Joseph L.-K. Chan

Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel

Metastasis, the cardinal feature of malignant tumors, is an important clinical variable in patient prognosis. To understand the basis for metastasis, we systematically selected for highly invasive cells from breast cancer cell lines, MCF7 and MDA-MB-453, with moderate to low invasive ability using Boyden chamber invasion assay. The four-cycle selected invasive lines, named MCF7-I4 and MDA-MB-453-I4, respectively, displayed epithelial-mesenchymal transition (EMT) and dramatically enhanced invasive ability. EMT changes were corroborated with decreased level of E-cadherin and increased vimentin, fibronectin, and beta(1) integrin. Twist, a basic helix-loop-helix transcription factor, and AKT2, a known proto-oncogene, were found to be elevated in the invasive cells compared with the parental. Ectopic expression and knockdown of Twist by short interference RNA resulted in significant increase and reduction, respectively, of AKT2 protein and mRNA expression. Twist bound to E-box elements on AKT2 promoter and enhanced its transcriptional activity. Moreover, silencing AKT2 decreased Twist-promoted migration, invasion, and paclitaxel resistance. Reintroducing AKT2 largely rescued the phenotype resulted from knockdown of Twist in I4 cells, suggesting that AKT2 is a downstream target and functional mediator of Twist. Finally, we observed a 68.8% correlation of elevated Twist and AKT2 expression in late-stage breast cancers as oppose to 13% in early-stage breast cancers. Our study identifies Twist as a positive transcriptional regulator of AKT2 expression, and Twist-AKT2 signaling is involved in promoting invasive ability and survival of breast cancer cells.

Vav3 Mediates Receptor Protein Tyrosine Kinase Signaling, Regulates GTPase Activity, Modulates Cell Morphology, and Induces Cell Transformation

ABSTRACT A recently reported new member of the Vav family proteins, Vav3 has been identified as a Ros receptor protein tyrosine kinase (RPTK) interacting protein by yeast two-hybrid screening. Northern analysis shows that Vav3 has a broad tissue expression profile that is distinct from those of Vav and Vav2. Two species of Vav3 transcripts, 3.4 and 5.4 kb, were detected with a differential expression pattern in various tissues. Transient expression of Vav in 293T and NIH 3T3 cells demonstrated that ligand stimulation of several RPTKs (epidermal growth factor receptor [EGFR], Ros, insulin receptor [IR], and insulin-like growth factor I receptor [IGFR]) led to tyrosine phosphorylation of Vav3 and its association with the receptors as well as their downstream signaling molecules, including Shc, Grb2, phospholipase C (PLC-γ), and phosphatidylinositol 3 kinase. In vitro binding assays using glutathione S-transferase-fusion polypeptides containing the GTPase-binding domains of Rok-α, Pak, or Ack revealed that overexpression of Vav3 in NIH 3T3 cells resulted in the activation of Rac-1 and Cdc42 whereas a deletion mutant lacking the N-terminal calponin homology and acidic region domains activated RhoA and Rac-1 but lost the ability to activate Cdc42. Vav3 induced marked membrane ruffles and microspikes in NIH 3T3 cells, while the N-terminal truncation mutants of Vav3 significantly enhanced membrane ruffle formation but had a reduced ability to induce microspikes. Activation of IR further enhanced the ability of Vav3 to induce membrane ruffles, but IGFR activation specifically promoted Vav3-mediated microspike formation. N-terminal truncation of Vav3 activated its transforming potential, as measured by focus-formation assays. We conclude that Vav3 mediates RPTK signaling and regulates GTPase activity, its native and mutant forms are able to modulate cell morphology, and it has the potential to induce cell transformation.

Synergistic effect between EGF and TGF-β1 in inducing oncogenic properties of intestinal epithelial cells

