The major source of cancer morbidity and mortality is uncontrolled metastasis. In the past two years, it has been established that a major determinant of metastasis in melanoma, lung cancer, breast cancer, and glioblastoma is elevated expression of the protein HEF1/NEDD9/Cas-L. HEF1 has been a major focus of our laboratory since we first described it in 1996. Using functional genomics as a screening method, we identified human HEF1 based on its ability to convert yeast from a normal to pseudohyphal growth pattern (Law et al., MCB 16:3327, 1996). In yeast, conversion to pseudohyphal growth requires coordinate alteration of cell polarity and cell cycle. Following a line of investigation based on this requirement, we have demonstrated that HEF1 indeed controls these functions in human cells, suggesting mechanisms through which increased HEF1 levels lead to metastasis. HEF1 has no catalytic activity, but has a complex modular structure that allows it to dock multi-protein complexes. This modularity allows HEF1 to act as an important signaling hub. Our work on HEF1 addresses the complex biology of this protein in mitotic regulation, function of cilia, and in signal transduction. We have also begun to probe the requirement of HEF1 in in vivo metastasis using a recently developed HEF1-/- mouse knockout model, in a Drosophila model, and in cell culture-based functional tests. Top
Seo and coworkers developed a strain of HEF1 knockout (HEF1-/-) mice, and in initial studies demonstrated that these animals were viable and fertile, although with measurable defects in immune system development. In collaborative studies, we have used these animals to study development of breast cancer and metastasis in a HEF1-/- background. We have crossed HEF1-/- mice to both the MMTV-polyoma virus T antigen (PyVT) and the MMTV-neu breast cancer models. Results of this analysis suggest a major influence of HEF1 status on the ability of breast tumors both to progress from early stages and to metastasize. Interestingly, a significant fraction of HEF1-/- mice also have signs of an enhanced immune system reaction to tumor. To provide context for this work, we have also begun to analyze spontaneous tumor generation and immune system function in aging HEF1-/- mice. Our results further support the idea of an altered immune system function relevant to cancer progression. Top
HEF1 is a member of a 4-member family of proteins known as the Cas proteins (Figure 1; see project 1D). The study of the biological consequences of deleting individual Cas proteins in mammals is complicated by the fact that there are 3 remaining paralogous members of the family, which may partially mask phenotypes. However, Drosophila melanogaster has only one Cas gene, which makes it a convenient genetic system to study Cas function. To knock out Cas in Drosophila, we created a fly strain that carries two transposable elements containing flippase recombination target (FRT) sites positioned at 5' and 3' ends of Cas. This recently developed technology makes it possible to use flippase, an enzyme that acts at the FRT sites, to induce homologous recombination. Homozygous mutants lacking Cas do not have severe developmental or other defects, and are viable and fertile; Over the past year, we have studied genetic interactions between Cas and selected genes which are predicted to interact with Cas based on known biology of this protein family in mammals, and also between Cas and a set of genes we hypothesized might be relevant to Cas function. This work in progress has yielded some potent synthetic lethal interactions between Cas and genes not only involved in integrin signaling, but also in cell-cell adhesion. As overexpression of Cas proteins promotes metastasis and correlates with poor prognosis for some human cancers, we have also created transgenic flies that overexpress Cas in the whole fly or various tissues. While high-level overexpression of Cas is lethal, Cas is tolerated at lower levels; we are currently analyzing the phenotypic and genetic properties of these flies. Top
A substantial component of our work is focused on understanding the cellular mechanism of HEF1 action. There are currently two major themes to this work. The first focuses on HEF1 interactions with Aurora-A, a cellular kinase and proto-oncogene. In G2 cells, HEF1 directly associates with Aurora-A and promotes its auto-phosphorylation, a necessary step for mitosis initiation. Recently we have identified Ajuba as a second bi-functional attachment/cell cycle control protein that binds HEF1 and synergizes to activate Aurora-A. We hypothesize HEF1 and Ajuba cooperate to allow a multi-step gradual activation of Aurora-A, timing mitotic entry. In cancer cells, overexpression or hyperactivation of Aurora-A induces aneuploidy and genomic instability, based on inappropriate exit from mitosis. We hypothesize that cancers overexpressing HEF1 are likely to have similar defects, a topic currently under investigation.
Extending studies of Aurora-A, we have found HEF1 also helps activate Aurora-A at an important cellular organelle called the cilium, which protrudes like an antenna from the apical surface of cells. Primary cilia are considered as sensory organs implicated in cell proliferation, polarity and differentiation. A number of human diseases are associated with ciliary dysfunction, including polycystic kidney disease (PKD) and Bardet-Biedl Syndrome (BBS). We have found that a chain of signal transmission involving HEF1, Aurora-A, and an effector protein, HDAC6, is necessary and sufficient for cilium disassembly in human cells (Figure 2). In recent work, we have also begun to map out direct connections between HEF1, Aurora-A, and the polycystin proteins mutated in PKD. We believe better understanding of these connections may help elucidate the cell polarity and cell signaling disruptions that mark PKD.
