Heide L. Ford - US grants

neoplasm /cancer genetics

DESCRIPTION (provided by applicant): Over the past several years it has become increasingly evident that cancer and normal development share many properties. Among other things, both processes involve alterations in cell proliferation and differentiation, alterations in cell death, neovascularization, cell motility, and invasion of surrounding tissue. Genes involved in these processes during normal development may therefore contribute to tumorigenesis if misexpressed. HSIX1 belongs to the superfamily of homeobox genes that encode transcription factors important for normal development, Its overexpression can attenuate the DNA damage-induced G2 cell cycle checkpoint in mammary carcinoma cells, providing evidence for its role in proliferative processes and suggesting a means through which it may affect tumorigenesis. Indeed, the gene is upregulated in 44 percent of primary breast cancers and 90 percent of metastatic lesions examined. Overexpression of HSIX1 in MCF7 cells significantly increases tumor burden in nude mice, suggesting that its role in cancer is causative, and not merely correlative. This proposal addresses the regulation of HSIX1, as well as the genes it regulates, in an effort to dissect the pathway (both upstream and downstream) through which HSIX1 affects cell cycle control and tumorigenesis. Specific aims 1 and 2 address the post-translational mechanisms by which the HSIX1 protein is regulated in the cell cycle in an effort to demonstrate whether this regulation is critical for its role in the G2 checkpoint and in tumorigenesis. Specifically, we will investigate how (a) mitotic phosphorylation and (b) proteasome-mediated degradation affect the role of HSIX1 in the G2 checkpoint and in tumorigenesis. This will be done using a variety of molecular biological, biochemical, and cell biological approaches, including irradiation assays in cell culture and nude mouse tumor assays. The last specific aim extends the proposal to identify genes transcriptionally regulated by HSIX1 in the G2 phase or at the G2/M boundary, with the goal of elucidating pathways important in HSIX1 control of the cell cycle and tumorigenesis. Methods will include examining known regulators of the G2 cell cycle checkpoint as well as microarray analysis. HSIX1 provides us with a unique opportunity to examine the relationship between developmental genes, cell cycle control, and cancer. A homeobox gene that is overexpressed in cancer cells but is normally absent or expressed at low levels in noncancerous, differentiated cells from the same tissue may serve as an ideal drug target, assuming that development of the organ is not essential at the time the cancer arises.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): During development, certain genes are activated to stimulate cell proliferation, survival, migration, invasion, and neovascularization. These genes are often downregulated once organ development is complete. During carcinogenesis, the same genes are often re-activated, stimulating the same properties, but now out of context. Transcribed by one such gene, the homeobox protein and transcription factor Six1 is expressed during embryogenesis where it mediates the proliferation and survival of progenitor cells. It is lost in most differentiated tissues but re-expressed in a number of cancers including breast cancer. We have shown that when Six1 is expressed out of context in adult cells, it aberrantly promotes both proliferation and survival, contributing to tumorigenesis. Our preliminary evidence shows that Six1 also brings about an epithelial-to-mesenchymal-like transition (EMT) in cultured cells and promotes metastasis in vivo. Our proposed experiments are therefore directed at testing the following tri-partite hypothesis: Misexpression of Six1 after development is complete imparts not only a pro-proliferative and survival phenotype, but also a pro-migratory and invasive phenotype, contributing to metastatic disease. Acquisition of the invasive phenotype is due to a Six1-dependent increase in TGF- signaling. The resulting aggressive, metastatic mammary tumors remain dependent on Six1 for survival and for their aggressive behavior. In the proposed research we will provide definitive tests of these hypotheses both in vitro and in vivo and begin to decipher the molecular mechanism by which Six1 causes EMT and metastasis, focusing on the TGF- signaling pathway. Our aims are: 1) To determine whether TR-I expression and signaling is required for Six1-mediated oncogenic EMT in cultured mammary epithelial cells;2) To determine in xenograft models whether Six1 overexpression is necessary and sufficient to cause metastatic breast disease, through upregulation of TGF- signaling;3) To use inducible transgenic models of Six1 overexpression to determine whether the invasive behavior of the resulting mammary tumors is due to TGF- signaling, and whether this behavior remains dependent on Six1. Relevance: Six1 is overexpressed in a much higher percentage of metastatic (90%) compared to primary breast cancers (50%) and its overexpression is often correlated with advanced tumor stage and worsened survival. Further, Six1 influences multiple stages of the tumorigenic process. For this reason therapeutic agents targeting Six1 have the potential to inhibit breast cancer at both early and later stages of disease progression. Because Six1 is lost in most adult tissues such therapeutic agents should have limited side effects. Project Narrative: Expression of the Six1 homeobox gene causes both tumor initiation and metastasis. Thus, therapeutic agents targeting Six1 have the potential to inhibit breast cancer at both early and later stages of disease progression. Further, because Six1 is expressed only during embryogenesis, and not in the adult, therapies that target Six1 should inhibit tumor cell growth and metastasis with limited side effects.

