Cell Signaling and Receptor/Membrane Traffic

Fosters studies on the pathways and mechanisms through which receptor endocytic activity is coupled to cell signaling pathways.

Investigator: Alan Fields, Ph.D.

Dr. Fields has previously shown that the atypical protein kinase C isozyme PKCι is a critical cancer gene that plays a requisite role in lung and colon cancer. His laboratory demonstrated that PKCι is a target for frequent tumor-specific gene amplification that drives PKCι expression in human lung cancers. Oncogenic PKCι functions by stimulating an Rac1-dependent signaling pathway required for transformed growth and invasions of lung cancer cells. Taken together, the studies provide compelling evidence that PKCι is an oncogene in lung cancer and represent the first demonstration, since the discovery of PKC 30 years ago, that a PKC isozyme is a human oncogene.

More recently, Dr. Fields' team designed and implemented a novel high-throughput assay to identify small molecule compounds that disrupt the ability of PKCι to couple to its downstream effector Rac1. As a result of this screen, they have identified a novel compound, aurothiomalate – currently in clinical use for rheumatoid arthritis – that exhibits potent anti-tumor activity by disrupting oncogenic PKCι signaling (Cancer Research. 66:1767-74, 2006). Dr. Fields is the primary investigator of a Phase I clinical trial currently underway at Mayo Clinic to determine a maximal tolerable dose of aurothiomalate and to assess clinical response in patients with advanced lung adenocarcinoma with elevated PKCι expression.

Investigator: Bruce Horazdovsky, Ph.D.

The Horazdovsky Laboratory focuses on the regulation of intracellular protein trafficking events and how these events impact cell growth and differentiation. Studies were initiated in yeast where a genetic approach was used to define several of the basic molecular mechanisms involved in conserved protein transport events. The insights gained from the yeast system have been applied to trafficking events that occur in mammalian cells, specifically the movement of growth factor receptors through the endocytic pathway. Current studies are focused on how the regulated trafficking of growth factor receptors serves to modulate the signal transduction pathways activated by these receptors.

For example, Dr. Horazdovsky's team has identified a large number of proteins that contain a specific, highly conserved domain that catalyzes Rab5 guanine nucleotide exchange (Trends in Cell Biology. 16(1):27-35, 2006). Activation of Rab5 by nucleotide exchange is required for protein trafficking through the early stages of the endocytic pathway. One of these Rab5 exchange factors, Rin1, is itself activated by binding Ras GTP. This Ras GTP mediated activation serves as a link between activated receptor endocytosis and EGF signaling cascade attenuation.

Like Rin1, other members of this protein family contain many signaling motifs in addition to their Rab5 exchange domain but have been implicated in regulating distinct receptor systems. For instance, Rin2 has been linked to regulation of HGF signaling via the c-Met receptor, while Alsin, a protein whose loss leads to presentation of juvenile amyotrophic lateral sclerosis (ALS, or Lou Gherig's disease), has been linked to regulation of IGF-1 receptor trafficking and signaling. By employing genetic, cell biological, biochemical and structural approaches, the Horazdovsky laboratory is addressing the functional role this family of proteins plays in linking endocytic protein trafficking with normal cellular homeostasis and disease.

Investigator: David Katzmann, Ph.D.

The Katzmann Laboratory utilizes genetics and biochemistry to study endosomal function and receptor downregulation in the model organism saccharomyces cerevisiae. These studies have lead to the identification of a group of gene products whose function is required for the proper function of the multivesicular body (MVB) pathway, critical for receptor downregulation.

Mutants in this pathway are referred to as class E vacuolar protein sorting mutants, a group comprised of 16 genes. Class E gene products are involved in protein sorting at the endosome, where they are responsible for recognizing and concentrating cargoes destined for entry into the MVB pathway and eventual degradation within the lysosome/vacuole. Function of the MVB pathway is required for a variety of cellular processes, including lysosome function, receptor downregulation, developmental patterning, immune response and even the budding of certain retroviruses (such as HIV-1) from a host cell (The Journal of Cell Biology. 172:705-17, 2006).

Investigator: Edward Leof, Ph.D.

Dr. Leof is investigating the signaling and trafficking behavior of the TGFβ receptor complex. Previous work from his laboratory has demonstrated that TGFβ receptor endocytosis is not simply a way to dampen the signaling response; instead it is required to propagate signaling via the Smad pathway.

Studies of this sort have direct cancer relevance as the normal progression of most epithelial tumors results in a loss of the growth inhibitory response to TGFβ with little or no change in TGFβ receptor expression. As such, these findings have direct implications to how "signaling impaired" TGFβ pathologies are to be targeted - the defect may not be in signaling, per se, but in appropriate receptor endocytosis and trafficking.

More recently, in a collaborative study with Richard Pagano, Ph.D., Dr. Leof's laboratory has determined that PI3K represents a branchpoint in cell type-specific TGFβ signaling leading to Pak2 or Akt activation (Cancer Research. 65(22):10431-40, 2005). These studies have been extended where it was shown that TGFβ receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures downstream of Pak2 (The Journal of Biological Chemistry. 281(38): 27846-54, 2006). As tumor development and progression is dependent upon the synergistic interplay of cancer cells and an activated stroma, further defining the role of TGFβ in both the "seed and the soil" is essential for effective intervention strategies.

