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The balance between phosphoinositides distributed at specific sites in the plasma membrane causes polarized actin polymerization. Oncogenic transformations affect this balance by regulating phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN), causing metastatic behavior in cancer cells. Here, we show that the PTEN tumor suppressor gene is required for epithelial cancer cell invasion. Loss of PTEN in Ras-transformed MDCK cells suppressed their migratory phenotype in collagen gel and invasion through Matrigel. Rescue experiments showed a requirement for the C2 domain-mediated membrane recruitment of PTEN, which is typically observed at the rear side of invading cancer cells. These findings support the role of PTEN in suppression of unwanted leading edges necessary for efficient migration of epithelial cancer cells.

Introduction Our previous clinical studies have demonstrated the short-term efficacy and safety of the sirolimus gel for patients with tuberous sclerosis complex (TSC). However, long-term clinical evidence is lacking. Our objective was to assess the safety and efficacy of long-term treatment with the sirolimus gel for the skin lesions of TSC patients. Methods We conducted a multicenter, open-label, uncontrolled clinical trial in 94 Japanese patients with TSC. Patients applied the 0.2% sirolimus gel on their face or head twice daily for > 52 weeks (maximum 136 weeks for safety). The safety endpoints were the rate of adverse event (AE)-caused discontinuation (primary endpoint) and the incidence of AEs. The efficacy endpoint was the response rate of angiofibromas, cephalic plaques, and hypomelanotic macules. Results Among 94 enrolled patients (mean age, 21 years; range 3–53 years), the rate of AE-caused discontinuation was 2.1% (2/94 patients). Although application site irritation and dry skin occurred relatively frequently, none of the drug-related AEs were serious; most of the drug-related AEs resolved rapidly. The major drug-related AEs (≥ 5% in incidence) were application site irritation (30.9%), dry skin (27.7%), acne (20.2%), eye irritation (8.5%), pruritus (8.5%), erythema (7.4%), dermatitis acneiform (6.4%), and dermatitis contact (5.3%). The response rates of angiofibromas, cephalic plaques, and hypomelanotic macules were 78.2% [95% confidence interval (CI) 68.0–86.3%], 66.7% (95% CI 51.1–80.0%), and 72.2% (95% CI 46.5–90.3%), respectively. Conclusions The gel was well tolerated for a long time by patients with TSC involving facial skin lesions and continued to be effective. Trial Registration ClinicalTrials.gov identifier: NCT02634931.

Cell competition is a biological process by which unfit cells are eliminated from "cell society." We previously showed that cultured mammalian epithelial Madin-Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by "normal" MDCK cells. However, the molecular mechanism underlying the elimination of active YAP-expressing cells was unknown. Here, we used high-throughput chemical compound screening to identify cyclooxygenase-2 (COX-2) as a key molecule triggering cell competition. Our work shows that COX-2-mediated PGE2 secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase-cyclic AMP-PKA pathway that, in the presence of active YAP, induces E-cadherin internalization leading to apical extrusion. Thus, COX-2-induced PGE(2) appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.

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.

Objective To determine whether vaginal progesterone (VP) reduces the rate of preterm birth in pregnant women after abdominal trachelectomy (AT) for early-stage cervical cancer Design Prospective cohort study with a historical cohort Setting University hospital Population Twelve pregnancies in ten women were included in the VP group between October 2016 and September 2020. By contrast, 19 pregnancies in 17 women were included in the historical control group between January 2007 and September 2016. Methods For the interventional study participants, the administration of vaginal progesterone was started between 16+0 and 19+6 weeks of gestation and discontinued at 34 weeks of gestation or at the time of delivery, rupture of membranes, or massive uterine bleeding, whichever occurred first. We investigated obstetric and neonatal outcomes among the study participants and compared them with outcomes of the historical control group participants. Main Outcome Measures The gestational age at delivery and incidence of preterm birth before 37 weeks and 34 weeks of gestation Results The incidence of preterm birth at

Cell competition is a process by which unwanted cells are eliminated from tissues. Apical extrusion is one mode whereby normal epithelial cells remove transformed cells, but it remains unclear how this process is mechanically effected. In this study, we show that autophagic and endocytic fluxes are attenuated in RasV12-transformed cells surrounded by normal cells due to lysosomal dysfunction, and that chemical manipulation of lysosomal activity compromises apical extrusion. We further find that RasV12 cells deficient in autophagy initiation machinery are resistant to elimination pressure exerted by normal cells, suggesting that non-degradable autophagic vacuoles are required for cell competition. Moreover, in vivo analysis revealed that autophagy-ablated RasV12 cells are less readily eliminated by cell competition, and remaining transformed cells destroy ductal integrity, leading to chronic pancreatitis. Collectively, our findings illuminate a positive role for autophagy in cell competition and reveal a homeostasis-preserving function of autophagy upon emergence of transformed cells.

