Jian-ang Li

BCR/ABL induces multiple abnormalities of cytoskeletal function.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.

The Related Adhesion Focal Tyrosine Kinase Forms a Complex with Paxillin in Hematopoietic Cells

Related adhesion focal tyrosine kinase (RAFTK), also known as proline-rich tyrosine kinase 2 and cellular adhesion kinase β, has been recently cloned and characterized as a member of the focal adhesion kinase (FAK) subfamily. RAFTK has an overall 48% amino acid homology to p125FAK and contains a kinase domain but lacks a transmembrane region, myristylation sites, and Src homology region 2 and 3 domains. By Northern blot analysis, RAFTK is expressed in myeloid, lymphoid, and megakaryocytic hematopoietic cells. Like p125FAK, we found that RAFTK interacts with the focal adhesion protein paxillin. In the lymphoid cell line BaF3 and the myeloid cell line 32Dcl3, RAFTK coprecipitates with paxillin. Using in vitro binding assays, RAFTK and paxillin were shown to bind directly, through a segment of paxillin that required amino acids 100-227 and a domain in the C terminus of RAFTK. In vitro, RAFTK could phosphorylate paxillin on tyrosine residues. These results suggest that RAFTK, as well as p125FAK, may be important in phosphotyrosine-signaling events within the focal adhesion.

p130CAS Forms a Signaling Complex with the Adapter Protein CRKL in Hematopoietic Cells Transformed by the BCR/ABL Oncogene

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210BCR/ABL. CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120CBL. Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130CAS. p130CAS was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130CAS was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125FAK, and paxillin constitutively associated with p130CAS. However, in BCR/ABL transformed cells, the interaction between p130CAS and tensin was disrupted, while the associations between p130CAS, p125FAK, and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130CAS in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130CAS containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130CAS with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells.

Mastermind-like transcriptional co-activators: emerging roles in regulating cross talk among multiple signaling pathways

A family of Mastermind-like (MAML) genes encodes critical transcriptional co-activators for Notch signaling, an evolutionarily conserved pathway with numerous roles in both development and human diseases. Notch receptors are cleaved upon ligand engagement and the intracellular domain of Notch shuttles to the nucleus. MAMLs form a functional DNA-binding complex with the cleaved Notch receptor and the transcription factor CSL, thereby regulating transcriptional events that are specific to the Notch pathway. Here, we review recent studies that have utilized molecular, cellular and physiological model system strategies to reveal the pivotal roles of the MAML proteins in Notch signaling. Unexpectedly, however, emerging evidence implicate MAML proteins as exciting key transcriptional co-activators in other signal transduction pathways including: muscle differentiation and myopathies (MEF2C), tumor suppressor pathway (p53) and colon carcinoma survival (β-catenin). Thus, the MAML family appears to function in transcriptional co-activation in a multitude of cellular processes. It is hypothesized that MAML proteins mediate cross-talk among the various signaling pathways and the diverse activities of the MAML proteins converge to impact normal biological processes and human diseases, including cancers.

The Bovine Papillomavirus E6 Protein Binds to the LD Motif Repeats of Paxillin and Blocks Its Interaction with Vinculin and the Focal Adhesion Kinase

The bovine papillomavirus type 1 (BPV-1) E6 oncoprotein can transform fibroblasts and induce anchorage-independent growth and disassembly of the actin stress fibers. We have previously shown that the E6 protein interacts with the focal adhesion protein, paxillin, suggesting a direct role of E6 in the disruption of the actin cytoskeleton. We have now mapped the E6 binding sites on paxillin to the LD motif repeats region, which has been implicated in mediating paxillin binding to two other focal adhesion proteins, vinculin and the focal adhesion kinase. The five LD motif repeats identified in paxillin do not contribute equally to its interaction with E6. The first LD repeat is most critical for paxillin binding to E6 both in vitro and in vivo. Furthermore, the binding of recombinant wild-type E6 protein to paxillin blocked the interaction of several cellular proteins with paxillin, including vinculin and the focal adhesion kinase. A mutant E6 protein (H105) which does not bind to paxillin had no effect on the binding of these cellular proteins to paxillin. These data suggest that E6 disruption of the actin stress fibers occurs through blocking the interaction of paxillin with its cellular effectors such as vinculin and the focal adhesion kinase.

Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility.

