Faculty Publications

A core set of oncoproteins is overexpressed or functionally activated in many types of cancer, and members of this group have attracted significant interest as subjects for development of targeted therapeutics. For some oncoproteins such as EGFR/ErbB1, both small molecule and antibody agents have been developed and applied in the clinic for over a decade. Analysis of clinical outcomes has revealed an initially unexpected complexity in the response of patients to these agents. Diverse factors, including developmental lineage of the tumor progenitor cell, co-mutation or epigenetic modulation of genes encoding proteins in an extended EGFR signaling network or regulating core survival responses in individual tumors, and environmental factors including inflammatory agents and viral infection, all have been identified as modulating response to treatment with EGFR-targeted drugs. Second and third generation therapeutic strategies increasingly incorporate knowledge of cancer type-specific signaling environments, in a more personalized treatment approach. This review takes squamous cell carcinoma of the head and neck (SCCHN) as a specific example of an EGFR-involved cancer with idiosyncratic biological features that influence design of treatment modalities, with particular emphasis on commonalities and differences with other cancer types. (C) 2009 Elsevier Inc. All rights reserved.

Background: Much effort has been devoted to development of cancer therapies targeting EGFR, based on its role in regulating cell growth. Small-molecule and antibody EGFR inhibitors have clinical roles based on their efficacy in a subset of cancers, generally as components of combination therapies. Many cancers are either initially resistant to EGFR inhibitors or become resistant during treatment, limiting the efficacy of these reagents. Objective/methods: To review cellular resistance mechanisms to EGFR-targeted therapies. Results/conclusions: The best validated of these mechanisms include activation of classic ATP-binding casette (ABC) multidrug transporters; activation or mutation of EGFR; and overexpression or activation of signaling proteins operating in relation to EGFR. We discuss current efforts and potential strategies to override these sources of resistance. We describe emerging systems-biology-based concepts of alternative resistance to EGFR-targeted therapies, and discuss their implications for use of EGFR-targeted and other targeted therapies.

The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.

In mammals, most cell types have primary cilia, protruding structures involved in sensing mechanical and chemical signals from the extracellular environment that act as major communication hubs for signaling controlling cell differentiation and polarity. The list of clinical disorders associated with ciliary dysfunction has expanded from polycystic kidney disease to include many others. Transformed cells commonly lack cilia, but whether this lack is cause or consequence of transformation is not well understood. Here we discuss work addressing recently identified actions of the cancer-promoting proteins Aurora A and HEF1/NEDD9/CAS-L at cilia. Together with older studies, this work suggests that loss of cilia in cancer may contribute to the insensitivity of cancer cells to environmental repressive signals, based in part on derangement of cell cycle checkpoints governed by cilia and centrosomes.

Recent studies have emphasized the importance of cellular microenvironment in modulating cell growth and signaling. In vitro, collagen matrices, Matrigel, and other synthetic support systems have been used to simulate in vivo microenvironments, and epithelial cells grown in these matrices manifest significant differences in proliferation, differentiation, response to drugs, and other parameters. However, these substrates do not closely resemble the mesenchymal microenvironment that is typically associated with advanced carcinomas in vivo, which is produced to a large extent by fibroblasts. In this study, we have evaluated the ability of a fibroblast-derived three-dimensional matrix to regulate the growth of a panel of 11 human tumor epithelial cell lines. Although proliferative and morphological responses to three-dimensional cues segregated independently, general responsiveness to the matrix correlated with the ability of matrix to influence drug responses. Fibroblast-derived three-dimensional matrix increased beta1-integrin-dependent survival of a subset of human cancer cell lines during taxol treatment, while it sensitized or minimally influenced survival of other cells. beta1-integrin-dependent changes in cell resistance to taxol did not correlate with the degree of modulation of FAK and Akt, implying that additional signaling factors are involved. Based on these results, we propose that these matrices potentially have value as in vitro drug screening platforms.

For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.

In mammals, most cell types have primary cilia, protruding structures involved in sensing mech. and chem. signals from the extracellular environment that act as major communication hubs for signaling controlling cell differentiation and polarity. The list of clin. disorders assocd. with ciliary dysfunction has expanded from polycystic kidney disease to include many others. Transformed cells commonly lack cilia, but whether this lack is cause or consequence of transformation is not well understood. Here we discuss work addressing recently identified actions of the cancer-promoting proteins Aurora A and HEF1/NEDD9/CAS-L at cilia. Together with older studies, this work suggests that loss of cilia in cancer may contribute to the insensitivity of cancer cells to environmental repressive signals, based in part on derangement of cell cycle checkpoints governed by cilia and centrosomes. [Cancer Res 2008;68(7):2058-61]. [on SciFinder (R)]

The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.

The mammalian cilium protrudes from the apical/lumenal surface of polarized cells and acts as a sensor of environmental cues. Numerous developmental disorders and pathological conditions have been shown to arise from defects in cilia-associated signaling proteins. Despite mounting evidence that cilia are essential sites for coordination of cell signaling, little is known about the cellular mechanisms controlling their formation and disassembly. Here, we show that interactions between the prometastatic scaffolding protein HEF1/Cas-L/NEDD9 and the oncogenic Aurora A (AurA) kinase at the basal body of cilia causes phosphorylation and activation of HDAC6, a tubulin deacetylase, promoting ciliary disassembly. We show that this pathway is both necessary and sufficient for ciliary resorption and that it constitutes an unexpected nonmitotic activity of AurA in vertebrates. Moreover, we demonstrate that small molecule inhibitors of AurA and HDAC6 selectively stabilize cilia from regulated resorption cues, suggesting a novel mode of action for these clinical agents.

