Cell-cell Contact Inhibition Research Articles

Abstract 4389: The role of Rab5 in epithelial growth factor stimulated E-cadherin internalization in spheroid growth of colon cancer cells

Abstract In epithelial based cancer formation, such as colon cancer, the tumor growth is accompanied with malfunction of cell-cell contact inhibition which is regulated by E-cadherin. However, the dynamics of E-cadherin is complicated, the underlying mechanism in tumor nest growth and collective cell migration is still not clear. Rab5 is the endocytic vesicle protein for the membrane receptor internalization into early endosome. Therefore, this study is to investigate the role of Rab5 in E-cadherin turnover of spheroid formation in colon cancer cells. HT-29 colon cancer cells were used in this study. The cells with less anchorage ability were isolated for spheroid formation. The distribution of Rab5, E-cadherin and focal adhesion kinase were observed by immuno-confocal microscopy. The molecular expressions were analyzed by western blot. HT-29 cells showed well defined epithelial phenotype. In the result of immunostaining, Rab5 was distributed in the cytosol and the cell-cell contacts where it colocalized with E-cadherin. Rab5 was also observed in the cell leading edge, however, not much associated with focal adhesions. When HT-29 cell clusters were stimulated with epidermal growth factor, Rab5 was found abundant in the cell-cell contacts. In cell spheroid that derived from HT-29 cells, Rab5 and E-cadherin co-localization at cell-cell contacts was also increased. Furthermore, when cells were treated with Nocodazole, the tubulin inhibitor blocking tubulin-mediated endocytosis, Rab5 accumulated and colocalized with E-cadherin in the cell leading edge. The results of this study demonstrated Rab5 plays the novel role in E-cadherin turnover upon epidermal growth factor stimulation in the progress of spheroid formation and Rab5 may mediate E-cadherin endocytosis through the cell leading edge. Citation Format: Yu-Chun Wang, Yu-Chien Ching, Hui-Chun Chen, Chun-Pu Cheng, Chia-En Ku, Shin-Ru Lee, Wei-Ting Chao. The role of Rab5 in epithelial growth factor stimulated E-cadherin internalization in spheroid growth of colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4389.

Autocrine Factors Produced by Mesenchymal Stem Cells in Response to Cell-Cell Contact Inhibition Have Anti-Tumor Properties.

Recently, mesenchymal stem cell (MSC) therapies have been questioned as MSCs are capable of both promoting and inhibiting tumorigenesis. Both MSCs and tumor cells replicate to increase their population size; however, MSCs, but not tumor cells, stop dividing when they reach confluence due to cell-cell contact inhibition and then differentiate. We hypothesized that contact inhibition results in the production of effector molecules by confluent MSCs and these effectors are capable of suppressing tumor cell growth. To test this hypothesis, we co-cultured breast cancer cells (MDA-MB-231) with either confluent or sub-confluent bone-marrow-derived MSCs (BM-MSCs); in addition, we treated various tumor cells with conditioned media (CM) obtained from either confluent or sub-confluent BM-MSCs. The results showed that the growth of tumor cells co-cultured with confluent BM-MSCs or treated with CM obtained from confluent BM-MSCs was inhibited, and this effect was significantly stronger than that seen with tumor cells co-cultured with sub-confluent BM-MSCs or CM obtained from sub-confluent BM-MSCs. Subcutaneous tumor formation was completely prevented by the inoculation of tumor cells mixed with CM. In the future, soluble anti-tumor effectors, produced by confluent MSCs, may be used as cell-free therapeutics; this approach provides a solution to current concerns associated with cell-based therapies.

