ST14 is up-regulated and its cognate ST14 inhibitor Hai1 is down-regulated in multiple human carcinomas. However, no efficient clinically relevant ST14 inhibitors have been identified thus far, while EGFR and PLD are current targets of clinical or preclinical cancer therapies.

The double face of ST14 and its mediators EGFR and PLD revealed in our studies is likely to complicate such therapies. Therefore, combinatorial treatments including a re-activation of the tumor-suppressive branch should be advantageous over current anti-carcinoma therapies.

In addition, given the double face of tumor-associated macrophages, promoting entosis to phagocytose tumor cells might be a therapeutic alternative to interfering with macrophage polarisation.

The power of anti-cancer therapies is often compromised by the fact that targeted cells, such as tumor-associated macrophages, as well as targeted molecules, can display a combination of oncogenic and tumor-suppressive effects. Our former work has unravelled a similar double face for Matriptase 1, also named ST14, a type II transmembrane serine protease, and its downstream mediators EGFR and phospholipase D (PLD). In zebrafish mutant embryos lacking the cognate ST14 inhibitor Hai1, the hyper-stimulated ST14 – EGFR – PLD pathway promotes both mTORC1-mediated epidermal hyperplasia and sphinogosine-1-phosphate (S1P)-mediated epidermal cell loss. The latter involves entosis of preneoplastic basal keratinocytes by neighboring surface keratinocytes and the subsequent apical extrusion of these cell complexes.

Accordingly, combined treatment with the PLD inhibitor FIPI and S1P leads to a perfect rescue and survival of otherwise lethal zebrafish hai1 null mutants. In the new funding period, we will test this combinatorial treatment in two different transgenic mouse carcinoma models and in human carcinoma cell lines. In addition, applying single cell sequencing and using mosaic hai1 zebrafish mutants and mammalian cell co-culture systems, we will elucidate the mechanisms by which healthy cells recognize, phagocytose and squeeze out hyper-proliferative cells during the entosis / apical cell extrusion process.

Finally, we will study the impact of two other known double-face regulators, p53 and Notch signalling, during ST14-dependent carcinogenesis in zebrafish hai1 mutants. In mammalian systems, p53 and Notch (at least in skin) are mainly known as tumor suppressors, whereas zebrafish hai1 mutants can be rescued by p53 and Notch inhibition, pointing to oncogenic effects.

1. Validation of combinatorial FIPI-S1P treatment in mouse and human systems of ST14-dependent carcinogenesis

2. Elucidation of the molecular mechanisms underlying cell heterogeneity among epithelial cells and their recognition and targeting by the entosis / apical extrusion system in zebrafish and mammalian models of ST14-dependent carcinogenesis

3. Molecular characterization of the roles of p53 and Notch signaling downstream of the oncogenic and / or tumor-suppressive effects of ST14

Zebrafisch mutants lacking the ST14 inhibitor Hai1 (Carney et al., 2007) display epidermal hyperplasia (oncogenic), but also entosis and apical cell extrusion of hyperplastic cells (tumor-suppressive). We could show that these two counteracting effects are mediated via a shared pathway involving EGFR and phospholipase D (PLD), which branches at the level of the PLD product phosphatidic acid (PA). PA in turn promotes mTORC1 activity and thereby basal cell proliferation.

In parallel, it promotes sphingosine kinase and thereby sphingosine-1-phosphate (S1P)-dependent loss of preneoplastic basal cells, involving an entosis-like engulfment of basal by outer keratinocytes and the subsequent apical extrusion of these cell complexes. Based on these findings, we designed a combinatorial pharmacological approach to block PLD from the shared part of the branched pathway, and to apply S1P to reconstitute the tumor-suppressive branch, which perfectly rescued the neoplasia of otherwise lethal hai1 mutant embryos (Armistead et al., 2020; Figure 1).

This was the first demonstration of a tumor-suppressive effect of entosis in vivo, opening the door for the proposed follow-up experiments to elucidate the molecular mechanisms underlying the selection of cells to be entosed. In addition, we have preliminary unpublished data according to which – similar to ST14 – inhibition of the transcription factor p53 and the cell surface receptor Notch lead to a perfect rescue of epidermal hyperplasia in zebrafish hai1a mutants. This suggests that in this scenario, p53 and Notch act downstream of ST14 and have predominan oncogenic, rather than the primarily published tumor-suppressive effects, opening the door for follow-up experiments to look into the molecular mechanisms underlying the regulation of the two factors by ST14.