Transforming growth factor (TGF)-β1 has a biphasic effect on rat intestinal epithelial (RIE) cells. By itself, TGF-β1 functions as a tumor suppressor by inhibiting the growth, migration and invasion of RIE cells. We show in this study that in conjunction with epidermal growth factor (EGF), TGF-β1 helped to augment migration, invasion and anchorage-independent growth (AIG) compared to that by EGF alone. EGF plus TGF-β1 induced a dramatic morphological change characteristic of epithelial–mesenchymal transition (EMT). The mechanism for this enhanced effect of TGF-β1 and EGF on oncogenic properties was explored by analysis of EGF- and TGF-β1-mediated signaling pathways and complementary DNA arrays. TGF-β1 augmented EGF-mediated signaling of mitogen-activated protein kinase (MAPK) and AKT by enhancing and prolonging the activation of the former and prolonging the activation of the latter. Inhibition of MAPK, but not phosphoinositide-3 kinase (PI3K), abolished TGF-β1 plus EGF-induced EMT and downregulation of E-cadherin at mRNA and protein levels. By contrast, cell migration and invasion were sensitive to inhibition of either MAPK or PI3 kinase. TGF-β1 plus EGF-induced AIG was significantly more resistant to inhibition of PI3K and MAPK compared to that induced by EGF alone. EGF and TGF-β1 synergistically induced the expression of a series of proteases including matrix metalloproteinase (MMP) 1 (collagenase), MMP3, MMP9, MMP10, MMP14 and cathepsin. Among them, the expression of MMP1, MMP3, MMP9 and MMP10 was MAPK dependent. Inhibition of the MMPs or cathepsin significantly blocked EGF plus TGF-β1-induced invasion, but had no effect on colony formation. Phospholipase C (PLC) and Cox2 induced by EGF plus TGF-β1 also played a significant role in invasion, whereas PLC was also important for colony formation. Our study reveals specific signaling functions and induction of genes differentially required for enhanced effect of EGF- and TGF-β1-induced oncogenic properties, and helps to explain the tumor-promoting effect of TGF-β1 in human cancer with elevated expression or activation of TGF-β1 and receptor protein tyrosine kinases.

A Phase 0 Trial of Riluzole in Patients with Resectable Stage III and IV Melanoma

Purpose: Ectopic expression of GRM1 in murine melanocytes results in transformation into a form of melanoma, and more than 60% of human melanoma samples tested ectopically express GRM1. Stimulation of this receptor in vitro results in up-regulation of activated extracellular signal–regulated kinase (ERK). Furthermore, a xenograft model of melanoma treated with riluzole, an oral GRM1 blocking agent, showed decreased tumor growth compared with the untreated controls. We have now completed a phase 0 trial of riluzole in patients with melanoma. Experimental Design: Patients enrolled on this trial underwent a pretreatment biopsy, took 200 mg of oral riluzole per day for 14 days, and then underwent resection of their remaining tumor. We compared the levels of pERK and pAKT in the pretreatment and post-treatment samples and assessed the metabolic activity of pretreatment and post-treatment tumors using fluorodeoxyglucose positron emission tomography (FDG-PET) scanning. Results: We accrued 12 patients and all expressed GRM1. We found a significant decrease in pAKT and/or pERK in post-treatment tumor samples as compared with pretreatment samples in 4 (34%) patients. These four patients had a significant decrease in FDG-PET intensity post-treatment as well. Two other patients had a clinical response with no corresponding metabolic response; five patients had similar pretreatment and post-treatment FDG-PET scan findings; and one patient had progressive disease. Conclusions: Our data show that glutamate blockade with riluzole can inhibit signaling through the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT pathways and suppress the metabolic activity of melanoma. The ectopic expression of metabotropic glutamate receptors may be important in the pathogenesis of human melanoma, and targeting this pathway may be an effective therapy.

Protein Kinase A Represses Skeletal Myogenesis by Targeting Myocyte Enhancer Factor 2D

ABSTRACT Activation of protein kinase A (PKA) by elevation of the intracellular cyclic AMP (cAMP) level inhibits skeletal myogenesis. Previously, an indirect modulation of the myogenic regulatory factors (MRFs) was implicated as the mechanism. Because myocyte enhancer factor 2 (MEF2) proteins are key regulators of myogenesis and obligatory partners for the MRFs, here we assessed whether these proteins could be involved in PKA-mediated myogenic repression. Initially, in silico analysis revealed several consensus PKA phosphoacceptor sites on MEF2, and subsequent analysis by in vitro kinase assays indicated that PKA directly and efficiently phosphorylates MEF2D. Using mass spectrometric determination of phosphorylated residues, we document that MEF2D serine 121 and serine 190 are targeted by PKA. Transcriptional reporter gene assays to assess MEF2D function revealed that PKA potently represses the transactivation properties of MEF2D. Furthermore, engineered mutation of MEF2D PKA phosphoacceptor sites (serines 121 and 190 to alanine) rendered a PKA-resistant MEF2D protein, which efficiently rescues myogenesis from PKA-mediated repression. Concomitantly, increased intracellular cAMP-mediated PKA activation also resulted in an enhanced nuclear accumulation of histone deacetylase 4 (HDAC4) and a subsequent increase in the MEF2D-HDAC4 repressor complex. Collectively, these data identify MEF2D as a primary target of PKA signaling in myoblasts that leads to inhibition of the skeletal muscle differentiation program.

