Victoria Seewaldt - US grants

DESCRIPTION (Provided by applicant): Abnormal p53 expression in benign breast tissue is associated with the subsequent development of breast cancer and may represent a very early event in breast carcinogenesis. While the importance of p53 as a tumor suppressor is well established, little is known about the fate of normal human mammary epithelial cells (HMECs) that acutely loose p53 function in the context of extracellular matrix-derived growth and differentiation signals. Loss of epithelial polarity and dysregulated growth (mammary hyperplasia) are the first morphologic abnormalities observed during the progression from normal mammary architecture to invasive breast cancer. Interactions between mammary epithelial cells and extracellular matrix (ECM) play a critical role in maintaining normal tissue homeostasis and are likely disrupted in mammary hyperplasia. We developed an in vitro system to investigate how ECM-signal transduction might act in HMECs that have acutely loss p53 function. We observe in Preliminary Data, that ECM-growth arrest and polarity signals may act to initiate apoptosis in HMECs with HPV-16 E6-suppressed p53 expression. Resistance to ECM-mediated growth arrest and epithelial polarity is associated with resistance to apoptosis. We hypothesize that ECM-derived signals may play an important role in preventing the clonal expansion of abnormal mammary epithelial cells by promoting apotosis of cells that acquire p53 mutations and therefore, resistance to ECM-signaling might promote breast carcinogenesis by inhibiting apoptosis. Our model would predict that 1) HMECs acquiring a p53 mutation in the context of a normal polarized epithelium will be eliminated by apoptosis but 2) when HMECs acquire a p53 mutation in the absence of normal ECM growth regulation and polarity signals (mammary hyperplasia) aberrant cells will not be eliminated by apoptosis. Specific Aim I, will test the requirement for p53 loss in ECM-mediated apoptosis: 1) p53 function in HMECs will be suppressed by non-viral approaches and 2) suppression of the p53-regulatory proteins CBP/p300. Specific Aim II, will test whether loss of epithelial polarity and resistance to ECM-mediated growth arrest results in resistance to apoptosis utilizing two in vitro human models of mammary hyperplasia. Suppression of ECM-mediated growth arrest and epithelial polarity will be accomplished by retroviral-mediated expression of dominant-negative retinoid receptor and by treatment of three dimensional ECM cultures with alpha6- and beta4-integrin blocking antibodies.

DESCRIPTION (provided by applicant): Although the molecular mechanism of tamoxifen action in estrogen receptor-"positive" (ER+) breast cancer cells is well studied, there is little information on how tamoxifen may act in normal ER-"poor" human mammary epithelial cells (HMECs). This information is critical for developing breast cancer prevention strategies with defined endpoints. To model p53 loss in the context of tamoxifen chemoprevention, we tested in vitro whether tamoxifen was able to eliminate HMECs immediately after suppression of p53 function. Surprisingly, when p53 was acutely suppressed, the resultant p53(-) HMECs underwent apoptosis when treated with clinically relevant concentrations oftamoxifen, p53(+) controls underwent growth arrest only. Induction of apoptosis correlated with a rapid reduction of Akt-1 phosphorylation, decreased expression of Bcl-2, mitochondrial depolarization, and caspase-activation. The "classic" mechanism of steroid action requires the presence of ER and both transcription and translation. However, there is evidence that estrogen and perhaps antiestrogens may act through "non-classic" signaling pathways. In Preliminary Data, we observed that induction of apoptosis was not induced by 4-OHT or ICI 182,780. Based on Preliminary Data we hypothesize that induction of apoptosis by tamoxifen in p53(-) HMECs may occur via a "non-classic" Akt-regulated pathway. While early passage p53(-) HMECs initially exhibited tamoxifen-sensitivity, resistance rapidly developed and correlated with persistent Akt-phosphorylation, lack of mitochondrial depolarization and loss of expression of the CREB-binding protein, CBP. Observations in our model system predict that if HMECs loose p53 function in the absence of tamoxifen chemoprevention there is a high likelihood that tamoxifen-resistance will subsequently develop. SA I will test l) whether ER modulates sensitivity to tamoxifen-induced apoptosis in early passage p53(-) HMECs utilizing phage display, ER-selective peptide ligand and 2) whether tamoxifen induces apoptosis in p53 (-/-) ER-"poor" mammary cells in vivo. SA II will test whether l) inhibition of Akt phosphorylation in p53(-) HMECs promotes tamoxifen-induced apoptosis and 2) Akt mediates CBP-phosphorylation. SA III will test whether tamoxifen promotes apoptosis in early passage p53(-) HEMCs by altering the ratio of Bcl-2/Bad and test the relationship between Akt phosphorylation and Bcl-2/Bad activity. Studies proposed in SAs will characterize this novel mechanism of tamoxifen-signaling and define potential biologic markers of response. Our long-term goal is to utilize this signal-transduction pathway to model novel combinations of hormonal and non-hormonal agents and then pilot these novel strategies in small-scale prevention trials utilizing markers developed in this proposal to test for response.

