Browsing by Subject "Breast cancer"

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Adjacent long non-coding RNA PVT1 and MYC co-operate in breast cancer with gain of 8q24
(2013-08) Tseng, Yuen-Yi
Copy number gain of 8q24 is a common structural abnormality in human cancers. Although MYC is usually assumed to be responsible for 8q24 gain cancer, the role of the other genes in the 8q24 region remains mostly unknown. We derived chromosome engineered mice with an extra copy of Myc-Gsdmc region which is syntenic to the common region gained in human 8q24-associated cancer. These mice show aberrant differentiation and loss of proliferation arrest of mammary epithelial cells, excessive branching of mammary ducts, and increased sensitization to mammary tumors. In contrast, mice carrying a duplication of either Myc or Pvt1-Gsdmc were found to be insufficient for neoplasia. We show that the long non-coding RNA Pvt1 and adjacent Myc can co-operate in tumorigenesis. Furthermore, PVT1 can regulate MYC protein stability in human breast cancer cells. Our study reveals a novel mechanism of MYC regulation by a long non-coding RNA in cancer cells and could provide therapeutic targets.
Antibodies In Cancer Therapy: New Targets, Applications And Combination Strategies
(2019-07) Khanna, Vidhi Devendra
Over the last decade, antibodies have become an important component in the arsenal of cancer therapeutics. Their high-specificity, low-off target effects, desirable pharmacokinetics and high success rate are a few of the many attributes that make them amenable for development as drugs. The work presented here explores the targeting, mechanisms and use of antibody-based cancer therapy. In the first chapter, we used a phage display-based cell panning procedure to develop two fully humanized antibodies, Tw1S4_6 and Tw1S4_AM6, that bind specifically to HSPG2/perlecan, a protein found to be overexpressed on tumor cells. Immunohistochemistry studies revealed high HSPG2 expression across various tumor sub-types including melanoma, bladder cancer, glioblastoma and ovarian cancer. There was significant correlation between high HSPG2 expression and poor survival in triple negative breast cancer, bladder and ovarian cancers. The data presented here points towards the relevance of HSPG2 as a novel target for not only triple negative breast cancer but other malignancies as well. Based on its over-expression in different solid tumors, we evaluated HSPG2 as a therapeutic target in the second chapter. We observed significant tumor growth inhibition with Tw1S4_AM6 in the triple negative MDA-MB-231-LM2 breast cancer xenograft model. This efficacy was reduced in NSG mice, suggesting NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) as a potential mechanism of action. In vitro studies using human PBMCs confirmed induction of ADCC with anti-HSPG2 antibodies. In addition, conjugation of Tw1S4_AM6 on the surface of polymeric nanoparticles enabled increased tumor cell uptake of nanoparticles, suggesting Tw1S4_AM6 could be valuable as a targeting ligand for drug delivery systems. There is a significant interest in designing therapeutic agents that can enhance ADCC and thereby improve clinical responses with approved antibodies. We have developed a suite of highly substituted imidazoquinolines, which activate TLR 7 and/or 8 and induce significantly higher levels of cytokines compared to the FDA-approved TLR7 agonist, imiquimod. In the third chapter, we evaluated our series of TLR7/8 agonists for their ability to improve ADCC. Our studies show that the second generation TLR 7/8 agonists induce robust pro-inflammatory cytokine secretion and activate NK cells. These agonists also enhanced ADCC in vitro. Finally, we found that these agonists significantly improved the anticancer efficacy of two monoclonal antibodies in vivo. Thus, the work presented here encompasses the three critical aspects of antibody therapeutics: identifying the target, understanding their mechanisms, and leveraging these mechanisms to improve their efficacy.
Association Between Adjuvant Chemotherapy and Nephrotoxicity and Kidney Function Monitoring in Elderly Breast Cancer Patients
(2013-05) Li, Shuling
Background: Chronic kidney disease (CKD) and cancer are major public health problems in the elderly population. In elderly cancer patients, little is known about chemotherapy-related nephrotoxicity or patterns of CKD screening. The purpose of this dissertation was to evaluate the association between adjuvant chemotherapy (CHEMO) and risks of acute kidney injury (AKI) and CKD and rate of CKD screening in elderly women diagnosed with stages I-III breast cancer. Methods: The study was a 1:1 individually matched, retrospective cohort design using Surveillance, Epidemiology, and End Results (SEER)-Medicare linked data. Matching was performed at the day of CHEMO initiation based on propensity score. The assembled matched cohorts were used in the analyses for all three objectives with different follow-up periods and statistical methods for each objective. HASH(0x307f974) Results: A total of 28,048 patients were included. CHEMO was associated with a 2.7-fold increased risk of AKI within 6 months after initiation (HR 2.7, 95% CI 1.8-4.1). To find a possible explanation to this association, the distribution of other diseases coded on hospital claims for AKI was examined and showed that septicemia occurred in 40% of CHEMO treated patients with AKI and in only 17% of untreated patients with AKI. No significant association was found between CHEMO and risk of CKD in the maximum 18 years follow-up (HR 1.00, 95% CI 0.93-1.07). The rate of CKD screening after treatment completion was low regardless of CHEMO status. HASH(0x2faf9d4) Conclusion: CHEMO is associated with increased risk of AKI. This association may be partially explained by septicemia caused by infection/neutropenia due to use of myelosuppressive chemotherapeutic agents, which highlights the importance of preventing serious complications of CHEMO in preventing AKI. The finding of no association between CHEMO and risk of CKD may not suggest a late nephrotoxic effect of chemotherapeutic agents commonly used to treat breast cancer in the adjuvant setting, or provide evidence to recommend a clinical practice guideline for CKD screening specifically in elderly breast cancer patients treated with CHEMO. Future studies of CKD as a late effect of cancer treatment for other solid tumors commonly treated with known or potential nephrotoxic agents are warranted.
