Pubertal exposure to dietary advanced glycation end products disrupts ductal morphogenesis and induces atypical hyperplasia in the mammary gland.

BACKGROUND: Advanced glycation end products (AGEs) are reactive metabolites intrinsically linked with modern dietary patterns. Processed foods, and those high in sugar, protein and fat, often contain high levels of AGEs. Increased AGE levels are associated with increased breast cancer risk, however their significance has been largely overlooked due to a lack of direct cause-and-effect relationship. METHODS: To address this knowledge gap, FVB/n mice were fed regular, low AGE, and high AGE diets from 3 weeks of age and mammary glands harvested during puberty (7 weeks) or adulthood (12 weeks and 7 months) to determine the effects upon mammary gland development. At endpoint mammary glands were harvested and assessed histologically (n ≥ 4). Immunohistochemistry and immunofluorescence were used to assess cellular proliferation and stromal fibroblast and macrophage recruitment. The Kruskal-Wallis test were used to compare continuous outcomes among groups. Mammary epithelial cell migration and invasion in response to AGE-mediated fibroblast activation was determined in two-compartment co-culture models. In vitro experiments were performed in triplicate. The nonparametric Wilcoxon rank sum test was used to compare differences between groups. RESULTS: Histological analysis revealed the high AGE diet delayed ductal elongation, increased primary branching, as well as increased terminal end bud number and size. The high AGE diet also led to increased recruitment and proliferation of stromal cells to abnormal structures that persisted into adulthood. Atypical hyperplasia was observed in the high AGE fed mice. Ex vivo fibroblasts from mice fed dietary-AGEs retain an activated phenotype and promoted epithelial migration and invasion of non-transformed immortalized and tumor-derived mammary epithelial cells. Mechanistically, we found that the receptor for AGE (RAGE) is required for AGE-mediated increases in epithelial cell migration and invasion. CONCLUSIONS: We observed a disruption in mammary gland development when mice were fed a diet high in AGEs. Further, both epithelial and stromal cell populations were impacted by the high AGE diet in the mammary gland. Educational, interventional, and pharmacological strategies to reduce AGEs associated with diet may be viewed as novel disease preventive and/or therapeutic initiatives during puberty.

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

PURPOSE: Lifestyle 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. METHODS: We 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. RESULTS: An 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. CONCLUSIONS: There 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.

miR-145 Antagonizes SNAI1-Mediated Stemness and Radiation Resistance in Colorectal Cancer.

Epithelial-to-mesenchymal transition (EMT) has been closely linked with therapy resistance and cancer stem cells (CSCs). However, EMT pathways have proven challenging to therapeutically target. MicroRNA 145 (miR-145) targets multiple stem cell transcription factors and its expression is inversely correlated with EMT. Therefore, we hypothesized that miR-145 represents a therapeutic target to reverse snail family transcriptional repressor 1 (SNAI1)-mediated stemness and radiation resistance (RT). Stable expression of SNAI1 in DLD1 and HCT116 cells (DLD1-SNAI1; HCT116-SNAI1) increased expression of Nanog and decreased miR-145 expression compared to control cells. Using a miR-145 luciferase reporter assay, we determined that ectopic SNAI1 expression significantly repressed the miR-145 promoter. DLD1-SNAI1 and HCT116-SNAI1 cells demonstrated decreased RT sensitivity and, conversely, miR-145 replacement significantly enhanced RT sensitivity. Of the five parental colon cancer cell lines, SW620 cells demonstrated relatively high endogenous SNAI1 and low miR-145 levels. In the SW620 cells, miR-145 replacement decreased CSC-related transcription factor expression, spheroid formation, and radiation resistance. In rectal cancer patient-derived xenografts, CSC identified by EpCAM+/aldehyde dehydrogenase (ALDH)+ demonstrated high expression of SNAI1, c-Myc, and Nanog compared with non-CSCs (EpCAM+/ALDH-). Conversely, patient-derived CSCs demonstrated low miR-145 expression levels relative to non-CSCs. These results suggest that the SNAI1:miR-145 pathway represents a novel therapeutic target in colorectal cancer to overcome RT resistance.

MicroRNA-510 promotes cell and tumor growth by targeting peroxiredoxin1 in breast cancer.

