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Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor.

Simple Summary: Colorectal cancer (CRC) belongs to the top three most common malignancies and is one of the deadliest cancers worldwide. Advancements in the understanding of CRC pathophysiology can lead to the development of novel treatments preventing cancer progression while prolonging overall survival. Numerous studies have shown a role for the biological clock in the regulation of cancer hallmarks and in CRC. However, the mechanistic link between the circadian clock and CRC progression is not fully understood. In the current study, we aimed to investigate the effects of a genetically disrupted clock on cancer properties using different CRC cell lines, with a focus on metastasis-related components. Our results demonstrate a reciprocal interplay between the circadian clock and the metastasis associated gene MACC1 (metastasis-associated in colon cancer 1), pointing to the circadian clock-regulation of CRC invasiveness. A circadian MACC1 expression, as shown by our data, may be considered to optimize MACC1-targeted CRC treatment. Abstract: The circadian clock coordinates the timing of several cellular processes including transcription, the cell cycle, and metabolism. Disruptions in the clock machinery trigger the abnormal regulation of cancer hallmarks, impair cellular homeostasis, and stimulate tumourigenesis. Here we investigated the role of a disrupted clock by knocking out or knocking down the core-clock (CC) genes ARNTL, PER2 or NR1D1 in cancer progression (e.g., cell proliferation and invasion) using colorectal cancer (CRC) cell lines HCT116, SW480 and SW620, from different progression stages with distinct clock phenotypes, and identified mechanistic links from the clock to altered cancer-promoting cellular properties. We identified MACC1 (metastasis-associated in colon cancer 1), a known driver for metastasis and an EMT (epithelial-to-mesenchymal transition)-related gene, to be significantly differentially expressed in CC manipulated cells and analysed the effect of MACC1 manipulation (knockout or overexpression) in terms of circadian clock phenotype as well as cancer progression. Our data points to a bi-directional MACC1-circadian clock interplay in CRC, via CC genes. In particular, knocking out MACC1 reduced the period of oscillations, while its overexpression increased it. Interestingly, we found the MACC1 protein to be circadian expressed in HCT116 WT cells, which was disrupted after the knockout of CC genes, and identified a MACC1-NR1D1 protein-protein interaction. In addition, MACC1 manipulation and CC knockout altered cell invasion properties of HCT116 cells, pointing to a regulation of clock and cancer progression in CRC, possibly via the interaction of MACC1 with core-clock genes.

Additional file 14: Supplemental Results. Estimation of cell-cell adhesion from 3D aggregate formation.

Treatment failure of solid cancers, represented by the development of drug resistance in the primary tumor or later outgrowth of drug resistant metastases, is the major cause of death for cancer patients. It represents an urgent clinical need for predictive biomarkers which indicate the success or failure of standard treatment regimens. Besides treatment prediction, interfering with cellular processes associated with drug resistance might improve treatment response by applying combination therapies. Metastasis-associated in colon cancer (MACC) 1 was identified in our group as a prognostic biomarker in human colorectal cancer, and has been established as key player, prognostic, and predictive biomarker for tumor progression and metastasis in a variety of solid cancers. Besides increased cell proliferation and motility, subsequently contributing to growth and metastatic spread of the primary tumor, MACC1 has also been shown to dysregulate apoptosis and is contributing to treatment resistance. Here we report the MACC1 dependent treatment resistance of colorectal cancer (CRC) cells to standard therapeutics like doxorubicin by upregulating ATP-binding cassette subfamily B member 1 (ABCB1) protein. Overexpression of MACC1 in CRC cells increased both its presence on the ABCB1 promoter and its transcriptional activity, resulting in elevated ABCB1 expression and thus treatment resistance to standard therapeutics. In contrast, depleting MACC1 increased intracellular drug concentrations, leading to better treatment response. We already identified the first MACC1 transcriptional inhibitors, such as lovastatin, by high-throughput screening of clinically approved small molecule drugs. These compounds inhibited cell motility in vitro but also restricted metastasis development in xenograft mouse models by reducing MACC1 expression. Here we report, that treating high MACC1 expressing CRC cells with a combination of statins and standard therapeutics increased the rate of cytotoxicity and resulted in higher treatment response.

