California Breast Cancer Research Program Research Articles

Californians Linking Action with Science for Prevention of Breast Cancer (CLASP-BC)—Phase 2

Californians Linking Action with Science for Prevention of Breast Cancer (CLASP-BC) is part of California Breast Cancer Research Program’s (CBCRP) Initiative strategic priority to disseminate and implement high-impact, population-based primary prevention interventions. CLASP-BC is informed by six years of funded program dissemination and implementation (D&I) research and evaluation conducted by the Canadian Partnership Against Cancer (CPAC) through its Coalitions Linking Action and Science for Prevention (CLASP). In its second phase, CLASP-BC will fund multi-sector, multi-jurisdictional initiatives that integrate the lessons learned from science with the lessons learned from practice and policy to reduce the risk of developing breast cancer and develop viable and sustainable infrastructure models for primary prevention breast cancer programs and research evidence implementation. Applications will be solicited from research, practice, policy, and community teams to address one or more of the intervention goals for the 23 risk factors identified in Paths to Prevention: The California Breast Cancer Primary Prevention Plan (P2P), expanding upon existing primary prevention efforts into two or more California jurisdictions, focused on disadvantaged, high risk communities with unmet social needs. The lessons learned from CLASP-BC will be widely disseminated within the participating jurisdictions, across California and, where applicable, to jurisdictions outside the state.

Abstract LB-391: Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) antagonist with anti-PD-L1 antibody blocks triple-negative breast cancer (TNBC) progression

Abstract Triple-negative breast cancer (TNBC) affects only about 10-15% of women with breast cancer (BC) but accounts for almost 50% of all BC deaths. At this time, development of targeted treatments for TNBC is urgently needed. Insulin-like growth factor-2 (IGF2) is highly expressed in TNBCs in African-American and Latina patients. Since hormone receptors, like insulin-like growth factor-1/insulin (IGF1R/IR, are known to be enriched and to cross-communicate in subtypes of TNBC, we assessed effects of IGF1R/IR antagonists on proliferation of human and murine TNBC cells in vitro. Combination treatment of BMS-754807 (IGF1R/IR) with NVP-AEW541 (IGF1R inhibitor) reduced proliferation of human MDA-MB-231, BT549, HCC1937 and murine 4T1 TNBC cells in vitro (P<0.001). Moreover, combination treatment using BMS-754807 and NVP-AEW541 significantly reduced MDA-MB-231 tumor xenograft progression in vivo (P<0.001). IHC study of tumor tissues from in vivo experiments confirmed reduced Ki-67 expression with combination treatments. Among breast cancer subtypes, TNBC is characterized by significant numbers of tumor infiltrating lymphocytes (TILs), a predictive marker for response to immunotherapy. Further, recent reports from clinical trials using immune checkpoint inhibitors demonstrate that a subset of TNBC patients respond to treatment with immune checkpoint inhibitors (ICIs). It is also notable that increased expression of IGF1Rs are identified on immune cells responsible for mounting adaptive and humoral immune responses. Using murine 4T1 TNBC implants in syngeneic, immune-competent BALB/c mice, we find that combination treatment with BMS-754807 and anti-PD-L1 antibody significantly blocks tumor progression as compared to both controls and with either agent used alone (P<0.0001). In addition, single cell analysis of the tumor microenvironment after treatment with BMS-754807 alone or combined with nti-PD-L1 antibody combination therapy shows a significant reduction in myeloid-derived suppressor cells (MDSCs). Moreover, BMS-754807 combined with anti-PD-L1 antibody increased infiltrating effector T cells and significantly enhanced the T central memory population (Tcm) (P<0.01). Functionally, it is reported that Tcm subsets of immune cells produce effector cytokines in response to antigens and can readily proliferate and differentiate into effector cells. Use of immunotherapy is a promising management option for a subset of TNBC patients, and combination treatments using IGF1R antagonists with immune checkpoint inhibitor may constitute a new treatment strategy to combat this deadly disease. [Funding by NCI U54 CA1433930; California Breast Cancer Research Program; UCLA Jonsson Citation Format: Nalo M. Hamilton, Diana C. Marquez-Garban, Lorena P. Burton, Begonya Comin-Anduix, Alejandro J. Garcia, Jaydutt V. Vadgama. Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) antagonist with anti-PD-L1 antibody blocks triple-negative breast cancer (TNBC) progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-391.

Doing What We Know, Knowing What to Do: Californians Linking Action with Science for Prevention of Breast Cancer (CLASP-BC).

Given the lack of progress in breast cancer prevention, the California Breast Cancer Research Program (CBCRP) plans to apply current scientific knowledge about breast cancer to primary prevention at the population level. This paper describes the first phase of Californians Linking Action with Science for Prevention of Breast Cancer (CLASP-BC). The foci of Phase 1 are building coalitions and coalition capacity building through community engagement in community-based participatory research (CBPR) and dissemination and implementation (D&I) research training. Based on the successful implementation and evaluation of Phase 1, the foci of Phase 2 (presented separately in this special issue) will be to translate the California Breast Cancer Prevention Plan overarching goal and specific intervention goals for 23 breast cancer risk and protective factors strategies into evidence-informed interventions (EIIs) that are disseminated and implemented across California. CLASP-BC is designed to identify, disseminate and implement high-impact, population-based prevention approaches by funding large scale EIIs, through multi-jurisdictional actions, with the intent to decrease the risk of breast cancer and other chronic diseases (sharing common risk factors), particularly among racial/ethnic minorities and medically underserved populations in California.

