Susan Kadlubar

JAK/STAT/SOCS-signaling pathway and colon and rectal cancer

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway is involved in immune function and cell growth. We evaluated the association between genetic variation in JAK1 (10 SNPs), JAK2 (9 SNPs), TYK2 (5 SNPs), suppressors of cytokine signaling (SOCS)1 (2 SNPs), SOCS2 (2 SNPs), STAT1 (16 SNPs), STAT2 (2 SNPs), STAT3 (6 SNPs), STAT4 (21 SNPs), STAT5A (2 SNPs), STAT5B (3 SNPs), STAT6 (4 SNPs) with risk of colorectal cancer. We used data from population‐based case‐control studies (colon cancer n = 1555 cases, 1,956 controls; rectal cancer n = 754 cases, 959 controls). JAK2, SOCS2, STAT1, STAT3, STAT5A, STAT5B, and STAT6 were associated with colon cancer; STAT3, STAT4, STAT6, and TYK2 were associated with rectal cancer. Given the biological role of the JAK/STAT‐signaling pathway and cytokines, we evaluated interaction with IFNG, TNF, and IL6; numerous statistically significant associations after adjustment for multiple comparisons were observed. The following statistically significant interactions were observed: TYK2 with aspirin/NSAID use; STAT1, STAT4, and TYK2 with estrogen status; and JAK2, STAT2, STAT4, STAT5A, STAT5B, and STAT6 with smoking status and colon cancer risk; JAK2, STAT6, and TYK2 with aspirin/NSAID use; JAK1 with estrogen status; STAT2 with cigarette smoking and rectal cancer. JAK2, SOCS1, STAT3, STAT5, and TYK2 were associated with colon cancer survival (hazard rate ratio (HRR) of 3.3 95% CI 2.01,5.42 for high mutational load). JAK2, SOCS1, STAT1, STAT4, and TYK2 were associated with rectal cancer survival (HRR 2.80 95% CI 1.63,4.80). These data support the importance of the JAK/STAT‐signaling pathway in colorectal cancer and suggest targets for intervention.

Induction of cell proliferation and survival genes by estradiol-repressed microRNAs in breast cancer cells

BackgroundIn estrogen responsive MCF-7 cells, estradiol (E2) binding to ERα leads to transcriptional regulation of genes involved in the control of cell proliferation and survival. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression. The aim of this study was to explore whether miRNAs were involved in hormonally regulated expression of estrogen responsive genes.MethodsWestern blot and QPCR were used to determine the expression of estrogen responsive genes and miRNAs respectively. Target gene expression regulated by miRNAs was validated by luciferase reporter assays and transfection of miRNA mimics or inhibitors. Cell proliferation was evaluated by MTS assay.ResultsE2 significantly induced bcl-2, cyclin D1 and survivin expression by suppressing the levels of a panel of miRNAs (miR-16, miR-143, miR-203) in MCF-7 cells. MiRNA transfection and luciferase assay confirmed that bcl-2 was regulated by miR-16 and miR-143, cyclinD1 was modulated by miR-16. Importantly, survivin was found to be targeted by miR-16, miR-143, miR-203. The regulatory effect of E2 can be either abrogated by anti-estrogen ICI 182, 780 and raloxifene pretreatment, or impaired by ERα siRNA, indicating the regulation is dependent on ERα. In order to investigate the functional significance of these miRNAs in estrogen responsive cells, miRNAs mimics were transfected into MCF-7 cells. It revealed that overexpression of these miRNAs significantly inhibited E2-induced cell proliferation. Further study of the expression of the miRNAs indicated that miR-16, miR-143 and miR-203 were highly expressed in triple positive breast cancer tissues, suggesting a potential tumor suppressing effect of these miRNAs in ER positive breast cancer.ConclusionsThese results demonstrate that E2 induces bcl-2, cyclin D1 and survivin by orchestrating the coordinate downregulation of a panel of miRNAs. In turn, the miRNAs manifest growth suppressive effects and control cell proliferation in response to E2. This sheds a new insight into the integral post-transcriptional regulation of cell proliferation and survival genes by miRNAs, a potential therapeutic option for breast cancer.

