David A. Foster - US grants

The objective of the proposed research is to understand how protein- tyrosine kinase oncongenes transform cells. The major emphasis is on the fps gene of Fujinami sarcoma virus. We will characterize signal transduction pathways used by v-fps to transform cells. We have developed a pharmaco-genetic approach for identifying signal transduction intermediates used by the v-fps gene product to transduce signals to the nucleus. Elevating the protein-tyrosine kinase activity of a temperature-sensitive derivative of v-fps, leads to the rapid transcriptional activation of the recently characterized 9E3 gene whose expression correlates with transformation (Sugano et al., Cell 49, 1321,-328, 1987). Drugs that interfere with known signal transduction intermediates are used to block the induction of 9E3 gene expression. In this way, we can determine if specific signal transduction intermediates are required for v-fps to induce expression of the transformation-related 9E3 gene. The pharmacological approach involves the generation of detailed drug- sensitivity profiles (DSPs) for inhibitors of signal transduction that are diagnostic for the involvement of specific signal transduction intermediates. Using several protein kinase C requirement. In addition, we have preliminary data suggesting a requirement for protein kinase C, a cholera toxin-sensitive G-protein, phospholipase C, and phospholipase A2 in the v-fps induction of 9E3 gene expression. Experiments proposed here will directly demonstrate the involvement of these implicated signal transduction intermediates in transformation by v-fps. The data to be generated from these studies may provide new targets and strategies for cancer chemotherapy in cases where protein-tyrosine kinase activity has been implicated.

The objective of this proposal is to understand how phospholipid metabolism, activated by the oncogenic protein-tyrosine kinase (PTK) v- Src, contributes to the complex set of intracellular signals initiated by v-Src that ultimately lead to transformation. The mechanisms by which v-Src and other PTKs generate the complex intracellular signals that frequently lead to cell proliferation is not well understood. To generate the signals necessary to induce a process as cell proliferation is not well understood. To generate the signals necessary to induce a process as complex as cell proliferation, many intracellular signalling molecules ar likely recruited by PTKs. In recent years it has become increasingly apparent that the complexity of the lipid components in membranes far exceeds that required for function as a biological barrier. Phospholipids, which comprise the majority of membrane lipids, can be metabolized to variety of biologically active molecules by many distinct enzymatic activities. In this proposal experiments are described that will follow up on our observation that v-Src-induced increases in diglycerides results not from the more established mechanism of phospholipase C-mediated hydrolysis of phosphoinositides, but rather by a phospholipase D-mediated hydrolysis of phosphatidylcholine (Song et al., 1991). In this proposal, experiments are described that will 1) characterize the mechanism by which v-Src activities phospholipid metabolism and 2) characterize the phospholipid metabolites including diglycerides, monoglycerides, phosphatidic acid and possibly lyso- phosphatidic acid generated in response to v-Src. Malignant transformation involves a progressive loss of control of intracellular signalling mechanisms. The many enzymes involved in the complex signals generated by metabolism of membrane lipids provides many potential targets for interfering with the intracellular signals that may contribute to malignant transformation. The studies proposed here will identify potential targets for interfering with the PTK-initiated intracellular signals that contribute to transformation.

Phospholipase D Activation by v-Src and v-Ras: In response to the oncogenic stimuli of v-Src, there is an activation of phospholipase D (PLD) that is dependent upon a GTPase cascade of Ras and Ral. Transformation by v-Src is also dependent upon both Ras and Ral, suggesting a role for PLD in cell transformation. PLD hydrolyzes phosphatidylcholine to phosphatidic acid (PA) and choline. The best understood effects of PA are mediated by the PA metabolite diacylglycerol, which leads to the activation of protein kinase C. However, PA is also biologically active and has been implicated in regulating a variety of signaling molecules including Raf-l, phosphatidylinositol kinases, and the GTPase activating proteins (GAPs) for Ras, Rac and Arf. PLD activity and PA have also been implicated in vesicle transport. Although RalA is required for v-Src-induced PLD activity and PLD exists in a complex with Ral, an activated RalA is not sufficient for PLD activation. Biochemical and genetic evidence suggest at least two factors in addition to RalA are required for PLD activation by v-Src. The objective of the proposed studies is to characterize the interaction between RalA and PLD and to identify factors contributing to the activation of PLD via the newly emerging Ras/Ral signaling pathway. Candidate proteins implicated in the activation of PLD by v-Src are the Rho family GTPases Rho, Rac, Cdc42 and the RalA binding protein Ral-BP1, which is a GAP protein for Rho family GTPases. Arf (ADP ribosylation factor), which activates the PLD associated with RalA is another candidate. Proposed studies will test whether these and other implicated factors contribute to activation of PLD by v-Src. Because of the many extracellular stimuli that activate PLD via tyrosine kinases and the many intracellular responses to the PLD-generated PA, understanding of the mechanism of PLD activation by tyrosine kinases will provide several new targets for therapeutic intervention in diseases such as human breast cancer where altered regulation of tyrosine kinase activity has been implicated.