Abstract D073: Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) and androgen receptor (AR) antagonists with metformins inhibits triple-negative breast cancer (TNBC) progression in vitro and in vivo

Abstract Triple-negative breast cancer (TNBC) affects only about 15% of women with breast cancer (BC) but accounts for almost 50% of all BC deaths. At this time, development of targeted treatments for TNBC is urgently needed. Insulin-like growth factors and diabetes mellitus type 2 are noted to be associated with increased incidence of estrogen receptor-negative BCs such as TNBC particularly among African American patients. Since receptors for insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) and androgen receptor (AR) are known to be enriched and to cross-communicate in subtypes of TNBC (Hamilton N et al., Int J Mol Sci 2017;18:2305 [PMCID: PMC5713274]; Hamilton N et al., Biomed Res Int. 2015;2015:925703 [PMCID: PMC4385615]), we first assessed effects of these receptor antagonists on proliferation of human and murine TNBC cells in vitro. Combination treatment with BMS-754807 and/or NVP-AEW541 (IGF1R/IR inhibitors) plus enzalutamide (AR inhibitor) effectively reduced proliferation of human MDA-MB-231, BT549, HCC1937 and murine 4T1 TNBC cells in vitro (P<0.001). Moreover, metformin and analogues of metformin that inhibit TNBC progression in part by interactions with AMPK and with IGFR1/IR pathways elicited further suppression of TNBC cell proliferation when combined with the IGFR1/IR-AR antagonists (P<0.001). Importantly, the combination treatments also reduced the migration in vitro of human and murine TNBC cells to a greater extent than any single agent given alone. Calculations to determine the coefficient of drug interaction (CDI) revealed therapeutic synergy in drug combinations predominantly in those containing BMS-754807. Of note, combination treatments containing BMS-754807 were found to significantly stimulate excess reactive oxygen species (ROS) in vitro, a process that may promote tumor cell apoptosis. In ongoing experiments, novel metformin analogues are also found to inhibit human MDA-MB-231 xenograft progression in vivo (P<0.001). Our findings suggest that combination treatments containing IGF1R/IR and AR antagonists alone and combined with metformins suppress TNBC progression, with potential to impact patient outcome in the future. [Funding by NCI U54 CA1433930; California Breast Cancer Research Program]. Citation Format: Nalo M. Hamilton, PhD, APRN-BC, Diana C. Márquez-Garbán, MD, Michael E. Jung, PhD, Jaydutt Vadgama, Richard J. Pietras, MD, PhD. Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) and androgen receptor (AR) antagonists with metformins inhibits triple-negative breast cancer (TNBC) progression in vitro and in vivo [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D073.

An Expanded Agenda for the Primary Prevention of Breast Cancer: Charting a Course for the Future.

Advances in breast cancer science, early detection, and treatment have resulted in improvements in breast cancer survival but not in breast cancer incidence. After skin cancer, breast cancer is the most common cancer diagnosis in the United States. Each year, nearly a quarter million U.S. women receive a breast cancer diagnosis, and the number continues to rise each year with the growth in the population of older women. Although much remains to be understood about breast cancer origins and prevention, action can be taken on the existing scientific knowledge to address the systemic factors that drive breast cancer risk at the population level. The California Breast Cancer Research Program funded a team at Breast Cancer Prevention Partners (BCPP) to convene leaders in advocacy, policy, and research related to breast cancer prevention from across the state of California. The objective was the development of a strategic plan to direct collective efforts toward specific and measurable objectives to reduce the incidence of breast cancer. The structured, innovative approach used by BCPP to integrate scientific evidence with community perspectives provides a model for other states to consider, to potentially change the future trajectory of breast cancer incidence in the United States.

Targeted Transdermal Delivery of Curcumin for Breast Cancer Prevention.

N.B. This manuscript is based on the research concept submitted to the “Global Challenge to Prevent Breast Cancer” idea showcase and competition, launched in 2018 by the California Breast Cancer Research Program (CBCRP), which was subsequently selected for publication. The hypothesis, methods, and discussion put forth here are thus proposed concept studies, which could eventually be elucidated in the future. Curcumin is an herbal supplement, shown in preclinical studies to have antioxidant, anti-inflammatory, and antitumoral properties that we believe can be harnessed for breast cancer prevention. However, due to its poor absorption when consumed orally, curcumin’s anticancer effects have not yet been exploited to their full therapeutic potential. Incorporating existing research that focuses on the optimization of curcumin’s bioavailability and the latest transdermal delivery technology, we propose, below, a hypothetical in vivo study to test whether a targeted daily dose of bioavailable curcumin has a cytotoxic effect on cancer cells, potentially reducing the incidence of breast cancer over time. Our ultimate objective is to adopt innovative methods to create curcumin-infused bio-textiles offering transdermal, targeted drug delivery, simply through contact with the skin. We would use this fabric to create disposable bra inserts for an effortless, daily breast cancer prevention regimen for healthy women. It would be essential that the cost of these inserts remain reasonable, but if successful, curcumin is readily available, affordable and non-toxic, and could thus be a preventive measure that would be beneficial for women from all socio-economic backgrounds.

