Monitoring Signal Transduction in Cancer: Tyrosine Kinase Gene Expression Profiling

Abstract

Abnormal expression of tyrosine kinase (TK) genes is common in tumors, in which it is believed to alter cell growth and response to external stimuli such as growth factors and hormones. Although the etiology and pathogenesis of carcinomas of the thyroid or breast remain unclear, there is evidence that the expression of TK genes, such as receptor tyrosine kinases, or mitogen-activated protein kinases, is dysregulated in these tumors, and that overexpression of particular TK genes due to gene amplification, changes in gene regulation, or structural alterations leads to oncogenic transformation of epithelial cells. We developed a rapid scheme to measure semiquantitatively the expression levels of 50–100 TK genes. Our assay is based on RT-PCR with mixed based primers that anneal to conserved regions in the catalytic domain of TK genes to generate gene-specific fragments. PCR products are then labeled by random priming and hybridized to DNA microarrays carrying known TK gene targets. Inclusion of differently labeled fragments from reference or normal cells allows identification of TK genes that show altered expression levels during malignant transformation or tumor progression. Examples demonstrate how this innovative assay might help to define new markers for tumor progression and potential targets for disease intervention. (J Histochem Cytochem 49:673–674, 2001)

Because the malignant transformation of epithelial cells and progression of carcinomas are accompanied by changes in the expression of receptor and cytosolic tyrosine kinase (TK) genes, we set out to develop an innovative DNA microarray-based assay to simultaneously determine the relative expression level of 50–100 TK genes using just a few cells.

A number of studies have shown that tumor development is accompanied by at least two changes: (a) a change in the way cells interact with their environment via membrane-bound receptors, and (b) a change in how signals originating from these receptors are transduced from the cell membrane to the cytoplasm and the nucleus. Among the hundreds of genes involved in receptor-mediated signal transduction, only a few are aberrantly expressed in tumors. A major motif in signal transduction is the selective phosphorylation and dephosphorylation of tyrosine residues in protein factors involved in signal processing. Proteins that phosphorylate tyrosine residues are products of genes belonging to the family of TK genes. The number of known TK genes has grown steadily in recent years, and the temporal and tissue-specific expression of ∼100 different TK genes in normal cells is carefully orchestrated. The level of expression of certain TK genes is increased in many human tumors (Luttrell et al. 1994; Liu et al. 1995). Simple overexpression of TK genes due to gene amplification or to changes in the regulation of gene expression may lead to oncogenic transformation. This has been clearly documented for the erbB2 protein, the product of the Her-2/neu proto-oncogene and other members of the erb B family (Tse et al. 1997). In addition, many tumors have acquired structurally altered TK proteins or abnormal expression patterns through de novo mutational events. When chromosomes became rearranged, the catalytic domain of a TK gene was found fused to the amino terminal of another protein, thus creating a new transforming activity as well as a new expression pattern. Well-known examples of this mechanism of oncogene activation are the bcr/abl-fusion protein in chronic myeloid leukemia with a translocation, t(9;22), and the activation of the receptor TKs ret and trk in papillary thyroid cancer (Sozzi et al. 1992; Jhiang and Mazzaferri 1994).

Various protein factors can be mis-expressed and, in combination with other events, might constitute one of several factors leading to the onset and/or progression of cancer. Factors including cell cycle-specific enzymes, hormone receptors, and peptide growth factors have been reported as having prognostic significance in some cases of prostate cancer. Overexpression of particular receptor TK genes such as the insulin-like growth factor receptors (IGF-IRs), the epidermal growth factor receptor (EGFR or erb B) family of receptors, focal adhesion kinase (FAK), or the protooncogenes ret and Nyk/mer have been shown to correlate with progression to a more malignant phenotype in a variety of tumors (Resnik et al. 1998), among them carcinoma of the prostate (Ling and Kung 1995; Dawson et al. 1998). Detailed knowledge about TK gene expression and its relation to tumor progression might increase our understanding of how tumors grow and help us design assays to more accurately stage tumors.

Our project targets the development of a novel assay format that enables us to determine the level of expression of many different genes. A rapid assay uses DNA microarrays carrying small amounts of individual TK gene-specific targets to simultaneously determine the expression level of up to 100 TK genes using a small number of cells. We cloned and characterized TK genes expressed in thyroid cancers and in seven different breast cancer cell lines. Using mixed-base oligonucleotides specific for conserved domains in the catalytic domains of TK genes, our PCR assays amplified ∼159–171-bp fragments of expressed TK genes. We size-selected and cloned the PCR products into plasmids. As of August 2000, we had identified more than 50 TK genes expressed in thyroid cancers plus an additional 172 TK fragment clones derived from breast cancer cell lines and ∼250 TK fragment clones from radiation-induced thyroid cancers in the prescreening and sequencing steps. Previously, we finished the construction of a robotic system to print DNA microarrays with about 100 sequences on glass slides (unpublished data). The performance of the system was tested by hybridization of fluorochrome-labeled TK gene-specific PCR fragments onto the TK-specific DNA microarrays. All experiments involving human cells or cell lines were approved by the U.C. Berkeley Human Subject Use Institutional Review Board. These experiments enabled us to optimize hybridization and washing conditions and to generate data regarding the relative level of gene expression. For example, the 184A1 and 184A1TH cell lines are closely related non-tumorigenic and tumorigenic human mammary epithelial cell lines derived from the same normal breast tissue specimen and transformed in vitro. After hybridizing Cy5-labeled TK fragments prepared from the cell line 184A1 and Cy3-labeled TK fragments prepared from cell line 184A1TH to a DNA microarray carrying 58 TK gene fragments and displaying Cy3 fluorescence signals in green and Cy5 signals in red, differences between the cell lines became readily apparent. Genes expressed at a higher level in 184A1TH cells led to increased green signals on the array, while those genes whose expression level is lower in 184A1TH cells compared to 184A1 cells generated spots that exhibited stronger red fluorescence. Similarly, differences between thyroid tumor cell lines could be demonstrated in simple dual-color hybridization experiments.