The MACC1 Dimerization and its Function in Tumor Metastasis

Colorectal cancer, a highly heterogeneous cancer, continues to be a leading cause of mortality worldwide. While the 5-year survival rates for patients with Stage I and II are high, there has been little or no improvement of survival for patients with metastases. To make matters worse, 20% of the patients already present with metastasis at the time of diagnosis. Therefore, early detection of patients who are at high risk of developing metastases using biomarkers is key to improving patient survival. Metastasis-associated in colon cancer 1 (MACC1) is one such biomarker that has been directly linked to metastasis development, reduced survival, and worse overall outcomes. In addition to identifying high-risk patients, MACC1 is biologically linked to tumor and metastasis development. Specifically, the MACC1 structure contains diverse domains and several tyrosine sites capable of versatile interactions. Therefore, the aim of the first part of the project was to study the role of tyrosine sites close to the N-terminus of MACC1. Employing computational tyrosine phosphorylation prediction tools, site Tyr379 and SRC kinase as one of the promising kinases responsible for its phosphorylation were identified. Preliminary examination reveals an association between MACC1 and SRC. Despite extensive evidence describing the functional diversity of MACC1, little is known about the structural features and self-association property of MACC1. To address this gap in knowledge, the goal of the second part of this project was to systematically evaluate the structural properties of MACC1 and the self-association capability of MACC1. Using AlphaFold2, the structures of MACC1 and MACC1 dimer were revealed. Val212, Ileu214, and Cys216 present in the ZU5 domain of MACC1 were found to be critical for dimerization. The knowledge gained from the AI prediction was transferred to set up a bioluminescence resonance energy transfer (BRET) assay to analyze MACC1 dimerization and the effect of mutation on dimerization. In addition to validating the presence of MACC1 dimer in living cells, the BRET assay confirmed reduced MACC1 self-association when the above residues were mutated. Ultimately, the impact of these mutations on MACC1 signaling and metastasis properties was verified using an in vitro metastasis assay. In summary, these results shed new light on the MACC1 structural characteristics particularly the presence of MACC1 homodimer, and reveal the residues important for dimerization, thus providing a framework for future development of intervention strategies

Biomarker based therapies in high risk cancer patients - MACC1 as molecular target

Das metastasierende kolorektale Karzinom stellt eine große Herausforderung in der Krebstherapie dar. Verlässliche und effiziente Biomarker zur Prognose des Krankheitsverlaufes oder der Therapieantwort (Prädiktion) sind rar. Metastasis-associated in colon cancer 1 (MACC1) ist ein prognostischer, prädiktiver und kausaler Biomarker für verschiedene Tumorentitäten. Durch die Induzierung von Zielgenen, wie z.B. MET, beeinflusst es Signalwege wie MEK/ERK und AKT/β-catenin und fördert so Zellproliferation und -motilität sowie Tumorprogression und Metastasierung in vivo. Diese Arbeit sollte neue Strategien erforschen diese Prozesse durch die Inhibition von MACC1 auf Transkriptions- und Signaltransduktionsebene zu unterbinden. Mit zwei verschiedenen Screeningmethoden konnten wir Statine als potente transkriptionelle Inhibitoren von MACC1 als auch phosphotyrosin (pY)-abhängige Interaktionen von MACC1 mit essentiellen Signalmolekülen identifizieren: SHP2, GRB2, SHC1, PLCG1 und STAT5B. Statine verringerten MACC1-spezifische Proliferation und Koloniebildung in vitro als auch Tumor Wachstum und Metastasierung in vivo bei Dosen äquivalent der humanen Standardtherapie zur Blutlipidsenkung. Mutation der pY-Bindungsstellen reduzierte die Aktivität des MACC1-induzierten ERK Signalwegs sowie Zellmigration und -proliferation. Anhand unserer Daten orchestriert MACC1, abhängig von MET und EGFR, neue SHP2/SRC/ERK und PKA/SRC/CREB Signalkaskaden zu einem malignen Phänotyp. Gezielte Intervention restringierte die MACC1-abhängige Koloniebildung, was neue therapeutische Interventionspunkte identifiziert und eine hervorragende Basis für Untersuchungen zur Kombinationstherapie darstellt. Die weitere Erforschung der spatiotemporalen Organisation des MACC1 Signalosoms und assoziierter Signalkaskaden soll das volle therapeutische Potential von MACC1 ausschöpfen. Wir empfehlen zudem Statine in der Krebstherapie bzw. -prävention, besonders bei MACC1-stratifzierten Patienten, anzuwenden.Metastatic colorectal cancer still represents a major challenge in therapy. Reliable and efficient biomarkers for early prognosis of disease course or treatment response (prediction) remain scarce. Metastasis-associated in colon cancer 1 (MACC1) has been established as prognostic, predictive and causal biomarker for several tumor entities. Its induction of target genes such as MET affects several signaling pathways including MEK/ERK and AKT/β-catenin. Thus, it promotes cellular proliferation and motility as well as tumor progression and metastasis formation in vivo. This study intended to explore new strategies to inhibit these processes by targeting MACC1 on transcriptional and signaling level. By two distinct screening methods, we identified statins as potent MACC1 transcriptional inhibitors as well as phosphotyrosine (pY)-dependent interactions of MACC1 with crucial signaling molecules: SHP2, GRB2, SHC1, PLCG1 and STAT5B. Statins showed MACC1-specific reduction of proliferation and colony formation in vitro as well as restriction of tumor growth and metastasis formation in vivo at doses equivalent to human standard lipid reduction therapy. Mutation of the pY-interaction sites abrogated MACC1-dependent ERK signaling as well as cell migration and proliferation. Our data further suggest that MACC1 governs SHP2/SRC/ERK and PKA/SRC/CREB axes conferring a malignant phenotype in response to MET and EGFR. Targeted intervention restricted MACC1-dependent colony formation which indicates new drug intervention points for MACC1 signaling and provides an excellent baseline for further investigations of combinatorial treatments. Additional research about the spatiotemporal organization of MACC1 signalosome formation and downstream signaling will reveal the entire potential of MACC1 as therapeutic target, whereas statins should already be considered for cancer therapy or prevention, especially in patients stratified for MACC1 expression