Lourdes M. Nogueira

Mesocortical Dopamine Neurons Operate in Distinct Temporal Domains Using Multimodal Signaling

In vivo extracellular recording studies have traditionally shown that dopamine (DA) transiently inhibits prefrontal cortex (PFC) neurons, yet recent biophysical measurements in vitro indicate that DA enhances the evoked excitability of PFC neurons for prolonged periods. Moreover, although DA neurons apparently encode stimulus salience by transient alterations in firing, the temporal properties of the PFC DA signal associated with various behaviors is often extraordinarily prolonged. The present study used in vivo electrophysiological and electrochemical measures to show that the mesocortical system produces a fast non-DA-mediated postsynaptic response in the PFC that appears to be initiated by glutamate. In contrast, short burst stimulation of mesocortical DA neurons that produced transient (<4 s) DA release in the PFC caused a simultaneous reduction in spontaneous firing (consistent with extracellular in vivo recordings) and a form of DA-induced potentiation in which evoked firing was increased for tens of minutes (consistent with in vitro measurements). We suggest that the mesocortical system might transmit fast signals about reward or salience via corelease of glutamate, whereas the simultaneous prolonged DA-mediated modulation of firing biases the long-term processing dynamics of PFC networks.

Long-Term Neuroadaptations Produced by Withdrawal from Repeated Cocaine Treatment: Role of Dopaminergic Receptors in Modulating Cortical Excitability

Dopamine (DA) modulates neuronal activity in the prefrontal cortex (PFC) and is necessary for optimal cognitive function. Dopamine transmission in the PFC is also important for the behavioral adaptations produced by repeated exposure to cocaine. Therefore, we investigated the effects of repeated cocaine treatment followed by withdrawal (2–4 weeks) on the responsivity of cortical cells to electrical stimulation of the ventral tegmental area (VTA) and to systemic administration of DA D1 or D2 receptor antagonists. Cortical cells in cocaine- and saline-treated animals exhibited a similar decrease in excitability after the administration of D1 receptor antagonists. In contrast, cortical neurons from cocaine-treated rats exhibited a lack of D2-mediated regulation relative to saline rats. Furthermore, in contrast to saline-treated animals, VTA stimulation did not increase cortical excitability in the cocaine group. These data suggest that withdrawal from repeated cocaine administration elicits some long-term neuroadaptations in the PFC, including (1) reduced D2-mediated regulation of cortical excitability, (2) reduced responsivity of cortical cells to phasic increases in DA, and (3) a trend toward an overall decrease in excitability of PFC neurons.

AGE Metabolites: A Biomarker Linked to Cancer Disparity?

Socioeconomic and environmental influences are established factors promoting cancer disparity, but the contribution of biologic factors is not clear. We report a mechanistic link between carbohydrate-derived metabolites and cancer that may provide a biologic consequence of established factors of cancer disparity. Glycation is the nonenzymatic glycosylation of carbohydrates to macromolecules, which produces reactive metabolites called advanced glycation end products (AGE). A sedentary lifestyle and poor diet all promote disease and the AGE accumulation pool in our bodies and also increase cancer risk. We examined AGE metabolites in clinical specimens of African American and European American patients with prostate cancer and found a higher AGE concentration in these specimens among African American patients when compared with European American patients. Elevated AGE levels corresponded with expression of the receptor for AGE (RAGE or AGER). We show that AGE-mediated increases in cancer-associated processes are dependent upon RAGE. Aberrant AGE accumulation may represent a metabolic susceptibility difference that contributes to cancer disparity. Cancer Epidemiol Biomarkers Prev; 23(10); 2186–91. ©2014 AACR.

SNAI2 modulates colorectal cancer 5-fluorouracil sensitivity through miR145 repression

Epithelial-to-mesenchymal transition (EMT) has been associated with poor treatment outcomes in various malignancies and is inversely associated with miRNA145 expression. Therefore, we hypothesized that SNAI2 (Slug) may mediate 5-fluorouracil (5FU) chemotherapy resistance through inhibition of miR145 in colorectal cancer and thus represents a novel therapeutic target to enhance current colorectal cancer treatment strategies. Compared with parental DLD1 colon cancer cells, 5FU-resistant (5FUr) DLD1 cells demonstrated features of EMT, including >2-fold enhanced invasion (P < 0.001) and migration, suppressed E-cadherin expression, and 2-fold increased SNAI2 expression. DLD1 and HCT116 cells with stable expression of SNAI2 (DLD1/SNAI2; HCT116/SNAI2) also demonstrated EMT features such as the decreased E-cadherin as well as significantly decreased miR145 expression, as compared with control empty vector cells. On the basis of an miR145 luciferase promoter assay, we demonstrated that SNAI2 repressed activity of the miR145 promoter in the DLD1 and HCT116 cells. In addition, the ectopic expressing SNAI2 cell lines demonstrated decreased 5FU sensitivity, and, conversely, miR145 replacement significantly enhanced 5FU sensitivity. In the parental SW620 colon cancer cell line with high SNAI2 and low miR145 levels, inhibition of SNAI2 directly with short hairpin sequence for SNAI2 and miR145 replacement therapy both decreased vimentin expression and increased in vitro 5FU sensitivity. In pretreatment rectal cancer patient biopsy samples, low miR145 expression levels correlated with poor response to neoadjuvant 5FU-based chemoradiation. These results suggested that the SNAI2:miR145 pathway may represent a novel clinical therapeutic target in colorectal cancer and may serve as a response predictor to chemoradiation therapy. Mol Cancer Ther; 13(11); 2713–26. ©2014 AACR.

