Kirby I. Bland, M.D.
The strategic plan of the University of Alabama at Birmingham (UAB) SPORE in Breast Cancer is to understand the biology of breast cancer and to translate this knowledge into the clinic for early detection, prevention, prognosis, and development of new therapies. The SPORE is investing in several translational research areas and to achieve its goals, basic and clinical scientists have been assembled, including molecular/cell biologists, pathologists, medical/surgical oncologists, experts in the development of new technologies, and biostatisticians. The SPORE consists of five major projects and three supporting cores. The projects are: 1) UAB Rexinoids for Breast Cancer Prevention; 2) BRMS1: Prognostic Marker and Therapeutic Target; 3) Breast Cancer Biomarkers in the KLF4 Signaling Pathway; 4) Peptides Blocking Hormonal Control in Breast Cancer; and 5) DR5 Antibody therapy for Breast Cancer. The translational research focus on the individual projects include: cancer biology (#2-4); therapy/survival (# 2, # 4-5); diagnosis/prognosis (#2, #3); and prevention (#1). The Cores are A) Administrative/Clinical Trials; B) Biostatistics/Database; and C) Tissue Resources/Immunopathology/Molecular Assays. All of the projects focus on human breast cancer, involve basic and clinical scientists, interact with the other projects, and utilize core resources. Currently, ten SPORE-initiated clinical trials are underway or will begin accruing subjects within the next few months. The SPORE has developed and is maintaining a comprehensive breast cancer database that correlates patient, tumor, and treatment data with patient-specific tissue and blood specimens. Innovative Developmental Research and Cancer Development Programs bring new investigators into and stimulate the SPORE. The strong support of these programs by UAB and the UAB Comprehensive Cancer Center increase their impact. The SPORE participates in the inter-SPORE efforts of the Breast SPORE "round-table" and the annual NCI-sponsored SPORE workshop. Achievement of the aims and objectives of this proposal will result in a major decrease in the incidence, morbidity, and mortality of breast cancer.
Donald D. Muccio, Ph.D.
The goal of cancer research is to reduce the morbidity and mortality associated with the cancer. This can be achieved either by cure of existing cancer or prevention of new cases. The problem being confronted for breast cancer is enormous. It is estimated that 40,110 women will die from this cancer in 2004.
Cancer progression is the result of a multistep process in which the cumulative effect of successive discrete genetic alterations leads to a gradual transition from normal through premalignant to frankly malignant tissue and ultimately to metastasis. This process provides many opportunities for intervention. These include increased surveillance, chemoprevention for patients with increased risk for breast cancer (genetic predisposition, premalignancy, prior breast cancer), and therapy for early stage invasive breast cancer based on specific prognostic and predictive indicators. This proposal focuses on chemoprevention, specifically the synthesis, preclinical analysis, and clinical evaluation of synthetic RXR-selective retinoid agonists.
From the previous funding cycle, we have now shown that the most promising retinoids for prevention of human breast cancer are those that are selective agonists for the nuclear retinoid X receptors (RXRs), named rexinoids, and we have designed a novel series of UAB rexinoids, which 1) produce less toxicity than 9-cis-retinoic acid (9cRA) or the rexinoid, Targretin (LGD1069), and 2) enhance the efficacy of tamoxifen in combination therapy in vivo. One of our novel UAB rexinoids, 9-cis-UAB30 (9cUAB30), is currently undergoing evaluation in the NCI RAPID program where initial toxicity and mutagenesis assays show very encouraging results. Most notably we have demonstrated that 9cUAB30 does not elevate triglycerides, which is a significant dose-limiting toxicity of other retinoids. We have also demonstrated synergy between 9cUAB30 and tamoxifen when evaluated in combination in the MNU breast cancer model. An optimal combination of tamoxifen (or other anti-estrogens) and a rexinoid such as 9cUAB30 will minimize toxicity and enhance breast cancer chemoprevention efficacy. In this proposal we will conduct human Phase I clinical trials on 9cUAB30, and we will design and preclinically develop new second-generation UAB rexinoids. These agents would be used clinically to prevent breast cancer in single or combination regimens with minimum toxicity.
Aim 1. Phase I Clinical Study of 9cUAB30: Following completion of ongoing preclinical analyses funded by the UAB Breast SPORE and the NCI RAPID program, 9cUAB30, a novel rexinoid with low toxicity, will be analyzed in a Phase I study. The start date is projected to be in 2006. Oral bioavailability, pharmacokinetics, and toxicity will be analyzed in single and multiple dose trials. Core B will provide statistical analyses of data.
