Joyce M. Slingerland - US grants

DESCRIPTION (provided by applicant): Transforming growth factor-beta (TGF-beta) mediates cell cycle arrest by activating cdk inhibitors, principle among which is p27[Kip1]. Loss of growth inhibition by TGF-beta occurs early in oncogenesis and contributes to tumor progression. We have studied cell cycle effects of TGF-beta and how they are lost in human tumors, p27 is often inactivated in human cancers due to accelerated p27 proteolysis or cytoplasmic mislocalization of p27. In cancer cells, progressive checkpoint loss makes p27 essential for TGF-beta mediated arrest. We showed activation of the phosphoinositol 3' kinase (PI3K) effector, protein kinase B (PKB) phosphorylates p27 at threonine 157, mislocalizating p27 in the cytoplasm. PKB activated cells also showed increased p27 in cyclin D1-cdk complexes. Preliminary data suggest that other effectors of the PI3K pathway may also inhibit p27 function. PI3K and its downstream effector pathways are often activated in cancers by oncogenic receptor tyrosine kinase overexpression, activating ras mutations or loss of the PI3K inhibitor, PTEN. Here we investigate how p27 function and G1 arrest by TGF-beta may be opposed by mitogenic PI3K signaling in cancers. Our hypothesis is that constitutive activation of the PI3K pathway leads to p27 phosphorylation events that reduce its affinity for cyclin E-cdk2, alter its stability and intraceltular localization, and abrogate its inhibitory function leading to TGF-beta resistance. This is pursued by the following specific aims: AIM 1) to assay if PKB activates p27 assembly function toward cyclin D1-cdk4 and reduces p27's inhibitory action toward cyclin E-cdk2; AIM 2) to determine whether PI3K effectors other than PKB can mediate TGF-beta resistance; AIM 3) to assay if other PI3K dependent kinases can phosphorylate p27 and alter its function and AIM 4) to investigate whether PI3K effectors impair nuclear import of p27. These studies may link TGF-beta resistance to oncogenic activation of mitogenic P13K signaling pathways commonly observed in human cancers. Elucidation of molecular mechanisms of p27 inactivation and TGF-beta resistance in cancers may yield new targets for therapeutic intervention.

DESCRIPTION (provided by applicant): Breast cancer is the most common cancer among women, accounting for one third of cancer diagnosis in the United States. Eradication of breast cancer is likely to require a partnership of diverse scientific disciplines and training of new breed of basic and translational researchers. The goal of this training grant is to train 6 postdoctoral fellows (including MD and PhD) to perform state-of-the-art research in hormonal regulation, signal transduction, cell cycle controls, cell structure and motility, and experimental therapeutics applied to breast cancer. The strength of the proposed grant is the training faculty. Drawn from both basic science departments and clinical departments (including Braman Family Breast Cancer Institute and Sylvester Comprehensive Cancer Center), the 13 faculty members are an excellent group whose well-funded research comprises a cross-section of structural, molecular and cell biological, basic and translational science approaches to breast cancer research. The faculty members have extensive experience in training graduate students and postdoctoral fellows (including MD/PhD) who have gone onto become successful independent researchers. The overall goal of this program is to provide guidance for trainees to acquire unique research skills and to mold their intellectual rigor to sustain an independent, clinically-informed research career in breast cancer. All trainees are required to attend formal didactic courses, symposia, breast cancer research seminars, grand rounds, workshops, journal clubs and an annual retreat, which provide forum for exchange of ideas and interaction among different disciplines. Progress of the trainee's research will be closely monitored in bi-weekly data reviews. Thus this training program fulfills a serious national need for breast cancer researchers with ample knowledge in basic, clinical and translational research. The proposed program has many unique strengths including : 1) the existence of a specialized breast cancer research institute (Braman Family Breast Cancer Institute) and a well-known cancer center (Sylvester Comprehensive Cancer Center), 2) the research productivity, research funding, training experience, and administrative capability and experience of program faculty in general and the program directors in particular, and 3) the opportunity and availability of infrastructure for fostering continued interactions between clinical and basic research in breast cancer.