TU‐CD‐303‐05: Unveiling Tumor Heterogeneity by Molecular Imaging

Recent advances in cancer research have shed new light on the complex processes of how therapeutic radiation initiates changes at cellular, tissue, and system levels that may lead to clinical effects. These new advances may transform the way we use radiation to combat certain types of cancers. For the past two decades many technological advancements in radiation therapy have been largely based on the hypothesis that direct radiation‐induced DNA double strand breaks cause cell death and thus tumor control and normal tissue damage. However, new insights have elucidated that in addition to causing cellular DNA damage, localized therapeutic radiation also initiates cascades of complex downstream biological responses in tissue that extend far beyond where therapeutic radiation dose is directly deposited. For instance, studies show that irradiated dying tumor cells release tumor antigens that can lead the immune system to a systemic anti‐cancer attack throughout the body of cancer patient; targeted irradiation to solid tumor also increases the migration of tumor cells already in bloodstream, the seeds of potential metastasis. Some of the new insights may explain the long ago discovered but still unexplained non‐localized radiation effects (bystander effect and abscopal effect) and the efficacy of spatially fractionated radiation therapy (microbeam radiation therapy and GRID therapy) where many “hot” and “cold” spots are intentionally created throughout the treatment volume. Better understanding of the mechanisms behind the non‐localized radiation effects creates tremendous opportunities to develop new and integrated cancer treatment strategies that are based on radiotherapy, immunology, and chemotherapy. However, in the multidisciplinary effort to advance new radiobiology, there are also tremendous challenges including a lack of multidisciplinary researchers and imaging technologies for the microscopic radiation‐induced responses.A better grasp of the essence of these advances in cancer biology research will give medical physicists a new perspective in daily clinical physics practice and in future radiation therapy technological development. Furthermore, academic medical physics should continue to be an integral part of the multidisciplinary cancer research community, harnessing our newly acquired understanding of radiation effects, and developing novel cost‐effective treatment strategies to better combat cancer.Learning Objectives: Understand that localized radiation can lead to non‐localized secondary effects such as radiation‐induced immune response, bystander effect, and abscopal effect. Understand that the non‐localized radiation effects may be harnessed to improve cancer treatment. Learn examples of physics participation in multidisciplinary research to advance cancer biology. Recognize the challenges and possibilities of physics applications in cancer research. Chang: NIH 5RC2CA148487‐02 and 1U54CA151652‐01Graves: IDEA award (19IB‐0106) from the California Breast Cancer Research Program (CBCRP), and by NIH P01 CA67166

TU‐CD‐303‐02: Beyond Radiation Induced Double Strand Breaks ‐ a New Horizon for Radiation Therapy Research

Recent advances in cancer research have shed new light on the complex processes of how therapeutic radiation initiates changes at cellular, tissue, and system levels that may lead to clinical effects. These new advances may transform the way we use radiation to combat certain types of cancers. For the past two decades many technological advancements in radiation therapy have been largely based on the hypothesis that direct radiation‐induced DNA double strand breaks cause cell death and thus tumor control and normal tissue damage. However, new insights have elucidated that in addition to causing cellular DNA damage, localized therapeutic radiation also initiates cascades of complex downstream biological responses in tissue that extend far beyond where therapeutic radiation dose is directly deposited. For instance, studies show that irradiated dying tumor cells release tumor antigens that can lead the immune system to a systemic anti‐cancer attack throughout the body of cancer patient; targeted irradiation to solid tumor also increases the migration of tumor cells already in bloodstream, the seeds of potential metastasis. Some of the new insights may explain the long ago discovered but still unexplained non‐localized radiation effects (bystander effect and abscopal effect) and the efficacy of spatially fractionated radiation therapy (microbeam radiation therapy and GRID therapy) where many “hot” and “cold” spots are intentionally created throughout the treatment volume. Better understanding of the mechanisms behind the non‐localized radiation effects creates tremendous opportunities to develop new and integrated cancer treatment strategies that are based on radiotherapy, immunology, and chemotherapy. However, in the multidisciplinary effort to advance new radiobiology, there are also tremendous challenges including a lack of multidisciplinary researchers and imaging technologies for the microscopic radiation‐induced responses.A better grasp of the essence of these advances in cancer biology research will give medical physicists a new perspective in daily clinical physics practice and in future radiation therapy technological development. Furthermore, academic medical physics should continue to be an integral part of the multidisciplinary cancer research community, harnessing our newly acquired understanding of radiation effects, and developing novel cost‐effective treatment strategies to better combat cancer.Learning Objectives: Understand that localized radiation can lead to non‐localized secondary effects such as radiation‐induced immune response, bystander effect, and abscopal effect. Understand that the non‐localized radiation effects may be harnessed to improve cancer treatment. Learn examples of physics participation in multidisciplinary research to advance cancer biology. Recognize the challenges and possibilities of physics applications in cancer research. Chang: NIH 5RC2CA148487‐02 and 1U54CA151652‐01Graves: IDEA award (19IB‐0106) from the California Breast Cancer Research Program (CBCRP), and by NIH P01 CA67166

TU‐CD‐303‐03: Localized Radiation Can Induce Systemic Anti‐Cancer Immune and Non‐Immune Responses and How We Might Utilize It

Recent advances in cancer research have shed new light on the complex processes of how therapeutic radiation initiates changes at cellular, tissue, and system levels that may lead to clinical effects. These new advances may transform the way we use radiation to combat certain types of cancers. For the past two decades many technological advancements in radiation therapy have been largely based on the hypothesis that direct radiation‐induced DNA double strand breaks cause cell death and thus tumor control and normal tissue damage. However, new insights have elucidated that in addition to causing cellular DNA damage, localized therapeutic radiation also initiates cascades of complex downstream biological responses in tissue that extend far beyond where therapeutic radiation dose is directly deposited. For instance, studies show that irradiated dying tumor cells release tumor antigens that can lead the immune system to a systemic anti‐cancer attack throughout the body of cancer patient; targeted irradiation to solid tumor also increases the migration of tumor cells already in bloodstream, the seeds of potential metastasis. Some of the new insights may explain the long ago discovered but still unexplained non‐localized radiation effects (bystander effect and abscopal effect) and the efficacy of spatially fractionated radiation therapy (microbeam radiation therapy and GRID therapy) where many “hot” and “cold” spots are intentionally created throughout the treatment volume. Better understanding of the mechanisms behind the non‐localized radiation effects creates tremendous opportunities to develop new and integrated cancer treatment strategies that are based on radiotherapy, immunology, and chemotherapy. However, in the multidisciplinary effort to advance new radiobiology, there are also tremendous challenges including a lack of multidisciplinary researchers and imaging technologies for the microscopic radiation‐induced responses.A better grasp of the essence of these advances in cancer biology research will give medical physicists a new perspective in daily clinical physics practice and in future radiation therapy technological development. Furthermore, academic medical physics should continue to be an integral part of the multidisciplinary cancer research community, harnessing our newly acquired understanding of radiation effects, and developing novel cost‐effective treatment strategies to better combat cancer.Learning Objectives: Understand that localized radiation can lead to non‐localized secondary effects such as radiation‐induced immune response, bystander effect, and abscopal effect. Understand that the non‐localized radiation effects may be harnessed to improve cancer treatment. Learn examples of physics participation in multidisciplinary research to advance cancer biology. Recognize the challenges and possibilities of physics applications in cancer research. Chang: NIH 5RC2CA148487‐02 and 1U54CA151652‐01Graves: IDEA award (19IB‐0106) from the California Breast Cancer Research Program (CBCRP), and by NIH P01 CA67166

