Robert M. Gemmill

Cyclooxygenase-2–Dependent Regulation of E-Cadherin: Prostaglandin E2 Induces Transcriptional Repressors ZEB1 and Snail in Non–Small Cell Lung Cancer

Elevated tumor cyclooxygenase-2 (COX-2) expression is associated with tumor invasion, metastasis, and poor prognosis in non-small cell lung cancer (NSCLC). Here, we report that COX-2-dependent pathways contribute to the modulation of E-cadherin expression in NSCLC. First, whereas genetically modified COX-2-sense (COX-2-S) NSCLC cells expressed low E-cadherin and showed diminished capacity for cellular aggregation, genetic or pharmacologic inhibition of tumor COX-2 led to increased E-cadherin expression and resulted in augmented homotypic cellular aggregation among NSCLC cells in vitro. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human lung adenocarcinoma tissue sections. Second, treatment of NSCLC cells with exogenous prostaglandin E(2) (PGE(2)) significantly decreased the expression of E-cadherin, whereas treatment of COX-2-S cells with celecoxib (1 mumol/L) led to increased E-cadherin expression. Third, the transcriptional suppressors of E-cadherin, ZEB1 and Snail, were up-regulated in COX-2-S cells or PGE(2)-treated NSCLC cells but decreased in COX-2-antisense cells. PGE(2) exposure led to enhanced ZEB1 and Snail binding at the chromatin level as determined by chromatin immunoprecipitation assays. Small interfering RNA-mediated knockdown of ZEB1 or Snail interrupted the capacity of PGE(2) to down-regulate E-cadherin. Fourth, an inverse relationship between E-cadherin and ZEB1 and a direct relationship between COX-2 and ZEB1 were shown by immunohistochemical staining of human lung adenocarcinoma tissue sections. These findings indicate that PGE(2), in autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in NSCLC. Thus, blocking PGE(2) production or activity may contribute to both prevention and treatment of NSCLC.

The TRC8 Ubiquitin Ligase Is Sterol Regulated and Interacts with Lipid and Protein Biosynthetic Pathways

TRC8/RNF139 encodes an endoplasmic reticulum–resident E3 ubiquitin ligase that inhibits growth in a RING- and ubiquitylation-dependent manner. TRC8 also contains a predicted sterol-sensing domain. Here, we report that TRC8 protein levels are sterol responsive and that it binds and stimulates ubiquitylation of the endoplasmic reticulum anchor protein INSIG. Induction of TRC8 destabilized the precursor forms of the transcription factors SREBP-1 and SREBP-2. Loss of SREBP precursors was proteasome dependent, required a functional RING domain, occurred without generating processed nuclear forms, and suppressed SREBP target genes. TRC8 knockdown had opposite effects in sterol-deprived cells. In Drosophila, growth inhibition by DTrc8 was genetically suppressed by loss of specific Mprlp, Padlp N-terminal domain–containing proteins found in the COP9 signalosome and eIF3. DTrc8 genetically and physically interacted with two eIF3 subunits: eIF3f and eIF3h. Coimmunoprecipitation experiments confirmed these interactions in mammalian cells, and TRC8 overexpression suppressed polysome profiles. Moreover, high–molecular weight ubiquitylated proteins were observed in eIF3 immunoprecipitations from TRC8-overexpressing cells. Thus, TRC8 function may provide a regulatory link between the lipid and protein biosynthetic pathways. Mol Cancer Res; 8(1); 93–106

The Philadelphia chromosome: A model of cancer and molecular cytogenetics

Recent developments in molecular biology related to the Ph chromosome lead us to an evaluation of knowledge regarding this chromosome. The molecular advances are related to two cellular oncogenes, c-abl and c-sis, and also to the identification and molecular cloning of specific areas of DNA (e.g., band 22q11), permitting the isolation of a probe specific for the translocation breakpoint domain. In the preponderant number of cases examined, it was found that the breakpoints at 22q11 occur within a limited region of up to 5-6 kb, for which the term breakpoint cluster region (bcr) has been suggested. In contrast, breaks at 9q34 seem to occur within a much larger region at the molecular level. Yet to be established is the exact genetic composition of the bcr and a determination as to whether or not the breaks leading to the disease occur preferentially within specific areas. In spite of this level of knowledge, we do not understand how the Ph chromosome participates in CML. If Ph-positive CML is ultimately associated with a cascade of gene activations, the unraveling of their nature and chronology will undoubtedly tell us much of their contribution to the biology of CML, in particular, and to neoplasia, in general. In this respect, the rather clear description of CML in cytogenetic, clinical, and laboratory terms, the relatively long chronic phase of the disease, and the association of the blastic phase with nonrandom chromosome changes (at least in the initial phases of the disease) make Ph-positive CML an excellent candidate for a model for the study of molecular events in human neoplasia.

Construction of long-range restriction maps in human DNA using pulsed field gel electrophoresis

Pulsed field gel electrophoresis (PFGE) is a powerful new tool for genetic analysis that can be applied to a variety of problems concerning genome structure and organization. This technique uses an agarose gel matrix to separate DNA molecules in a size range from 40 kb to 2,000 kb, molecules far larger than the maximum separable using standard agarose gel electrophoresis. The PFGE method can be used to separate the intact chromosomes from lower eukaryotes or to separate very large DNA fragments from higher eukaryotes generated by digestion with restriction endonucleases whose cleavage sites are rare. This paper describes the use of PFGE for construction of long-range restriction maps in the human genome and includes detailed methods for all steps. A pulsed field gel device that utilizes a rotating platform for altering the applied electric field is also described. Map construction is illustrated using a cloned DNA fragment (D3S2) from human chromosome 3. Several technical problems specific for mammalian genomes are discussed.

