Control of glioma cell migration by IIp45 and HDAC6Background: Cell migration is a critical process for tissue remodeling during development, wound healing, and tumor invasion and metastasis. Compared with other types of human tumor, rapid spreading and highly invasive phenotype are the signatures for malignant brain tumor. The major type of human brain tumor is glioma, a type of primary central nervous system (CNS) tumor that arises from glial cells. Gliomas commonly affect middle-aged and older adults with -18,000 new patients diagnosed annually and -13,000 annual deaths in the United States alone (CBTRUS 2004-2005). Most of all primary brain tumor cases were unfortunately diagnosed as glioblastoma multiforme (GBM), the most advanced glioma with a median survival of less than a year (Behin 2003; Maher 2001; Ohgaki and Kleihues 2005). Apart from poor survival, this type of cancer also causes a variety of neurological disorders secondary to the unique anatomic origin within the central nervous system, which invariably leads to major issues of quality of life for both the patients and their families. Therefore there is no doubt that research for a better understanding of possible mechanisms behind glioma cell migration and invasion is needed to improve this situation.
Previous studies: Our group has a long standing interest in dissecting the molecular basis of glioma migration and invasion in order to find an effective way to intervene. Towards this goal, we first identified insulin-like growth factor binding protein 2 (IGFBP2) as a key gene that is elevated in high-grade gliomas and promotes glioma cell migration and invasion (Wang 2003, Wang 2006). IGFBP2 activates the expression of matrix metalloprotease 2 (MMP2) in glioma cells, which contributes to the increased invasion. We recently reported that IGFBP2 is an oncogene for glioma development and progression using a glial-specific mouse model (RCAS-nestin-tva, Dunlap, PNAS 2007). A few years ago, we discovered a novel gene we termed Invasion Inhibitory Protein with molecular weight 45kDa (IIp45). IIp45 encodes a protein that interacts with insulin-like growth factor binding protein 2 (IGFBP2) and inhibits migration and invasion of glioma cells (Song, PNAS, 2003). Loss of IIp45 expression was found in highly invasive GBM. In vitro knockdown of IIp45 expression by siRNA led to increased cell migration and invasion. The IIp45 gene is located on chromosome lp36, a region frequently deleted in human malignancies including brain tumors. Thus, IIp45 is likely an important tumor suppressor gene that is attenuated in glioma development. In order to gain insight into the mechanism of IIp45-mediated cell motility and invasion regulation, we carried out a number of preliminary studies to identify other binding partners for IIp45. Through yeast two-hybrid screening, pull-down, and co-immunoprecipitation assays, we discovered that IIp45 protein interacts with histone deacetylase 6 (HDAC6).
HDAC6 belongs to a subtype of the HDAC family, a class II histone deacetylase specifically for deacetylation of alpha tubulin, the most abundant microtubule component, rather than histone (Hubbert, 2002). HDAC6 also deacetylate cortactin (Zhang, 2007), a protein that promotes polymerization and rearrangement of the actin cytoskeleton and is important in promoting lamellipodia formation, invadopodia formation, and cell migration. A number of studies have shown that HDAC6 regulates microtubule dynamics/cytoskeleton structure and increases cell migration by reduction of alpha tubulin and cortactin acetylation (Hubbert, 2002; Zhang, 2007). Thus, we believe that we have identified a key mechanism through which IIp45 regulates cell migration. Because microtubule/cytoskeleton is critical for protein transport, we also hypothesize that IIp45 regulates MMP2 protein secretion through microtubules/cytoskeleton thus regulates invasion through this mechanism in addition to inhibition of IGFBP2.
Hypothesis: IIp45 inhibits cell migration and invasion by interacting with HDAC6 and modulating HDAC6-regulated microtubule/cytoskeleton acetylation and structure, which are important for cell migration and MMP2 protein transport/secretion.
Specific aim 1. To characterize the relationship between IIp45, HDAC6, and acetylated alpha tubulin and cortactin in glioma tissues which have different migratory and invasive ability (low grade vs. high grade). We will examine the protein expression patterns and their relationship between IIp45 and HDAC6 in glioma tissues using western blotting assay. We will further confirm that IIp45 and HDAC6 protein indeed interact in primary glioma tissues. We will determine the relationship between IIp45 and acetylated alpha tubulin and cortactin expression in glioma tissues. This aim will provide key information from clinical samples whether our proposed mechanism is physiologically relevant.
Specific aim 2. To functionally determine the relationship between IIp45 and HDAC6 expression in regulation of microtubule dynamics, cell migration, and MMP2 protein transport/secretion in glioma. We will use gain-of-function and loss-of-function approach to either increase or decrease IIp45 expression and determine the effect on the level and distribution of acetylated alpha tubulin and cortactin in glioma cells and MMP2 secretion to the conditioned media. We will modulate HDAC6 levels in glioma cells through either a molecular (siRNA) or pharmacologic approach (inhibitor Tubacin) and examine the effect on the levels of acetylated alpha tubulin and cortactin and their distribution in the cells, MMP2 secretion to the conditioned media, and cell migration and invasion. This aim will directly determine whether a key target of IIp45 function in cell migration and invasion is HDAC6-mediated microtubule regulation.
Specific aim 3. To delineate the domains on IIp45 and HDAC6 that interacts with each other. We will generate deletion constructs of IIp45 and HDAC6 and carry out protein binding assays to delineate the domains on IIp45 that interacts with HDAC6 and vice versa. Results from this aim will provide information for development of peptide-based HDAC6 inhibitors for therapeutic purpose in the future.
Novelty and Impact: Our previous studies identified a novel gene, IIp45, that inhibit glioma cell migration and invasion. Subsequent studies have unraveled a novel mechanism (via targeting HDAC6) through which IIp45 may regulate these two important processes. Characterization of this proposed mechanism will contribute to the fundamental knowledge of an important process that is critical for glioma pathobiology. This study will also allow us to begin to explore whether targeting HDAC6 is a viable approach for development of therapeutic intervention for glioma.
