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

Haraoka, Yukinari; Akieda, Yuki; Nagai, Yuri; Mogi, Chihiro; Ishitani, Tohru

doi:10.1038/s41467-022-29061-6

Download PDF

Article
Open access
Published: 18 March 2022

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

Nature Communications volume 13, Article number: 1417 (2022) Cite this article

8523 Accesses
10 Citations
7 Altmetric
Metrics details

Subjects

Abstract

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 Ras^G12V mutation becomes senescent and is eliminated from the epithelia, which is prevented by adding a TP53 gain-of-function mutation (TP53^R175H) into Ras^G12V cells. Surviving Ras^G12V-TP53^R175H 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.

KRas-transformed epithelia cells invade and partially dedifferentiate by basal cell extrusion

Article Open access 10 December 2021

John Fadul, Teresa Zulueta-Coarasa, … Jody Rosenblatt

Unmasking senescence: context-dependent effects of SASP in cancer

Article 24 June 2019

Douglas V. Faget, Qihao Ren & Sheila A. Stewart

The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo

Article Open access 29 January 2021

Briana Rackley, Chang-Soo Seong, … Melissa Gilbert-Ross

Introduction

Tumour cells harbour multiple mutations. Decades of human clinical sample analyses and animal model studies have revealed that mutations in oncogenes and tumour-suppressor genes synergistically promote tumorigenesis^1,2. The biochemical interactions between these multiple mutations in cancer cell proliferation, survival, and migration have also been widely investigated^3,4,5,6,7. Based on these studies, it is thought that most cancers originate from a sporadically emerged single oncogenic cell with a mutation in oncogenes or tumour-suppressor genes^8,9,10 and that mutation accumulation facilitates tumorigenesis^11,12,13. However, the processes by which a single transformed cell primes neoplastic development and the effects of additional mutations on these processes remain unclear. Moreover, although a newly emerged oncogenic cell in healthy tissue is surrounded by normal cells, the interaction between an oncogenic cell and neighbouring normal cells in the early stages of tumorigenesis has not been extensively studied.

Recent studies suggested that cellular senescence, the state of ultimate and irreversible cell cycle arrest, plays both tumour-suppressive and tumour-promoting roles depending on the cellular context. Cellular senescence is triggered by various stresses, including chromosomal instability, telomere shortening, oxidative stress, and DNA damage^14,15,16,17. Abnormal oncogenic activation, including hyperactivation of Ras signalling, can also induce cellular senescence to exert a tumour-suppressive role by inducing cell cycle arrest. In fact, senescence-associated cell cycle arrest has been detected in human premalignant tumours, and in vivo model studies using Ras signalling-activated or PTEN-deficient mice demonstrated that cellular senescence negatively regulates tumour initiation and progression^14,18,19,20. However, senescent cells can also mediate tumorigenic effects by secreting a variety of inflammatory cytokines and growth factors, referred to as the senescence-associated secretory phenotype (SASP)²¹. SASP secretion leads to various effects, including a pathological increase in proliferation of neighbouring premalignant and malignant cells^22,23,24. However, the roles of SASP in primary tumorigenesis, particularly in newly emerged oncogenic cells in living tissues, are unknown. In addition, how oncogenic cells decide to manipulate cellular senescence for tumour-suppressive or tumorigenic effects is unclear.

In this study, using imaging analyses, we show that a newly emerged Ras^G12V oncogenic cell is eliminated from living tissue through cellular senescence. An additional TP53 mutation alters the effects of cellular senescence in Ras^G12V cell from tumour-suppressive to SASP-mediated tumorigenic, and SASP secretion from the Ras-TP53 double mutant cell converts its neighbouring normal cells into senescent cells, thereby priming neoplastic development.

Results

Mosaically introduced Ras^G12V cells are eliminated

To understand the behaviour of a newly generated oncogenic cell, we used zebrafish larval skin as a model of human epithelial tissue. We previously established a method for oncogenic cell visualisation in zebrafish skin^25,26, in which fluorescent protein (e.g., mKO2, mCherry, or GFP)-expressing oncogenic cells were artificially introduced into the larval skin of the keratin4 (krt4)-gal4 zebrafish line²⁷ by injecting UAS promoter-driven expression plasmids (Fig. 1a). Low-dose injection of the UAS-driven plasmids induced a mosaic distribution of fluorescent cells, each of which was surrounded by normal cells in the larval skin (Supplementary Fig. 1a). Ras mutations, observed in ~30% of human cancers, are considered to occur in the early stages of tumorigenesis^28,29. Previous studies demonstrated that forced activation of oncogenic Ras signalling covering a wide area can induce excessive proliferation and dysplasia in the epithelial tissues of model animals, including mice, zebrafish, and flies^30,31,32. Interestingly, mosaically introduced Ras^G12V cells gradually disappeared and therefore did not induce any lesions (Fig. 1b). Ras^G12V cells increased their cytoplasmic and nuclear volumes and apically protruded (Fig. 1c, Supplementary Movie 1, 2), but the protruding Ras^G12V cells were nonapoptotic (Supplementary Fig. 1b). Thus, the newly generated Ras^G12V cells surrounded by normal cells were eliminated from the larval skin. This oncogenic cell elimination appears to occur in an immune cell-independent manner. Consistent with this idea, Ras^G12V cells were also eliminated from zebrafish larvae that lacked myeloid lineage cells (macrophages, monocytes, and neutrophil) established by injection of morpholino antisense oligo (MO) against spi1b (also known as pu.1)³³ (Supplementary Fig. 1c, d). In addition, zebrafish larvae have not developed an adaptive immune system, as this system is not functionally mature until approximately 4 weeks post-fertilization³⁴. Moreover, we found that the neighbouring cells accumulate F-actin at the contact sites with the protruding Ras^G12V cells (Supplementary Fig. 1e), suggesting that neighbouring epithelial cells are involved in Ras^G12V cell elimination. To determine whether similar elimination can occur in other types of oncogenic cells, we mosaically introduced cells overexpressing v-Src, Myc and TP53 hot-spot mutants (TP53^R175H, TP53^R248W or TP53^R273H), the abnormal activity of which can induce tumorigenesis^35,36,37. Mosaic v-Src cells were eliminated as observed for Ras^G12V cells (Supplementary Fig. 2a), whereas Myc-overexpressing cells and TP53 mutants-expressing cells remained (Supplementary Fig. 2b–e), suggesting that this elimination is triggered by a specific type of oncogenic activation.

**Fig. 1: Newly generated Ras^G12V cell surrounded by normal cells is eliminated from zebrafish epithelia.**

Ras^G12V cells undergo cellular senescence

Cytoplasmic and nuclear swelling is a common phenotype in senescent cells^38,39. In addition, activation of oncogenic Ras signalling can induce cellular senescence³⁸. Therefore, we tested whether cellular senescence occurred in swollen Ras^G12V cells in the zebrafish epithelia. The senescence phenotype is often characterised by several unique features: (i) activation of a chronic DNA damage response, such as phosphorylation of the histone H2AX (γH2AX), (ii) senescence-associated β-galactosidase (SA-β-gal) accumulation, (iii) epigenetic alternation such as H3K9 tri-methylation (H3K9me3), and (iv) irreversible cell-cycle arrest and its associated expression of various cyclin-dependent kinase inhibitors, including CDKN2A (p16^INK4a), CDKN2B (p15^INK4b), and CDKN1A (p21^CIP)^17,39. In the zebrafish genome, CDKN2A and CDKN2B are integrated into only one gene, cdkn2a/b. Accumulation of γH2AX in the nucleus (Fig. 2a), SA-β-gal accumulation (Fig. 2b), H3K9me3 upregulation (Supplementary Fig. 3a), cell-cycle arrest (reduction of EdU incorporation) (Fig. 2c), and high cdkn2a/b expression (Fig. 2d) were detected in mosaically introduced Ras^G12V cells, suggesting that the Ras^G12V cells were senescent. Interestingly, high cdkn2a/b expression and nuclear size enlargement were not detected in zebrafish larval skin ubiquitously expressing Ras^G12V (Supplementary Fig. 4a, b), indicating that Ras activation is not sufficient to induce cellular senescence in the skin. These results also suggest that neighbouring normal cells are required to induce senescence in mosaic Ras^G12V cells.

