Biosocial determinants inform on enduring cancer disparities.

Social, environmental, and biological risk factors influence exposures to newly termed 'biosocial determinants of health'. As molecular factors that lie at the intersection between lived experiences and individual biology, biosocial determinants may inform on the enduring complexity of cancer disparity across transdisciplinary studies.

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.

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.

The National Conference on Health Disparities Student Research Forum.

The annual National Conference on Health Disparities (NCHD) was launched in 2000. It unites health professionals, researchers, community leaders, and government officials, and is a catalyzing force in developing policies, research interventions, and programs that address prevention, social determinants, health disparities, and health equity. The NCHD Student Research Forum (SRF) was established in 2011 at the Medical University of South Carolina to build high-quality biomedical research presentation capacity in primarily underrepresented undergraduate and graduate/professional students. This paper describes the unique research training and professional development aspects of the NCHD SRF. These include guidance in abstract development, a webinar on presentation techniques and methods, a vibrant student-centric conference, and professional development workshops on finding a mentor and locating scholarship/fellowship funding, networking, and strategies for handling ethical issues in research with mentors. Between 2011 and 2018, 400 undergraduate and graduate/professional students participated in the NCHD SRF. Most students were women (80.5%). Approximately half were African American or black (52.3%), 18.0% were white, and 21.3% were of Hispanic/Latinx ethnicity. The NCHD SRF is unique in several ways. First, it provides detailed instructions on developing a scientific abstract, including content area examples. Second, it establishes a mandatory pre-conference training webinar demonstrating how to prepare a scientific poster. Third, it works with the research mentors, faculty advisors, department chairs, and deans to help identify potential sources of travel funding for students with accepted abstracts. These features make the NCHD SRF different from many other conferences focused on students' scientific presentations.

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.

Differential immune signatures in the tumor microenvironment are associated with colon cancer racial disparities.

BACKGROUND: Disparities in colon cancer (CC) outcomes may be due to a more aggressive phenotype in African American patients in the setting of a decreased tumor immunity, though the precise mechanism for this result has not been well elucidated. To explore the molecular factors underlying CC disparities, we compared the immunogenomic signatures of CC from African American and European American patients. METHODS: We identified all CC patients from the publicly available Cancer Genome Atlas for whom race and survival data are available. Immunophenotype signatures were established for African American and European American patients. Comparisons were made regarding survival and a multivariable linear regression model was created to determine the association of immune cellular components with race. Differential gene expression was also assessed. RESULTS: Of the 254 patients identified, 58 (23%) were African American and 196 (77%) were European American. African American patients had a decreased progression free survival (p = 0.04). Tumors from African American patients displayed a reduced fraction of macrophages and CD8+ T cells and an increased fraction of B cells compared with tumors from European Americans. Differences persisted when controlling for sex, age, and disease stage. Immunostimulatory and immunoinhibitory gene profiles including major histocompatibility complex expression differed by race. CONCLUSIONS: Differences in the tumor immune microenvironment of African American as compared to European American CC specimens may play a role in the survival differences between the groups. These differences may provide targeted therapeutic opportunities.

Pubertal mammary development as a "susceptibility window" for breast cancer disparity.

Factors such as socioeconomic status, age at menarche and childbearing patterns are components that have been shown to influence mammary gland development and establish breast cancer disparity. Pubertal mammary gland development is selected as the focus of this review, as it is identified as a "window of susceptibility" for breast cancer risk and disparity. Here we recognize non-Hispanic White, African American, and Asian American women as the focus of breast cancer disparity, in conjunction with diets associated with changes in breast cancer risk. Diets consisting of high fat, N-3 polyunsaturated fatty acids, N-6 polyunsaturated fatty acids, as well as obesity and the Western diet have shown to lead to changes in pubertal mammary gland development in mammalian models, therefore increasing the risk of breast cancer and breast cancer disparity. While limited intervention strategies are offered to adolescents to mitigate development changes and breast cancer risk, the prominent solution to closing the disparity among the selected population is to foster lifestyle changes that avoid the deleterious effects of unhealthy diets.

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.

Modeling rectal cancer to advance neoadjuvant precision therapy.