SummaryMalignant cancers emerge in epithelial tissues through a progressive process in which a single transformed mutant cell becomes tumorigenic and invasive. Although numerous genes involved in the malignant transformation of cancer cells have been described, how tumor cells launch an invasion into the basal side of epithelial tissues remains elusive. Here, using a Drosophila wing imaginal disc epithelia, we show that genetically mosaic clones of cells mutant for a neoplastic-tumor-suppressor gene (nTSG) in combination with the oncogenic Ras (RasV12) expression initiate invasion into the basal side of the epithelial layer at specific spots in the epithelial tissue. In this “invasion hotspot”, the oncogenic double-mutant cells activate c-Jun N-terminal kinase (JNK) signaling, which causes basal extrusion of the double-mutant cells and destruction of basement membrane through upregulation of a matrix metalloprotease, MMP1. Conversely, in other regions of the epithelial tissue, the double-mutant cells do not strongly activate JNK, deviate from the apical side of the epithelial layer, and show benign tumor growth in the lumen. These data indicate that the onset of tumor-cell invasion is highly dependent on the tissue-intrinsic local microenvironment. Given the conservation of genetic signaling pathways involved in this process, initiation of tumor-cell invasion from invasion hotspots in Drosophila wing imaginal epithelia could help us to understand the developmental mechanisms of invasive cancers.

Cell competition is a process by which cells with different properties compete with each other for survival and space, and consequently suboptimal/abnormal cells are often eliminated from, in particular, epithelial tissues. In the last few years, cell competition studies have been developing at an explosive speed, and the molecular mechanisms of cell competition have been considerably revealed. For instance, upon cell competition, loser cells are eliminated from tissues via a variety of loser phenotypes, including apoptosis, cell differentiation, and cell death-independent extrusion. In addition, upstream regulatory mechanisms for the induction of these phenotypes have been elucidated. Furthermore, it has become evident that cell competition is involved in various physiological and pathological processes and thus is a crucial and indispensable homeostatic machinery that is required for embryonic development and prevention of diseases and ageing. Moreover, cell competition now has a profound impact on other research fields such as regenerative medicine. In this review, we will summarize the development of these recent studies, especially focusing on cell competition in vertebrates.

Fast ignition (FI) is a promising approach for high-energy-gain inertial confinement fusion in the laboratory. To achieve ignition, the energy of a short-pulse laser is required to be delivered efficiently to the pre-compressed fuel core via a high-energy electron beam. Therefore, understanding the transport and energy deposition of this electron beam inside the pre-compressed core is the key for FI. Here we report on the direct observation of the electron beam transport and deposition in a compressed core through the stimulated Cu Kα emission in the super-penetration scheme. Simulations reproducing the experimental measurements indicate that, at the time of peak compression, about 1% of the short-pulse energy is coupled to a relatively low-density core with a radius of 70 μm. Analysis with the support of 2D particle-in-cell simulations uncovers the key factors improving this coupling efficiency. Our findings are of critical importance for optimizing FI experiments in a super-penetration scheme., Fast ignition is an interesting scheme for nuclear fusion reaction. Here the authors show electron generation using intense short laser pulses and energy transport by coupling the laser energy to the imploded plasma core as in the ICF conditions.

Gastric cancer involving the placenta during pregnancy is rare; however, we present 1 such case in this report. A 31-year-old Japanese woman was referred at 26 weeks of gestation for the evaluation of a swollen left supraclavicular lymph node. Biopsy revealed poorly differentiated adenocarcinoma, and esophagogastroduodenoscopy with biopsy of the stomach confirmed the diagnosis of gastric cancer. Her epigastric and back pain became more pronounced and her general status worsened, and we performed a cesarean delivery at 29 weeks. Microscopic examination of the placental specimen revealed poorly differentiated adenocarcinoma cells diffused into the intervillous space. Postpartum chemotherapy consisted of S-1 plus oxaliplatin. Unfortunately, this treatment was ineffective, and the patient died 3 months after delivery. The infant did well, without clinical or laboratory manifestations of metastasis. In patients with advanced gastric cancer during pregnancy, it is important to perform a microscopic examination of the placenta to evaluate for metastatic involvement.

For isolated single cells on a substrate, the intracellular stiffness, which is often measured as the Young’s modulus, E, by atomic force microscopy (AFM), depends on the substrate rigidity. However, little is known about how the E of cells is influenced by the surrounding cells in a cell population system in which cells physically and tightly contact adjacent cells. In this study, we investigated the spatial heterogeneities of E in a jammed epithelial monolayer in which cell migration was highly inhibited, allowing us to precisely measure the spatial distribution of E in large-scale regions by AFM. The AFM measurements showed that E can be characterized using two spatial correlation lengths: the shorter correlation length, l(S), is within the single cell size, whereas the longer correlation length, l(L), is longer than the distance between adjacent cells and corresponds to the intercellular correlation of E. We found that l(L) decreased significantly when the actin filaments were disrupted or calcium ions were chelated using chemical treatments, and the decreased l(L) recovered to the value in the control condition after the treatments were washed out. Moreover, we found that l(L) decreased significantly when E-cadherin was knocked down. These results indicate that the observed long-range correlation of E is not fixed within the jammed state but inherently arises from the formation of a large-scale actin filament structure via E-cadherin-dependent cell-cell junctions.