**Fig. 2: Newly generated oncogenic cells undergo cellular senescence.**

Senescence drives Ras^G12V cell elimination

To examine the involvement of cellular senescence in the Ras^G12V cell elimination, we inhibited both CDKN2A/B- and TP53-mediated senescence-inducing pathways¹⁷ by injecting MOs against cdkn2a/b and tp53. Injection of both MOs inhibited the swelling and elimination of mosaically introduced Ras^G12V cells (Fig. 2e, f), suggesting that senescence-induced cell swelling generates a driving force for oncogenic cell elimination. We also examined the status of cell adhesion, as mosaically introduced Ras^G12V cells were apically delaminated. As expected, the membrane-localizing activity of E-cadherin, a major component of cellular adhesion⁴⁰, was decreased in the Ras^G12V cells, whereas the surviving Ras^G12V cells in larvae injected with cdkn2a/b and tp53 MOs retained membrane E-cadherin (Supplementary Fig. 5a), suggesting that Ras^G12V-induced cellular senescence reduces adhesion between Ras^G12V cells and neighbouring normal cells. Consistent with the above findings showing that ubiquitous expression of Ras^G12V cells was insufficient to induce cellular senescence in the skin, a reduction of membrane E-cadherin levels was not detected in the skin ubiquitously expressing Ras^G12V (Supplementary Fig. 5b), suggesting that neighbouring normal epithelial cells are required to induce senescence-mediated E-cadherin reduction and mosaic Ras^G12V cell elimination. These results indicate that cellular senescence suppresses tumorigenesis through immune cell-independent oncogenic cell elimination.

Ras^G12V-TP53^R175H double mutant cells form a tumour-like cell mass

Although mutation accumulation is thought to facilitate tumorigenesis^11,12,13, the effects of additional mutations on the behaviour of a newly generated oncogenic cell remain unclear. Recently, we reported that a newly generated Wnt signalling-hyperactivated oncogenic cell is eliminated through apoptosis in zebrafish embryos, whereas loss of the tumour-suppressor gene smad4, which is associated with Wnt signalling hyperactivation in human cancers, blocked this elimination²⁵. Therefore, we tested whether Ras^G12V cell behaviour is influenced by additional mutations, including gain or loss of TP53, gain of Myc, loss of Smad4, and loss of adenomatous polyposis coli, events which are known to interact genetically and/or biochemically with oncogenic Ras mutation in tumorigenesis^4,5,6,41,42. Our analyses revealed that addition of TP53^R175H, TP53^R248W, or TP53^R273H, all of which are considered as TP53 gain-of-function hot-spot mutations³⁷, had the most significant effects on the Ras^G12V cells. We mosaically introduced oncogenic cells co-expressing Ras^G12V with each TP53 mutant into zebrafish larval skin (Supplementary Fig. 6a). Although cells with Ras^G12V alone were actively eliminated at 2 days postfertilisation (dpf), addition of TP53^R175H, TP53^R248W, or TP53^R273H mutation to Ras^G12V cells significantly increased their viability (Fig. 3a, Supplementary Fig. 6b). Consistent with these results, additional TP53^R175H mutation blocked Ras^G12V cell-induced F-actin accumulation in neighbouring cells (Supplementary Fig. 1e) and membrane E-cadherin reduction (Supplementary Fig. 5c). Interestingly, surviving Ras^G12V-TP53 double mutant cells formed heterogenous tumour-like cell masses containing a variety of cells, including surrounding skin cells lacking Ras^G12V-TP53 double mutations (Fig. 3b–d; Supplementary Fig. 6c; Supplementary Movie 3). E-cadherin levels in the cell mass were also heterogenous, and some double-mutant cells and their neighbours lost membrane E-cadherin (Supplementary Fig. 5d). In contrast, mosaically introduced cells with either Ras^G12V or TP53^R175H or with both Ras^G12V and TP53 wild-type did not efficiently form the cell mass (Fig. 3d, e). Further, knockdown of endogenous tp53 using tp53 MO injection did not enhance Ras^G12V cell-induced cell mass formation (Fig. 3d). Co-expression with TP53^DD, a TP53 dominant-negative mutant⁴³ that can inhibit the activity of TP53 wild-type (Supplementary Fig. 7a, b), in Ras^G12V cells did not reinforce cell mass formation (Fig. 3d). Thus, gain-of-function, but not loss-of-function, of TP53 cooperates with the Ras^G12V mutation to promote the evolution of a few oncogenic cells into a tumour-like cell mass. Notably, the introduction of cells co-expressing Ras^G12V with a transcriptionally-inactive TP53^R175H-QSQS mutant^44,45, did not induce cell mass formation (Fig. 3d), indicating that TP53^R175H requires its transcriptional activity to facilitate double-mutant-derived cell mass formation.

**Fig. 3: TP53^R175H mutation attenuates mosaic Ras^G12V cell elimination and stimulates heterogeneous cell mass formation.**

TP53^R175H addition enhances Ras^G12V-induced senescence

We next investigated how Ras^G12V and TP53^R175H mutations synergistically facilitate cell mass formation. Gain-of-function TP53 mutants, including TP53^R175H, acquire activities that elevate Ras^G12V-mediated signalling and gene expression⁴⁶, and TP53 positively regulates cellular senescence by controlling CDKN1A expression⁴⁷. These previous reports suggest that TP53^R175H affects Ras^G12V-mediated senescence. Consistent with this idea, co-expression of TP53^R175H significantly enhanced Ras^G12V-induced expression of senescent makers, γH2AX, SA-β-gal, cdkn2a/b, and H3K9me3 (Fig. 2a, b, d, Supplementary Fig. 3a), and SASP factors, reactive oxygen species (ROS), interleukin (IL)-1β (il1b), IL-6 (il6), IL-6-related cytokine IL-11 (il11b), and IL-8/CXCL8 (cxcl8a)^21,39,48,49, were synergistically induced by co-expression of TP53^R175H with Ras^G12V (Fig. 4a, b, Supplementary Fig. 3b, c). These SASP factors were detected inside the Ras^G12V-TP53^R175H double-mutant cells (Fig. 4a, white arrows in top panels and bottom left graph, and Fig. 4b, Supplementary Fig. 3b) and il1b expression was specifically activated in cell cycle-arrested senescent Ras^G12V-TP53^R175H cells (Supplementary Fig. 3d). In contrast, the effects of TP53^R175H mutation alone on the expression levels of these senescent makers and SASP factors were very minor (Figs. 2a, b, d, 4a, b, Supplementary Fig. 3a–c). In addition, depletion of endogenous TP53 proteins by tp53 MO injection did not affect Ras^G12V-induced cdkn2a/b expression (Supplementary Fig. 7c). Co-expression of exogenous TP53 wild-type or TP53^DD dominant-negative mutant with Ras^G12V did not activate the expression of il1b and il11b (Supplementary Fig. 7d). These results suggest that gain-of-function, but not loss-of-function, of TP53 cooperates with the Ras^G12V mutation to promote cellular senescence and SASP factor production.

**Fig. 4: Additional TP53^R175H mutation reinforces SASP.**

Double-mutant cells form tumour-like cell masses through SASP

Although active cell proliferation should occur during cell mass formation, almost all Ras^G12V-TP53^R175H double-mutant cells were EdU-negative non-proliferative cells (Fig. 2c) and EdU-positive double-mutant cells were not detected in the cell masses (Supplementary Fig. 8a). Interestingly, EdU-positive or phospho-histone H3 (pH3)-positive proliferating cells lacking Ras^G12V-TP53^R175H mutations were detected in the double-mutant cell-surrounding area (Fig. 5a, Supplementary Fig. 8b). Surviving double-mutant cells in zebrafish skin express SASP factors, suggesting that SASP factors secreted from these cells stimulate the proliferation of the neighbouring cells and consequent cell mass formation. To test this, we knocked down the mitogenic SASP factor il1b^50,51,52 using MO blocking il1b mRNA translation (Supplementary Fig. 9a). As expected, il1b knockdown blocked the double-mutant cell-induced increase in pH3-positive proliferating cells in the surrounding area (Fig. 5a) and cell mass formation (Fig. 5b), suggesting that the double-mutant cell-derived Il1b stimulates cell proliferation in neighbouring normal cells, leading to cell mass formation. We also found that treatment with the ROS scavenger N-acetyl-L-cysteine (NAC) reduced ROS production in the double-mutant cells (Fig. 4a) and cell mass formation (Fig. 5c), suggesting that, in addition to Il1b, other SASP factor ROS are involved in cell mass formation. To further evaluate the contribution of Il1b and ROS, we artificially increased the levels of Il1b and ROS in mosaic Ras^G12V cells. Interestingly, co-expression of exogenous Il1b in Ras^G12V cells reduced F-actin accumulation in the neighbouring cells (Supplementary Fig. 9b) and induced cell mass formation in a small number of larvae (Fig. 5d), indicating that addition of exogenous Il1b partially mimicked the effects of TP53^R175H mutation addition. Moreover, exposure to hydrogen peroxide (H₂O₂), an ROS that can be generated in senescent cells¹⁷, further facilitated cell mass formation induced by cells co-expressing Ras^G12V cells and exogenous Il1b (Fig. 5d). However, H₂O₂ treatment did not affect Ras^G12V cell-induced cell mass formation in the absence of exogenous Il1b (Fig. 5d). These results suggest that the double mutation-induced high-level production of Il1b and ROS in oncogenic cells enables cell mass formation in cooperation with neighbouring cells.

