Molecular evolution of the def6/swap70 gene family and functional analysis of swap70a in zebrafish embryogenesis

Rho GTPases including Rac1, RhoA, and Cdc42 are molecular switches as for signal transduction. Cycling between the GTP-bound active state and GDPbound inactive state is tightly controlled by regulatory proteins. The exchange of GDP for GTP is catalysed by guanine nucleotide exchange factors (GEFs). Upon the activation of Rho GTPases through GEFs, downstream effector molecules are activated and thus trigger cellular responses such as actin cytoskeletal reorganisation, membrane ruffling, cell migration, and gene expression. Based on homology, there are three main families of Rho GEFs, Dbl family, Dock family, and def6/swap70 family. The large Dbl family is characterised through an invariable domain arrangement of an N-terminal catalytic dbl homology (DH) and a C-terminal regulatory pleckstrin homology (PH) domain whereas Dock family members lack the DH domain but instead contain a Dock homology region 2 (DHR2) domain. The def6/swap70 GEFs on the other hand contain an atypical and unique PH-DH like domain arrangement. Mammalian DEF6 and SWAP70 that exhibit a high similarity in their N-terminal ends containing a putative Ca2+ - binding EF hand are crucial mediators of signal transduction in T and B cells, respectively. Phylogenetic sequence analysis revealed that the atypical domain structure as well as the primary amino acid sequences of def6 and swap70 family members has been highly conserved in vertebrates and invertebrates. Whereas invertebrates have only one def6/swap70 gene, two genes have been identified in tetrapod species, and four to five genes have been identified in teleosts species. In zebrafish, five paralogous genes were identified: def6a, def6b, swap70a, swap70b and def6-like. Remarkably, the predicted secondary and tertiary structure of all def6/swap70 family members including the five proteins identified in zebrafish are very similar; most of them folding into a ‘donut-shape’ structure. The expression profile of the def6/swap70 genes during zebrafish development indicated that def6a and swap70a are expressed maternally as well as zygotically during early development whereas expression of the other three genes was restricted to later stages. Morpholino-mediated knockdown of swap70a using two different splice morpholinos resulted in a delay of zebrafish development likely to be due to impaired convergence and extension cell movements during gastrulation. In addition, development of brain, eyes, ears number of otoliths and tail formation was affected. Preliminary data using the AUG morpholino to target maternal and zygotic expression of swap70a indicate a more severe phenotype and high mortality of the morphants. Co-injection of GFP-tagged swap70a mRNA in low dose resulted in a partial rescue of the splice morpholinomediated phenotype. Over-expression of GFP-swap70a in high dose however, resulted also in developmental defects in eyes, number of otoliths and tail formation. The observed phenotype of swap70a morphants described here is reminiscent of the phenotype of def6a morphants that was shown to be downstream of Wnt5b in the non-canonical Wnt/PCP signalling pathway (Goudevenou et al. in preparation) regulating convergence extension cell movement during gastrulation. It is therefore tempting to speculate that swap70a has a similar role acting either in conjunction with or in parallel to def6a in the non-canonical Wnt signalling pathway

Molecular evolution of the def6/swap70 gene family and functional analysis of swap70a in zebrafish embryogenesis