Abstract 5819: Src inhibitor suppresses rab11 mediated collective cell migration in colon cancer cells

Abstract Collective cell migration has been demonstrated the importance in cancer metastasis. Instead of single cell migration, collective cell migration requires E-cadherin for the cohort cell migration. Rab11 the recycling vesicle protein plays the key role in E-cadherin dynamics and mediates collective cell migration. In this study, Src was hypothesized to regulate Rab11 mediated collective cell migration. Our results demonstrated Src inhibitor Dasatinib has less toxicity in HT-29 colon cancer cells, instead, the cell morphology was turned aggregated. The immunofluorescence image also showed E-cadherin polarity was lost after Dasatinib treatment and increased the potential of cell-cell contact inhibition. In transwell assay, Rab11 was found colocalized with E-cadherin in collective migrated cells. Dasatinib treatment can suppress collective cell migration and decrease the interaction of Rab11 and E-cadherin. This study demonstrated the important role of Src in Rab11 mediated collective cell migration through E-cadherin dynamics in colon cancer. Citation Format: Yi-Wen Lu, Xiang-Ling Hou, Hui Min Koo, Wei-Ting Chao. Src inhibitor suppresses rab11 mediated collective cell migration in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5819.

Hallmarks of cancer: The insulin-like growth factors perspective.

The identification of a series of attributes or hallmarks that are shared by virtually all cancer cells constitutes a true milestone in cancer research. The conceptualization of a catalogue of common genetic, molecular, biochemical and cellular events under a unifying Hallmarks of Cancer idea had a major impact in oncology. Furthermore, the fact that different types of cancer, ranging from pediatric tumors and leukemias to adult epithelial cancers, share a large number of fundamental traits reflects the universal nature of the biological events involved in oncogenesis. The dissection of a complex disease like cancer into a finite directory of hallmarks is of major basic and translational relevance. The role of insulin-like growth factor-1 (IGF1) as a progression/survival factor required for normal cell cycle transition has been firmly established. Similarly well characterized are the biochemical and cellular activities of IGF1 and IGF2 in the chain of events leading from a phenotypically normal cell to a diseased one harboring neoplastic traits, including growth factor independence, loss of cell-cell contact inhibition, chromosomal abnormalities, accumulation of mutations, activation of oncogenes, etc. The purpose of the present review is to provide an in-depth evaluation of the biology of IGF1 at the light of paradigms that emerge from analysis of cancer hallmarks. Given the fact that the IGF1 axis emerged in recent years as a promising therapeutic target, we believe that a careful exploration of this signaling system might be of critical importance on our ability to design and optimize cancer therapies.

A LONG-TERM 10G-HYPERGRAVITY EXPOSURE PROMOTES CELL-CELL CONTACTS AND REDUCES ADHESIVENESS TO A SUBSTRATE, MIGRATION, AND INVASIVENESS OF MCF-7HUMAN BREAST CANCER CELLS.

G-force is a fundamental force controlling human cells. Cancer is one of the 4major health challenges in the Space missions. Cancer in Space project evaluates the reaction of human cancer cells to the conditions of the space flights, including an exposure to high g-forces. Explore an impact of 10g force on the oncogenic properties of human breast adenocarcinoma cells MCF-7. Cells were exposed to 10g force for 10days, as part of a 6-week simulation of conditions of a space flight. Then the cells were cultured for one week under normal culture conditions, before performing tests. Cell proliferation, cell viability, cell-cell contact inhibition, migration, and invasiveness were measured. Immunoblotting was used to evaluate expression of proteins. Proliferation, cell-cell interaction and formation of 3D structures, migration, and invasiveness of cells exposed to 10g were compared to parental cells cultured at 1g condition. 10g exposed cells showed a higher propensity for cell-cell contact inhibitions and lower for 3-dimensional growth in dense culture. This correlated with the decrease of proliferation in a dense culture as compared to the parental cells. The decrease of migration, adherence to a surface, and invasiveness was observed for cells subjected to the hypergravity, as compared to the parental MCF-7cells. Enhanced expression of E-cadherin and phosphorylated pY576-FAK were observed in 10g exposed cells but no impact on the expression of Erk, pErk, FAK and p53was detected. The prolonged exposure of MCF-7cells to 10g force targets cell-cell and cell-substrate interactions.

Regulatory T Cell (Treg) Cytotoxic T Lymphocyte-associated Antigen-4 Deficits in Biliary Atresia (BA) and Disease Rescue With Treg Augmentation in Murine BA.

