Sheng-Chieh Hsu

Epidermal Growth Factor Receptor Cooperates with Signal Transducer and Activator of Transcription 3 to Induce Epithelial-Mesenchymal Transition in Cancer Cells via Up-regulation of TWIST Gene Expression

Aberrant epidermal growth factor receptor (EGFR) signaling is a major cause of tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether deregulated EGFR pathway is involved in epithelial-mesenchymal transition (EMT), an early event that occurs during metastasis of cancers of an epithelial origin. Here, we show that EGF induces EGFR-expressing cancer cells to undergo a transition from the epithelial to the spindle-like mesenchymal morphology. EGF reduced E-cadherin expression and increased that of mesenchymal proteins. In search of a downstream mediator that may account for EGF-induced EMT, we focused on transcription repressors of E-cadherin, TWIST, SLUG, and Snail and found that cancer cells express high levels of TWIST and that EGF enhances its expression. EGF significantly increases TWIST transcripts and protein in EGFR-expressing lines. Forced expression of EGFR reactivates TWIST expression in EGFR-null cells. TWIST expression is suppressed by EGFR and Janus-activated kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) inhibitors, but not significantly by those targeting phosphoinositide-3 kinase and MEK/ERK. Furthermore, constitutively active STAT3 significantly activates the TWIST promoter, whereas the JAK/STAT3 inhibitor and dominant-negative STAT3 suppressed TWIST promoter. Deletion/mutation studies further show that a 26-bp promoter region contains putative STAT3 elements required for the EGF-responsiveness of the TWIST promoter. Chromatin immunoprecipitation assays further show that EGF induces binding of nuclear STAT3 to the TWIST promoter. Immunohistochemical analysis of 130 primary breast carcinomas indicates positive correlations between non-nuclear EGFR and TWIST and between phosphorylated STAT3 and TWIST. Together, we report here that EGF/EGFR signaling pathways induce cancer cell EMT via STAT3-mediated TWIST gene expression.

Nuclear-cytoplasmic transport of EGFR involves receptor endocytosis, importin β1 and CRM1

Many receptor tyrosine kinases (RTKs) can be detected in the cell nucleus, such as EGFR, HER‐2, HER‐3, HER‐4, and fibroblast growth factor receptor. EGFR, HER‐2 and HER‐4 contain transactivational activity and function as transcription co‐factors to activate gene promoters. High EGFR in tumor nuclei correlates with increased tumor proliferation and poor survival in cancer patients. However, the mechanism by which cell‐surface EGFR translocates into the cell nucleus remains largely unknown. Here, we found that EGFR co‐localizes and interacts with importins α1/β1, carriers that are critical for macromolecules nuclear import. EGFR variant mutated at the nuclear localization signal (NLS) is defective in associating with importins and in entering the nuclei indicating that EGFRs NLS is critical for EGFR/importins interaction and EGFR nuclear import. Moreover, disruption of receptor internalization process using chemicals and forced expression of dominant‐negative Dynamin II mutant suppressed nuclear entry of EGFR. Additional evidences suggest an involvement of endosomal sorting machinery in EGFR nuclear translocalization. Finally, we found that nuclear export of EGFR may involve CRM1 exportin as we detected EGFR/CRM1 interaction and markedly increased nuclear EGFR following exposure to leptomycin B, a CRM1 inhibitor. Collectively, these data suggest the importance of receptor endocytosis, endosomal sorting machinery, interaction with importins α1/β1, and exportin CRM1 in EGFR nuclear‐cytoplasmic trafficking. Together, our work sheds light into the nature and regulation of the nuclear EGFR pathway and provides a plausible mechanism by which cells shuttle cell‐surface EGFR and potentially other RTKs through the nuclear pore complex and into the nuclear compartment. J. Cell. Biochem.

Characterization of a Novel Tripartite Nuclear Localization Sequence in the EGFR Family

Aberrant expression of epidermal growth factor receptor (EGFR) is present in many human tumors. Several reports have shown that EGFR is translocated into the nucleus during liver regeneration and in several types of cells and tissues such as placenta and thyroid. Nuclear EGFR is associated with transcription, DNA synthesis, and DNA repair activity and serves as a prognostic marker in breast carcinoma and oropharyngeal squamous cell cancer. However, the nuclear localization sequence (NLS) of EGFR has not been extensively examined. In this study, we have shown that the juxtamembrane region of EGFR harbors a putative NLS with three clusters of basic amino acids (RRRHIVRKRTLRR (amino acids 645–657)) that mediates the nuclear localization of EGFR. We found that this newly characterized tripartite NLS is conserved among the EGFR family members (EGFR, ErbB2, ErbB3, and ErbB4) and is able to move each to the nucleus. Further, this tripartite NLS could also mediate the nuclear localization of other known cytoplasmic proteins such as pyruvate kinase. We have demonstrated that mutating one of the three basic amino acid clusters (R or K → A) leads to significant impairment of the nuclear localization of EGFR and that of a green fluorescent protein-pyruvate kinase-NLS reporter protein. Our results show that this tripartite NLS is distinct from the traditional mono- and bipartite NLS and reveal a mechanism that could account for the nuclear localization of membrane receptors.