Aim 2. Design and Synthesis of Second-Generation UAB Rexinoids Aided by Structure-Based Design. By classical structure-activity relationships, we identified three potent UAB rexinoids (9cUAB30; 4-methyl-9cUAB30; and 9cUAB76) that prevent mammary cancers alone, and, at suboptimal doses, they synergize with selective estrogen receptor modulators (SERMs) like tamoxifen. We hypothesize that UAB rexinoids with even greater potency/lower toxicity can be designed aided by structure-based methods. 3D structures of the target receptor, RXRa, which are bound to active UAB rexinoids, will be determined. New second-generation UAB rexinoids will be designed using these 3D structures. After synthesis, these rexinoids will undergo in vitro nuclear receptor binding/transactivational studies to establish their potency as agonists.
Aim 3: Determine the Efficacy for Cancer Chemoprevention of New UAB Rexinoids in the MNU-induced cancer model alone and in combination with SERMs. From previous funding we established the working hypothesis: UAB rexinoids with the most promising potential for chemoprevention are also potent RXR-agonists that (A) are delivered efficiently to mammary tissue to reduce cell proliferation, and (B) do not increase serum triglyceride levels. To validate this hypothesis each UAB rexinoid will be studied in a short-term screen consisting of: (A) changes in surrogate endpoint biomarkers for cell proliferation (e.g. BrdU) and apoptosis (e.g. TUNEL) on established cancers; and (B) serum levels of rexinoid, triglycerides, and cholesterol. Next second-generation UAB rexinoids (Aim 2) will be evaluated in the N-methylnitrosourea (MNU) model for mammary cancer chemopreventive efficacy and toxicity in rats. If the short-term screen(s) are capable of predicting chemoprevention, then only UAB rexinoids which pass the screen will be selected for study in the MNU mammary cancer prevention model. UAB rexinoids that are effective alone in this prevention model will next be evaluated in combination with SERMs (e.g., tamoxifen). Tissue distribution, oral bioavailability and metabolism will be determined in rats for potent (alone and in combination with SERMs) UAB rexinoids.
Aim 4: Study the Effects of UAB Rexinoids on Gene and Protein Expression Profiles. For the clinically promising UAB rexinoids, like 9cUAB30, the gene and protein expression patterns induced by rexinoids will be studied in normal and transformed mammary tissue in vivo. The hypothesis is that genomic and/or proteomic changes in these tissues induced by UAB rexinoids would become potential biomarkers for chemoprevention. Biomarkers would be useful in the clinical development of rexinoids, and they may provide important insight into mechanisms of action of rexinoids.
Jennifer De Los Santos
Metastasis is the most serious impediment to cancer cure. SEER (Surveillance Epidemiology and End Results) data suggests that approximately one-third of breast cancer patients diagnosed with stage I disease eventually develop metastases . Adjuvant therapies are therefore applied with the rationale of reducing risk of local, regional and/or distant recurrences. Yet, this means that ~70% of patients who would have otherwise have been cured by surgery alone undergo toxic, inconvenient and expensive chemotherapy and/or radiation treatments. The long term objectives of Project 2 are: (i) to more accurately identify the patients most likely to develop metastases so that adjuvant treatment might be more judiciously administered only to those needing it; and (ii) to prevent development of metastases or more effectively treat metastatic disease.
Toward those ends, we will take advantage of laboratory findings in which we cloned several genes which, when re-expressed in metastatic human breast carcinoma cell lines, suppress metastasis without blocking tumorigenicity. Because these genes, termed metastasis suppressors, show promise for distinguishing which patients' tumors are more or less likely to develop metastases. Likewise, they provide a target for therapeutic intervention, i.e., we hypothesize that, if one could restore metastasis suppressor expression and function, establishment of new metastases could be prevented and/or already established metastases could be eliminated or forced into quiescence. Project #2 will test two hypotheses in three aims. We will focus on the metastasis suppressor, BRMS1.
Hypothesis 1: Decreased expression or mutation of BRMS1 results in greater likelihood of metastasis.