This proposal seeks to understand the role that the RalA/phospholipase D (PLD) signaling pathway plays in mitogenic signaling. The applicant's laboratory has demonstrated that PLD activity is elevated in cells treated with a variety of mitogenic stimuli, and that this pathway is a critical mediator of transformation by v-Src, v-Raf, v-Ras, and overexpression of the EGF receptor. In normal quiescent cells, PLD is complexed with GDP-bound RalA and localized to cellular membranes. The enzyme becomes activated upon RalA GTP exchange and recruitment of Arf GTP to the complex, both events thought to be brought about by activation of Ras. However, Arf GTP does not bind the RalA/PLD complex directly, and elevated PLD activity in membranes of Ras-transformed cells requires the addition of cytosol. These findings suggest that an additional factor is needed for complete activation of PLD. PLD hydrolyzes phosphatidylcholine to phosphatidic acid (PA). PA is involved in many biological processes, including intracellular vesicle formation and activation of signaling molecules, but its exact role in promoting mitogenic signaling is not understood. Preliminary evidence from the applicant's lab suggests that PLD may play a role in EGF receptor endocytosis. Two aims are proposed to further investigate both the mechanism of PLD activation and its putative role in endocytosis. The first aim is to purify and characterize a recently discovered low molecular weight PLD-stimulating factor (PLD-SF) that is elevated in transformed and dividing cells. The second aim will test the hypothesis that PLD regulates intracellular signaling by facilitating receptor-mediated endocytosis to generate "signaling vesicles."

DESCRIPTION (provided by applicant): Protein kinase C delta (PKC d) negatively regulates cell cycle progression and has been proposed to be a tumor suppresser gene. Consistent with this hypothesis, the PKC d gene localizes to a region on chromosome 3p where several tumor suppresser genes are thought to reside. While a role for PKC d as a negative regulator of proliferation has been established, little is known as to how PKC d exerts this effect, nor whether PKC d function is suppressed or lost in human cancer. The major objective for this proposal is to determine how PKC d impacts upon cell proliferation and survival in human breast cancer cells. Preliminary studies with breast cancer cell lines indicate non-random differences in the level of PKC d expression in different breast cancer cell lines with different cancerous phenotypes. We propose that tumor-suppressing effects of PKC d can be exploited to negatively regulate cell proliferation and induce apoptosis in human breast cancer cells. Specifically, we propose to: Aim 1: To characterize PKC d expression in breast cancer cell lines with different genetic defects. We will determine whether expression of PKC d correlates with specific genetic alterations such as p53 status, loss of estrogen receptor, or tyrosine kinase expression. Aim 2: To characterize the impact of PKC d activity upon cell cycle progression and apoptosis in breast cancer cells. Aim 3: To determine whether p53 expression can be enhanced by elevated expression of PKC d or by PKC d agonists such as bryostatin1 and bistratene A. Aim 4: To characterize the role that PKC d plays in suppressing metastatic phenotypes. We will examine the effect of PKC d upon cell migration, invasion and protease secretion. The studies proposed here will characterize a potentially important indicator of tumor status--that being the expression of PKC d in breast cancer cells with different genetic backgrounds. Based on preliminary studies that have revealed a PKC d requirement for p53 expression, it is proposed that inhibiting PKC d would have tumor-promoting effects by preventing the expression of p53. And more importantly, activating PKC d with compounds like bryostatin1 could have tumor suppressing effects that could be exploited therapeutically.

