ATF2 loss promotes tumor invasion in colorectal cancer cells via upregulation of cancer driver TROP2.

In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.

Semantic clustering analysis of E3-ubiquitin ligases in gastrointestinal tract defines genes ontology clusters with tissue expression patterns.

BACKGROUND: Ubiquitin ligases (Ub-ligases) are essential intracellular enzymes responsible for the regulation of proteome homeostasis, signaling pathway crosstalk, cell differentiation and stress responses. Individual Ub-ligases exhibit their unique functions based on the nature of their substrates. They create a complex regulatory network with alternative and feedback pathways to maintain cell homeostasis, being thus important players in many physiological and pathological conditions. However, the functional classification of Ub-ligases needs to be revised and extended. METHODS: In the current study, we used a novel semantic biclustering technique for expression profiling of Ub-ligases and ubiquitination-related genes in the murine gastrointestinal tract (GIT). We accommodated a general framework of the algorithm for finding tissue-specific gene expression clusters in GIT. In order to test identified clusters in a biological system, we used a model of epithelial regeneration. For this purpose, a dextran sulfate sodium (DSS) mouse model, following with in situ hybridization, was used to expose genes with possible compensatory features. To determine cell-type specific distribution of Ub-ligases and ubiquitination-related genes, principal component analysis (PCA) and Uniform Manifold Approximation and Projection technique (UMAP) were used to analyze the Tabula Muris scRNA-seq data of murine colon followed by comparison with our clustering results. RESULTS: Our established clustering protocol, that incorporates the semantic biclustering algorithm, demonstrated the potential to reveal interesting expression patterns. In this manner, we statistically defined gene clusters consisting of the same genes involved in distinct regulatory pathways vs distinct genes playing roles in functionally similar signaling pathways. This allowed us to uncover the potentially redundant features of GIT-specific Ub-ligases and ubiquitination-related genes. Testing the statistically obtained results on the mouse model showed that genes clustered to the same ontology group simultaneously alter their expression pattern after induced epithelial damage, illustrating their complementary role during tissue regeneration. CONCLUSIONS: An optimized semantic clustering protocol demonstrates the potential to reveal a readable and unique pattern in the expression profiling of GIT-specific Ub-ligases, exposing ontologically relevant gene clusters with potentially redundant features. This extends our knowledge of ontological relationships among Ub-ligases and ubiquitination-related genes, providing an alternative and more functional gene classification. In a similar way, semantic cluster analysis could be used for studding of other enzyme families, tissues and systems.

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.

Bispecific antibody neutralizes circulating SARS-CoV-2 variants, prevents escape and protects mice from disease.

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19) 1,2 . We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C135 3 . CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 neutralizes SARS-CoV-2 and its variants of concern, as well as the escape mutants generated by the parental monoclonals. In a novel animal model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.