[unreadable] DESCRIPTION (provided by applicant): TNF-Related Apoptosis Inducing Ligand (TRAIL) kills tumor cells with little effect on normal tissues and recombinant TRAIL and antibodies that recognize TRAIL receptors are in clinical trials at the University of Colorado and elsewhere. In addition, TRAIL receptor signaling determines the efficiency with which other agents kill tumor cells. However, tumor cells are often resistant to TRAIL and the mechanisms by which this occurs and what this resistance means for tumor progression and clinical outcomes is poorly understood. We recently discovered that one mechanism by which breast and ovarian tumor cells can become selectively resistant to TRAIL is through the increased expression of the homeobox transcription factor Six1. We found that increased Six1 is common, occurring in >60% of metastatic ovarian cancers and 90% of metastatic breast cancers, and associated with poor clinical outcomes. We further found that Six1 expression is sufficient to make non-metastatic tumor cells metastasize in vivo. Because TRAIL signaling is known to suppress metastasis, we hypothesize that Six1 inhibits TRAIL by a specific mechanism and this leads to increased metastasis resulting in poor clinical outcomes in patients and resistance of patient's tumors to TRAIL. To test this hypothesis we propose an integrated project that will determine the molecular mechanism by which Six1 inhibits TRAIL receptor-induced apoptosis, test if these mechanisms are responsible for increased metastasis in mice and determine whether these effects apply in primary tumor cells from patients and if they lead to worse clinical outcomes. Because this work encompasses research on basic mechanisms at the cellular level, testing those mechanisms in animal models of cancer progression and metastasis and clinical and translational studies in ovarian and breast cancer patients' tumors, we have adopted a team approach that will involve a cell biologist with expertise in TRAIL signaling (Dr. Thorburn), a pioneer in the analysis of Six1 in cancer development and progression (Dr. Ford) and an oncologist with expertise in clinical and translational research (Dr. Behbakht). To achieve these goals we have the following aims: 1. Determine how Six1 alters signaling by TRAIL receptor-targeted therapeutic agonists in ovarian and breast cancer. 2. Test if Six1-induced metastasis involves the TRAIL resistance mechanism, and 3. Determine if Six1 expression predicts TRAIL sensitivity and prognosis in patient tumors. Together, these aims should allow us to understand how Six1 regulates TRAIL receptor signaling, determine the role of these mechanisms in tumor metastasis and test if the same mechanisms apply in breast and ovarian cancer patients and thus determines their clinical outcomes. PUBLIC HEALTH RELEVANCE: Breast and ovarian cancer are the second and fifth leading causes of cancer death in women. One exciting new approach to treating these (and other) cancers is to use therapies that directly activate TRAIL receptors, however many tumor cells are resistant to these therapies. This project focuses on one mechanism (Six1 expression) by which tumor cells become resistant to TRAIL that our data indicate affect a high proportion of metastatic cancer patients (~60-90%). By understanding how this mechanism causes resistance to TRAIL, how this affects disease progression and metastasis and how this alters clinical outcomes in patients, we should gain new insights into the value of Six1 as a prognostic marker and better understand how to use the TRAIL therapeutics that are being developed. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Eya family of proteins is essential co-activators of the Six1 transcription factor. Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. The Eya family of proteins was also found to contain a unique HAD phosphatase domain with protein Tyr phosphatase activity which can potentially play a role in Six1-mediated breast tumorigenesis. Recently, Eya was found to dephosphorylate the histone variant H2AX and direct cells to the DNA repair instead of apoptosis pathway in the event of DNA damage. We have setup a fluorescence-based HTS assay targeting the phosphatase activity of Eya. We propose to perform a large scale high throughput screening using the NIH MLPCN compounds to identify inhibitors of the Eya phosphatase. We plan to test these inhibitors for their potential as therapeutic tools to inhibit Six1-mediated breast tumorigenesis and to increase the efficiency of radiation and certain chemotherapy. These inhibitors can also be used as chemical probes to study the function of Eya's phosphatase activity and its role in Six1-mediated breast tumorigenesis. PUBLIC HEALTH RELEVANCE: The Eya family of proteins is essential co-activators of the Six1 transcription factor which is known to be critical for the onset and progression of a large percentage of breast tumors. In addition, Eya protein contains phosphatase activity that is essential for directing cells to the repair instead of apoptosis pathway upon DNA damage. This phosphatase may also play a role in Six1-mediated breast tumorigenesis. This project aims at identifying inhibitors of the Eya phosphatase activity, which can potentially serve as therapeutic tools to inhibit Six1-mediated breast tumorigenesis and/or to increase the efficiency of radiation and some chemotherapy.

DESCRIPTION (provided by applicant): Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. Since Six1 does not have its own activation or repression domain, the Eya family of Six1 co-activator proteins is essential for Six1-mediated breast tumorigenesis and metastasis. We have developed an AlphaScreen based HTS assay targeting the Six1/Eya interaction. We propose to perform a large scale high throughput screening using the NIH MLPCN compounds to identify inhibitors of the Six1/Eya interaction. We plan to test these inhibitors for their potential as therapeutic tools to inhibit Six1-mediated breast tumorigenesis and metastasis. These inhibitors can also be used as valuable chemical probes for studies involving the Six1/Eya interaction.

DESCRIPTION (provided by applicant): Breast cancer is the second leading cause of cancer-related deaths among women, with deaths occurring due to the metastatic spread of tumor cells to distant organs. Spread of cancer cells occurs via blood vessels or, particularly important for breast cancer, via the lymphatic system. Although tumor dissemination via the blood has been extensively studied, the mechanisms that lead to lymphatic dissemination are poorly understood. This proposal focuses on understanding a new mechanism of lymphangiogenesis and breast tumor metastatic spread, via control of VEGF-C levels by the Six1/Eya2 transcriptional complex. The hypothesis to be tested is that Six1 overexpression in breast cancers leads to upregulation of VEGF-C, resulting in increased lymphangiogenesis and enhancing the early stages of metastasis including lymphatic dissemination of tumor cells. The ability of Six1 to activate VEGF-C and stimulate lymphatic metastasis is dependent on its interaction with the Eya2 phosphatase co-factor, and on the enzymatic activity of Eya2. If this hypothesis is correct, it will provide important insights into the mechanism of breast cancer metastasis and provide a rationale for a new strategy to therapeutically target lymphatic metastasis by interfering with Six1/Eya2 function through the druggable approaches of inhibiting Six1/Eya interaction and/or Eya2 phosphatase activity. To address this hypothesis, we will: 1) Test the hypothesis that VEGF-C is a direct transcriptional target of Six1/Eya2, and that Six1 and Eya2 correlate with VEGF-C and lymphangiogenesis in human breast cancer; 2) Test the hypothesis that VEGF-C is a critical mediator of Six1-induced lymphangiogenesis and metastasis using in vivo mouse metastasis models; and 3) Test the hypothesis that Eya2 is required for Six1-induced lymphangiogenesis and metastasis in vivo, with a specific emphasis on the interaction between Six1 and Eya2, as well as the role of the Eya2 phosphatase activity. Targeting Six1/Eya2 has the potential to inhibit breast cancer both at early (studied in this proposal) and later stages of the disease. Due to the paucity of expression of the Six1/Eya2 developmental regulators in most normal adult tissues, and their re-expression in cancers, therapeutic agents targeting this complex should inhibit lymphangiogenesis and metastasis with limited side effects. Thus the Six1/Eya2 complex is an ideal breast cancer therapeutic target, and work within this proposal will lay the foundation for eventual targeting of the complex.

