Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma

BACKGROUND: Anaplastic lymphoma kinase (ALK), which is a receptor tyrosine kinase, is essentially and transiently expressed in the developing nervous system. Recently, the deregulated expression of full-length ALK has been observed in some primary solid tumors, but little is known about its involvement in the tumorigenesis of uterine carcinosarcomas (UCSs). Here we examined the functional role of the ALK gene in UCSs. METHODS: Regulation and function of the ALK gene were assessed using two endometrial carcinoma cell lines. Expression of ALK and its related molecules were also investigated using clinical samples of UCSs. RESULTS: In cell lines, ALK promoter activity was significantly increased by transfection of Sox11 and N-myc, which are known to contribute to neuronal properties. Cells stably overexpressing full-length ALK showed an enhancement of EMT properties mediated by TGF-β1 and HGF, along with an increase in phosphorylated (p) Akt and nuclear p65. Overexpression of p65 also led to transactivation of Twist1 gene, known as an EMT inducer. Finally, treatment of the stable ALK-overexpressing cells with doxorubicin resulted in inhibition of apoptosis with progressive increase in the expression ratio of both pAkt and bcl2 relative to total Akt and bax, respectively. In clinical samples, strong cytoplasmic ALK immunoreactivity and mRNA signals without rearrangement or amplification of the ALK locus were frequently observed in UCSs, particularly in the sarcomatous components. Further, ALK IHC score was found to be positively correlated with Sox11, N-myc, Twist1, and bcl2 scores. CONCLUSION: ALK-related signal cascades containing Akt, NF-κB, Twist1, and bcl2 may participate in initial signaling for divergent sarcomatous differentiation driven from carcinomatous components in UCSs through induction of the EMT process and inhibition of apoptotic features. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12943-017-0609-8) contains supplementary material, which is available to authorized users

Scalable generation of cerebellar neurons from pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, our ability to differentiate cerebellar neurons from pluripotent stem cells is still limited. Recently, we described the long-term culture of cerebellar neuroepithelium formed from human iPSCs, recapitulating the early developmental events of the cerebellum. Additionally, an efficient maturation of replated cerebellar progenitors into distinct types of functional cerebellar neurons was also achieved under defined and feeder-free conditions. However, developing a scalable protocol that allows to produce large numbers of organoids and high yields of mature neurons in a 3D bioreactor culture systems is still a difficult challenge. In this work, we present a new approach for the reproducible and scalable generation of mid-hindbrain organoids under chemically defined conditions by using the novel PBS 0.1 (100 mL) Vertical-Wheel single-use bioreactor. In this system, an efficient cell aggregation with shape and size-controlled aggregates can be obtained, which is important for homogeneous and efficient differentiation. Moreover, a larger amount of iPSC-derived aggregates can be generated without being excessively labour-intensive, achieving 431 ± 53.6 aggregates/mL at 24 hours after seeding. After differentiation, distinct types of cerebellar neurons were generated, including Purkinje cells (Calbindin+), Granule cells (BARHL1+ and Pax6+), Golgi cells (Neurogranin+ and GAD65+), Deep cerebellar nuclei projection neurons (TBR1+) and Non-Golgi-type interneurons (Parvalbumin+ and Calbindin-). These cells show signs of efficient maturation, staining positive for MAP2, and are able to change intracellular Ca2+ concentration following KCl stimulation. In this system, human iPSC-derived organoids are able to mature into different mature cerebellar neurons and to survive for up to 3 months, without replating and co-culture with feeder layers

Autosomal dominant pseudohypoaldosteronism type 1 with a novel splice site mutation in MR gene

<p>Abstract</p> <p>Background</p> <p>Autosomal dominant pseudohypoaldosteronism type 1 (PHA1) is a rare inherited condition that is characterized by renal resistance to aldosterone as well as salt wasting, hyperkalemia, and metabolic acidosis. Renal PHA1 is caused by mutations of the human mineralcorticoid receptor gene (<it>MR</it>), but it is a matter of debate whether <it>MR </it>mutations cause mineralcorticoid resistance via haploinsufficiency or dominant negative mechanism. It was previously reported that in a case with nonsense mutation the mutant mRNA was absent in lymphocytes because of nonsense mediated mRNA decay (NMD) and therefore postulated that haploinsufficiency alone can give rise to the PHA1 phenotype in patients with truncated mutations.</p> <p>Methods and Results</p> <p>We conducted genomic DNA analysis and mRNA analysis for familial PHA1 patients extracted from lymphocytes and urinary sediments and could detect one novel splice site mutation which leads to exon skipping and frame shift result in premature termination at the transcript level. The mRNA analysis showed evidence of wild type and exon-skipped RT-PCR products.</p> <p>Conclusion</p> <p>mRNA analysis have been rarely conducted for PHA1 because kidney tissues are unavailable for this disease. However, we conducted RT-PCR analysis using mRNA extracted from urinary sediments. We could demonstrate that NMD does not fully function in kidney cells and that haploinsufficiency due to NMD with premature termination is not sufficient to give rise to the PHA1 phenotype at least in this mutation of our patient. Additional studies including mRNA analysis will be needed to identify the exact mechanism of the phenotype of PHA.</p