**Fig. 5: SASP-mediated senescence propagation is involved in Ras^G12VTP53^R175H cell-driven cell mass formation.**

Propagation of senescence promotes tumour-like cell mass formation

Notably, the introduction of the double mutant cells upregulated ROS levels not only in themselves but also in neighbouring cells, and introduction of cells with either Ras^G12V or TP53^R175H mutation did not induce such ROS diffusion (Fig. 4a, yellow arrows in top panels and bottom right graph). ROS were also detected in cells with and without double mutations inside the growing cell mass (Fig. 5e). Given that ROS are known both SASP factors^17,48 and activators of senescence through DNA double-strand breaks induction⁵³, we hypothesised that diffused ROS induce senescence in neighbouring cells. Consistently, the growing cell mass contained γH2AX-, or cdkn2a/b-positive cells lacking Ras^G12V-TP53^R175H mutations (Fig. 5f, g, arrowheads). In addition, mosaically introduced Ras^G12V-TP53^R175H double-mutant cells, but not cells with either Ras^G12V or TP53^R175H, induced SA-β-gal activity and γH2AX in neighbouring cells (Fig. 2b, arrowheads, Fig. 5h). All γH2AX-positive neighbouring cells were pH3-negative non-proliferative cells (Supplementary Fig. 8b), and NAC treatment reduced the double-mutant-induced γH2AX in neighbours (Fig. 5h). These results suggest that senescent double-mutant cell-secreted ROS senesce neighbouring cells.

To further analyse neighbouring cell senescence, we generated cellular senescence reporter zebrafish Tg(cdkn2a/b-hs:Achilles) (Supplementary Fig. 10a), which express fast-maturing YFP variant protein, Achilles⁵⁴, in cells expressing endogenous cdkn2a/b (Supplementary Fig. 10b). As expected, the cdkn2a/b-hs:Achilles reporter was upregulated in mosaic Ras^G12V single-mutant or Ras^G12V-TP53^R175H double-mutant cells, and almost all of these cells were EdU-negative arrested (Supplementary Fig. 10c), suggesting that the cdkn2a/b-hs:Achilles reporter can be used to detect senescent cells. Time-lapse imaging of live Tg(cdkn2a/b-hs:Achilles) larvae captured the beginning of cdkn2a/b reporter expression in Ras^G12V-TP53^R175H cells (Supplementary Fig. 10d, Supplementary Movie 4) and subsequent expression of the cdkn2a/b reporter in the neighbours (Supplementary Fig. 10e, Supplementary Movie 5). These observations are consistent with our model that the senescent double-mutant cells propagate senescence to the neighbours. We also confirmed that both cdkn2a/b reporter-positive senescent cells and EdU-positive proliferating cells were present in the Ras^G12V-TP53^R175H cell neighbours, but these two populations were mutually exclusive (Supplementary Fig. 10f).

ROS and il1b and il6 transcripts were detected in both Ras^G12V-TP53^R175H double-mutant cells and cells lacking Ras^G12V-TP53^R175H mutations inside the cell mass (Fig. 5e, i, Supplementary Fig. 11a), indicating that SASP factors are generated not only in the double-mutant cells but also in the senesced neighbours. To test this possibility, we prevented senescence in neighbouring normal cells by treatment with NAC to inhibit ROS, a senescence inducer secreted from double-mutant cells, or by MO-mediated knockdown of tp53, which is a mediator of ROS-induced cellular senescence⁵⁵. NAC treatment reduced the expression of SASP factors, il1b and il11b, in larvae mosaically introduced with Ras^G12V-TP53^R175H double-mutant cells (Supplementary Fig. 3e). In TP53-knockdown zebrafish skin, mosaic introduction of the double-mutant cells did not efficiently induce ROS production and il1b expression in the surrounding area (Supplementary Fig. 11b, c) and cell mass formation (Fig. 5j), suggesting that neighbouring cell senescence and its associated production of SASP factors are required for cell mass formation. These findings also indicate that ROS generated in senescent double-mutant cells induce senescence and ROS production in the neighbours, suggesting that a senesced neighbouring cell also induces senescence in its neighbours, which may propagate senescence. Consistent with this idea, mosaic introduction of the double-mutant cells induced strong SA-β-gal activity in adjacent cells (~20 μm radius from the double-mutant cells) and moderate SA-β-gal activity in second adjacent cells (20–40 μm radius from the double-mutant cells) (Fig. 5k, Supplementary Fig. 11d). In the double-mutant cell-induced cell mass, the senescence markers γH2AX and cdkn2a/b were broadly expressed in not only the area immediately neighbouring the double-mutant cells but also 2–3 cells away from these cells (Fig. 5f, g). Thus, in addition to Il1b-mediated neighbouring cell proliferation, ROS-mediated senescence propagation may also contribute to cell mass formation.

Mosaic Ras^G12V cells form a cell mass in damaged skin

Next, we tested the behaviour of oncogenic cells on unhealthy epithelia with abnormal TP53 activity or senesced epithelia. Mosaically introduced of Ras^G12V mutation in larval skins ubiquitously expressing TP53^R175H efficiently generated tumour-like cell masses (Fig. 6a), in which γH2AX, ROS, and il1b were detected in cells with and without Ras^G12V mutation (Supplementary Fig. 12a–c); this cell mass formation was supressed by il1b MO or NAC treatment (Fig. 6b, c), suggesting that a newly generated Ras^G12V mutation can prime tumorigenesis by inducing Il1b-mediated proliferation and ROS-mediated senescence in its neighbours in the epithelia with abnormal TP53 activity. To produce senesced epithelia, we treated zebrafish larvae with doxorubicin, which induces DNA damage and consequent TP53 activation and cellular senescence^56,57. Doxorubicin treatment partially induced γH2AX-positive senescent cells in larval skin (Supplementary Fig. 12d), whereas it did not induce apoptotic cells (Supplementary Fig. 12e). Compared to mosaically introduced Ras^G12V cells in normal skin, those in doxorubicin-treated skin relatively strongly expressed cdkn2a/b, il1b, 1l11b, and ROS (Fig. 6d–g, Supplementary Fig. 12f) and efficiently induced tumour-like cell masses (Fig. 6h). Mosaic Ras^G12V cell-induced cell mass formation in doxorubicin-treated skin was blocked by MO-mediated tp53 knockdown (Fig. 6i). These results indicate that the mechanism of Ras^G12V cell-induced cell mass formation in senesced epithelia is similar to that of double-mutant cell-induced cell mass formation in the healthy epithelia, and that senesced epithelia lose the potential to eliminate oncogenic cell and become prone to tumorigenesis initiation compared to healthy epithelia.

**Fig. 6: Mosaic Ras^G12V cells stimulate cell mass formation in damaged skin.**

Discussion

We demonstrated the potential mechanisms of tumour initiation and roles of cellular senescence in this process. In healthy zebrafish epithelial tissue, a newly emerged oncogenic Ras^G12V cell becomes senescent, which stimulates cell swelling and loss of intercellular adhesion with neighbouring cells and consequent Ras^G12V cell elimination (Fig. 7, left). However, the addition of a TP53 gain-of-function mutation (TP53^R175H) prevents Ras^G12V cell elimination by restoring adhesion with neighbouring cells and reinforces Ras^G12V cell senescence and consequent production of SASP factors, including IL-1β and ROS (Fig. 7, middle). Additional TP53 mutation-mediated survival of mutant cells would assist in the continuous high-level secretion of IL-1β and ROS to the neighbours. Neighbouring cells undergo proliferation or senescence in response to IL-1β or ROS, respectively. Secondary senesced cells also secrete IL-1β and ROS, which can propagate cellular senescence and stimulate aberrant cell proliferation nearby, resulting in formation of a heterogeneous tumour-like cell mass. Similar heterogeneous cell mass formation depending on IL-1β and ROS occurs in abnormal zebrafish skin with TP53^R175H mutation or DNA damage (Fig. 7, right).

