Contact Inhibition Of Proliferation Research Articles

Transient proliferation by reversible YAP and mitogen-control of the cyclin D1/p27 ratio.

Hippo-YAP signaling orchestrates epithelial tissue repair and is therefore an attractive target in regenerative medicine. Yet it is unresolved how YAP integrates with mitogen signaling and contact inhibition to control the underlying transient proliferative response. Here we show that reduced contact inhibition, increased mitogen signaling, and YAP-TEAD activation converge on increasing the nuclear cyclin D1/p27 protein ratio during G1 phase, towards a threshold ratio that dictates whether individual cells enter or exit the cell cycle. YAP increases this ratio indirectly, in concert with mitogen signaling, by increasing EGFR and other receptors that signal primarily through ERK. After a delay, contact inhibition suppresses YAP activity which gradually downregulates mitogen signaling and the cyclin D1/p27 ratio. Increasing YAP activity by ablating the suppressor Merlin/NF2 reveals a balancing mechanism in which YAP suppression and contact inhibition of proliferation can be recovered but only at higher local cell density. Thus, critical for tissue repair, robust proliferation responses result from the YAP-induced and receptor-mediated prolonged increase in the cyclin D1/p27 ratio, which is only reversed by delayed suppression of receptor signaling after contact inhibition of YAP.

A ZO-2 scaffolding mechanism regulates the Hippo signalling pathway.

Contact inhibition of proliferation is a critical cell density control mechanism governed by the Hippo signalling pathway. The biochemical signalling underlying cell density-dependent cues regulating Hippo signalling and its downstream effectors, YAP, remains poorly understood. Here, we reveal that the tight junction protein ZO-2 is required for the contact-mediated inhibition of proliferation. We additionally determined that the well-established molecular players of this process, namely Hippo kinase LATS1 and YAP, are regulated by ZO-2 and that the scaffolding function of ZO-2 promotes the interaction with and phosphorylation of YAP by LATS1. Mechanistically, YAP is phosphorylated when ZO-2 brings LATS1 and YAP together via its SH3 and PDZ domains, respectively, subsequently leading to the cytoplasmic retention and inactivation of YAP. In conclusion, we demonstrate that ZO-2 maintains Hippo signalling pathway activation by promoting the stability of LATS1 to inactivate YAP.

Contact inhibition of proliferation is accompanied by expression of the PHF10D subunit of the chromatin remodeling complex PBAF in mouse and human cell lines

Contact inhibition of proliferation is accompanied by expression of the PHF10D subunit of the chromatin remodeling complex PBAF in mouse and human cell lines

Mechanical control of cell proliferation patterns in growing epithelial monolayers

Cell proliferation plays a crucial role in regulating tissue homeostasis and development. However, our understanding of how cell proliferation is controlled in densely packed tissues is limited. Here we develop a computational framework to predict the patterns of cell proliferation in growing epithelial tissues, connecting single-cell behaviors and cell-cell interactions to tissue-level growth. Our model incorporates probabilistic rules governing cell growth, division, and elimination, also taking into account their feedback with tissue mechanics. In particular, cell growth is suppressed and apoptosis is enhanced in regions of high cell density. With these rules and model parameters calibrated using experimental data for epithelial monolayers, we predict how tissue confinement influences cell size and proliferation dynamics and how single-cell physical properties influence the spatiotemporal patterns of tissue growth. In this model, mechanical feedback between tissue confinement and cell growth leads to enhanced cell proliferation at tissue boundaries, whereas cell growth in the bulk is arrested, recapitulating experimental observations in epithelial tissues. By tuning cellular elasticity and contact inhibition of proliferation we can regulate the emergent patterns of cell proliferation, ranging from uniform growth at low contact inhibition to localized growth at higher contact inhibition. We show that the cell size threshold at G1/S transition governs the homeostatic cell density and tissue turnover rate, whereas the mechanical state of the tissue governs the dynamics of tissue growth. In particular, we find that the cellular parameters affecting tissue pressure play a significant role in determining the overall growth rate. Our computational study thus underscores the impact of cell mechanical properties on the spatiotemporal patterns of cell proliferation in growing epithelial tissues.

The Role of Mechanotransduction in Contact Inhibition of Locomotion and Proliferation.