Rho GTPases including Rac1, RhoA, and Cdc42 are molecular switches as for signal transduction. Cycling between the GTP-bound active state and GDPbound inactive state is tightly controlled by regulatory proteins. The exchange of GDP for GTP is catalysed by guanine nucleotide exchange factors (GEFs). Upon the activation of Rho GTPases through GEFs, downstream effector molecules are activated and thus trigger cellular responses such as actin cytoskeletal reorganisation, membrane ruffling, cell migration, and gene expression. Based on homology, there are three main families of Rho GEFs, Dbl family, Dock family, and def6/swap70 family. The large Dbl family is characterised through an invariable domain arrangement of an N-terminal catalytic dbl homology (DH) and a C-terminal regulatory pleckstrin homology (PH) domain whereas Dock family members lack the DH domain but instead contain a Dock homology region 2 (DHR2) domain. The def6/swap70 GEFs on the other hand contain an atypical and unique PH-DH like domain arrangement. Mammalian DEF6 and SWAP70 that exhibit a high similarity in their N-terminal ends containing a putative Ca2+ - binding EF hand are crucial mediators of signal transduction in T and B cells, respectively. Phylogenetic sequence analysis revealed that the atypical domain structure as well as the primary amino acid sequences of def6 and swap70 family members has been highly conserved in vertebrates and invertebrates. Whereas invertebrates have only one def6/swap70 gene, two genes have been identified in tetrapod species, and four to five genes have been identified in teleosts species. In zebrafish, five paralogous genes were identified: def6a, def6b, swap70a, swap70b and def6-like. Remarkably, the predicted secondary and tertiary structure of all def6/swap70 family members including the five proteins identified in zebrafish are very similar; most of them folding into a ‘donut-shape’ structure. The expression profile of the def6/swap70 genes during zebrafish development indicated that def6a and swap70a are expressed maternally as well as zygotically during early development whereas expression of the other three genes was restricted to later stages. Morpholino-mediated knockdown of swap70a using two different splice morpholinos resulted in a delay of zebrafish development likely to be due to impaired convergence and extension cell movements during gastrulation. In addition, development of brain, eyes, ears number of otoliths and tail formation was affected. Preliminary data using the AUG morpholino to target maternal and zygotic expression of swap70a indicate a more severe phenotype and high mortality of the morphants. Co-injection of GFP-tagged swap70a mRNA in low dose resulted in a partial rescue of the splice morpholinomediated phenotype. Over-expression of GFP-swap70a in high dose however, resulted also in developmental defects in eyes, number of otoliths and tail formation. The observed phenotype of swap70a morphants described here is reminiscent of the phenotype of def6a morphants that was shown to be downstream of Wnt5b in the non-canonical Wnt/PCP signalling pathway (Goudevenou et al. in preparation) regulating convergence extension cell movement during gastrulation. It is therefore tempting to speculate that swap70a has a similar role acting either in conjunction with or in parallel to def6a in the non-canonical Wnt signalling pathway

A Perspective on Cell Therapy and Cancer Vaccine in Biliary Tract Cancers (BTCs).

Biliary tract cancer (BTC) is a rare, but aggressive, disease that comprises of gallbladder carcinoma, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma, with heterogeneous molecular profiles. Advanced disease has limited therapeutic options beyond first-line platinum-based chemotherapy. Immunotherapy has emerged as a viable option for many cancers with a similar unmet need. Therefore, we reviewed current understanding of the tumor immune microenvironment and recent advances in cellular immunotherapy and therapeutic cancer vaccines against BTC. We illustrated the efficacy of dendritic cell vaccination in one patient with advanced, chemorefractory, melanoma-associated antigen (MAGE)-positive gallbladder carcinoma, who was given multiple injections of an allogenic MAGE antigen-positive melanoma cell lysate (MCL)-based autologous dendritic cell vaccine combined with sequential anti-angiogenic therapy. This resulted in good radiological and tumor marker response and an overall survival of 3 years from diagnosis. We postulate the potential synergism of adding anti-angiogenic therapy, such as bevacizumab, to immunotherapy in BTC, as a rational scientific principle to positively modulate the tumor microenvironment to augment antitumor immunity

Comparison of Characteristics of Breast Cancer Detected through Different Imaging Modalities in a Large Cohort of Hong Kong Chinese Women: Implication of Imaging Choice on Upcoming Local Screening Program

Background. We compared the clinico-radio-pathological characteristics of breast cancer detected through mammogram (MMG) and ultrasound (USG) and discuss the implication of the choice of imaging as the future direction of our recently launched local screening program. Methods. Retrospective study of 14613 Hong Kong Chinese female patients with histologically confirmed breast cancer registered in the Hong Kong Breast Cancer Registry between January 2006 and February 2020. Patients were classified into four groups based on the mode of breast cancer detection (detectable by both mammogram and ultrasound (MMG+/USG+), mammogram only (MMG+/USG−), ultrasound only (MMG−/USG+), or not detectable by either (MMG−/USG−). Characteristics of breast cancer detected were compared, including patient demographics, breast density on MMG, mode of presentation, tumour size, histological type, and staging. Types of mammographic abnormalities were also evaluated for MMG+ subgroups. Results. 85% of the cancers were detectable by MMG, while USG detected an additional 9%. MMG+/USG+ cancers were larger, more advanced in stage, often of symptomatic presentation, and commonly manifested as mammographic mass. MMG+/USG− cancers were more likely of asymptomatic presentation, manifested as microcalcifications, and of earlier stage and to be ductal carcinoma in situ. MMG−/USG+ cancers were more likely seen in young patients and those with denser breasts and more likely of symptomatic presentation. MMG−/USG− cancers were often smaller and found in denser breasts. Conclusion. Mammogram has a good detection rate of cancers in our local population. It has superiority in detecting early cancers by detecting microcalcifications. Our current study agrees that ultrasound is one of the key adjunct tools of breast cancer detection