TU‐CD‐303‐04: Radiation‐Induced Long Distance Tumor Cell Migration Into and Out of the Radiation Field and Its Clinical Implication

Recent advances in cancer research have shed new light on the complex processes of how therapeutic radiation initiates changes at cellular, tissue, and system levels that may lead to clinical effects. These new advances may transform the way we use radiation to combat certain types of cancers. For the past two decades many technological advancements in radiation therapy have been largely based on the hypothesis that direct radiation‐induced DNA double strand breaks cause cell death and thus tumor control and normal tissue damage. However, new insights have elucidated that in addition to causing cellular DNA damage, localized therapeutic radiation also initiates cascades of complex downstream biological responses in tissue that extend far beyond where therapeutic radiation dose is directly deposited. For instance, studies show that irradiated dying tumor cells release tumor antigens that can lead the immune system to a systemic anti‐cancer attack throughout the body of cancer patient; targeted irradiation to solid tumor also increases the migration of tumor cells already in bloodstream, the seeds of potential metastasis. Some of the new insights may explain the long ago discovered but still unexplained non‐localized radiation effects (bystander effect and abscopal effect) and the efficacy of spatially fractionated radiation therapy (microbeam radiation therapy and GRID therapy) where many “hot” and “cold” spots are intentionally created throughout the treatment volume. Better understanding of the mechanisms behind the non‐localized radiation effects creates tremendous opportunities to develop new and integrated cancer treatment strategies that are based on radiotherapy, immunology, and chemotherapy. However, in the multidisciplinary effort to advance new radiobiology, there are also tremendous challenges including a lack of multidisciplinary researchers and imaging technologies for the microscopic radiation‐induced responses.A better grasp of the essence of these advances in cancer biology research will give medical physicists a new perspective in daily clinical physics practice and in future radiation therapy technological development. Furthermore, academic medical physics should continue to be an integral part of the multidisciplinary cancer research community, harnessing our newly acquired understanding of radiation effects, and developing novel cost‐effective treatment strategies to better combat cancer.Learning Objectives: Understand that localized radiation can lead to non‐localized secondary effects such as radiation‐induced immune response, bystander effect, and abscopal effect. Understand that the non‐localized radiation effects may be harnessed to improve cancer treatment. Learn examples of physics participation in multidisciplinary research to advance cancer biology. Recognize the challenges and possibilities of physics applications in cancer research. Chang: NIH 5RC2CA148487‐02 and 1U54CA151652‐01Graves: IDEA award (19IB‐0106) from the California Breast Cancer Research Program (CBCRP), and by NIH P01 CA67166

Folate deficiency affects mammary gland development in pre‐ and peri‐pubescent mice (827.12)

The ducts of mammary glands (MG) undergo phases of isometric and allometric growth during postnatal life. Folate is required for DNA synthesis and stability, where its effects are modulated by estrogen. Here we investigated the effects of folate deficiency on early MG development. Female FVB mice were assigned to either a control (2 mg folic acid/kg) or deficient (0 mg folic acid/kg) amino acid defined diet during in utero, postnatal, and early pubertal development. Tissues were assessed at weaning and during the peri‐pubescent period. The area occupied by the mammary ducts was reduced in folate deficient animals at weaning and during early puberty (p<0.0001). The slope of MG growth in the isometric phase, from birth to weaning, was less for the MG of deficient females compared with controls (95% confidence limits 0.23‐0.78 and 1.47‐1.82, respectively). Allometric growth was unaffected by diet. We conclude that folate deficiency impairs ductal elongation in the MGs of female mice prior to puberty. Alterations in pre‐pubertal development may fundamentally alter MG physiology throughout life, with potential impacts on lactation potential and risk of breast cancer.Grant Funding Source: Supported by California Breast Cancer Research Program

Abstract A04: The California Breast Cancer Survivorship Consortium (CBCSC): Prognostic factors associated with racial/ethnic differences in breast cancer survival