Contact inhibition (CI) represents a crucial tumor-suppressive mechanism responsible for controlling the unbridled growth of cells, thus preventing the formation of cancerous tissues. CI can be further categorized into two distinct yet interrelated components: CI of locomotion (CIL) and CI of proliferation (CIP). These two components of CI have historically been viewed as separate processes, but emerging research suggests that they may be regulated by both distinct and shared pathways. Specifically, recent studies have indicated that both CIP and CIL utilize mechanotransduction pathways, a process that involves cells sensing and responding to mechanical forces. This review article describes the role of mechanotransduction in CI, shedding light on how mechanical forces regulate CIL and CIP. Emphasis is placed on filamin A (FLNA)-mediated mechanotransduction, elucidating how FLNA senses mechanical forces and translates them into crucial biochemical signals that regulate cell locomotion and proliferation. In addition to FLNA, trans-acting factors (TAFs), which are proteins or regulatory RNAs capable of directly or indirectly binding to specific DNA sequences in distant genes to regulate gene expression, emerge as sensitive players in both the mechanotransduction and signaling pathways of CI. This article presents methods for identifying these TAF proteins and profiling the associated changes in chromatin structure, offering valuable insights into CI and other biological functions mediated by mechanotransduction. Finally, it addresses unanswered research questions in these fields and delineates their possible future directions.

Development of Novel Bioluminescent Biosensors Monitoring the Conformation and Activity of the Merlin Tumour Suppressor.

Solid tumours can universally evade contact inhibition of proliferation (CIP), a mechanism halting cell proliferation when cell-cell contact occurs. Merlin, an ERM-like protein, crucially regulates CIP and is frequently deactivated in various cancers, indicating its significance as a tumour suppressor in cancer biology. Despite extensive investigations into Merlin's role in cancer, its lack of intrinsic catalytic activity and frequent conformation changes have made it notoriously challenging to study. To address this challenge, we harnessed innovative luciferase technologies to create and validate a NanoBiT split-luciferase biosensor system in which Merlin is cloned between two split components (LgBiT and SmBiT) of NanoLuc luciferase. This system enables precise quantification of Merlin's conformation and activity both in vitro and within living cells. This biosensor significantly enhances the study of Merlin's molecular functions, serving as a potent tool for exploring its contributions to CIP and tumorigenesis.

Identification and partial characterization of new cell density-dependent nucleocytoplasmic shuttling proteins and open chromatin

The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP.

Contact Inhibition of Proliferation Is Accompanied by Expression of the PHF10D Subunit of the Chromatin Remodeling Complex PBAF in Mouse and Human Cell Lines.

PHF10 is a subunit of the PBAF complex, which regulates the expression of many genes in developing and maturing organisms. PHF10 has four isoforms that differ in domain structure. The PHF10A isoform, containing a DPF domain at the C-terminus and 46 amino acids at the N-terminus, is necessary for the expression of proliferation genes; the functions of the other isoforms are less studied. In this work, we have established that, upon contact inhibition of mouse and human cell proliferation caused by the establishment of a tight junction and adherence junction between cells, the expression of the PHF10A isoform stops and instead the PHF10D isoform is expressed, which does not contain DPF-domain and N-terminal sequence. The function of the PHF10D isoform may be associated with the establishment of intercellular contacts.

Collective heterogeneity of mitochondrial potential in contact inhibition of proliferation

In the epithelium, cell density and cell proliferation are closely connected to each other through contact inhibition of proliferation (CIP). Depending on cell density, CIP proceeds through three distinct stages: the free-growing stage at low density, the pre-epithelial transition stage at medium density, and the post-epithelial transition stage at high density. Previous studies have elucidated how cell morphology, motion, and mechanics vary in these stages. However, it remains unknown whether cellular metabolism also has a density-dependent behavior. By measuring the mitochondrial membrane potential at different cell densities, here we reveal a heterogeneous landscape of metabolism in the epithelium, which appears qualitatively distinct in three stages of CIP and did not follow the trend of other CIP-associated parameters, which increases or decreases monotonically with increasing cell density. Importantly, epithelial cells established a collective metabolic heterogeneity exclusively in the pre-epithelial transition stage, where the multicellular clusters of high- and low-potential cells emerged. However, in the post-epithelial transition stage, the metabolic potential field became relatively homogeneous. Next, to study the underlying dynamics, we constructed a system biology model, which predicted the role of cell proliferation in metabolic potential toward establishing collective heterogeneity. Further experiments indeed revealed that the metabolic pattern spatially correlated with the proliferation capacity of cells, as measured by the nuclear localization of a pro-proliferation protein, YAP. Finally, experiments perturbing the actomyosin contractility revealed that, while metabolic heterogeneity was maintained in the absence of actomyosin contractility, its ab initio emergence depended on the latter. Taken together, our results revealed a density-dependent collective heterogeneity in the metabolic field of a pre-epithelial transition-stage epithelial monolayer, which may have significant implications for epithelial form and function.