DESCRIPTION (provided by applicant): Estrogen receptor 1 (ER) is expressed in 70% of new breast cancers but de novo and acquired resistance to tamoxifen or aromatase inhibitors (AI) limit their efficacy. The cell cycle inhibitor, p27, is required for therapeutic effects of antiestrogens. p27 is reduced in 60% of human breast cancers and is a poor prognostic factor, particularly in ER+ cancers. Src phosphorylates p27 to promote its degradation. Her2 or Src activation in ER+ breast cancer cells alters p27 phosphorylation, impairs its Cdk2 inhibitory function and causes antiestrogen resistance. Expression profiling has revealed a Src activation gene signature in up to 40% of human breast cancers that powerfully predicts growth arrest by Src inhibitor drugs in vitro. We found Src activation in 39% of ER+ human breast cancers is associated with low p27. This may predict resistance to endocrine therapy. Antiestrogen resistance was reversed in ER+ breast cancers in vitro by combining the Src inhibitor drug, AZD0530, with tamoxifen or anastrozole. AZD0530 increased p27 and inhibited proliferation when added to anastrozole. AZD0530 also synergized with anastrozole to inhibit growth of orthotopic MCF-AROM5 xenograft tumors in vivo. Thus, AZD0530 may oppose resistance to the AI anastrozole in human breast cancers. We investigate the hypothesis that Src inhibition combined with anastrozole will lead to a greater reduction in tumor volume than anastrozole alone and increase in pCR rate following neoadjuvant treatment setting of locally advanced breast cancer. Moreover, we postulate that responsive breast cancers will show a greater increase in p27 with treatment and a Src activated protein or gene expression profile in the primary tumors that will change during treatment. We also postulate that changes in the tumor initiating cells (cancer stem cells) may be critically linked to both immediate responsiveness to neoadjuvant therapy and to long term outcome. AIM 1 We propose a Phase II study to test if anastrozole combined with the Src inhibitor AZD0530 is better able to reduce tumor volume than anastrozole alone in postmenopausal women with locally advanced ER+/or PR+ (HR) breast cancer. This trial will be carried out after a Phase I trial to test tolerability and bioavailability of daily oral anastrozole and AZD0530 co-administration in post-menopausal women with HR+ metastatic breast cancer. A secondary trial endpoint will evaluate the ability of serial MRI changes in tumor diameter, volume and blood flow to complement clinical MD measurement of response. To define biologic endpoints or predictors of treatment response that may assist in patient selection for subsequent studies, AIM 2 will assay Src targets and upstream regulators using protein and gene expression profiles and quantitate tumor initiating cells in diagnostic samples, tumor biopsies at 8 weeks and in cancers resected after 4-6 months of neoadjuvant treatment with anastrozole alone or anastrozole and AZD0530. Evidence of short term efficacy of this new Src inhibitor and identification of predictive biomarkers would support subsequent phase II-III trials and further refinement of the potential predictive value of molecular Src activation profiles. PUBLIC HEALTH RELEVANCE: This grant will support a clinical trial that combines, for the first time, a novel molecular targeted drug, AZD0530, that inhibits the Src oncogene, with the antiestrogen anastrozole for women with breast cancer. In addition to testing the safety and early efficacy of this drug combination, this study will permit molecular tests to help predict which patients are likely to have a good response and which will have resistant disease. This trial may form the basis for subsequent, larger clinical trials to enhance the efficacy of endocrine therapy for breast cancer.