**Fig. 7: Schematic illustration of the roles and regulation of cellular senescence during primary tumorigenesis.**

Previous studies revealed two tumour-suppressive mechanisms of cellular senescence. The first is induction of cyclin-dependent kinase inhibitor-mediated irreversible cell cycle arrest in oncogenic cells^14,15,16,17. The second is stimulation of immune cell-mediated senescent cell elimination^58,59. In this study, we found that immune cell-independent oncogenic cell elimination is a third antitumour mechanism of cellular senescence. Interestingly, similar elimination was triggered by activation of Src in the zebrafish epithelia. Cells with hyperactive Src may be eliminated through cellular senescence because these signalling pathways are also involved in senescence induction⁶⁰. In contrast, zebrafish epithelia could not eliminate oncogenic cells with TP53 hot-spot mutations (TP53^R175H, TP53^R248W and TP53^R273H), high Myc levels, and abnormally high Wnt/β-catenin activity²⁵, suggesting that this elimination system functions against a specific type of oncogenic activation.

Our study also showed that a newly generated Ras^G12V cell stimulates senescence neighbouring normal cells, indicating that communication with surrounding cells is involved in Ras^G12V cell elimination. Notably, mammalian epithelia can also eliminate sporadically emerging oncogenic cells by communicating with neighbouring normal cells; in Madin-Darby canine kidney cell culture, mosaically introduced oncogenic cells with abnormal Ras and Src activities were apically extruded through communication with neighbouring normal cells^61,62. This neighbouring cell-mediated oncogenic cell elimination system is known as cell competition or epithelial defence against cancer (EDAC)⁶³. A similar phenomenon occurs in cultured cells in the mouse small intestine, pancreas, and lung^64,65. The mechanisms of EDAC are relatively well-studied. For example, recent studies showed that neighbouring cell-mediated upregulation of pyruvate dehydrogenase kinase in Ras^G12V cells and the calcium wave, which is propagated from Ras^G12V cells to surrounding cells via the mechanosensitive calcium channel and IP3 receptor, promote Ras^G12V cell elimination^26,64. Interestingly, we found that treatment with 2-aminoethoxydiphenylborane (inhibitor of mechanosensitive calcium channel and IP3 receptor) or dichloroacetate (specific inhibitor of pyruvate dehydrogenase kinase family) significantly suppressed cellular senescence in Ras^G12V cells and their elimination (Supplementary Fig. 13a, b), suggesting that these EDAC mediators function upstream of Ras^G12V cell senescence and that senescence-driven Ras^G12V cell elimination in zebrafish is mediated by mechanisms similar to that of EDAC in mammals. In addition, our findings suggest that cellular senescence-mediated elimination functions in mammalian epithelial tissues.

We identified membrane E-cadherin localization as a key factor in mutant cell elimination. Mosaically introduced Ras^G12V cells blocked membrane localization of E-cadherin in a cell-nonautonomous manner, which drives apical extrusion of Ras^G12V cells from the zebrafish larval skin. Additional TP53 mutation recovers membrane E-cadherin and allows retention of mutant cells. However, the detailed mechanisms of how Ras^G12V and TP53 mutations control E-cadherin localization were unclear. Notably, Ras^G12V cells induced F-actin accumulation in its surrounding cells, whereas Ras^G12V-TP53^R175H double-mutant cells did not, indicating that the levels of F-actin accumulation in neighbouring cells are inversely correlated with E-cadherin levels in mutant cells. One possibility is that F-actin accumulation in neighbours is involved in regulating E-cadherin in the mutant cells. It will be interesting to study the neighbouring cell’s machinery controlling E-cadherin in mutant cell and its elimination.

We also showed that inside the growing cell mass, some double-mutant cells and neighbouring cells had reduced E-cadherin levels, whereas the remaining cells did not. It remains unclear how this E-cadherin heterogeneity is induced. Previous reports showed that a SASP factor, including ROS, can disrupt the membrane-localization of cadherin⁶⁶. ROS, which are continuously produced inside the cell mass, may be involved in reducing E-cadherin levels in this context.

Addition of a TP53 gain-of-function mutation prevented Ras^G12V cell elimination by restoring intercellular adhesion with neighbours and blocking F-actin accumulation in neighbours to reinforce Ras^G12V cell senescence and SASP factor production, resulting in tumour-like cell mass formation in zebrafish epithelia. Ras and TP53 mutations are common genetic mutations in various cancers, and human pancreatic ductal adenocarcinoma and colorectal cancer cells acquire both mutations during tumour progression^67,68. Interestingly, recent studies revealed that human TP53 hot-spot point mutations, including TP53^R175H, TP53^R248W, and TP53^R273H, are gain-of-function⁶⁹ and enhance oncogenic potential^70,71. In fact, mice with both Ras and TP53 gain-of-function mutations efficiently form more malignant tumours compared to in mice with either Ras or TP53 mutation^3,7,72,73. Notably, a previous study reported that an additional TP53^R175H mutation prevents the disappearance of senescent cells with oncogenic Ras mutations and stimulates tumorigenesis in the mouse pancreas⁷⁴. Although this previous study did not examine the mechanisms of senescent cell disappearance and involvement of SASP in tumorigenesis, the mechanisms may be similar to those we observed in the zebrafish model and in mammals.

We observed a new mode of Ras-TP53-mediated primary tumorigenesis. TP53 mutation stimulates senescent Ras^G12V cells not only to survive but also to induce proliferation and secondary senescence in the surrounding area. In mouse liver and human fibroblast cultures, senesced oncogenic cells also induced secondary senescence in neighbouring cells⁷⁵. In addition, in human patient specimens, signs of senescence, including oxidative damage accumulation and TP53 upregulation, were detected in normal tissues adjacent to the tumours^{76,77,78,79,80,81}, suggesting that secondary senescence occurs in human tumorigenesis. However, the role of secondary senescence in tumorigenesis has not been examined. Here, we demonstrated that secondary senescence in the neighbouring cells of oncogenic cells is required for tumour initiation in a zebrafish model. Thus, our work provides insight into the interactions between oncogenic cells and their surrounding cells during the early stages of cancer development.

Human tumours generally exhibit heterogeneity and include a diverse collection of cells harbouring distinct molecular signatures⁸². Although cancer heterogeneity is the largest barrier to cancer treatment, mainly because of its resistance to therapy⁸³, the mechanisms that generate heterogeneity in cancer have not been completely explained. The currently acceptable view is that differentiation and/or additional mutations in cancer cells generate tumour heterogeneity⁸². In our zebrafish model, mutant cells converted neighbouring cells to abnormal proliferative or senescent cells through the secretion of SASP factors, thereby forming a heterogeneous cell mass. Based on these findings, SASP-mediated propagation of senescence and abnormal proliferation in the neighbourhood may be a new model of heterogeneous tumour generation. Thus, by using zebrafish imaging analyses, we revealed potential mechanisms of primary tumorigenesis.

Methods

Zebrafish maintenance

Zebrafish wild-type strain (AB), Tg(krt4p:gal4; UAS:EGFP)²⁷, Tg(krt4p:gal4; UAS:mKO2), Tg(UAS:GAP43mKO2-T2A-H-Ras^G12V), Tg(UAS:GAP43EGFP-T2A-TP53^R175H), Tg(cdkn2a/b-hs:Achilles), Tg(UAS:zE-cadherin-GFP), and Tg(il1b:EGFP)⁸⁴ were raised and maintained under standard conditions. All experimental animal care was performed in accordance with institutional and national guidelines and regulations. The study protocol was approved by the Institutional Animal Care and Use Committee of the respective universities (Gunma University Permit# 17-051; Osaka University, RIMD Permit# R02-04). One-cell stage embryos were used for cell injection to generate transgenic fish lines or mosaic larvae, with the latter processed for up to 2 dpf. To avoid excess tumour formation, zebrafish larvae were sacrificed within 24 h after a day after the initial cell mass formation, in our study.