The emerging role of class-3 semaphorins and their neuropilin receptors in oncology.

The semaphorins, discovered over 20 years ago, are a large family of secreted or transmembrane and glycophosphatidylinositol -anchored proteins initially identified as axon guidance molecules crucial for the development of the nervous system. It has now been established that they also play important roles in organ development and function, especially involving the immune, respiratory, and cardiovascular systems, and in pathological disorders, including cancer. During tumor progression, semaphorins can have both pro- and anti-tumor functions, and this has created complexities in our understanding of these systems. Semaphorins may affect tumor growth and metastases by directly targeting tumor cells, as well as indirectly by interacting with and influencing cells from the micro-environment and vasculature. Mechanistically, semaphorins, through binding to their receptors, neuropilins and plexins, affect pathways involved in cell adhesion, migration, invasion, proliferation, and survival. Importantly, neuropilins also act as co-receptors for several growth factors and enhance their signaling activities, while class 3 semaphorins may interfere with this. In this review, we focus on the secreted class 3 semaphorins and their neuropilin co-receptors in cancer, including aspects of their signaling that may be clinically relevant.

Loss of heterozygosity on 3p in a renal cell carcinoma in von Hippel-Lindau syndrome

A renal cell carcinoma with an unbalanced t(X;3) in a patient with von Hippel-Lindau (VHL) syndrome has previously been reported. This rearrangement suggested loss of genetic material from the short arm of chromosome 3, which we are now able to confirm by restriction fragment length polymorphism analysis of tumor DNA using polymorphic probes derived from 3p. The VHL gene has recently been mapped to 3p, therefore loss of this region in this VHL-related renal cell carcinoma may have cogent significance for tumor development in this interesting cancer-predisposing syndrome.

Cytogenetic and fluorescence in situ hybridization studies on sporadic and hereditary tumors associated with von Hippel-Lindau syndrome (VHL).

We performed cytogenetic and fluorescence in situ hybridization (FISH) studies on 29 sporadic or familial tumors associated with von Hippel-Lindau [correction of Landau] disease. Four of five renal cell carcinomas with detectable alterations showed clones with chromosome 3 alterations. These changes led to loss of genetic material visible with cytogenetic resolution: either an unbalanced translocation involving 3p or loss of a whole homolog 3, resulting in monosomy of 3p. We have previously mapped the VHL gene to chromosomal region 3p25-p26. We applied FISH using the single copy probes cA233 and cA479, sequences close to the VHL gene, in a search for submicroscopic deletions of 3p. Use of FISH with differentially labeled probes indicated cA479 to be distal to cA233, but both were located within bands 3p25-26. FISH with single copy probes for interphase cytogenetics detected four subclones with deletions in the VHL region in 8/22 tumors, including four tumors which appeared cytogenetically normal. FISH proved to be a powerful tool in tumor genetic studies, especially helpful in detecting tumor subclones in benign and slowly growing tumors.

Loss of common 3p14 fragile site expression in renal cell carcinoma with deletion breakpoint at 3p14

The common fragile site in human chromosome band 3p14 is a constant cytogenetic marker present on every normal chromosome #3. Therefore, we selected a renal cell carcinoma with a deletion breakpoint in 3p14 for analysis of the 3p14 fragile site. Aphidicolin was used to induce the expression of the 3p14 fragile site. The fragile sites expressed in the renal carcinoma cells generally mirrored those expressed in lymphocytes. The normal chromosome #3 in the renal carcinoma cells expressed the common 3p14 fragile site. The partially deleted #3 did not. The deletion breakpoint, therefore, cannot be beyond the 3p14 fragile site. The common fragile site in 3p14 must be at or very near the deletion breakpoint in 3p14 in renal cell carcinoma. These results are consistent with this fragile site causing this cancer chromosome deletion.

Epigenetic Regulation of the Epithelial to Mesenchymal Transition in Lung Cancer

Lung cancer is the leading cause of cancer deaths worldwide. It is an aggressive and devastating cancer because of metastasis triggered by enhanced migration and invasion, and resistance to cytotoxic chemotherapy. The epithelial to mesenchymal transition (EMT) is a fundamental developmental process that is reactivated in wound healing and a variety of diseases including cancer where it promotes migration/invasion and metastasis, resistance to treatment, and generation and maintenance of cancer stem cells. The induction of EMT is associated with reprogramming of the epigenome. This review focuses on major mechanisms of epigenetic regulation mainly in lung cancer with recent data on EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit ), the catalytic subunit of the PRC2 (Polycomb Group PcG), that behaves as an oncogene in lung cancer associated with gene repression, non-coding RNAs and the epitranscriptome.

613 Homozygous deletions of human chromosome 3p in lung tumors

Cytogenetic and loss of heterozygosity (LOH) studies have demonstrated that deletions of chromosome 3p occur at a high frequency in all forms of lung cancer. To clarify the role of 3p in lung tumorigenesis and to more precisely identify targets for positional cloning efforts, we have performed 3p deletion analyses (microsatellite and fluorescence in situ hybridization) in a series of lung cancer cell lines and uncultured tumor samples. Importantly, we identified homozygous deletions in four uncultured tumors and one cell line. Homozygous deletions were found in three squamous tumors within a region of 3p21 which had previously been described only in cell lines, a 1-2-megabase homozygous deletion in a small cell tumor at 3p12, and a 3p14.2 homozygous deletion in a non-small cell lung carcinoma cell line. The detection of homozygous deletions affecting these multiple regions in uncultured tumor cells substantiates the belief (previously based on deletions found only in tumor cell lines) that these sites contain important tumor suppressor genes. Along with previously reported homozygous deletions in a distal portion of 3p21.3, we now have evidence for four separate regions of 3p which undergo homozygous deletions in either uncultured lung tumors or cell lines.