Biliary atresia (BA) entails an inflammatory injury of the biliary tree, leading to fibrosis of the extrahepatic and intrahepatic bile ducts. The chronic inflammatory biliary injury may be due to lack of appropriate regulatory T cell (Treg) suppression of inflammation. The aims of the study were to characterize Treg deficits in human BA and to determine if Treg augmentation therapy improved outcomes in the rhesus rotavirus (RRV)-induced mouse model of BA. Immunophenotyping of human peripheral blood and liver Tregs was performed with flow cytometry, Vectra-6 multicolor immunohistochemistry (IHC), and real-time polymerase chain reaction. Measured outcomes of Treg augmentation with the interleukin-2 monoclonal antibody JES6-1/interleukin-2 in the RRV-induced mouse model of BA included survival, direct bilirubin, IHC, and liver flow cytometry. Patients with BA had decreased peripheral blood Treg frequency and lack of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) upregulation despite a highly activated, effector Treg phenotype. IHC revealed decreased liver Treg frequency and Treg CTLA-4 expression. Treg augmentation in the murine model led to increased survival, decreased direct bilirubin levels and liver inflammation, and expansion of resident macrophages. In addition to the M2 phenotype of resident macrophages, these cells adopted an inflammatory M1 phenotype in response to RRV infection, which was inhibited with Treg augmentation. Patients with BA have Treg deficiencies associated with lack of sufficient CTLA-4 expression that is necessary for cell-cell contact inhibition of inflammatory responses. Treg augmentation therapy in murine BA protected from disease. Future treatment trials for BA should include agents that enhance Treg number or function, mimic CTLA-4 function, and promote anti-inflammatory M2 macrophage phenotypes.

Examining the Role of HIPPO/YAP and AMOT Dysregulation and Imbalance in Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder, most commonly caused by mutations in Polycystin-1 and Polycystin-2 (PC1 and PC2), in which many large lumenal cysts develop in the kidney epithelium. The disease is progressive and ultimately leads to renal dialysis and/or kidney transplant given the lack of efficacious therapeutic intervention. Current research has implicated HIPPO/YAP pathway dysregulation and AMOT imbalance following defects in cell-cell adhesion and contact inhibition as the molecular basis for ADPKD. To test this hypothesis as to the minimum cellular changes necessary to produce cystogenesis, the Bacallao lab microinjected normal human kidney epithelial (HK2) cells in order to drive ectopic expression of Cadherin-8 (CAD-8). Our Imaging with confocal microscopy and subsequent image analysis indicates CAD-8, a cadherin abnormally expressed in renal epithelial cells of ADPKD patients, is sufficient to cause formation of cysts albeit with uncharacteristically small lumens compared to typical ADPKD cysts. However, these cells exhibit significantly larger cyst expansion when they are transduced for coexpression of CAD-8 and constitutively active YAP5SA. Cells modified for heterozygosity with respect to a defective PC1 gene (PKD) also exhibited significantly larger lumen cysts when transduced with YAP5SA reinforcing the role of Yap in lumen size control. Finally, PKD cells transduced with AMOTL1 exhibited significantly smaller lumen sizes. AMOTL1 belongs to a family of adapter proteins which bind and inhibit Yap activity thus showing conclusively that positive and negative modulation of Yap activity correlates with cyst lumen size expansion and reduction respectively.
 “This project was funded, in part, with support from the O’Brien Center for Advanced Renal Microscopic Analysis funded, in part by P30 DK079312 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

