Mei-Jung Wang

Osteopontin promotes integrin activation through outside-in and inside-out mechanisms: OPN-CD44V interaction enhances survival in gastrointestinal cancer cells.

Osteopontin (OPN) and splice variants of CD44 (CD44(V)) have independently been identified as markers for tumor progression. In this study, we show that both OPN and CD44(V) are frequently overexpressed in human gastric cancer and that OPN-engaged CD44(V) ligation confers cells an increased survival mediated through integrin activation. First, we show that OPN treatment confers cells an increased resistance to UV-induced apoptosis. The OPN-mediated antiapoptosis is dependent on the expression of the variant exon 6 (V6)- or V7-containing CD44 as shown by overexpression of individual CD44(V) in gastric AZ521 cells that express no or very low level of endogenous CD44 and by knockdown of the constitutively expressed V6-containing CD44 isoforms in colon HT29 cells. Although OPN also interacts with RGD integrins, OPN-RGD sequence is dispensable for OPN-mediated antiapoptosis. OPN-induced antiapoptosis is mainly attributed to the engagement of CD44(V) isoforms and the relay of an inside-out signaling via Src activity, leading to robust integrin activation. Furthermore, OPN-elicited antiapoptosis was observed when cells were plated on fibronectin but not on poly-D-lysin, and preincubation of cells with anti-integrin beta(1) antibody to block integrin-extracellular matrix (ECM) interaction or ectopic expression of the dominant-negative forms of focal adhesion kinase to block ECM-derived signal abolished OPN-induced survival, suggesting that OPN-elicited antiapoptotic function is propagated from matrix transduced by integrin. Taken together, we showed that OPN-CD44(V) interaction promotes ECM-derived survival signal mediated through integrin activation, which may play an important role in the pathogenic development and progression of gastric cancer.

Acetylation and activation of STAT3 mediated by nuclear translocation of CD44

Expression of the type I transmembrane glycoprotein CD44 has recently been recognized as a signature for cancer stem cells. In this study, we demonstrate that CD44, once engaged, is internalized and translocated to the nucleus, where it binds to various promoters, including that of cyclin D1, leading to cell fate change through transcriptional reprogramming. In regulating cyclin D1 expression, the internalized CD44 forms a complex with STAT3 and p300 (acetyltransferase), eliciting STAT3 acetylation at lysine 685 and dimer formation in a cytokine- and growth factor–independent manner. A bipartite nuclear localization signal (NLS) was mapped to the cytoplasmic tail of CD44, which mediates its nuclear translocation. Expression of CD44(NLS) mutant sequesters STAT3 in cytosol. In the nucleus, the acetylated STAT3 dimer remains associated with CD44 and binds to the cyclin D1 promoter, leading to increased cyclin D1 expression and cell proliferation. This study describes a novel function for CD44 in transcriptional modulation through nuclear translocation of the internalized CD44 and complex formation with transcription factors.

Rho/Rhotekin-mediated NF-kappaB activation confers resistance to apoptosis

Rhotekin (RTKN), the gene coding for the Rho effector, RTKN, was shown to be overexpressed in human gastric cancer (GC). In this study, we further showed that RTKN is expressed at a low level in normal cells and is overexpressed in many cancer-derived cell lines. The function of RTKN as an effector protein in Rho GTPase-mediated pathways regulating apoptosis was investigated. By transfection and expression of RTKN in cells that expressed endogenous RTKN at a low basal level, we showed that RTKN overexpression conferred cell resistance to apoptosis induced by serum deprivation or treatment with sodium butyrate, and the increased resistance correlated to the level of RTKN. Conversely, reducing RTKN expression by small interfering RNAs greatly sensitized cells to apoptosis. The RTKN-mediated antiapoptotic effect was blocked by the nuclear factor-κB (NF-κB) inhibitors, curcumin or parthenolide, but not by the phosphatidylinositol 3′-OH-kinase inhibitor, LY294002, or the MAP kinase inhibitor, PD98059. Reporter gene assays and electrophoretic mobility shift assay confirmed that RTKN overexpression led to constitutive activation of NF-κB through the phosphorylation of IκB by IKKβ. By using the RTKN truncation mutants, we showed that RTKN mediated Rho activity eliciting signaling pathway to activate NF-κB, with a concomitant induction of expression of the NF-κB antiapoptotic genes, cIAP-2, BCl-xL, A1, and A20. Consistent with these data, RTKN-expressing cells showed increased chemoresistance to 5-fluorouracil and paclitaxol, and the resistance was greatly attenuated by NF-κB inhibitor. In conclusion, overactivated Rho/RTKN/NF-κB signaling pathway through overexpression of RTKN may play a key role in gastric tumorigenesis by conferring cells resistance to apoptosis, and this signaling pathway may serve as an important target for novel therapeutic approaches to the treatment of human GC.

CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts.

ABSTRACT CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin β1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.

SOCS6, down-regulated in gastric cancer, inhibits cell proliferation and colony formation

Members of the suppressor of cytokine-induced signaling (SOCS) family are negative regulators of cytokine signaling pathways. By mRNA differential display, we showed that SOCS6 was frequently down-regulated in gastric cancer (GC). Our data showed that allelic loss and promoter hypermethylation may account for the major mechanisms leading to SOCS6 inactivation. Ectopic expression of SOCS6 suppressed cell growth and colony formation, in part through eliciting intrinsic apoptotic pathway, accompanied with decreased mitochondrial membrane potential. Taken together, this study provides molecular and functional data supporting the importance of loss-of-function of SOCS6 as a frequent event in gastric tumorigenesis.

Suppressor of cytokine signaling 6 (SOCS6) promotes mitochondrial fission via regulating DRP1 translocation

Mitochondria are highly motile organelles that constantly undergo fission and fusion. Impairment of mitochondrial dynamics is associated with mitochondrial dysfunction and is frequently linked to the pathogenesis of neurodegenerative diseases and cancer. We have previously shown that biallelic inactivation of the suppressor of cytokine signaling 6 (SOCS6) gene is a frequent event in human gastric cancer. In this study, we recapitulated the event of SOCS6 loss using a Lentivirus-based knockdown approach, and demonstrated the linkage between SOCS6 depletion and the suppression of programmed cell death. SOCS6 promotes intrinsic apoptosis, with increased Bax conformational change, mitochondrial targeting, and oligomerization. Most importantly, SOCS6 is targeted to mitochondria and induces mitochondrial fragmentation mediated through an increase in DRP1 fission activity. Here, we show that SOCS6 forms complex with DRP1 and the mitochondrial phosphatase PGAM5, attenuates DRP1 phosphorylation, and promotes DRP1 mitochondrial translocation. Based on mutation analyses, SOCS6-mediated apoptosis is tightly coupled to its ability to induce mitochondrial fission. This study demonstrates an important role for SOCS6 in modulating mitochondrial dynamics and apoptosis.

Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

Background Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale. Methodology/Principal Findings By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (nu200a=u200a77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells. Conclusions/Significance This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

Label-free quantitative proteomics and N-glycoproteomics analysis of KRAS-activated human bronchial epithelial cells

Mutational activation of KRAS promotes various malignancies, including lung adenocarcinoma. Knowledge of the molecular targets mediating the downstream effects of activated KRAS is limited. Here, we provide the KRAS target proteins and N-glycoproteins using human bronchial epithelial cells with and without the expression of activated KRAS (KRASV12). Using an OFFGEL peptide fractionation and hydrazide method combined with subsequent LTQ-Orbitrap analysis, we identified 5713 proteins and 608 N-glycosites on 317 proteins in human bronchial epithelial cells. Label-free quantitation of 3058 proteins (≥2 peptides; coefficient of variation (CV) ≤ 20%) and 297 N-glycoproteins (CV ≤ 20%) revealed the differential regulation of 23 proteins and 14 N-glycoproteins caused by activated KRAS, including 84% novel ones. An informatics-assisted IPA-Biomarker® filter analysis prioritized some of the differentially regulated proteins (ALDH3A1, CA2, CTSD, DST, EPHA2, and VIM) and N-glycoproteins (ALCAM, ITGA3, and TIMP-1) as cancer biomarkers. Further, integrated in silico analysis of microarray repository data of lung adenocarcinoma clinical samples and cell lines containing KRAS mutations showed positive mRNA fold changes (p < 0.05) for 61% of the KRAS-regulated proteins, including biomarker proteins, CA2 and CTSD. The most significant discovery of the integrated validation is the down-regulation of FABP5 and PDCD4. A few validated proteins, including tumor suppressor PDCD4, were further confirmed as KRAS targets by shRNA-based knockdown experiments. Finally, the studies on KRAS-regulated N-glycoproteins revealed structural alterations in the core N-glycans of SEMA4B in KRAS-activated human bronchial epithelial cells and functional role of N-glycosylation of TIMP-1 in the regulation of lung adenocarcinoma A549 cell invasion. Together, our study represents the largest proteome and N-glycoproteome data sets for HBECs, which we used to identify several novel potential targets of activated KRAS that may provide insights into KRAS-induced adenocarcinoma and have implications for both lung cancer therapy and diagnosis.