Specific Aim 1 will test whether BRMS1 expression in breast cancers predicts the likelihood of breast cancers to metastasize. We will use the UAB Breast SPORE collections of frozen and archival formalin-fixed, paraffin-embedded (FFPE) specimens to assess whether mRNA (using real-time RT-PCR, hereafter RTQ) and protein (using immunohistochemistry, IHC) expression correlates metastatic potential. Laser capture microdissection (LCM) will be used to isolate primary tumor cells, adjacent normal cells and cancer cells that spread to regional lymph nodes. RNA will be isolated and BRMS1 expression will be determined relative to control gene (S9) expression. Decreased expression is predicted to correlate with development of metastases. As a corollary, we plan to eventually ask whether differential expression of BRMS1 predicts the location of metastases (lymph node vs. viscera vs. bone) using archived advanced tissue specimens collected at UAB and other Breast SPOREs. Specific Aim 2 will test whether BRMS1 is wild-type or mutated. While functional data demonstrate that BRMS1 expression is inversely correlated with metastasis in breast cancer cell lines, existing data do not distinguish decreased expression and loss-of-function mutations. DNA will be isolated from LCM primary tumor cells, adjacent normal tissues and regional lymph node specimens and subjected to PCR-SSCP (single-strand conformational polymorphism) and sequenced.
Hypothesis 2: BRMS1 expression can be restored in metastatic breast carcinoma cells by treatment with 5-aza-deoxycytidine (5-azaC), a potent DNA hypomethylating agent. We also found that BRMS1 has a large (>1 kb) CpG island 5' to the start site and spanning exon 1. The open reading frame was not mutated, suggesting that acquisition of metastatic potential may be due to down-regulation of BRMS1. Although the prevalence of decreased expression vs. mutation are not known in breast cancers per se, our findings (and precedence from other metastasis suppressors) support the hypothesis that restoring BRMS1 expression and function, could reverse the metastatic phenotype. Furthermore, strategies to re-induce expression of metastasis suppressors could be developed to prevent and/or treat metastases, which is the concept to be tested in Specific Aim 3.
Specific Aim 3 is a series of pre-clinical studies to provide proof-of-principle that treatment with DNA hypomethylating agents (5-azaC and Zebularine) could restore BRMS1 metastasis suppressor expression, resulting in either loss of already-established metastases or preventing establishment of new metastases. These experiments will utilize cell lines that have no mutations in the protein-coding region but which have low/no expression. We propose pre-clinical experiments that we will translate into a Phase I/II trial during years 3 or 4 of the SPORE funding period.
Kirby I. Bland
The goal of this proposal is to analyze the KLF4/GKLF pathway as a marker and potential effector of an aggressive phenotype in early stage infiltrating ductal carcinoma (IDC). Functional studies in vitro and in mice, and analysis of human tumors identified KLF4 as a candidate breast cancer and squamous cell carcinoma (SCC) oncogene (1-3). In early stage breast cancers, preferential nuclear localization of KLF4 (termed Type 1 tumors) was associated with eventual death due to breast cancer (4). Based on these results, KLF4 was identified by the SPORE Biomarkers Working Group as a priority for further investigation (Appendix).
An inducible strategy enabled identification of genes that are directly regulated by KLF4, including Notch1 (Ntc1), p21/Waf1, Hes-1, c-Myc, TGF?, and others, leading to a model in which KLF4 functions to maintain the expression pattern of critical cell fate and patterning molecules that function in concert with KLF4 during morphogenesis of normal epithelium (5). Ntc1 has been identified as an integration site in mouse mammary tumor virus (MMTV)-induced breast cancer (6), and is frequently upregulated in human breast cancer, including T1-Type 1 tumors. Induction of Ntc1 is i) necessary for KLF4-induced transformation of rat RK3E epithelial cells, and ii) sufficient on its own to transform RK3E. Thus, KLF4 directly regulates oncogenes with compelling links to breast cancer pathogenesis (e.g., Ntc1) or clinical outcome (e.g., p21/Waf1) (7). Deregulation of KLF4 may explain how oncogenes such as Ntc1 or p21/Waf1 are activated in human tumors. In addition, the studies proposed may support a role for Ntc antagonists as therapeutic agents in Type 1 tumors.
Aim 1 To correlate expression of KLF4 and KLF4 regulated genes (target genes) in primary tumors.
Hypothesis: Type 1 tumors (i.e., tumors with elevated nuclear KLF4) have increased KLF4 transcriptional activity, as indicated by expression of the target genes Ntc1 and p21/Waf1.