Axillary Web syndrome ongoing medical evaluation
(2013-01) Koehler, Linda Ann
Background: Movement loss, reduced function, pain, and lymphedema are frequent problems following breast cancer surgery. Axillary web syndrome (AWS) is a condition that develops after breast surgery and appears as a cord causing pain and movement loss and has been considered a possible risk factor for lymphedema. There are no reported imaging correlates of AWS in the literature. This study ascertained the clinical and ultrasonographic characteristics of AWS. Methods: Patients with surgical breast cancer (n=36) were assessed for shoulder range of motion (ROM), lymphedema, function, pain, and psychological issues at 2 weeks, 4 weeks, and 3 months. Subjects with AWS had an 18 MHz ultrasound of the cord. Analysis: A repeated measures ANOVA compared the AWS and non AWS groups across visits. Univariable and multivariable logistic regression identified AWS risk factors. A sign test was used for ultrasound analysis. Results: Seventeen subjects were identified with AWS with ten subjects having AWS at 3 months. There was an interaction effect in shoulder abduction with active ROM and passive ROM being statistically lower in the AWS group at 2 and 4 weeks. There was an interaction effect in upper extremity lymphedema tissue dielectric constant (TDC) measures with the AWS group having initially higher measures then decreasing which was opposite of the non AWS group. Both groups indicated trunk edema on the chest wall using TDC measures. Functional, psychological, and pain measures had no group or interaction effect. Younger age, low BMI, and higher number of lymph nodes removed were identified as significant risk factors for AWS. The strongest predictor was identified as BMI. There were no significant differences in ultrasonographic characteristics. Conclusion: The onset of AWS is often within weeks after surgery but later onset is possible. Cords do not resolve in all subjects by 3 months. Greater early movement restriction is evident in subjects with AWS though neither group achieved full pain free motion. There were no differences in early edema measurements between groups, but both groups indicated trunk edema at 3 months. Low BMI was a risk factor for AWS. An identifiable structure was not found using an 18MHz ultrasound.
Biological functions of cytochrome P450 1A1 in the proliferation and survival of breast cancer cells
(2013-04) Rodriguez Pantoja, Mariangellys
Cytochrome P450 1A1 (CYP1A1) is an extrahepatic phase I metabolizing enzyme whose expression is suppressed under physiologic conditions, but can be induced by substrates via the aryl hydrocarbon receptor (AhR). Nonetheless, recent studies show that the majority of breast tumors constitutively express CYP1A1. These findings led us to test the hypothesis that CYP1A1 promotes breast cancer progression by evaluating the effects of CYP1A1 knockdown on the proliferation and survival of the MCF7 and MDA-MB-231 lines. Independent of estrogen receptor status, CYP1A1 knockdown decreases cell proliferation, decreases colony formation, blocks the cell cycle at G0/G1 associated with reduction of cyclin D1, and increases apoptosis associated with reduction of survivin. CYP1A1knockdown markedly increases phosphorylation of AMP-activated protein kinase (AMPK) and decreases phosphorylation of AKT, extracellular signal-regulated kinases (ERK)-1 and 2, and 70 kDa ribosomal protein S6 kinase (P70S6K). AMPK inhibition by compound C partially abrogates the proapoptotic effects of CYP1A1siRNA, suggesting that CYP1A1siRNA effects are mediated, in part, through AMPK signaling. Consistent with CYP1A1 knockdown results, pharmacologic reduction of CYP1A1 levels by the phytopolyphenol carnosol also correlates with impaired proliferation and induced AMPK phosphorylation. These results indicate that reduction of basal CYP1A1 expression is critical for inhibition of proliferation, which is neither affected by alpha-naphthoflavone-mediated inhibition of CYP1A1 activity nor modulated by AhR silencing. Growth complementation experiments were performed to identify the exact mechanism by which CYP1A1 regulates these signaling pathways. Our results show that CYP1A1 regulates cell signal transduction through mechanisms other than synthesis of 8,9- epoxyeicosatrienoic acid, 11,12- epoxyeicosatrienoic acid, 14,15- epoxyeicosatrienoic acid, 20-hydroxyeicosatetraenoic acid, and 17,18-epoxyeicosatetraenoic acid or metabolism of all-trans-retinoic acid, 9-cis retinoic acid, 13-cis retinoic acid and pregnenolone. These studies support that CYP1A1 may promote breast cancer proliferation and survival, at least in part, through AMPK signaling and that reduction of CYP1A1 levels is a potential strategy for breast cancer therapeutics.