Abnormal/cancerous cells within healthy epithelial tissues undergo apical extrusion to protect against carcinogenesis, although they acquire invasive capacity once carcinogenesis progresses. However, the molecular mechanisms by which cancer cells escape from apical extrusion and invade surrounding tissues remain elusive. In this study, we demonstrate a molecular mechanism for cell fate switching during epithelial cell competition. We found that during competition within epithelial cell layers, Src transformation promotes maturation of focal adhesions and degradation of extracellular matrix. Src-transformed cells underwent basal delamination by Src activation within sphingolipid/cholesterol-enriched membrane microdomains/lipid rafts, whereas they were apically extruded when Src was outside of lipid rafts. A comparative analysis of contrasting phenotypes revealed that activation of the Src-STAT3-MMP axis through lipid rafts was required for basal delamination. CUB-domain-containing protein 1 (CDCP1) was identified as an Src-activating scaffold and as a Met regulator in lipid rafts, and its overexpression induced basal delamination. In renal cancer models, CDCP1 promoted epithelial-mesenchymal transition-mediated invasive behavior by activating the Src-STAT3-MMP axis through Met activation. Overall, these results suggest that spatial activation of Src signaling in lipid rafts confers resistance to apical extrusion and invasive potential on epithelial cells to promote carcinogenesis.

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.

Infants with congenital diaphragmatic hernia (CDH) are at risk of neurodevelopmental disabilities. This study aimed to investigate the association between lung to thorax transverse area ratio (LTR) and neurodevelopmental outcomes at 3 years of age in fetuses with CDH.We performed a retrospective study of infants with prenatally diagnosed isolated left-sided CDH born in Kyushu University Hospital between 2008 and 2016. We examined the association between prenatal ultrasound findings including LTR and development quotient (DQ) at 36 to 42 months of chronological age.We identified 34 live-born fetuses with isolated left-sided CDH, of which 30 survived and four died before discharge. The median LTR in the survivors was higher than in the non-survivors (p 0.01). Among the survivors, 26 had available data on LTR (median 0.12, range 0.08-0.18) and overall DQ at 3 years of age (93, 61-112). Their median gestational age and birth weight were 37.6 (range 34.4-39.1) weeks and 2716 (2.256-3494) grams, respectively. There was no significant difference in overall DQ scores between the two groups divided according to the median LTR values (p = 0.62). LTR values were not associated with overall DQ scores after adjusting for gestational age (p = 0.39). In addition, no association was observed between LTR values and any subscale DQ scores.In fetuses with isolated left-sided CDH, prenatal LTR predicts the mortality but not neurodevelopmental outcomes at 3 years of age.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening mucocutaneous adverse drug reactions characterized by massive epidermal detachment. Cytotoxic T cells and associated effector molecules are known to drive SJS/TEN pathophysiology, but the contribution of innate immune responses is not well understood. We describe a mechanism by which neutrophils triggered inflammation during early phases of SJS/TEN. Skin-infiltrating CD8+ T cells produced lipocalin-2 in a drug-specific manner, which triggered the formation of neutrophil extracellular traps (NETs) in early lesional skin. Neutrophils undergoing NETosis released LL-37, an antimicrobial peptide, which induced formyl peptide receptor 1 (FPR1) expression by keratinocytes. FPR1 expression caused keratinocytes to be vulnerable to necroptosis that caused further release of LL-37 by necroptotic keratinocytes and induced FPR1 expression on surrounding keratinocytes, which likely amplified the necroptotic response. The NETs-necroptosis axis was not observed in less severe cutaneous adverse drug reactions, autoimmune diseases, or neutrophil-associated disorders, suggesting that this was a process specific to SJS/TEN. Initiation and progression of SJS/TEN keratinocyte necroptosis appear to involve a cascade of events mediated by innate and adaptive immune responses, and understanding these responses may contribute to the identification of diagnostic markers or therapeutic targets for these adverse drug reactions.

Epidermolysis bullosa (EB) is a group of genodermatoses characterized by generalized blisters from mutations in the genes encoding the basement membrane zone (BMZ) proteins.1 The infusion of allogeneic cells such as mesenchymal stem/stromal cells (MSCs), which have intact BMZ genes, is a promising treatment.

Due to the recent trend of women having children at an older age, the number of pregnancies complicated by cervical cancer has increased. In the present study, the clinical characteristics of patients with cervical cancer complicated by pregnancy were analyzed. The clinicopathological characteristics and prognosis of patients with cervical cancer during pregnancy who underwent treatment at Kyushu University Hospital from January 2008 to December 2017 were assessed retrospectively from their medical records. The medical information of patients diagnosed within 6 months after parturition was also evaluated as these patients were considered to be affected by cervical cancer during pregnancy. A total of 19 women were diagnosed with cervical cancer during pregnancy (median age, 33 years), three of whom were diagnosed as negative for intraepithelial lesion or malignancy at the initial visit to a previous clinic after pregnancy. The tumor stage was IA1 in one patient, IB1 in 16 patients, IB2 in one patient and IVB in one patient. The median gestational age at the time of cervical cancer diagnosis was 13 weeks. One patient died of cervical cancer during the follow-up period. An additional 12 patients were diagnosed within 6 months after parturition. The median age of these patients was 35.5 years. Cancer screening was performed in 10 patients during pregnancy, none of whom were diagnosed with cervical cancer, including three patients who were negative for intraepithelial lesion or malignancy. Of the 12 patients, one had tumor stage IA1, eight had IB1, two had IB2 and one had IIB. Three patients experienced recurrence, of whom one died of cervical cancer. Advanced cervical cancer was diagnosed both during pregnancy and within 6 months after parturition. Diagnosis of cervical cancer during pregnancy is difficult even at an advanced stage; however, rapid diagnosis and prompt multidisciplinary treatment are critical. Therefore, it is necessary to improve the accuracy of cervical cancer diagnosis, and to characterize tumor cells and their microenvironment, during pregnancy.