DESCRIPTION (provided by applicant): Breast cancer is the most frequent womens'cancer. One third of new breast cancers are estrogen receptor a (ER) protein negative and have a worse prognosis than ER positive breast cancers. The ER is a ligand activated transcription factor. Estrogen:ER binding stimulates rapid Src activation that feeds back to phosphorylate ER and increases its transcriptional activity. Estrogen rapidly activates ubiquitin-dependent ER proteolysis which in turn regulates ER activity. Our data suggest that Src-stimulates ER proteolysis and this is linked to activation of certain ER target genes. Src induction increased both ER-activated gene expression and ER proteolysis. Src inhibition increased ER stability. The weakly ER +, MDA-MB-361 and ER negative, BT-20 breast cancer lines both have high Src, and while ER protein synthesis was easily detected, the ER t1/2 was reduced. ER was increased by both estrogen deprivation and proteasome inhibitors in both ER + and ER- line studied. SiRNA to Src increased ER protein in BT-20. E6AP is an ubiquitin ligase that also acts as an ER- coactivator. We show E6AP acts as ubiquitin ligase for ER in vitro and E6AP-mediated ER ubiquitylation was increased by ER pre-treatment with Src kinase. Src inhibitors impaired ubiquitylation and degradation of ER in vivo and in vitro. Src activity was increased in primary ER negative breast cancers compared to ER positive. We further investigate if Src, when recruited by activated ER, regulates transcription-coupled ER proteolysis. Our Hypothesis is that a subset of ER negative breast cancers are estrogen responsive: they express ER mRNA but ER protein is undetectable due to accelerated Src and E6AP-mediated ER proteolysis. Oncogenic Src activation may phosphorylate the ER or key co-regulators to activate both ER proteolysis and ER target gene transcription. This is pursued in Aims 1-3: 1. To further test how Src activation affects ER levels in primary human breast cancers and in breast cancer lines;2. To test whether Src mediated phosphorylation of the ER or of a key co-activator can promote ER ubiquitylation by E6AP and ER degradation in vitro and in vivo;and 3. To assay if Src activation modulates ER transcriptional activity. Oncogenic Src promotes breast cancer proliferation and survival, and may also accelerate ER proteolysis in breast cancers. Elucidation of mechanisms underlying the ER negative status of breast cancer may indicate why they are so aggressive and yield new therapeutic targets for this treatment-resistant breast cancer type. PUBLIC HEALTH RELEVANCE: One third of new breast cancers are estrogen receptor a (ER) protein negative and have a worse prognosis than ER positive breast cancers. This grant pursues the hypothesis that a subset of ER negative breast cancers are estrogen responsive: they express ER mRNA but ER protein is undetectable due to accelerated Src and E6AP-mediated ER proteolysis. Oncogenic Src activation may phosphorylate the ER or key co- regulators to activate both ER proteolysis and ER target gene transcription. Oncogenic Src promotes breast cancer proliferation and survival. Our work suggests that it may also accelerate ER proteolysis in breast cancers. Elucidation of mechanisms underlying the ER negative status of breast cancer may indicate why they are so aggressive and yield new therapeutic targets for this treatment-resistant breast cancer type.

DESCRIPTION (provided by applicant): While nuclear p27 is often reduced, p27 gene deletion and complete p27 loss is rare in human cancers. Cytoplasmic p27, seen in many cancers, is associated with a poor prognosis. Cytoplasmic p27 acquires an oncogenic gain of function to promote cell motility by binding RhoA to inhibit RhoA-ROCK activation needed for cytoskeletal stability. p27CK- knock- in mice show cytoplasmic p27, and increased cell motility, progenitor/stem cell populations and lung tumor formation. Thus, p27 regulates both cell proliferation and migration, and has a pro-oncogenic action to promote cell motility independent of its cell cycle effect, which may explain why p27 is rarely entirely lost in human cancers. Here we investigate how p27 phosphorylation regulates its motility function. We showed that p27 is phosphorylated by AGC family kinases downstream of PI3K at T157, T198, or both, which impairs nuclear p27 import, stabilizes p27 in the cytoplasm and stimulates cell motility. Cells overexpressing AGC kinases show a p27-dependent increased cell motility which is reversed by p27 knock- down via shRNAp27. T198 phosphorylation increased p27:RhoA binding in vitro and p27T157AT198A bound RhoA poorly, suggesting that T198 phosphorylation