Differential biophysical mechanisms driving cancer stem cell migration
(2022-01) Heussner, Rachel
Cancer stem cells (CSCs) are known to have a high capacity for tumor initiation and are likely a key player in the formation of metastases. We have previously shown that in aligned collagen constructs similar to in vivo structures indicative of disease progression, breast CSCs demonstrate enhanced directional and total motility compared to the carcinoma population as a whole (WP). Here, we show that increased motility is maintained by CSCs in diverse environments including elastic, nonaligned 2D polyacrylamide gels at various stiffness; 3D randomly oriented collagen matrices; and ectopic cerebral slices representative of common metastatic sites. The consistency of CSCs’ enhanced motility across diverse environments suggests a general shift in cell migration mechanics between well differentiated carcinoma cells and their stem-like counterparts. To further elucidate the source of differences in migration, we demonstrate that CSCs are less contractile than the carcinoma population as a whole and concomitantly produce fewer and smaller focal adhesions. This shift in CSC biophysical behavior can be tuned via contractility. The WP can be shifted to a CSC-like enhanced migratory phenotype using partial myosin II inhibition. Inversely, CSCs can be shifted to a less migratory WP-like phenotype using microtubule destabilizing drugs to increase contractility. This work begins to elucidate the mechanistic differences driving CSC migration and raises important implications regarding the potentially disparate effects of microtubule-targeting agents on the motility of different cell populations.
Effects of dietary fat and omega-3 fatty acids on eicosanoids, endogenous sex hormones and the insulin-like growth factor pathway
(2010-06) Orr, Lindsay Rae
This dissertation details a clinical trial that investigated the effects of three controlled, 8-week duration test diets: a high fat diet (HF; 40% of energy from fat), a low fat diet (LF; 20% of energy from fat), and a low fat diet high in omega-3 (n-3) fatty acids (LFn3; 23% energy from fat including 3% of energy from n-3 fatty acids) on breast cancer risk markers including plasma and urinary sex hormones, urinary eicosanoids, and insulin-like growth factor (IGF) pathway endpoints in postmenopausal women. Chapter 1 contains a review of the literature providing context for the clinical trial. Chapter 2 describes the effects of the three test diets on plasma phospholipid fatty acids (PLFA), urinary eicosanoids, and plasma sex hormones. The LFn3 diet significantly increased plasma n-3 PLFA and the HF diet significantly increased estradiol and urinary eicosanoids. These results indicate that high fat diet increases breast cancer risk markers, but are inconclusive with respect to n-3 fatty acids. Chapter 3 describes the effect of the three test diets on urinary sex hormones and metabolites. Urinary excretion of estrone was significantly greater after the LF and LFn3 compared to the HF; however in the context of all the urinary hormones and metabolites measured, this indicates that no clinically significant alterations were observed following the test diets. Chapter 4 details the effects of the test diets on IGF pathway endpoints. LFn3 increased IGF-I and IGF binding protein-3 (IGFBP-3) and the LF increased IGFBP-3. These results indicate that low fat diet may reduce free IGF-I while the addition of n-3 fatty acids to the low fat diet may increase free IGF-I concentrations. The impact on breast cancer risk mediated by the increase in IGF-I with the LFn3 is unknown, but an increase in circulating IGF-I may have an impact on reducing the effects of aging. In conclusion, the test diets had pronounced effects on PLFA but modest effects on plasma and urinary sex hormones. The LFn3 unexpectedly increased IGF-I concentrations, which may demonstrate a role of n-3 in preventing the effects of aging.
Effects of Green Tea Extract on Biomarkers of Breast Cancer Risk Including Reproductive Hormones and IGF axis Proteins
(2015-08) Samavat, Hamed
Objective: To investigate the effects of daily intake of a highly concentrated green tea extract (GTE) for one year on circulating sex hormones and insulin-like growth factor (IGF) proteins as well as urinary estrogens and estrogen metabolites in postmenopausal women with different catechol-O-methyltransferase (COMT) genotypes. Method: The Minnesota Green Tea Trial (MGTT) is a randomized, double-blind, placebo-controlled, phase II clinical trial. Healthy postmenopausal women with heterogeneously or extremely dense breast tissue (age = 59.78 ± 5.02 years; body mass index= 25.70 ± 8.21 kg/m2) were randomly assigned to the GTE group (n=538) and were given 4 capsules a day, each containing 200 mg epigallocatechin gallate (EGCG) and the others (n=537) to the placebo group. Participants were 93% non-Hispanic white and non-current hormone users. Twenty-four hour urine samples were collected at month 0 and at the end of the study, and fasting blood samples were drawn at months 0, 6 and 12. Circulating and urinary estrogens, as well as urinary estrogen metabolites were quantified by the liquid chromatography-tandem mass spectrometry method. Blood IGF axis proteins were analyzed by ELISA. Results: GTE supplementation was associated with reduced urinary estriol levels (P= 0.02) and higher urinary 2-hydroxyestrone (P= 0.02) compared to the placebo. There was also less of a reduction in the urinary levels of 16α-hydroxyestrone in the GTE versus placebo group. Intake of the GTE resulted in significant increase of circulating estradiol and testosterone and their corresponding free and bioavailable fractions, whereas these measures were reduced in the placebo group. Additionally, COMT genotype did not modify the GTE effect on either circulating sex hormones and IGF proteins or urinary estrogens. Conclusion: Daily intake of high-dose of green tea extract for 12 month exerts modest effects on urinary excretion of estrogen metabolites, yet these effects are not modified by the COMT polymorphisms. Potential breast cancer protective effects of GTE are not mediated by alterations in circulating sex hormones or IGF axis proteins.