DESCRIPTION (provided by applicant): Interactions between normal human mammary epithelial cells (HMECs) and extracellular matrix (ECM) are critical for maintaining normal mammary gland homeostasis and loss ECM-signaling is observed during early mammary carcinogenesis. During the original funding period of this grant, we tested the hypothesis that 1) ECM-mediated growth regulation signals target the elimination of acutely damaged HMECs and 2) loss of ECM-signaling may provide a local environment or "high-risk epithelial field" that promotes the survival of a second genetic "hit". We identified an important role for the CREB-binding protein, CBP, in regulating apoptosis in HMECs that had acutely lost p53 function (*p53(-)HMECs). CBP promoted apoptosis in conjunction with 1) loss of CBP phosphorylation at Thr1871, 2) recruitment of CBP to the interferon regulatory factor-1 (IRF-1) GAS element, and 3) induction of IRF-1. These observations suggest that CBP acts to target the elimination of HMECs that have acutely lost p53 function and loss of CBP expression may provide an environment that promotes the survival of a second genetic "hit". Random Periareolar Fine Needle Aspiration (RPFNA) is a research tool developed to test for "high-risk" epithelial field effects in women at high-risk for breast cancer. In Preliminary Data, we used RPFNA to show that loss of CBP predicted atypia in mammary epithelial cells but only in specimens that expressed low levels of ER. Based on these observations, we will test the hypothesis that 1) CBP targets the elimination of acutely damaged p53(-) HMECs through recruitment of CBP to the IRF-1 GAS-element and 2) in high-risk women that loss of CBP promotes ER(-) mammary carcinogenesis. Aim I will test whether loss of CBP phosphorylation at Thr1873 promote apoptosis in *p53(-)HMECs through recruitment of CBP to the IRF-1 GAS-element enhanced affinity of the CBP CHS domain for STAT1 and 2) recruitment of CBP to the IRF-1 GAS element. Aim 2 will test whether the survival factor Akt regulate phosphorylation of in vitro and in vivo phosphorylation studies of tryptic digests of either expressed or isolated CBP by HPLC-TOF-MS. Aim 3 will test whether loss of CBP in high-risk women predict ER(-) cytological progression. Significance: Completions of these aims will provide insight into early events in ER(-) mammary carcinogenesis and provide novel targets for breast cancer chemoprevention.

DESCRIPTION (provided by applicant): Biomarkers are needed to accurately predict short-term breast cancer risk, so that 1) women who are most likely to benefit from preventive therapy can be identified, and 2) response to chemoprevention can be accurately assessed. This proposal aims to characterize a signal-transduction pathway that we hypothesize plays an important role in targeting the elimination of acutely damaged mammary epithelial cells. In preclinical models, we observe that 1) the retinoic acid receptor-beta 2 (RARbeta2) and the CREB-binding protein, CBP, regulate proliferation and promote apoptosis in acutely damaged mammary epithelial cells, 2) RARbeta2 regulates the expression of CBP in a positive feedback loop, and 3) suppression of RARbeta2 or CBP inhibits apoptosis. Based on these observations we will test the hypothesis that 1) RARbeta2 and CBP promote apoptosis in acutely damaged HMECs through IRF-1-induction and 2) RARbeta2 P2 methylation predicts loss of CBP expression in mammary epithelial cells obtained from high-risk women. Specific Aim I will test whether RARbeta2 and CBP promote apoptosis in acutely damaged HMECs through recruitment to the IRF-1 promoter and indution of IRF-1. Specific Aim II will test whether the presence of RARbeta2 P2 promoter methylation predicts loss of CBP expression in mammary epithelial cell strains obtained from women at high-risk from breast cancer. Specific Aim III will use a research technique called Random Periareolar Fine Needle Aspirate (RPFNA) to test whether methylation inactivation of RARbeta2 in high-risk women promotes 1) loss of RARbeta2 and CBP expression and 2) predicts short-term cytological progression. The correlation between RARbeta2 P2 methylation and CBP expression in RPFNA will be tested by MS-PCR and quantitative RT-PCR in microdissected epithelial cell clusters. Cytological progression will be tested in high-risk women in our chemoprevention cohort. Relevance to public health: Information obtained in this proposal can be immediately used to 1) improve our ability to predict short-term breast cancer-risk and 2) develop makers to test whether women who are at high-risk for breast cancer are responding to prevention agents.