Abstract A47: MicroRNA-510 as a predictive marker for response to platinum-based chemotherapy in triple negative breast cancer

Breast cancer is a heterogeneous disease with multiple subtypes, which are clinically classified based on the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Associated with the poorest prognosis of all subtypes, triple negative breast cancer (TNBC) is defined as ER-/PR-/HER2-; without these molecular targets, these cancers are insensitive to highly effective targeted therapies and systemic chemotherapy remains the mainstay of treatment for these women. Resent research has shown that platinum chemotherapy agents are particularly active in TNBC, including a clinical trial that demonstrated single agent cisplatin alone can induce response in a subset of TNBC patients. The identification of biomarkers to predict response is required to distinguish patients most likely to benefit from this agent from resistant ones, whom may respond better from other forms of chemotherapy. We have published studies examining the role of miR-510 in breast cancer and observed that miR-510 expression is elevated in tumors when compared to matched non-tumor samples. Drug cytotoxicity assays indicate miR-510 positively correlates with sensitivity to cisplatin in vitro. Furthermore, inhibition of miR-510 causes sensitive breast cancer cell lines to be more resistant in vitro, while overexpression of miR-510 restores sensitivity to cisplatin in resistant breast cancer cell lines both in vitro and in vivo. Recent mechanistic studies have revealed that cisplatin activates the ΔNp63/TAp73 apoptotic pathway specifically in in TNBC with p53 mutations (∼60% of all TNBCs). We have validated peroxiredoxin 1 (Prdx1) as a direct target of miR-510 and studies have also indicated that Prdx1 is a negative regulator of the ΔNp63/Tap73 pathway, suggesting that miR-510 may mediate sensitivity to cisplatin through the negative regulation of Prdx1. We demonstrate an increase in the activation of this pathway in TNBC cells expressing miR-510 upon cisplatin treatment. Based on these data we propose that elevated levels of miR-510 mediates cisplatin sensitivity and that it may serve as a non-invasive biomarker to predict response to cisplatin in TNBC patients. To this end, we have shown by qPCR that miR-510 levels are detectable in serum samples from a subset of human breast cancer patients, and further studies will assess positive correlations between miR-510 expression and cisplatin sensitivity. Citation Format: Qi Jin Guo, Jamie N. Miils, Natalie Mason, Savannah G. Bandurraga, Lourdes M. Nogueira, Rita Kramer, David P. Turner, Victoria J. Findlay. MicroRNA-510 as a predictive marker for response to platinum-based chemotherapy in triple negative breast cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A47.

Strong somatic stimulation differentially regulates the firing properties of prefrontal cortex neurons.

Among the brain structures involved in processing affective stimuli, the roles of the prefrontal cortex (PFC) and the mesocorticolimbic dopaminergic (DA) innervation are well established. In contrast to our understanding of the reward stimuli, less is known about how strong somatic stimulation is processed within the PFC. Here, we examined the effects of a strong pinch delivered to the rat posterior paw on spontaneous and current-evoked activity of PFC neurons using intracellular recordings in anesthetized rats. Following the paw pinch, pyramidal cells exhibited a significant decrease in spontaneous activity along with a significant increase in the current-evoked firing. The increase in current-evoked firing elicited by the paw pinch was inversely correlated with the baseline firing rate. Systemic administration of a selective dopamine D2 receptor antagonist partially blocked the effects elicited by the paw pinch on cortical excitability, whereas systemic administration of a D1 antagonist seems to facilitate paw-mediated increases in evoked firing. These results suggest that strong somatic stimuli decrease spontaneous firing while increasing depolarization-evoked firing in a DA receptor dependent manner. These mechanisms may help in the control of the signal to noise ratio or the salience of information processing in the PFC following strong somatic stimulation.