FGFR1-induced soluble factors promote mammary tumorigenesis and chemoresistance
(2013-08) Bade, Lindsey Kay
The fibroblast growth factor receptor (FGFR) family consists of four receptor tyrosine kinases that are known regulators of cellular processes such as proliferation, migration, survival, and angiogenesis. Anomalous expression or uncontrolled activation of these receptors or their ligands has been correlated with progression of various types of cancer, including breast cancer. Specifically, the chromosomal locus of FGFR1, 8p11-12, is found to be aberrantly amplified in approximately 10% of patients diagnosed with breast cancer. Patients who harbor the FGFR1 amplification do not respond well to current therapies and develop resistance to hormone-based therapies. Therefore, understanding the molecular mechanisms of how FGFR1 overexpression promotes tumorigenesis may provide insights into better targets for novel, more effective therapies. The work presented here shows that FGFR1 activation significantly upregulates expression of the ligands AREG and EREG at the transcript and protein levels both in vitro and in vivo, which then activate EGFR signaling. AREG is critical for normal ductal morphogenesis in the mammary gland and has also been linked to breast cancer progression. Studies examining AREG expression in human breast cancers have found AREG expression to significantly correlate with regional lymph node metastases, large tumor size, and high-grade tumors. While EREG promotes proliferation of several normal and cancerous cell types, the role of EREG has not been extensively characterized in the mammary gland. However, recent studies have demonstrated that EREG is a potent mediator of metastasis of breast cancer cells to the lung and that overexpression of EREG is an indicator of poor prognosis for inflammatory breast cancer patients. EGFR, a member of the ErbB receptor tyrosine kinase family, has been well studied in the mammary gland, and it is known that EGFR is required for normal mammary gland ductal morphogenesis. Alternatively, overexpression or constitutive activation of EGFR in the mammary gland has been linked to mammary tumorigenesis. Additionally, overexpression of EGFR in the breast is associated with recurrence of earlier stage breast cancers and decreased disease-free and overall survival in later stage breast cancer patients. Notably, we demonstrate that EGFR activation is at least in part required for FGFR1-induced proliferation and migration and ERK1/2 activation, as inhibition of EGFR with the small molecule kinase inhibitor erlotinib significantly blocks these processes. Moreover, we show that FGFR1 and EGFR are co-expressed in TNBC cell lines and that both FGFR1 and EGFR can mediate Doxorubicin chemoresistance. We further show that FGFR1 upregulates expression of the cytokine LIF, which then signals through gp130/JAK to activate STAT3 in vitro. Directly inhibiting either FGFR1 or STAT3 significantly reduces chemoresistance and increases apoptosis in vitro. Furthermore, inhibition of FGFR1 with the small molecule inhibitor PD173074 results in increased chemosensitivity and apoptosis in a mouse model of mammary tumorigenesis. These results are significant because they are the first to show that FGFR1 signals through EGFR and that FGFR1 mediates chemoresistance through activation of STAT3. This study furthers our understanding of FGFR1-amplified mammary tumorigenesis and presents alternative factors for targeted therapies for patients with FGFR1-amplified breast cancers.
Fibroblast growth factor Receptor 1-induced osteopontin regulates proinflammatory molecules to mediate cross-talk between breast cancer cells and the surrounding tumor microenvironment
(2012-06) Reed, Johanna Rae
Tumor formation is an extensive process requiring complex interactions that involve both tumor cell-intrinsic pathways and soluble mediators within the microenvironment. Tumor cells exploit the intrinsic functions of many soluble molecules, including cytokines and chemokines and their receptors, to regulate pro-tumorigenic phenotypes that are required for development of the primary tumor. Previous studies demonstrated that activation of inducible FGFR1 (iFGFR1) in mammary epithelial cells resulted in increased proliferation, migration, and invasion in vitro and tumor formation in vivo. Early studies also indicated that iFGFR1 activation stimulated recruitment of macrophages to the epithelium resulting in increased epithelial cell proliferation and angiogenesis. Our current studies further examined this model to identify novel mechanisms that regulate early stage tumorigenesis with a specific emphasis on the soluble mediators that are regulated by iFGFR1 to promote epithelial and stromal cell migration. Results from this study elucidate a novel role for iFGFR1-induced osteopontin in promoting a proinflammatory tumor microenvironment through the regulation of proinflammatory molecules such as IL-1beta; and activation of the COX-2/PGE2 pathway as well as by promoting recruitment of CX3CR1-expressing macrophages through regulation of the chemokine CX3CL1. Defining the role of osteopontin-regulated proinflammatory, secreted molecules in promoting iFGFR1-mediated mammary tumorigenesis is important for understanding how initiating oncogenic events drive tumor growth and progression through the secretion of soluble mediators. Moreover, results from these studies will aid in identifying potential novel molecular targets for therapeutic intervention.