Greater understanding of metastasis is required to improve cancer treatment outcomes. Recently, changes in expression of the scaffold protein IIEF1/CAS-L/NEDD9 were found to be a potent prometastatic stimulus in melanoma and other cancers. Mechanistic studies suggest diverse cellular roles of HEF1 and highlight its importance in the response to extracellular cues that drive invasion and metastasis. As a metastatic "hub" for signaling in cancer, HEF1 may provide a useful target for drug discovery efforts.

Human enhancer of invasion, clone 10 (HEI10) (CCNB1IP1) was first described as a RING-finger family ubiquitin ligase that regulates cell cycle by interacting with cyclin B and promoting its degradation. Subsequently, other studies suggested specific upregulation of HEI10 in metastatic melanoma and demonstrated direct interaction between HEI10 and the tumor suppressor Merlin, encoded by the neurofibromatosis 2 gene. These and other results led us to hypothesize that HEI10 also influences the processes of cell migration and metastasis. We here show that cells with depleted HEI10 both migrate more rapidly and invade more effectively than control cells. HEI10 depletion post-transcriptionally increases the expression of a group of promotility regulatory proteins including p130Cas, paxillin, Cdk1 and cyclin B2, but excluding Merlin. Among these, only inhibition of Cdk1/cyclin B activity specifically reversed the motility and invasion of HEI10-depleted cells. Finally, HEI10 is abundantly transcribed in many human tissues, and particularly abundant in some tumor cell lines, suggesting that it may be commonly involved in coordinating cell cycle with cell migration and invasion.

An important clinical task is to coherently integrate the use of protein-targeted drugs into preexisting therapeutic regimens, with the goal of improving treatment efficacy. Constitutive activation of Ras-dependent signaling is important in many tumors, and agents that inhibit this pathway might be useful in numerous therapeutic combinations. The MCP compounds were identified as inhibitors of Ras-Raf interactions and previously shown to inhibit multiple Ras-dependent transformation phenotypes when used as monoagents in cell culture analyses. In this study, we investigate the ability of the MCP110 compound to synergistically enhance the activity of other therapeutic agents. In both a defined K-Ras-transformed fibroblast model and in human tumor cell lines with mutationally activated Ras, MCP110 selectively synergizes with other agents targeting the mitogen-activated protein kinase pathway, and with multiple agents (paclitaxel, docetaxel, and vincristine) targeting the microtubule network. The synergistic activity of MCP110 and paclitaxel was further established by experiments showing that in Kaposi's sarcoma oncogenically transformed cell lines, cellular models for tumors treated with taxanes in the clinic and in which Raf-dependent signaling plays an important role, MCP110 synergizes with paclitaxel and limit growth. Finally, in vivo testing indicate that MCP110 is bioavailable, inhibits the growth of LXFA 629 lung and SW620 colon carcinoma cells in xenograft models, and again strongly synergizes with paclitaxel. Together, these findings indicate that MCP compounds have potential to be effective in combination with other anticancer agents. [Mol Cancer Ther 2007;6(3):898-906].

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene have frequently been detected not only in schwannomas and other central nervous system tumors of NF2 patients but also in their sporadic counterparts and malignant tumors unrelated to the NF2 syndrome such as malignant mesothelioma, indicating a broader role for the NF2 gene in human tumorigenesis. However, the mechanisms by which the NF2 product, merlin or schwannomin, is regulated and controls cell proliferation remain elusive. Here, we identify a novel GTP-binding protein, dubbed NGB (referring to NF2-associated GTP binding protein), which binds to merlin. NGB is highly conserved between Saccharomyces cerevisiae, Caenorhabditis elegans, and human cells, and its GTP-binding region is very similar to those found in R-ras and Rap2. However, ectopic expression of NGB inhibits cell growth, cell aggregation, and tumorigenicity in tumorigenic schwanomma cells. Down-regulation and infrequent mutation of NGB were detected in human glioma cell lines and primary tumors. The interaction of NGB with merlin impairs the turnover of merlin, yet merlin does not affect the GTPase nor GTP-binding activity of NGB. Finally, the tumor suppressor functions of NGB require merlin and are linked to its ability to suppress cyclin D1 expression. Collectively, these findings indicate that NGB is a tumor suppressor that regulates and requires merlin to suppress cell proliferation.

Over the past 5 years, the Raf kinase family has emerged as a promising target for protein-directed cancer therapy development. The goal of this review is to first provide a concise summary of the data validating Raf proteins as high-interest therapeutic targets. The authors then outline the mode of action of Raf kinases, emphasizing how Raf activities and protein interactions suggest specific approaches to inhibiting Raf. The authors then summarize the set of drugs, antisense reagents and antibodies available or in development for therapeutically targeting Raf or Raf-related proteins, as well as existing strategies combining these and other therapeutic agents. Finally, the authors discuss recent results from systems biology analyses that have the potential to increasingly guide the intelligent selection of combination therapies involving Raf-targeting agents and other therapeutics.