Plasmids

To prepare UAS promoter-driven plasmids, the UAS promoter was sub-cloned into the pTol2 vector (a gift from Dr. K. Kawakami). Subsequently, membrane-tagged (GAP43-fused) GFP (or GAP43mKO2, mCherry) and T2A were sub-cloned into pTol2-UAS promoter plasmids. These plasmids expressed GAP43GFP (or GAP43mKO2, mCherry) alone in response to Gal4 activation. To generate plasmids expressing such fluorescent proteins with oncogenic proteins, PCR-amplified cDNAs encoding oncogenic proteins were sub-cloned into the downstream site of T2A of pTol2-UAS-GAP43GFP (or GAP43mKO2, mCherry)-T2A plasmids. Oncogenic protein cDNA were obtained as follows: an oncogenic constitutively active mutant of human H-Ras (Ras^G12V), in which Gly12 was substituted to Val, was a gift from Dr. A. Yoshimura; Rous sarcoma virus src (v-Src), which is a constitutively active Src kinase, was a gift from Dr. Y. Fujita⁶²; human c-Myc is a gift from Dr. W. El-Deiry (Addgene #16011)⁸⁵; zebrafish il1b is a gift from Dr. A. Kawakami⁸⁴; human TP53^WT and TP53^R175H was a gift from Dr. B. Vogelstein (Addgene #16543, #16544)⁸⁶; TP53^R248W and TP53^R273H and TP53^DD (TP53 p53 aa 1–11, 305–393)⁴³ were generated by PCR; transactivation deficient mutants of TP53^R175H (TP53^QSQS), in which Leu22, Trp23, Trp53, and Phe54 were substituted to Gln, Ser, Gln, and Ser, respectively^44,45, were established in the lab. TP53 luciferase reporter with p53 binding sites, PG13-luc, was a gift from Dr. B. Vogelstein (Addgene # 16442)⁸⁷. Zebrafish E-cadherin-GFP fusion gene was a gift from Dr. E. Raz⁸⁸. IL-1β5′ untranslated regions were annealed by DNA oligos and cDNA for GFP were PCR-amplified, and these two DNA fragments were cloned into the multi-cloning site of the pCS2p+vector by In-Fusion® HD Cloning Kit (Takara, Kusatsu, Japan). cDNA for α-Bungarotoxin (a gift from Dr. S. Megason, Addgene #69542)⁸⁹ was also PCR-amplified and cloned into the multi-cloning site of the pCS2p+vector. Probes for in situ hybridisation were prepared from templates encoding GFP⁹⁰, mKO2, zebrafish-codon-optimized Achilles, lcp1⁹¹, npsn⁹², il1b, il11b, il6, cxcl8a, and cdkn2a/b and specific primers are described in Supplementary Table 1.

Generation of transgenic zebrafish

To generate Tg(UAS:G43mKO2-T2A-H-Ras^G12V), Tg(UAS:G43EGFP-T2A-TP53^R175H), or Tg(UAS:zE-cadherin-GFP), each plasmid DNA along with Tol2 transposase mRNA were co-injected into one-cell stage wild-type zebrafish (AB) embryos.

Mosaic introduction of oncogenic cells

UAS promoter-driven plasmids (plasmid doses were as follows: fluorescent protein alone; 7.5–15 pg, mosaic Ras^G12V: 12.5–25 pg, TP53^WT: 15 pg, TP53^R175H: 35–50 pg (except for Fig. 3e where 15 pg was used), TP53^R248W: 35–50 pg, TP53^R273H: 35–50 pg, TP53^R175H-QSQS: 35–50 pg, TP53^DD: 35–50 pg, zebrafish Il1b: 20 pg) were injected into one-cell-stage embryos and maintained at 25.5–28.5 °C until 26–28 h post-fertilisation (hpf) (1 dpf). To immobilise zebrafish larvae, we co-injected α-Bungarotoxin mRNA (10 pg)⁸⁹.

mRNA and antisense oligo MO microinjection

Capped mRNA was synthesised using the SP6 mMessage mMachine kit (Ambion, Austin, TX, USA) and purified using Micro Bio-Spin columns (Bio-Rad, Hercules, CA, USA). We injected synthesised mRNA at the one-cell stage of zebrafish embryos. To perform knockdown experiments in zebrafish embryos, antisense oligo MOs (Gene Tools, Philomath, OR, USA) were injected into one-cell and/or two-cell stage embryos. MOs against IL-1β (il1b) (5 ng), pu.1 (spi1b) (5 ng)³³, tp53 (2.5–5 ng)⁹³, and cdkn2a/b (2.5 ng)⁹⁴ were used. MO sequences were shown in Supplementary Table 1.

Chemical treatment

To evaluate the effects of ROS on cell mass formation, zebrafish larvae were treated with 150 μM N-acetyl-L-cysteine NAC (Sigma-Aldrich, St. Louis, MO, USA) at 18 hpf (approximately 18 somite) for 8 h at 25.5–26.5 °C. For ROS detection (described below), 150 μM NAC solution was added at 10 hpf (approximately bud somite) overnight at 25.5–26.5 °C.

Doxorubicin hydrochloride (FUJIFILM Wako, Osaka, Japan; 040-21521) was dissolved in phosphate-buffered saline (PBS) at 5 mM and stored at -30 °C. Doxorubicin (50 μM) was added at 13 hpf (approximately 10 somite stage) for 10 h at 25.5–26.5 °C. Hydrogen peroxide (300 μM) was added at 13 hpf (approximately 10 somite stage) for 10 h at 25.5–26.5 °C.

2-Aminoethoxydiphenylborane (Sigma-Aldrich; D9754) was dissolved in dimethyl sulphoxide (DMSO) at 150 mM and stored at −30 °C. 2-Aminoethoxydiphenylborane (6.25 μM) was added at 18 hpf (approximately 18 somite) (Supplementary Fig. 13a) or at 24 hpf (approximately Prim-6 stage) (Supplementary Fig. 13b) at 28.5 °C. Dichloroacetate (Selleck Chemicals, Houston, TX; S8615) was dissolved in sterilized distilled water at 150 mM and stored at -30 °C. Dichloroacetate (30 mM) was added at 18 hpf (approximately 18 somite) (Supplementary Fig. 13a) or at 24 hpf (approximately Prim-6 stage) (Supplementary Fig. 13b) at 28.5 °C.

Detection of ROS

To detect ROS production, zebrafish larvae were incubated with 2.5 μM CellROX DeepRed (Thermo Fisher Scientific, Waltham, MA, USA) in egg water for 2 h, or with 5 μM CellROX Green (Thermo Fisher Scientific) in egg water for 5 h at 25.5-26.5 °C. After loading, embryos were washed three times in egg water and confocal images were obtained.

Detection of SA-β-gal-positive senescent cells using SPiDER-βGal

SA-β-gal using SPiDER-βGal was performed according to the manufacturer’s instructions. In detail, larvae were fixed with 4% paraformaldehyde in PBS (4% PFA) for 1 h at RT. After washing three times with PBS, to detect senescent cells, larvae were incubated with SPiDER-βGal (DOJINDO, Kumamoto, Japan, SG03), which enables specific detection of SA-β-gal activity-positive cells in living tissue at single-cell resolution⁹⁵, in Mcllvaine buffer (pH 6.0, dilution 1/1000) for 30 min at 37 °C. After loading, larvae were washed three times in 0.1% Tween 20 in PBS, and then immunostaining was performed.

Detection of proliferating cells using EdU

To detect proliferating cells, zebrafish larvae were incubated with 500 μM EdU/10% DMSO in Egg water for 1 h, and larvae were washed three times in egg water. Larvae were fixed with 4% PFA in PBS for 1 h at RT. The larvae were rinsed in double-distilled water, placed in acetone at 20 °C for 7 min, rinsed in double-distilled water, and permeabilized for 1 h in PBS/1%DMSO/1%Triton. For EdU detection, larvae were processed according to the Click-iT EdU Cell Proliferation Assay Kit (Invitrogen, Carlsbad, CA, USA; Alexa Fluor 488: C10637 and Alexa Fluor 647: C10640). After loading, larvae were washed three times in 0.1% Tween 20 in PBS, and then immunostaining was performed.

Whole-mount immunostaining

At 26–32 hpf, Tg(krt4p:gal4) larvae with mosaically introduced fluorescent cells were manually dechorionated and then fixed with 4% PFA overnight at 4 °C. As for H3K9me3, antigen retrieval was performed⁹⁶. The larvae were washed four times with 0.5% Triton X-100 (PBST) and incubated with blocking reagent (10% foetal bovine serum, 4% Block Ace (Megmilk Snow Brand, Tokyo, Japan), and 1% DMSO in 0.1% PBST) for 1 h (γH2AX-stained larvae were incubated with blocking reagent without Block Ace). The larvae were incubated with primary antibodies overnight at 4 °C, washed, and incubated with AlexaFluor-conjugated secondary antibodies and with Hoechst33342 (Invitrogen) overnight at 4 °C. Immunostained larvae were visualised with a M205FA fluorescent stereomicroscope (Leica), LSM700, or FV1000 (Olympus, Tokyo, Japan) confocal laser-scanning microscope. Images were prepared and analysed using ImageJ software (NIH, Bethesda, MD, USA). A list of primary and secondary antibodies is shown in Supplementary Table 1.

Whole-mount in situ hybridisation

Whole-mount in situ hybridisation was performed according to a standard protocol. Fluorescence in situ hybridisation was performed according to a previously described protocol⁹⁷. Digoxigenin- or FITC-labelled RNA antisense probes were prepared from plasmids containing GFP, Achilles, mKO2, lcp1, npsn, il1b, il6, il11b, cxcl8a, and cdkn2a/b. Images were taken using a M205A stereomicroscope (Leica) and FV3000 confocal laser scanning microscope.

Haematoxylin and eosin staining

The fixed larvae were embedded in paraffin and serially sectioned. Sections of 4-μm thickness were stained with haematoxylin and eosin according to a standard procedure.