High-Resolution Breast Diffusion Weighted Imaging with Improved Nyquist Ghost Correction and Simultaneous Multislice Imaging
(2020-07) McKay, Jessica
Diffusion-weighted imaging (DWI) is a quantitative MRI method that measures the apparent diffusion coefficient (ADC) of water molecules, which reflects cell density and serves as an indication of malignancy. Unfortunately, however, the clinical value of DWI is severely limited by the undesirable features in images that common clinical methods produce, including large geometric distortions, ghosting and chemical shift artifacts, and insufficient spatial resolution. Thus, in order to exploit information encoded in diffusion characteristics and fully assess the clinical value of ADC measurements, it is first imperative to achieve technical advancements of DWI. The purpose of this work is to improve DWI methods for breast imaging at 3 Tesla to robustly provide diffusion-weighted images and ADC maps with anatomical quality and resolution. This dissertation will first lay out the background information to provide clinical motivation for this work and explain the current standard in breast DWI, as well as some alternatives proposed throughout the literature. The main work of this project has two major parts: Nyquist ghost correction and the use of simultaneous multislice imaging (SMS) to achieve high resolution. Exploratory work was completed to characterize the Nyquist ghost in breast DWI, showing that, although the ghost is mostly linear, the three-line navigator is unreliable, especially in the presence of fat. A novel referenceless ghost correction, Ghost/Object minimization was developed that reduced the ghost in standard SE-EPI and advanced SMS. An advanced SMS method with axial reformatting (AR) is presented for high resolution breast DWI. In a reader study, AR-SMS was preferred by three breast radiologists compared to the standard SE-EPI and readout-segmented-EPI. Finally, future directions are suggested, including some preliminary work explored throughout this project.
Humanized antibody development using phage display: applications to solid tumor metastasis
(2016-07) Kalscheuer, Stephen
The outlook for cancer patients who present with evidence of metastasis is best characterized as a precipitous decline in prognosis and overall survival. This dramatic reduction in survival suggests a need to focus on the development of therapeutic and diagnostic reagents that are tailored to cancer in its disseminated form. In pursuit of this goal, a phage display based phenotype screening platform was developed to generate humanized antibodies for use in both circulating tumor cell detection, and therapeutic intervention, using in vivo models of breast cancer metastasis. A broader perspective on this work is that it highlights methods that focus on biologic drug development based on disease phenotype, as opposed to conventional target-based methods. In the context of metastasis, the present work focused on the relevant cancer cell phenotype, termed epithelial to mesenchymal transition, which is believed to be the driver phenotype of cancer dissemination. Phenotype screening approaches do not require prior knowledge of potential targets, and are thus amenable to cancer biomarker discovery, which in turn can lead to innovative, first-in-class approaches to cancer management. A broadly applicable method for deriving humanized antibodies from cell based phenotype screening was developed. Target deconvolution approaches to identify the binding partner of candidate antibodies were also explored. Finally, the fine tuning of antibody binding affinity via affinity maturation methods was also explored through physiologically based pharmacokinetic modelling, in an attempt to establish optimal targeting affinities for both solid tumors and metastases. The work concludes with in vitro and in vivo characterization of two candidate antibodies as therapeutic agents.
IGF-1 stimulates glycolytic ATP production in MCF-7L cells
(2023-07) Rajoria, Bhumika
The Insulin-like Growth Factor (IGF) system in breast cancer progression has been a matter of interest for decades, but targeting this system did not result in a successful clinical strategy. The system’s complexity, and homology of its two receptors - insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) are possible causes. The IGF system maintains cell proliferation and regulates metabolism, making it a pathway to explore. To understand the metabolic phenotype of breast cancer cells, we quantified their real-time ATP production rate upon acute stimulation with ligands – insulin-like growth factor 1 (1GF-1) and insulin. MCF-7L cells express both IGF-1R and IR, while tamoxifen-resistant MCF-7L (MCF-7L TamR) cells have downregulated IGF-1R with unchanged IR levels. Treating MCF-7L cells with 5nM IGF-1 increased the glycolytic ATP production rate while 10nM insulin did not affect metabolism when compared with control. Neither treatment altered ATP production in MCF-7L TamR cells. The study provides evidence of the relationship between metabolic dysfunction, cancer, and the IGF axis. In these cells, IGF-1R, and not IR, regulates ATP production.
