Kevin S. Kimbro

Interaction among apoptosis-associated sequence variants and joint effects on aggressive prostate cancer

BackgroundMolecular and epidemiological evidence demonstrate that altered gene expression and single nucleotide polymorphisms in the apoptotic pathway are linked to many cancers. Yet, few studies emphasize the interaction of variant apoptotic genes and their joint modifying effects on prostate cancer (PCA) outcomes. An exhaustive assessment of all the possible two-, three- and four-way gene-gene interactions is computationally burdensome. This statistical conundrum stems from the prohibitive amount of data needed to account for multiple hypothesis testing.MethodsTo address this issue, we systematically prioritized and evaluated individual effects and complex interactions among 172 apoptotic SNPs in relation to PCA risk and aggressive disease (i.e., Gleason score ≥ 7 and tumor stages III/IV). Single and joint modifying effects on PCA outcomes among European-American men were analyzed using statistical epistasis networks coupled with multi-factor dimensionality reduction (SEN-guided MDR). The case-control study design included 1,175 incident PCA cases and 1,111 controls from the prostate, lung, colo-rectal, and ovarian (PLCO) cancer screening trial. Moreover, a subset analysis of PCA cases consisted of 688 aggressive and 488 non-aggressive PCA cases. SNP profiles were obtained using the NCI Cancer Genetic Markers of Susceptibility (CGEMS) data portal. Main effects were assessed using logistic regression (LR) models. Prior to modeling interactions, SEN was used to pre-process our genetic data. SEN used network science to reduce our analysis from > 36 million to < 13,000 SNP interactions. Interactions were visualized, evaluated, and validated using entropy-based MDR. All parametric and non-parametric models were adjusted for age, family history of PCA, and multiple hypothesis testing.ResultsFollowing LR modeling, eleven and thirteen sequence variants were associated with PCA risk and aggressive disease, respectively. However, none of these markers remained significant after we adjusted for multiple comparisons. Nevertheless, we detected a modest synergistic interaction between AKT3 rs2125230-PRKCQ rs571715 and disease aggressiveness using SEN-guided MDR (p = 0.011).ConclusionsIn summary, entropy-based SEN-guided MDR facilitated the logical prioritization and evaluation of apoptotic SNPs in relation to aggressive PCA. The suggestive interaction between AKT3-PRKCQ and aggressive PCA requires further validation using independent observational studies.

Chemokine Ligand 5 (CCL5) and chemokine receptor (CCR5) genetic variants and prostate cancer risk among men of African Descent:a case-control study

BackgroundChemokine and chemokine receptors play an essential role in tumorigenesis. Although chemokine-associated single nucleotide polymorphisms (SNPs) are associated with various cancers, their impact on prostate cancer (PCA) among men of African descent is unknown. Consequently, this study evaluated 43 chemokine-associated SNPs in relation to PCA risk. We hypothesized inheritance of variant chemokine-associated alleles may lead to alterations in PCA susceptibility, presumably due to variations in antitumor immune responses.MethodsSequence variants were evaluated in germ-line DNA samples from 814 African-American and Jamaican men (279 PCA cases and 535 controls) using Illumina’s Goldengate genotyping system.ResultsInheritance of CCL5 rs2107538 (AA, GA+AA) and rs3817655 (AA, AG, AG+AA) genotypes were linked with a 34-48% reduction in PCA risk. Additionally, the recessive and dominant models for CCR5 rs1799988 and CCR7 rs3136685 were associated with a 1.52-1.73 fold increase in PCA risk. Upon stratification, only CCL5 rs3817655 and CCR7 rs3136685 remained significant for the Jamaican and U.S. subgroups, respectively.ConclusionsIn summary, CCL5 (rs2107538, rs3817655) and CCR5 (rs1799988) sequence variants significantly modified PCA susceptibility among men of African descent, even after adjusting for age and multiple comparisons. Our findings are only suggestive and require further evaluation and validation in relation to prostate cancer risk and ultimately disease progression, biochemical/disease recurrence and mortality in larger high-risk subgroups. Such efforts will help to identify genetic markers capable of explaining disproportionately high prostate cancer incidence, mortality, and morbidity rates among men of African descent.

