Cancer Genetics and Dissection of Signaling Networks in Normal and Malignant Cells
We are using insertional mutagenesis and other genetic strategies to identify genes that are involved in oncogenesis or regulate phenotypic changes in tumor cells. Genes identified via these strategies are used as probes to explore function from the molecular to the animal level. Some of the genes we have identified and exploited to date include Akt1, Tpl2, Gfi-1 and Gfi-1B. Our work highlights the value of genetic studies in animal models for understanding the molecular basis of cancer and other diseases in humans.
Akt1 is the founding member of a protein kinase family composed of three members, Akt1, Akt2 and Akt3. The Akt kinases are activated by phosphorylation via a P1-3 kinase-dependent process, and they regulate a diverse array of cellular functions including apoptosis, cellular proliferation, differentiation and intermediary metabolism. Akt activation depends on PtdIns-3,4,5-P3, and to a lesser extent on PtdIns-3,4-P2, both of which are products of phosphoinositide 3-kinase (PI-3K). The interaction of PtdIns-3,4,5-P3 with the PH domain of Akt, promotes the translocation of Akt to the plasma membrane where it undergoes phosphorylation at two sites: Thr308 in the activation loop and Ser473 in the carboxy-terminal tail. The kinase that phosphorylates Akt at Thr308 is PI-3K-dependent kinase-1 (PDK-1). The identity of the kinase that phosphorylates Akt at Ser473 (PDK-2) remain elusive, although it appears to be membrane-associated. Several kinases, including ILK-1, PDK1, Akt itself and perhaps other AGC kinases, have the potential to phosphorylate Akt at this site. Phosphorylated Akt translocates from the plasma membrane to the cytosol or the nucleus. Activated Akt ultimately undergoes dephosphorylation by phosphatases and returns to the inactive state (Figure 1). Other regulatory molecules include proteins that interact with the kinase. Such Akt interactors may regulate Akt phosphorylation, subcellular localization or stability.
Figure 1. The figure illustrates the mechanisms by which Akt family members are activated.
Akt plays a critical role in the induction and/or progression of a variety of human neoplasms. Its role may be direct, as in the case of Akt2 amplification in epithelial neoplasms, or indirect, consisting in the transduction of oncogenic signals initiated by mutations in other oncogenes or tumor suppressor genes. Because of its role in human cancer and other diseases, Akt has been recognized as an important target for the development of antineoplastic drugs.
Our current studies on Akt focus on the function of the three Akt isoforms. Researchers are working on the identification of isoform-specific Akt targets and the role of the three isoforms in the development of the immune system and in the regulation of the immune response. Finally, Daniel Tiber focuses on the role of the three Akt isoforms in the transduction of oncogenic signals initiated by mutated oncogenes or tumor suppressor genes involved in Akt regulation.
Tpl2 is also a serine-threonine protein kinase that is activated by provirus insertion in retrovirus-induced T cell lymphomas and mammary adenocarcinomas in rodents. Overexpression of Tpl-2 in a variety of cell types, activates ERK, JNK, p38 MAPK, and the transcription factors NF-AT and NF-kB. Moreover, transgenic mice expressing a constitutively-active form of Tpl-2 under the control of a T cell specific promoter develop thymic lymphomas. Finally, Tpl2 contributes to the transduction of oncogenic signals induced by LMP1. Our studies using Tpl-2 knockout mice, revealed that Tpl-2 plays an obligatory role in the transduction of Toll-like receptor and death receptor signals that activate ERK and regulate the expression of cytokines, chemokines and other molecules involved in the regulation of innate and adaptive immunity, inflammation and oncogenesis. Interestingly, the mechanism by which the Tpl2/ERK pathway regulates the expression of different genes is variable. Thus, Tpl2 regulates the induction of TNF-α by LPS, by transducing ERK activation signals that target the 3’ARE of the TNF-α mRNA and regulate its transport from the nucleus to the cytoplasm.
Figure 2. The model depicts the pathway by which LPS activates Tpl2.
Tpl2 is also required for the induction of COX-2 by LPS. COX-2 induction however, depends on Tpl2-transduced signals that control COX-2 mRNA levels by regulating COX-2 transcription and mRNA stability. Because of its role in oncogenesis and inflammation, Tpl2 has been recognized as an important target for the development of antineoplastic and anti-inflammatory drugs.
Figure 3. Model of Tpl2-mediated COX-2 transactivation and PGE2 production in response to LPS.
Our current studies on Tpl2 focus on the regulation of the Tpl2 kinase activity by Toll-like receptor and Death receptor signals. Along these lines, lab members are addressing the role of posttranslational modifications in Tpl2 regulation. In addition, we are interested on the role of Tpl2 in the transduction of signals that regulate innate and adaptive immunity, inflammation and oncogenesis. Along these lines, we are focusing on the role of Tpl2 in tumor induction by a variety of oncogenic signals and on the role of Tpl2 in immune regulation and its role in the regulation of gene expression.
Gfi-1 was identified as a gene that is involved in the transition of IL-2-dependent T cell lymphoma lines to IL-2 independence. Gfi-1, and its homolog Gfi-1B, are nuclear proteins that function as transcriptional repressors. Both proteins contain a DNA binding zinc finger domain and an N terminal repressor domain SNAG that is shared by the Gfi-1 and the Snail and Slug families of transcription factors (Snail-Gfi1). Our earlier studies had shown that Gfi-1 promotes cell cycle progression and inhibits apoptosis of T cells deprived of IL-2 and that Gfi-1B controls the differentiation of hematopoietic cell lines in culture. More recent studies revealed that the anti apoptotic and proliferative properties of Gfi-1 are required for the survival and proliferation of Th2 T cells. In addition both Gfi-1 and Gfi-1B are required for hematopoietic and neuronal cell differentiation in animals.
Our current studies focus on the regulation of Gfi-1 by upstream signals and on the identification of genes that co-operate with Gfi-1 in oncogenesis.
More recent studies, using genome wide screens for loci of common integration in retrovirus induced lymphomas in rodents, revealed that more than 50% of provirus integrations target genes that may be involved in tumor induction and progression. Such screens led to the identification of several novel oncogenes, which we are presently characterizing.