Kinase shRNA screening reveals that TAOK3 enhances microtubule-targeted drug resistance of breast cancer cells via the NF-κB signaling pathway.

BACKGROUND: Chemotherapy is currently one of the most effective treatments for advanced breast cancer. Anti-microtubule agents, including taxanes, eribulin and vinca-alkaloids are one of the primary major anti-breast cancer chemotherapies; however, chemoresistance remains a problem that is difficult to solve. We aimed to discover novel candidate protein targets to combat chemoresistance in breast cancer. METHODS: A lentiviral shRNA-based high-throughput screening platform was designed and developed to screen the global kinome to find new therapeutic targets in paclitaxel-resistant breast cancer cells. The phenotypes were confirmed with alternative expression in vitro and in vivo. Molecular mechanisms were investigated using global phosphoprotein arrays and expression microarrays. Global microarray analysis was performed to determine TAOK3 and genes that induced paclitaxel resistance. RESULTS: A serine/threonine kinase gene, TAOK3, was identified from 724 screened kinase genes. TAOK3 shRNA exhibited the most significant reduction in IC50 values in response to paclitaxel treatment. Ectopic downregulation of TAOK3 resulted in paclitaxel-resistant breast cancer cells sensitize to paclitaxel treatment in vitro and in vivo. The expression of TAOK3 also was correlated to sensitivity to two other anti-microtubule drugs, eribulin and vinorelbine. Our TAOK3-modulated microarray analysis indicated that NF-κB signaling played a major upstream regulation role. TAOK3 inhibitor, CP43, and shRNA of NF-κB both reduced the paclitaxel resistance in TAOK3 overexpressed cells. In clinical microarray databases, high TAOK3 expressed breast cancer patients had poorer prognoses after adjuvant chemotherapy. CONCLUSIONS: Here we identified TAOK3 overexpression increased anti-microtubule drug resistance through upregulation of NF-κB signaling, which reduced cell death in breast cancer. Therefore, inhibition of the interaction between TAOK3 and NF-κB signaling may have therapeutic implications for breast cancer patients treated with anti-microtubule drugs. Video abstract.

Loss of RUNX3 expression correlates with differentiation, nodal metastasis, and poor prognosis of gastric cancer.

BACKGROUND: RUNX3 is a major growth regulator of gastric epithelial cells that is involved in gastric tumorigenesis in both humans and mice. In this study, we investigated the involvement of RUNX3 in tumor progression, and in the prognosis of human gastric cancer. METHODS: We analyzed the extent of RUNX3 protein expression by immunohistochemistry in 95 primary gastric adenocarcinomas, and correlated expression levels with clinicopathological parameters. We examined the effects of pFlag/RUNX3 on cell growth, apoptosis, and caspase-3 expression in AGS and SNU1 gastric cancer cell lines by colony-forming assay, terminal deoxynucleotidyl transferase (TdT)-mediate deoxyuridine triphosphatase (dUTP) nick-end labeling (TUNEL) assay, and Western blot analysis, respectively. The pFlag/RUNX3 effects on AGS invasion and migration potentials were also evaluated. RESULTS: RUNX3 expression was lost in 37 (39%) cases of gastric cancer. The expression of RUNX3 in diffuse- and mixed-type cancers was less frequent than expression in intestinal-type cancer (P < 0.001 and P = 0.001, respectively). In addition, the loss of RUNX3 expression was associated with lymph node metastasis (P = 0.02), and correlated with poor gastric cancer survival (P = 0.018). The growth of gastric cancer cells was suppressed after pFlag/RUNX3 transfection. The re-expression of RUNX3 resulted in the upregulation of caspase-3 and promoted apoptosis. Furthermore, Re-expression of RUNX3 induced significant inhibitions of AGS cell invasion and migration in vitro. CONCLUSIONS: This work shows that loss of RUNX3 expression is highly associated with lymph node metastasis and poor prognosis of gastric cancer. The re-expression of RUNX3 may induce apoptosis and inhibit the growth as well as invasion/migration of cancer cells. These results indicate that the targeting of the RUNX3 pathway could represent a potential modality for treating gastric cancer.

Polyethylenimine-mediated PUMA gene delivery to orthotopic oral cancer: suppression of tumor growth through apoptosis induction in situ and prolonged survival.