Oxidized LDL Receptor 1 (OLR1) as a Possible Link between Obesity, Dyslipidemia and Cancer

Recent studies have linked expression of lectin-like ox-LDL receptor 1 (OLR1) to tumorigenesis. We analyzed microarray data from Olr1 knockout (KO) and wild type (WT) mice for genes involved in cellular transformation and evaluated effects of OLR1 over-expression in normal mammary epithelial cells (MCF10A) and breast cancer cells (HCC1143) in terms of gene expression, migration, adhesion and transendothelial migration. Twenty-six out of 238 genes were inhibited in tissues of OLR1 KO mice; the vast majority of OLR1 sensitive genes contained NF-κB binding sites in their promoters. Further studies revealed broad inhibition of NF-kB target genes outside of the transformation-associated gene pool, with enrichment themes of defense response, immune response, apoptosis, proliferation, and wound healing. Transcriptome of Olr1 KO mice also revealed inhibition of de novo lipogenesis, rate-limiting enzymes fatty acid synthase (Fasn), stearoyl-CoA desaturase (Scd1) and ELOVL family member 6 (Elovl6), as well as lipolytic phospholipase A2 group IVB (Pla2g4b). In studies comparing MCF10A and HCC1143, the latter displayed 60% higher OLR1 expression. Forced over-expression of OLR1 resulted in upregulation of NF-κB (p65) and its target pro-oncogenes involved in inhibition of apoptosis (BCL2, BCL2A1, TNFAIP3) and regulation of cell cycle (CCND2) in both cell lines. Basal expression of FASN, SCD1 and PLA2G4B, as well as lipogenesis transcription factors PPARA, SREBF2 and CREM, was higher in HCC1143 cells. Over-expression of OLR1 in HCC1143 cells also enhanced cell migration, without affecting their adherence to TNFα-activated endothelium or transendothelial migration. On the other hand, OLR1 neutralizing antibody inhibited both adhesion and transmigration of untreated HCC1143 cells. We conclude that OLR1 may act as an oncogene by activation of NF-kB target genes responsible for proliferation, migration and inhibition of apoptosis and de novo lipogenesis genes.

In vitro and in vivo oxidative metabolism and glucuronidation of anastrozole

AIMS Little information is available regarding the metabolic routes of anastrozole and the specific enzymes involved. We characterized anastrozole oxidative and conjugation metabolism in vitro and in vivo. METHODS A sensitive LC-MS/MS method was developed to measure anastrozole and its metabolites in vitro and in vivo. Anastrozole metabolism was characterized using human liver microsomes (HLMs), expressed cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs). RESULTS Hydroxyanastrozole and anastrozole glucuronide were identified as the main oxidative and conjugated metabolites of anastrozole in vitro, respectively. Formation of hydroxyanastrozole from anastrozole was markedly inhibited by CYP3A selective chemical inhibitors (by >90%) and significantly correlated with CYP3A activity in a panel of HLMs (r= 0.96, P= 0.0005) and mainly catalyzed by expressed CYP3A4 and CYP3A5. The K(m) values obtained from HLMs were also close to those from CYP3A4 and CYP3A5. Formation of anastrozole glucuronide in a bank of HLMs was correlated strongly with imipramine N-glucuronide, a marker of UGT1A4 (r= 0.72, P < 0.0001), while expressed UGT1A4 catalyzed its formation at the highest rate. Hydroxyanastrozole (mainly as a glucuronide) and anastrozole were quantified in plasma of breast cancer patients taking anastrozole (1 mg day⁻¹); anastrozole glucuronide was less apparent. CONCLUSION Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4 (and to some extent by CYP3A5 and CYP2C8). Once formed, this metabolite undergoes glucuronidation. Variable activity of CYP3A4 (and probably UGT1A4), possibly due to genetic polymorphisms and drug interactions, may alter anastrozole disposition and its effects in vivo.

Expression and localization of cytosolic sulfotransferase (SULT) 1A1 and SULT1A3 in normal human brain.

Cytosolic sulfotransferases (SULTs) are a family of Phase II drug-metabolizing enzymes that catalyze the transfer of a sulfonate group from 3′-phosphoadenosine 5′-phosphosulfate to endogenous and xenobiotic compounds. Several SULT isoform messages have been detected in the human brain; however, protein expression patterns have not been characterized. Immunoblot analysis of the SULT1A1 and 1A3 isoforms was carried out with cytosolic fractions isolated from superior temporal gyrus, hippocampus, cerebellum, occipital pole, frontal pole, and temporal pole regions of normal adult human brains. SULT1A1 expression was highest in cytosolic fractions isolated from cerebellum, occipital, and frontal lobes, whereas, SULT1A3 expression was highest in cytosol from superior temporal gyrus, hippocampus, and temporal lobe. SULT1A1 and SULT1A3 immunoreactivities were found in both neurons and glial cells by immunohistochemical analysis in all brain regions studied. SULT1A1 is known to catalyze the metabolism of small phenols, whereas SULT1A3 sulfates catecholamine neurotransmitters. Because SULT1A1 and 1A3 have distinct substrate specificities, the differences in expression pattern and cellular localization of the SULT1A isoforms are probably associated with the distribution and function of their selective substrates in the different brain regions.

