Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells.

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

Basal extrusion of single-oncogenic mutant cells induces dome-like structures with altered microenvironments.

At the initial stage of carcinogenesis, oncogenic transformation occurs in single cells within epithelial layers. However, the behavior and fate of the newly emerging transformed cells remain enigmatic. Here, using originally established mouse models, we investigate the fate of RasV12-transformed cells that appear in a mosaic manner within epithelial tissues. In the lung bronchial epithelium, most majority of RasV12-transformed cells are apically extruded, whereas noneliminated RasV12 cells are often basally delaminated leading to various noncell-autonomous changes in surrounding environments; macrophages and activated fibroblasts are accumulated, and normal epithelial cells overlying RasV12 cells overproliferate and form a convex multilayer, which is termed a 'dome-like structure'. In addition, basally extruded RasV12 cells acquire certain features of epithelial-mesenchymal transition (EMT). Furthermore, the expression of COX-2 is profoundly elevated in RasV12 cells in dome-like structures, and treatment with the COX inhibitor ibuprofen suppresses the recruitment of activated fibroblasts and moderately diminishes the formation of dome-like structures. Therefore, basal extrusion of single-oncogenic mutant cells can induce a tumor microenvironment and EMT and generate characteristic precancerous lesions, providing molecular insights into the earlier steps of cancer development.

The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia.

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

Calcium Wave Promotes Cell Extrusion.

When oncogenic transformation or apoptosis occurs within epithelia, the harmful or dead cells are apically extruded from tissues to maintain epithelial homeostasis. However, the underlying molecular mechanism still remains elusive. In this study, we first show, using mammalian cultured epithelial cells and zebrafish embryos, that prior to apical extrusion of RasV12-transformed cells, calcium wave occurs from the transformed cell and propagates across the surrounding cells. The calcium wave then triggers and facilitates the process of extrusion. IP3 receptor, gap junction, and mechanosensitive calcium channel TRPC1 are involved in calcium wave. Calcium wave induces the polarized movement of the surrounding cells toward the extruding transformed cells. Furthermore, calcium wave facilitates apical extrusion, at least partly, by inducing actin rearrangement in the surrounding cells. Moreover, comparable calcium propagation also promotes apical extrusion of apoptotic cells. Thus, calcium wave is an evolutionarily conserved, general regulatory mechanism of cell extrusion.

Mutant p53-Expressing Cells Undergo Necroptosis via Cell Competition with the Neighboring Normal Epithelial Cells.

p53 is a tumor suppressor protein, and its missense mutations are frequently found in human cancers. During the multi-step progression of cancer, p53 mutations generally accumulate at the mid or late stage, but not in the early stage, and the underlying mechanism is still unclear. In this study, using mammalian cell culture and mouse ex vivo systems, we demonstrate that when p53R273H- or p53R175H-expressing cells are surrounded by normal epithelial cells, mutant p53 cells undergo necroptosis and are basally extruded from the epithelial monolayer. When mutant p53 cells alone are present, cell death does not occur, indicating that necroptosis results from cell competition with the surrounding normal cells. Furthermore, when p53R273H mutation occurs within RasV12-transformed epithelia, cell death is strongly suppressed and most of the p53R273H-expressing cells remain intact. These results suggest that the order of oncogenic mutations in cancer development could be dictated by cell competition.

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes.

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.