Proteomics uncover EPHA2 in cetuximab resistant colorectal cancer cell lines as a potential novel therapeutic target

Colorectal cancer (CRC) affects more than 50 000 people yearly in Germany alone, with one fifth of patients presenting with UICC stage IV, metastatic disease (mCRC). These patients are typically treated with polychemotherapy combined with targeted anti-EGFR, anti-BRAF or anti-VEGF targeted therapies. Patients with left-sided mCRC and KRAS, NRAS and BRAF wildtype disease are typically treated with double chemotherapy (FOLFOX or FOLFIRI) combined with the anti-EGFR targeted monoclonal antibodies cetuximab (CET) or panitumumab. Despite the intensive therapy regimens, 5-year survival rates in metastatic disease are as low as 16%, owing to primary treatment resistance or the rapid development of acquired (or secondary) treatment resistance. Both primary and secondary resistance to anti-EGFR therapies, such as CET, have been linked to alterations in the RAS genes (mainly KRAS and NRAS), as these cause self-sufficient mitogenic signalling. RAS small GTPase proteins sit at a crossroad of multiple signalling pathways and constitutively active RAS signalling affects many cellular functions besides mitogenic signalling, making it difficult to assess the full biological implications of oncogenic RAS signalling. This study therefore used mass-spectrometric based proteomics to identify kinome reprogramming in mCRC cell lines with KRAS associated, acquired CET resistance in order to investigate the molecular implications of RAS oncogenic signalling and uncover potential second-line therapy options to overcome acquired CET resistance. The cell line-based model of acquired CET resistance used in this study, comprising two CET sensitive parental cell lines and four isogenic CET resistant cell lines, was characterised by significant changes in the kinome associated with resistance, most of which were specific to individual cell lines. Chemical proteomics identified Ephrin type-A receptor 2 (EPHA2) overexpression as a common feature in all CET resistant cell lines. These resistant cells displayed significantly stronger migration than their parental counterparts (p<0,01) and EPHA2 was found to be a molecular driver of cell migration. Migration rates could be significantly reduced by targeting EPHA2 using RNA interference (RNAi) (p<0,001), ligand stimulation (p<0,001), tyrosine kinase inhibitor treatment (p<0,01) and anti-EPHA2 antibody treatment (p<0,001). These results identify EPHA2 as a potential actionable target for second-line targeted therapies in CET resistant mCRC. This study supported by recent findings emphasises the potential role of EPHA2 as a biomarker and target for precision medicine approaches in mCRC.In Deutschland erkranken jährlich über 50 000 Menschen am kolorektalen Karzinom (KRK), und etwa jede fünfte Erkrankung manifestiert sich im metastasierten Stadium (UICC Stadium IV). Die Behandlung des metastasiertem KRK (mKRK) umfasst typischerweise eine Polychemotherapie in Kombination mit einer zielgerichteten anti-EGFR, anti-BRAF oder anti-VEGF zielgerichteter Behandlung. Patient*innen mit linksseitigem mKRK und KRAS, NRAS und BRAF Wildtyp werden typischerweise mit einer Chemotherapie Doublette (FOLFOX oder FOLFIRI) und einem der monoklonalen anti-EGFR Antikörper Cetuximab (CET) oder Panitumumab behandelt. Trotz intensiver Therapie beträgt das 5-Jahres Überleben im metastasierten Stadium lediglich 16%, geschuldet durch das Vorhandensein von primäre Resistenzmechanismen oder durch die rasche Entwicklung von sekundären Resistenzmechanismen. Sowohl primäre als auch sekundäre Resistenzen gegen anti-EGFR Therapeutika werden durch Alterationen der RAS-Gene (hauptsächlich KRAS und NRAS) stark begünstigt. Onkogenes RAS hält in Krebszellen mitotische Signalwege aufrecht. Diese kleine GTPasen sitzen an einem molekularen Schaltwerk verschiedener Signalwege und ihre konstitutive Aktivierung beeinflusst viele weitere zelluläre Funktionen als nur mitotische Signalwege, sodass die genaue Wirkung von onkogenem RAS in Krebszellen schwer zu beurteilen ist. Daher untersuchte dieses Forschungsprojekt Änderungen des Kinoms in mKRK-Zelllinien mit sekundärer, KRAS assoziierter CET-Resistenz, um die molekulare Auswirkung von onkogenem RAS zu beurteilen und um potenzielle alternative Zielstrukturen für zielgerichtete Therapien zu finden. Das in diesem Projekt benützte Zelllinien-basierte Model sekundärer CET-Resistenz bestand aus zwei CET-sensitiven parentalen- und vier isogenen CET-resistenten Zelllinien. CET-resistente Zellen wiesen eine stark individuelle Alteration des Kinoms auf. Eine Gemeinsamkeit aller resistenten Zelllinien war die Überexpression von Ephrin Typ-A Rezeptor 2 (EPHA2). Resistente Zellen migrierten signifikant stärker als parentale Zellen (p<0,01) und EPHA2 konnte als molekularer Treiber zellulärer Migration identifiziert werden, denn die Migrationsrate konnte durch spezifisches Eingreifen in den EPHA2 Signalweg signifikant reduziert werden. Dies konnte durch RNA-Interferenz (RNAi) (p<0,001), Ligandenstimulation (p<0,001), Tyrosinkinaseinhibition (p<0,01) und anti-EPHA2 Antikörperbehandlung (p<0,001) erzielt werden. Diese Ergebnisse identifizieren EPHA2 als potentiell spezifisches Ziel für eine zielgerichtete Therapie in CET-resistentem mKRK. Dieses Projekt, gestützt durch aktuelle wissenschaftliche Erkenntnisse, zeigt die Rolle von EPHA2 als Biomarker und potenzielles Ziel für eine zielgerichtete Behandlungsstrategie in Patient*innen mit mKRK auf

