Glioma invasion into the CNS involves the interaction of tumor cells with the host's cells and extracellular matrix (ECM) molecules. In this study, the expression of ECM-associated and cell-associated proteins such as the transmembrane CD44 adhesion molecule and neuro-glial proteoglycan 2 (NG2), a member of the chondroitin sulfate proteoglycan family, were evaluated during glioma progression, in vitro and in vivo, using a model of a highly invasive and aggressive intracerebral mouse G-26 glioma. We found a marked increase in CD44 and NG2 expression in brain tissue containing glioma. The glioma levels of these proteins gradually increased over time to reach 3-15 times the levels in the contralateral control. NG2 and CD44 expression paralleled progression of the glioma, being higher on days 14 and 21 than on day 2 post-glioma implant. In addition, when invading glioma crossed the midline in the advanced tumor stage, levels of each of these proteins in the contralateral tissue were elevated, but were still significantly lower than in the ipsilateral, tumor-bearing hemisphere. Immunohistochemistry of advanced stage G-26 glioma (day 21) showed CD44 expression to be most prominent at the front of the glioma invasion line, sharply separated from normal brain parenchyma which expressed glial fibrillary acidic protein (GFAP). However, single CD44 positive cells that escaped the tumor mass penetrated between the astrocytes that encased the tumor at its periphery. In contrast, NG2 was expressed on nearly all glioma cells within the tumor mass but less so at the leading edge of the tumor. The NG2 positive cells were clearly demarcated and morphologically distinguishable from GFAP positive cells and only sporadic, small groups of NG2 positive cells were seen in the GFAP positive zone of the neuropil. Taken together, these data show that during glioma progression in the brain, the level and pattern of glioma-associated molecules such as CD44 and NG2 may aid in tracing and targeting the invading glioma cells.

Activation of the extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in mediating a diverse array of cellular functions including cell differentiation, proliferation, and inflammatory responses. In this review, we will discuss approaches to identify inhibitors of ERK proteins through targeting ATP-dependent and ATP-independent mechanisms. Given the diversity of ERK substrates and the importance of ERK signaling in normal cell functions, emphasis will be placed on the methods for identifying small molecular weight compounds that are substrate selective through ATP-independent interactions and potentially relevant to inflammatory processes. The approach for selective targeting of ERK substrates takes advantage of the basic understanding of unique ERK docking domains that are thought to interact with specific amino acid sequences on substrate proteins. Computer aided drug design (CADD) can facilitate the high throughput screening of millions of compounds with the potential for selective interactions with ERK docking domains and disruption of substrate interactions. As such, the CADD approach significantly reduces the number of compounds that will be evaluated in subsequent biological assays and greatly increases the hit rate of biologically active compounds. The potentially active compounds are evaluated for ERK protein binding using spectroscopic and structural biology methods. Compounds that show ERK interactions are then tested for their ability to inhibit substrate interactions and phosphorylation as well as ERK-dependent functions in whole organism or cell-based assays. Finally, the relevance of substrate-selective ERK inhibitors in the context of inflammatory disease will be discussed.

Serum embryonic neural cell adhesion molecule (eNCAM) levels were measured at diagnosis in 92 patients with plasma cell disorders. Significantly elevated levels of serum eNCAM were detected in patients with multiple myeloma when compared to both normal controls and patients with monoclonal gammopathy of uncertain significance (MGUS). Very high levels of serum eNCAM were seen in patients with high tumour burdens. There was a significant correlation between serum eNCAM and beta 2-microglobulin (beta 2m) (r = 0.33; P = 0.002), but not between serum eNCAM and C-reactive protein or serum albumen. There was a trend towards longer survival for patients with low serum NCAM. The median survival of the low serum eNCAM group (eNCAM < 20 U/ml) was 36 months compared to 16 months for the high serum eNCAM group (log rank test chi 2 = 2.42, P = 0.1). Serum eNCAM is a new marker of tumour mass in multiple myeloma and correlates with clinical stage and beta 2m. Patients with low serum eNCAM levels may have a survival advantage. Serum eNCAM warrants further evaluation as a tumour marker and prognostic factor in multiple myeloma.

The ERK proteins are potent mediators of cell proliferation and survival. Unregulated activation of the ERK proteins plays a role in the progression of a variety of cancers. Thus, targeted inhibition of ERK's function in promoting cell proliferation and survival is viewed as a promising approach for anti‐cancer therapy. Using in silico modeling, we have recently developed small molecular weight compounds that target ERK domains involved in specific substrate interactions. Compounds designed to target the common docking (CD) domain on ERK2 were tested for their ability to block anti‐apoptotic mechanisms involving ERK phosphorylation of p90RSK‐1, which promotes cell survival by phosphorylating and inactivating the pro‐apoptotic protein BAD. HeLa cells treated with 50 μM of one compound (termed 76) caused induction of apoptotic signaling pathways as measured by cleavage of poly (ADP‐ribose) polymerase (PARP), which was observed within 3 hours. PARP cleavage induced by 76 required activation of the intrinsic apoptotic pathway, specifically caspase 9 and caspase 3. Compound 76 inhibited p90RSK‐1 phosphorylation of BAD in a manner that correlates with caspase activation. Furthermore, inhibition of BAD phosphorylation in the presence of compound 76 was not due to off‐target effects on other pro‐survival pathways. These findings suggest that substrate selective inhibition of ERK signaling promotes apoptosis in transformed cells, illustrating a novel approach to the development of anti‐cancer therapies. (Supported by NIH CA120215)