Abstract Background: Racial/ethnic disparities in mortality among US breast cancer patients are well documented. Using 2000-2006 data from Surveillance, Epidemiology, and End Results (SEER) registries, relative risk [95% confidence interval (CI)] of breast cancer-specific mortality relative to non-Latina Whites was significantly higher for African Americans (1.5 [1.4,1.6]) and Latinas (1.1 [1.0,1.2]), and non-significantly lower (0.9 [0.8,1.0]) for Asian Americans after adjusting for diagnosis age, SEER registry, disease stage, estrogen/progesterone receptor (ER/PR) status, county level poverty/education index, and other factors (Breast Cancer Res Treat, 127:729–738). Reasons for these differences are not well-understood; individual lifestyle factors may explain these differences, but current knowledge of the role of lifestyle in disease prognosis is limited for African Americans, Latinas and Asian Americans. Methods: The CBCSC assembled interview and cancer registry data from six California-based breast cancer etiology or prognosis studies, resulting in 12,210 breast cancer patients diagnosed with primary invasive breast cancer (6501 non-Latina Whites, 2060 African Americans, 2032 Latinas, 1505 Asian Americans, 112 others) between 1993 and 2007. Clinical characteristics and mortality data were obtained from the California Cancer Registry (CCR). Demographics (education, birthplace, migration), family history of breast cancer, parity, smoking, alcohol consumption, and other interview data were harmonized and pooled. Cox proportional hazards regression of overall and breast-cancer specific mortality were used to estimate racial/ethnic differences in stage-specific survival. Results: Frequency of adverse tumor characteristics (stage, differentiation, ER/PR status, nodal status, tumor size) were highest in African Americans, followed by Latinas, Asian Americans and non-Latina Whites, generally in that order. Latinas had lower education and higher parity than other racial/ethnic groups, whereas family history of breast cancer and alcohol consumption were highest in non-Latina Whites, and smoking was highest in African Americans. In total, 3047 deaths (1570 breast cancer-related) were observed after a mean (SD) follow-up of 8.3 (3.5) years. Compared to non-Latina Whites, the hazard ratio (HR) and 95% CI for breast cancer specific mortality was 1.15 [0.98,1.34] for African Americans, 0.77 [0.64,0.92], for Latinas, and 0.57 [0.36,0.92], for Asian Americans (adjusting for age and characteristics available from the tumor registry). HRs were slightly attenuated by further adjustment for education, migration, smoking and other factors (1.12 [0.95,1.31], 0.84 [0.69,1.02], 0.61 [0.38,0.99], respectively). Analyses are ongoing to determine the effect of neighborhood contextual factors, physical activity, body size, and comorbidities on racial/ethnic disparities in breast cancer survival. Conclusions: The CBCSC is a unique resource of a large and racially/ethnically diverse breast cancer cohort that will allow joint consideration of a variety of clinical, lifestyle, and contextual factors in explaining the long-standing disparities in breast cancer survival. Funded by: California Breast Cancer Research Program Special Research Initiative (16ZB-8004). Citation Format: Cheryl LP Vigen, Scarlett Lin Gomez, Richard Sposto, Yani Lu, Marilyn L. Kwan, Esther M. John, Kristine R. Monroe, Theresa HM Keegan, Salma Shariff-Marco, Allison W. Kurian, Iona Cheng, Bette J. Caan, Valerie S. Lee, Janise M. Roh, Jane Sullivan-Halley, Leslie Bernstein, Anna H. Wu. The California Breast Cancer Survivorship Consortium (CBCSC): Prognostic factors associated with racial/ethnic differences in breast cancer survival. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A04.

Abstract LB-48: The survivorship and advocacy task force model at Breast Cancer Connections

Abstract This poster demonstrates that an advocacy program can facilitate effective collaboration between a non-profit cancer support organization and the cancer research community. The evolution and scaling of the Survivorship and Advocacy Task Force Model is illustrated so that it can be replicated by other non-profit disease support organizations. Breast Cancer Connections (BCC), is a non-profit breast cancer resource center serving people touched by breast cancer in the San Francisco Bay Area and globally via the Internet and the telephone Helpline. BCC provides personalized information, support, and connections to other local and national resources in an atmosphere of warmth, sensitivity, and understanding. BCC was founded in 1993, by a surgeon and her patient who saw a need to augment what was being offered through the medical model. With more than 35 programs and services, all offered at no charge, BCC remains a vital and unique resource. Prompted by the requirement for patient advocate and survivor involvement in the research grant application process (California Breast Cancer Research Program, DoD, Susan G. Komen for the Cure) as well as by the increasing needs of breast cancer survivors, BCC formed the Survivorship and Advocacy Task Force (SATF) in 2008. The dual purpose of the SATF is to provide input and review to researchers applying for research grants and to assist BCC in developing services and programs that will improve the lives of breast cancer survivors. Task Force members who participate in reviewing research grants must be active research advocates and have formal training in the language and concepts of breast cancer research (such as Project LEAD Institute) or a Ph.D. in a discipline relevant to breast cancer research.The poster author chairs the BCC's SATF with five active members. The Task Force's efforts include meeting the PI to hear about the research, providing input to the PI on the research from the patient/survivor perspective, reviewing the grant proposal, refining the language of the lay abstract, writing a letter of support to accompany the grant proposal, and participating in the research if the research application is accepted. Current collaborators include breast cancer researchers from Stanford University, Palo Alto Medical Foundation, and University of California, San Francisco. As funding agencies increase their requirements for advocate involvement and as awareness of the importance of survivorship continues to grow, requests for SATF participation continues to increase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-48. doi:1538-7445.AM2012-LB-48