Meta-analysis: the diagnostic accuracy of critical flicker frequency in minimal hepatic encephalopathy

BACKGROUND: Minimal hepatic encephalopathy (MHE) reduces quality of life, increases the risk of road traffic incidents and predicts progression to overt hepatic encephalopathy and death. Current psychometry-based diagnostic methods are effective, but time-consuming and a universal ‘gold standard’ test has yet to be agreed upon. Critical Flicker Frequency (CFF) is a proposed language-independent diagnostic tool for MHE, but its accuracy has yet to be confirmed. AIM: To assess the diagnostic accuracy of CFF for MHE by performing a systematic review and meta-analysis of all studies, which report on the diagnostic accuracy of this test. METHODS: A systematic literature search was performed to locate all publications reporting on the diagnostic accuracy of CFF for MHE. Data were extracted from 2 × 2 tables or calculated from reported accuracy data. Collated data were meta-analysed for sensitivity, specificity, diagnostic odds ratio (DOR) and summary receiver operator curve (sROC) analysis. Prespecified subgroup analysis and meta-regression were also performed. RESULTS: Nine studies with data for 622 patients were included. Summary sensitivity was 61% (95% CI: 55–67), specificity 79% (95% CI: 75–83) and DOR 10.9 (95% CI: 4.2–28.3). A symmetrical sROC gave an area under the receiver operator curve of 0.84 (SE = 0.06). The heterogeneity of the DOR was 74%. CONCLUSIONS: Critical Flicker Frequency has a high specificity and moderate sensitivity for diagnosing minimal hepatic encephalopathy. Given the advantages of language independence and being both simple to perform and interpret, we suggest the use of critical flicker frequency as an adjunct (but not replacement) to psychometric testing

Proteomics uncover EPHA2 as a potential novel therapeutic target in colorectal cancer cell lines with acquired cetuximab resistance.

BACKGROUND: In metastatic colorectal cancer (mCRC), acquired resistance against anti-EGFR targeted monoclonal antibodies, such as cetuximab (CET), was shown to be frequently caused by activating alterations in the RAS genes KRAS or NRAS. To this day, no efficient follow-up treatment option has emerged to treat mCRC in such a setting of resistance. METHODS: To uncover potential targets for second-line targeted therapies, we used mass-spectrometric proteomics to shed light on kinome reprogramming in an established cellular model of acquired, KRAS-associated CET resistance. RESULTS: This CET resistance was reflected by significant changes in the kinome, most of them individual to each cell line. Interestingly, all investigated resistant cell lines displayed upregulation of the Ephrin type-A receptor 2 (EPHA2), a well-known driver of traits of progression. Expectedly resistant cell lines displayed increased migration (p < 0.01) that was significantly reduced by targeting the EPHA2 signalling axis using RNA interference (RNAi) (p < 0.001), ephrin-A1 stimulation (p < 0.001), dasatinib (p < 0.01), or anti-EPHA2 antibody treatment (p < 0.001), identifying it as an actionable target in mCRC with acquired CET resistance. CONCLUSION: These results highlight EPHA2 and its role in mCRC with KRAS-gene mutated acquired CET resistance and support its use as a potential actionable target for the development of future precision medicine therapies

Willkommen in München: a HyperCard database for use in teaching German

Willkommen in München: a HyperCard database for use in teaching Germa

Subchondral Bone Plate Thickening Precedes Chondrocyte Apoptosis and Cartilage Degradation in Spontaneous Animal Models of Osteoarthritis

Osteoarthritis (OA) is the most common joint disorder characterised by bone remodelling and cartilage degradation and associated with chondrocyte apoptosis. These processes were investigated at 10, 16, 24, and 30 weeks in Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs that develop OA spontaneously. Both strains had a more pronounced chondrocyte apoptosis, cartilage degradation, and subchondral bone changes in the medial than the lateral side of the tibia, and between strains, the changes were always greater and faster in DH than BS2. In the medial side, a significant increase of chondrocyte apoptosis and cartilage degradation was observed in DH between 24 and 30 weeks of age preceded by a progressive thickening and stiffening of subchondral bone plate (Sbp). The Sbp thickness consistently increased over the 30-week study period but the bone mineral density (BMD) of the Sbp gradually decreased after 16 weeks. The absence of these changes in the medial side of BS2 may indicate that the Sbp of DH was undergoing remodelling. Chondrocyte apoptosis was largely confined to the deep zone of articular cartilage and correlated with thickness of the subchondral bone plate suggesting that cartilage degradation and chondrocyte apoptosis may be a consequence of continuous bone remodelling during the development of OA in these animal models of OA