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

Additional file 1: Figure S1. of Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma

(A) ChIP assay shows that N-myc is bound to the proximal region (−126 to +12 bp) of the ALK promoter. (B) The ALK promoter sequence containing two putative E-boxes (E1 and E2). (C) Various promoter constructs were used for evaluating transcriptional regulation of the ALK promoter by N-myc. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate. (TIF 725 kb

Additional file 2: Figure S2. of Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma

(A) Two independent Hec251 cell lines stably overexpressing ALK (H251-ALK#8 and #16) and mock cells were seeded at low density and monitored for growth. The cell numbers presented are means ± SDs. P0, P3, P5, and P7: 0, 3,5, and 7 days after passage. (B) Western blot analysis of expression of cyclin A, p21waf1, and p27kip1 at P6 of cell growth in stable ALK-overexpressing cell lines. (C) The pNF-κB reporter construct was transfected into H251-ALK#16 cells treated with 2.5 ng/ml TGF-β1 or 50 ng/ml HGF for 48 h. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate. (D) Various promoter constructs were used for evaluating transcriptional regulation of the ALK promoter by TNF-α. (E) The pNF-κB reporter construct, together with the ALK expression vector, were transfected into Ishikawa cells. (TIF 843 kb

Utility of Adrenal Vein Sampling With and Without Ultra-Low Dose ACTH Infusion in the Diagnostic Evaluation of Primary Aldosteronism

BACKGROUND: Adrenal vein sampling (AVS), integral to identifying surgically remediable unilateral primary aldosteronism (PA), is technically challenging and subject to fluctuations in cortisol and aldosterone secretion. Intra-procedural adrenocorticotropic hormone (ACTH), conventionally administered as a 250-μg bolus and/or 50 μg per hour infusion, increases cortisol and aldosterone secretion and can improve AVS success, but may cause discordant lateralisation compared to unstimulated AVS. AIMS: To assess if AVS performed with ultra-low dose ACTH infusion causes discordant lateralisation. METHODS: Here, we describe our preliminary experience using an ultra-low dose ACTH infusion AVS protocol. We retrospectively reviewed the results of consecutive AVS procedures (n = 37) performed with and without ultra-low dose ACTH (1-μg bolus followed by 1.25 μg per hour infusion). RESULTS: Bilateral AV cannulation was successful in 70% of procedures pre-ACTH and 89% post-ACTH (p < 0.01). Sixty-nine percent of studies lateralised pre-ACTH and 55% post-ACTH, improving to 79% when both groups were combined. Lateralisation was discordant in 11 cases, including eight in which lateralisation was present only on basal sampling, and three in which lateralisation occurred only with ACTH stimulation. DISCUSSION: Overall, the decrease in lateralisation rates with ACTH was higher than previously reported for some protocols utilising conventional doses of ACTH. Our results suggest that AVS performed with ultra-low dose ACTH can cause discordant lateralisation similar to AVS performed with conventional doses of ACTH. CONCLUSION: Prospective studies directly comparing low and conventional dose ACTH AVS protocols and long-term patient outcomes are needed to help define the optimal ACTH dose for accurate PA subtyping