in tumors and the prognostic significance are incomplete and needs to be further studied. Recent studies indicate that NR4A2 protein-protein interactions as well as its subcellular localization are of vital importance when it comes to the specific cellular response. The aim of this study was to characterize interacting partners of NR4A2 in cellular model systems. In the present study, the expression of the CCK2 receptor and gastrin induced NR4A2 expression in gastric carcinoma cell lines were examined. The MKN45 and HEK293 cells were used to characterize protein-protein interactions of NR4A2. No interactions were observed between NR4A2 and the proteins initially hypothesized to bind to NR4A2. However, Mass Spectrometry in combination with immunoprecipitation identified several potential binding partners. This thesis provides an overview of potential interactions partners of NR4A2 that may be important in gastric cancer and can provide candidate proteins for further work

Extracellular Vesicles in Colorectal Cancer: Mediators of tumor aggressiveness

Extracellular vesicles (EVs) are small vesicles released from cells. EVs represent an important mode of intercellular communication by serving as vehicles for transfer of proteins and nucleic acids between cells. Interestingly, the exosome composition differs in healthy and diseased individuals, making these vesicles attractive as a non-invasive source of biomarkers. The aims of this thesis were to i) analyze the miRNA-cargo of EVs from hypoxic CRC cell lines and investigate their impact on tumor aggressiveness and resistance to therapy in rectal cancer patients, ii) identify plasma exosomal miRNAs associated with rectal cancer aggressiveness and outcome, iii) and investigate the modulatory role of cancer derived EVs on immune cells. Well established CRC-cell lines and primary monocytes isolated from healthy individuals were used for the in vitro studies, and plasma samples were collected from patients with locally advanced rectal cancer involved in the OxyTarget study and LARC-RRP study. We observed a cell-line specific profile of exosomal miRNAs regulated by hypoxia in vitro where three miRNAs were differentially expressed in the circulation of patients with high-risk and aggressive rectal cancer. Furthermore, we investigated baseline characteristics of rectal cancer patients and identified two exosomal miRNAs predictive of metastasis in two cohorts. Finally, functional studies of EV-uptake and responses by monocytes supported that CRC-derived EVs could be mediators of immune responses by regulating monocyte gene transcription and cytokine release. Overall, the results contribute to increased knowledge of the cargo and functional role of CRC-derived EVs. Vesicle-associated differences in miRNA expressions can serve as markers for tumor hypoxia and metastasis, however, the findings must be validated in larger patient cohorts

Pro-survival responses to the dual inhibition of anti-apoptotic Bcl-2 family proteins and mTOR-mediated signaling in hypoxic colorectal carcinoma cells

Background The use of targeted agents to impel dual inhibition of anti-apoptotic mechanisms and mTOR-mediated pro-survival signaling in colorectal carcinoma (CRC) cell lines with KRAS or BRAF mutation has been shown to induce apoptosis, a timely result given CRC entities harboring such mutations are in need of new therapies. Since CRC comprises heterogeneous tumors with predominant hypoxic components, we investigated effects of an inhibitor of anti-apoptotic Bcl-2 family proteins (ABT-737) in combination with an mTOR inhibitor (AZD8055)—collectively referred to as combo-Rx, in hypoxic CRC cell lines. Methods Cell viability measures, expression of proteins implicated in apoptosis and MAPK/PI3K-AKT/mTOR pathway signaling, and profiling of composite kinase activities were undertaken in a panel of 14 cell lines. Results In hypoxic conditions, combo-Rx suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. Hypoxic KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis but with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. In contrast, in hypoxic KRAS-mutant LoVo (devoid of PIK3CA mutation), BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines (all with high endogenous Mcl-1 expression and being resistant to the additional effect of ABT-737 to AZD8055), combo-Rx did not elicit apoptotic or pro-survival responses. Conclusions The concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic KRAS/PIK3CA-mutant CRC cell lines resulted in pro-survival responses in parallel with the intended anti-proliferative effects, a finding that should be of note if considering combinatory targeting of multiple pathways in this CRC entity

An experimental strategy unveiling exosomal microRNAs 486-5p, 181a-5p and 30d-5p from hypoxic tumour cells as circulating indicators of high-risk rectal cancer

Tumour hypoxia contributes to poor treatment outcome in locally advanced rectal cancer (LARC) and circulating extracellular vesicles (EVs) as potential biomarkers of tumour hypoxia and adverse prognosis have not been fully explored. We examined EV miRNAs from hypoxic colorectal cancer cell lines as template for relevant miRNAs in LARC patients participating in a prospective biomarker study (NCT01816607). Five cell lines were cultured under normoxia (21% O2) or hypoxia (0.2% O2) for 24 h, and exosomes were isolated by differential ultracentrifugation. Using a commercial kit, exosomes were precipitated from 24 patient plasma samples collected at the time of diagnosis. Exosome size distribution and protein cargo were determined by cryo-electron microscopy, nanoparticle tracking analysis, immunoblotting and flow cytometry. The vesicles harboured strong cell line-specific miRNA profiles with 35 unique miRNAs differentially expressed between hypoxic and normoxic cells. Six of these miRNAs were considered candidate-circulating markers of tumour hypoxia in the patients based on the frequency or magnitude of variance in hypoxic versus normoxic cell line experiments and prevalence in patient plasma. Of these, low plasma levels of exosomal miR-486-5p and miR-181a-5p were associated with organ-invasive primary tumour (p = 0.029) and lymph node metastases (p = 0.024), respectively, both attributes of adverse LARC prognosis. In line with this, the plasma level of exosomal miR-30d-5p was elevated in patients who experienced metastatic progression (p = 0.036). Our strategy confirmed that EVs from colorectal cancer cell lines were exosomes containing the oxygen-sensitive miRNAs 486-5p, 181a-5p and 30d-5p, which were retrieved as circulating markers of high-risk LARC