Advanced glycation end products are elevated in estrogen receptor-positive breast cancer patients, alter response to therapy, and can be targeted by lifestyle intervention

PurposeLifestyle factors associated with personal behavior can alter tumor-associated biological pathways and thereby increase cancer risk, growth, and disease recurrence. Advanced glycation end products (AGEs) are reactive metabolites produced endogenously as a by-product of normal metabolism. A Western lifestyle also promotes AGE accumulation in the body which is associated with disease phenotypes through modification of the genome, protein crosslinking/dysfunction, and aberrant cell signaling. Given the links between lifestyle, AGEs, and disease, we examined the association between dietary-AGEs and breast cancer.MethodsWe evaluated AGE levels in bio-specimens from estrogen receptor-positive (ER+) and estrogen receptor-negative (ER−) breast cancer patients, examined their role in therapy resistance, and assessed the ability of lifestyle intervention to reduce circulating AGE levels in ER+ breast cancer survivors.ResultsAn association between ER status and AGE levels was observed in tumor and serum samples. AGE treatment of ER+ breast cancer cells altered ERα phosphorylation and promoted resistance to tamoxifen therapy. In a proof of concept study, physical activity and dietary intervention was shown to be viable options for reducing circulating AGE levels in breast cancer survivors.ConclusionsThere is a potential prognostic and therapeutic role for lifestyle derived AGEs in breast cancer. Given the potential benefits of lifestyle intervention on incidence and mortality, opportunities exist for the development of community health and nutritional programs aimed at reducing AGE exposure in order to improve breast cancer prevention and treatment outcomes.

Abstract P4-05-01: Diet, development and predisposition to breast cancer: The impact of sugar derived metabolites (AGEs) on pubertal mammary gland development

The mammary gland is one of the few organs that continues to develop postnatally through stages including puberty, pregnancy, lactation, and involution. The gland is composed of epithelial and stromal cells that include fibroblasts, adipocytes, endothelial cells, nerve cells, and macrophages. Terminal end bud (TEB) structures are found exclusively in the pubertal developmental stage. The formation of TEBs and side branching drives mammary gland epithelial cell invasion into the mammary fat pad, continuing until the entire fat pad is filled. Pubertal mammary gland morphogenesis integrates a balance of epithelial cell proliferation, differentiation, and apoptosis. Several studies have shown that the interaction between mammary epithelial and stromal cells is crucial for the proper postnatal development of the mammary ductal tree. Interestingly, studies have shown that processes important in mammary gland development are often deregulated during breast cancer tumorigenesis. Thus, understanding the complex signaling network as well as the interactions between the different cell types during mammary gland development will be vital for elucidating the mechanisms underlying breast cancer progression and metastasis. Glycation is the non-enzymatic glycosylation of sugar moieties to biological macromolecules such as protein and DNA which produces reactive metabolites known as advanced glycation end products (AGE9s). AGE content in the Western Diet has consistently increased over the last 50 years due to increased consumption of sugar laden and cheap processed/manufactured foods which are high in reactive AGE metabolites. AGE containing food can lead to the accumulation of AGEs in the body overtime leading to pro-inflammatory and pro-oxidant effects when signaling through receptor for advanced glycation end products (RAGE). Leading too many complications associated with diseases including diabetes, Alzheimer9s, heart disease and cancer. Preliminary data in our lab has shown that AGEs also have an effect on phosphorylation and signaling of estrogen receptor α (ERα), a key receptor and signaling pathway in the regulation of mammary gland development during puberty. This observation, together with the links between diet, mammary gland development and immune cell recruitment lead us to examine the biological effects of a diet high in AGEs on pubertal mammary gland development in mice. We observed a significant disruption of normal pubertal mammary gland development in mice fed a high AGE diet when compared to mice fed a control diet. Mice fed the high AGE diet showed increases in TEB number as well as width, length and area. We also observed an increase in ductal branching and a decrease in ductal extension. Future studies will assess the role of macrophage recruitment to the developing gland, specifically around the TEBs based on its reported role in normal TEB function. We also plan to assess ERa signaling in mice fed the high AGE diet based on the reported role of estrogen signaling in ductal elongation. Citation Format: Krisanits BA, Nogueira LM, Findlay VJ, Turner DP. Diet, development and predisposition to breast cancer: The impact of sugar derived metabolites (AGEs) on pubertal mammary gland development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-05-01.

Abstract PR11: The molecular implications of lifestyle associated metabolites (AGEs) to prostate cancer disparity