may increase cell motility via Rho-ROCK1 inhibition. p27 also appears to mediate PI3K driven metastasis. High metastatic MDA-MB-231-derivatives (4175 and 1833) showed PI3K activation, increased cytoplasmic p27 and increased cell motility that were reversed by p27 knockdown. p27 knock-down reversed the high lung metastasis of MDA-MB-231-4715 to levels similar to those in parental MDA-MB-231. Thus, PI3K may stimulate invasion and metastasis via oncogenic effects of cytoplasmic p27. Our hypothesis is that p27 phosphorylation at T198 promotes its association with RhoA to increase tumor cell motility and potentiate tumor invasion and metastasis. This grant investigates further how p27 phosphorylation by PI3K effector kinases increases cell motility and tests if p27pT157pT198 promotes tumorigenesis and metastasis in vivo. AIM1 will identify the p27:RhoA binding site and test the importance of phosphorylation to RhoA binding and increased cell motility. AIM 2 will test effects of pT198 and cytoplasmic p27 on local tumor invasion and metastasis in xenografts. AIM 3 will create p27CK-T198D and p27CK-T198A knock-in mice and test their susceptibility to tumorigenesis. Cytoplasmic p27pT198 and its cytoskeleton effects may be major drivers of tumorigenicity and metastasis activated by RTK/PI3K in human cancers. Therapeutic targeting agents that disrupt p27:RhoA interaction may prevent tumor invasion and metastasis. In cancers, cytoplasmic p27 may predict potential for response to PI3K/mTOR inhibitor drugs.

Project Summary Obesity increases the risk and adverse prognosis of postmenopausal estrogen receptor-positive (ER+) breast cancer. Paradoxically, although estrogens stimulate breast cancers, risk rises markedly after menopause, when estrogens decrease. After menopause, estradiol falls and estrone is produced largely in fat by aromatase. Obesity women have high estrone, and both obesity and high estrone, but not estradiol, increase ER+ breast cancer risk after menopause. Low intra-tumor levels of enzymes converting estrone into estradiol, and elevation of enzymes that produce estrone both confer worse ER+ breast cancer outcome. Obesity mediates chronic inflammation through NF-?B driven cytokine expression. We showed contact between invading breast cancer cells and obese adipose tissue induces pro-inflammatory cytokines in both cell types that stimulate cancer stem cells (CSC) and drive metastasis. Our data suggest that cytokine induction after breast fat:cancer cell contact is estrogen:ER dependent, since blocking estrogen synthesis with the aromatase inhibitor, letrozole, reduced cytokine induction upon co-culture. While estradiol is known to oppose NF-?B mediated inflammation, the role of estrone in inflammation is not known and may differ from that of estradiol. We will study how estrone and estradiol, and changes in the ratios thereof before and after- menopause, may influence NF-?B activity and the pro-inflammatory state in obese postmenopausal women. We hypothesize that increased estrone:estradiol ratio after menopause shifts ER from an NF-?B co- repressor to a co-activator to up-regulate cytokines in obese adipocytes and cancer cells that drive CSC expansion and metastasis. We also posit that enzymes that convert estradiol to estrone may contribute to the poor outcome of ER+ cancers in obesity. Aim 1 will test if estradiol:ER decreases, estrone:ER increases or different estradiol:estrone ratios alter ER effects on NF-?B mediated induction of pro-inflammatory cytokine gene drivers of CSCs in ER+ breast cancer cells. To test in Aim 2 if estrone cooperates with obesity to drive ER+ breast cancer initiation and growth, we will co-culture human ER+ breast cancer cells with mammary adipocytes from women with different body mass index (BMI) +/- aromatase inhibition and test consequences on cytokine levels and CSC. We will also implant syngeneic breast cancers into lean or obese wild-type or aromatase knock-out mice to elucidate if host estrone mediates tumor promoting effects of obesity. Aim 3 will test if overexpression of HSD17B14, that converts estradiol to estrone, increases ER+ breast cancer cytokine expression and CSC in vitro, and increases tumor initiation and metastasis in vivo. We will also compare levels of estradiol/estrone interconversion enzymes in breast adipocytes and cancers from lean, overweight and obese women. A better understanding of the roles of estradiol and estrone may lead to new strategies for breast cancer prevention and treatment, and to changes in hormone replacement therapies. Shifting toward a higher estradiol: estrone ratio in serum or in breast cancer cells may prove to have therapeutic potential.