Aim 2 To validate KLF4 as a prognostic marker in breast cancer (inter-SPORE study).
Hypothesis: Type 1 tumors represent a high-risk subset of early stage breast cancer
Aim 3 Examine the mechanism of transformation of human mammary epithelial cells by KLF4.
Hypothesis: Ntc1 mediates KLF4-induced transformation of MCF10A cells.
The transcription factor, Sp1, plays a key role in determining growth factor expression [1, 2]. As Sp1 is O-glycosylated, we have analyzed the effects on Sp1 of this modification. In summary, we have found that the enzyme that catalyzes this O-GlcNAc modification (OGT) of Sp1 resides in corepressor complexes that bind to those nuclear receptors that are not ligand-bound [3, 4]. Estrogen and other lipid-soluble hormones utilize OGT to repress the expression of genes that they target. In order to activate these same genes, the opposing enzyme, O-GlcNAcase must be present to remove the repressive sugars. Through an interaction domain, O-GlcNAcase binds to the OGT in the corepressor complexes, providing the means for the escape from repression to activation. Thus, when ligands, such as estrogen, bind to their receptors, the activation of genes requires O-GlcNAcase. Not only must O-GlcNAcase be physically present but it must be activated in order to catalyze the removal of these sugars. We have established that activation of the O-GlcNAcase enzyme occurs downstream of hormone signals, In ongoing studies, we are further determining the molecular mechanisms that govern this regulatory system. In this Project, we propose to develop and test a therapeutic approach in which the escape from repression is blocked, thereby permanently repressing the genes that are normally activated by estrogen. Notably, as the intervention is downstream of estrogen, then the growth of even estrogen-insensitive tumors may be blocked.
A. Specific Aims
A peptide was designed with two protein transduction domains (PTDs) which would cause the peptide to be internalized into cells to which it is targeted. For proof of principal, we first will target cells that express high levels of EGF receptors but the modularity of the peptide will allow other targeting strategies. The peptide also contains an interaction domain (ID) but no enzymatic activities. This ID allows the peptide to displace wildtype NCOAT, which has histone acetyltransferase and O-GlcNAcase activities , from corepression complexes. This displacement can occur because the peptide contains the domain for interaction . Thus, the peptide acts like a dominant negative on the basis of location. Expression of proteins with similarities to this peptide prevents estrogen-responsive genes from transitioning from a repressed state to an activated one. In preliminary results shown in the original proposal, that are being revised for publication as a paper, we have shown estrogen-responsive genes cannot be expressed by human breast cancer cells and estrogen-responsive development of transgenic mouse mammary glands is impaired. A diagram of the proposed peptide is shown in Fig 1 for clarity.
The first peptide made only had the N-terminal PTD (left PTD). We have since created a peptide with the C-terminal PTD as diagramed in Fig 1 so that the peptide has two PTDs. Two PTDs seems to cause more efficient internalization. This peptide is being expressed and purified at the present time. Nevertheless, we have used the peptide with one PTD on a human breast cancer cell line that expresses high levels of EGF receptors (MDA468 cells).
Using a peptide with one PTD, we showed inhibition of growth of MDA468 cells. Fig 2 show this inhibition (100 ?g/ml is equivalent to 5 ?M). The relatively high concentration of peptide might relate to the lower efficiency of cellular uptake that results from one PTD. Other controls are to replace the targeting module and ID each with green fluorescent protein. The presence and location of the peptide can be monitored by fluorescence microscopy. If the peptide cannot be targeted by TGF? it will be far less toxic. It should have no effect on growth if there is no ID (replaced with GFP). These new peptides are in the process of being produced right now.
1 The peptide design to be used in hormone-unresponsive breast cancer. Targeting is modular but for now will be to cells that express many EGF receptors. The "ID" is explained in the text.
2 Cells were exposed to the indicated peptide concentration for 4 days then counted. Since the peptide was cleaved from GST with thrombin, the peptidase was added without peptide (Thr).