Membrane and nuclear progesterone receptors activate tumor suppressive signaling mechanisms in cell line models of human ovarian cancer
(2010-07) Charles, Nathan Jon
Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States. Mortality rates for ovarian cancer have been unchanged for more than 70 years even though surgical and chemotherapeutic strategies have become considerably more sophisticated. While a lack of clinical success is largely due to a poor etiologic understanding, recent observations suggest that the ovarian steroid hormone progesterone may be an endogenous ovarian cancer tumor suppressor. Therefore, the goal of our studies was to define progesterone receptor action in ovarian cancer and identify the signaling mechanisms responsible for its tumor suppressive function. Membrane progesterone receptors (mPRs) represent a newly defined class of ~40 kDa GPCR-like progesterone receptors belonging to the adipoQ receptor ( PAQR ) gene family. Never studied in cancerous cells of ovarian surface epithelial origin, we identified positive expression of each isoform (mPRα/PAQR7, mPRβ/PAQR8, and mPRγ/PAQR5) in a panel of ovarian cancer cell lines. Contrary to breast cancer cells, progesterone stimulation of these receptors in ovarian cancer cells increased intracellular cAMP levels and cAMP response element (CRE) transcriptional activity, but required high, pregnancy equivalent levels of progesterone as well as β 1,2 -adrenergic receptor co-stimulation. High-dose progesterone exposure also increased phosphorylation of the stress-activated JNK1/2 and p38 mitogen activated protein kinases (MAPKs). In particular, mPR-mediated p38 activation was responsible for increasing BAX mRNA expression; a pro-apoptotic Bcl family member. These results demonstrate that functionally active mPRs are capable of activating signaling pathways associated with tumor suppression in ovarian cancer cells. Clinical observations have shown that nuclear progesterone receptor (PR) expression is downregulated as ovarian tumors become progressively more malignant. Overexpression of PR in the ES-2 ovarian cancer cell line inhibited cellular proliferation and increased cell survival. As a result, prolonged ligand stimulation caused PR-expressing cells to undergo cellular senescence and exit the cell-cycle into G 0 . Senescent cells expressed significantly higher mRNA levels of the cell-cycle inhibitor p21. When the inhibition of PR-induced p21 expression was removed by stably downregulating STAT3 expression, PR-mediated senescence occurred more quickly and p21 induction was enhanced as cells arrested in the G1 phase of the cell-cycle. However, when increased p21 expression was prevented, ligand-stimulated PR-expressing cells also exhibited a heightened senescence response. These findings, along with the observation that ligand-stimulated PR-positive primary human ovarian cancer cells also become senescent, support our conclusion that PR-mediated cellular senescence is an endogenous ovarian cancer tumor suppressive mechanism. Taken together, these results demonstrate that progesterone receptors possess tumor suppressing characteristics in ovarian cancer cells, and warrant further investigation into the use of progesterone as an ovarian cancer chemopreventive and chemotherapeutic agent.
Modeling Brk/PTK6 expression in the mammary epithelium.
(2010-12) Lofgren, Kristopher Andrew
Protein tyrosine kinases (PTKs) regulate cellular proliferation and differentiation during development and homeostasis of normal tissues. Additionally, PTKs are frequently overexpressed in cancers. As such, they have become promising targets for new therapies. Breast tumor kinase (Brk/PTK6) is a non-receptor tyrosine kinase initially cloned from a metastatic breast tumor, and is overexpressed in ~86% of human breast tumors and several breast cancer cell lines. While expressed in differentiating cells in the skin and intestine, Brk is notably absent from normal mammary tissue and non-transformed mammary epithelial cell lines. The role of Brk/PTK6 in breast pathology is unclear. We hypothesized that Brk expression in non-transformed mammary epithelium would promote tumorigenesis. To determine the effects Brk expression in the normal mammary gland, we sought to create a mouse model for tissue-specific Brk expression. We expressed a WAP-driven Brk/PTK6 transgene in FVB/n mice, and analyzed wild type and transgenic mammary glands after forced weaning. Brk-transgenic dams exhibited delayed mammary gland involution and infrequent tumors in aged mice. Transgenic mammary glands displayed decreased STAT3 phosphorylation, a marker of early stage involution. Signaling mediators downstream of Brk, including STAT5 and p38 MAPK were activated relative to wild-type mice. Brk-mediated signaling to STAT5 and p38 MAPK was recapitulated with Brk expression in non-transformed HC11 murine mammary epithelial cells. Additionally, non-transformed HMEC and HC11 cells expressing Brk exhibited increased anchorage-independent survival when cultured on PolyHEMA coated dishes. Samples from breast tumor biopsies were subjected to IHC analysis for co-expression of Brk, phospho-STAT5, and phospho-p38 MAPK. Ductal and lobular carcinomas expressing Brk exhibited elevated phospho-STAT5 and phospho-p38 MAPK, respectively, whereas non-malignant tissues were Brk-null with dramatically less phospho-STAT5 and phospho-p38 expression. HMEC cells expressing Brk underwent luminal hollowing (reliant on cell death) similar to normal mammary alveologenesis when cultured on Matrigel, but are partially resistant to doxorubicin treatment, suggesting a context dependent component to Brk-mediated survival. HGF, EGF, or IGF treatment in cells that endogenously express Brk (non-transformed HaCaT keratinocytes and MDA-MB-231 breast cancer cells), resulted in growth factor specific changes in Brk localization, but only in MDA-MB-231 cells. Brk-knockdown in breast cancer cells (T47D and SKBR3) identified differential phosphorylation of Erk5, and notably, p38 MAPK in response to EGF and heregulin-β1 treatment. These studies illustrate that forced expression of Brk/PTK6 in non-transformed mammary epithelial cells in vivo mediates STAT5 and p38 MAPK phosphorylation and promotes increased cellular survival, delayed involution, and latent tumor formation. Brk expression in human breast tumors may contribute to progression by inducing these same pathways, as evidenced in clinical samples of invasive breast carcinoma. Models of Brk expression in non-transformed mammary epithelial cells in vitro promote cellular survival in a context dependent manner, however, remain a strong tool for identifying molecules contributing to Brk mediated breast tumor progression. Inhibition of STAT5 and/or p38 MAPK may provide strong therapeutic potential in Brk-positive breast cancer.