Testing Chemicals for Effects on Breast Development, Lactation, and Cancer

Factors across the life cycle, beginning before birth, influence breast cancer risk. We know this from epidemiologic studies of characteristics self-reported by older women or gleaned from medical records. For example, larger babies have higher breast cancer risk decades later, and preeclampsia is associated with lower risk in daughters (Ruder et al. 2008; Xue and Michels 2007). Earlier puberty increases risk, and women with fewer or no pregnancies have higher risk later on (Bernstein 2002). These factors span sensitive periods of breast development, prenatally and during puberty and pregnancy (Rudel et al. 2011). If environmental chemicals have comparable effects during sensitive developmental periods, identifying these risks could lead to prevention. However, epidemiologic data to assess the effects of chemicals in early life are rarely attainable. Exceptional events or decades-long cohort studies can provide some information. For example, studies of atom bomb survivors revealed breast cancer risk was highest from radiation exposure to young girls (Land 1995). A unique study using blood collected in the 1960s and stored for more than 40 years found higher breast cancer risk in women who were younger than 14 years of age when DDT was first put into use, taking advantage of a natural “experiment” (Cohn et al. 2007). But we cannot rely on observational chances like these to evaluate breast cancer risks for the many chemicals in use today. Thus, improving test methods in animals and cells is essential to identify chemicals that may interfere with breast development and contribute to cancer, so we can use this knowledge for primary prevention. These methods will extend recent changes in toxicity testing, which were designed to respond to research on endocrine-disrupting compounds by evaluating how exposures in utero and during other windows of development set the stage for chronic diseases later in life. New procedures include dosing in utero and throughout development, adding morphological and functional assessments of reproductive organs, and including longer-term follow-up, none of which are included in traditional toxicity testing. For example, new protocols recently demonstrated that in utero antiandrogen exposure in rodents leads to altered male reproductive development, as well as reduced fertility and possibly increased cancer later on. These observations are thought to parallel human “testicular dysgenesis syndrome,” which includes decreased fertility and increased incidence of cryptorchidism, hypospadias, and testicular cancer (Luccio-Camelo and Prins 2011). Mammary gland development has usually not been assessed in the new protocols, though. Yet, mounting evidence supports the importance of testing for breast effects for several reasons. The breast develops over a long period, with vulnerability beginning in utero and extending through the first pregnancy. For some chemicals tested to date, mammary gland development in males and females is altered at lower doses than the levels that cause changes in other tissues. The effects that have been observed after disrupted mammary gland development include impaired lactation and increased susceptibility to cancer, so they are of potentially great public health significance. In this issue, three articles address these points: Makris (2011) reviews standard chemical testing protocols to identify gaps in mammary gland assessment. Key gaps include lack of early life exposure in tests for carcinogenic effects; lack of assessment of lactation function other than nonspecific measures; and inadequate examination of mammary gland morphology and pathology. Rudel et al. (2011) present a comprehensive review of hormone and chemical effects on mammary gland development, lactation, and cancer. Emerging from a meeting of 60 international experts, this article reports the majority opinion of this group that normal mammary gland development and carcinogenesis are similar in rodents and humans; that chemical and hormone exposure in utero or early in life leads to altered mammary gland development; and that these changes may be risk factors for impaired lactation and cancer. Effects on mammary gland development are not limited to estrogenic endocrine disruptors but are induced by diverse chemicals, including perfluorinated compounds and the herbicide atrazine, in addition to the soy phytoestrogen genistein and synthetic estrogens such as bisphenol A. White et al. (2011) demonstrate persistent effects of perfluorooctanoic acid (PFOA) in drinking water on mouse mammary gland development at exposures lower than in some contaminated drinking water supplies. Research needs identified in the articles include a call for dosing during development; improved assessment of mammary gland development, structure, and function; and increased assessment of the male mammary gland. These recommendations have been implemented by the National Center for Toxicological Research in several studies done in conjunction with the U.S. National Toxicology Program (NTP) (Delclos et al. 2009; Latendresse et al. 2009), but they still need to be integrated into standard protocols at the NTP, the U.S. Environmental Protection Agency (EPA), and the Organisation for Economic Co-operation and Development (OECD). Research is also needed to determine the relationship of mammary gland changes to effects on lactation and cancer susceptibility. The $5 million California Breast Cancer Research Program initiative “Making Chemicals Testing Relevant to Breast Cancer” is a model for stimulating research in this field, and the National Institute of Enironmental Health Sciences (NIEHS) Breast Cancer and the Environment Research Program is contributing knowledge about puberty as a critical window of development and susceptibility. Although traditional cancer bioassays have identified many common pollutants that increase mammary gland tumors, including common air pollutants, drinking water disinfection by-products, and chlorinated solvents (Rudel et al. 2007), research reported in these three articles suggests that traditional tests that neglect developmental effects may be missing many more. Given the magnitude of potential public health impacts on breast-feeding and breast cancer, it is critical to strengthen testing methods and give more weight to them in policy decisions. Good decisions about pollution limits, pesticide approvals, and chemicals in consumer products and food rely on a full and accurate understanding of risks associated with exposure.

Abstract 4747: Twist1-induced invadopodia formation promotes tumor metastasis

Abstract Tumor cells acquire the ability to degrade basement membrane and extracellular matrix (ECM), thus allowing them to invade and metastasize to distant organs. The molecular triggers for ECM degradation and invasion are largely unknown. The Twist1 transcription factor plays key roles in tumor metastasis and is known to induce Epithelial-Mesenchymal Transition (EMT). In addition to Twist1, a number of studies show that other EMT-inducing transcription factors, including Snail1/Snail2 and ZEB1/ZEB2, are also important players in tumor metastasis. One question unanswered is what are the specific cellular functions and transcriptional targets of individual EMT-inducing transcription factors required for EMT and tumor metastasis. Here we report a novel function of Twist1 in promoting extracellular matrix degradation by inducing the formation of invadopodia. Invadopodia are specialized actin-based membrane protrusions that degrade ECM via clustering of proteases. Invadopodia contain a F-actin core and actin regulatory proteins, such as cortactin, the unique adaptor proteins Tks4 and Tks5 in clustering components of invadopodia, and a large number of proteases for matrix degradation. Twist1 induces PDGFRα expression, which in turn activates Src kinase in various breast tumor cells. Phosphorylation of multiple invadopodia components by Src kinase promotes invadopodia assembly and matrix degradation. We show that Twist1 and PDGFRα are central mediators of invadopodia formation and ECM degradation in response to various EMT-inducing signals, including TGFβ and Snail1. Induction of PDGFRα and invadopodia is essential for Twist1 to promote invadopodia formation, local tumor invasion, and distant metastasis in vivo. Consistent with PDGFRα being a direct transcriptional target of Twist1, expression of Twist1 and PDGFRα is highly correlated and associated with poor survival in breast cancer patients. Together, our study demonstrates that invadopodia-mediated matrix degradation is a key function of Twist1 in promoting tumor metastasis. Furthermore, it reveals that matrix degradation and loss of cell adhesion are regulated by different EMT-inducing transcription factors. This explains why multiple factors need to be activated coordinately to promote carcinoma cells to undergo EMT and invade. We also identify PDGFRα as a direct transcriptional target of Twist1 in promoting invadopodia formation and tumor metastasis, therefore suggesting that PDGFRs might be potential targets for anti-metastasis therapies. Grant acknowledgements: NIH Director's New Innovator Award (DP2 OD002420-01), Kimmel Scholar Award, and California Breast Cancer Research Program(12IB-0065) to J.Y. a DOD Breast Cancer Pre-doctoral fellowship to M.A.E, a DOD Breast Cancer Era of Hope Postdoctoral Fellowship to E.D., Susan G. Komen Foundation grant FAS0703850 to J.K. and L.O. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2011-4747

