DEREGULATION BY HU PROTEINS IN LUNG CANCER

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Hu proteins are a family of RNA-binding proteins containing three RNA- binding motifs. HuD, HuC and Hel-N1 are expressed in neuronal tissue, while HuR is expressed ubiquitously. The neuronal Hu proteins are also expressed in all small cell lung cancers (SCLCs) and about half of all neuroblastomas. They can bind to the destabilizing sequences of labile proto-oncogene mRNAs, such as those of the family of myc genes. myc genes are frequently oncogenically activated in SCLC and neuroblastoma by events that increase the levels of Myc protein. The proposed natural function of Hu proteins in RNA degradation and neuronal-specific splicing would allow these proteins to interfere with proper mRNA decay and splicing in cancer cells. The specific aims of this project are to test the following hypotheses: 1) Hu protein expression in SCLC plays a role in post-transcriptional gene deregulation by altering the degradation and/or splicing pattern of mRNAs encoding proteins that affect the cell's growth or adhesion properties. 2) Increasing the levels of Hu proteins inside a cell with no or low endogenous levels of these proteins will lead to changes in gene regulation that are similar to some of the abnormalities observed in SCLC. To test our two hypotheses, we propose studies with the following specific aims: 1a) To determine the levels and identities of Hu proteins expressed in lung cancer cell lines, both of the SCLC and non-SCLC type. 1b) To measure the level of expression and half-life of mRNAs of the myc family of proto-oncogenes genes in cells lacking and expressing Hu proteins in order to determine whether a correlation exists between expression of one or more Hu proteins and improper myc mRNA degradation. 1c) To determine whether specific Hu proteins show a binding preference for particular myc mRNA targets, such as the c-, N-, or L-myc untranslated regions in vitro, and to determine whether Hu proteins interact with myc mRNA in vivo. 1d) To analyze the expression and splicing pattern of mRNAs encoding extracellular matrix proteins and growth stimulatory proteins in cell lines of the SCLC and non-SCLC type, in order to determine whether splicing changes are observed in cells expressing one or more Hu proteins. 2a) to express Hu proteins in cells that normally lack them, and 2b) to determine the consequences of Hu expression in these cells as outlined under 1b and 1d above. 2c) To use the cell lines established in 2a to obtain genes that are affected by Hu protein expression. The study of the role of Hu proteins in SCLC offers a unique opportunity to begin to understand the role of RNA- binding proteins in the development and progression of cancer.