DESCRIPTION (provided by applicant): During development, Six1 plays a critical role in the expansion of progenitor cell populations via regulating proliferation, survival, migration, and invasion. While not expressed in most differentiated tissues, Six1 is re- expressed in 50% of primary and 90% of metastatic breast lesions. In animal models, Six1 overexpression causes both mammary tumorigenesis and metastasis. Because Six1 is a transcription factor and therefore not amenable to targeting for anti-cancer therapy, we must understand the mechanism by which it mediates tumor progression in an effort to identify means to target the pathways it activates. We have found that Six1 induces an epithelial to mesenchymal transition (EMT), a tumor initiating cell (TIC) phenotype, and metastasis via its ability to induce TGF? signaling. While inhibition of TGF? signaling in Six1-overexpressing breast cancer cells reverses metastasis, inhibition of TGF? signaling in control cells promotes metastasis. These data indicate that Six1 is able to shift TGF? signaling from tumor suppressive to promotional, and may provide a novel mechanism to explain the elusive TGF? paradox. Although Six1 is expressed in all subtypes of breast cancer, high expression of Six1 significantly correlates with adverse outcomes particularly in the aggressive and hard to treat luminal B subtype. Similarly, when breast cancer datasets are queried with a signature that examines only the tumor suppressive arm of TGF? signaling, low expression of this signature is associated with poor prognosis specifically in ER+ (luminal) breast cancers, and the luminal B subtype expresses the lowest levels of the TGF? tumor suppressive signature. These data imply that tumors within this subgroup escape the growth suppressive effects of TGF?. Importantly, TGF? inhibitors are currently in clinical trials, yet because TGF? signaling can be tumor suppressive or promotional, one of the greatest concerns surrounding their use in cancer is how to predict which patients will benefit. In this proposal we focus on a novel mechanism by which Six1 may mediate the switch in TGF? signaling, particularly in luminal B tumors: activation of the miR106b-25 cluster of microRNAs (miRs). This cluster of miRs is known to overcome the growth suppressive effects of TGF? signaling, and we demonstrate that the same cluster can also activate the tumor promotional arm of TGF? signaling. Thus, we will test the hypothesis that induction of the miR106b-25 cluster by Six1 provides a mechanism by which Six1 executes the switch in TGF? signaling from tumor suppressive to promotional, thus leading to increased EMT and TIC capacity and increased metastasis, and that inhibition of miR106b-25 may thus be a means to therapeutically target luminal B breast cancers. We will also test whether Six1/miR-106b-25 expression may provide a means to distinguish between patients who will benefit rather than be harmed by treatment with TGF? inhibitors. To address these hypotheses, we will utilize cell culture, xenograft, and transgenic mouse models in which the Six1/miR106b-25/ TGF? axis is modulated by genetic means and/or drug treatment, as well as by experimentation on human breast cancer samples.