INTRODUCTION: MicroRNAs are small non-coding RNAs that are involved in the post-transcriptional negative regulation of mRNAs. MicroRNA 510 (miR-510) was initially shown to have a potential oncogenic role in breast cancer by the observation of its elevated levels in human breast tumor samples when compared to matched non-tumor samples. Few targets have been identified for miR-510. However, as microRNAs function through the negative regulation of their direct targets, the identification of those targets is critical for the understanding of their functional role in breast cancer. METHODS: Breast cancer cell lines were transfected with pre-miR-510 or antisense miR-510 and western blotting and quantitative real time PCR were performed. Functional assays performed included cell growth, migration, invasion, colony formation, cytotoxicity and in vivo tumor growth. We performed a PCR assay to identify novel direct targets of miR-510. The study focused on peroxiredoxin 1 (PRDX1) as it was identified through our screen and was bioinformatically predicted to contain a miR-510 seed site in its 3' untranslated region (3'UTR). Luciferase reporter assays and site-directed mutagenesis were performed to confirm PRDX1 as a direct target. The Student's two-sided, paired t-test was used and a P-value less than 0.05 was considered significant. RESULTS: We show that miR-510 overexpression in non-transformed and breast cancer cells can increase their cell growth, migration, invasion and colony formation in vitro. We also observed increased tumor growth when miR-510 was overexpressed in vivo. We identified PRDX1 through a novel PCR screen and confirmed it as a direct target using luciferase reporter assays. The reintroduction of PRDX1 into breast cancer cell lines without its regulatory 3'UTR confirmed that miR-510 was mediating its migratory phenotype at least in part through the negative regulation of PRDX1. Furthermore, the PI3K/Akt pathway was identified as a positive regulator of miR-510 both in vitro and in vivo. CONCLUSIONS: In this study, we provide evidence to support a role for miR-510 as a novel oncomir. We show that miR-510 directly binds to the 3'UTR of PRDX1 and blocks its protein expression, thereby suppressing migration of human breast cancer cells. Taken together, these data support a pivotal role for miR-510 in breast cancer progression and suggest it as a potential therapeutic target in breast cancer patients.

MicroRNA-510 mediated negative regulation of Caveolin-1 in fibroblasts promotes aggressive tumor growth.

Introduction: In the US, despite the recent decline in breast cancer deaths, a persistent mortality disparity exists between black and white women with breast cancer, with black women having a 41% higher death rate. Several studies are now reporting that racial disparities can exist independent of socioeconomic and standard of care issues, suggesting that biological factors may be involved. Caveolin-1 (Cav1) loss in the tumor stromal compartment is a novel clinical biomarker for predicting poor outcome in breast cancer including triple negative subtype, however the mechanism of Cav1 loss is unknown. We previously identified miR-510-5p as a novel oncomir and propose here that the high levels observed in patients is a novel mechanism leading to stromal Cav1 loss and worse outcomes. Methods: Cav1 was identified as a direct target of miR-510-5p through luciferase, western blot and qPCR assays. Stromal cross talk between epithelial cells and fibroblasts was assessed in vitro using transwell co-culture assays and in vivo using xenograft assays. Results: We found that Cav1 is a direct target of miR-510-5p and that expression in fibroblasts results in an 'activated' phenotype. We propose that this could be important in the context of cancer disparities as we also observed increased levels of circulating miR-510-5p and reduced levels of stromal Cav1 in black women compared to white women with breast cancer. Finally, we observed a significant increase in tumor growth when tumor cells were co-injected with miR-510-5p expressing cancer associated fibroblasts in vivo. Conclusion: We propose that miR-510-5p mediated negative regulation of Cav1 in fibroblasts is a novel mechanism of aggressive tumor growth and may be a driver of breast cancer disparity.

Non-enzymatic glycoxidation linked with nutrition enhances the tumorigenic capacity of prostate cancer epithelia through AGE mediated activation of RAGE in cancer associated fibroblasts.