Malignancy is associated with changes in cell mechanics that contribute to extensive cell deformation required for metastatic dissemination. We hypothesized that the cell-intrinsic physical factors that maintain epithelial cell mechanics could function as tumor suppressors. Here we show, using optical tweezers, genetic interference, mechanical perturbations, and in vivo studies, that epithelial cells maintain higher plasma membrane (PM) tension than their metastatic counterparts and that high PM tension potently inhibits cancer cell migration and invasion by counteracting membrane curvature sensing/generating BAR family proteins. This tensional homeostasis is achieved by membrane-to-cortex attachment (MCA) regulated by ERM proteins, whose disruption spontaneously transforms epithelial cells into a mesenchymal migratory phenotype powered by BAR proteins. Consistently, the forced expression of epithelial–mesenchymal transition (EMT)-inducing transcription factors results in decreased PM tension. In metastatic cells, increasing PM tension by manipulating MCA is sufficient to suppress both mesenchymal and amoeboid 3D migration, tumor invasion, and metastasis by compromising membrane-mediated mechanosignaling by BAR proteins, thereby uncovering a previously undescribed mechanical tumor suppressor mechanism., Changes in cell mechanics contribute to cancer cell dissemination. Here the authors show that high plasma membrane (PM) tension inhibits cancer dissemination by counteracting mechanosensitive BAR family protein assembly, while restoration of PM tension phenotypically convert malignant cells into a non-motile epithelial cell state.

Objective: To determine whether vaginal progesterone (VP) reduces the rate of preterm birth in pregnant women after abdominal trachelectomy (AT) for early-stage cervical cancer / Study Design: This is an interventional study with a historical cohort. For the interventional study participants who had singleton pregnancies after AT between October 2016 and September 2020, the administration of vaginal progesterone was started between 16+ and 19+6 weeks of gestation and discontinued at 34 weeks of gestation or at the time of delivery, rupture of membranes, or massive uterine bleeding. We investigated obstetric and neonatal outcomes among the study participants and compared them with outcomes of the historical control group participants, included women with singleton pregnancies after AT who were managed without VP at our institution between January 2007 and September 2016, using Fisher’s exact test and the Mann–Whitney U test The main outcomes were the gestational age at delivery and incidence of preterm birth before 37 weeks and 34 weeks of gestation. / Result: Twelve pregnancies in ten women were included in the VP group. In contrast, 19 pregnancies in 17 women were included in the historical control group. The incidence of preterm birth at

Purpose The role of activated retinal microglia in driving retinal degeneration has been implicated in a number of in vivo disease models. Here, we investigated the primary consequences of microglial activation by the specific expression of constitutively active Ras in microglia in a transgenic mouse model before the onset of any degenerative changes in the retina. Methods The double transgenic lines CAG-LSL-RasV12-IRES-EGFP; Cx3cr1CreER/+ (Cx3cr1-RasV12 mice) and CAG-LSL-EGFP; Cx3cr1CreER_+ (control mice) were generated. The expression of RasV12 was induced in microglia by tamoxifen administration, and the retinas were examined by immunohistochemistry of frozen sections, RT-qPCR, and live imaging. Results RasV12 expression in retinal microglial cells promoted cell proliferation, cytokine expression, and phagocytosis. RasV12-expressing microglia migrated toward the inner and outer layers of the retina. Examination of glial fibrillary acidic protein (GFAP) expression revealed activation of Muller glia in the retina. We also observed loss of the photoreceptors in the outer nuclear layer in close proximity to microglial cells. However, no significant neurodegeneration was detected in the inner nuclear layer (INL) or ganglion cell layer (GCL). The morphology of RasV12-expressing microglia in the GCL and INL retained more ramified features compared with the predominantly-ameboid morphology found in outer retinal microglia. Conclusions The expression of RasV12 is sufficient to activate microglia and lead to photoreceptor degeneration. Neurons in the inner side of the retina were not damaged by the RasV12-activated microglia, suggesting that microenvironment cues may modulate the microglial phenotypic features and effects of microglial activation.

