Itraconazole Exerts Its Antitumor Effect in Esophageal Cancer By Suppressing the HER2/AKT Signaling Pathway.

Itraconazole, an FDA-approved antifungal, has antitumor activity against a variety of cancers. We sought to determine the effects of itraconazole on esophageal cancer and elucidate its mechanism of action. Itraconazole inhibited cell proliferation and induced G1-phase cell-cycle arrest in esophageal squamous cell carcinoma and adenocarcinoma cell lines. Using an unbiased kinase array, we found that itraconazole downregulated protein kinase AKT phosphorylation in OE33 esophageal adenocarcinoma cells. Itraconazole also decreased phosphorylation of downstream ribosomal protein S6, transcriptional expression of the upstream receptor tyrosine kinase HER2, and phosphorylation of upstream PI3K in esophageal cancer cells. Lapatinib, a tyrosine kinase inhibitor that targets HER2, and siRNA-mediated knockdown of HER2 similarly suppressed cancer cell growth in vitro Itraconazole significantly inhibited growth of OE33-derived flank xenografts in mice with detectable levels of itraconazole and its primary metabolite, hydroxyitraconazole, in esophagi and tumors. HER2 total protein and phosphorylation of AKT and S6 proteins were decreased in xenografts from itraconazole-treated mice compared to xenografts from placebo-treated mice. In an early phase I clinical trial (NCT02749513) in patients with esophageal cancer, itraconazole decreased HER2 total protein expression and phosphorylation of AKT and S6 proteins in tumors. These data demonstrate that itraconazole has potent antitumor properties in esophageal cancer, partially through blockade of HER2/AKT signaling.

GEAMP, a novel gastroesophageal junction carcinoma cell line derived from a malignant pleural effusion.

Gastroesophageal junction (GEJ) cancer remains a clinically significant disease in Western countries due to its increasing incidence, which mirrors that of esophageal cancer, and poor prognosis. To develop novel and effective approaches for prevention, early detection, and treatment of patients with GEJ cancer, a better understanding of the mechanisms driving pathogenesis and malignant progression of this disease is required. These efforts have been limited by the small number of available cell lines and appropriate preclinical animal models for in vitro and in vivo studies. We have established and characterized a novel GEJ cancer cell line, GEAMP, derived from the malignant pleural effusion of a previously treated GEJ cancer patient. Comprehensive genetic analyses confirmed a clonal relationship between GEAMP cells and the primary tumor. Targeted next-generation sequencing identified 56 nonsynonymous alterations in 51 genes including TP53 and APC, which are commonly altered in GEJ cancer. In addition, multiple copy-number alterations were found including EGFR and K-RAS gene amplifications and loss of CDKN2A and CDKN2B. Histological examination of subcutaneous flank xenografts in nude and NOD-SCID mice showed a carcinoma with mixed squamous and glandular differentiation, suggesting GEAMP cells contain a subpopulation with multipotent potential. Finally, pharmacologic inhibition of the EGFR signaling pathway led to downregulation of key downstream kinases and inhibition of cell proliferation in vitro. Thus, GEAMP represents a valuable addition to the limited number of bona fide GEJ cancer cell lines.

Molecular cloning and identification of mouse Cklfsf2a and Cklfsf2b, two homologues of human CKLFSF2.

Human chemokine-like factor superfamily (CKLFSF) is a novel gene family comprising CKLF and CKLFSF1-8. Among them, CKLFSF2 is highly expressed in testis and may play important roles in male reproduction. Besides, it is very active during evolution and has two counterparts in mouse. For further study, we cloned the two mouse genes by EST assembly and RT-PCR methods and designated them as mouse Cklfsf2a and Cklfsf2b. Their predicted open-reading frames (ORFs) that encode 169 and 210 amino acids, respectively, were obtained; and their predicted full-length molecular sizes that are approximately 1.2 kb for mCklfsf2a and 0.9 kb for mCklfsf2b were confirmed by Northern blot analysis. Mouse Cklfsf2a and Cklfsf2b show similarities with human CKLFSF2 in the expression patterns that are abundant in testis, hematopoietic and immune tissues; as well as in the chromosome localizations that neighbor CKLFSF1 and 3. Their putative protein products have 47.6 and 45.5% identities with hCKLFSF2, respectively; both of them contain four potential transmembrane regions and MARVEL domains, which are also similar with hCKLFSF2. Functionally, they all can affect the transcriptional activity of androgen receptor in PC-3 and HeLa cells, but mCklfsf2a is a repressor while mCklfsf2b and hCKLFSF2 are enhancers. Taken together, we conclude that mouse Cklfsf2a and Cklfsf2b are two homologues of human CKLFSF2. Studies on them would provide much help in further investigation of the latter.

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction.

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca(2+)/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca(2+)/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.

Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8.

It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression.

Accumulating reports demonstrate that apoptosis does not explain all the forms of programmed cell death (PCD), particularly in individual development and neurodegenerative disease. Recently, a novel type of PCD, designated 'paraptosis', was described. Here, we show that overexpression of TAJ/TROY, a member of the tumor necrosis factor receptor superfamily, induces non-apoptotic cell death with paraptosis-like morphology in 293T cells. Transmission electron microscopy studies reveal extensive cytoplasmic vacuolation and mitochondrial swelling in some dying cells and no condensation or fragmentation of the nuclei. Characteristically, cell death triggered by TAJ/TROY was accompanied by phosphatidylserine externalization, loss of the mitochondrial transmembrane potential and independent of caspase activation. In addition, TAJ/TROY suppressed clonogenic growth of HEK293 and HeLa cells. Interestingly, overexpression of Programmed cell death 5 (PDCD5), an apoptosis-promoting protein, enhanced TAJ/TROY-induced paraptotic cell death. Moreover, cellular endogenous PDCD5 protein was significantly upregulated in response to TAJ/TROY overexpression. These results provide novel evidence that TAJ/TROY activates a death pathway distinct from apoptosis and that PDCD5 is an important regulator in both apoptotic and non-apoptotic PCD.

Identification of eight genes encoding chemokine-like factor superfamily members 1-8 (CKLFSF1-8) by in silico cloning and experimental validation.

TM4SF11 is only 102 kb from the chemokine gene cluster composed of SCYA22, SCYD1, and SCYA17 on chromosome 16q13. CKLF maps on chromosome 16q22. CKLFs have some characteristics associated with the CCL22/MDC, CX3CL1/fractalkine, CCL17/TARC, and TM4SF proteins. Bioinformatics based on CKLF2 cDNA and protein sequences in combination with experimental validation identified eight novel genes designated chemokine-like factor superfamily members 1-8 (CKLFSF1-8). CKLFSF1-8 form gene clusters; the sequence identities between CKLF2 and CKLFSF1-8 are from 12.5 to 39.7%. Most of the CKLFSFs have alternative RNA splicing forms. CKLFSF1 has a CC motif and higher sequence similarity with chemokines than with any of the other CKLFSFs. CKLFSF8 shares 39.3% amino acid identity with TM4SF11. CKLFSF1 links the CKLFSF family with chemokines, and CKLFSF8 links it with TM4SF. The characteristics of CKLFSF2-7 are intermediate between CKLFSF1 and CKLFSF8. This indicates that CKLFSF represents a novel gene family between the SCY and the TM4SF gene families.