EGFR signaling pathway in breast cancers: from traditional signal transduction to direct nuclear translocalization.

SummaryAberrant epidermal growth factor receptor (EGFR) signaling is a major characteristic of many human malignancies including breast cancer. Since the discovery of EGF in 1960’s and its receptor in 1980’s, our understanding of the EGF/EGFR pathway has been significantly advanced and consequently, EGFR is considered as a major oncogenic factor and an attractive therapeutic target. The well-established traditional function of EGFR is known to transmit extra-cellular mitogenic signals, such as EGF and transforming growth factor-α (TGF-α), through activating a number of downstream signaling cascades. These include signaling modules that involve phospholipase C-γ, Ras, and phosphatidylinositol-3 kinase (PI-3K). In cancer cells, the common outcomes following the activation of the EGFR-mediated downstream pathways are altered gene activities, leading to un-controlled tumor proliferation and apoptosis. Interestingly, emerging evidences suggest the existence of a direct mode of the EGFR pathway that is distinct from the traditional transduction pathway. This new mode of EGFR signaling involves cellular transport of EGFR from the cell-surface to the cell nucleus, association of nuclear EGFR complex with gene promoters, and transcriptional regulation of the target genes. Although the nature and pathological consequences of the nuclear EGFR pathway remain elusive, accumulating evidences suggest its association with increased tumor cell proliferation and poor survival rate in breast cancer patients. While several anti-EGFR agents are being tested in breast cancer patients clinically and others under pre-clinical development, a better understanding of the traditional and the nuclear EGFR pathways will facilitate the identification of patients that are likely to respond to these agents as well as future development of more effective anti-EGFR therapeutic interventions.

RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFR-induced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR–RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.

Varied assembly and RNA editing efficiencies between genotypes I and II hepatitis D virus and their implications

The mechanisms that link genotypes of hepatitis D virus (HDV) with clinical outcomes have not yet been elucidated. Genotypic variations are unevenly distributed along the sequences of hepatitis delta antigens (HDAgs). Of these variations, the packaging signal at the C-terminus has a divergence of 74% between genotypes I and II. In this report, we address the issue of whether these high variations between genotypes affect assembly efficiency of HDV particles and editing efficiency of RNA. Viral package systems of transfection with expression plasmids of hepatitis B surface antigen and HDAgs or whole genomes of HDV consistently indicate that the package efficiency of genotype I HDV is higher than that of genotype II. Segment swapping of large-form HDAg indicates that the C-terminal 19-residue region plays a key role for the varied assembly efficiencies. Also, the editing efficiency of genotype I HDV is higher than that of genotype II. The nucleotide and structural changes surrounding the editing site may explain why genotype II HDV has a low RNA editing efficiency. The findings of in vitro assembly systems were further supported by the observations that patients infected with genotype II had significantly lower alanine transaminase (ALT) levels, more favorable outcomes (P <.05), and a trend to have lower serum HDV RNA levels as compared with those infected with genotype I HDV (P =.094). In conclusion, genotype II HDV secretes fewer viral particles than genotype I HDV does, which in turn may reduce the extent of infection of hepatocytes and result in less severe hepatic inflammation.

Localization of isoprenylated antigen of hepatitis delta virus by anti-farnesyl antibodies.

Hepatitis delta virus (HDV) is a subviral pathogen that requires pre-existing or concurrent infection with hepatitis B virus (HBV). HDV expresses two forms of a single protein, the delta antigen (HDAg), which are identical except for an additional 19 residues at the C terminus of the large form. Within this C-terminal extension a cysteine residue is isoprenylated; this isoprenylation is critical for interaction with HBV envelope proteins to enable virus assembly and release into the medium. Therefore, large HDAg must be recruited to an extracellular compartment. However, immuno-staining with HDAg-specific antibodies has localized the large antigen mainly to the nucleus and supports the notion that large HDAg suppresses virus replication in the nucleus. Since isoprenylation would increase the hydrophobicity of the protein and may favour transport towards specific membranes, the question remains whether the large HDAg detected in the nucleus carries an isoprenyl group. To address this issue, antibodies against the farnesyl modification were generated to allow direct visualization of the antigen by immunofluorescence microscopy. The anti-farnesyl antibodies specifically stained large HDAg expressed in Huh-7 cells, and the signal was largely restricted to the nucleus; the staining pattern could be superimposed on those of cells stained for large HDAg. The large HDAg translocated into the nucleus was therefore isoprenylated. In addition, antibodies specific for the farnesyl modification should be applicable to the study of other similarly isoprenylated proteins.

