The long term goals of this project are to identify the mechanisms by which cancer cells become growth factor-independent. We hope to identify novel proteins that act as tumor suppressors or oncoproteins by regulating nutrient transporter internalization and endocytic trafficking. Proteins that regulate this process may represent potential targets for cancer chemotherapy.
Specific Aim 1 : Determine which stages of nutrient transporter internalization and trafficking are affected by PKCd induction. Hypothesis: PKCd induction following growth factor withdrawal increases nutrient transporter endocytosis, decreases recycling, and increases ubiquitination. Rates of nutrient transporter internalization and recycling will be measured in the presence or absence of growth factor using antibodies specific for nutrient transporters by flow cytometry. The role of PKCd will be assessed using cells expressing shRNA, a dominant negative, and a chemical inhibitor of PKCd (rottlerin). Ubiquitination will be assessed by immunoprecipitation of the transporters followed by Western blot for ubiquitin.
Specific Aim 2 : Identify the PKCd substrates responsible for growth factor induced nutrient transporter down-regulation. Hypothesis: PKCd induction following growth factor withdrawal leads to phosphorylation of a novel PKCd substrate responsible for nutrient transporter down-regulation. Candidate substrates identified by Scansite as potential PKCd substrates will be analyzed for PKCd phosphorylation by: 1) orthophosphate labeling, 2) in vitro kinase assays and 3) 2-D electrophoresis. Results from Aim 1 will help to determine which substrates we will focus on. We will also use a biochemical approach to identify novel PKCd substrates, scaffolding proteins and binding partners. These studies seek to identify new chemotherapeutic targets that would be useful in treating numerous types of cancer. Our approach to cancer control is novel: by understanding how cancer cells grow while normal cells lose asses to nutrients we may learn how to kill cancer cell by starving them to death. Blocking individual pathways may kill cancer cells while affecting normal cells that have back-up pathways for nutrient uptake. Many types of cancer should be sensitive to this type of treatment because all cancer cells need nutrients to survive. ? ? ?
| Romero Rosales, Kimberly; Singh, Gurpreet; Wu, Kevin et al. (2011) Sphingolipid-based drugs selectively kill cancer cells by down-regulating nutrient transporter proteins. Biochem J 439:299-311 |
| Siskind, Leah J; Mullen, Thomas D; Romero Rosales, Kimberly et al. (2010) The BCL-2 protein BAK is required for long-chain ceramide generation during apoptosis. J Biol Chem 285:11818-26 |
| Romero Rosales, Kimberly; Peralta, Eigen R; Guenther, Garret G et al. (2009) Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis. Mol Biol Cell 20:2831-40 |
| Guenther, Garret G; Peralta, Eigen R; Rosales, Kimberly Romero et al. (2008) Ceramide starves cells to death by downregulating nutrient transporter proteins. Proc Natl Acad Sci U S A 105:17402-7 |