Objective We sought to investigate the efficacy of human bone marrow mesenchymal stem/stromal cell (hBM-MSC) in a murine spinal cord ischemia/reperfusion (SCIR) model. Methods C57BL/6J mice were subjected to SCIR by crossclamping the aortic arch and left subclavian artery for 5.5 minutes. Two hours after reperfusion, hBM-MSCs (hBM-MSC group) or phosphate-buffered saline (control group) were intravenously injected without immunosuppressant. Hindlimb motor function was assessed until day 28 after reperfusion using the Basso Mouse Scale (BMS). The lumbar spinal cord was harvested at hour 24 and day 28, and the histologic number of NeuN-positive motor neurons in 3 cross-sections of each lumbar spinal cord and the gene expression were evaluated. Results BMS score was 0 throughout the study period in all control mice. BMS score was significantly greater in the hBM-MSC group than the control group from hour 8 (P Conclusions hBM-MSC therapy may attenuate SCIR injury by preserving motor neurons, at least in part, through inhibition of proinflammatory cytokines and upregulation of proangiogenic factors in the reperfusion-injured spinal cord.

SummaryRevertant mosaicism, or ‘natural gene therapy’, refers to the spontaneous in vivo reversion of an inherited mutation in a somatic cell1. Only ∼50 human genetic disorders exhibit revertant mosaicism, implicating a distinctive role played by mutant proteins in somatic correction of a pathogenic germline mutation2. However, the process by which mutant proteins induce somatic genetic reversion in these diseases remains unknown. Here we show that heterozygous pathogenic CARD14 mutations causing autoinflammatory skin diseases, including psoriasis and pityriasis rubra pilaris, are repaired mainly via homologous recombination. Rather than altering the DNA damage response to exogenous stimuli such as X-irradiation or etoposide treatment, mutant CARD14 increased DNA double-strand breaks under conditions of replication stress. Furthermore, mutant CARD14 suppressed new origin firings without promoting crossover events in the replication stress state. Together, these results suggest that mutant CARD14 alters the replication stress response and preferentially drives break-induced replication (BIR), which is generally suppressed in eukaryotes3. Our results highlight the involvement of BIR in reversion events, thus revealing a previously undescribed role of BIR that could potentially be exploited to develop therapeutics for currently intractable genetic diseases.

Summary Previous studies have revealed that, at the initial step of carcinogenesis, transformed cells are often eliminated from epithelia via cell competition with the surrounding normal cells. In this study, we performed cell competition-based high-throughput screening for chemical compounds using cultured epithelial cells and confocal microscopy. PLX4720 was identified as a hit compound that promoted apical extrusion of RasV12-transformed cells surrounded by normal epithelial cells. Knockdown/knockout of ZAK, a target of PLX4720, substantially enhanced the apical elimination of RasV12 cells in vitro and in vivo. ZAK negatively modulated the accumulation or activation of multiple cell competition regulators. Moreover, PLX4720 treatment promoted apical elimination of RasV12-transformed cells in vivo and suppressed the formation of potentially precancerous tumors. This is the first report demonstrating that a cell competition-promoting chemical drug facilitates apical elimination of transformed cells in vivo, providing a new dimension in cancer preventive medicine., Graphical Abstract, Highlights • A cell competition-based high-throughput screen identifies chemical enhancers • PLX4720 enhances the apical elimination of RasV12 cells via ZAK inhibition • ZAK negatively modulates cell competition regulators • PLX4720 treatment promotes apical elimination of RasV12 cells in vivo, Chemistry; Biological Sciences; Cancer

Cell competition is a social cellular phenomenon in which unfit cells are selectively eliminated to maintain tissue homeostasis.1-3 Recent studies have revealed that mechanical forces induce competitive cell-cell interactions in Drosophila.4-6 This mechanical cell competition has also been reported to play an important role in mammalian cells, using Madin-Darby canine kidney (MDCK) cells depleted of a polarity regulator Scribble in a tetracycline-inducible manner (scribKD cells).7scribKD cells are hypersensitive to crowding due to the lower homeostatic density than wild-type (WT) cells,7,8 and in the context of cell competition, scribKD cells are compacted and eliminated by WT cells.7-10 Although p38 and p53 are involved in this process,7,10 the molecular mechanism by which WT cells recognize and mechanically eliminate scribKD cells remains unclear. Here, we report that scribKD cells secrete fibroblast growth factor 21 (FGF21) to drive cell competition. Knockdown of FGF21 in scribKD cells or loss of FGFR1 in WT cells suppresses cell competition, suggesting that WT cells recognize scribKD cells through FGF21. FGF21-containing culture medium of scribKD cells activates cell motility. Moreover, FGF21 promotes the compression and elimination of scribKD cells by attracting surrounding WT cells. We also demonstrate that activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway in scribKD cells induces FGF21 to drive cell competition. Our findings reveal a mechanism whereby WT cells mechanically eliminate scribKD cells and propose a new function for FGF21 in cell-cell communication.