The molecular implications of food consumption on cancer etiology are poorly defined. The rate of nutrition associated non-enzymatic glycoxidation, a reaction that occurs between reactive carbonyl groups on linear sugars and nucleophilic amino, lysyl and arginyl groups on fats and proteins, is rapidly increased by food cooking and manufacturing processes. In this study, we assign nutrition-associated glycoxidation with significant oncogenic potential, promoting prostate tumor growth, progression, and metastasis in vivo. Advanced glycation end products (AGEs) are the final irreversible product of non-enzymatic glycoxidation. Exogenous treatment of prostate tumor cells with a single AGE peptide replicated glycoxidation induced tumor growth in vivo. Mechanistically, receptor for AGE (RAGE) deficiency in the stroma inhibited AGE mediated tumor growth. Functionally, AGE treatment induced RAGE dimerization in activated fibroblasts which sustained and increased the migratory potential of tumor epithelial cells. These data identify a novel nutrition associated pathway that can promote a tissue microenvironment conducive for aggressive tumor growth. Targeted and/or interventional strategies aimed at reducing AGE bioavailability as a consequence of nutrition may be viewed as novel chemoprevention initiatives.

BMI, physical activity, and breast cancer subtype in white, black, and Sea Island breast cancer survivors.

Higher BMI, lower rates of physical activity (PA), and hormone receptor-negative breast cancer (BC) subtype are associated with poorer BC treatment outcomes. We evaluated the prevalence of high BMI, low PA level, and BC subtype among survivors with white/European American (EA) and African American (AA) ancestry, as well as a distinct subset of AAs with Sea Island/Gullah ancestry (SI). We used the South Carolina Central Cancer Registry to identify 137 (42 EAs, 66 AAs, and 29 SIs) women diagnosed with BC and who were within 6-21 months of diagnosis. We employed linear and logistic regression to investigate associations between BMI, PA, and age at diagnosis by racial/ethnic group. Most participants (82%) were overweight/obese (P=0.46). BMI was highest in younger AAs (P=0.02). CDC PA guidelines (≥150min/week) were met by only 28% of participants. The frequency of estrogen receptor (ER)-negative BC subtype was lower in EAs and SIs than in AAs (P<0.05). This is the first study to identify differences in obesity and PA rates, and BC subtype in EAs, AAs, and SIs. BMI was higher, PA rates were lower, and frequency of ER-negative BC was higher in AAs as compared to EAs and SIs. This study highlights the need to promote lifestyle interventions among BC survivors, with the goal of reducing the likelihood of a BC recurrence. Integrating dietary and PA interventions into ongoing survivorship care is essential. Future research could evaluate potential differential immune responses linked to the frequency of triple negative BC in AAs.

Tolerance to non-opioid analgesics in PAG involves unresponsiveness of medullary pain-modulating neurons in male rats.

Opiate analgesia can be hampered by a reduction in pharmacological effectiveness (tolerance), and this crucially depends on the periaqueductal gray matter (PAG). Non-opioids like metamizol (dipyrone) or aspirin also induce PAG-dependent analgesia and tolerance, but the neuronal bases of this tolerance are unknown. Metamizol is a pyrazolon derivative and cyclooxygenase inhibitor with widespread use as an analgesic in Europe and Latin America. Metamizol was microinjected into the PAG of awake male rats, and antinociception was assessed by the tail flick (TF) and hot plate (HP) tests. Microinjection twice daily for 2.5 days caused tolerance to metamizol. The rats were then anesthetized and recordings from pain-facilitating on-cells and pain-inhibiting off-cells of the rostral ventromedial medulla (RVM) were performed. PAG microinjection of morphine or metamizol depresses on-cells, activates off-cells and thus inhibits nociception, including TF and HP. In metamizol-tolerant rats, however, PAG microinjection of metamizol failed to affect on- or off-cells, and this is interpreted as the reason for tolerance. In metamizol-tolerant rats morphine microinjection into PAG also failed to affect RVM neurons or nociception (cross-tolerance). In naïve, non-tolerant rats the antinociceptive effect of PAG-microinjected metamizol or morphine was blocked when CTOP, a mu-opioid antagonist, was previously microinjected into the same PAG site. These results emphasize a close relationship between opioid and non-opioid analgesic mechanisms in the PAG and show that, like morphine, tolerance to metamizol involves a failure of on- and off-cells to, respectively, disfacilitate and inhibit nociception. Cross-tolerance between non-opioid and opioid analgesics should be important in the clinical setting.

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.

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.

Involvement of cholecystokinin in the opioid tolerance induced by dipyrone (metamizol) microinjections into the periaqueductal gray matter of rats.