Differential requirements of the MAP kinase and PI3 kinase signaling pathways in Src- versus insulin and IGF-1 receptors-induced growth and transformation of rat intestinal epithelial cells

There have been few studies on the specific signaling pathways involved in the transformation of epithelial cells by oncogenic protein tyrosine kinases. Here we investigate the requirement of MAP (MAPK) and phosphatidylinositol 3- (PI3K) kinases in the transformation of rat intestinal epithelial (RIE) cells by oncogenic forms of insulin receptor (gag-IR), insulin-like growth factor-1 receptor (gag-IGFR), and v-Src. MAPK is not significantly activated in cells transformed by gag-IR and gag-IGFR but is activated in v-Src transformed cells. Treatment with PD98059, a MEK inhibitor, at concentrations where MAPK activity was reduced below the basal level showed that MAPK is partially required for the monolayer growth of parental and transformed RIE cells. However, MAPK is not essential for the focus forming ability of the three oncogene-transformed cells. It is also not necessary for the colony forming ability of gag-IR- and gag-IGFR-, but is partially required for v-Src-transformed cells. PI3K is significantly activated in all three oncogene transformed RIE cells. LY294002, a PI3K inhibitor, potently inhibited monolayer growth of all three oncogene-transformed cells. However, at concentrations of LY294002 where activated forms of Akt, a downstream component of the PI3K pathway, were undetectable, colony and focus forming abilities of the v-Src-RIE cells were only slightly affected whereas those of gag-IR/IGFR-RIE cells were greatly inhibited. These results were confirmed using a different pharmacological inhibitor, wortmannin, and a dominant negative form of PI3K, Δp85. Similarly, rapamycin, known to inhibit p70S6 kinase, a downstream component of the PI3K-Akt pathway, also inhibited gag-IR/IGFR-induced, but not v-Src-induced, focus and colony formation. We conclude that the MAPK and PI3K signaling pathways are differentially required for transformation of RIE cells by oncogenic IR and IGFR versus Src and the pattern of requirements is different from that of fibroblast transformation.

RACK1 and CIS Mediate the Degradation of BimEL in Cancer Cells

RACK1 is a 7-WD motif-containing protein with numerous downstream effectors regulating various cellular functions. Using a yeast two-hybrid screen, we identified dynein light chain 1 as a novel interacting partner of RACK1. Additionally, we demonstrated that RACK1 formed a complex with DLC1 and Bim, specifically BimEL, in the presence of apoptotic agents. Upon paclitaxel treatment, RACK1, DLC1, and CIS mediated the degradation of BimEL through the ElonginB/C-Cullin2-CIS ubiquitin-protein isopeptide ligase complex. We further showed that RACK1 conferred paclitaxel resistance to breast cancer cells in vitro and in vivo. Finally, we observed an inverse correlation between CIS and BimEL levels in both ovarian and breast cancer cell lines and specimens. Our study suggests a role of RACK1 in protecting cancer cells from apoptosis by regulating the degradation of BimEL, which together with CIS could play an important role of drug resistance in chemotherapy.

Glutamatergic Pathway Targeting in Melanoma; Single Agent and Combinatorial Therapies