Background Microglial activation is commonly observed in neurodegenerative diseases. However, its role in the complex inflammatory network remains unclear. In this study, we generated a microglial activation mouse model by inducing the expression of a constitutively active form of Ras in microglia.Methods The double transgenic lines CAG-LSL-RasV12-IRES-EGFP; Cx3cr1CreER (Cx3cr1-RasV12 mice) and CAG-LSL-EGFP;Cx3cr1CreER (control mice) were generated. The expression of RasV12 was induced in microglia by tamoxifen administration. Effects of the expression of RasV12 in the retina were examined by immunohistochemistry of frozen sections, RT-qPCR, and live imaging.Results RasV12 expression in retinal microglial cells promoted cell proliferation, cytokine expression, and phagocytosis. RasV12-expressing microglia migrated towards the apical and basal regions of the retina. Examination of GFAP expression revealed activation of Müller glia in the retina. We also observed loss of the photoreceptors in the outer nuclear layer (ONL) in close proximity to microglial cells. However, no neurodegeneration was observed in the inner nuclear layer (INL) or ganglion cell layer (GCL). Furthermore, we found that RasV12-expressing microglia in the ONL were smaller in size and engulfed photoreceptors. In contrast, the morphology of RasV12-expressing microglia in the GCL and INL resembled resting microglia. In conclusion, RasV12-induced microglial activation impaired rod photoreceptor survival in the ONL, but not in other regions of the retina.Conclusion The expression of RasV12 is sufficient to activate microglia, and our results suggested that microenvironment cues may modulate the microglial phenotypic features and effects of microglial activation.

Background The optimal timing of elective repeat caesarean delivery has yet to be determined. One of the reasons to schedule an elective repeat caesarean delivery before 39 weeks gestation is to avoid emergency caesarean delivery due to spontaneous onset of labour. Aims By ascertaining maternal characteristics and neonatal outcomes associated with early-term onset of spontaneous labour, we aim to determine the optimal timing for each individual repeat caesarean delivery. Materials and methods We performed a retrospective analysis of women with repeat caesarean deliveries planned at 38 weeks gestation between 2005 and 2019 at a tertiary referral hospital in Japan. A multivariate logistic regression analysis was adopted to identify independent contributing factors for early-term spontaneous labour onset. We also compared the rate of neonatal adverse events between women who underwent emergency repeat caesarean deliveries due to the onset of early-term labour and the ones who underwent elective repeat caesarean deliveries at 38 weeks. Results We included 1152 women. History of vaginal deliveries (adjusted odds ratio (AOR), 2.12; 95% confidence interval (95% CI), 1.21-3.74), history of preterm deliveries (AOR, 2.28; 95% CI, 1.38-3.77), and inadequate maternal weight gain during pregnancy (AOR, 1.78; 95% CI, 1.15-2.75) significantly increased the risk of early-term spontaneous labour onset. In terms of occurrence rate of neonatal complications, we found no significant difference between the groups. Conclusion These maternal factors are significant predictors for early-term labour onset of repeat caesarean deliveries. The onset of early-term labour did not increase the likelihood of neonatal complications.

At the initial stage of carcinogenesis, when RasV12-transformed cells are surrounded by normal epithelial cells, RasV12 cells are apically extruded from epithelia through cell competition with the surrounding normal cells. In this study, we demonstrate that expression of cyclooxygenase (COX)−2 is upregulated in normal cells surrounding RasV12-transformed cells. Addition of COX inhibitor or COX-2-knockout promotes apical extrusion of RasV12 cells. Furthermore, production of Prostaglandin (PG) E2, a downstream prostanoid of COX-2, is elevated in normal cells surrounding RasV12 cells, and addition of PGE2 suppresses apical extrusion of RasV12 cells. In a cell competition mouse model, expression of COX-2 is elevated in pancreatic epithelia harbouring RasV12-exressing cells, and the COX inhibitor ibuprofen promotes apical extrusion of RasV12 cells. Moreover, caerulein-induced chronic inflammation substantially suppresses apical elimination of RasV12 cells. These results indicate that intrinsically or extrinsically mediated inflammation can promote tumour initiation by diminishing cell competition between normal and transformed cells., Sato et al find that in an epithelial cell sheet containing some RasV12-transformed cells, expression of cyclooxygenase-2 and production of its downstream product prostaglandin E2 are increased in normal cells surrounding transformed cells, and suppress extrusion of the latter. This study sheds light on how transformed cells are eliminated from epithelia.

Cell competition is a biological process by which unfit cells are eliminated from “cell society.” We previously showed that cultured mammalian epithelial Madin‐Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by “normal” MDCK cells. However, the molecular mechanism underlying the elimination of active YAP‐expressing cells was unknown. Here, we used high‐throughput chemical compound screening to identify cyclooxygenase‐2 (COX‐2) as a key molecule triggering cell competition. Our work shows that COX‐2‐mediated PGE2 secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase‐cyclic AMP‐PKA pathway that, in the presence of active YAP, induces E‐cadherin internalization leading to apical extrusion. Thus, COX‐2‐induced PGE2 appears a warning signal to both abnormal and surrounding normal cells to drive cell competition., Mammalian epithelial Madin‐Darby canine kidney (MDCK) cells expressing constitutively active YAP are eliminated by apical extrusion when surrounded by “normal” MDCK cells. However, the molecular mechanism underlying it was unknown. We show that COX‐2‐induced PGE2 activates adenylyl cyclase‐cyclic AMP‐PKA signaling pathway that, in the presence of active YAP, induces E‐cadherin internalization leading to apical extrusion.