Acetylation of EGF Receptor Contributes to Tumor Cell Resistance to Histone Deacetylase Inhibitors

Alteration of epidermal growth factor receptor (EGFR) is involved in various human cancers and has been intensively investigated. A plethora of evidence demonstrates that posttranslational modifications of EGFR play a pivotal role in controlling its function and metabolism. Here, we show that EGFR can be acetylated by CREB binding protein (CBP) acetyltransferase. Interestingly, EGFR acetylation affects its tyrosine phosphorylation, which may contribute to cancer cell resistance to histone deacetylase inhibitors (HDACIs). Since there is an increasing interest in using HDACIs to treat various cancers in the clinic, our current study provides insights and rationale for selecting effective therapeutic regimen. Consistent with the previous reports, we also show that HDACI combined with EGFR inhibitors achieves better therapeutic outcomes and provides a molecular rationale for the enhanced effect of combination therapy. Our results unveil a critical role of EGFR acetylation that regulates EGFR function, which may have an important clinical implication.

Varied Immunity Generated in Mice by DNA Vaccines with Large and Small Hepatitis Delta Antigens

ABSTRACT Whether the hepatitis delta virus (HDV) DNA vaccine can induce anti-HDV antibodies has been debatable. The role of the isoprenylated motif of hepatitis delta antigens (HDAg) in the generation of immune responses following DNA-based immunization has never been studied. Plasmids p2577L, encoding large HDAg (L-HDAg), p2577S, expressing small HDAg (S-HDAg), and p25L-211S, encoding a mutant form of L-HDAg with a cysteine-to-serine mutation at codon 211, were constructed in this study. Mice were intramuscularly injected with the plasmids. The anti-HDV antibody titers, T-cell proliferation responses, T-helper responses, and HDV-specific, gamma interferon (IFN-γ)-producing CD8+ T cells were analyzed. Animals immunized with p2577S showed a strong anti-HDV antibody response. Conversely, only a low titer of anti-HDV antibodies was detected in mice immunized with p2577L. Epitope mapping revealed that the anti-HDV antibodies generated by p2577L vaccination hardly reacted with epitope amino acids 174 to 194, located at the C terminus of S-HDAg. All of the HDAg-encoding plasmids could induce significant T-cell proliferation responses and generate Th1 responses and HDV-specific, IFN-γ-producing CD8+ T cells. In conclusion, HDAg-specific antibodies definitely exist following DNA vaccination. The magnitudes of the humoral immune responses generated by L-HDAg- and S-HDAg-encoding DNA vaccines are different. The isoprenylated motif can mask epitope amino acids 174 to 195 of HDAg but does not interfere with cellular immunity following DNA-based immunization. These findings are important for the choice of a candidate HDV DNA vaccine in the future.

Characterization of a strain-specific monoclonal antibody to hepatitis delta virus antigen.

Sequences of the hepatitis delta virus (HDV) vary to different degrees among isolates. A monoclonal antibody, designated as HP6A1, against the antigen of HDV (HDAg) has been characterized for its specificity. HP6A1 bound to HDAg of isolate 25 (genotype I) that was used for immunization, but not to others of both genotypes I and II. The epitope recognized by HP6A1 was then determined by a phage library displaying various heptapeptides. A consensus peptide deduced has the best match with that of residues 4-10 of HDAg (isolate 25). To confirm the phage mapping result, Escherichia coli recombinant proteins containing different lengths and various segments of HDAg (isolate 25) were constructed. The shortest HDAg segment contained in the fusion protein that reacted with HP6A1 was residues 1-10. When this peptide was added to the N-terminus of a heterologous protein engineered for eucaryotic expression, the fusion protein was detected by HP6A1. It is concluded that HP6A1 recognizes an epitope located at the N-terminus of HDAg (isolate 25). Since viruses of quasi-species exist in natural infections, a question of how different viral strains interact in vivo remains to be explored. The highly specific MAb opens a possibility to examine the fate of one strain in the presence of other related species in a cell transfection system.