Progress in rectal cancer therapy has been hindered by the lack of effective disease-specific preclinical models that account for the unique molecular profile and biology of rectal cancer. Thus, we developed complementary patient-derived xenograft (PDX) and subsequent in vitro tumor organoid (PDTO) platforms established from preneoadjuvant therapy rectal cancer specimens to advance personalized care for rectal cancer patients. Multiple endoscopic samples were obtained from 26 Stages 2 and 3 rectal cancer patients prior to receiving 5FU/RT and implanted subcutaneously into NSG mice to generate 15 subcutaneous PDXs. Second passaged xenografts demonstrated 100% correlation with the corresponding human cancer histology with maintained mutational profiles. Individual rectal cancer PDXs reproduced the 5FU/RT response observed in the corresponding human cancers. Similarly, rectal cancer PDTOs reproduced significant heterogeneity in cellular morphology and architecture. PDTO in vitro 5FU/RT treatment response replicated the clinical 5FU/RT neoadjuvant therapy pathologic response observed in the corresponding patient tumors (p < 0.05). The addition of cetuximab to the 5FU/RT regiment was significantly more sensitive in the rectal cancer PDX and PDTOs with wild-type KRAS compared to mutated KRAS (p < 0.05). Considering the close relationship between the patient's cancer and the corresponding PDX/PDTO, rectal cancer patient-derived research platforms represent powerful translational research resources as population-based tools for biomarker discovery and experimental therapy testing. In addition, our findings suggest that cetuximab may enhance RT effectiveness by improved patient selection based on mutational profile in addition to KRAS or by developing a protocol using PDTOs to identify sensitive patients.

Eukaryotic initiation factor 4E-binding protein as an oncogene in breast cancer.

BACKGROUND: Eukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy. METHODS: Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1. RESULTS: Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes. CONCLUSIONS: These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.

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.

Hematopoietic stem cell-derived cancer-associated fibroblasts are novel contributors to the pro-tumorigenic microenvironment.

Targeting the tumor microenvironment is critical toward improving the effectiveness of cancer therapeutics. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types of the tumor microenvironment, playing an important role in tumor progression. Multiple origins for CAFs have been proposed including resident fibroblasts, adipocytes, and bone marrow. Our laboratory previously identified a novel hematopoietic stem cell (HSC) origin for CAFs; however, the functional roles of HSC-derived CAFs (HSC-CAFs) in tumor progression have not yet been examined. To test the hypothesis that HSC-CAFs promote tumor progression through contribution to extracellular matrix (ECM) and paracrine production of pro-angiogenic factors, we developed a method to isolate HSC-CAFs. HSC-CAFs were profiled on the basis of their expression of hematopoietic and fibroblastic markers in two murine tumor models. Profiling revealed production of factors associated with ECM deposition and remodeling. Functional in vivo studies showed that co-injection of HSC-CAFs with tumor cells resulted in increased tumor growth rate and significantly larger tumors than tumor cells alone. Immunohistochemical studies revealed increased blood vessel density with co-injection, demonstrating a role for HSC-CAFs in tumor vascularization. Mechanistic in vitro studies indicated that HSC-CAFs play a role in producing vascular endothelial growth factor A and transforming growth factor-ß1 in endothelial tube formation and patterning. In vitro and in vivo findings suggest that HSC-CAFs are a critical component of the tumor microenvironment and suggest that targeting the novel HSC-CAF may be a promising therapeutic strategy.

FLI1 expression is correlated with breast cancer cellular growth, migration, and invasion and altered gene expression.

ETS factors have been shown to be dysregulated in breast cancer. ETS factors control the expression of genes involved in many biological processes, such as cellular proliferation, differentiation, and apoptosis. FLI1 is an ETS protein aberrantly expressed in retrovirus-induced hematological tumors, but limited attention has been directed towards elucidating the role of FLI1 in epithelial-derived cancers. Using data mining, we show that loss of FLI1 expression is associated with shorter survival and more aggressive phenotypes of breast cancer. Gain and loss of function cellular studies indicate the inhibitory effect of FLI1 expression on cellular growth, migration, and invasion. Using Fli1 mutant mice and both a transgenic murine breast cancer model and an orthotopic injection of syngeneic tumor cells indicates that reduced Fli1 contributes to accelerated tumor growth. Global expression analysis and RNA-Seq data from an invasive human breast cancer cell line with over expression of either FLI1 and another ETS gene, PDEF, shows changes in several cellular pathways associated with cancer, such as the cytokine-cytokine receptor interaction and PI3K-Akt signaling pathways. This study demonstrates a novel role for FLI1 in epithelial cells. In addition, these results reveal that FLI1 down-regulation in breast cancer may promote tumor progression.

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.