Abstract 858: Development of ultrasensitive split luciferase biosensors monitoring activity of the merlin tumor suppressor

Abstract Understanding features common to all forms of cancer is a vital component of treatment and research into malignant disease. All solid tumours share the ability to overcome contact inhibition of proliferation (CIP), the process by which intercellular contacts engage signalling to stop proliferation. Merlin, a tumour suppressor protein that is inactivated in a wide variety of cancers, plays a crucial role in CIP; merlin-deficient cells lose the ability to be contact-inhibited and subsequently form tumours. Although the role of merlin in cancer has been investigated extensively over the past 28 years, this protein has been notoriously difficult to study. To address this issue, we develop and utilize 2 split-luciferase biosensor systems that enable accurate quantification of merlin activity in real time. Merlin undergoes a conformational change that is functionally important in tumour suppression. Phosphorylation at a key C-terminal residue promotes transition of the protein from an open, active conformation to a closed, N-to-C terminal autoinhibited conformation. Therefore, monitoring this conformation change in real time could provide unique insights into merlin function. To do this, we apply NanoBiT split-luciferase technology to develop an intramolecular merlin biosensor (intra-Mer-BS). In brief, 2 split-luciferase components, LgBiT and SmBiT, are fused to the N- and C-terminus of merlin, respectively. Upon open-to-closed conformation change of merlin, LgBiT and SmBiT complement to reconstitute a functional luciferase and emit light. This enables accurate quantification of merlin’s functionally relevant conformation changes in real time. Importantly, cotransfection of the intra-Mer-BS alongside PAK1, an upstream merlin regulator that promotes transition to the closed conformation, significantly increases luminescent activity of the intra-Mer-BS, indicating that the biosensor faithfully reports on merlin’s conformation. Moreover, merlin has been shown to exert its tumour suppressive function through activation of LATS, the central mediator of the Hippo signalling pathway. In addition to the intra-Mer-BS, we develop and validate a NanoBiT biosensor to monitor the interaction between merlin and LATS (Mer-LATS-BS). This Mer-LATS-BS is used to quantify the effect of merlin activators and inhibitors on merlin/LATS tumour suppressive activity in cancer cells. In summary, we develop and validate 2 novel bioluminescent biosensors to monitor merlin’s conformation changes and activity in cancer cells. The intra-Mer-BS and Mer-LATS-BS provide real time information with high sensitivity and excellent reproducibility. Ultimately, these biosensors enable high throughput screening to discover novel upstream regulators of merlin in cancer and provide mechanistic insight into how contact inhibitive signalling is propagated through merlin and the Hippo pathway. Citation Format: Alexander J. Pipchuk, Xiaolong Yang. Development of ultrasensitive split luciferase biosensors monitoring activity of the merlin tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 858.

Air pollution-regulated E-cadherin mediates contact inhibition of proliferation via the hippo signaling pathways in emphysema

Air pollution has been linked to emphysema in chronic obstruction pulmonary disease (COPD). However, the underlying mechanisms in the development of emphysema due to air pollution remain unclear. The objective of this study was to investigate the role of components of the Hippo signaling pathway for E-cadherin-mediated contact inhibition of proliferation in the lungs after air pollution exposure. E-Cadherin-mediated contact inhibition of proliferation via the Hippo signaling pathway was investigated in Sprague-Dawley (SD) rats whole-body exposed to air pollution, and in alveolar epithelial A549 cells exposed to diesel exhaust particles (DEPs), E-cadherin-knockdown, and high-mobility group box 1 (HMGB1) treatment. Underlying epithelial differentiation, apoptosis, and senescence were also examined, and the interaction network among these proteins was examined. COPD lung sections were used to confirm the observations in rats. Expressions of HMGB1 and E-cadherin were negatively regulated in the lungs and A549 cells by air pollution, and this was confirmed by knockdown of E-cadherin and by treating A549 cells with HMGB1. Depletion of phosphorylated (p)-Yap occurred after exposure to air pollution and E-cadherin-knockdown, which resulted in decreases of SPC and T1α. Exposure to air pollution and E-cadherin-knockdown respectively downregulated p-Sirt1 and increased p53 levels in the lungs and in A549 cells. Moreover, the protein interaction network suggested that E-cadherin is a key activator in regulating Sirt1 and p53, as well as alveolar epithelial cell differentiation by SPC and T1α. Consistently, downregulation of E-cadherin, p-Yap, SPC, and T1α was observed in COPD alveolar regions with particulate matter (PM) deposition. In conclusion, our results indicated that E-cadherin-mediated cell-cell contact directly regulates the Hippo signaling pathway to control differentiation, cell proliferation, and senescence due to air pollution. Exposure to air pollution may initiate emphysema in COPD patients.