Project Summary Obesity increases the risk and adverse prognosis of postmenopausal estrogen receptor-positive (ER+) breast cancer. After menopause, estradiol falls and estrone is produced largely in fat by aromatase. Obesity women have high estrone, and both obesity and high estrone, but not estradiol, increase ER+ breast cancer risk after menopause. Obesity mediates chronic inflammation through NF-?B driven cytokine expression. We showed contact between invading breast cancer cells and obese adipose tissue induces pro-inflammatory cytokines in both cell types that stimulate cancer stem cells (CSC) and drive metastasis. Our data suggest that cytokine induction after breast fat:cancer cell contact is estrogen:ER dependent, since blocking estrogen synthesis with the aromatase inhibitor, letrozole, reduced cytokine induction upon co-culture. While estradiol is known to oppose NF-?B mediated inflammation, the role of estrone in inflammation is not known and may differ from that of estradiol. We will study how estrone and estradiol, and changes in the ratios thereof before and after- menopause, may influence NF-?B activity and the pro-inflammatory state in obese postmenopausal women. We hypothesize that increased estrone:estradiol ratio after menopause shifts ER from an NF-?B co- repressor to a co-activator to up-regulate cytokines in obese adipocytes and cancer cells that drive CSC expansion and metastasis. We also posit that enzymes that convert estradiol to estrone may contribute to the poor outcome of ER+ cancers in obesity. Aim 1 will test if estradiol:ER decreases, estrone:ER increases or different estradiol:estrone ratios alter ER effects on NF-?B mediated induction of pro-inflammatory cytokine drivers of CSCs in ER+ breast cancer cells. To test in Aim 2 if estrone cooperates with obesity to drive ER+ breast cancer growth, we will co-culture human ER+ breast cancer cells with mammary adipocytes from women with BMI +/- aromatase inhibition and test effects on cytokine levels and CSC. We will also implant syngeneic breast cancers into lean or obese wild-type or aromatase knock-out mice to elucidate if host estrone mediates tumor promoting effects of obesity. Aim 3 will test if overexpression of HSD17B14, that converts estradiol to estrone, increases ER+ breast cancer cytokine expression and CSC in vitro, and tumor initiation and metastasis in vivo. A better understanding of the roles of estradiol and estrone may lead to new strategies for breast cancer prevention and treatment, and to changes in hormone replacement therapies. Shifting toward a higher estradiol: estrone ratio in serum or in breast cancer cells may prove to have therapeutic potential. This SUPPLEMENT will test if estrone is more effective in stimulating ER+ breast cancer patient derived xenografts than estradiol. This will test our original hypothesis and help meet objectives of AIMS 1 & 2. It may also elucidate why ER+ breast cancer PDX have been so difficult to establish in the past. This work will also increase the number of ER+ PDX models for the research community to use for therapeutic research.