Additional file 1: Fig. S1. of Pro-survival responses to the dual inhibition of anti-apoptotic Bcl-2 family proteins and mTOR-mediated signaling in hypoxic colorectal carcinoma cells

Cell viability of hypoxic colorectal carcinoma (CRC) cell lines. a. Four CRC cell lines were treated for 24 h with the indicated concentrations of ABT-737 (inhibitor of anti-apoptotic Bcl-2 family proteins) or AZD8055 (mTOR inhibitor) under normoxic or hypoxic culture conditions. Cell viability (measured by the MTS assay) value for each condition relative to the corresponding control cell (vehicle-treated) value is shown as mean ± SD from at least three independent experiments, each plated at least in triplicate. Statistically significant changes are indicated (asterisk, p < 0.05; cross, p < 0.01; circle, p < 0.001). b. Cultures of the CRC cell line were left in the hypoxic chamber at 0.2 % O2 and harvested after the indicated time periods. Expression of the hypoxia-inducible factor type 1α (HIF-1α) and its target gene carbonic anhydrase IX (CAIX) was examined by Western blot analysis, with α-tubulin expression as loading control, and with 24 h of normoxia and 4 h of 100 μM CoCl2 exposure in normoxia to generate negative and positive biological controls for HIF-1α expression, respectively. (DOCX 369 kb

Circulating Exosomal miR-141-3p and miR-375 in Metastatic Progression of Rectal Cancer

As many as 30% to 40% of locally advanced rectal cancer (LARC) patients experience metastatic progression of the disease. Recognizing the potential of the genetic cargo in tumor-derived exosomes, we hypothesized that plasma exosomal microRNA (miRNA) may reflect biological aggressiveness in LARC and provide new markers for rectal cancer aggressiveness and risk stratification. In a prospective LARC cohort (NCT01816607), plasma samples were collected from 29 patients at the time of diagnosis, before neoadjuvant therapy and surgery. Exosomes, precipitated from plasma using a commercial kit, were verified by cryo-electron microscopy, nanoparticle tracking analysis, and western blotting. Expression of exosomal miRNAs was profiled using a miRCURY LNA miRNA microarray and validation of six miRNAs associated with pathological and clinical end-points was undertaken in plasma collected at the time of diagnosis from 64 patients in an independent prospective LARC cohort (NCT00278694). In both cohorts, exosomal miR-141-3p and miR-375 were higher in patients with synchronous liver metastasis than in those without (P = .010 and P = .017 respectively in the investigative cohort, and P < .001 for both in the validation cohort). Further, high exosomal miR-141-3p was associated with post-operative metastatic liver progression in the investigative cohort (P = .034). Because both miRNAs are associated with tumor angiogenesis and immune modulation, we propose that these miRNAs in circulating exosomes may reflect rectal cancer aggressiveness and accordingly be candidate biomarkers for further investigations

Additional file 4: Table S4. of Pro-survival responses to the dual inhibition of anti-apoptotic Bcl-2 family proteins and mTOR-mediated signaling in hypoxic colorectal carcinoma cells

Tyrosine Kinase PamChip® Array substrates—phosphorylation levels. The color map visualizes normalized log2-transformed signal intensities from kinase substrate arrays incubated with lysates from the three colorectal carcinoma cell lines treated for 24 h with ABT-737 (inhibitor of anti-apoptotic Bcl-2 family proteins; 10 μM), AZD8055 (mTOR inhibitor; 10 μM), or combo-Rx (10 μM of both compounds in combination). Red corresponds to higher and blue to lower substrate phosphorylation levels relative to levels from the corresponding control cells. a Retrieved from UniProtKB/SwissProt ( http://www.uniprot.org/ ). b Position(s) of the tyrosine phosphorylation site(s) within the protein. c Retrieved from PathCard ( http://pathcards.genecards.org/ ). Super-pathway definitions were ‘PI3K-AKT signaling pathway’ and ‘MAPK signaling pathway’. (DOC 536 kb

Circulating Exosomal miR-141-3p and miR-375 in Metastatic Progression of Rectal Cancer

As many as 30% to 40% of locally advanced rectal cancer (LARC) patients experience metastatic progression ofthe disease. Recognizing the potential of the genetic cargo in tumor-derived exosomes, we hypothesized thatplasma exosomal microRNA (miRNA) may reflect biological aggressiveness in LARC and provide new markers forrectal cancer aggressiveness and risk stratification. In a prospective LARC cohort (NCT01816607), plasma sampleswere collected from 29 patients at the time of diagnosis, before neoadjuvant therapy and surgery. Exosomes,precipitated from plasma using a commercial kit, were verified by cryo-electron microscopy, nanoparticle trackinganalysis, and western blotting. Expression of exosomal miRNAs was profiled using a miRCURY LNA miRNAmicroarray and validation of six miRNAs associated with pathological and clinical end-points was undertaken inplasma collected at the time of diagnosis from 64 patients in an independent prospective LARC cohort(NCT00278694). In both cohorts, exosomal miR-141-3p and miR-375 were higher in patients with synchronousliver metastasis than in those without (P= .010 andP= .017 respectively in the investigative cohort, andPb.001for both in the validation cohort). Further,high exosomal miR-141-3p was associated with post-operativemetastatic liver progression in the investigative cohort (P= .034). Because both miRNAs are associated withtumor angiogenesis and immune modulation, we propose that these miRNAs in circulating exosomes may reflectrectal cancer aggressiveness and accordingly be candidate biomarkers for further investigations