Interleukin genes and associations with colon and rectal cancer risk and overall survival.

Interleukins are a group of cytokines that contribute to growth and differentiation, cell migration, and inflammatory and anti‐inflammatory responses by the immune system. In our study, we examined genetic variation in genes from various anti‐inflammatory and proinflammatory interleukins to determine association with colon and rectal cancer risk and overall survival. Data from two population‐based incident studies of colon cancer (1,555 cases and 1,956 controls) and rectal cancer (754 cases and 954 controls) were used. After controlling for multiple comparisons, single nucleotide polymorphisms (SNPs) from four genes, IL3, IL6R, IL8, IL15, were associated with increased colon cancer risk, and CXCR1 and CXCR2 were significantly associated with increased rectal cancer risk. Only SNPs from genes within the IL‐8 pathway (IL8, CXCR1 and CXCR2) showed a significant association with both colon and rectal cancer risk. Several SNPs interacted significantly with IL8 and IFNG SNPs and with aspirin/non‐steroidal anti‐inflammatory drug (NSAID), cigarette smoking, estrogen use and BMI. For both colon and rectal cancer, increasing numbers of risk alleles were associated with increased hazard of death from cancer; the estimated hazard of death for colon cancer for the highest category of risk alleles was 1.74 (95% confidence interval [CI] 1.18–2.56) and 1.96 (95% CI 1.28–2.99) for rectal cancer. These data suggest that interleukin genes play a role in risk and overall survival for colon and rectal cancer.

Suppression of CYP2C9 by MicroRNA hsa-miR-128-3p in Human Liver Cells and Association with Hepatocellular Carcinoma

Published studies have identified genetic variants, somatic mutations, and changes in gene expression profiles that are associated with hepatocellular carcinoma (HCC), particularly involving genes that encode drug metabolizing enzymes (DMEs). CYP2C9, one of the most abundant and important DMEs, is involved in the metabolism of many carcinogens and drugs and is down-regulated in HCC. To investigate the molecular mechanisms that control CYP2C9 expression, we applied integrative approaches including in silico, in vitro, and in vivo analyses to elucidate the role of microRNA hsa-miR-128-3p in the regulation of CYP2C9 expression and translation. RNA electrophoresis mobility shift assays demonstrated a direct interaction between hsa-miR-128-3p and its cognate target, the CYP2C9 transcript. Furthermore, the expression of a luciferase reporter gene containing the 3′-UTR of CYP2C9 and the endogenous expression of CYP2C9 were suppressed by transfection of hsa-miR-128-3p. Importantly, chemically-induced up- or down-regulation of hsa-miR-128-3p correlated inversely with the expression of CYP2C9. Finally, an association analysis revealed that the expression of hsa-miR-128-3p is inversely correlated with the expression of CYP2C9 in HCC tumor tissues. Altogether, the study helped to elucidate the mechanism of CYP2C9 regulation by hsa-miR-128-3p, and the inverse association in HCC.

Genetic variation in bone morphogenetic protein and colon and rectal cancer

Bone morphogenetic proteins (BMP) are part of the TGF‐β‐signaling pathway; genetic variation in these genes may be involved in colorectal cancer. In this study, we evaluated the association between genetic variation in BMP1 (11 tagSNPs), BMP2 (5 tagSNPs), BMP4 (3 tagSNPs), BMPR1A (9 tagSNPs), BMPR1B (21 tagSNPs), BMPR2 (11 tagSNPs) and GDF10 (7 tagSNPs) with risk of colon and rectal cancer and tumor molecular phenotype. We used data from population‐based case‐control studies (colon cancer n = 1,574 cases, 1,970 controls; rectal cancer n = 791 cases, 999 controls). We observed that genetic variation in BMPR1A, BMPR1B, BMPR2, BMP2 and BMP4 was associated with risk of developing colon cancer, with 20 to 30% increased risk for most high‐risk genotypes. A summary of high‐risk genotypes showed over a twofold increase in colon cancer risk at the upper risk category (OR = 2.49 95% CI = 1.95, 3.18). BMPR2, BMPR1B, BMP2 and GDF10 were associated with rectal cancer. BMPR2 rs2228545 was associated with an almost twofold increased risk of rectal cancer. The risk associated with the highest category of the summary score for rectal cancer was 2.97 (95% CI = 1.87, 4.72). Genes in the BMP‐signaling pathway were consistently associated with CIMP+ status in combination with both KRAS‐mutated and MSI tumors. BMP genes interacted statistically significantly with other genes in the TGF‐β‐signaling pathway, including TGFβ1, TGFβR1, Smad 3, Smad 4 and Smad 7. Our data support a role for genetic variation in BMP‐related genes in the etiology of colon and rectal cancer. One possible mechanism is via the TGF‐β‐signaling pathway.

