Identification of Metastasis-related Genes in a Mouse Model Using a Library of Randomized Ribozymes

Abstract

Libraries of randomized ribozymes have considerable potential as tools for the identification of functional genes critically involved in a biological phenotype of interest in vitro.We have used a ribozyme library in an in vivo mouse model to identify genes related to metastasis. We injected weakly metastatic melanoma cells that had been treated with the library intravenously into mice. We then isolated ribozymes that accelerated metastasis from pulmonary tumors that had developed from metastasizing cells. As candidates for metastasisrelated genes that were targets of the isolated ribozymes, we identified five unknown and three known genes: stromal interaction molecule 1 (STIM1), polymerase ␥2 accessory subunit (Polg2), and cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22). Repression of four of these by small interfering RNAs indeed resulted in the accelerated mobility of cells in in vitro scratch-wound assay. The further characterization of these candidate genes would provide clues to the complex mechanism(s) of metastasis.Hammerhead ribozymes (referred to herein after as ribozymes) that specifically cleave the transcripts of particular genes can be produced when the nucleotide sequences of the substrate-recognition arms are designed such that they are complementary to the sequences of individual target mRNAs. Such ribozymes have been used successfully to interfere with the expression of specific genes via cleavage of the respective target mRNAs in mammalian cells (1-6). The use of a library of ribozymes with randomized substrate-recognition arms allowed the identification of genes that are involved directly in certain phenomena, such as the transformation of NIH3T3 fibroblasts, anchorage-independent cell growth, tumor necrosis factor-␣-induced apoptosis and tumor invasion (7-15).The metastatic spread of tumor cells around the body is a key target for cancer therapy (16,17). An experimental assay of metastasis in vivo that involves intravenous injection of tumor cells into mice was developed by Fidler (18) for the analysis of the metastatic properties of melanoma cells (Fig. 1). In this assay, cells of interest are injected into the tail vein of mice and allowed to proliferate and/or metastasize. After several weeks, pulmonary metastases are observed in the case of strongly metastatic melanoma cells, while relatively few metastases are formed after injection of cells with limited metastatic potential. This assay allows the selection of strongly metastatic cells from a heterogeneous population of weakly metastatic tumor cells (18,19). Thus, the assay allows not only the study of the metastatic characteristics of melanoma cells but also the isolation of strongly metastatic cells. In the present study, we used this mouse model and mouse B16F0 melanoma cells, a weakly metastatic melanoma cell line, to investigate the applicability of a library of ribozymes in vivo, to isolate ribozymes that converted weakly metastatic melanoma cells to strongly metastatic cells by the apparent disruption of expressi...

Novel Protein Kinase C-Mediated Control of Orai1 Function in Invasive Melanoma

“…Considered collectively, these studies show that Ca 2ϩ entry supports the migration and invasion by melanoma cells. However, in 2004, an unbiased screen for metastasis-related genes in an invasive melanoma model revealed STIM1 as a repressor of metastasis (16). This apparent inconsistency with current thinking is supported by the surprising findings of the current study, in which we examined the expression and function of STIM1 and Orai1 in a series of melanoma cell lines exhibiting marked differences in Wnt5A expression with corresponding differences in invasive character (17,18).…”

Ganglioside GD1a regulation of caveolin-1 and Stim1 expression in mouse FBJ cells:Augmented expression of caveolin-1 and Stim1 in cells with increased GD1a content

“…Using rhabdomyosarcoma cells in vitro and rhabdoid tumors in vivo, Stim1 was shown to inhibit the growth of tumor cells [32] and be expressed in many human primary and transformed cell lines [30]. The selective suppression of stim1 mRNA brought about rhabdomiasarcoma tumorigenesis [32] and with the application of randomized ribozymes to metastasizing cells, Stim1 mRNA broke down to promote the pulmonary metastasis of B16F0 cells [33]. Stim1 and Stim2 have recently been shown essential to Ca 2+ -store-depletion-mediated Ca 2+ -influx or storeoperated Ca 2+ (SOC) influx, based on data obtained using siRNA techniques [34,35].…”

Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis

“…STIM1 mRNA is widely expressed in different human tissues, and strongly presented in lymphoid and myeloid cells (9). STIM1 knockdown accelerates the cell motility of melanoma cells and is defined as an antimetastasis gene (12). However, Yang et al (13) demonstrated that STIM1 or Orai1 silencing inhibits the migration and metastasis of breast cancer cells by suppressing focal adhesion turnover.…”