The protein Grb2 plays a central role in signalling by receptor protein-tyrosine kinases, where its SH2 domain binds to the receptor and its two SH3 domains link to effectors. One target effector is Sos, so Grb2 links receptor protein-tyrosine kinases with the Ras signalling pathway. The SH3 domains can also couple to other signalling proteins, including Vav, c-Abl and dynamin. We have identified several bands in glial and medulloblastoma tumours that are recognized by Grb2 but these did not correspond to any known protein. Here we use recombinant Grb2 to isolate a complementary DNA called Gab1 (for Grb2-associated binder-1). Gab1 shares amino-acid homology and several structural features with IRS-1 (insulin-receptor substrate-1; refs 6,7), is a substrate of the EGF and insulin receptors, and can act as a docking protein for several SH2-containing proteins. Over-expression of Gab1 enhances cell growth and results in transformation. We conclude that Gab1 is a new protein in EGF and insulin receptor signalling which could integrate signals from different systems.

The epidermal growth factor receptor (EGFR) gene is amplified in 40% of lignant gliomas, and the amplified genes are frequently rearranged. We have characterized the genetic alterations asoated with these rearrangements in five malignant gliomas. In one tumor the rearrangement resulted in the deletion of most of the extracytoplasmic domain of the receptor, resulting in a hybrid mRNA between new sequences and the truncated EGFR sequence. The predicted amino acid sequence of the protein from this tumor was remarkably similar to that described for several viral erbB oncogenes. Four other tumors were noted to have internal deletions of the EGFR gene. These rearrangements brought about in-frame deletions affecting either of two cysteine-rich domains in the extracytoplasmic portion of the molecule. The clonal nature of these alterations, and the fact that Identical alterations were seen in more than one tumor, suggests a role for these mutant receptor proteins in tumorigenesis. Further, these studies document the existence of tumor-specific cell surface molecules resulting from somatic mutation.

Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.In vitro experiments have shown that greatly increased expression of some protooncogenes can lead to neoplastic transformation (1-3). In naturally occurring tumors, increases in gene expression have been postulated to occur through two kinds of mechanism (4-6). One class of mechanism involves structural changes within or surrounding the expressed gene, either through DNA amplification (7,8) or rearrangement (9). The other class of mechanism includes changes in DNA-binding proteins (10) or DNA methylation (11) in the absence of structural alterations of the expressed gene. In several human tumors, increased expression of protooncogenes apparently takes place in the absence of genetic changes at the protooncogene locus (see, e.g., refs. 12-15) and these increases have been suggested to play an active role in tumor formation. The presence of genomic alterations of a protooncogene in a tumor provides strong evidence for involvement of the protooncogene in formation of the tumor. However, in the absence of such structural alterations, it is difficult to know whether increased expression of a protooncogene is causally related to the tumorigenic process or simply reflects the abnormal growth status or unusual microenvironment present in tumors.The epidermal growth factor receptor (EGFR) is a protooncogene that has been extensively studied (reviewed in ref. purified from frozen blocks of tissue, 1.5-to 4-ptg samples were cleaved with EcoRI, separated by electrophoresis through a 1% agarose gel, and blotted on nitrocellulose. Prehybridization, hybridization, and washing conditions were as described (19). The EGFR probe used was the 1.6-kilobase EcoRI fragment of pE7 (21), a cDNA clone of EGFR mRNA generously provided by G. Merlino and I. Pastan (National Institutes of Health). Filters were rehybridized sequentially with three other probes: a 1.0-kb EcoRI/ BamHI fragment of pNB-1 (22), containing part of the second exon of the N-myc (human, NMYC) gene; a 1.6-kb Sst I fragment of pHSR-1, containing the second exon of c-myc (human, MYC) (23); a 1.55-kb Pst I insert of pKK36P1, containing gli sequences from chromosome 12 (24); and a 5.0-kb EcoRI insert of pAW101, containing sequences from chromosome 14 (25).In Situ Hybridization. Tissue sections of 6 ,um thickness were cut from paraffin...

A gene, termed gli, was identified that is amplified more than 50-fold in a malignant glioma. The gene is expressed at high levels in the original tumor and its derived cell line and is located at chromosome 12 position (q13 to q14.3). The gli gene is a member of a select group of cellular genes that are genetically altered in primary human tumors.

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CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate–responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG selfrenewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.Hedgehog pathway | cancer stem cells | brainstem glioma

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