The second major theme reflects the observation that metastasis induction by HEF1 is intimately linked to the activation of signaling by the Ras oncogene. Shc proteins are members of a multidomain adapter protein family that helps activate Ras/MAPK signaling, pro-survival PI3K/NF-kappa B signaling, and signaling involving FAK and Src that impacts cell adhesion, motility and survival. Increased expression of Shc family proteins marks many brain, ovarian, gastric and thyroid tumors. There is a growing appreciation that there may be important cross-signaling between Cas and Shc family proteins through intermediates such as the HEF1-binding proteins FAK, Src, and Crk (see Figure 3). We have now demonstrated that HEF1 directly interacts with Shc proteins, using structure-function analysis to define specific sites of protein interaction. Although previous reports indicate that HEF1 function in cancer depends on Ras activation, we have now found that HEF1 overexpression can reciprocally activate Ras, indicating bidirectional signaling. Ongoing studies probe the role of Shc proteins in coordinating HEF1-Ras interactions in cancer, using both cell culture and the mouse model. Top
For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas 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 have now identified 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 (Figure 1). 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. However, depletion of HEPL causes some unusual and unexpected phenotypes in relation to cell migration, suggesting this protein may not merely echo the function of other family members. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, while 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 better inform prediction of cancer incidence and prognosis. Top
A second part of our work seeks to better understand the factors leading to drug resistance, with the goals of reversing these resistance mechanisms to improve cancer therapies, and of better stratifying patients as likely or unlikely to respond to specific treatments. For this work, we are applying bioinformatics approaches coupled with siRNA screening to identify resistance genes, focusing on the epidermal growth factor receptor (EGFR) signaling network.
In brief overview, hyperactivity of a central EGFR-Ras-MAPK signaling axis is an essential aspect of the growth, metastasis, and drug resistance of many types of tumor (Figure 3). Given the importance of increased EGFR signaling in many tumor types, a number of clinical agents have been developed that bind and block the activity of EGFR and its family members (e.g, ErbB2/HER2). These agents include antibodies (cetuximab, panitumumab), and small molecules (erlotinib, lapatinib). Although some of these agents are emerging as effective in a subset of patients, a larger group of patients with apparently comparable derangement of EGFR do not respond to treatment.
The primary overall goal of this project is to better understand why some tumors respond effectively to drugs targeting the EGFR pathway while others are resistant, and then to exploit this knowledge to improve breast cancer treatment. We propose that non-responsive cancers contain additional malignant changes (either genetic mutations or epigenetic modifications influencing gene expression) that provide alternative signaling routes to downstream essential EGFR signaling targets. Identifying these "rescue" routes would allow the design of combined therapeutic approaches in which both EGFR and the rescue pathway were simultaneously inhibited, improving clinical response.
We have based our project on several key observations. First, systems biology studies in model organisms have begun to establish that synthetic lethal relationships commonly involve genes that are involved in redundant, parallel pathways, or are vertically linked in the same pathway. Further, in the design of combination therapies in the clinic, the selection strategy for drugs to combine frequently involves common principles: that is, identifying two drugs that 1) inhibit the same target, 2) inhibit functionally linked and/or semi-redundant targets, or 3) inhibit vertically linked targets. Together, these observations suggest that generation of a mid-throughput siRNA library that is large enough to represent genes functionally linked to the drug target by no more than 2-3 degrees of separation, and small enough to be probed at low cost under multiple informative experimental conditions will greatly increase the useful "hit" rate for genes that chemosensitize EGFR family-targeted therapies.
We extensively mined public access databases containing information about protein-protein interactions and mRNA expression profiles generated in humans. We extracted a set of proteins that either directly bind EGFR and its proximal effectors, or are purified in complexes including EGFR; a set of genes transcriptionally upregulated by EGFR-pathway stimulation and downregulated by EGFR inhibition; and a set of genes otherwise involved in EGFR signaling. We also incorporated data generated from genetic interactions reported in model organisms, for strongly conserved evolutionary orthologs of genes in this pathway. We identified a core high value set of genes that fell into at least two of these linkage categories, and based on other weighting functions. We note, in spite of such linkage, the vast majority of these genes have never been tested for ability to modulate EGFR family-targeted therapies.
We have completed initial reiterative screens of a library representing such selected genes. The screening hits are in the progress of validation, and are being remapped to the network to reveal "hubs" or clusters that are particularly rich in sensitizing hits. The further investigation and exploitation of the drug-sensitizing network is a major laboratory focus for 2008-2009. Top