Joseph Locker

Classifying collective cancer cell invasion

Most invasive solid tumours display predominantly collective invasion, in which groups of cells invade the peritumoral stroma while maintaining cell-cell contacts. As the concepts and experimental models for functional analysis of collective cancer cell invasion are rapidly developing, we propose a framework for addressing potential mechanisms, experimental strategies and technical challenges to study this process.

Indolent mantle cell lymphoma with nodal involvement and mutated immunoglobulin heavy chain genes

Mantle cell lymphoma (MCL) is typically considered an aggressive but incurable neoplasm composed of cyclin D1+ monoclonal B-cells with a t(11;14)(q13;q32) and usually unmutated immunoglobulin (Ig) genes. Although it has been suggested that a more indolent leukemic disorder exists with the same phenotype and genotype but with mutated Ig genes, others have considered these cases to be variants of chronic lymphocytic leukemia. We present a case of an indolent MCL that was documented with cyclin D1 expression in a lymph node biopsy performed more than 12 years ago. The patient has peripheral blood involvement with a lymphocyte count in the reference range, variable thrombocytopenia, and minimal adenopathy but is otherwise well, never having received any antineoplastic therapy. Study of peripheral blood samples from 2002 revealed a CD5-variable B-cell monoclonal proliferation with a t(11;14)(q13;q32) plus other karyotypic abnormalities, positive fluorescence in situ hybridization studies for the CCND1/IgH translocation, and clonal Ig gene rearrangement with mutated Ig genes (95.7% homology to VH 4-31). The subtle but diagnostic lymph node biopsy in this case helps to further support that an indolent t(11;14) monoclonal lymphocytosis with mutated Ig genes can represent an MCL variant rather than chronic lymphocytic leukemia.

Skp2 is required for survival of aberrantly proliferating Rb1-deficient cells and for tumorigenesis in Rb1+/- mice

Heterozygosity of the retinoblastoma gene Rb1 elicits tumorigenesis in susceptible tissues following spontaneous loss of the remaining functional allele. Inactivation of previously studied retinoblastoma protein (pRb) targets partially inhibited tumorigenesis in Rb1+/− mice. Here we report that inactivation of pRb target Skp2 (refs. 7,8) completely prevents spontaneous tumorigenesis in Rb1+/− mice. Targeted Rb1 deletion in melanotrophs ablates the entire pituitary intermediate lobe when Skp2 is inactivated. Skp2 inactivation does not inhibit aberrant proliferation of Rb1-deleted melanotrophs but induces their apoptotic death. Eliminating p27 phosphorylation on T187 in p27T187A knock-in mice reproduces the effects of Skp2 knockout, identifying p27 ubiquitination by SCFSkp2 ubiquitin ligase as the underlying mechanism for Skp2s essential tumorigenic role in this setting. RB1-deficient human retinoblastoma cells also undergo apoptosis after Skp2 knockdown; and ectopic expression of p27, especially the p27T187A mutant, induces apoptosis. These results reveal that Skp2 becomes an essential survival gene when susceptible cells incur Rb1 deficiency.

ErbB3-Dependent Motility and Intravasation in Breast Cancer Metastasis

A better understanding of how epidermal growth factor receptor family members (ErbBs) contribute to metastasis is important for evaluating ErbB-directed therapies. Activation of ErbB3/ErbB2 heterodimers can affect both proliferation and motility. We find that increasing ErbB3-dependent signaling in orthotopic injection models of breast cancer can enhance intravasation and lung metastasis with no effect on primary tumor growth or microvessel density. Enhanced metastatic ability due to increased expression of ErbB2 or ErbB3 correlated with stronger chemotaxis and invasion responses to heregulin beta1. Suppression of ErbB3 expression reduced both intravasation and metastasis. A human breast cancer tumor tissue microarray showed a significant association between ErbB3 and ErbB2 expression and metastasis independent of tumor size. These results indicate that ErbB3-dependent signaling through ErbB3/ErbB2 heterodimers can contribute to metastasis through enhancing tumor cell invasion and intravasation in vivo and that ErbB-directed therapies may be useful for the inhibition of invasion independent of effects on tumor growth.

Rybp/DEDAF Is Required for Early Postimplantation and for Central Nervous System Development

ABSTRACT The Rybp/DEDAF protein has been implicated in both transcriptional regulation and apoptotic signaling, but its precise molecular function is unclear. To determine the physiological role of Rybp, we analyzed its expression during mouse development and generated mice carrying a targeted deletion of Rybp using homologous recombination in embryonic stem cells. Rybp was found to be broadly expressed during embryogenesis and was particularly abundant in extraembryonic tissues, including trophoblast giant cells. Consistent with this result, rybp homozygous null embryos exhibited lethality at the early postimplantation stage. At this time, Rybp was essential for survival of the embryo, for the establishment of functional extraembryonic structures, and for the execution of full decidualization. Through the use of a chimeric approach, the embryonic lethal phenotype was circumvented and a role for Rybp in central nervous system development was uncovered. Specifically, the presence of Rybp-deficient cells resulted in marked forebrain overgrowth and in localized regions of disrupted neural tube closure. Functions for Rybp in the brain also were supported by the finding of exencephaly in about 15% of rybp heterozygous mutant embryos, and by Rybps distinct neural expression pattern. Together, these findings support critical roles for Rybp at multiple stages of mouse embryogenesis.

