NTSR1 glycosylation and MMP dependent cleavage generate three distinct forms of the protein.

NTSR1 abnormal expression by cancer cells makes it a strategic target for antitumoral therapies, such as compounds that use NTSR1 binding probes to deliver cytotoxic agents to tumor cells. Success of these therapies relies on NTSR1 protein availability and accessibility; therefore, understanding the protein's biology is crucial. We studied NTSR1 protein in exogenously and endogenously expressing non-tumoral and tumoral cells. We found NTSR1 to be expressed as three distinct protein forms: the NTSR1-high form, a glycosylated protein; the NTSR1-low form, a N-terminally cleaved and de-glycosylated protein; and the NTSR1-LP protein with the MW size predicted by its NTSR1 amino acid sequence. We show that the NTSR1-high form is cleaved by MMPs to generate the NTSR1-low form, a process that is promoted by the Neurotensin (NTS) ligand. In addition, NTS induced the internalization of plasma membrane localized NTSR1 and degradation of NTSR1-low form via the proteasome. Importantly, we found NTSR1-low form to be the most abundant form in the tumoral cells and in PDAC Patient Derived Xenograft, demonstrating its physiopathological relevance. Altogether, our work provides important technical and experimental tools as well as new crucial insights into NTSR1 protein biology that are required to develop clinically relevant NTSR1 targeting anti-tumoral therapies.

ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinomas.

The metastatic progression of cancer is a multi-step process initiated by the local invasion of the peritumoral stroma. To identify the mechanisms underlying colorectal carcinoma (CRC) invasion, we collected live human primary cancer specimens at the time of surgery and monitored them ex vivo. This revealed that conventional adenocarcinomas undergo collective invasion while retaining their epithelial glandular architecture with an inward apical pole delineating a luminal cavity. To identify the underlying mechanisms, we used microscopy-based assays on 3D organotypic cultures of Caco-2 cysts as a model system. We performed two siRNA screens targeting Rho-GTPases effectors and guanine nucleotide exchange factors. These screens revealed that ROCK2 inhibition triggers the initial leader/follower polarization of the CRC cell cohorts and induces collective invasion. We further identified FARP2 as the Rac1 GEF necessary for CRC collective invasion. However, FARP2 activation is not sufficient to trigger leader cell formation and the concomitant inhibition of Myosin-II is required to induce invasion downstream of ROCK2 inhibition. Our results contrast with ROCK pro-invasive function in other cancers, stressing that the molecular mechanism of metastatic spread likely depends on tumour types and invasion mode.

Tumour spheres with inverted polarity drive the formation of peritoneal metastases in patients with hypermethylated colorectal carcinomas.

Metastases account for 90% of cancer-related deaths; thus, it is vital to understand the biology of tumour dissemination. Here, we collected and monitored >50 patient specimens ex vivo to investigate the cell biology of colorectal cancer (CRC) metastatic spread to the peritoneum. This reveals an unpredicted mode of dissemination. Large clusters of cancer epithelial cells displaying a robust outward apical pole, which we termed tumour spheres with inverted polarity (TSIPs), were observed throughout the process of dissemination. TSIPs form and propagate through the collective apical budding of hypermethylated CRCs downstream of canonical and non-canonical transforming growth factor-ß signalling. TSIPs maintain their apical-out topology and use actomyosin contractility to collectively invade three-dimensional extracellular matrices. TSIPs invade paired patient peritoneum explants, initiate metastases in mice xenograft models and correlate with adverse patient prognosis. Thus, despite their epithelial architecture and inverted topology TSIPs seem to drive the metastatic spread of hypermethylated CRCs.

KIF17 regulates RhoA-dependent actin remodeling at epithelial cell-cell adhesions.

The kinesin KIF17 localizes at microtubule plus-ends where it contributes to regulation of microtubule stabilization and epithelial polarization. We now show that KIF17 localizes at cell-cell adhesions and that KIF17 depletion inhibits accumulation of actin at the apical pole of cells grown in 3D organotypic cultures and alters the distribution of actin and E-cadherin in cells cultured in 2D on solid supports. Overexpression of full-length KIF17 constructs or truncation mutants containing the N-terminal motor domain resulted in accumulation of newly incorporated GFP-actin into junctional actin foci, cleared E-cadherin from cytoplasmic vesicles and stabilized cell-cell adhesions to challenge with calcium depletion. Expression of these KIF17 constructs also increased cellular levels of active RhoA, whereas active RhoA was diminished in KIF17-depleted cells. Inhibition of RhoA or its effector ROCK, or expression of LIMK1 kinase-dead or activated cofilin(S3A) inhibited KIF17-induced junctional actin accumulation. Interestingly, KIF17 activity toward actin depends on the motor domain but is independent of microtubule binding. Together, these data show that KIF17 can modify RhoA-GTPase signaling to influence junctional actin and the stability of the apical junctional complex of epithelial cells.