Purpose: Melanoma is a heterogeneous disease where monotherapies are likely to fail due to variations in genomic signatures. B-RAF inhibitors have been clinically inadequate but response might be augmented with combination therapies targeting multiple signaling pathways. We investigate the preclinical efficacy of combining the multikinase inhibitor sorafenib or the mutated B-RAF inhibitor PLX4720 with riluzole, an inhibitor of glutamate release that antagonizes metabotropic glutamate receptor 1 (GRM1) signaling in melanoma cells. Experimental Design: Melanoma cell lines that express GRM1 and either wild-type B-RAF or mutated B-RAF were treated with riluzole, sorafenib, PLX4720, or the combination of riluzole either with sorafenib or with PLX4720. Extracellular glutamate levels were determined by glutamate release assays. MTT assays and cell-cycle analysis show effects of the compounds on proliferation, viability, and cell-cycle profiles. Western immunoblotting and immunohistochemical staining showed apoptotic markers. Consequences on mitogen-activated protein kinase pathway were assessed by Western immunoblotting. Xenograft tumor models were used to determine the efficacy of the compounds in vivo. Results: The combination of riluzole with sorafenib exhibited enhanced antitumor activities in GRM1-expressing melanoma cells harboring either wild-type or mutated B-RAF. The combination of riluzole with PLX4720 showed lessened efficacy compared with the combination of riluzole and sorafenib in suppressing the growth of GRM1-expressing cells harboring the B-RAFV600E mutation. Conclusions: The combination of riluzole with sorafenib seems potent in suppressing tumor proliferation in vitro and in vivo in GRM1-expressing melanoma cells regardless of B-RAF genotype and may be a viable therapeutic clinical combination. Clin Cancer Res; 17(22); 7080–92. ©2011 AACR.

The Role of Phosphatidylinositol 3-Kinase, Rho Family GTPases, and STAT3 in Ros-induced Cell Transformation

Using loss-of-function mutants of Ros and inducible epidermal growth factor receptor-Ros chimeras we investigated the role of various signaling pathways in Ros-induced cell transformation. Inhibition of the mitogen-activated protein kinase (MAPK) pathway with the MEK (MAP/extracellular signal-regulated kinase kinase) inhibitor PD98059 had little effect on the Ros-induced monolayer and anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells even though more than 70% of the MAPK was inhibited. In contrast, inhibiting the phosphatidylinositol 3-kinase (PI3K) pathway with the drug LY294002, a dominant negative mutant of PI3K, Δp85, or the phosphatidylinositol phosphatase PTEN (phosphatase and tensin homologue deleted in chromosome ten) resulted in a dramatic reduction of v-Ros- and epidermal growth factor receptor-Ros-promoted anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells, respectively. Parallel and downstream components of PI3K signaling such as the Rho family GTPases (Rac, Rho, Cdc42) and the survival factor Akt were all shown to contribute to Ros-induced anchorage-independent growth, although Rac appeared to be less important for Ros-induced colony formation in NIH3T3 cells. Furthermore, the transformation-attenuated v-Ros mutants F419 and DI could be complemented by constitutively active mutants of PI3K and Akt. Finally, we found that overexpressing a constitutively active mutant of STAT3 (STAT3C) conferred a resistance to the inhibition of Ros-induced anchorage-independent growth by LY294002, suggesting a possible overlap of functions between PI3K and STAT3 signaling in mediating Ros-induced anchorage-independent growth.

Direct Interaction between Myocyte Enhancer Factor 2 (MEF2) and Protein Phosphatase 1α Represses MEF2-Dependent Gene Expression

ABSTRACT The myocyte enhancer factor 2 (MEF2) transcription factors play important roles in neuronal, cardiac, and skeletal muscle tissues. MEF2 serves as a nuclear sensor, integrating signals from several signaling cascades through protein-protein interactions with kinases, chromatin remodeling factors, and other transcriptional regulators. Here, we report a novel interaction between the catalytic subunit of protein phosphatase 1α (PP1α) and MEF2. Interaction occurs within the nucleus, and binding of PP1α to MEF2 potently represses MEF2-dependent transcription. The interaction utilizes uncharacterized domains in both PP1α and MEF2, and PP1α phosphatase activity is not obligatory for MEF2 repression. Moreover, a MEF2-PP1α regulatory complex leads to nuclear retention and recruitment of histone deacetylase 4 to MEF2 transcription complexes. PP1α-mediated repression of MEF2 overrides the positive influence of calcineurin signaling, suggesting PP1α exerts a dominant level of control over MEF2 function. Indeed, PP1α-mediated repression of MEF2 function interferes with the prosurvival effect of MEF2 in primary hippocampal neurons. The PP1α-MEF2 interaction constitutes a potent locus of control for MEF2-dependent gene expression, having potentially important implications for neuronal cell survival, cardiac remodeling in disease, and terminal differentiation of vascular, cardiac, and skeletal muscle.