TGM1 is the most common gene responsible for lamellar ichthyosis. Previous studies have suggested that patients with lamellar ichthyosis carrying two missense mutations in TGM1 show significantly less severe phenotypes than those with at least one truncating mutation in TGM1. Here, we report a patient with severe lamellar ichthyosis who was compound heterozygous for TGM1 missense mutations, including a novel one. A 22-year-old Japanese man presented with large, dark brown, plate-like scales on the extremities and small adherent scales on the face and trunk. His other clinical findings included ectropion, hair loss, hypohidrosis, hyperthermia in summer, palmoplantar keratoderma and constriction of the fingers. Dermoscopy revealed accentuated sulci cutis with numerous large keratotic plugs in the cristae cutis. Histologically, orthohyperkeratosis and mild acanthosis were noted. Electron microscopy showed reduced cornified envelope thickness and numerous lipid droplets in the stratum corneum. Mutation analysis revealed the patient to be compound heterozygous for missense mutations, c.620T>C (p.Leu207Pro) and c.1631A>G (p.Tyr544Cys), in TGM1. Furthermore, we showed that TGM1 enzymatic activity was largely absent in his epidermis. These findings led us to diagnose him as having lamellar ichthyosis. This study has two important notions. First, even two missense mutations in TGM1 can cause severe lamellar ichthyosis. Second, this is the first report of dermoscopic findings of lamellar ichthyosis, implicating the obstruction of sweat glands by keratotic plugs in the pathogenesis of hypohidrosis in the disease. In conclusion, this study provides further insights into genotype-phenotype correlations and pathogenesis in lamellar ichthyosis.

Background Epidermolysis bullosa (EB) is a group of hereditary disorders caused by mutations in the genes encoding structural molecules of the dermal-epidermal junction (DEJ). Cell-based therapies such as allogeneic mesenchymal stem/stromal cell (MSC) transplantation have recently been explored for severe EB types, such as recessive dystrophic EB (RDEB). However, hurdles exist in current MSC-based therapies, such as limited proliferation from a single cell source and limited cell survival due to potential allogenic rejection. Objectives We aimed to develop MSCs from keratinocyte-derived induced pluripotent stem cells (iPSCs). Methods Keratinocyte-derived iPSCs (KC-iPSCs) of a healthy human and an RDEB patient were cultured with activin A, 6-bromoindirubin-3′-oxime and bone morphogenetic protein 4 to induce mesodermal lineage formation. These induced cells were subjected to immunohistochemical analysis, flow cytometric analysis and RNA microarray analysis in vitro, and were injected subcutaneously and intravenously to wounded immunodeficient mice to assess their wound-healing efficacy. Results After their induction, KC-iPSC-induced cells were found to be compatible with MSCs. Furthermore, with the subcutaneous and intravenous injection of the KC-iPSC-induced cells into wounded immunodeficient mice, human type VII collagen was detected at the DEJ of epithelized areas. Conclusions We successfully established iPSC-derived MSCs from keratinocytes (KC-iPSC-MSCs) of a normal human and an RDEB patient. KC-iPSC-MSCs may have potential in therapies for RDEB.

Critical limb ischemia (CLI) is a devastating disease in patients undergoing hemodialysis (HD). Based on the unsatisfactory results of autologous mononuclear cell transplantation for patients with CLI undergoing HD, we conducted a phase II clinical trial to evaluate the safety and efficacy of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood-derived autologous purified CD34 positive (CD34+) cell transplantation for CLI in patients undergoing HD. Six patients with CLI (two with Rutherford category 4 and four with Rutherford category 5) were enrolled. As for primary endpoint, there were no major adverse events related to this therapy. As for efficacy, the amputation-free survival rate was 100% at 1 year after cell therapy. Both rest pain scale and ulcer size were significantly improved as early as 4 weeks after therapy compared with baseline (p < .01), and three out of five ulcers completely healed within 12 weeks after cell transplantation. Clinical severity, including Fontaine scale and Rutherford category, significantly improved at 24 weeks after cell transplantation (p < .05), and further improved at 52 weeks (p < .01) compared with baseline. The improvement rate from CLI stage to non-CLI stage was 83.3% at 52 weeks. Toe skin perfusion pressure and absolute claudication distance were also significantly improved. In conclusion, G-CSF-mobilized peripheral blood CD34+ cell transplantation was safe, feasible, and effective for patients with CLI undergoing HD.

Aortic pulse waveforms are composed of a forward wave from the heart and a reflection wave from the periphery. We focused on this forward wave and suggested a new parameter, the maximum slope of aortic pulse waveforms (max dD/dt), for fetal cardiac contractility. Max dD/dt was calculated from fetal aortic pulse waveforms recorded with an echo-tracking system. A normal range of max dD/dt was constructed in 105 healthy fetuses using linear regression analysis. Twenty-two fetuses with suspected fetal cardiac dysfunction were divided into normal and decreased max dD/dt groups, and their clinical parameters were compared. Max dD/dt of aortic pulse waveforms increased linearly with advancing gestational age (r = 0.93). The decreased max dD/dt was associated with abnormal cardiotocography findings and short- and long-term prognosis. In conclusion, max dD/dt calculated from the aortic pulse waveforms in fetuses can substitute for max dP/dt, an index of cardiac contractility in adults.