[unreadable] DESCRIPTION (provided by applicant): Elevated phospholipase D (PLD) activity has been reported in several types of human cancer including breast, kidney, colon and gastric cancer. Recent work has revealed that elevated PLD activity in human breast cancer cells can suppress apoptosis and promote cell migration - two critical steps in progression to a malignant cancer. The survival and migration signals generated by PLD are mediated - at least in part - by mTOR (the mammalian target of rapamycin), which has been widely implicated in cancer survival signals. While it is clear that PLD is capable of contributing to tumorigenesis and that PLD activity is elevated in a large number of human cancers, it is not known how and in what context elevated PLD activity contributes to the transformation of human cells or tumorigenesis in an animal. There is also much to be learned about the signaling pathways that activate PLD in human cancer cells. The Central Hypothesis of the proposal is that: PLD generates signals that suppress apoptosis and enhance cell migration in human cancer cells. Specifically, we propose to: 1) Characterize signals regulating PLD activity in human cancer cell lines and to evaluate targeting these signals pharmacologically both in vitro and in vivo; 2) Investigate a role for PLD survival signals in cell migration and metastasis; and 3) Evaluate the ability of PLD to cooperate with oncogenes to transform human cells in culture and to stimulate cell migration. The ability of PLD to both suppress apoptosis and enhance cell migration makes PLD an ideal target for the development of therapeutic strategies. As the era of molecular medicine and pathology evolves and individual tumors are examined at the molecular level, elevated PLD activity could be easily determined and the signals generated by PLD activity could then be targeted specifically. The studies proposed here will provide a conceptual framework for rational targeting of the apparent large number of human cancers with elevated PLD activity. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Default apoptotic programs have been adapted as a first line of defense against cancer, and virtually all cancer cells have mutations that activate survival signals in order to suppress apoptosis. A central node in survival signaling is mTOR (the mammalian target of rapamycin). mTOR is activated in response to signals mediated by the phosphatidylinositol-3-kinase (PI3K) signaling pathway. However, more recently it has become apparent that mTOR is also targeted by signals that activate phospholipase D (PLD). PLD generates phosphatidic acid (PA), a lipid second messenger that interacts directly with mTOR in a manner that is competitive with rapamycin - and PA is required for the activation of mTOR. Importantly, PLD activity is elevated in several types of human cancer. PLD activity is elevated in many human cancer cells and is required for mTOR-mediated signals that are critical for survival and promote cell cycle progression. The CENTRAL HYPOTHESIS of the proposal is - Elevated PLD activity in human cancer cells promotes passage through a late G1 Cell Growth Checkpoint and suppresses default apoptotic programs. We are proposing that virtually all cancer cells must activate signals that allow passage through this checkpoint. SPECIFICALLY, we propose: 1) To determine how PLD-mTOR signaling impacts on cell cycle progression through a proposed Cell Growth Checkpoint; 2) To determine the mechanism by which PLD-generated PA regulates mTORC1 and mTORC2 in concert with other signaling inputs; and 3) To characterize signals that lead to elevated PLD activity in human cancer cell lines and to evaluate targeting these signals pharmacologically both in vitro and in vivo. We are proposing that a PLD-mTOR signaling pathway in human cancer cells represents a widely employed strategy by cancer cells to promote cell cycle progression and suppress default apoptotic programs. The studies proposed here will provide a framework for the rational targeting of an apparent large number of cancers that depend upon elevated PLD activity for G1 cell cycle progression and suppression of apoptosis.

DESCRIPTION (provided by applicant): Dysregulated Metabolic Cell Cycle Checkpoints in Human Cancer The decision by a cell to divide or enter quiescence is made in early G1 after mitosis. Cells need instructions from growth factors to continue cycling through G1 into S-phase. In the absence of appropriate growth factor signals, cells enter a resting quiescent state referred to as G0. The site in G1 where growth factor signals are required has been mapped to a site early in G1. This site is commonly referred to as the Restriction Point (R). However, many reports describe R as a site much later in G1 where the cell makes a final commitment to replicate its DNA and divide. This site resembles a site in the yeast cell cycle known as START. However, START is dependent upon nutrient sufficiency rather than growth factors. We are proposing a set of metabolic checkpoints late in G1 that are dependent on nutrient input prior to committing to replicate the genome and dividing. This late G1 cell cycle control site is likely integrated into signals mediated by mTOR - the mammalian target of rapamycin, which senses nutrient and energy sufficiency. We hypothesize that these late G1 cell cycle control sites collectively represent a Cell Growth checkpoint where levels of essential nutrients are evaluated prior to committing to doubling in mass and replicating the genome. It is proposed that these metabolic checkpoints, along with R, need to be dysregulated in virtually all human cancers and that complementary genetic changes in human cancer cells cooperate to overcome both R and the Cell Growth checkpoints. This study addresses long-running misconceptions about of G1 cell cycle progression and investigates the feasibility of therapeutic exploitation of dysregulated cell cycle checkpoints in human cancers. The Central Hypothesis of the proposal is that there are Metabolic Cell Cycle Checkpoints late in G1 that can be distinguished from the growth factor-dependent Restriction Point (R). Thus, cells decide first whether they should divide at R, and then prior to replicating the genome, they decide whether they are capable of dividing at a set of Metabolic Cell Growth checkpoints that monitor nutrient sufficiency. It is proposed that mTOR is the final arbiter of whether to commit to replicating the genome and dividing. A series of experiments are proposed that will: 1) Distinguish the impact of growth factors, nutrients and mTOR on G1 cell cycle progression - most significantly, a newly identified lipid-sensitive G1 checkpoint; 2) Distinguish requirements of cells for G1 cell cycle progression when originating from mitosis or from quiescence; and 3) Characterize cell cycle checkpoint(s) mediated by glutamine and evaluate the feasibility of therapeutically exploiting dysregulated checkpoints in human cancer cell lines. Many of the signals that promote progression through late G1 of the cell cycle are commonly referred to as survival signals because they suppress apoptotic programs that kick in if the cell is not capable of replicating the genome and dividing. These signals are ideal targets for therapeutic intervention because, in principle, suppression of survival signals leads to either apoptosis or senescence. This study will investigate a proposed set of Metabolic checkpoints in late G1 that are overcome by survival signals in virtually all cancer cells. Therefore, the study will have relevance - and impact - for a large percentage of human cancers because of the need to dysregulate the control of progression through late G1 and avoid the cell death and senescence programs that prevent cancer.