Abstract 10754: Disrupted N-Cadherin Expression Leads to Sarcomeric Disassembly and Cell Cycle Activation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: Induction of cardiomyocyte proliferation holds great promise for cardiac regenerative therapy. We showed previously that in the presence of Wnt signaling agonist, the removal of cell-cell contact between cardiomyocytes is the key to drive massive expansion of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the molecular mechanism regulating cell contact-mediated suppression of hiPSC-CM proliferation remains unknown. Hypothesis: We hypothesized that N-cadherin, the major adherens junction molecule expressed in cardiomyocytes, mediates contact inhibition of hiPSC-CM proliferation. Methods/Results: By qPCR and Western blotting, we showed comparable transcript and protein expression level of N-cadherin when hiPSC-CMs were cultured either densely (with cell contact) or sparsely (without cell contact), despite the observed N-cadherin localization at intercellular junction in densely cultured cells only. We disrupted N-cadherin expression with transient transfection of siRNA and found that the loss of N-cadherin expression resulted in 1.75-fold increase in Ki67 expression in densely but not sparsely cultured day 30 hiPSC-CM, suggesting that junction-localized N-cadherin may affect hiPSC-CM proliferation. Furthermore, we found that the loss of N-cadherin expression led to wide-spread sarcomeric disassembly. To examine whether sarcomeric disassembly alone is sufficient to stimulate cell cycle activity in densely cultured hiPSC-CMs, we knocked down α-actinin expression which led to sarcomere disruption and an increase in Ki67+ expression by 1.6-fold in day 30 hiPSC-CMs. Conclusions: Taken together, our results suggest that N-cadherin mediates the previously observed cell-cell contact inhibition of hiPSC-CM proliferation and the disruption of N-cadherin expression in densely cultured hiPSC-CMs leads to sarcomeric disassembly and hiPSC-CM proliferation. Furthermore, we showed that sarcomeric disassembly alone stimulates hiPSC-CM proliferation. These findings support the need for inducing cardiomyocyte dedifferentiation to achieve in situ cardiomyocyte proliferation for developing novel regenerative therapy to treat congenital and adult heart diseases in the future.

Massive expansion of human induced pluripotent stem cells resulting in efficient biobanking and functional 3D tissue analysis of genetic cardiomyopathies

Abstract Introduction Over the past decade, various protocols were established to ensure efficient differentiation of hiPSC into cardiomyocytes (CMs). A major limitation, however, remained the batch-to-batch variability of hiPSC-CM efficiency and cell number. Here, we suggest an approach in which concomitant GSK-3β inhibition and removal of cell-cell contact inhibition, resulted in a massive proliferative response of hiPSC-CMs1–3. This efficient method allows expansion and passaging of functional hiPSC-CMs, that routinely can be cryopreserved and subsequently used as a stable cell source for the downstream applications, such 3D in vitro models for the disease modelling of dilated cardiomyopathy (DCM). We focussed on the deletion of arginine 14 in the PLN gene (R14del), which is associated with severe heart failure in DCM patients, associated with arrhythmias, cardiac fibrosis and premature death. Methods Subsequent expansion of hiPSC-CM cultures is generally modest (<10 fold). Here, we describe a cost-effective strategy for massive expansion (up to 250-fold) of high-purity hiPSC-CMs relying on two aspects; 1) inhibition of cell-cell contact via low-density seeding and serial passaging in culture flask-format, 2)small molecular glycogen synthase kinase-3β inhibition with CHIR99021 (CHIR). Patient-specific hiPSC-CMs harbouring a PLNR14del mutation were generated and used for EHT formation and functional follow-up. Results We observed that proliferating hiPSC-CMs, especially within the first 2 passages, can routinely be cryopreserved and subsequently further expanded or utilized in downstream applications. Moreover, using this strategy, it is possible to produce ultimately >1 billion CMs within 3–5 weeks starting with one differentiation batch of day 11 hiPSC-CMs, without the need for cell sorting or selection. Expanded hiPSC-CMs retain their capacity to mature and allows fibrin-based engineered heart tissues (EHTs) formation. Previously expanded CMs from PLNR14del patient-specific hiPSC were used to generate EHT and displayed a reduced force phenotype (0.137±0.012 mN) vs healthy control (0.229±0.030 mN) and isogenic control (0.224±0.008 mN) in previously expanded CMs. Conclusion We provpresent a novel strategy for the massive expansion of functional hiPSC-CMs with concomitant GSK-3β inhibition and low cell density culture that ultimately generates up to a 250-fold increase in hiPSC-CM numbers. Expansion healthy control hiPSC-CMs does not limit the subsequent maturation process, and moreover cells remain fully functional such as required for downstream tissue engineering approaches. Therefore, CM expansion forms a well-controlled platform for upscaling hiPSC-CM production for functional 3-dimensionale PLN cardiac disease models, large drug screenings and multiple translational/regenerative applications. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): PLN Foundation

A Short SOX9 Peptide Mimics SOX9 Tumor Suppressor Activity and Is Sufficient to Inhibit Colon Cancer Cell Growth.