Lung cancer can lead to abnormalities of the actin cytoskeleton structure which may be important in transformation. In this study, we have investigated the expression of the cytoskeletal associated protein paxillin in lung cancer. Paxillin is a 68 kDa focal adhesion protein, with four tandem LIM domains at the C-terminus, involved in growth factor receptor, integrin and oncogenic signaling such as v-src, BCR/ABL, and E6 of the papilloma virus. In non-small cell lung cancer (NSCLC) cell lines, paxillin localized to the focal adhesions. The possible role of paxillin in lung cancer cells was assessed by overexpressing green fluorescence protein (GFP)-paxillin construct in two separate NSCLC cell lines (Calu-1 and H661). Over the course of 48 h, GFP-paxillin consistently caused the cells to become round and to decrease cell motility as compared to normal controls, GFP-N-terminus paxillin, or GFP-LIM transfected cells. Because some lung cancers may be quite aggressive and metastasize quickly, which may be related to the cytoskeleton, we determined the expression of paxillin in NSCLC and small cell lung cancer (SCLC) cell lines and patient tumor tissues. Expression of paxillin in NSCLC and SCLC cell lines were determined by Northern blot and Western blot analysis. The expression of paxillin was consistently low in SCLC cell lines, whereas there was paxillin expression in NSCLC cell lines. There was a variability of expression of paxillin in NSCLC tumor tissue as compared to normal lung tissue. In contrast, by immunohistochemistry, we show that there was no detectable expression of paxillin in 5/5 SCLC patients. This data suggests that absence or low level of paxillin protein expression may cause certain lung cancers, such as SCLC, to be more motile and possibly more aggressive.

Altered LKB1/CREB-regulated transcription co-activator (CRTC) signaling axis promotes esophageal cancer cell migration and invasion.

LKB1 is a tumor susceptibility gene for the Peutz–Jeghers cancer syndrome and is a target for mutational inactivation in sporadic human malignancies. LKB1 encodes a serine/threonine kinase that has critical roles in cell growth, polarity and metabolism. A novel and important function of LKB1 is its ability to regulate the phosphorylation of CREB-regulated transcription co-activators (CRTCs) whose aberrant activation is linked with oncogenic activities. However, the roles and mechanisms of LKB1 and CRTC in the pathogenesis of esophageal cancer have not been previously investigated. In this study, we observed altered LKB1–CRTC signaling in a subset of human esophageal cancer cell lines and patient samples. LKB1 negatively regulates esophageal cancer cell migration and invasion in vitro. Mechanistically, we determined that CRTC signaling becomes activated because of LKB1 loss, which results in the transcriptional activation of specific downstream targets including LYPD3, a critical mediator for LKB1 loss-of-function. Our data indicate that de-regulated LKB1–CRTC signaling might represent a crucial mechanism for esophageal cancer progression.

DDX5 is a positive regulator of oncogenic NOTCH1 signaling in T cell acute lymphoblastic leukemia

Notch signaling is a highly conserved cell–cell communication pathway regulating normal development and tissue homeostasis. Aberrant Notch signaling represents an important oncogenic mechanism for T cell acute lymphoblastic leukemia (T-ALL), an aggressive subset of the most common malignant childhood cancer ALL. Therefore, understanding the molecular regulation of Notch signaling is critical to identify new approaches to block aberrant Notch oncogenic activity. The family of three MAML transcriptional coactivators is crucial for Notch signaling activation. The prototypic member MAML1 is the major coactivator that regulates Notch oncogenic activities in leukemic cells. However, the molecular basis underlying MAML1 coactivator function that contributes to Notch signaling remains unclear. In this study, we performed proteomic studies and identified DDX5, an ATP-dependent DEAD-box RNA helicase, as a component of the MAML1 protein complex. DDX5 interacts with MAML1 in vitro and in vivo, and is associated with the endogenous NOTCH1 transcription activation complex in human T-ALL leukemic cells. Lentivirus-mediated short-hairpin RNA knock-down of DDX5 resulted in decreased expression of Notch target genes, reduced cell proliferation and increased apoptosis in cultured human leukemic cells with constitutive activation of Notch signaling. Also, DDX5 depletion inhibited the growth of human leukemia xenograft in nude mice. Moreover, DDX5 is highly expressed in primary human T-ALL leukemic cells based on the analyses of Oncomine and GEO databases, and Immunohistochemical staining. Our overall findings revealed a critical role of DDX5 in promoting efficient Notch-mediated transcription in leukemic cells, suggesting that DDX5 might be critical for NOTCH1-mediated T-ALL pathogenesis and thus is a potential new target for modulating the Notch signaling in leukemia.

Aberrantly activated AREG-EGFR signaling is required for the growth and survival of CRTC1-MAML2 fusion-positive mucoepidermoid carcinoma cells

Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1–MAML2 fusion oncogene, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a frequent genetic alteration found in >50% of mucoepidermoid carcinomas (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1–MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1–MAML2 fusion gene and its downstream AREG–EGFR signaling in human MEC cancer cell growth and survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1–MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and in vivo. The CRTC1–MAML2 oncoprotein induced the upregulation of the epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) by co-activating the transcription factor CREB, and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1–MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG–EGFR signaling is required for CRTC1–MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with advanced, unresectable CRTC1–MAML2-positive MEC.

Genetic inactivation or pharmacological inhibition of Pdk1 delays development and inhibits metastasis of Braf V600E ::Pten –/– melanoma

Phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and protein kinase C (PKC), and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, owing to auto-activation, or elevated phosphorylation by phosphoinositide 3′-OH-kinase (PI3K). Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten–/– or BrafV600E::Cdkn2a–/–::Pten–/– mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-hydroxytamoxifen. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten–/– mice. Pdk1–/– melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten wild-type (WT) genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes, and inhibition of FOXO3a restored proliferation and colony formation of Pdk1–/– melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.