The impact of genetic variants in inflammatory-related genes on prostate cancer risk among men of African Descent: a case control study

PurposeAlthough case–control studies have evaluated the role of variant inflammatory-related loci in prostate cancer, their impact is virtually unknown among men of African descent. To address this, we evaluated the impact of inflammatory cytokine single nucleotide polymorphisms (SNPs) on prostate cancer risk for men of African descent.MethodsForty-four SNPs in inflammatory cytokine-associated genes were evaluated among 814 African-American and Jamaican men (279 prostate cancer cases and 535 controls) using Illumina’s Golden gate genotyping system. Individual SNP effects were evaluated using logistic regression analysis.ResultsFour SNPs were modestly associated with prostate cancer after adjusting for age. In the total population, inheritance of the IL1R2 rs11886877 AA, IL8RB rs11574752 AA, TNF rs1800629 GA + AA, and TNF rs673 GA genotypes modestly increased prostate cancer risk by 1.45 to 11.7-fold relative to the referent genotype. Among U.S. men, age-adjusted dominant, recessive and additive genetic models for the IL1R2 rs11886877 locus were linked to an increase in prostate cancer susceptibility. However, these main effects did not persist after adjusting for multiple hypothesis testing.ConclusionOur preliminary data does not strongly support the hypothesis that inflammatory-related sequence variants influence prostate cancer risk among men of African descent. However, further evaluation is needed to assess whether other variant inflammatory-related genes may contribute to prostate cancer risk and disease progression in larger and ethnically diverse multi-center studies.

Abstract A70: Chemokine-associated genetic variants as predictors of prostate cancer outcomes among men of African descent

Background: African-American men in the U.S. are 1.6 and 2-fold more likely to receive a prostate cancer (PCA) diagnosis and die from the disease relative to their Caucasian counterparts, respectively. This health disparity extends beyond the U.S. For instance, men from Kingston, Jamaica have higher cancer mortality and morbidity rates compared to African-Americans. Reasons for these disparities may be attributed to differences in cancer screening practices, lifestyle factors, clinical management of the disease, detection of aggressive/advance tumors that are not responsive to available treatments, and genetic susceptibilities. Recent research efforts have focused on the identification of genetic determinants of PCA. Emphasis has been placed on genes that encode chemokines and their receptors, since they play an essential role in tumorigenesis. For instance, over expression of CCR6 is associated with lung, pancreatic, and PCA, presumably by triggering leukocyte production and promoting cell survival and metastasis. Moreover sequence variants detected in CCL5 (rs2280078, rs2107538 at positions −28 and −403, respectively) and CCR6 (rs9459883, rs3798315) were significantly associated with oral cancer, lymphoma or brain cancer in three independent studies. Research Goal and Objective: With the ultimate goal of improving PCA detection/prognosis predictions and clinical management practices, this study seeks to systematically evaluate the individual and combined effects of 41 single nucleotide polymorphisms (SNPs) in chemokine associated genes in relation to PCA risk and disease progression among 1,237 African-American, African and Caribbean men. Hypothesis: We hypothesized that individuals inheriting one or multiple chemokine associated loci (linked to increased inflammation, immune response surveillance, metastasis and cell survival) will have an increased risk of developing PCA relative to those of referent genotypes. Materials and Methods: Forty-one sequence variants detected in chemokine associated genes will be evaluated in germ-line DNA samples collected from 420 PCA cases and 735 controls using Illumina9s Veracode genotyping system. Study participants were recruited from cancer screening programs, hospitals, or cancer centers located in the Washington D.C., South Carolina, and Kingston, Jamaica. Main effects and complex SNP interactions will be evaluated using logistic regression analysis and multi-factor dimensionality reduction (MDR), respectively. All risk models will be adjusted for potential confounders (i.e., age and West African Ancestry) and multiple hypothesis testing. Results: In a pilot study, four SNPs detected in CCR6, CCL5, and CCR4 were significantly associated with PCA among 230 men of African Descent from the United States (124 PCA cases, 106 controls). Inheritance of at least one minor CCR6 rs2023305 A allele was associated with a 1.92 fold increase in the risk of developing PCA. However, a 48–63% decrease in PCA was observed among carriers of the CCL5 (rs2107538 AG+AA, rs3817655 AT+AA) and CCR4 rs6550178 AG. With the exception of the CCR4 marker, these markers remained statistically after adjusting for age and West African ancestry. Discussion: In summary, polymorphisms in chemokine associated genes modify PCA susceptibility among men of African descent in the current study. Our findings will undergo further evaluation and validation in a larger and ethnically diverse study population, including 420 PCA cases and 735 controls from the U.S. and Jamaica. Such efforts will help to identify genetic markers capable of explaining disproportionately high prostate cancer incidence, mortality, and morbidity rates among men of African descent. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A70.

