FOXC2 promotes vasculogenic mimicry and resistance to anti-angiogenic therapy.

Vasculogenic mimicry (VM) describes the formation of pseudo blood vessels constructed of tumor cells that have acquired endothelial-like properties. VM channels endow the tumor with a tumor-derived vascular system that directly connects to host blood vessels, and their presence is generally associated with poor patient prognosis. Here we show that the transcription factor, Foxc2, promotes VM in diverse solid tumor types by driving ectopic expression of endothelial genes in tumor cells, a process that is stimulated by hypoxia. VM-proficient tumors are resistant to anti-angiogenic therapy, and suppression of Foxc2 augments response. This work establishes co-option of an embryonic endothelial transcription factor by tumor cells as a key mechanism driving VM proclivity and motivates the search for VM-inhibitory agents that could form the basis of combination therapies with anti-angiogenics.

Combining long-term circuit mapping and network transcriptomics with SiR-N2c.

An exciting frontier in circuit neuroscience lies at the intersection between neural network mapping and single-cell genomics. Monosynaptic rabies viruses provide a promising platform for the merger of circuit mapping methods with -omics approaches. However, three key limitations have hindered the extraction of physiologically meaningful gene expression profiles from rabies-mapped circuits: inherent viral cytotoxicity, high viral immunogenicity and virus-induced alteration of cellular transcriptional regulation. These factors alter the transcriptional and translational profiles of infected neurons and their neighboring cells. To overcome these limitations we applied a self-inactivating genomic modification to the less immunogenic rabies strain, CVS-N2c, to generate a self-inactivating CVS-N2c rabies virus (SiR-N2c). SiR-N2c not only eliminates undesired cytotoxic effects but also substantially reduces gene expression alterations in infected neurons and dampens the recruitment of innate and acquired immune responses, thus enabling open-ended interventions on neural networks and their genetic characterization using single-cell genomic approaches.

Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System.

PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.