Differently from cytotoxic chemotherapies, targeted therapies do not necessarily drive cancer cells toward death, but reduce cell proliferation, angiogenesis, and/or prevent metastasis without affecting healthy cells. Oncogenic proteins that are hyperactivated and/or overexpressed in cancer cells are prime targets for such therapies. On the other hand, the activity of tumor suppressor proteins is more difficult to harness. Here, we identified a short SOX9 sequence (S9pep) located at the hinge between the HMG DNA-binding domain and the SOX-E central conserved domain that mimics SOX9 tumor-suppressive properties. Doxycycline-induced S9pep expression in DLD-1 colorectal cancer cells inhibited the growth potential of these cells, including colorectal cancer stem cells, restored cell-cell contact inhibition, and inhibited the activity of the oncogenic Wnt/β-catenin signaling pathway. It also significantly decreased tumor growth in BALB/cAnNCrl mice grafted with mouse doxycycline-inducible CT26 colorectal cancer cells in which S9pep was induced by treating them with doxycycline. As the Wnt/β-catenin signaling pathway is constitutively activated in 80% of colorectal cancer and SOX9-inactivating mutations are present in up to 11% of colorectal cancer, S9pep could be a promising starting point for the development of a peptide-based therapeutic approach to restore a SOX9-like tumor suppressor function in colorectal cancer.

Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells

The field cancerization effect has been proposed to explain bladder cancer's multifocal and recurrent nature, yet the mechanisms of this effect remain unknown. In this work, using cell biology, flow cytometry, and qPCR analyses, along with a xenograft mouse tumor model, we show that chronic exposure to tumor-derived extracellular vesicles (TEVs) results in the neoplastic transformation of nonmalignant human SV-HUC urothelial cells. Inhibition of EV uptake prevented this transformation. Transformed cells not only possessed several oncogenic properties, such as increased genome instability, loss of cell-cell contact inhibition, and invasiveness, but also displayed altered morphology and cell structures, such as an enlarged cytoplasm with disrupted endoplasmic reticulum (ER) alignment and the accumulation of smaller mitochondria. Exposure of SV-HUC cells to TEVs provoked the unfolded protein response in the endoplasmic reticulum (UPRER). Prolonged induction of UPRER signaling activated the survival branch of the UPRER pathway, in which cells had elevated expression of inositol-requiring enzyme 1 (IRE1), NF-κB, and the inflammatory cytokine leptin, and incurred loss of the pro-apoptotic protein C/EBP homologous protein (CHOP). More importantly, inhibition of ER stress by docosahexaenoic acid prevented TEV-induced transformation. We propose that TEVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress-induced unfolded protein response and inflammation. This study provides detailed insight into the mechanisms underlying the bladder cancer field effect and tumor recurrence.

Improvement of sulfamethoxazole (SMX) elimination and inhibition of formations of hydroxylamine-SMX and N4-acetyl-SMX by membrane bioreactor systems.

Sulfamethoxazole (SMX) has frequently been detected in aquatic environments. In natural environment, not only individual microorganism but also microbial consortia are involved in some biotransformation of pollutants. The competition for space under consortia causing cell-cell contact inhibition changes the cellular behaviors. Herein, the membrane bioreactor system (MBRS) was applied to improve SMX elimination thorough exchanging the cell-free broths (CFB). The removal efficiency of SMX was increased by more than 24% whether under the pure culture of A. faecalis or under the co-culture of A. faecalis and P. denitrificans with MBRS. Meanwhile, MBRS significantly inhibited the formation of HA-SMX, and Ac-SMX from parent compound. Additionally, the cellular growth under MBRS was obviously enhanced, indicating that the increases in the cellular growth under MBRS arepossibly related to the decreases in the levels of HA-SMX and Ac-SMX compared to that without MBRS. The intracellular NADH/NAD+ ratios of A. faecalis under MBRS were increased whether thorough itself-recycle of CFB or exchanging CFB between the pure cultures of A. faecalis and P. denitrificans, suggesting that the enhancement in the bioremoval efficiencies of SMX under MBRS by A. faecalis is likely related to the increases in the NADH/NAD+ ratio. Taken together, the regulation of cell-to-cell communication is preferable strategy to improve the bioremoval efficiency of SMX.