Slug expression inhibits calcitriol-mediated sensitivity to radiation in colorectal cancer.

Recently, a reciprocal relationship between calcitriol and epithelial-to-mesenchymal transition has been described. Therefore, we hypothesized that calcitriol (1α,25-dihydroxyvitamin D3) would enhance radiation sensitivity in colorectal cancer regulated by epithelial mesenchymal transition. Vitamin-D receptor, E-cadherin and vimentin protein as well as E-cadherin, Snail and Slug mRNA levels were assessed in a panel of human colorectal cancer cell lines at baseline and in response calcitriol. We defined cell lines as calcitriol sensitive based on demonstrating an enhanced epithelial phenotype with increased E-cadherin, reduced vimentin and decreased expression of Snail and Slug as well as decreased cellular migration in response to calcitriol. In calcitriol sensitive cells, including DLD-1 and HCT116, 24 h calcitriol pre-treatment enhanced the radiation sensitivity by 2.3- and 2.6-fold, respectively, at 4 Gy (P < 0.05). In contrast, SW620 cells with high baseline mesenchymal features including high Slug and vimentin expression with low E-cadherin expression demonstrated no significant radiation sensitizing response to calcitriol treatment. Similarly, transfection of Slug in the calcitriol sensitive colon cancer cell lines, DLD-1 and HCT 116, completely inhibited the radiation sensitizing effect of calcitriol. Collectively, we demonstrate that calcitriol can enhance the therapeutic effects of radiation in colon cancer cells and Slug expression mitigates this observed effect potentially representing an effective biomarker for calcitriol therapy.

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.

Understanding the role of ETS-mediated gene regulation in complex biological processes.

Ets factors are members of one of the largest families of evolutionarily conserved transcription factors, regulating critical functions in normal cell homeostasis, which when perturbed contribute to tumor progression. The well-documented alterations in ETS factor expression and function during cancer progression result in pleiotropic effects manifested by the downstream effect on their target genes. Multiple ETS factors bind to the same regulatory sites present on target genes, suggesting redundant or competitive functions. The anti- and prometastatic signatures obtained by examining specific ETS regulatory networks will significantly improve our ability to accurately predict tumor progression and advance our understanding of gene regulation in cancer. Coordination of multiple ETS gene functions also mediates interactions between tumor and stromal cells and thus contributes to the cancer phenotype. As such, these new insights may provide a novel view of the ETS gene family as well as a focal point for studying the complex biological control involved in tumor progression. One of the goals of molecular biology is to elucidate the mechanisms that contribute to the development and progression of cancer. Such an understanding of the molecular basis of cancer will provide new possibilities for: (1) earlier detection, as well as better diagnosis and staging of disease; (2) detection of minimal residual disease recurrences and evaluation of response to therapy; (3) prevention; and (4) novel treatment strategies. Increased understanding of ETS-regulated biological pathways will directly impact these areas.

Kaposi's sarcoma-associated herpesvirus suppression of DUSP1 facilitates cellular pathogenesis following de novo infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), and KSHV activation of mitogen-activated protein kinases (MAPKs) initiates a number of key pathogenic determinants of KS. Direct inhibition of signal transduction as a therapeutic approach presents several challenges, and a better understanding of KSHV-induced mechanisms regulating MAPK activation may facilitate the development of new treatment or prevention strategies for KS. MAPK phosphatases, including dual-specificity phosphatase-1 (DUSP1), negatively regulate signal transduction and cytokine activation through MAPK dephosphorylation or interference with effector molecule binding to MAPKs, including the extracellular signal-regulated kinase (ERK). We found that ERK-dependent latent viral gene expression, the induction of promigratory factors, and cell invasiveness following de novo infection of primary human endothelial cells are in part dependent on KSHV suppression of DUSP1 expression during de novo infection. KSHV-encoded miR-K12-11 upregulates the expression of xCT (an amino acid transporter and KSHV fusion/entry receptor), and existing data indicate a role for xCT in the regulation of 14-3-3ß, a transcriptional repressor of DUSP1. We found that miR-K12-11 induces endothelial cell secretion of promigratory factors and cell invasiveness through upregulation of xCT-dependent, 14-3-3ß-mediated suppression of DUSP1. Finally, proof-of-principle experiments revealed that pharmacologic upregulation of DUSP1 inhibits the induction of promigratory factors and cell invasiveness during de novo KSHV infection. These data reveal an indirect role for miR-K12-11 in the regulation of DUSP1 and downstream pathogenesis.