P190A RhoGAP induces CDH1 expression and cooperates with E-cadherin to activate LATS kinases and suppress tumor cell growth

The ARHGAP35 gene encoding p190A RhoGAP (p190A) is significantly altered by both mutation and allelic deletion in human cancer, but the functional implications of such alterations are not known. Here, we demonstrate for the first time that p190A is a tumor suppressor using a xenograft mouse model with carcinoma cells harboring defined ARHGAP35 alterations. In vitro, restoration of p190A expression in carcinoma cells promotes contact inhibition of proliferation (CIP) through activation of LATS kinases and phosphorylation of the proto-oncogenic transcriptional co-activator YAP. In contrast, p190A forms harboring recurrent cancer mutations exhibit loss of function in modulating the Hippo pathway, inducing CIP, as well as attenuated suppression of tumor growth in mice. We determine that p190A promotes mesenchymal to epithelial transition (MET) and elicits expression of a cassette of epithelial adherens junction-associated genes in a cell density-dependent manner. This cassette includes CDH1 encoding E-cadherin, which amplifies p190A-mediated LATS activation and is necessary for CIP. Oppositely, we establish that p190A is obligatory for E-cadherin to activate LATS kinases and induce CIP. Collectively, this work defines a novel mechanism by which p190A and E-cadherin cooperate in modulating Hippo signaling to suppress tumor cell growth.

Close Encounters of the Cell Kind: The Impact of Contact Inhibition on Tumour Growth and Cancer Models

Cancer is a complex phenomenon, and the sheer variation in behaviour across different types renders it difficult to ascertain underlying biological mechanisms. Experimental approaches frequently yield conflicting results for myriad reasons, and mathematical modelling of cancer is a vital tool to explore what we cannot readily measure, and ultimately improve treatment and prognosis. Like experiments, models are underpinned by certain biological assumptions, variation of which can lead to divergent predictions. An outstanding and important question concerns contact inhibition of proliferation (CIP), the observation that proliferation ceases when cells are spatially confined by their neighbours. CIP is a characteristic of many healthy adult tissues, but it remains unclear to which extent it holds in solid tumours, which exhibit regions of hyper-proliferation, and apparent breakdown of CIP. What precisely occurs in tumour tissue remains an open question, which mathematical modelling can help shed light on. In this perspective piece, we explore the implications of different hypotheses and available experimental evidence to elucidate the implications of these scenarios. We also outline how erroneous conclusions about the nature of tumour growth may be arrived at by looking selectively at biological data in isolation, and how this might be circumvented.

Boolean model of anchorage dependence and contact inhibition points to coordinated inhibition but semi-independent induction of proliferation and migration

Epithelial cells respond to their physical neighborhood with mechano-sensitive behaviors required for development and tissue maintenance. These include anchorage dependence, matrix stiffness-dependent proliferation, contact inhibition of proliferation and migration, and collective migration that balances cell crawling with the maintenance of cell junctions. While required for development and tissue repair, these coordinated responses to the microenvironment also contribute to cancer metastasis. Predictive models of the signaling networks that coordinate these behaviors are critical in controlling cell behavior to halt disease. Here we propose a Boolean regulatory network model that synthesizes mechanosensitive signaling that links anchorage to a matrix of varying stiffness and cell density sensing to contact inhibition, proliferation, migration, and apoptosis. Our model can reproduce anchorage dependence and anoikis, detachment-induced cytokinesis errors, the effect of matrix stiffness on proliferation, and contact inhibition of proliferation and migration by two mechanisms that converge on the YAP transcription factor. In addition, we offer testable predictions related to cell cycle-dependent anoikis sensitivity, the molecular requirements for abolishing contact inhibition, and substrate stiffness dependent expression of the catalytic subunit of PI3K. Moreover, our model predicts heterogeneity in migratory vs. non-migratory phenotypes in sub-confluent monolayers, and co-inhibition but semi-independent induction of proliferation vs. migration as a function of cell density and mitogenic stimulation. Our model serves as a stepping-stone towards modeling mechanosensitive routes to the epithelial to mesenchymal transition, capturing the effects of the mesenchymal state on anoikis resistance, and understanding the balance between migration versus proliferation at each stage of the epithelial to mesenchymal transition.