DESCRIPTION (provided by applicant): Triple-negative breast cancers [ER/PR-/-, HER2/neu wt, EGFR+] frequently occur in young women and carry a poor prognosis. While not all triple-negative breast cancers are lethal, triple-negative breast cancers have 86% 5-year mortality in pre-menopausal African American women. Since many triple-negative breast cancers are chemotherapy-resistant at diagnosis, there is a great need for early detection. Here we aim to investigate a novel signaling pathway that holds promise for early detection of triple-negative breast cancer.TASK3 is a pH sensitive potassium channel protein that regulates mitochondrial membrane potential m). Overexpression of TASK3 increases m, and thereby promotes apoptosis resistance and tolerance of hypoxia. In Preliminary Data, we show that TASK3 is overexpressed in 1) chemotherapy-resistant metastatic triple-negative breast cancers and 2) premalignant breast disease in high-risk African American women. KCNK9 (TASK3 gene) is regulated by imprinting: loss of imprinting predicts chemotherapy resistance and subsequent metastasis of triple-negative breast cancer: The gene coding for the TASK3 protein, KCNK9, is regulated by methylation imprinting. Imprinting is a normal regulatory process where one copy of the gene is inactivated resulting in mono-allelic gene expression. Loss of normal imprinting results in a functional diploid state and overexpression of the target gene. One copy of the KCNK9 gene is normally imprinted, making the normal functional state of KCNK9 haploid (one gene copy expressed, one copy not expressed). In Preliminary Data, we identified a differentially methylated region (DMR) in the KCNK9 promoter that was imprinted in normal mammary epithelial cells but not in primary chemotherapy-resistant triple-negative breast cancers. Loss of DMR imprinting predicted TASK3 overexpression, chemotherapy-resistance, and subsequent metastasis. Hypothesis: Loss of methylation imprinting of KCNK9 DMR and hypoxia synergistically promote TASK3 overexpression and metastasis in triple-negative breast cancer. Aim 1 will test whether hypoxia transcriptionally activates TASK3 in triple-negative breast cancers that lack imprinting of the KCNK9 DMR. We will perform in vivo multi-parametric analysis of KCNK9 HRE activity, TASK3 expression, tumor growth and metastasis, and hemoglobin saturation in normoxic and hypoxic conditions. Aim 2 will test whether loss of normal KCNK9 DMR imprinting promote initiation, progression, and metastasis of triple-negative breast cancer. Significance: Successful completion of the aims of this proposal will improve our ability to identify biologically aggressive metastatic triple-negative breast cancer. Triple-negative breast cancers have a high mortality in premenopausal African American women. Our established high-risk cohort will allow us to rapidly test whether loss of KCNK9 DMR imprinting promotes chemotherapy-resistant metastatic triple- negative breast cancers.

DESCRIPTION (provided by applicant): Currently, targeted agents are in clinical testing for treatment of ER- breast cancer, but have not been adequately tested for prevention. This is because, without a better understanding of the biology of human breast cancer initiation, Phase I/II testing of targeted agents for preventing ER- breast cancer will be too risky and expensive. To meet these challenges, we aim to identify key signaling networks activated during initiation of ER- breast cancer in women in our high-risk cohort and use these networks to target prevention. Normal mammary gland homeostasis requires the coordinated regulation of signaling networks. Currently we lack an understanding of 1) whether the signaling networks that are activated in aggressive ER- breast cancer are also activated in mammary atypia, and 2) if so, whether activation of specific signaling networks predicts cancer initiation and progression. Here we will investigate whether signaling network activation in atypia predicts subsequent cancer etiology. Activation of Akt/mTOR, IL6/Stat3, EGRF/MEK/ERK, and mitochondrial survival pathways are known to predict aggressive biology in ER- breast cancer. Our Preliminary Data provide evidence that activation of these poor-prognosis signaling networks can be detected in mammary atypia from high risk women. However, just because we can identify signatures with high inter-class coefficients of variation between subjects, does not necessarily imply those processes are important for cancer etiology. Here will test the hypothesis that signaling pathways that underlie the aggressive behavior of ER- breast cancer can be detected in mammary atypia from high-risk women, and whether these signaling pathways can be used to predict cancer initiation and guide targeted prevention strategies. Aim 1 will test whether phosphoprotein signatures identified in ER- breast cancers are present in premalignant lesions in high-high risk women. Aim 2 will prospectively investigate whether phosphoprotein signatures predict initiation of ER- breast cancer. Aim 3 will investigate whether RPPM signatures predict in vitro sensitivity to targeted agents. Aim 4 will perform Pilot Testing and use phosphoprotein signatures to select and track response to targeted agents to prevent breast cancer in high-risk women with mammary atypia. Significance: Here we will identify protein signaling pathways that are activated during breast cancer initiation. Information gained in this proposal will allow us to identify activated signaling pathways in atypia, test whether pathway activation predicts cancer etiology, and use this information to select, and track response to, targeted agents.