One-carbon metabolism and breast cancer
(2013-02) Inoue-Choi, Maki
Breast cancer is the most common cancer among women in the United States. Nutrients in one-carbon metabolism (OCM) have been examined as potential modifiable risk factors because of OCM’s important role in DNA methylation and DNA synthesis. However, biologic mechanisms between OCM and carcinogenesis are still not clarified. The first manuscript tested the hypothesis that OCM nutrient status and genetic variation in methionine adenosyltransferases (MAT1A, MAT2A and MAT2B) are associated with plasma S-adenosylmethionine (SAM) levels in a cross-sectional analysis among healthy Singapore Chinese adults. Choline and methionine were strongly and positively associated with plasma SAM levels (ptrend<0.0001), and folate and betaine were positively associated with plasma SAM only in men (ptrend=0.02). The association between MAT1A rs2993763 and plasma SAM was modified by gender and plasma methionine levels. In the same study population, the second manuscript cross-sectionally tested the hypothesis that plasma SAM levels alone or in combination with genetic variation in DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) are associated with global DNA methylation measured at long interspersed nucleotide element-1 (LINE-1). The LINE-1 methylation index was positively associated with plasma SAM levels (p≤0.01), with a plateau at approximately 78% and 77% methylation in men and women, respectively. Among men, there were statistically significant positive or negative associations between DNMT1 rs2114724 or DNMT3A rs758127 genotype and the LINE 1 methylation index, respectively (ptrend<0.01). The SAM-LINE-1 methylation association was modulated by DNMT1 rs2114724 genotype in men only. In a prospective cohort study, the third manuscript attempted to replicate increased breast cancer risk related to higher dietary nitrate intake only among those with low folate intake, which was previously reported by a case-control study. Opposite my hypothesis, among women with total folate intake 400 μg/day or above, breast cancer risk was statistically significantly higher among women in the highest quintile of nitrate intake from public water (HR=1.40, 95%CI=1.05-1.87) and private well users (HR=1.38, 95%CI=1.05-1.82) than those with the lowest nitrate intake from public water. The projects described in this dissertation contribute evidence describing how OCM may be related to cancer risk, and are a step in pursuit of my long-term goal to enhance our understanding of cancer etiology through OCM.
Out-Of-Pocket Costs, Subsidies, And The Delivery Of Breast Cancer Care Among Elderly Women
(2020-11) Qin, Xuanzi
Out-of-pocket (OOP) costs can affect patients’ access to care and clinical outcomes. This dissertation takes advantage of the natural experiment provided by the combination of the introduction of generic aromatase inhibitors (AIs) and Medicare Part D low-income subsidy (LIS) policy to understand the role of OOP costs and subsidies in the delivery of breast cancer care. Guidelines suggest postmenopausal women diagnosed with hormone receptor-positive (HR+) breast cancer initiate adjuvant hormonal therapy with either AIs or tamoxifen. Switching to another therapy drug is an important strategy to manage treatment related side effects. Those diagnosed at early stages should also receive surgery and/or radiation before or after the initiation of hormonal therapy. The three AIs, anastrozole, exemestane and letrozole, went off patent sequentially in 2010 and 2011. Generic entry lowered OOP costs for AIs. Medicare Part D beneficiaries receiving LIS (subsidized) have substantial lower OOP costs for prescription drugs than those without LIS (unsubsidized), and thus are unlikely to be affected by changes in OOP costs due to generic entry. Medicare and Medicaid dually eligible beneficiaries (duals) receive LIS and Medicaid support for other medical services. This dissertation uses the Surveillance, Epidemiology and End Results (SEER)-Medicare linked database to identify the main study cohort that consists of women first diagnosed with HR+ breast cancer at age 65 years and older between 2007 and 2013 (N=93,650). I find the introduction of generic AIs was associated with improved pharmaceutical access indicated by increased probability of initiating any hormonal therapy drugs, increased timeliness of initiation and increased probability of receiving AIs over tamoxifen. Despite the minimal reduction in OOP costs for AIs after generic entry among the subsidized group, the subsidized experienced similar changes in outcomes like the unsubsidized group who had large reductions in OOP costs after generic entry. Thus, reduced OOP costs due to generic entry can only partially explain the improved access after generic entry. I also find that generic entry increased therapy adherence and reduced early discontinuation both directly through reduced OOP costs and through intermediary changes in switching behaviors resulted from reduced OOP costs. Generic entry affected therapy adherence and continuation without drug switches directly through reduced OOP costs, and thus increased adherence and continuation without drug switches after generic entry were only observed in the unsubsidized group. Generic entry might affect adherence and continuation with drug switches through both reduced OOP costs and improved management of side effects, and thus increased adherence and continuation with drug switches were observed in both the subsidized and unsubsidized groups. Finally, I find that although duals have their medical service use and prescription drug use subsidized, duals were less likely to be diagnosed at early stages, receive guideline-compliant treatment and initiate hormonal therapy than non-duals. Among duals, those with Medicaid coverage gaps or coverage changes were less likely to be diagnosed at early stages and more likely to discontinue their hormonal therapy than duals without any coverage changes. These results demonstrate that interventions simply reducing OOP costs may be not enough for improving access to care. The inconsistent relationships between costs and health care use and outcomes among the subsidized group highlight the importance to understand non-financial barriers to access, such as health insurance literacy, physician and pharmacist behaviors.