Summary: 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. : Watanabe et al. demonstrate that mutant p53 cells undergo necroptosis and are eliminated from epithelia via cell competition with surrounding normal cells. Moreover, the eradication of mutant p53 cells is suppressed within RasV12-transformed epithelia, suggesting that the order of oncogenic mutations in cancer development could be dictated by cell competition. Keywords: cell competition, p53, necroptosis, p53R273H, p53R175H, RasV12, multi-step cancer progression, MDCK cells, mouse intestinal organoids

SUMMARY Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine., In Brief Sasaki et al. demonstrate using a cell competition mouse model that high-fat diet feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. These results indicate that obesity can profoundly influence competitive interaction between normal and transformed cells at the initial stage of carcinogenesis., Graphical Abstract

Nagashima-type palmoplantar keratosis (NPPK) is an autosomal recessive skin disorder with a high, unmet medical need that is caused by mutations in SERPINB7. Almost all NPPK patients carry the founder nonsense mutation c.796C>T (p.Arg266Ter) in the last exon of SERPINB7. Here we sought to determine whether topical nonsense-suppression (readthrough) therapy using gentamicin is applicable to NPPK. First, we demonstrated that gentamicin enhanced readthrough activity in cells transfected with SERPINB7 cDNA carrying the mutation and promoted full-length SERPINB7 protein synthesis in NPPK keratinocytes. We next conducted an investigator-blinded, randomized, bilaterally controlled compassionate use study of topical gentamicin in which five NPPK patients with c.796C>T were enrolled. Patients' self-reported improvement of hyperkeratosis was significantly greater on the gentamicin side than the control side (P = 0.0349). In two patients, hyperkeratosis was improved on the gentamicin side, as determined by a blinded-investigator assessment. These results indicate the therapeutic potential of topical gentamicin for NPPK. Unexpectedly, we also found that mutant SERPINB7 mRNAs harboring r.796c>u were degraded by nonsense-mediated mRNA decay. Furthermore, the truncated SERPINB7 protein was degraded via a proteasome-mediated pathway. These findings provide important insights into the mRNA/protein quality-control system in humans, which could be a potential therapeutic target for genetic diseases.

Although fibrosis is one of the most prominent pathologic features of preeclamptic (PE) placentas, its mechanism remains largely unknown. Consistent with previous reports, we observed overexpression of collagen; actin, α2, smooth muscle, aorta; connective tissue growth factor; and fibronectin in PE placentas compared with control ones. To investigate the mechanism of fibrosis in PE placentas, placental fibroblasts were isolated from PE placentas or normal pregnancies at delivery. The expression of fibrosis-related factors in fibroblasts was evaluated by real-time RT-PCR, Western blotting, enzyme-linked immunosorbent assay, and gene microarrays. An in vitro collagen gel contraction assay was also performed. Fibroblasts isolated from PE placentas showed higher expression levels of fibrosis-related factors compared with those from control ones. Global gene expression profiling of PE fibroblasts was contrasted with that of control ones and indicated an intimate association with transforming growth factor-β1 (TGFB1) signaling. Furthermore, the PE fibroblasts expressed abundant phosphorylated SMAD family member 2 and showed higher expression levels of target genes of TGFB1 signaling compared with the control ones. The PE fibroblasts also had a greater ability to contract compared with the control ones. Contractility also depended on TGFB1 signaling. Our results suggest that TGFB1 signaling is activated in the fibroblasts in PE placentas and that these active fibroblasts contribute to fibrosis.

Background Induced pluripotent stem cell (iPSC) technology enables patient-specific pluripotent stem cells to be derived from adult somatic cells without the use of an embryonic cell source. To date, recessive dystrophic epidermolysis bullosa (RDEB)-specific iPSCs have been generated from patients using integrating retroviral vectors. However, vector integration into the host genome can endanger the biosafety and differentiation propensities of iPSCs. Although various integration-free reprogramming systems have been reported, their utility in reprogramming somatic cells from patients remains largely undetermined. Objective Our study aims to establish safe iPSCs from keratinocytes of RDEB patients using non-integration vector. Method We optimized and infected non-integrating Sendai viral vectors to reprogram keratinocytes from healthy volunteers and RDEB patients. Results Sendai vector infection led to the reproducible generation of genomic modification-free iPSCs from these keratinocytes, which was proved by immunohistochemistry, reverse transcription polymerase chain reaction, methylation assay, teratoma assay and embryoid body formation assay. Furthermore, we confirmed that these iPSCs have the potential to differentiate into dermal fibroblasts and epidermal keratinocytes. Conclusion This is the first report to prove that the Sendai vector system facilitates the reliable reprogramming of patient keratinocytes into transgene-free iPSCs, providing another pluripotent platform for personalized diagnostic and therapeutic approaches to RDEB.