Abstract P4-02-09: Extranuclear Estrogen Receptors in Human Breast Cancer

Abstract Estrogens are well known to promote growth and progression of human breast cancer. These effects are exerted by binding of estradiol to estrogen receptors (ER), ER-alpha and ER-beta. Immunohistochemical (IHC) detection of nuclear ER-alpha in breast tumors is a standard assessment in the clinic to plan patient management decisions. However, ERs also occur at extranuclear sites, and numerous studies now show that extranuclear ER (exER) play important roles in regulating downstream signaling by activating kinase cascades that impact gene expression and tumor progression. Nonetheless, we still have poor understanding of the relative contributions of nuclear ER versus extranuclear ER in mediating estrogen action. Based on current IHC assays for nuclear estrogen receptors, only about half of advanced breast cancers with expression of ER and/or progesterone receptor respond to endocrine therapy, indicating a need for improved assays designed to correlate not only nuclear ER but possibly also exER with patient outcome. In this study we focused on development of innovative methods for IHC detection of exER-alpha as well as nuclear ER-alpha and correlation of expression with clinical outcome. IHC results depend greatly on the type of antibody used, the detection procedure, subcellular densities of ER and problems due to long-term formalin fixation (antigen masking). We have tested and compared different methods and antibodies that specifically detect nuclear and extranuclear forms of ER in formalin-fixed, paraffin-embedded tissues. Although some exER staining is apparent in clinical samples that test positive for nuclear ER with antibodies commonly used for standard ER assays, our findings suggest that carboxy-terminal antibodies are more sensitive in the detection of specific exER staining. Such C-terminal antibodies can also detect exER staining even in the absence of significant nuclear staining in about one third of 16 breast cancer cases analyzed from the clinic. Extranuclear ER staining is specific, but exhibits lower intensity than nuclear ER staining, and it does not occur in breast tumor cells negative for ER by Western blot or when antibodies are pre-absorbed with specific peptides. Further, subsets of human breast tumor cells selected for membrane-associated exER in vitro using flow cytometry and ligand-affinty binding methods (about 4-5% of the total tumor cell population) show enhanced growth responses to estradiol (P<0.001) and increased expression of CD133, polycomb gene Bmi-1 and ALDH-1. These biomarkers are often associated with high tumor-initiating activity and poor clinical outcome. The findings suggest that tumor cell subpopulations with relatively high expression of exER may represent tumor progenitor cells with a capacity to drive tumor progression. Despite advances indicating the role of exER in regulating cancer progression, there is no current assay in the clinic to detect exER, nor to consider its contribution in managing hormonal therapy. Assay of breast tumor exER may prove important in predicting endocrine response and resistance as well as in development of new therapies to target these tumor cell subsets. [Supported by California Breast Cancer Research Program and Stiles Program funding] Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-09.

Abstract 35: Metabolomic analysis of MCF-7 and MCF-10 cells after thiaminase I exposure

Abstract We have recently shown that recombinant thiaminase I enzyme has cytotoxic activity against breast cancer cell lines (Liu, S., Monks, N.R., Hanes, J.W., Begley, T.P., Yu, H. and Moscow, J.A. Sensitivity of breast cancer cell lines to recombinant thiaminase I. Cancer Chemotherapy and Pharmacology, 2009). Because thiamine is a cofactor in several key metabolic enzymes, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and transketolase, information regarding the metabolic consequences of thiaminase I treatment may increase our understanding of the cytotoxic mechanism-of-action of thiaminase I. We analyzed the metabolomes of the MCF-7 breast cancer cell line and the immortalized but non-malignant breast cell line MCF-10A with or without incubation for 4 days in medium containing thiaminase I at the predetermined cytotoxic IC50 concentrations (0.125 U/ml for MCF-7 and 0.25 U/ml for MCF-10A), with the addition of [U-13C]-labeled glucose in the final 2 days. By tracking the metabolism of [U-13C]-glucose using GCMS and NMR we were able to quantify intracellular metabolite pools and calculate both relative and absolute flux rates through several key pathways. Thiaminase I treatment decreased the amount of protein per MCF-7 cell by 30% (163 pg/cell to 115 pg/cell) while the total protein remained essentially unchanged in the MCF-10A cells (141 pg/cell vs 152 pg/cell). While both cell lines displayed increased glucose consumption and lactate production during thiaminase I treatment, these responses were greater in the MCF-7 cell line. Metabolite analysis revealed significant alterations to glycolysis and the TCA cycle in both cell lines, with glycolysis-linked metabolites being depleted in the MCF-7 cell line and changes in the equilibrium of TCA cycle intermediates in the MCF-10A cell line. The 13C- labeling patterns suggest that the MCF-10A cell line responds to thiaminase I treatment by increasing the catabolism of alternate, non-glucose carbon sources through the activation of specific metabolic pathways. In contrast, the MCF-7 cell line appears to have a less adaptable metabolic program and must down-regulate its overall metabolic throughput. Therefore, the possibility is raised that differential metabolic effects of thiaminase I could be exploited therapeutically. Supported by the Susan G. Komen For the Cure Foundation (JAM) and the California Breast Cancer Research Program (ADR). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 35.

