Faculty Bibliography

Targeted therapies that inhibit receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol 3-kinase (PI3K) signaling pathway have shown promising anticancer activity, but their efficacy in the brain tumor glioblastoma multiforme (GBM) and other solid tumors has been modest. We hypothesized that multiple RTKs are coactivated in these tumors and that redundant inputs drive and maintain downstream signaling, thereby limiting the efficacy of therapies targeting single RTKs. Tumor cell lines, xenotransplants, and primary tumors indeed show multiple concomitantly activated RTKs. Combinations of RTK inhibitors and/or RNA interference, but not single agents, decreased signaling, cell survival, and anchorage-independent growth even in glioma cells deficient in PTEN, a frequently inactivated inhibitor of PI3K. Thus, effective GBM therapy may require combined regimens targeting multiple RTKs.

The genetic paradigm of cancer, focused largely on sequential molecular aberrations and associated biological impact in the neoplastic cell compartment of malignant tumors, has dominated our view of cancer pathogenesis. For the most part, this conceptualization has overlooked the dynamic and complex contributions of the surrounding microenvironment comprised of non-tumor cells (stroma) that may resist, react to, and/or foster tumor development. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease in which a prominent tumor stroma compartment is a defining characteristic. Indeed, the bulk of PDAC tumor volume consists of non-neoplastic fibroblastic, vascular, and inflammatory cells surrounded by immense quantities of extracellular matrix, far exceeding that found in most other tumor types. Remarkably, little is known about the composition and physiology of the PDAC tumor microenvironment, in particular, the role of stroma in tumor initiation and progression. This review attempts to define key challenges, opportunities and state-of-knowledge relating to the PDAC microenvironment research with an emphasis on how inflammatory processes and key cancer pathways may shape the ontogeny of the tumor stroma. Such knowledge may be used to understand the evolution and biology of this lethal cancer and may convert these insights into new points of therapeutic intervention.

R-Ras3/M-Ras is a member of the RAS superfamily of small-molecular-weight GTP-binding proteins. Previous studies have demonstrated high levels of expression in several regions of the central nervous system, and a constitutively active form of M-Ras promotes cytoskeletal reorganization, cellular transformation, survival, and differentiation. However, the physiological functions of M-Ras during embryogenesis and postnatal development have not been elucidated. By using a specific M-Ras antibody, we demonstrated a high level of M-Ras expression in astrocytes, in addition to neurons. Endogenous M-Ras was activated by several trophic factors in astrocytes, including epidermal growth factor (EGF), basic fibroblast growth factor, and hepatocyte growth factor. Interestingly, M-Ras activation by EGF was more sustained compared to prototypic Ras. A mouse strain deficient in M-Ras was generated to investigate its role in development. M-Ras null mice appeared phenotypically normal, and there was a lack of detectable morphological and neurological defects. In addition, primary astrocytes derived from Mras(-/-) mice did not appear to display substantial alterations in the activation of both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in response to trophic factors.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with a median survival of <6 mo and a dismal 5-yr survival rate of 3%-5%. The cancer's lethal nature stems from its propensity to rapidly disseminate to the lymphatic system and distant organs. This aggressive biology and resistance to conventional and targeted therapeutic agents leads to a typical clinical presentation of incurable disease at the time of diagnosis. The well-defined serial histopathologic picture and accompanying molecular profiles of PDAC and its precursor lesions have provided the framework for emerging basic and translational research. Recent advances include insights into the cancer's cellular origins, high-resolution genomic profiles pointing to potential new therapeutic targets, and refined mouse models reflecting both the genetics and histopathologic evolution of human PDAC. This confluence of developments offers the opportunity for accelerated discovery and the future promise of improved treatment.

The Kruppel-like transcription factor KLF6 is a novel tumor-suppressor gene mutated in a significant fraction of human prostate cancer. It is localized to human chromosome 10p14-15, a region that displays frequent loss of heterozygosity in glioblastoma multiforme (GBM). Indeed, mutations of the KLF6 gene have recently been reported in this tumor type. In this study, we report that the expression of KLF6 is attenuated in human GBM when compared with primary astrocytes. Expression of KLF6 in GBM cells reverts their tumorigenicity both in vitro and in vivo, which is correlated with its transactivation of the p21/CIP1/WAF1 promoter. Additionally, KLF6 inhibits cellular transformation induced by several oncogenes (c-sis/PDGF-B, v-src, H-Ras, and EGFR) that are components of signaling cascades implicated in GBM. Our results provide the first evidence of functional tumor suppression by KFL6, and its loss may contribute to glial tumor progression.