Glioblastoma multiforme is the most aggressive primary brain tumor of adults, but lacks reliable and liquid biomarkers. We evaluated circulating plasma transcripts of metastasis-associated in colon cancer-1 (MACC1), a prognostic biomarker for solid cancer entities, for prediction of clinical outcome and therapy response in glioblastomas. MACC1 transcripts were significantly higher in patients compared to controls. Low MACC1 levels clustered together with other prognostically favorable markers. It was associated with patients' prognosis in conjunction with the isocitrate dehydrogenase (IDH) mutation status: IDH1 R132H mutation and low MACC1 was most favorable (median overall survival (OS) not yet reached), IDH1 wildtype and high MACC1 was worst (median OS 8.1 months), while IDH1 wildtype and low MACC1 was intermediate (median OS 9.1 months). No patients displayed IDH1 R132H mutation and high MACC1. Patients with low MACC1 levels receiving standard therapy survived longer (median OS 22.6 months) than patients with high MACC1 levels (median OS 8.1 months). Patients not receiving the standard regimen showed the worst prognosis, independent of MACC1 levels (low: 6.8 months, high: 4.4 months). Addition of circulating MACC1 transcript levels to the existing prognostic workup may improve the accuracy of outcome prediction and help define more precise risk categories of glioblastoma patients.

Die Steuerung biochemischer Reaktionen und die Lokalisierung von Proteinen erfolgt in der Zelle oft über Signalmoleküle. Neben Phosphorylierung, Methylierung und Acetylierung von Ami­ nosäureresten, werden zelluläre Prozesse durch eine Modifikation der beteiligten Faktoren mit Ubiquitin oder Ubiquitin-ähnlichen Proteinen reguliert. Die gemeinsame Grundlage dieser Prote­ infamilie stellt die Faltung des gesamten Proteins oder einer seiner Domänen dar. Ubl5 ist ein Mit­ glied dieser Familie, dessen Homologe in eukaryontischen Lebewesen stark konserviert sind. Ab­ gesehen von der strukturellen Ähnlichkeit mit Ubiquitin besitzt es wenig Übereinstimmung in der Aminosäuresequenz und wird, soweit bekannt, nicht kovalent an Zielproteine gebunden. In dieser Arbeit wurde das homologe Protein der Bäckerhefe Hub1, auf dessen Interaktionspartner sowohl auf genetischer als auch auf Proteinebene untersucht. So konnten eine synthetisch letale Interak­ tion zwischen Hub1 und einer P1484L-Mutation im Splicing-Faktor PRP8, sowie weitere genetische Interaktionen nicht-essentieller Komponenten des Spliceosoms und des prä-mRNS- Retentions­ systems charakterisiert werden. Sowohl die Prp8-Mutante, als auch die Deletion von Hub1 führen zu Splicing-Defekten von Introns mit ungewöhnlichen 5'-Spleißstellen. In Zellen ohne Hub1 wur­ de zusätzlich ein Anstieg der aus dem Kern exportierten, ungespleißten prä-mRNS festgestellt.Da bisher keine Informationen über funktionale Aminosäurereste von Hub1 bekannt waren, wurden durch PCR-basierte Mutagenese Hub1-Mutanten generiert und deren Funktion durch die charakterisierten genetischen Interaktionen (in vivo), als auch durch die Bindung an Snu66 (in vi­ tro) überprüft. Dabei konnten Bereiche von Hub1 festgestellt werden, bei denen Mutationen ent­ weder die Bindung an Snu66 störten, aber für die Komplementation der genetischen Interaktion nicht essentiell waren, und umgekehrt. Hub1 beeinflusst demnach die Effizienz, mit der bestimmte Transkripte gespleißt und im Zell­ kern zurückgehalten werden. Beide Mechanismen sind auf die korrekte Erkennung der 5'-Spleiß­ stelle angewiesen, wodurch eine Funktion von Hub1 bei diesem Prozess vermutet werden kann., Biochemical reactions and localization of proteins or other cellular factors are often regulated by signaling molecules. In addition to phosphorylation, methylation or acetylation of amino acid residues, modification of proteins with ubiquitin or ubiquitin-like modifiers is a key process of regulation. This protein family is characterized by a similar structural fold of either the whole protein or one of their domains. Ubl5 is a member of this protein family, that is highly conserved within eucaryotes. Aside from its tertiary structure, it shares only weak similarity with ubiquitin and seems not to be attached covalently to other proteins. The focus of this work was the S. cere­ visiae homologue Hub1 and its physical and genetic interactions. The deletion of Hub1 leads to genetic interactions with several splicing factors, including a P1384L mutation in Prp8 and members of the U4/U6.U5 tri-snRNP and the complexes containing Prp19 and Cef1. Moreover, the deletion of Hub1 also leads to an increased leakage of unspliced pre-mRNA to the cytoplasm. Since there was no information available, which amino acid residues of Hub1 were essential for its function, a set of Hub1 mutants with substitutions of single amino acids was generated and tested for their protein stability, their ability to bind the only known direct interaction partner Snu66 and their function to complement the genetic interactions. Distinct areas at the surface of Hub1 were found, that showed reduced binding of Snu66 in vitro, but kept their function to com­ plement the genetic interactions, and vice versa. The deletion of Hub1 showed a decrease in the splicing efficiency of certain transcripts and their retention in the nucleus. Both mechanisms depend on the correct identification of the 5'- splice site and Hub1 may have a distinct function in this process.