Estimation of cytoplasm and nuclear size

The cytoplasm/nuclear areas in each cell were measured using ImageJ software. To estimate the size of the cytoplasm/nuclear area, the sum of the measured areas was multiplied by the thickness between the slices.

Quantification of membrane zebrafish E-cadherin-GFP levels

In Supplementary Fig 5a, b, UAS:zE-Cadherin-GFP plasmid (10 pg) were co-injected with UAS:mCherry-T2A-H-Ras^G12V into one-cell-stage Tg embryos (krt4p: gal4). In Supplementary Fig 5c, d, UAS:mCherry-T2A-H-Ras^G12V were injected into one-cell-stage Tg embryos (krt4p: gal4; UAS:zE-cadherin-GFP). The fluorescence intensity of E-cadherin-GFP in the membrane and cytosolic areas in each region was measured after subtracting background signals using ImageJ software (NIH, Bethesda, MD, USA).

Detection of F-actin

To detect F-actin, 1 dpf zebrafish larvae were incubated with Alexa Fluor488-conjugated phalloidin (Invitrogen, Carlsbad, CA, USA; A12379, 1:50 dilution) overnight at 4°C.

Quantitative PCR (qPCR)

UAS promoter-driven plasmids (Ras expression plasmid doses were as follows: 25 pg in Supplementary Fig. 3c, e, 15 pg in Supplementary Fig. 7d, 12.5 pg in Supplementary Fig. 12 f) were injected into one-cell-stage embryos. Total RNA from 25 larvae was purified using TRIzol reagents (Invitrogen), and cDNAs were synthesised using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). qPCR was performed on an Mx3000P QPCR system (Agilent Technologies, Santa Clara, CA, USA) with THUNDERBIRD SYBR qPCR Mix (Toyobo); the specific primers are described in Supplementary Table 1; actb1 was used as a loading control. qPCR cycling conditions were as follows: 95 °C for 1 min, [95 °C for 10 s, 60 °C for 30 s] (45 cycles), followed by dissociation curve analysis. The primer list is shown in Supplementary Table 1.

Generation of Tg(cdkn2a/b-hs:Achilles) zebrafish

To generate a donor plasmid containing a heat-shock promoter and cDNA encoding zebafish-codon-optimized and nuclear-localization-signal-tagged Achilles (Tbait-hs-zAchilles-NLS), zAchilles-NLS cDNA was synthesised (FASMAC, Atsugi, Japan) and replaced with GFP sequence in Tbait-hs-GFP plasmid (a gift from Dr. S. Higashijima)⁹⁸. Tbait-hs-zAchilles-NLS was co-injected with a single-guide RNA (sgRNA) targeted for digestion of cdkn2a/b, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA into one-cell stage wild-type embryos. A transgenic fish was outcrossed with a wild-type fish to produce the founder line. sgRNA sequences are described in Supplementary Table 1.

Luciferase assays

HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and a 100 U/ml penicillin-streptomycin mixed solution. Cells were seeded into 35 mm-diameter plates and transfected with the expression plasmids and TP53 reporter plasmids using polyethylenimine MW 25000 (Polysciences, Warrington, PA, USA). After 24 h, firefly and Renilla luciferase activities were determined with the Promega Dual Luciferase Assay System (Promega, Madison, WI, USA). The pRL-EF vector, which expresses Renilla luciferase under the control of the EF-1α promoter, was used to normalize the transfection efficiency of the luciferase reporters. The values shown are the averages of experiments repeated three times, with each transfection performed in duplicate for each experiment.

Time-lapse imaging

For time-lapse live imaging, Tg(krt4p:gal4) larvae with mosaically introduced Ras^G12V cells were manually dechorionated using forceps and then mounted in 2% methylcellulose onto glass-bottom dishes (Matsunami Glass, Osaka, Japan). Images were collected using an MZ16FA stereomicroscope (Leica, Wetzlar, Germany) equipped with Objective plan-apo 1.0x (10447157) at 28 °C for approximately 2 h, 30 min, or 6 h (Supplementary Movie 1, 2, or 3, respectively). The time interval was 5 min.

For time-lapse confocal live imaging, larvae were manually dechorionated by forceps and mounted in 1% low melting agarose with egg water onto glass-bottom dishes. Live imaging was performed using an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 20 × 1.0 NA objective. Two laser lines, 488 and 561 nm were used. The recording interval was 20 min. At each time point, 40–50 confocal slices through the z-axis were acquired. Collected image data were processed using ImageJ.

Statistics and Reproducibility

Differences between groups were examined using a two-tailed unpaired Student’s t-test, one-way analysis of variance, and Kruskal-Wallis test in Excel (Microsoft, Redmond, WA, USA), R (The R Project for Statistical Computing), or Prism8 (GraphPad, Inc., San Diego, CA, USA). p values < 0.05 were considered to indicate significant results. Figures of representative images or plots were reproduced in at least two (Figs. 2a–e, 4a, 5c–i, 5k, 6a–i, Supplementary Fig. 1e, 3a–e, 4b, 5b–d, 6b–c, 7c–d, 8a, 9a–b, 10b, 11a–c, 12a–d, 12f, 13a–b), or three or more (Figs. 1b, c, 2f, 3a–d, 4b, 5a–b, 5j Supplementary Fig. 1a–d, 2a–e, 4a, 5a, 6a, 7b, 8b, 10c–f, 11d, 12e) independent experiments.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Source data are provided with this paper. All the other data are available within the article and its Supplementary Information. Source data are provided with this paper.