Composition of lectin receptors in serous and mucinous ovarian cystadenomas

The most common group of benign cystic ovarian tumors in women of all ages are epithelial tumors (cystadenomas) that make up about a third of ovarian tumors and half of benign lesions. The growth of tumor proliferative activity accompanied by increased number of lectins receptors in cells. Lectins are non-immune origin proteins that have a common property of binding carbohydrates and carbohydrate determinants of biopolymers strictly defined structure and intact their covalent structure. It was established that oncogenesis changes of the structure of glycoconjugates membrane as cancer and immune cells, and mark faster than glycoconjugates variability in tumor cells. This is the basis for the use of labeled lectins to study changes in cell membrane and secretory structures of glycoconjugates during tumor growth. The aim of the study was to determine the composition of the receptors in serous and mucinous cystadenomas. Operating material was studied from 60 women operated on for ovarian serous cystadenomas, and from 60 patients – on for ovarian mucinous cystadenomas. To identify the set of glycoconjugates it were used peroxidaselabeled lectins with different carbohydrate specificity. Characteristic of serous and mucinous cystadenomas was the accumulation of lectins receptors in their cells of wheat germs (WGA), groundnut seed (PNA) and soybean seeds (SBA). Papillary serous cystadenomas differed moderate accumulation in the epithelium papillae throughout the cytoplasm lectins receptors of SBA, mistletoe (VAL) and WGA. The most intense and uniform expression in mucinous cystadenomas was one of lectin receptors of bark elderberry (SNA) and SBA. The authors conclude that changes in the molecular-spatial structure of glycoconjugates of the plasmolemma cells surfaces and increase of the mobility of membrane lectins receptors in serous and mucinous cystadenomas cause lack of contact inhibition of proliferation. In their opinion, further study of lectins can contribute to the elucidation of the pathogenesis of ovarian formations, their differential diagnosis, the development of tumor markers and drugs lectins.

Merlin/NF2-Lin28B-let-7 Is a Tumor-Suppressive Pathway that Is Cell-Density Dependent and Hippo Independent

Contact inhibition of proliferation is critical for tissue organization, and its dysregulation contributes to tumorigenesis. Merlin/NF2 is a tumor suppressor that governs contact inhibition. Although Merlin/NF2 inhibits YAP1 and TAZ, which are paralogous Hippo pathway transcriptional co-activators and oncoproteins, it is not fully understood how Merlin/NF2-mediated signal transduction triggered by cell-cell contact exerts tumor suppression. Here, we identify Lin28B, an inhibitor of let-7 microRNAs (miRNAs), as an important downstream target of Merlin/NF2. Functional studies revealed that, at low cell density, Merlin/NF2 is phosphorylated and does not bind to Lin28B, allowing Lin28B to enter the nucleus, bind to pri-let-7 miRNAs, and inhibit their maturation in aYAP1/TAZ-independent manner. This inhibition of pri-let-7 maturation then promotes cell growth. However, cell-cell contact triggers Merlin/NF2 dephosphorylation, which sequesters Lin28B in the cytoplasm and permits pri-let-7 maturation. Our results reveal that Merlin/NF2-mediated signaling drives atumor-suppressive pathway that is cell-density dependent and Hippo independent.

Abstract 3520: TAZ (WWTR1), a key transcription co-activator of hippo-pathway, promotes hepatocellular carcinoma progression via PI3K/Akt/mTOR pathway

Abstract Introduction: The contact inhibition of proliferation is crucial for well-controlled organogenesis. Its disruption results in sustained cell proliferation, which is a hallmark of solid tumors. The Hippo signaling pathway has been implicated in contact inhibition of proliferation. Here, we investigated the role of TAZ (WWTR1), a key transcription co-activator of hippo-pathway, in HCC progression. Methods and Results: In HCC patients, the high TAZmRNA expression examined by real time PCR showed worsen survival outcomes compared with low expression group. Additionally, the tumor size in High TAZmRNA expression group was significantly larger than that in low expression group, and its high protein expression in HCC was deeply associated with the cell proliferative activity. A knockdown of TAZ expression in HCC cell lines attenuated the cell growth accompanying the downstream gene expressions (CTGF and CYR61). As the TAZ-mediated signaling pathway in cell growth, a knockdown of TAZ expression using RNAi inactivated the PI3K/Akt/mTOR pathway. Conclusion: Thus, TAZ plays a crucial role in tumor progression by accelerating cell growth via a PI3K/Akt/mTOR pathway, and tumoral TAZmRNA expression level may be a useful prognostic biomarker in HCC. Citation Format: Hiromitsu Hayashi, Hideyuki Kuroki, Shigeki Nakagawa, Takaaki Higashi, Keita Sakamoto, Naomi Yokoyama, Takatoshi Ishiko, Toru Beppu, Hideo Baba. TAZ (WWTR1), a key transcription co-activator of hippo-pathway, promotes hepatocellular carcinoma progression via PI3K/Akt/mTOR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2014-3520