Abstract 700: Prolactin negates the ability of BRCA1 to increase p21 expression and the ability of already synthesized p21 to inhibit the cell cycle

Abstract The literature supports a role for prolactin (PRL) in the development of some cancers through PRL's promotion of the cell cycle and/or inhibition of apoptosis. The selective PRL receptor modulator, S179D PRL, on the other hand, inhibits cancer cell proliferation and promotes differentiation and/or apoptosis. BRCA1 is an important tumor suppressor, shown to play a role in breast (B), ovarian (O), and prostate (P) cancer. The purpose of the current study was to determine the effect of the functionally distinct ligands for the PRL receptor on aspects of BRCA1 biology. Contrary to expectation, both PRL and S179D PRL (at 500 ng/ml) elevated levels of BRCA1 protein (∼3 fold in 24h and up to 10 fold in 48h) in all cancer types examined (B, O, &P). The promoter for the cell cycle inhibitory molecule, p21, has a BRCA1 binding site, but only S179D PRL increased levels of p21 protein as a result of BRCA1 elevation. Using deletion analysis of a p21 promoter-luciferase construct, we confirmed that the BRCA1 binding site (−84 to −129) was crucial to p21 expression in response to S179D PRL. An important question therefore was how elevated BRCA1 in response to each ligand had a different end result in terms of p21 expression. Examination of nuclear translocation of BRCA1 in response to cell stimulation with each ligand showed that both promoted movement into the nucleus. Moreover, both ligands similarly increased BRCA1 interaction with the important region (−84 to −129) of the endogenous promoter of the p21 gene (ChIP assay). A major difference in the signaling resulting from receptor engagement by PRL versus S179D PRL is that only PRL activates Stat5. With PRL, but not S179D PRL, BRCA1 formed a complex with phospho-Stat5 and this complex was present in the nucleus. Since both ligands increase BRCA1 interaction with the p21 promoter, but only PRL results in a BRCA1-phospho-Stat5 complex, it appears that binding of phospho-Stat5 to BRCA1, while not inhibiting its ability to bind to the promoter, negates the ability of BRCA1 to transactivate the p21 promoter. For already synthesized p21, additional studies demonstrated that stimulation of cells with PRL, but not S179D PRL, resulted in phosphorylation of p21, a posttranslational modification that inhibits nuclear translocation and therefore the ability of p21 to inhibit the cell cycle. This study has uncovered two ways in which p21 function is hampered by PRL and hence additional ways in which PRL promotes cell proliferation. At the same time, since S179D PRL inhibits Stat5 activation in response to PRL in addition to itself signaling to p21 elevation, this work also furthers our understanding of the anti-cancer activities of this selective PRL receptor modulator. Supported by California Breast Cancer Research program # 10PB-0127 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 700.

RETHINKING BREAST CANCER TOXICOLOGY

EVALUATING CHEMICALS for breast cancer risk should focus more on identifying critical disease pathways and high-throughput screening of compounds and less on traditional toxicology tests on adult animals, according to an expert panel. The panel was convened as part of the Breast Cancer & Chemicals Policy Project (BCCPP), which was funded by the California Breast Cancer Research Program. One goal of the project was to develop a toxicity testing approach to identify chemicals that contribute to breast cancer. Another was to recommend ways to close gaps in toxicological methods and scientific knowledge related to identifying chemicals involved in breast cancer. One in eight women develops breast cancer in her lifetime, and the disease is the second leading cause of death in adult women, said Sarah Janssen in a presentation at the February meeting of the American Association for the Advancement of Science. Janssen is one of the directors of BCCPP and a staff ...

Abstract C21: A novel protein microarray analysis for the identification of protein ubiquitination in cancer

Abstract Ubiquitination is a post-translational modification wherein a small (8 kDa) and highly abundant protein called ubiquitin is covalently attached to specific protein substrates in cells. This “glinkage” plays an important role in the regulation of many cellular functions including division, differentiation, protein trafficking, response to DNA damage, and gene transcription, among others. A lysine 48-linked chain of four or more ubiquitins targets a substrate for degradation by the 26S proteasome. A wealth of experimental evidence supports the notion that derangement of ubiquitin-mediated proteolysis contributes to various human malignancies including breast cancer. During metastasis, breast tumor cells undergo a process that is similar to the epithelial-mesenchymal-transition (EMT). This process is characterized by multiple steps ultimately enabling breast tumor cells to leave their place of origin and to colonize new organs. Little is known about the intrinsic ubiquitination activity during this process and how the ubiquitination pattern differs in metastatic cells compared to their non-malignant counterparts. Furthermore, there are no adequate experimental tools to identify the ubiquitination of protein substrates on a proteome-wide scale. The ubiquitin-proteasome system has been recognized as a valid target for the treatment of some malignancies (e.g. Bortezomib), thus deciphering the change of tumor cells' intrinsic enzymatic activity could warrant for the development of enhanced diagnostic tools and effective treatment of breast cancer patients. To overcome this shortcoming, we have developed an innovative approach that combines protein microarrays (where >8,300 human recombinant proteins are spotted onto glass microscope slides) with an ubiquitination reaction to determine novel ubiquitinated substrates during EMT. We obtained breast tumor material using the 4T1 mouse model of breast cancer, which consists of five isogenic murine mammary carcinoma cells lines (67NR, 168FARN, 4TO7, 4T1, and 66cl4) that spontaneously arose from a single mammary tumor in BALB/c mice and exhibit differing metastatic abilities resembling the multi-step process of EMT. Lysed tumor tissue was subsequently analyzed for a change of the ubiquitination signature. More than 230 alterations in specific target protein ubiquitination were detected in the cell lines used, indicating a possible regulatory role of protein uniquitination during the process of breast cancer progression. The identification of the biological consequences of some of these protein modifications will aid in gaining more insight into how the cells' intrinsic enzymatic activity has become distorted and how this could drive the metastatic process of breast cancer. Our novel identification of target protein ubiquitination could ultimately serve as a diagnostic and prognostic tool and refine the regimens for breast cancer therapies. (This work is supported by California Breast Cancer Research Program grant CBCRP-14FB-0114 to S.G.) Citation Information: Cancer Res 2009;69(23 Suppl):C21.