Overexpression of Rho Effector Rhotekin Confers Increased Survival in Gastric Adenocarcinoma

Like many epithelial-derived cancers, gastric cancer (GC) results from a multistep tumorigenic process. However, the detailed mechanisms involved in GC formation are poorly characterized. Using an ordered differential display method, we have identified rhotekin (RTKN), the gene coding for the Rho effector, RTKN, as one of the genes differentially expressed in human GC. Northern analysis using human multiple tissue blots showed that RTKN is predominantly expressed in the kidney and spinal cord, and, to a lesser degree, in the thyroid, tongue, liver, brain, prostate, trachea, and stomach. RT-PCR analysis confirmed that RTKN was overexpressed in most (5/7; 71%) GC examined. By analyzing the Stanford Microarray Database for the expression profiles of gastric tissues, we also found a progressional increase in RTKN expression in nonneoplastic mucosa, GC, and then lymph node metastases (p < 0.005 by Jonckheere-Terpstra test), suggesting that RTKN expression correlates with GC progression. The role of RTKN in the pathogenic development of GC was investigated by transfection and expression of RTKN in AGS gastric cells, which express endogenous RTKN at a low basal level. Flow-cytometric analysis showed that RTKN-transfected AGS cells were significantly more resistant than vector-transfected cells to apoptosis upon treatment with sodium butyrate. To explore the mechanisms underlying RTKN-mediated cell survival, a reporter assay was performed. Since the NF-ĸB activation is known to promote cell survival and Rho GTPase may lead to NF-ĸB activation, we transfected AGS cells with the RTKN expression vector along with a pNF-ĸB-Luc reporter plasmid. Our results showed that overexpression of RTKN induced robust activation of NF-ĸB, and RTKN-mediated NF-ĸB activation was suppressed significantly by C3 transferase, an inhibitor of the small GTPase Rho. We conclude that Rho/RTKN-mediated NF-ĸB activation leading to cell survival may play a key role in gastric tumorigenesis. This study provides original documentation for the overrepresentation of the Rho GTPase effector rhotekin in human cancer and its links to cancer formation.

Genomic organization and functional characterization of the promoter for the human suppressor of cytokine signaling 6 gene.

In this study, we report the expression and genomic structure of the gene encoding human suppressor of cytokine signaling 6 (SOCS6), and the characterization of the functional promoter region. The human SOCS6 gene, spanning 40 kb on chromosome 18q22.2, is composed of two exons separated by an intron of 35 kb. Two transcripts are ubiquitously expressed, and both encode the full-length open reading frame of SOCS6. A primer extension assay revealed that the major transcription initiation site is located 469 bp upstream the ATG codon. Luciferase promoter analysis demonstrated that the 5-flanking region is able to drive transcription, and the CpG-rich sequences near the transcription initiation site are important for the TATA-less SOCS6 promoter activity. Analogous to SOCS1 and SOCS3, which are down-regulated in several human cancers, SOCS6 is expressed at lower levels in carcinomas of stomach and colon. We demonstrated that hypermethylation of the SOCS6 promoter is one of the mechanisms for the epigenetic regulation of SOCS6 expression. Firstly, in vitro methylation of the reporter promoter plasmid significantly suppressed the promoter activity. Secondly, SOCS6 expression in vivo was enhanced by treating cells with a methyltransferase inhibitor. The SOCS6 gene from various species shares significant homology in amino acid sequences, transcription factor binding motifs in promoter regions and the two-exon genomic structure, suggesting that the SOCS6 gene is highly conserved.