Arylamine N-acetyltransferase 1 protects against reactive oxygen species during glucose starvation: Role in the regulation of p53 stability.

Human arylamine N-acetyltransferase 1 (NAT1) has been associated with cancer cell growth and invasion, but the underlying molecular mechanisms remain unknown. NAT1 is located on the short arm of chromosome 8 (8p21), a region that is commonly deleted in colon cancer. Previously, it was reported that HT-29 colon cancer cells, which have a large deletion at 8p21-22, show marked morphological changes, increased E-cadherin expression and altered cell-cell contact inhibition following down-regulation of NAT1 with shRNA. By contrast, no effects on growth were observed in HeLa cells. In the present study, cellular changes following knockout of NAT1 with CRISPR/Cas9 in HT-29 and HeLa cells were compared in the presence and absence of glucose. Cell growth decreased in both cell-lines during glucose starvation, but it was enhanced in HT-29 cells following NAT1 deletion. This was due to an increase in ROS production that induced cell apoptosis. Both ROS production and cell death were prevented by the glutathione precursor N-acetylcysteine. NAT1 knockout also resulted in a loss of the gain-of-function p53 protein in HT-29 cells. When p53 expression was inhibited with siRNA in parental HT-29 cells, ROS production and apoptosis increased to levels seen in the NAT1 knockout cells. The loss of p53 may explain the decreased colony formation and increased contact inhibition previously reported following NAT1 down-regulation in these cells. In conclusion, NAT1 is important in maintaining intracellular ROS, especially during glucose starvation, by stabilizing gain-of-function p53 in HT-29 cells. These results suggest that NAT1 may be a novel target to decrease intracellular gain-of -function p53.

Bladder Cancer Extracellular Vesicles Drive Tumorigenesis by Inducing Endoplasmic Reticulum Stress

The field cancerization effect has been proposed to explain bladder cancer's multifocal and recurrent nature, yet its mechanisms remain unknown. In this work we show that chronic exposure to tumor-derived extracellular vesicles (TEVs) resulted in the neoplastic transformation of non-malignant human SV-HUC urothelial cells. Inhibition of EV uptake prevented transformation. Transformed cells not only possessed several oncogenic properties, such as genome instability, loss of cell-cell contact inhibition, and invasiveness, but also displayed altered morphology where cells show enlarged cytoplasm with disrupted ER alignment and the accumulation of smaller mitochondria. Treatment of SV-HUC cells with TEVs provoked the unfolded protein response of endoplasmic reticulum (UPRER). Prolonged induction of UPRER signaling led to the activation of the survival branch of the UPRER pathway, where cells had elevated expression of the IRE1, NFi«B and the inflammatory cytokine leptin, and loss of CHOP, a pro-apoptotic protein. More importantly, inhibition of ER stress by docosahexaenoic acid prevented TEV-induced transformation. We propose that TEVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress-induced unfolded protein response and inflammation. This study provides insight into the mechanisms of the bladder cancer field effect and tumor recurrence. Funding Statement: This work is supported by NCI R01 CA173986 (YF Lee, PI). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: The study was approved by the University of Rochester Committee on Animal Resources, and the mice were kept in a specific pathogen-free environment.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-mediated deregulation of myeloid and sebaceous gland stem/progenitor cell homeostasis.