Secretory Stanniocalcin 1 promotes metastasis of hepatocellular carcinoma through activation of JNK signaling pathway

© 2017 Elsevier B.V. The hypoxic microenvironment is well-characterized in hepatocellular carcinoma (HCC). Delineation of hypoxia-responsive events is an integral part to understand the pathogenesis of HCC. We studied the functional role and clinical relevance of Stanniocalcin 1 (STC1), a hypoxia-induced molecular target, in HCC. In our clinical cohort, STC1 transcript was up-regulated in HCC tumor tissues. Moreover, STC1 protein was detected in the sera of HCC patients. A higher serum STC1 level was correlated with larger tumor size and poorer 5-year disease-free survival. Functionally, recombinant STC1 protein (rhSTC1) promoted cell migration and cell invasion in vitro; and the effect was abolished by co-treatment of anti-STC1 neutralizing antibody. By in vivo mouse model, silencing of STC1 in HCC cells downregulated secretory STC1 level and suppressed lung metastasis. Furthermore, we found that rhSTC1 activated the JNK pathway, as evidenced by altered expression of the key molecular targets pJNK and p-c-Jun. The functional effects conferred by rhSTC1 were abrogated by co-treatment of JNK inhibitor. In summary, secretory STC1 enhances metastatic potential of HCC via JNK signaling. It potentially serves as a prognostic serum biomarker and a therapeutic target for HCC.Link_to_subscribed_fulltex

A Perspective on Cell Therapy and Cancer Vaccine in Biliary Tract Cancers (BTCs)

Biliary tract cancer (BTC) is a rare, but aggressive, disease that comprises of gallbladder carcinoma, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma, with heterogeneous molecular profiles. Advanced disease has limited therapeutic options beyond first-line platinum-based chemotherapy. Immunotherapy has emerged as a viable option for many cancers with a similar unmet need. Therefore, we reviewed current understanding of the tumor immune microenvironment and recent advances in cellular immunotherapy and therapeutic cancer vaccines against BTC. We illustrated the efficacy of dendritic cell vaccination in one patient with advanced, chemorefractory, melanoma-associated antigen (MAGE)-positive gallbladder carcinoma, who was given multiple injections of an allogenic MAGE antigen-positive melanoma cell lysate (MCL)-based autologous dendritic cell vaccine combined with sequential anti-angiogenic therapy. This resulted in good radiological and tumor marker response and an overall survival of 3 years from diagnosis. We postulate the potential synergism of adding anti-angiogenic therapy, such as bevacizumab, to immunotherapy in BTC, as a rational scientific principle to positively modulate the tumor microenvironment to augment antitumor immunity

NF-κB p65 Subunit Is Modulated by Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2) in Nasopharyngeal Carcinoma HONE1 and HK1 Cells.

NF-κB is a well-characterized transcription factor, widely known as a key player in tumor-derived inflammation and cancer development. Herein, we present the functional and molecular relevance of the canonical NF-κB p65 subunit in nasopharyngeal carcinoma (NPC). Loss- and gain-of-function approaches were utilized to reveal the functional characteristics of p65 in propagating tumor growth, tumor-associated angiogenesis, and epithelial-to-mesenchymal transition in NPC cells. Extracellular inflammatory stimuli are critical factors that trigger the NF-κB p65 signaling; hence, we investigated the components of the tumor microenvironment that might potentially influence the p65 signaling pathway. This led to the identification of an extracellular matrix (ECM) protein that was previously reported as a candidate tumor suppressor in NPC. Our studies on the Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2) protein provides substantial evidence that it can modulate the p65 transcriptional activity. Re-expression of LTBP2 elicits tumor suppressive effects that parallel the inactivation of p65 in NPC cells. LTBP2 was able to reduce phosphorylation of p65 at Serine 536, inhibit nuclear localization of active phosphorylated p65, and impair the p65 DNA-binding ability. This results in a consequential down-regulation of p65-related gene expression. Therefore, the data suggest that the overall up-regulation of p65 expression and the loss of this candidate ECM tumor suppressor are milestone events contributing to NPC development