PROJECT SUMMARY The University of Miami (UM) Calabresi Clinical Oncology Research Career Development program is designed to launch the careers of clinical researchers committed to pursuing patient-oriented cancer research. Identifying such individuals and facilitating their career development will ensure that a new generation of clinician scholars can effectively translate scientific discovery to clinical research opportunities that improve the delivery of cancer care and accordingly, patient outcomes. As of recent, the University of Miami Sylvester Comprehensive Cancer Center has experienced substantial growth in its basic, translational and population science programs and has necessary infrastructure to support a successful K12 Program. UM has an outstanding cadre of Translational and Clinical Mentors, as well as, a large pool of strong and remarkably diverse potential K12 scholars to launch the proposed career development program. those radiation, faculty and University's practicums such of Scholars will include who recently completed postgraduate training in a subspecialty oncology program (pediatric, urologic, surgical, hematology, head and neck, gynecologic) and those who have recently assumed their first appointment. Scholars will choose a mentoring dyad composed of a Translational and a Clinical Mentor, will have the option of obtaining a Masters in Clinical and Translational Investigation, supported by our Clinical Translational Science Institute. Scholars will choose from a wide range of courses and designed to assist them in translating research discoveries into clinical practice. They will then use knowledge to design, implement and manage a clinical cancer therapy trial , which draws from the findings ongoing translational and population-based research initiatives led by K12 mentors.The UM K12 Program will fulfill the major objectives of the Calabresi Award. It will provide, for a two-year period, dedicated time, optimal mentorship, formal education, and practical experience in developing novel, patient centric clinical trials to a diverse group of talented, and highly-motivated clinical Scholars. The Program will evaluate short and long-term outcomes of scholars' professional trajectory and use corresponding data to adjust the program to appropriately meet the dynamic and rapidly changing landscape of clinical oncology research. An important feature of the UM K12 Program is its geographic location in South Florida, which is characterized by unparalleled multiculturalism. Such diversity provides a unique opportunity to develop trials responsive to the unique needs of racial/ethnic minorities, recent immigrants, and other population sub-groups, who contribute to cancer disparity.

Project Summary p27 is a CDK inhibitor that limits normal cell proliferation. We showed p27 C-terminal phosphorylation at T157 and T198 by AKT increases in mid G1 and p27pT157pT198 (p27pTpT) facilitates interaction with novel protein partners. We recently showed C-terminal p27pTpT phosphorylation promotes its association with cJun. Genomic profiling showed p27 is co-recruited with cJun to over half of cJun chromatin binding sites to either activate or repress target genes. p27/cJun activated targets include TGFB2, and are associated with EMT, and programs that upregulate stem cells and alter cell adhesion and migration. Profiles of target genes repressed by p27/cJun suggest that p27pTpT opposes tissue differentiation. A subset of p27/cJun target genes bind to a STAT3 consensus motif and p27, cJun and STAT3 all appear to bind and co-regulate cMYC. Notably, p27pTpT is a driver of stem cell potency: cellular p27pTpT upregulates spheres, SOX2, NANOG and cMYC and tumor initiating cells in vivo. Our TG-p27CK-DD mice transgenic for a p27 phosphomimetic mutant that fails to bind cyclin/CDKs, show multi-organ overgrowth and increased size, suggesting that p27CK-DD abrogates WTp27 actions to restrain normal stem and progenitor expansion. This grant investigates the role of p27 as a transcriptional regulator in normal cells and development. Our data support the hypothesis that WTp27 plays important transcriptional roles during differentiation, to limit stem and progenitor cell expansion in various tissues. In contrast, upon C-terminal phosphorylation, p27 interacts with cJun, STAT3 and other factors to expand or maintain tissue stem or progenitor cells and oppose differentiation. We will compare p27WT, p27CDK and p27CK-DD MEFS in AIM1 to evaluate how p27 interacts with cJun and STAT3 on chromatin to govern gene expression across the cell cycle from G0, to mid-G1 and the G1/S phase transition. We will investigate if increased C-terminal p27 phosphorylation alters co-regulator and target gene selection and abrogates the co-repressive functions of p27 in quiescent cells. In AIM2, we will separate actively transcribed euchromatin from transcriptionally inactive heterochromatin and carry out ChIP-seq and ChIP-Mass Spec to identify the chromatin associated p27 interactome involved in transactivator versus repressor complexes. AIM3 will study mouse development, tissue differentiation, and adult stem and progenitor populations in TG-p27CK- DD and TG-p27CK- mice. We will test if p27CK-DD confers stronger reprogramming potential on MEFs and disrupts differentiation of iPSC into embryoid bodies. This work will elucidate a novel function of p27 as a phosphorylation-dependent transcriptional regulator with implications for tissue stem cell control and potential applications in regenerative medicine. Controlled modulation of p27, or of p27 target genes identified herein, might offer potential to expand or regenerate hematologic and other tissues. Understanding how p27/cJun transcriptional programs regulate tissue development might identify new therapeutic targets to be exploited for tissue regeneration and illuminate other human diseases at the interface of differentiation and growth control.