Studies of TCDD toxicitystimulated identification of the responsible aryl hydrocarbon receptor (AHR), a multifunctional, ligand-activated transcription factor of the basic helix-loop-helix/Per-Arnt-Sim family. Accumulating evidence suggests a role of this receptor in homeostasis of stem/progenitor cells, in addition to its known role in xenobiotic metabolism. (1) Regulation of myelopoiesis is complex. As one example, AHR-mediated downregulation of human CD34+ progenitor differentiation to monocytes/macrophages is discussed. (2) Accumulation of TCDD in sebum leads to deregulation of sebocyte differentiation via Blimp1-mediated inhibition of c-Myc signaling and stimulation of Wnt-mediated proliferation of interfollicular epidermis. The resulting sebaceous gland atrophy and formation of dermal cysts may explain the pathogenesis of chloracne, the hallmark of TCDD toxicity. (3) TCDD treatment of confluent liver stem cell-like rat WB-F344 cells leads to release from cell-cell contact inhibition via AHR-mediated crosstalk with multiple signaling pathways. Further work is needed to delineate AHR function in crosstalk with other signaling pathways.

Sex Chromosome Analysis of Postmortem Corneal Endothelium After Sex-Mismatch Descemet Membrane Endothelial Keratoplasty.

To identify the origin of corneal endothelial cells (host or donor) present on grafts at various time points after Descemet membrane endothelial keratoplasty (DMEK), using fluorescence in situ hybridization (FISH) of sex chromosomes on post mortem corneas with sex mismatch between the donor and host. Corneoscleral buttons of 6 post mortem DMEK eyes of 4 patients, operated for Fuchs endothelial dystrophy, with an average postoperative time of 2.6 (±1.8) years (range, 7 months-4.5 years), of 2.5 (±1.7) years (range, 7 months-4 years), were processed for FISH detection of XX (female) or XY (male)-labeling signals in corneal endothelial cells in the central area of the DMEK graft. Two male patients underwent bilateral DMEK with grafts from female donors, and 2 female patients underwent unilateral DMEK and received a graft from a male donor. FISH consistently showed the presence of donor endothelial cells across the graft area, with signaling of sex chromosomes opposite to the sex of the host. Donor endothelial cells may survive up to 4.5 years after DMEK. If so, the lower incidence of allograft rejection in DMEK may not be explained by early host cell replacement. Potential host cell migration may be limited by donor/recipient cell-cell contact inhibition.

Prostate cancer cells specifically reorganize epithelial cell-fibroblast communication through proteoglycan and junction pathways

ABSTRACTMicroenvironment and stromal fibroblasts are able to inhibit tumor cell proliferation both through secreted signaling molecules and direct cell-cell interactions but molecular mechanisms of these effects remain unclear. In this study, we investigated a role of cell-cell contact-related molecules (protein ECM components, proteoglycans (PGs) and junction-related molecules) in intercellular communications between the human TERT immortalized fibroblasts (BjTERT fibroblasts) and normal (PNT2) or cancer (LNCaP, PC3, DU145) prostate epithelial cells. It was shown that BjTERT-PNT2 cell coculture resulted in significant decrease of both BjTERT and PNT2 proliferation rates and reorganization of transcriptional activity of cell-cell contact-related genes in both cell types. Immunocytochemical staining revealed redistribution of DCN and LUM in PNT2 cells and significant increase of SDC1 at the intercellular contact zones between BjTERT and PNT2 cells, suggesting active involvement of the PGs in cell-cell contacts and contact inhibition of cell proliferation. Unlike to PNT2 cells, PC3 cells did not respond to BjTERT in terms of PGs expression, moderately increased transcriptional activity of junctions-related genes (especially tight junction) and failed to establish PC3-BjTERT contacts. At the same time, PC3 cells significantly down-regulated junctions-related genes (especially focal adhesions and adherens junctions) in BjTERT fibroblasts resulting in visible preference for homotypic PC3-PC3 over heterotypic PC3-BjTERT contacts and autonomous growth of PC3 clones. Taken together, the results demonstrate that an instructing role of fibroblasts to normal prostate epithelial cells is revoked by cancer cells through deregulation of proteoglycans and junction molecules expression and overall disorganization of fibroblast-cancer cell communication.