Poor diet, low income, obesity, and a lack of exercise are established lifestyle factors that are known to increase cancer burden and are often more prevalent in African American communities. As our understanding of tumor biology advances, it is becoming increasingly clear that these inter-related lifestyle factors have distinct molecular consequences on the biologic make up of tumors, altering cell signaling events and gene expression profiles to contribute to cancer disparity outcomes such as its earlier development or its progression to more aggressive disease. Advanced glycation end-products (AGEs), are reactive metabolites produced endogenously as a consequence of glucose uptake during glycolysis. AGEs accumulate in tissues and organs as we grow older to promote multiple chronic disease phenotypes. AGE pathogenic effects are mediated through modification of protein function, genetic fidelity, stress responses and cellular signaling pathways. Critically, cancer disparity factors such as a sedentary lifestyle, obesity and an unhealthy diet are external influences that also contribute to the AGE accumulation pool in the body. The investigators studies support the concept that AGE metabolites represent a biological consequence of the socioeconomic and environmental factors that promote cancer health disparity. This research group examined circulating and tumor AGE levels in clinical specimens of prostate cancer and identified a race specific, tumor-dependent pattern of accumulation. The aberrant activation and recruitment of immune cells is a major pathogenic consequence of AGE accumulation and a series of studies have highlighted the tumor associated immune response as a critical pathway contributing to cancer disparity. Using patient derived primary tumor cells, the investigators found that AGEs released into the extracellular matrix can recapitulate the tumor associated immune response observed in race specific prostate tumor tissues. Activated immune cells show a similar metabolic profile as a glycolytic tumor cell with a shift towards increased glucose metabolism and aerobic glycolysis (i.e. the Warburg effect). Evidence suggests that abnormal glucose uptake may occur earlier in African American cancer patients with aggressive disease. Further preliminary studies indicate that AGE treatment of prostate cancer cells can alter how cancer cells metabolize glucose to promote an aggressive phenotype. Based on associations between active metabolism, lifestyle, and race, increases in AGE accumulation may represent a novel biologic mechanism contributing to cancer disparity and may represent a new paradigm to explaining the increased cancer incidence and mortality figures observed within health disparity populations. Given the potential benefits of lifestyle changes and the potential biological role of AGEs in promoting cancer, opportunities exist for collaborations impacting basic, translational, epidemiological and cancer prevention initiatives. Citation Format: Danzell Smith, Dion Foster, Laura Spruill, Lourdes Nogueira, Bradley Krisanits, Scott Cramer, Marvella Ford, Stephen Savage, Thomas Keane, Victoria Findlay, David Turner. The molecular implications of lifestyle associated metabolites (AGEs) to prostate cancer disparity. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr PR11.

Abstract LB-043: Improving rectal cancer therapy using a patient-derived tumor xenograft model

Mechanisms of locally advanced rectal cancer (LARC) neoadjuvant chemoradiation resistance are poorly understood. Current research models to study this issue are incomplete and restricted by the limited supply of untreated tissue samples. Patient-derived xenografts (PDX) are commonly developed from surgical specimens and can serve as effective tools to investigate therapy. We previously demonstrated that the SNAI2/ miR-145 signaling axis enhanced the tumor-initiating cell (TIC) and chemotherapy resistance. We developed a unique PDX model from pre-neoadjuvant therapy LARC biopsy samples to study chemoradiation resistance. Further, we hypothesized that TICs isolated from LARC patient-derived xenografts would demonstrated increased SNAI2/ miR-145 signaling. Methods: Fresh LARC endoscopic biopsy samples were digested using Liberase DH to yield a dissociated single-cell solution. All in vivo experiments consisted of injecting cells with Matrigel subcutaneously into NSG NODSCID/IL2Rgnull immunocompromised mice. Xenograft (Passage 1, P1) single cells were resuspended in ALDEFLUOR assay buffer. ALDEFLUOR and EPCAM positive cells (ALDH+/EPCAM+) were then detected using a flow cytometer to identify the TICs. Using the negative control ALDH inhibitor, DEAB (1.5 μM), identify the ALDH+ cells compared with background. For limited dilution (LD) in vitro spheroid assay, 5000 EPCAM+/ALDH+ and ALDH- cells were cultured in ultra-low attachment plates in cancer stem-cell medium (DMEM-F12; 1X B-27, βEGF, βFGF, 1% penicillin/streptomycin, BSA, heparin, β-mercaptoethanol). For LD in vivo study, EPCAM+ ALDH sorted cells were implanted subcutaneously with Matrigel and closely observed. Droplet Digital PCR was conducted using the QX200 Droplet Digital PCR System. miR-145 PCR was performed using TaqMan MicroRNA Assays (Applied Biosystems) and normalized to RNU6B. Results: Our LARC PDX model had high outgrowth and engraftment rates of 70 and 85% respectively and maintained critical histologic features. Both LD in vitro and in vivo experiments demonstrated that EPCAM+/ALDH+ cells were highly tumorigenic suggestive of TICs (p Conclusions: The lack of novel strategies plays a central role in the inability to overcome therapeutic resistance in LARC. Our results demonstrate a novel model to investigate LARC and suggest that a novel miR-145 therapeutic strategy may be able to target the TIC in LARC patients. Citation Format: Ernest R. Camp, Cindy Wang, Katie Hurst, Lourdes M. Nogueira, Victoria J. Findlay. Improving rectal cancer therapy using a patient-derived tumor xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-043.