DESCRIPTION (provided by applicant): In the United States obesity rates are increasing, and understanding the molecular and cellular basis for breast cancer associated with obesity is of significant clinical relevance and impact. Obese women are more likely to be diagnosed with non-familial estrogen receptor negative tumors than lean women, and these tumors are more likely to be associated with nodal metastases. Since breast stromal tissue is a reservoir of subcutaneous fat the stromal tissue microenvironment is known to have a profound effects on breast cancer development and progression, the changes that take place within the fat depots of obese individuals may play a significant role in the pathogenesis of breast cancer. Therefore, elucidating the mechanistic role of adipocytes and adipose tissue biology in breast cancer is critical for prevention and treatment of obesity-related cancer. Fat cells within the breast tissue of obese women secrete a monocyte chemo-attractant protein-1 (MCP-1) that promotes the recruitment of macrophages into breast adipose tissue. These cells when recruited induce local tissue inflammation, but role of macrophages in obesity-associated inflammation during is currently unknown. Here, we aim to test from bench to human clinical trials, the hypothesis that breast tissue in obese women exhibits increased neoangiogenesis and inflammation prior to overt tumor formation due to the recruitment of macrophages by MCP-1 producing adipocytes, thereby leading to increased vascularity and malignancy. In Aim 1 of this project, we will investigate the molecular mechanism by which bone marrow-derived macrophages promote obesity-induced angiogenesis and cancer initiation. Aim 2 will test whether bone marrow-derived macrophages are necessary for obesity-induced tumor progression, and Aim 3 will determine whether obesity-induced preneoplastic changes can be reversed. Our studies provide innovative insight as to why obesity is associated with increased cancer incidence and aggressiveness. In addition, this work will provide a novel paradigm for the prophylactic treatment of high risk obese patients.

? DESCRIPTION (provided by applicant): Triple-negative breast cancers (TNBC) exhibit aggressive tumor biology and carry a poor prognosis, particularly in premenopausal African American (AA) women who carry a disproportionate burden of breast cancer mortality. The precursor lesion for TNBC is poorly characterized and the pathophysiology of TNBC is not well understood so therapies often fail to achieve complete pathological response and the disease is frequently non-curative. This proposal aims to clarify the molecular pathology of TNBC so that biomarkers for early diagnosis, prevention strategies and curative therapies can be developed. In our high-risk, multi-institutional cohort, with a high percent of AA women, we found that during breast cancer initiation, pStat3 is high as is ECM stiffness and integrin/YAP mechanosignaling, and miRNAs implicated in tumor progression/aggression. TNBCs had the highest inflammation, pStat3 and miR-18a, the stiffest ECM and the lowest miR-203. Mouse studies indicated preventing inflammation decreases fibrosis and that reducing ECM stiffening lower pStat3 and inflammation and EMT and metastasis. Driving mammary mechanosignaling induced miR-18a and EMT and enhanced tumor aggression/metastasis. This suggests that an activated Stat3/tissue tension feedback loop, linked to tissue inflammation, promotes TNBC by engaging mechanosignaling pathways that alter miRs and induce an EMT and tumor aggression. While some breast cancers arise from focal lesions, TNBCs often appear to arise diffusely. We predict that in women at high-risk for TNBC (familial association, BRCA1 mutation) there is a dynamic and reciprocal relationship between the at risk epithelium and tissue tension that activates mechano-signaling pathways and induces Stat3/miRNA to 1) initiate TNBC, 3) induce an EMT and/or enhance tumor aggression, that 3) can be used to idenify precancerous lesions that have a high likelihood of progression to TNBC, and 4) could be used to monitor efficacy of prevention strategies and identify targets to improve TNBC treatment. We will use preclinical models to test: 1) if there is a reciprocal relationship between inflammation, pStat3 and tissue tension that promotes TNBC progression/aggression and 2) if this is mediated through miRs and EMT. We will examine a clinical cohort of high risk women who rapidly develop TNBCs to 3) test the prevalence of this signaling circuit in biopsies from women with TNBC and determine whether these biomarkers can identify precancerous lesions that have a high likelihood of progression to TNBC. Significance: Our studies could transform concepts of breast cancer by demonstrating that tissue tension could molecularly-prime tissue to malignancy. Markers that identify preneoplastic changes in TNBC, that could be used to monitor efficacy of risk reduction strategies, would have a transformative impact on TNBC mortality rates and particularly AA women.