Pantomeshes: kinematics, synthesis, and applications of closed pantograph-style linkage systems.
(2010-12) Larson, Blake Timothy
This research describes the kinematics, analysis, and synthesis of a pantomesh. A pantomesh is a patchwork assembly of pantograph elements (known elsewhere as scissor pairs or duplets) that obey certain mobility requirements. A pantomesh, as described in this thesis, has scissor-like elements connected to one another by spherical joints to allow a wide variety of motions. Previous pantograph-style linkages, such as the Hoberman Sphere, use special geometry restrictions and have elements joined with gussets, thereby limiting the variety of shapes possible. The thesis begins with examining the kinematics of pantomeshes and their constituent parts. First, the kinematics of the individual pantograph elements are detailed for further use. The mobility of a closed pantomesh is ensured by the mobility of its constituent pantopatches, two-wide by two-high sub-assemblies of pantograph elements that must be mobile themselves for the entire pantomesh to be mobile. A new method for mobility of spatial linkages is presented relating the use of polygonal elements. Next, two methods for pantomesh synthesis are presented. A graphical method is presented to use a computer-aided design system to create a mobile pantomesh that meets specified requirements. A computational method for synthesis is also presented, using a numerical optimization method to create pantomeshes to certain specifications. Practical considerations of manufacturing are considered in the discussion of multi-link spherical joints, including past work and new approaches. The new approaches include a compliant multi-link spherical joint and a crossed-tendon system that acts a a spherical joint. Finally, an application is presented: a new linkage which provides radial pressure for the purpose of stabilizing a human breast during cancer-related diagnosis and treatment procedures.
Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression.
(2012-07) Knutson, Todd Philip
Introduction: Progesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by MAPK and CDK2. Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (i.e. a repressive modification). Antagonism of PR SUMOylation by mitogenic protein kinases provides a mechanism for derepression (i.e. transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression. Methods: Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by MTT and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature. Results: “SUMO-sensitive” PR target genes (i.e. repressed by PR SUMOylation) primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (i.e. MSX2, RGS2, MAP1A, and PDK4) along with the steroid receptor coactivator, CBP, and MLL2, a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at “closed” enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with ERBB2-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR targetgenes and inhibited proliferation in BT-474 (ER+/PR+/ERBB2+) breast cancer cells. Conclusions: We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (i.e. derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin. Supplementary files: The supplementary files presented in this dissertation are fully described in the appendices. They include: (A) Antibodies used in this study, (B) PCR primer sets used in this study, (C) Genes differentially regulated by wild-type and SUMO-deficient PR, (D) Overlapping lists of PR-dependent target genes from previously described gene expression microarrays, (E) The PR ligand-dependent (LD) and ligand-independent (LI) KR>WT gene signatures, (F) Breast tumor Oncomine concepts associated with the LD KR>WT gene signature.
The role of Krüppel-like factors in breast cancer cell mechanobiology
(2023) Marocci Lima Pimenta, Rafaela
Mechanobiology explores the relationship between mechanical forces and biological systems, and is increasingly being realized to play fundamental roles in human health and disease. There is constant cross-talk between cells and their surrounding stroma, in which cells can perceive physical cues and modify their behavior - as well as their environment - in response. Mechanosensitive transcription factors (MSTFs) function as important effectors in this process, transforming physical stimuli into changes in gene expression. In cancer, physical changes in the tumor microenvironment can influence the activity of genes involved in proliferation, migration, and invasion, and thereby impact disease progression. This research focuses on Krüppel-like factors (KLFs), a group of transcription factors initially recognized for their importance in embryo patterning and stem cell plasticity. More recently, however, it has been shown that KLFs can also function as MSTFs in a number of diseases, including cancer. The aim of this project was to investigate whether a select group of KLFs (KLF2, KLF4, KLF5, and KLF6) function as MSTFs in breast cancer cells. To do this, we looked at patterns of KLF expression in response to both long-term adaption to different physical environments, as well as to rapid physical changes, using two-dimensional (2D) and three-dimensional (3D) cell culture model systems and the 4T1 series of heterogeneous breast cancer subclones. Signaling pathways potentially involved in transducing physical events into KLF expression changes were examined, and siRNA KLF knockdown experiments were conducted to assess the possible impacts of altered KLF expression on tumor cell morphology, adhesion, proliferation, invasion, and the ability to exert tension on the extracellular matrix. RT-qPCR results indicate that the expression of the KLFs examined exhibit both short-term (rapid), as well as longer-term (adaptive) responses to changes in the physical microenvironment. Some of the expression patterns were common among the different KLFs and tumor cell subclones, whereas certain others diverged. Pharmacological inhibition studies implicated mechanosensitive Piezo1 calcium channels and focal adhesion kinase activity in coupling physical changes and KLF expression. Cell-cell and cell-substrate adhesion, and the ability of cells to invade a collagen gel were identified as behaviors markedly affected by KLF5 and KLF6 knockdown, respectively. Overall, these results support the hypothesis that KLFs function as mechanosensitive transcription factors in breast cancer cells.

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