Gadd45β is induced through a CAR‐dependent, TNF‐independent pathway in murine liver hyperplasia

We previously observed that Gadd45β/MyD118, a member of the Gadd45 family of inducible factors, showed the strongest immediate‐early induction common to two distinctive proliferation responses of the liver: (1) regeneration induced by surgical partial hepatectomy and (2) hyperplasia induced by the primary mitogen TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Gadd45β is known to be stimulated by nuclear factor (NF) κB, which is activated by tumor necrosis factor alpha (TNFα) in the early response to partial hepatectomy. We therefore investigated whether TNFα and NFκB also stimulated Gadd45β as part of the response to CAR ligands, or whether activation occurred by an alternative pathway. TCPOBOP effects were characterized in three mouse genotypes: wild‐type, TNFR1−/−, and TNFR1−/−TNFR2−/−. The results showed that TCPOBOP did not activate NFκB in any of the mice, but a strong induction of Gadd45β messenger RNA was observed in all three genotypes, where TCPOBOP also induced CyP2b10, a classical target gene of activated CAR, and cyclin D1, a proliferation linked gene. Thus, the absence of TNFR signaling and induction of NFκB did not impair CAR‐mediated gene induction. Moreover, hepatocyte proliferation was strongly induced, and at significantly higher levels than wild type, in both TNFR1−/− and TNFR1−/−TNFR2−/− mice. Further studies evaluated TCPOBOP‐induced gene expression in CAR−/− mice, by microarray expression profiling and Northern blot. The induced changes in gene expression, including the stimulation of Gadd45β, were almost completely abolished—hence all were mediated via CAR activation. In conclusion, in the liver, Gadd45β can be induced by a distinctive pathway that requires CAR and is independent of TNFα‐NFκB. The greater induction of proliferation in TNFR‐null mice suggests negative cross‐talk between the CAR and TNFα‐NFκB controls that regulate proliferation. (HEPATOLOGY 2005.)

Gadd45beta is induced through a CAR-dependent, TNF-independent pathway in murine liver hyperplasia.

We previously observed that Gadd45β/MyD118, a member of the Gadd45 family of inducible factors, showed the strongest immediate‐early induction common to two distinctive proliferation responses of the liver: (1) regeneration induced by surgical partial hepatectomy and (2) hyperplasia induced by the primary mitogen TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Gadd45β is known to be stimulated by nuclear factor (NF) κB, which is activated by tumor necrosis factor alpha (TNFα) in the early response to partial hepatectomy. We therefore investigated whether TNFα and NFκB also stimulated Gadd45β as part of the response to CAR ligands, or whether activation occurred by an alternative pathway. TCPOBOP effects were characterized in three mouse genotypes: wild‐type, TNFR1−/−, and TNFR1−/−TNFR2−/−. The results showed that TCPOBOP did not activate NFκB in any of the mice, but a strong induction of Gadd45β messenger RNA was observed in all three genotypes, where TCPOBOP also induced CyP2b10, a classical target gene of activated CAR, and cyclin D1, a proliferation linked gene. Thus, the absence of TNFR signaling and induction of NFκB did not impair CAR‐mediated gene induction. Moreover, hepatocyte proliferation was strongly induced, and at significantly higher levels than wild type, in both TNFR1−/− and TNFR1−/−TNFR2−/− mice. Further studies evaluated TCPOBOP‐induced gene expression in CAR−/− mice, by microarray expression profiling and Northern blot. The induced changes in gene expression, including the stimulation of Gadd45β, were almost completely abolished—hence all were mediated via CAR activation. In conclusion, in the liver, Gadd45β can be induced by a distinctive pathway that requires CAR and is independent of TNFα‐NFκB. The greater induction of proliferation in TNFR‐null mice suggests negative cross‐talk between the CAR and TNFα‐NFκB controls that regulate proliferation. (HEPATOLOGY 2005.)

Structure and Function of the Virulence-Associated High-Temperature Requirement A of Mycobacterium tuberculosis

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.

Hypermethylation of the human proton-coupled folate transporter (SLC46A1) minimal trancriptional regulatory region in an antifolate-resistant HeLa cell line

This laboratory recently identified a novel proton-coupled folate transporter (PCFT) that mediates intestinal folate absorption and transport of folates into the central nervous system. The present study focuses on the definition of the minimum transcriptional regulatory region of this gene in HeLa cells and the mechanism(s) underlying the loss of PCFT expression in the methotrexate-resistant HeLa R1-11 cell line. The PCFT transcriptional regulatory controls were localized between −42 and +96 bases from the transcriptional start site using a luciferase-reporter gene system. The promoter is a G + C rich region of 139 nucleotides contained in a CpG island. HeLa R1-11 cells have no mutations in the PCFT open reading frame and its promoter; the transcription/translation machinery is intact because transient transfections in HeLa R1-11 and wild-type HeLa cells produced similar luciferase activities. Hypermethylation at CpG sites within the minimal transcriptional regulatory region was shown in HeLa R1-11 cells as compared with the parental PCFT-competent HeLa cells, using bisulfite conversion and sequence analysis. Treatment with 5-aza-2′-deoxycytidine resulted in a substantial restoration of transport and PCFT mRNA expression and small but significant decreases in methylation in the promoter region. In vitro methylation of the transfected reporter plasmid inhibited luciferase gene expression. Cytogenetics/fluorescence in situ hybridization indicated a loss of half the PCFT gene copies in HeLa R1-11 as compared with PCFT-competent HeLa cells. Taken together, promoter silencing through methylation and gene copy loss accounted for the loss of PCFT activity in antifolate-resistant HeLa R1-11 cells. [Mol Cancer Ther 2009;8(8):2424–31]

Differential tissue and subcellular expressionof ERM proteins in normal and malignant tissues: Cytoplasmic ezrin expression has prognostic signficance for head and neck squamous cell carcinoma†

Members of the ezrin‐radixin‐moesin (ERM) protein family regulate cellular shape, motility, and proliferation and potentially influence ability to metastasize. We investigated the correlation between ERM subcellular localization and survival in patients with squamous cell carcinoma (SCC)