A cadherin switch underlies malignancy in high-grade gliomas.

Although the infiltrative behavior of malignant gliomas is one of their most critical aspects, the mechanisms underlying it have not yet been elucidated. To migrate in the brain parenchyma, malignant glioma cells need to bypass the cell-cell contact inhibitory signals. Here we propose that the blinding of cell-cell contact sensing in gliomas is caused by an unusual mechanism of cadherin switch, involving the replacement of N-cadherin with R-cadherin (Rcad) at the cell-cell junctions and the activation of ERK and p27. In our model of malignant glioma, we found that Rcad expression is necessary and sufficient to release cells from contact inhibition of proliferation, and is necessary, although not sufficient, for overriding contact inhibition of migration and for tumorigenicity. Altogether, these observations suggest that Rcad is a potential target for malignant glioma therapies.

Abstract 1160: Inactivated hippo-pathway as a prognostic biomarker in hepatocellular carcinoma.

Abstract The contact inhibition of proliferation and cell movement is crucially important in embryonic development, tissue regeneration, and wound healing for the well-controlled organogenesis. The disruption of contact inhibition results in sustained cell proliferation and uncontrolled cell movement, which is a hallmark of solid tumors. Loss of contact inhibition allows cancer cells to facilitate the invasion of neighboring tissues and metastasis to remote organs. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. Indeed, the conditional inactivation of hippo-pathway in liver leads to enlarged liver (massive hepatomegaly) and tumorigenesis (Cell, 2007). Clinically, HCC often shows the huge tumor more than size in 10cm, strongly speculating that inactivation of hippo-pathway plays an important role in HCC progression. However, clinical significance of hippo-pathway in HCC is largely unknown. Here, we investigated the clinical significance of inactivated hippo-pathway, which induce overexpressed oncogenes (YAP/TAZ), using resected 80 HCC samples, and suggested the tumoral-high TAZmRNA expression was a worse prognostic biomarker for HCC patients represented by high proliferative activity. The tumor size in High TAZmRNA expression group (n =24) was significantly larger than that in low expression group (n = 56) (P < 0.05). YAP/TAZ protein over-expression in HCC was associated with high proliferative activity by Ki-67 positive cells. The high TAZmRNA expression group showed significantly worsen disease free survival and overall survival compared with that in low expression group (P = 0.01 and 0.02, respectively). Thus, inactivation of Hippo-pathway plays a crucial role in tumor progression by accelerating proliferative activity, and tumoral TAZmRNA expression level may be a useful prognostic biomarker in HCC. Citation Format: Hiromitsu Hayashi, Hideyuki Kuroki, Naomi Yokoyama, Shigeki Nakagawa, Hidetoshi Nitta, Katsunori Imai, Daisuke Hashimoto, Yoshiaki Ikuta, Akira Chikamoto, Toru Beppu, Hideo Baba. Inactivated hippo-pathway as a prognostic biomarker in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1160. doi:10.1158/1538-7445.AM2013-1160

Growth characteristics of CHO cells in culture

The dependence of the growth characteristics and monolayer formation on the initial cell plating concentration were studied on a permanent CHO cell line. The cells were cultivated under standard conditions on plastic substrate. Initial plating concentrations were varied as 1000, 2000, 3000, 4000, 5000, and 6000 cells/cm2. It was shown that the cell growth can be formally described by a standard S-shaped dependence. However, a more detailed analysis revealed inconsistency of the experimental and expected data. Specifically, the cell growth termination produced by monolayer formation does not coincide with the time when the theoretical curves approach a plateau. It is concluded that cell proliferation and monolayer formation are independent processes (at least in CHO cells). Both processes may be considered as analogs of proliferation and morphogenesis in metazoa. In addition, it is shown that the cessation of cell division is induced by reduction in the cell size to some limiting dimension and increasing of the cell polarization rather than contact inhibition of proliferation after the monolayer formation.