Project 5 is a new project being initiated in the competitive renewal of the UAB SPORE in Breast Cancer. The project has developed from the Career Development Program of the Breast SPORE in collaboration with the UAB SPORE in Ovarian Cancer (both of which operate in the Women's Cancer Program of the Cancer Center). The overall goal of this proposal is to develop an effective therapeutic strategy for treatment of breast cancer by selectively targeting death receptor 5 (DR5; TNFRSF10B) with a novel monoclonal antibody (TRA-8) in combination with chemotherapy. The central hypothesis is that breast and other cancer cells may differentially express increasing levels of DR5 during malignant transformation and that DR5 can be selectively targeted with an agonistic monoclonal antibody to directly induce apoptosis of cancer cells. As TRA-8 and chemotherapy agents may utilize different but complementary pathways to trigger apoptosis, the susceptibility of breast cancer cells to TRA-8-mediated apoptosis can be enhanced by chemotherapy agents. While the hTRA-8 as a clinical candidate is moving toward clinical trials in cancer patients, there are several fundamental questions that need to be addressed in support of future clinical trials. The essential questions are what are the biomarkers for the susceptibility or resistance of breast cancer cells to DR5-mediated apoptosis and can we identify these biomarkers a priori to predict the patients' response to TRA-8 therapy? These biomarkers would be critical for selection of responsive breast cancer patients in phase II and III clinical trials. Second, our preliminary studies demonstrate a remarkable and enhanced anti-tumor efficacy when TRA-8 is combined with chemotherapy and suggest that TRA-8 increases chemosensitivity of cancer cells in part by preventing and/or reversing chemoresistance. Thus, an understanding of the key molecular regulatory elements that are involved in the synergistic induction of apoptosis of cancer cells by TRA-8 and chemotherapy and identification of the potentially distinct mechanisms for reversing chemoresistance are significant issues in the development of a more effective combination therapy.
AIM 1 (Pre-Clinical): To develop murine xenograft models with implanted primary breast carcinoma tissues and to examine the therapeutic efficacy of TRA-8 alone and in combination with chemotherapy.
1a. To develop murine xenograft models with implanted primary breast carcinoma tissues.
1b. To determine the expression levels of DR5 and susceptibility to TRA-8-mediated apoptosis in primary breast cancer cells.
1c. To determine the therapeutic efficacy of TRA-8 alone or in combination with chemotherapy in primary breast carcinoma xenograft models.
AIM 2 (Pre-Clinical): To elucidate the apoptosis signaling and regulatory mechanisms that determine the susceptibility of breast cancer cells to TRA-8-mediated apoptosis, and to identify the biomarkers for predicting tumor cell response to TRA-8 therapy.
2a. To locate DR5 signaling blockade by analysis of sequential activation of caspases.
2b. To identify the molecules causing DR5 signaling blockade by analysis of protein trafficking at the death domain of DR5 during TRA-8-mediated apoptosis.
2c. To compare the apoptosis regulatory network in TRA-8 sensitive and resistant breast cancer cells by analysis of expression and modification of the proteins of the Bcl-2 and IAP families.
AIM 3 (Pre-Clinical): To determine in vivo synergistic mechanisms of combination therapy with TRA-8 and chemotherapy.
3a. To determine the therapeutic efficacy of TRA-8 and chemotherapy combination in TRA-8 resistant breast cancer cells.
3b. To determine the therapeutic efficacy of TRA-8 and chemotherapy combination in chemoresistant breast cancer cells.
3c. To determine the synergistic mechanisms by analyzing the apoptosis proteome profiles of xenograft tumors treated with combination therapy.
AIM 4 (Clinical): To start clinical development of TRA-8.
4a. Phase I Trial. We will carry out a phase I dose escalation trial to determine safety and maximal tolerated dose (MTD) of hTRA-8 (CS-1008) given intravenously (IV) as a single agent in patients with relapsed/refractory malignancies resistant to standard therapies.
4b. Phase II Trial in Breast Cancer. We will carry out a phase II trial to determine efficacy of hTRA-8 (CS-1008) given intravenously (IV) as a single agent in patients with relapsed/refractory metastatic breast cancer who have failed at least 2 standard chemotherapy regimens for metastatic breast cancer.
4c. Phase I/II Trial in Metastatic Breast Cancer Patients using a Combination of TRA-8 (CS-1008) and Chemotherapy. Once we have completed our phase I dose escalation trial and initiated the phase II single agent inter-SPORE trial, we will carry out a phase I/II trial to determine safety and the maximal tolerated dose of hTRA-8 (CS-1008) given intravenously (IV) in combination with chemotherapy (phase I) and later on to establish efficacy (phase II) of the combination in patients receiving initial chemotherapy for metastatic breast cancer.