The analgesic effect of non-steroidal anti-inflammatory drugs (NSAIDs) is partly due to an action upon the periaqueductal gray matter (PAG), which triggers the descending pain control system and thus inhibits nociceptive transmission. This action of NSAIDs engages endogenous opioids at the PAG, the nucleus raphe magnus and the spinal cord. Repeated administration of NSAIDs such as dipyrone (metamizol) and acetylsalicylate thus induces tolerance to these compounds and cross-tolerance to morphine. Since cholecystokinin plays a key role in opioid tolerance, the present study in rats investigated whether PAG cholecystokinin is also responsible for tolerance to PAG-microinjected dipyrone. Microinjection of cholecystokinin (1 ng/0.5 microl) into PAG blocked the antinociceptive effect of a subsequent microinjection of dipyrone (150 microg/0.5 microl) into the same site, as evaluated by the tail flick and hot plate tests. Microinjection of proglumide (0.4 microg/0.5 microl), a non-selective cholecystokinin antagonist, into PAG prevented the development of tolerance to subsequent microinjections of dipyrone, as well as cross-tolerance to microinjection of morphine (5 microg/0.5 microl) into the same site. In rats tolerant to PAG dipyrone, a PAG microinjection of proglumide restored the antinociceptive effect of a subsequent microinjection of dipyrone or morphine. These results suggest that PAG-microinjected dipyrone triggers and/or potentiates local opioidergic circuits leading to descending inhibition of nociception, on the one hand, and to a local antiopioid action by cholecystokinin, on the other. Reiteration of these events would then result in an enhancement of cholecystokinin's antiopioid action and thus tolerance to opioids and dipyrone in the PAG.

Involvement of local cholecystokinin in the tolerance induced by morphine microinjections into the periaqueductal gray of rats.

The ventrolateral periaqueductal gray (PAG) is a key structure for the development of opioid tolerance. An increased activity of 'anti-opioids' like cholecystokinin (CCK) has been proposed as a possible mechanism for opioid tolerance. The present study evaluates the role of PAG-located CCK in the opioid tolerance induced by repeated microinjections of morphine (MOR) into PAG. Male rats were implanted with chronic guide cannulae aimed at the PAG. Microinjection of MOR (0.5 microg in 0.5 microl) into PAG caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if each MOR microinjection was preceded (within 15 min) by a microinjection of the non-selective CCK receptor antagonist proglumide (PRO), (0.4 microg in 0.5 microl) into the same PAG site, the microinjections of MOR always produced antinociception and did not induce tolerance. If PRO microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single PRO microinjection into the same PAG site was enough to restore the antinociceptive effect of MOR. On the other hand, if CCK (1 ng in 0.5 microl) was microinjected into PAG, then MOR microinjection administered 15 min later into the same PAG site did not elicit antinociception. These results show that CCK has anti-opioid activity in PAG and that tolerance to MOR in PAG can be prevented or reversed if CCK receptors are blocked with PRO. Finally, opioid tolerance induced by repeated systemic MOR injections (5mg/kg intraperitoneal ) was reversed by a single microinjection of PRO into PAG. This emphasizes the central importance of PAG in the MOR/CCK interactions that lead to opioid tolerance.

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.

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.

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.

Synaptic basis of persistent activity in prefrontal cortex in vivo and in organotypic cultures.

Persistent activity is observed in many cortical and subcortical brain regions, and may subserve a variety of functions. Within the prefrontal cortex (PFC), neurons transiently maintain information in working memory via persistent activity patterns; however, the mechanisms involved are largely unknown. The present study used intracellular recordings from deep layer PFC neurons in vivo and patch-clamp recordings from PFC neurons in organotypic brain slice cultures to examine the ionic mechanisms underlying persistent activity states evoked by various inputs. Persistent activity had consistent features regardless of the initiating stimulus; it was driven by non-NMDA glutamate receptors yet consisted of an initial GABA mediated component, followed by a prolonged synaptically mediated inward current that maintained the sustained depolarization on which rode many asynchronous GABA-mediated events. The stereotyped nature of the multiple-component persistent activity pattern reported here might be a common feature of interconnected cortical networks but within PFC could be related to the persistent activity required for working memory.