Inflammation polymorphisms and prostate cancer risk in Jamaican men: Role of obesity/body size

African ancestry and obesity are associated with higher risk of prostate cancer (PC). In a pilot study, we explored interactions between obesity (as measured by waist to hip ratio (WHR)) and inflammatory SNPs in relation to PC risk among Jamaican men. This study evaluated 87 chemokine and cytokine associated SNPs in obese and normal weight cases (N=109) and controls (N=102) using a stepwise penalized logistic regression approach in multivariable analyses. Upon stratification by WHR (normal weight (WHR<0.90) or obese (WHR≥0.90)), inheritance of CCR6 rs2023305 AG+GG (OR=1.75, p=0.007), CCR9 rs7613548 AG+GG (OR=1.71, p=0.012) and IL10ra rs2229113 AG+GG (OR=1.45, p=0.01) genotypes was associated with increase in overall or low grade (Gleason score<7) PC risk among normal weight men. These odds were elevated among obese men who possessed the CCR5 rs1799987 AG+GG (OR=1.95, p=0.003) and RNASEL rs12135247 CT+TT genotypes (OR=1.59, p=0.05). CCR7 rs3136685 AG+GG (p=0.032) was associated with a 1.52-1.70 fold increase in the risk of high grade cancer (Gleason score≥7) among obese men. CCR7 variant emerged as an important factor associated with high grade PC risk among obese men in our analyses. Overall, genetic loci found significant in normal weight men were not significant in obese men and vice-versa, partially explaining the role of obesity on PC risk among black men. Also, older age was an important risk factor both in normal weight and obese men but only with regard to low grade PC. Associations of inflammatory SNPs with obesity are suggestive and require further validation in larger cohorts to help develop an understanding of PC risk among obese and non-obese men of African descent.

Abstract 1921: Low doses of metformin increase expression of microRNAs that regulate innate immune genes in breast cancer cells

Background: Metformin (MF), the most widely used anti-diabetic drug, has also been shown to impact the incidence of breast cancer (BrCa), although the molecular mechanisms responsible for the therapeutic effects of MF are poorly understood. Unfortunately, until now in vitro studies have used high doses of MF (5-20 mM) and may not accurately model MF mechanisms of action at clinical doses. Objectives & Hypothesis: Metformin is thought to inhibit cancer cell growth by decreasing available cellular energy, creating a fatal metabolic deficit in cancer cells. Recent evidence suggests that innate immunity may play a role in the regulation of metabolism. We hypothesize that: 1) metabolic pathways targeted by MF impact innate immune pathways modulated by IRAK4 in BrCa cells and 2) metabolic and innate immune pathways in BrCa are regulated by common microRNAs (miRs). Therefore, we examined the effects of a) pharmacologically relevant MF concentrations on the expression of selected miRs and proteins associated with innate immunity and metabolism, and b) various concentrations of MF on BrCa cell lines that differ in their metastatic potential. Methods: Highly metastatic 231s and moderately metastatic 468s were cultured in low glucose medium and 15uM MF for 0, 2, 6, 12 and 24hrs. Changes in the expression of 43 miRs associated with innate immune and/or metabolic pathways were determined from total RNA by qRT-PCR using a miR array. MF-induced changes in IRAK4 protein expression were compared with known MF targets AMPK and its upstream regulator LKB1. The effects of MF concentrations up to 1 mM on BrCa cell growth and migration were also tested. Results: MF enhanced levels of hsa-miR-124-3p (which targets LKB1and AMPK) >2-fold at 2 and 6hrs in 468s. In contrast, hsa-miR-302c (which targets IRAK4) was up-regulated (>2-fold) by MF in 231s but not 468s at 2 and 6hrs. Expression of total IRAK4 protein was reduced after 12hrs of exposure to MF in 231s and after 6 and 12 hours in 468s. As reported previously, LKB1 was not expressed in 231s, whereas LKB1 was constitutively expressed in 468s. AMPK activation was inhibited at 2 and 12hrs in 231s, while in 468s AMPK activation was highly variable. Neither cell line showed changes in proliferation or migration in the presence of MF. Conclusions: Low concentrations of MF were sufficient to increase levels of two miRs known to regulate innate immune transcripts. MF treatment reduced IRAK4 levels by 6hrs (468s) to 12hrs (231s). Cell-specific differences in AMPK and LKB1 activation likely reflect differences in their metabolic rates and programs. Our data suggests that MF-sensitive metabolic pathways are involved in IRAK4-dependent innate immunity in breast cancer cells. Future studies will continue to unravel the effects of low doses of MF on BrCa. Citation Format: Jonne S. Woodard, Susan Yeyeodu, Kevin S. Kimbro. Low doses of metformin increase expression of microRNAs that regulate innate immune genes in breast cancer cells. [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 1921.