References

Ashworth, A., Lord, C. J. & Reis-Filho, J. S. Genetic interactions in cancer progression and treatment. Cell 145, 30–38 (2011).
Article CAS PubMed Google Scholar
Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).
Article CAS PubMed Google Scholar
Hingorani, S. R. et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7, 469–483 (2005).
Article CAS PubMed Google Scholar
Janssen, K.-P. et al. APC and oncogenic KRAS are synergistic in enhancing Wnt signaling in intestinal tumor formation and progression. Gastroenterology 131, 1096–1109 (2006).
Article CAS PubMed Google Scholar
Buganim, Y. et al. p53 Regulates the Ras circuit to inhibit the expression of a cancer-related gene signature by various molecular pathways. Cancer Res. 70, 2274–2284 (2010).
Article CAS PubMed Google Scholar
Zhao, S., Wang, Y., Cao, L., Ouellette, M. M. & Freeman, J. W. Expression of oncogenic K-ras and loss of Smad4 cooperate to induce the expression of EGFR and to promote invasion of immortalized human pancreas ductal cells. Int. J. Cancer 127, 2076–2087 (2010).
Article CAS PubMed PubMed Central Google Scholar
Acin, S. et al. Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras. J. Pathol. 225, 479–489 (2011).
Article CAS PubMed PubMed Central Google Scholar
Nowell, P. C. The clonal evolution of tumor cell populations. Science 194, 23–28 (1976).
Article ADS CAS PubMed Google Scholar
Greaves, M. & Maley, C. C. Clonal evolution in cancer. Nature 481, 306–313 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Balani, S., Nguyen, L. V. & Eaves, C. J. Modeling the process of human tumorigenesis. Nat. Commun. 8, 15422 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Stratton, M. R., Campbell, P. J. & Futreal, P. A. The cancer genome. Nature 458, 719–724 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Vogelstein, B. et al. Cancer genome landscapes. Science 339, 1546–1558 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Garraway, L. A. & Lander, E. S. Lessons from the cancer genome. Cell 153, 17–37 (2013).
Article CAS PubMed Google Scholar
Collado, M. & Serrano, M. Senescence in tumours: evidence from mice and humans. Nat. Rev. Cancer 10, 51–57 (2010).
Article CAS PubMed PubMed Central Google Scholar
Rodier, F. & Campisi, J. Four faces of cellular senescence. J. Cell Biol. 192, 547–556 (2011).
Article CAS PubMed PubMed Central Google Scholar
Ohtani, N. & Hara, E. Roles and mechanisms of cellular senescence in regulation of tissue homeostasis. Cancer Sci. 104, 525–530 (2013).
Article CAS PubMed PubMed Central Google Scholar
Gorgoulis, V. et al. Cellular Senescence: Defining a Path Forward. Cell 179, 813–827 (2019).
Article CAS PubMed Google Scholar
Bartkova, J. et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870 (2005).
Article ADS CAS PubMed Google Scholar
Michaloglou, C. et al. BRAF^E600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720–724 (2005).
Article ADS CAS PubMed Google Scholar
Braig, M. et al. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436, 660–665 (2005).
Article ADS CAS PubMed Google Scholar
Coppé, J.-P. et al. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 6, 2853–2868 (2008).
Article PubMed Google Scholar
Coppé, J.-P., Kauser, K., Campisi, J. & Beauséjour, C. M. Secretion of vascular endothelial growth factor by primary human fibroblasts at senescence. J. Biol. Chem. 281, 29568–29574 (2006).
Article PubMed Google Scholar
Wang, B., Hendricks, D. T., Wamunyokoli, F. & Parker, M. I. A growth-related oncogene/CXC chemokine receptor 2 autocrine loop contributes to cellular proliferation in esophageal cancer. Cancer Res. 66, 3071–3077 (2006).
Article CAS PubMed Google Scholar
Neves, J., Demaria, M., Campisi, J. & Jasper, H. Of flies, mice, and men: evolutionarily conserved tissue damage responses and aging. Dev. Cell 32, 9–18 (2015).
Article CAS PubMed PubMed Central Google Scholar
Akieda, Y. et al. Cell competition corrects noisy Wnt morphogen gradients to achieve robust patterning in the zebrafish embryo. Nat. Commun. 10, 4710 (2019).
Article ADS PubMed PubMed Central Google Scholar
Takeuchi, Y. et al. Calcium Wave Promotes Cell Extrusion. Curr. Biol. 30, 4 (2020).
Article Google Scholar
Wada, H. et al. Wnt/Dkk negative feedback regulates sensory organ size in zebrafish. Curr. Biol. 23, 1559–1565 (2013).
Article CAS PubMed Google Scholar
Bos, J. L. The ras gene family and human carcinogenesis. Mutat. Res. 195, 255–271 (1988).
Article CAS PubMed Google Scholar
Prior, I. A., Lewis, P. D. & Mattos, C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 72, 2457–2467 (2012).
Article CAS PubMed PubMed Central Google Scholar
Adams, J. M. & Cory, S. Transgenic models of tumor development. Science 254, 1161–1167 (1991).
Article ADS CAS PubMed Google Scholar
White, R., Rose, K. & Zon, L. Zebrafish cancer: the state of the art and the path forward. Nat. Rev. Cancer 13, 624–636 (2013).
Article CAS PubMed PubMed Central Google Scholar
Brumby, A. M. & Richardson, H. E. Using Drosophila melanogaster to map human cancer pathways. Nat. Rev. Cancer 5, 626–639 (2005).
Article CAS PubMed Google Scholar
Rhodes, J. et al. Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish. Dev. Cell 8, 97–108 (2005).
Article CAS PubMed Google Scholar
Trede, N. S., Langenau, D. M., Traver, D., Look, A. T. & Zon, L. I. The use of zebrafish to understand immunity. Immunity 20, 367–379 (2004).
Article CAS PubMed Google Scholar
Gabay, M., Li, Y. & Felsher, D. W. MYC activation is a hallmark of cancer initiation and maintenance. Cold Spring Harb. Perspect. Med. 4, a014241 (2014).
Article PubMed PubMed Central Google Scholar
Yeatman, T. J. A renaissance for SRC. Nat. Rev. Cancer 4, 470–480 (2004).
Article CAS PubMed Google Scholar
Muller, P. A. J. & Vousden, K. H. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell 25, 304–317 (2014).
Article CAS PubMed PubMed Central Google Scholar
Kobayashi, Y. et al. Nuclear swelling occurs during premature senescence mediated by MAP kinases in normal human fibroblasts. Biosci. Biotechnol. Biochem. 72, 1122–1125 (2008).
Article CAS PubMed Google Scholar
Hernandez-Segura, A., Nehme, J. & Demaria, M. Hallmarks of Cellular Senescence. Trends Cell Biol. 28, 436–453 (2018).
Article CAS PubMed Google Scholar
van Roy, F. & Berx, G. The cell-cell adhesion molecule E-cadherin. Cell. Mol. Life Sci. 65, 3756–3788 (2008).
Article CAS PubMed Google Scholar
Freed-Pastor, W. A. & Prives, C. Mutant p53: one name, many proteins. Genes Dev. 26, 1268–1286 (2012).
Article CAS PubMed PubMed Central Google Scholar
Podsypanina, K., Politi, K., Beverly, L. J. & Varmus, H. E. Oncogene cooperation in tumor maintenance and tumor recurrence in mouse mammary tumors induced by Myc and mutant Kras. Proc. Natl Acad. Sci. U. S. A. 105, 5242–5247 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Bowman, T. et al. Tissue-specific inactivation of p53 tumor suppression in the mouse. Genes Dev. 10, 826–835 (1996).
Article CAS PubMed Google Scholar
Zhu, J., Zhou, W., Jiang, J. & Chen, X. Identification of a novel p53 functional domain that is necessary for mediating apoptosis. J. Biol. Chem. 273, 13030–13036 (1998).
Article CAS PubMed Google Scholar
Brady, C. A. et al. Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression. Cell 145, 571–583 (2011).
Article CAS PubMed PubMed Central Google Scholar
Solomon, H. et al. Various p53 mutant proteins differently regulate the Ras circuit to induce a cancer-related gene signature. J. Cell Sci. 125, 3144–3152 (2012).
CAS PubMed Google Scholar
Kastenhuber, E. R. & Lowe, S. W. Putting p53 in Context. Cell 170, 1062–1078 (2017).
Article CAS PubMed PubMed Central Google Scholar
Correia-Melo, C., Hewitt, G. & Passos, J. F. Telomeres, oxidative stress and inflammatory factors: partners in cellular senescence? Longev. Healthspan 3, 1 (2014).
Article PubMed PubMed Central Google Scholar
Krizhanovsky, V. et al. Senescence of activated stellate cells limits liver fibrosis. Cell 134, 657–667 (2008).
Article CAS PubMed PubMed Central Google Scholar
Faget, D. V., Ren, Q. & Stewart, S. A. Unmasking senescence: context-dependent effects of SASP in cancer. Nat. Rev. Cancer 19, 439–453 (2019).
Article CAS PubMed Google Scholar
Ingram, J. L., Rice, A. B., Geisenhoffer, K., Madtes, D. K. & Bonner, J. C. IL‐13 and IL‐1β promote lung fibroblast growth through coordinated up‐regulation of PDGF‐AA and PDGF‐Rα. FASEB J. 18, 1132–1134 (2004).
Article CAS PubMed Google Scholar
Bent, R., Moll, L., Grabbe, S. & Bros, M. Interleukin-1 Beta-A Friend or Foe in Malignancies? Int. J. Mol. Sci. 19, 2155 (2018).
Schieber, M. & Chandel, N. S. ROS function in redox signaling and oxidative stress. Curr. Biol. 24, R453–R462 (2014).
Article CAS PubMed PubMed Central Google Scholar
Yoshioka-Kobayashi, K. et al. Coupling delay controls synchronized oscillation in the segmentation clock. Nature 580, 119–123 (2020).
Article ADS CAS PubMed Google Scholar
Liu, D. & Xu, Y. p53, Oxidative Stress, and Aging. Antioxid. Redox Signal. 15, 1669–1678 (2011).
Article CAS PubMed PubMed Central Google Scholar
Ewald, J. A., Desotelle, J. A., Wilding, G. & Jarrard, D. F. Therapy-induced senescence in cancer. J. Natl Cancer Inst. 102, 1536–1546 (2010).
Article CAS PubMed PubMed Central Google Scholar
Forrest, R. A. et al. Activation of DNA damage response pathways as a consequence of anthracycline-DNA adduct formation. Biochem. Pharmacol. 83, 1602–1612 (2012).
Article CAS PubMed Google Scholar
Xue, W. et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 445, 656–660 (2007).
Article CAS PubMed PubMed Central Google Scholar
Kang, T.-W. et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 479, 547–551 (2011).
Article ADS CAS PubMed Google Scholar
Honda, T. et al. v-Src-driven transformation is due to chromosome abnormalities but not Src-mediated growth signaling. Sci. Rep. 8, 1063 (2018).
Article ADS PubMed PubMed Central Google Scholar
Hogan, C. et al. Characterization of the interface between normal and transformed epithelial cells. Nat. Cell Biol. 11, 460–467 (2009).
Article CAS PubMed Google Scholar
Kajita, M. et al. Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Src-transformed cells. J. Cell Sci. 123, 171–180 (2010).
Article CAS PubMed Google Scholar
Kajita, M. & Fujita, Y. EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals. J. Biochem. 158, 15–23 (2015).
Article CAS PubMed Google Scholar
Kon, S. et al. Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes. Nat. Cell Biol. 19, 530–541 (2017).
Article CAS PubMed Google Scholar
Sasaki, A. et al. Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues. Cell Rep. 23, 974–982 (2018).
Article CAS PubMed PubMed Central Google Scholar
van Wetering, S. et al. Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells. J. Cell Sci. 115, 1837–1846 (2002).
Article PubMed Google Scholar
Fearon, E. R. & Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 61, 759–767 (1990).
Article CAS PubMed Google Scholar
Maitra, A. & Hruban, R. H. Pancreatic cancer. Annu. Rev. Pathol. 3, 157–188 (2008).
Article CAS PubMed PubMed Central Google Scholar
Oren, M. & Rotter, V. Mutant p53 gain-of-function in cancer. Cold Spring Harb. Perspect. Biol. 2, a001107 (2010).
Article PubMed PubMed Central Google Scholar
Lang, G. A. et al. Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119, 861–872 (2004).
Article CAS PubMed Google Scholar
Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell 119, 847–860 (2004).
Article CAS PubMed Google Scholar
Caulin, C. et al. An inducible mouse model for skin cancer reveals distinct roles for gain- and loss-of-function p53 mutations. J. Clin. Invest. 117, 1893–1901 (2007).
Article CAS PubMed PubMed Central Google Scholar
Jiang, D. et al. Comparison of effects of p53 null and gain-of-function mutations on salivary tumors in MMTV-Hras transgenic mice. PLoS One 10, e0118029 (2015).
Article PubMed PubMed Central Google Scholar
Morton, J. P. et al. Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. Proc. Natl Acad. Sci. U. S. A 107, 246–251 (2010).
Article ADS CAS PubMed Google Scholar
Acosta, J. C. et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat. Cell Biol. 15, 978–990 (2013).
Article CAS PubMed PubMed Central Google Scholar
Deng, G., Lu, Y., Zlotnikov, G., Thor, A. D. & Smith, H. S. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science 274, 2057–2059 (1996).
Article ADS CAS PubMed Google Scholar
Lakhani, S. R. et al. Genetic alterations in “normal” luminal and myoepithelial cells of the breast. J. Pathol. 189, 496–503 (1999).
Article CAS PubMed Google Scholar
Jüngst, C. et al. Oxidative damage is increased in human liver tissue adjacent to hepatocellular carcinoma. Hepatology 39, 1663–1672 (2004).
Article PubMed Google Scholar
Chandran, U. R. et al. Differences in gene expression in prostate cancer, normal appearing prostate tissue adjacent to cancer and prostate tissue from cancer free organ donors. BMC Cancer 5, 45 (2005).
Article PubMed PubMed Central Google Scholar
Liu, G.-Y. et al. Novel cancerization marker, TP53, and its role in distinguishing normal tissue adjacent to cancerous tissue from normal tissue adjacent to benign tissue. World. J. Surg. Oncol. 10, 252 (2012).
Google Scholar
Aran, D. et al. Comprehensive analysis of normal adjacent to tumor transcriptomes. Nat. Commun. 8, 1077 (2017).
Article ADS PubMed PubMed Central Google Scholar
Beck, B. & Blanpain, C. Unravelling cancer stem cell potential. Nat. Rev. Cancer 13, 727–738 (2013).
Article CAS PubMed Google Scholar
Dexter, D. L. & Leith, J. T. Tumor heterogeneity and drug resistance. J. Clin. Oncol. 4, 244–257 (1986).
Article CAS PubMed Google Scholar
Hasegawa, T. et al. Transient inflammatory response mediated by interleukin-1β is required for proper regeneration in zebrafish fin fold. Elife 6, e22716 (2017).
Article PubMed PubMed Central Google Scholar
Ricci, M. S. et al. Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity. Mol. Cell. Biol. 24, 8541–8555 (2004).
Article CAS PubMed PubMed Central Google Scholar
Yu, J. et al. Identification and classification of p53-regulated genes. Proc. Natl Acad. Sci. U. S. A 96, 14517–14522 (1999).
Article ADS CAS PubMed PubMed Central Google Scholar
el-Deiry, W. S. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825 (1993).
Article CAS PubMed Google Scholar
Kardash, E. et al. A role for Rho GTPases and cell-cell adhesion in single-cell motility in vivo. Nat. Cell Biol. 12, 47–53 (2010).
Article CAS PubMed Google Scholar
Swinburne, I. A., Mosaliganti, K. R., Green, A. A. & Megason, S. G. Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin. PLoS One 10, e0134005 (2015).
Article PubMed PubMed Central Google Scholar
Shimizu, N., Kawakami, K. & Ishitani, T. Visualization and exploration of Tcf/Lef function using a highly responsive Wnt/β-catenin signaling-reporter transgenic zebrafish. Dev. Biol. 370, 71–85 (2012).
Article CAS PubMed Google Scholar
Herbomel, P., Thisse, B. & Thisse, C. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126, 3735–3745 (1999).
Article CAS PubMed Google Scholar
Di, Q. et al. Zebrafish nephrosin helps host defence against Escherichia coli infection. Open Biol. 7, 170040 (2017).
Article PubMed PubMed Central Google Scholar
Robu, M. E. et al. p53 activation by knockdown technologies. PLoS Genet 3, e78 (2007).
Article PubMed PubMed Central Google Scholar
Mochida, G. H. et al. CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development. Nat. Genet. 44, 1260–1264 (2012).
Article CAS PubMed PubMed Central Google Scholar
Doura, T. et al. Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution. Angew. Chem. Int. Ed. Engl. 55, 9620–9624 (2016).
Article CAS PubMed Google Scholar
Inoue, D. & Wittbrodt, J. One for all–a highly efficient and versatile method for fluorescent immunostaining in fish embryos. PLoS One 6, e19713 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Brend, T. & Holley, S. A. Zebrafish whole mount high-resolution double fluorescent in situ hybridization. J. Vis. Exp. 25, 1229 (2009).
Kimura, Y., Hisano, Y., Kawahara, A. & Higashijima, S.-I. Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering. Sci. Rep. 4, 6545 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank E. Hara for his helpful discussion, H. Wada, K. Kawakami, A. Yoshimura, B. Vogelstein, W. El-Deiry, Y. Fujita, E. Raz, S. Higashijima, and A. Miyawaki for providing the plasmids, H. Wada and NBRP for providing transgenic zebrafish, and Ishitani lab members for their helpful discussion, technical support, and fish maintenance. This research was supported by the Takeda Foundation (T.I.), Mitsubishi Foundation (T.I.), Kao Foundation (Y.A.), Nagase Science and Technology Foundation (T.I.), and Kawano Foundation (T.I.), Daiichi Sankyo Foundation (T.I.), Uehara Memorial Foundation (T.I.), Mochida Memorial Foundation (T.I.), AMED (21gm5010001h0005) (T.I.), and a Grant-in-Aid for Scientific Research on Innovative Areas (25117720, 20H05365) (T.I.) (20H05534) (Y.A.), Young Scientists (20770514) (Y.A.), Exploratory Research (20K21502) (T.I.), Transformative Research Areas(A) (21H05287) (T.I.), JST SPRING (Y.H.), and Scientific Research (B) (19H03412) (T.I.).