Potential Role of UGT1A4 Promoter SNPs in Anastrozole Pharmacogenomics

Anastrozole belongs to the nonsteroidal triazole-derivative group of aromatase inhibitors. Recently, clinical trials demonstrated improved antitumoral efficacy and a favorable toxicity with third-generation aromatase inhibitors, compared with tamoxifen. Anastrozole is predominantly metabolized by phase I oxidation with the potential for further phase II glucuronidation. It also, however, is subject to direct N-glucuronidation by UDP-glucuronosyltransferase 1A4 (UGT1A4). Anastrozole pharmacokinetics vary widely among patients, but pharmacogenomic studies of patients treated with anastrozole are sparse. In this study, we examined individual variability in the glucuronidation of anastrozole and its association with UGT1A4 promoter and coding region polymorphisms. In vitro assays using liver microsomal preparations from individual subjects (n = 96) demonstrated 235-fold variability in anastrozole glucuronidation. Anastrozole glucuronidation was correlated (r = 0.99; P < 0.0001) with lamotrigine glucuronidation (a diagnostic substrate for UGT1A4) and with UGT1A4 mRNA expression levels in human liver microsomes (r = 0.99; P < 0.0001). Recombinant UGT1A4 catalyzed anastrozole glucuronidation, which was inhibited by hecogenin (IC50 = 15 µM), a UGT1A4 specific inhibitor. The promoter region of UGT1A4 is polymorphic, and compared with those homozygous for the common allele, lower enzymatic activity was observed in microsomes from individuals heterozygous for −163G<A, −219T<G, and −217C<T (P = 0.009, P = 0.014, and P = 0.009, respectively). These results indicate that variability in glucuronidation could contribute to response to anastrozole in the treatment of breast cancer.

Potent Inhibition of Human Sulfotransferase 1A1 by 17α-Ethinylestradiol: Role of 3′-Phosphoadenosine 5′-Phosphosulfate Binding and Structural Rearrangements in Regulating Inhibition and Activity

Sulfotransferase (SULT) 1A1 is the major drug/xenobiotic-conjugating SULT isoform in human liver because of its broad substrate reactivity and high expression level. SULT1A1 sulfates estrogens with low micromolar Km values consistent with its affinity for sulfation of many small phenolic compounds. Binding studies showed the unexpected ability of 17α-ethinylestradiol (EE2) to bind and inhibit SULT1A1 activity toward p-nitrophenol and β-naphthol at low nanomolar concentrations, whereas EE2 was not sulfated until significantly higher concentrations were reached. EE2 had a Ki of 10 nM for inhibiting p-nitrophenol and β-naphthol sulfation and inhibited 17β-estradiol (E2) sulfation in intact human MCF-7 breast cancer cells with a Ki of 19 nM. In contrast, the Km for EE2 sulfation by SULT1A1 was 700 nM. The Kd for EE2 binding of pure SULT1A1 was 0.5 ± 0.15 μM; however, the Kd for EE2 binding to the SULT1A1-PAP complex was >100-fold lower (4.3 ± 1.7 nM). The Kd for E2 binding to SULT1A1 changed from 2.3 ± 0.9 to 1.2 ± 0.56 μM in the presence of PAP. Docking studies with E2 indicate that E2 binds in a competent orientation in the resolved structure of SULT1A1 in the both presence and absence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). However, EE2 binds in a catalytically competent orientation in the absence of PAPS but in a noncompetent orientation via formation of a charge interaction with Tyr108 if PAPS is bound first. In conclusion, EE2 is a potent inhibitor, but not a substrate, of SULT1A1 at low nanomolar concentrations, indicating the possibility of drug-drug interactions during contraceptive therapy.