BACKGROUND: PUMA (a p53 up-regulated modulator of apoptosis) is induced by p53 tumor suppressor and other apoptotic stimuli. It was found to be a principal mediator of cell death in response to diverse apoptotic signals, implicating PUMA as a likely tumor suppressor. METHODS: In this study, we examined the efficacy of targeted PUMA gene therapy in human oral cancer (SAS) cells using polyethylenimine (PEI)-mediated transfection for gene delivery. RESULTS: Exogenous expression of PUMA in SAS cells resulted in apoptosis with cytochrome c release, activation of caspase-3 and -9, and cleavage of PARP. Gene delivery of PEI/PUMA in SAS xenografts induced apoptosis and resulted in significant reductions (∼60%) of tumor growth in vivo. Furthermore, we have shown that PEI-mediated PUMA gene therapy prolonged survival of animals with orthotopic SAS oral cancers. CONCLUSIONS: Taken together, these results indicated that PUMA gene therapy via PEI delivery could be a promising method for the treatment of oral squamous cell carcinoma.

S100 protein translocation in response to extracellular S100 is mediated by receptor for advanced glycation endproducts in human endothelial cells.

The extracellular functions of S100 proteins have attracted more attention in recent years. S100 proteins are a group of calcium-binding proteins which exhibit cell- and tissue-specific expression, and different expression levels of members from this family have been observed in various pathological conditions. The reported extracellular functions of S100 proteins include the ability to enhance neurite outgrowth, involvement in inflammation, and motility of tumour cells. In our previous study, we reported translocation of S100A13 in response to the elevated intracellular calcium levels induced by angiotensin II. In order to investigate potential effects of extracellular S100A13, recombinant S100A13 was used here to stimulate human endothelial cells. Addition of extracellular S100A13 to the cells resulted in both endogenous protein translocation and protein uptake from the extracellular space. To test specificity of this effect, addition of various other S100 proteins was also performed. Interestingly, translocation of specific S100 proteins was only observed when the cells were stimulated with the same extracellular S100 protein. Since the receptor for advanced glycation end products (RAGE) is a putative cell surface receptor for S100 proteins and is involved in various signal transduction pathways, we next investigated the interaction between the receptor and extracellular S100 proteins. We show here that NF-kappaB which is a downstream regulator in RAGE-mediated transduction pathways can be activated by addition of extracellular S100 proteins, and translocation of S100 proteins was inhibited by soluble RAGE. These experiments suggest a common cell surface receptor for S100 proteins on endothelial cells even though intracellular translocation induced by extracellular S100 proteins is specific.

Expression analysis of S100 proteins and RAGE in human tumors using tissue microarrays.

Microarray technology provides important information for diagnostic, prognostic, and even therapeutic applications. Several S100 proteins have been proposed to play important roles in tumor progression and are recognized as potential tumor markers. To substantiate these limited earlier findings, we screened hundreds of tumor specimens from patients of eight different tumor types using tissue microarrays. The results validated the expression of S100A4, S100A6, and S100B in specific tumor types. A significant S100A2 expression was observed in lymphoma biopsies, which implies a possible link between this S100 protein and lymphoma development. In contrast, S100A5 and S100A12 were not significantly expressed in any of the tumor tissues tested. Interestingly, expression of RAGE (receptor for advanced glycation end products) was found in breast and lung tumor tissues where abundant S100A4 and S100A6 expression was also observed. This suggests a possible role of RAGE-mediated signal transduction in the development of these particular cancers.

S100A13 and S100A6 exhibit distinct translocation pathways in endothelial cells.

S100 proteins have attracted great interest in recent years because of their cell- and tissue-specific expression and association with various human pathologies. Most S100 proteins are small acidic proteins with calcium-binding domains - the EF hands. It is thought that this group of proteins carry out their cellular functions by interacting with specific target proteins, an interaction that is mainly dependent on exposure of hydrophobic patches, which result from calcium binding. S100A13, one of the most recently identified members of the S100 family, is expressed in various tissues. Interestingly, hydrophobic exposure was not observed upon calcium binding to S100A13 even though the dimeric form displays two high- and two low- affinity sites for calcium. Here, we followed the translocation of S100A13 in response to an increase in intracellular calcium levels, as protein translocation has been implicated in assembly of signaling complexes and signaling cascades, and several other S100 proteins are involved in such events. Translocation of S100A13 was observed in endothelial cells in response to angiotensin II, and the process was dependent on the classic Golgi-ER pathway. By contrast, S100A6 translocation was found to be distinct and dependent on actin-stress fibers. These experiments suggest that different S100 proteins utilize distinct translocation pathways, which might lead them to certain subcellular compartments in order to perform their physiological tasks in the same cellular environment.