Author information

Authors and Affiliations

Department of Homeostatic Regulation, Division of Cellular and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan
Yukinari Haraoka, Yuki Akieda, Yuri Nagai & Tohru Ishitani
Institute for Molecular & Cellular Regulation, Gunma University, Gunma, 371-8512, Japan
Chihiro Mogi & Tohru Ishitani
Center for Infectious Disease Education and Research (CiDER), Osaka University, Suita, Osaka, 565-0871, Japan
Tohru Ishitani

Authors

Yukinari Haraoka
View author publications
You can also search for this author in PubMed Google Scholar
Yuki Akieda
View author publications
You can also search for this author in PubMed Google Scholar
Yuri Nagai
View author publications
You can also search for this author in PubMed Google Scholar
Chihiro Mogi
View author publications
You can also search for this author in PubMed Google Scholar
Tohru Ishitani
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Conception and design: Y.H., Y.A., and T.I.; Investigation: Y.H., Y.A., Y.N., C.M., and T.I.; Writing and review: Y.H. and T.I.; Writing contribution and review: Y.H., Y.A., and T.I.

Corresponding author

Correspondence to Tohru Ishitani.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Manuel Collado, Valery Krizhanovsky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Peer Review File

Description of Additional Supplementary Files

Supplementary Movie 1

Supplementary Movie 2

Supplementary Movie 3

Supplementary Movie 4

Supplementary Movie 5

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Haraoka, Y., Akieda, Y., Nagai, Y. et al. Zebrafish imaging reveals TP53 mutation switching oncogene-induced senescence from suppressor to driver in primary tumorigenesis. Nat Commun 13, 1417 (2022). https://doi.org/10.1038/s41467-022-29061-6

Download citation

Received: 02 September 2020
Accepted: 24 February 2022
Published: 18 March 2022
DOI: https://doi.org/10.1038/s41467-022-29061-6

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.