Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells.

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition.

Zebrafish imaging reveals TP53 mutation switching oncogene-induced senescence from suppressor to driver in primary tumorigenesis.

Most tumours are thought to arise through oncogenic cell generation followed by additional mutations. How a new oncogenic cell primes tumorigenesis by acquiring additional mutations remains unclear. We show that an additional TP53 mutation stimulates primary tumorigenesis by switching oncogene-induced senescence from a tumour suppressor to a driver. Zebrafish imaging reveals that a newly emerged oncogenic cell with the RasG12V mutation becomes senescent and is eliminated from the epithelia, which is prevented by adding a TP53 gain-of-function mutation (TP53R175H) into RasG12V cells. Surviving RasG12V-TP53R175H double-mutant cells senesce and secrete senescence-associated secretory phenotype (SASP)-related inflammatory molecules that convert neighbouring normal cells into SASP factor-secreting senescent cells, generating a heterogeneous tumour-like cell mass. We identify oncogenic cell behaviours that may control the initial human tumorigenesis step. Ras and TP53 mutations and cellular senescence are frequently detected in human tumours; similar switching may occur during the initial step of human tumorigenesis.

A novel role for PRL in regulating epithelial cell density by inducing apoptosis at confluence.

Maintaining proper epithelial cell density is essential for the survival of multicellular organisms. Although regulation of cell density through apoptosis is well known, its mechanistic details remain elusive. Here, we report the involvement of membrane-anchored phosphatase of regenerating liver (PRL), originally known for its role in cancer malignancy, in this process. In epithelial Madin-Darby canine kidney cells, upon confluence, doxycycline-induced expression of PRL upregulated apoptosis, reducing cell density. This could be circumvented by artificially reducing cell density via stretching the cell-seeded silicon chamber. Moreover, small interfering RNA-mediated knockdown of endogenous PRL blocked apoptosis, leading to greater cell density. Mechanistically, PRL promoted apoptosis by upregulating the translation of E-cadherin and activating the TGF-ß pathway. Morpholino-mediated inhibition of PRL expression in zebrafish embryos caused developmental defects, with reduced apoptosis and increased epithelial cell density during convergent extension. Overall, this study revealed a novel role for PRL in regulating density-dependent apoptosis in vertebrate epithelia. This article has an associated First Person interview with the first author of the paper.

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.

Cell competition corrects noisy Wnt morphogen gradients to achieve robust patterning in the zebrafish embryo.

Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength-dependent manner to pattern developing tissues. However, developing tissues also undergo dynamic morphogenesis, which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradients. Here we show that a cell competition-related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/ß-catenin signalling gradient, which acts as a morphogen to establish embryonic anterior-posterior patterning, identify that unfit cells with abnormal Wnt/ß-catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/ß-catenin gradient formation and consequent anterior-posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues, this system may support tissue robustness and disease prevention.

[Live-imaging Analyses Using Small Fish Models Reveal New Mechanisms That Regulate Primary Tumorigenesis].

Since the 1980s, zebrafish (Danio rerio) have been used as a valuable model system to investigate developmental processes because they: 1) grow outside their mothers; 2) are transparent during the embryonic stage; and 3) have organs similar to those in humans. Recently, zebrafish have emerged as a powerful model animal for studying not only developmental biology but also human diseases, especially cancer. Owing to the significant advantages of zebrafish, such as low-cost breeding, high efficiency of transgenesis, and ease of in vivo imaging and oncogenic/tumor cell induction, zebrafish offer a unique opportunity to unveil novel mechanisms of cancer progression, invasion, and metastasis. In addition, the small size of zebrafish larvae enables high-throughput chemical screening, and this advantage contributes to generating useful platforms for antitumor drug discovery. Owing to these various merits, which other model animals (such as fly, mouse, and rat) do not possess, zebrafish could achieve a unique status in cancer research. In this review, we discuss the availability of zebrafish for studying cancer and introduce recent cancer studies that have used zebrafish.

CD28 co-stimulation is dispensable for the steady state homeostasis of intestinal regulatory T cells.

It is well-established that CD28 co-stimulation is required for the development and the proliferation of thymus-derived regulatory T cells (tTregs). Meanwhile, the role of CD28 co-stimulation in the homeostasis of peripherally derived Tregs (pTregs) remains unclear. To clarify this issue, we analyzed Tregs in small and large intestines (SI and LI), the principle sites of pTreg development. Interestingly, and different from in the thymus, Tregs were abundant in the intestines of CD28-/- mice, and most of them were phenotypically pTregs. We showed that CD28-/- naive T cells differentiated into pTregs in the LI after oral exposure to antigens and that CD28-/- pTregs in the LI had the same highly proliferative activity as CD28+/- cells. CD28-/- pTregs acquired these Treg-specific features at transcriptional and epigenetics levels. On the other hand, some immune suppressive molecules were down-regulated in CD28-/- pTregs. Correspondingly, the suppressive activity of CD28-/- pTregs was weaker than CD28+/+ cells. These results indicate that the homeostasis of pTregs in the intestines is maintained even in the absence of CD28, whereas CD28 is required for the maximal suppressive activity of intestinal pTregs.

Defects in regulatory T cells due to CD28 deficiency induce a qualitative change of allogeneic immune response in chronic graft-versus-host disease.

In patients receiving allogeneic hematopoietic cell transplantation, chronic graft-versus-host disease (cGVHD) remains a frequent complication and resembles autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. Our previous work demonstrated the critical role of CD28 costimulation of donor T cells for GVHD induction. In this study, we investigate the role of CD28 costimulation of host T cells in cGVHD. CD28-intact mice as hosts showed systemic lupus erythematosus-type cGVHD, whereas CD28-deficient mice developed a distinct phenotype of cGVHD, with fibrotic damage in skin and internal organs, resembling systemic sclerosis. This phenotype was due to a lack of signaling through the C-terminal proline-rich motif within host CD28's cytoplasmic tail, a motif previously shown to be required for development of regulatory T cells (Tregs) and function of conventional T cells. Adoptive transfer experiments demonstrated that a defect in host CD4(+)CD25(+) Tregs, but not in conventional T cells, was responsible for disease phenotype. Host Treg deficiency altered the cytokine pattern of donor CD4(+) T cells and the Ag specificity of autoantibodies, and these might lead to phenotypic change. Thus, host CD28 signaling controlled the pathogenesis of cGVHD through effects on host Tregs, whose status impacts qualitatively on the allogeneic immune responses.