Abstract 2992: Analysis of divergent hypoxic gene expression data in prostate cancer cell lines suggests novel tumor progression biomarkers

Decreased oxygen tension which can be caused by isolation of solid tumor cells from vascularization is hypothesized to lead cause migration, adaptation, or death. These cellular endpoints require transcriptional regulation of genes that are involved in numerous metabolic and physiological processes. We hypothesize those differences in transcriptional regulation account for variation of cellular response to a hypoxic environment (i.e. metastasis). To test this hypothesis, we characterized the difference in hypoxic gene expression of two isogenic cell lines (LnCap and C4-2) which differ in metastatic potential. In three independent experiments, cell lines (LnCAP and C-42) were incubated in a hypoxic or normoxic environment and pathway analysis was performed on the list of genes whose expression was significantly different. Analysis of the divergent hypoxic gene expression data (i.e. 641 genes) supported significant changes in starch and sucrose pathways (i.e. UDP glucoronosyltransferases), and cardiac arteriolopathy (i.e. DPP4). Differences in gene expression (i.e. 300 genes) between LNCaP and C4-2 were noted under normoxic conditions and included genes related to cell cycle (i.e. CDKN1B, JUNB, and CYR61) and cell differentiation (i.e. ID2, ID3, and HBP1). C4-2 cells are much more metastatic than LNCaP cells from which they were derived. Differences in response to hypoxia may reflect a strategy used by the C4-2 cell line to escape or survive in a hypoxic environment. Transcript profiles may have prognostic or diagnostic value for distinguishing the propensity for prostate cells to metastasize, and those genes that assist in hypoxic survival of C4-2 may be therapeutic targets for PCa. Continued investigation will identify the contributions of these genes to the metastatic process and clarify the downstream transcriptional networks involved in this facet of tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2992. doi:1538-7445.AM2012-2992

Abstract 2780: Chemokine and chemokine receptor genetic variants as predictors of aggressive prostate cancer among European-American men

Background: Chemokines are essential mediators in the pathogenesis of inflammatory, autoimmune, vascular and neoplastic diseases. Consequently, investigators have evaluated the relationship between sequence variants in chemokines in relation to autoimmune or inflammatory disorders. However, the impact of inheriting multiple chemokine-chemokine receptor genetic susceptibilities on prostate cancer (PCa) clinical outcomes remains uncharacterized. Objectives & Hypothesis: Consequently, we systematically evaluated the joint modifying effects of 78 single nucleotide polymorphisms (SNPs) detected in 43 chemokine and chemokine receptor-associated genes. We hypothesized that individuals inheriting multiple chemokine-associated alleles linked with increased tumor metastasis, cell survival, or inflammation would have an increased risk of developing PCa or aggressive disease relative to those with the referent genotypes. Methods: To test the hypothesis, we used SNP profile data collected from 2277 European-American male participants of the Cancer Genetic Markers of Susceptibility (CGEMS) project, involving 1176 PCa cases and 1101 controls. Using a case-control and case-only designs, we evaluated the independent and joint modifying effects of 78 SNPs detected in chemokines, chemokine receptors and downstream signaling targets in relation to PCa risk and aggressive disease (tumor stage >2 and tumor grade >2). Main effects and complex interactions were assessed using logistic regression analysis, multi-factor dimensionality reduction (MDR) with covariate adjustment (i.e., age-group and family history of prostate cancer), and hierarchical entropy graphs. To control for multiple comparisons, we used 1000-fold permutation testing. Results: A three-SNP interaction [CCL21 promoter SNP at position -191 (rs11574914), CCR6 promoter SNP at position -397 (rs2023305), CCL17 intron 1+2217 (rs223895)] was the best prediction model in relation to aggressive disease, following restriction of the data analysis to the top 25 th percentile of markers based on ReliefF scores (TBA = 56%, CVC = 90%, p = 0.024). Verification of this interaction using other bioinformatic techniques, including symbolic modeling, classification and regression trees (CART) and computational evolutionary systems modeling is currently underway. Conclusions: Our preliminary findings support the role of chemokine-related markers as significant predictors of aggressive prostate cancer among European-American men. Future Directions: Additional studies are needed to determine whether CCL21-CCR6-CCL17 interactions are predictive of disease prognosis within ethnically diverse sub-populations. In addition, the biological implications of the interaction require further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2780. doi:10.1158/1538-7445.AM2011-2780