DESCRIPTION (provided by applicant): Currently, targeted agents are in clinical testing for treatment of ER- breast cancer, but have not been adequately tested for prevention. This is because, without a better understanding of the biology of human breast cancer initiation, Phase I/II testing of targeted agents for preventing ER- breast cancer will be too risky and expensive. To meet these challenges, we aim to identify key signaling networks activated during initiation of ER- breast cancer in women in our high-risk cohort and use these networks to target prevention. Normal mammary gland homeostasis requires the coordinated regulation of signaling networks. Currently we lack an understanding of 1) whether the signaling networks that are activated in aggressive ER- breast cancer are also activated in mammary atypia, and 2) if so, whether activation of specific signaling networks predicts cancer initiation and progression. Here we will investigate whether signaling network activation in atypia predicts subsequent cancer etiology. Activation of Akt/mTOR, IL6/Stat3, EGRF/MEK/ERK, and mitochondrial survival pathways are known to predict aggressive biology in ER- breast cancer. Our Preliminary Data provide evidence that activation of these poor-prognosis signaling networks can be detected in mammary atypia from high risk women. However, just because we can identify signatures with high inter-class coefficients of variation between subjects, does not necessarily imply those processes are important for cancer etiology. Here will test the hypothesis that signaling pathways that underlie the aggressive behavior of ER- breast cancer can be detected in mammary atypia from high-risk women, and whether these signaling pathways can be used to predict cancer initiation and guide targeted prevention strategies. Aim 1 will test whether phosphoprotein signatures identified in ER- breast cancers are present in premalignant lesions in high-high risk women. Aim 2 will prospectively investigate whether phosphoprotein signatures predict initiation of ER- breast cancer. Aim 3 will investigate whether RPPM signatures predict in vitro sensitivity to targeted agents. Aim 4 will perform Pilot Testing and use phosphoprotein signatures to select and track response to targeted agents to prevent breast cancer in high-risk women with mammary atypia. Significance: Here we will identify protein signaling pathways that are activated during breast cancer initiation. Information gained in this proposal will allow us to identify activated signaling pathways in atypia, test whether pathway activation predicts cancer etiology, and use this information to select, and track response to, targeted agents.

ABSTRACT Cancer drugs in the United States are almost exclusively developed by, tested in, and optimized for European- Americans. Disparities in U.S. drug development are the result of long-standing inequalities that occur throughout the entire drug discovery pipeline. Only a small number of basic, translational and clinical scientists are Latino/Hispanic or Black/African-American. Less than 2-5% of trial participants are Latino/Hispanic or Black/African-American. Yet, in spite of a lack of data, new drugs are approved by the U.S. Food and Drug Administration (FDA) and subsequently prescribed for Latino/Hispanic or Black/African-American patients, without sufficient testing. It is unacceptable, especially in this era of the Precision Medicine Initiative (PMI), that currently our drugs are developed by, and optimized for, only for an exclusive segment of our citizens. We seek to shift this paradigm by strengthening the existing partnership between University of California at Riverside (UCR) and City of Hope Comprehensive Cancer Center (CoH-CCC). In this proposed P20, UCR and CoH-CCC will partner to develop the resources, infrastructure, and training programs necessary to develop the next generation of therapeutics researchers that reflect the ethnic diversity of Inland Southern California. We anticipate that our P20 program will drive new collaborative R01 grants and K01 and T-type training grants. .