? DESCRIPTION (provided by applicant): Most anti-cancer drugs target binding pockets in enzymes or on protein receptors. These drugs often target upstream pathways that can cripple tumors, but may fail to hit the central node of the tumor. For some time, it has been known that transcription factors can serve as these central nodes. Indeed, de-regulated transcription has been associated with most, if not all, properties that are critical to the cancer cell, including uncontrolled growth, unlimited replicative potential, migration and invasion. Yet few investigators have taken on the more difficult task of targeting transcription factors. The Six1 homeobox gene encodes a transcription factor that is critical for embryonic development. In most tissues, Six1 expression is lost once development is complete. However, Six1 is re-activated in as many as 50% of primary breast tumors and 90% of metastatic lesions, as well as in many other tumor types. Six1 plays a causal role in tumor initiation, tumor growth, and metastasis of breast and other cancers, and its inhibition dramatically diminishes the aforementioned properties in several mouse models of cancer. Six1 has no intrinsic activation or repression domains, and thus requires cofactors to mediate its transcriptional effects. We have demonstrated that Six1 requires the Eya cofactor to mediate its pro-proliferative and pro-metastatic effects in breast cancer cells. Examination of public breast cancer microarray datasets reveals that high expression levels of Six1 and Eya together, but not either gene alone, correlate with shortened time to relapse, shortened time to metastasis, and shortened overall survival in breast cancer. Importantly, we recently determined the crystal structure of the Six1/Eya2 complex, which reveals that Six1 uses a single alpha helix to interact with Eya, resembling two well-known protein- protein interactions (p53/HDM2 and Bak/Bcl-xL) that have been successfully targeted by small molecules. We further demonstrated that a single amino acid mutation on the Six1 helix abrogates the binding of Six1 to Eya and Six1-mediated tumor initiation and metastasis. The goal of this project is to identify and develop novel anti- cancer agents that target the Six1/Eya complex through characterizing and optimizing hit compounds identified through the use of a high throughput screen (Aim 1). Identified inhibitors will be tested in both cell culture and animal models for their ability to inhibit tumorigenicity and metastasis (Aim 2). Since the Six1/Eya complex influences multiple stages of the tumorigenic and metastatic processes and is not expressed in most adult tissues, targeting this never before targeted complex has the therapeutic potential to inhibit breast and other cancers both at early and later stages of disease progression with limited side effects, something most current therapies do not do.

Summary Rhabdomyosarcoma (RMS) accounts for ~3% of all pediatric cancers. Unfortunately, the overall 5-year survival rate for children diagnosed with metastatic RMS is less than 30%. Sarcoma patients experience higher rates of morbidity and mortality than other cancer patients, and this is particularly evident in children. As a result of their therapies, 42% of childhood cancer survivors experience severe, disabling, or life threatening conditions (including secondary tumors). Thus, there is clearly a need to develop new, more targeted treatment strategies for pediatric tumors; treatments that inhibit tumor progression yet confer limited side effects. The pro-metastatic transcription factor SIX1 is overexpressed in RMS, where it is plays a critical role in metastasis. During embryonic development, Six1 promotes precursor cell activation and migration to enable proper formation of muscle, kidney and the inner ear. However, after development is complete, Six1 expression is silenced or reduced in most tissues. Thus, Six1 is upregulated in the setting of RMS, where it is known to be important for progression of the disease. These data suggest that gaining an understanding of the mechanisms controlling Six1 downregulation during muscle development may provide a novel means by which to target Six1 in the setting of RMS. Such targeting may be anticipated to inhibit the tumor, yet have limited toxicity due to the paucity of Six1 expression in adult tissue. Throughout embryogenesis and myogenesis, microRNAs (miRs) have been shown to coordinate complex temporal and tissue-specific patterns of protein expression, including regulation of many homeobox genes. In tumor models, miRs have been shown to inhibit RMS tumor growth, and miR-mediated downregulation of Six1 can prevent kidney tumor progression. In zebrafish, there are two orthologs of Six1, six1a and six1b. Our recently published data demonstrate that miR30a negatively regulates both six1a and six1b in zebrafish muscle development, and that miR30a-mediated downregulation of Six1 is required for proper muscle development to occur. Thus, in this proposal we will test the following Hypothesis: Upregulation of Six1 is required for RMS tumor onset and progression. We will further test whether zebrafish will be an ideal model to rapidly and inexpensively test whether inhibitors of Six1 (beginning with miR30a, but in the future adding in additional miRs or novel Six1 small molecule inhibitors) will inhibit RMS progression. Specific aims: 1) To investigate the role of the Six1 transcriptional complex in RMS initiation and progression in zebrafish models, and 2) To identify potential therapeutic options for RMS through downregulation of Six1. The second of these aims will test, as proof of principal, whether miR30a has the potential to inhibit RMS progression. This R21 mechanism, by allowing us to develop the zebrafish models, will set the stage for testing novel small molecule inhibitors that target the Six1 transcriptional complex in the context of a whole organism. As we are currently developing such inhibitors, these models will allow for more efficient screening of a large number of compounds in vivo than could be done in other model systems.