YAP Nuclear Localization in the Absence of Cell-Cell Contact Is Mediated by a Filamentous Actin-dependent, Myosin II- and Phospho-YAP-independent Pathway during Extracellular Matrix Mechanosensing

Cell-cell contact inhibition and the mechanical environment of cells have both been shown to regulate YAP nuclear localization to modulate cell proliferation. Changes in cellular contractility by genetic, pharmacological, and matrix stiffness perturbations regulate YAP nuclear localization. However, because contractility and F-actin organization are interconnected cytoskeletal properties, it remains unclear which of these distinctly regulates YAP localization. Here we show that in the absence of cell-cell contact, actomyosin contractility suppresses YAP phosphorylation at Ser(112), however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. We find that actin cytoskeletal integrity is essential for YAP nuclear localization, and can override phosphoregulation or contractility-mediated regulation of YAP nuclear localization. This actin-mediated regulation is conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced cytoskeletal integrity leading to nuclear exclusion of both YAP and Ser(P)(112)-YAP. These data provide evidence for two actin-mediated pathways for YAP regulation; one in which actomyosin contractility regulates YAP phosphorylation, and a second that involves cytoskeletal integrity-mediated regulation of YAP nuclear localization independent of contractility. We suggest that in non-contact inhibited cells, this latter mechanism may be important in low stiffness regimes, such as may be encountered in physiological environments.

Alteration of cell-cell and cell-matrix adhesion in urothelial cells: an oncogenic mechanism for mutant FGFR3.

Activating mutations of FGFR3 are a common and early event in bladder cancer. Ectopic expression of mutant FGFR3 in normal urothelial cells has both pro-proliferative and antiapoptotic effects at confluence, suggesting that mutant cells are insensitive to cell-cell contact inhibition. Herein, detailed analysis revealed that these cells have reduced cell-cell adhesion, with large intercellular spaces observable at confluence, and diminished cell-substrate adhesion to collagen IV, collagen I, and fibronectin. These phenotypic alterations are accompanied by changes in the expression of genes involved in cell adhesion and extracellular matrix remodeling. Silencing of endogenous mutant FGFR3 in bladder cancer cells induced converse changes in transcript levels of CDH16, PLAU, MMP10, EPCAM, TNC, and HAS3, confirming them as downstream gene targets of mutant FGFR3. Overexpression of EPCAM, HAS3, and MMP10 transcripts was found in a large fraction of primary bladder tumors analyzed, supporting their key role in bladder tumorigenesis in vivo. However, no correlation was found between their protein and/or mRNA expression and FGFR3 mutation status in tumor specimens, indicating that these genes may be targeted by several converging oncogenic pathways. Overall, these results indicate that mutant FGFR3 favors the development and progression of premalignant bladder lesions by altering key genes regulating the cell-cell and cell-matrix adhesive properties of urothelial cells. The ability of mutant FGFR3 to drive transcriptional expression profiles involved in tumor cell adhesion suggests a mechanism for expansion of premalignant urothelial lesions.

N-terminal truncation of Lats1 causes abnormal cell growth control and chromosomal instability

The tumor suppressors Lats1 and Lats2 are mediators of the Hippo pathway that regulates tissue growth and proliferation. Their N-terminal non-kinase regions are distinct except for Lats conserved domains 1 and 2 (LCD1 and LCD2), which may be important for Lats1/2-specific functions. Lats1 knockout mice were generated by disrupting the N-terminal region containing LCD1 (Lats1(ΔN/ΔN)). Some Lats1(ΔN/ΔN) mice were born safely and grew normally. However, mouse embryonic fibroblasts (MEFs) from Lats1(ΔN/ΔN) mice displayed mitotic defects, centrosomal overduplication, chromosomal misalignment, multipolar spindle formation, chromosomal bridging and cytokinesis failure. They also showed anchorage-independent growth and continued cell cycles and cell growth, bypassing cell-cell contact inhibition similar to tumor cells. Lats1(ΔN/ΔN) MEFs produced tumors in nude mice after subcutaneous injection, although the tumor growth rate was much slower than that of ordinary cancer cells. Yap, a key transcriptional coactivator of the Hippo pathway, was overexpressed and stably retained in Lats1(ΔN/ΔN) MEFs in a cell density independent manner, and Lats2 mRNA expression was downregulated. In conclusion, N-terminally truncated Lats1 induced Lats2 downregulation and Yap protein accumulation, leading to chromosomal instability and tumorigenesis.