R-Ras3/M-Ras is a novel member of the Ras subfamily of GTP-binding proteins which has a unique expression pattern highly restricted to the mammalian central nervous system. In situ hybridization using an R-Ras3 cRNA probe revealed high levels of R-Ras3 transcripts in the hippocampal region of the mouse brain as well as a pattern of expression in the cerebellum that was distinct from that of H-Ras. We found that R-Ras3 was activated by nerve growth factor (NGF) and basic fibroblast growth factor as well as by the guanine nucleotide exchange factor GRP but not by epidermal growth factor. Ectopic expression of either R-Ras3 or GRP in PC12 cells induced efficient neuronal differentiation. The ability of NGF as well as GRP to promote differentiation of PC12 cells was attenuated by an R-Ras3 dominant-negative mutant. Furthermore, the biological action of R-Ras3 in PC12 cells was dependent on the mitogen-activated protein kinase (MAPK). Interestingly, whereas R-Ras3 was unable to mediate efficient activation of MAPK activity in NIH 3T3 cells, it was able to do so in PC12 cells. This cell-type specificity is in stark contrast to that of H-Ras, which can stimulate the MAPK pathway in both cell types. Indeed, this pattern of MAPK activation could be explained by the fact that R-Ras3 was unable to activate c-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf, in PC12 cells. Thus, R-Ras3 is implicated in a novel pathway of neuronal differentiation by coupling specific trophic factors to the MAPK cascade through the activation of B-Raf.

Phosphoinositide 3-kinase (PI3K) type IA is a heterodimer of a catalytic subunit, p110, and a regulatory subunit, p85. Here we show that p85 contains a GTPase-responsive domain and an inhibitory domain, which together form a molecular switch that regulates PI3K. H-Ras and Rac1 activate PI3K by targeting the GTPase-responsive domain. The stimulatory effect of these molecules, however, is blocked by the inhibitory domain, which functions by binding to tyrosine-phosphorylated molecules and is neutralized by tyrosine phosphorylation. The complementary effects of tyrosine kinases and small GTPases on the p85 molecular switch result in synergy between these two classes of molecules toward the activation of the PI3K/Akt pathway.

Kruppel-like factor 6 (KLF6) is a zinc finger transcription factor of unknown function. Here, we show that the KLF6 gene is mutated in a subset of human prostate cancer. Loss-of-heterozygosity analysis revealed that one KLF6 allele is deleted in 77% (17 of 22) of primary prostate tumors. Sequence analysis of the retained KLF6 allele revealed mutations in 71% of these tumors. Functional studies confirm that whereas wild-type KLF6 up-regulates p21 (WAF1/CIP1) in a p53-independent manner and significantly reduces cell proliferation, tumor-derived KLF6 mutants do not. Our data suggest that KLF6 is a tumor suppressor gene involved in human prostate cancer.

We have reported previously that the PTEN COOH-terminal 33 amino acids play a role in the maintenance of PTEN protein stability (Tolkacheva and Chan, Oncogene, 19: 680-689, 2000). By site-directed mutagenesis, we identified two threonine residues within this COOH-terminal region at codon 382 and 383 that may be targets for phosphorylation events. Interestingly, PTEN mutants rendered phosphorylation-incompetent at these two sites, T382A/T383A, and were found to have drastically reduced expression in cultured cells. The enhanced degradation of PTEN was most likely mediated by the proteosome-dependent pathway, we have evidence that PTEN was polyubiquitinated. More interestingly, the non-phosphorylated forms of PTEN displayed significantly greater binding affinity than the wild-type protein to a previously identified PTEN interacting partner, MAGI-2/ARIP1. On the basis of all these data, we propose that PTEN recruitment to the cell-cell junction may be regulated through the phosphorylation of its COOH terminus.

Familial cancer syndromes can involve nervous system tumors. Such syndromes are important in that they identify patients and families that are at higher risk for developing neoplastic disease and also because of their contribution in delineating basic mechanisms of cancer. As our abilities as clinicians to diagnose these patients increases with technological advances in medicine, the identification of such patients and their families takes on additional relevance for their scientific value and as appropriate monitoring and therapy.