Abstract The Eya proteins (Eya 1-4) were initially discovered as essential co-activators of the Six family of homeoproteins that regulate embryonic development via controlling proliferation, survival, epithelial versus mesenchymal fates, and overall cell fate specification in numerous tissues. The Six/Eya complex is downregulated after development is complete, but is re-expressed in numerous cancers, including breast cancer. In addition to acting as transcriptional cofactors for the Six family of proteins, Eyas also contain a Tyr phosphatase activity in their C-terminal domains that has been implicated not only in transcriptional activation, but also in motility, DNA repair and survival in response to damage, angiogenesis, and inhibition of the anti- tumor activity of estrogen receptor-b. Currently, inhibitors are being developed against the transcriptional and Tyr phosphatase activities of Eyas to dissect its different functions, and to potentially develop anti-cancer agents. The Eyas also contain a Ser/Thr phosphatase activity in their N-terminal domain, that is completely separable from the Tyr phosphatase activity. The function of this activity in cancer is completely unknown and it is not targeted by any of the Eya inhibitors under development. Intriguingly, the N-terminal domain of Eya does not contain any recognizable phosphatase motifs and our data suggest that the Ser/Thr phosphatase activity of Eya is not intrinsic, but is instead due to an interaction with the protein phosphatase 2A (PP2A). In addition, using two different immune competent mouse models, we show for the first time that Eya3, which is overexpressed in triple negative breast cancers (TNBC) as compared to other subtypes, and contains the highest Ser/Thr phosphatase activity, influences tumor progression via its effects on adaptive immunity. We further demonstrate that Eya3, through its PP2A-associated Ser/Thr phosphatase activity, regulates the key immune checkpoint protein programmed death-ligand 1 (PD-L1). Thus, we identified a novel tumor promoting function for Eya3 that is only revealed in the context of a functional adaptive immune system. In this proposal we test the hypothesis that Eya3 regulates breast tumor growth and progression via its ability to recruit PP2A, leading to increased levels of PD-L1 and a diminished tumor specific T-cell response. Results obtained in this project will significantly advance our understanding of the role of Eya3 in breast tumor progression, and may also reveal that Eya3, through its previously unknown ability to interact with PP2A, is an important new player in adaptive tumor immunity. Further, we may uncover a novel regulatory mechanism of a key immune checkpoint protein, PD-L1, whose regulation has remained largely elusive. Thus our studies may result in novel biomarker or therapeutic strategies to enhance efficacy of PD-1/PD-L1 mediated therapies.

Project Summary: Medulloblastoma (MB) is the most common malignant brain tumor in children. Treatment for MB includes surgery, radiation and chemotherapy. Unfortunately, long-term morbidity, including lifelong cognitive deficiencies, endocrine dysfunction, neurological defects, emotional and social problems, and secondary tumors are associated with current treatments. Such treatment associated side effects are particularly evident in children, as their brains are still growing, and they can survive many years after treatment. MB is a heterogeneous disease with at least 12 different subgroups identified. Patients with high MYC expressing MB (25-30% of cases; referred to as Group3) have the worst prognosis (survival at 5 years is 41.9% in the Group 3? subtype), and MYC is known to be a major driver of this MB subtype. Thus, for Group 3 MB there is a pressing need to develop novel targeted therapies that confer limited toxicities. However, MYC has remained ?undruggable?. To identify novel therapeutic targets in high MYC expressing MBs, we have begun to examine the role of the EYA2 transcriptional co-factor and dual phosphatase (Tyr and Ser/Thr in separable domains) in MB progression. Intriguingly, EYA2 has been shown to control MYC, both transcriptionally and post- translationally, during embryonic development, but is normally downregulated after development is complete. Our preliminary data show that EYA2 is overexpressed in Group3 MB compared to normal cerebellum and other subtypes of MB, that it controls MYC levels in the context of Group3 MB, and that KO of EYA2 dramatically diminishes in vivo growth of Group3 MB. Our main objective in this proposal