Investigator: Richard Pagano, Ph.D.

Dr. Pagano's Laboratory team studies lipid trafficking in normal and disease cell types. One lipid class of interest is the glycosphingolipids (GSLs) — plasma membrane constituents that play important roles in normal cell adhesion, migration, and proliferation, as well as in pathological conditions such as tumorigenesis and atherosclerosis.

Recently, Dr. Pagano's group demonstrated that caveolar endocytosis, critical for the uptake of some bacterial toxins and viruses, is regulated by syntaxin 6-dependent delivery of membrane components to the cell surface (Nature Cell Biology. 8:317-28, 2006). They have now discovered a non-natural GSL stereoisomer that inhibits caveolar uptake, SV40 infection, and β1-integrin signaling by blocking the clustering of lipids and proteins into GSL- and cholesterol-enriched microdomains at the plasma membrane (Journal of Cell Biology. 176:895-901, 2007). Experiments are currently underway to further study the mechanism of action of this lipid and to learn whether it also modulates cell migration, perhaps as a result of altering integrin signaling.

Investigator: Derek Radisky, Ph.D.

Most investigations of cancer development have pursued how genetic mutations stimulate tumor development, either through activation of oncogenes or loss of tumor suppressor genes. However, there is an increasing awareness that signals from the cellular microenvironment can induce the genetic alterations that underlie tumor formation, can stimulate tumor growth and progression, and can dictate both therapeutic response and ultimate clinical outcome. This principle is particularly clear in breast cancer, Dr. Radisky's primary area of investigation.

His team of investigators uses sophisticated 3-dimensional cell culture models and transgenic animals to define how altered signals from the microenvironment contribute to breakdown of tissue structure, increased cellular proliferation, and transition to the malignant phenotype. Their research largely focuses on defining key signaling pathways in cultured mammary epithelial to determine how alterations in the microenvironment stimulate the malignant phenotype, and also uses transgenic mouse models to define how breakdown of tissue structure through expression of matrix-degrading metalloproteinases causes mammary hyperproliferation, progression to fibrosis, and development of neoplasia, and how inhibition of these processes may provide insight into potential therapeutic avenues.

Investigator: Peter Storz, Ph.D.

Reactive oxygen species profoundly affect numerous cellular functions and inefficient cellular detoxification has been implicated in the onset or progression of several human cancers. Exerting their specific functions, reactive oxygen species activate cellular signaling cascades, which then control responses such as cell detoxification, DNA repair, cell survival and motility. The activation of protein kinase D (PKD)-driven signaling pathways by RhoA and oxidative stress suggests a role for this kinase in endothelial tumor formation and progression. Dr. Storz' research team is investigating this in breast, cervix and lung cancer.

The researchers found that PKD enhances cell proliferation and survival, but inhibits cell migration and invasion. Their working hypothesis is that PKD on one hand is tumorigenic, but on the other hand inhibits metastasis and thus contributes to benign tumor formation. Ongoing projects in the laboratory are to:

• Investigate the regulation of tumor cell motility by RhoA- and ROS-activated PKD signaling pathways, and;
• Determine the role of the PKD-regulated transcription factors NF-κB and FOXO in lung and breast cancer progression.

Dr. Storz' team uses in vitro 3-dimensional cell culture and in vivo orthotopic animal models to answer their questions. They also plan more studies to determine the importance of PKD in the development and progression of epithelial cancers.

Investigator: E. Aubrey Thompson, Ph.D.

Dr. Thompson has a long standing interest in lipid signaling mechanisms and their role in physiology and disease. His research team is working on a number of projects, including studying peroxisome proliferator-activate receptor-gamma and protein kinase C-betaII, which are regulated by dietary lipids, and phospholipase D1 (PLD1), which converts phosphatidyl choline to phosphatidic acid (PA).

Regulation of PLD1 is complex and involves Ras superfamily GTPases and phosphatidylinositol-bis-phosphate. PA likewise signals to a diverse array of pathways and conveys survival signals through mTOR and proliferative signals through MAPK. The investigators have focused upon the role of PLD1 in lung squamous cell carcinoma (LSCC).

About two-thirds of LSCC tumors exhibit PLD1 amplification, and shRNA-mediated knockdown of PLD1 in LSCC cells inhibits invasion in vitro. Dr. Thompson's team is testing the hypothesis that the etiology of LSCC depends upon simultaneous co-amplification of PLD1 and protein kinase C-iota, which is resides immediately adjacent to the PLD1 locus. Their data indicate the PKCiota activates PLD1, likely through a Rho-family GTPase-dependent mechanism. Furthermore, the data suggest that the ability of PKCiota to stimulate invasion of LSCC cells requires PLD1. The investigators are testing the hypothesis that PKCiota activates PLD1 through Rac1 and that coordinate amplification of the PLD1 and PLCiota loci is required for invasion in vitro and promotes metastasis in vivo.