NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

αKlotho Regulates Age-Associated Vascular Calcification and Lifespan in Zebrafish.

The hormone αKlotho regulates lifespan in mice, as knockouts die early of what appears to be accelerated aging due to hyperphosphatemia and soft tissue calcification. In contrast, the overexpression of αKlotho increases lifespan. Given the severe mouse phenotype, we generated zebrafish mutants for αklotho as well as its binding partner fibroblast growth factor-23 (fgf23). Both mutations cause shortened lifespan in zebrafish, with abrupt onset of behavioral and degenerative physical changes at around 5 months of age. There is a calcification of vessels throughout the body, most dramatically in the outflow tract of the heart, the bulbus arteriosus (BA). This calcification is associated with an ectopic activation of osteoclast differentiation pathways. These findings suggest that the gradual loss of αKlotho found in normal aging might give rise to ectopic calcification.

mTORC1 signaling suppresses Wnt/ß-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level.

Wnt/ß-catenin signaling plays pivotal roles in cell proliferation and tissue homeostasis by maintaining somatic stem cell functions. The mammalian target of rapamycin (mTOR) signaling functions as an integrative rheostat that orchestrates various cellular and metabolic activities that shape tissue homeostasis. Whether these two fundamental signaling pathways couple to exert physiological functions still remains mysterious. Using a genome-wide CRISPR-Cas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/ß-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/ß-catenin target gene expression, which can be rescued by RAD001. Mechanistically, mTORC1 signaling regulates the cell surface level of Wnt receptor Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influencing the association of DVL and clathrin AP-2 adaptor. Sustained mTORC1 activation impairs Wnt/ß-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/ß-catenin-dependent liver metabolic zonation gene expression program is also down-regulated by mTORC1 activation. Our study provides a paradigm that mTORC1 signaling cell autonomously regulates Wnt/ß-catenin pathway to influence stem cell maintenance.

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling.

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.

Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation.

OBJECTIVES: We report histological analysis of hearts from patients with end-stage heart disease who were transplanted with autologous skeletal myoblasts concurrent with left ventricular assist device (LVAD) implantation. BACKGROUND: Autologous skeletal myoblast transplantation is under investigation as a means to repair infarcted myocardium. To date, there is only indirect evidence to suggest survival of skeletal muscle in humans. METHODS: Five patients (all male; median age 60 years) with ischemic cardiomyopathy, refractory heart failure, and listed for heart transplantation underwent muscle biopsy from the quadriceps muscle. The muscle specimen was shipped to a cell isolation facility where myoblasts were isolated and grown. Patients received a transplant of 300 million cells concomitant with LVAD implantation. Four patients underwent LVAD explant after 68, 91, 141, and 191 days of LVAD support (three transplant, one LVAD death), respectively. One patient remains alive on LVAD support awaiting heart transplantation. RESULTS: Skeletal muscle cell survival and differentiation into mature myofibers were directly demonstrated in scarred myocardium from three of the four explanted hearts using an antibody against skeletal muscle-specific myosin heavy chain. An increase in small vessel formation was observed in one of three patients at the site of surviving myotubes, but not in adjacent tissue devoid of engrafted cells. CONCLUSIONS: These findings represent demonstration of autologous myoblast cell survival in human heart. The implanted skeletal myoblasts formed viable grafts in heavily scarred human myocardial tissue. These results establish the feasibility of myoblast transplants for myocardial repair in humans.

Comparison of fresh and cryopreserved porcine ventral mesencephalon cells transplanted in A rat model of Parkinson's disease.

To evaluate whether cryopreservation of porcine ventral mesencephalon cells influences graft survival and function in vivo, we have transplanted either freshly prepared or cryopreserved cells into the striatum of 6-hydroxydopamine-lesioned rats. A single cell suspension of porcine ventral mesencephalon cells from the same isolation either was stored at 4 degrees C and transplanted the next day or was cryopreserved for 4 weeks in liquid nitrogen vapor. The cryopreserved cells were then rapidly thawed, rinsed, and transplanted in the same manner as the fresh cells, with the same dose of viable cells. All animals received daily injections of cyclosporin A to prevent xenograft rejection. To monitor graft function, amphetamine-induced rotation was measured every 3 weeks between 6 and 15 weeks posttransplantation. After sacrifice at 15 weeks posttransplantation, histological methods were used to compare fresh cell and cryopreserved cell transplants with respect to graft survival, differentiation and integration, and host immune response. Cryopreserved cells were found to be either equivalent or in some cases superior to fresh cells with respect to rotational correction, graft survival, graft volume, numbers of graft-derived dopaminergic neurons, and host immune responses. In conclusion, the results indicate that it is feasible to cryopreserve porcine ventral mesencephalon cells for long-term storage of cells prior to transplantation in an animal model of Parkinson's disease.