is to identify novel druggable targets in Group 3 MB; targets that when inhibited will not lead to the significant side effects associated with current MB therapies. To this end, we will test the hypothesis that the SIX1/EYA2 transcriptional complex and/or EYA2 phosphatase plays a critical role in Group 3 MB progression via transcriptionally activating and/or stabilizing MYC, and that novel inhibitors targeting the activity of EYA2 can diminish disease progression while conferring limited side effects. To address this hypothesis, we will carry out three aims: 1) Determine which activity of EYA2 regulates MYC and contributes to the aggressive nature of Group 3 MBs, 2) Determine if regulation of MYC by EYA2 is required for its effects on MB growth and progression, 3) Determine whether EYA2 inhibition, genetically or pharmacologically (using novel small molecule inhibitors targeting either its transcriptional activity with SIX1 or its Tyr phosphatase activity), will provide a unique means to inhibit the critical oncogene, MYC, preventing MB progression in vivo. If our hypothesis is correct, and EYA2 is a key, druggable regulator of MYC, we will have identified an achilles heel not only for Group3 MB, but potentially for the many other MYC-dependent tumors. Targeting MYC remains a ?holy grail? in cancer research, and our studies seek to do so via a novel means anticipated to have limited toxicity due to the paucity of EYA2 expression in normal tissues after development is complete.

Virtually all breast cancer (BC) related deaths result from metastatic burden rather than the primary tumor. It has long been thought that primary tumors are heterogeneous, with only some cells having the capacity to metastasize. Our work suggests that this view is oversimplified, and that non-metastatic cells can become more metastatic due to paracrine-mediated signaling from other cancer cells. Understanding this mechanism will uncover new strategies to inhibit metastatic disease. To understand crosstalk between cells of varying metastatic potential in heterogeneous tumors, we examined whether metastatic cells expressing the EMT-inducing transcription factors (TFs), Twist1 and Snail1, can promote metastatic properties in intrinsically non-metastatic cells. Our data show that, in a manner dependent on a third EMT TF, Six1, the EMT TFs can non-cell autonomously enhance metastatic properties. Mechanistically, we show that cells expressing Six1 mediate paracrine activation of GLI-mediated signaling in intrinsically non-metastatic cells, leading to increased aggressiveness and metastasis of these cells. Importantly, paracrine mediated activation of GLI can occur through non-canonical pathways independent of Hedgehog (Hh) ligands and Smoothened (SMO)-mediated Hedgehog signaling. Our strong data lead us to hypothesize that SMO inhibitors, which are in clinical trials, will fail in a large percentage of BC patients. We hypothesize that targeting GLI directly will be effective in a broader range of breast tumors (encompassing subtypes that show features of EMT), and that determining the molecular mechanism of non-canonical paracrine GLI activation will lead to additional therapies that are effective in a broad range of BCs. Further, we hypothesize that targeting the central mediator, Six1, using our novel small molecule inhibitor, will be efficacious in the broadest range of BCs due to its cell and non-cell autonomous role in tumor progression. To test these related hypotheses, we will first determine the paracrine mechanism by which EMT/metastatic cells increase the aggressiveness of intrinsically non-metastatic cells, focusing on VEGF-C and its downstream signaling as key mediators. We will then perform a large scale head-to-head comparison of inhibitors of EMT/metastatic and non-metastatic cell crosstalk in breast patient derived xenograft (PDX) models that encompass various breast cancer subtypes and are metastatic, to determine which inhibitors are efficacious in a broader range of tumors, and to characterize the types of tumors that will respond to specific inhibitors. Our work may provide a partial explanation for why SMO inhibitors have been ineffective as single agents in tumors such as BCs. Further, by uncovering a new mechanism by which EMT promotes metastasis, and by deciphering the molecular components of that mechanism, we may not only provide an explanation for the controversy in the field, but may more importantly identify highly efficacious means to target the many BCs in which a percentage of cells have undergone an EMT.