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Supplementary Table 1

Genistein, the major isoflavone in soybean, has been reported to exert anticancer effects on various types of cancer including ovarian cancer; however, its chemopreventive effects and mechanisms of action in ovarian cancer have not been fully elucidated in spontaneously developing ovarian cancer models. In this study, we demonstrated the preventive effects and mechanisms of genistein in the laying hen model that develops spontaneous ovarian cancer at high incidence rates. Laying hens were randomized to three groups: control (3.01 mg/hen, n = 100), low (52.48 mg/hen n = 100), and high genistein supplementation (106.26 mg/hen/day; per group). At the end of 78 weeks, hens were euthanized and ovarian tumors were collected and analyzed. We observed that genistein supplementation significantly reduced the ovarian tumor incidence (P = 0.002), as well as the number and size of the tumors (P = 0.0001). Molecular analysis of the ovarian tumors revealed that genistein downregulated serum malondialdehyde, a marker for oxidative stress and the expression of NFκB and Bcl-2, whereas it upregulated Nrf2, HO-1, and Bax expression at protein level in ovarian tissues. Moreover, genistein intake decreased the activity of mTOR pathway as evidenced by reduced phosphorylation of mTOR, p70S6K1, and 4E-BP1. Taken together, our findings strongly support the potential of genistein in the chemoprevention of ovarian cancer and highlight the effects of the genistein on the molecular pathways involved in ovarian tumorigenesis.

Supplemental Figure Legends

Supplemental Figure 1 Figure S1. Breeding scheme for generation of prostate epithelium-specific Sox4 and Pten conditional double-knockout mice. Male PSA-CRE-ER/PTENfl/fl mice (1) were crossed with female Sox4fl/fl mice (2) and bred to homozygosity.

Understanding remains incomplete of the mechanisms underlying initiation and progression of prostate cancer, the most commonly diagnosed cancer in American men. The transcription factor SOX4 is overexpressed in many human cancers, including prostate cancer, suggesting it may participate in prostate tumorigenesis. In this study, we investigated this possibility by genetically deleting Sox4 in a mouse model of prostate cancer initiated by loss of the tumor suppressor Pten. We found that specific homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibited tumor progression initiated by homozygous loss of Pten. Mechanistically, Sox4 ablation reduced activation of AKT and β-catenin, leading to an attenuated invasive phenotype. Furthermore, SOX4 expression was induced by Pten loss as a result of the activation of PI3K–AKT–mTOR signaling, suggesting a positive feedback loop between SOX4 and PI3K–AKT–mTOR activity. Collectively, our findings establish that SOX4 is a critical component of the PTEN/PI3K/AKT pathway in prostate cancer, with potential implications for combination-targeted therapies against both primary and advanced prostate cancers. Cancer Res; 76(5); 1112–21. ©2015 AACR.

Supplemental Figure 3 Figure S3. Low power H&E images of wild-type, PTEN-/-, and PTEN-/-/SOX4-/- prostates.

Supplemental Figure 2 Figure S2. Probasin-Cre mice (3) obtained from the NCI mouse repository were crossed to ROSA-lox-stop-lox-GFP mice (C57/BL6;129-ROSA26-Lox-Stop-Lox-EGFP (Charles River Laboratories)). Tissues were harvested from male mice after reaching sexual maturity at 8 weeks of age. GFP imaging of organs was performed on an Olympus OV-100 In Vivo Imaging System. Representative tissues from double transgenic mice are shown on the left, and from control mice on the right. Strong GFP signal was detected in pancreas and all three lobes of the prostate.

Treatment of cancer with chemotherapeutic drugs is associated with numerous adverse effects as well as the eventual development of resistance to chemotherapy. There is a great need for complementary therapies such as botanicals and nutritional supplements with little or no side effects that prevent resistance to chemotherapy and reduce its adverse effects. Inflammation plays a major role in the development of chemoresistance and the adverse effects of chemotherapy. Phytochemicals have well-established anti-inflammatory effects; thus, they could be used as complementary therapies along with chemotherapy to increase its efficacy and reduce its toxicity. Botanical compounds inhibit the NF-κB signaling pathway, which plays an important role in the generation of inflammation, chemotherapy resistance, and modulation of cell survival and apoptosis. Botanicals have previously been studied extensively for their cancer chemopreventive activities and are generally considered safe for human consumption. The present review focuses on the modulation of inflammation by phytochemicals and their role in increasing the efficacy and reducing the toxicity of cancer chemotherapy.

Genistein, the major isoflavone in soybean, has been reported to exert anticancer effects on various types of cancer including ovarian cancer; however, its chemopreventive effects and mechanisms of action in ovarian cancer have not been fully elucidated in spontaneously developing ovarian cancer models. In this study, we demonstrated the preventive effects and mechanisms of genistein in the laying hen model that develops spontaneous ovarian cancer at high incidence rates. Laying hens were randomized to three groups: control (3.01 mg/hen, n = 100), low (52.48 mg/hen n = 100), and high genistein supplementation (106.26 mg/hen/day; per group). At the end of 78 weeks, hens were euthanized and ovarian tumors were collected and analyzed. We observed that genistein supplementation significantly reduced the ovarian tumor incidence (P = 0.002), as well as the number and size of the tumors (P = 0.0001). Molecular analysis of the ovarian tumors revealed that genistein downregulated serum malondialdehyde, a marker for oxidative stress and the expression of NFκB and Bcl-2, whereas it upregulated Nrf2, HO-1, and Bax expression at protein level in ovarian tissues. Moreover, genistein intake decreased the activity of mTOR pathway as evidenced by reduced phosphorylation of mTOR, p70S6K1, and 4E-BP1. Taken together, our findings strongly support the potential of genistein in the chemoprevention of ovarian cancer and highlight the effects of the genistein on the molecular pathways involved in ovarian tumorigenesis.

Objective Pantothenate kinase-associated neurodegeneration (PKAN) is caused by mutations of the pantothenate kinase 2 ( PANK2 ) gene. The major clinical sign of PKAN is dystonia and the eye-of-the-tiger pattern on the MRI has been a clue for the diagnosis. We aim to discuss clinical and genetic findings of 22 PKAN patients from 13 families. Methods Twenty-two patients were clinically diagnosed with PKAN and screened for PANK2 mutations. The patients were classified according to their onset age and progression rate. Results Mutation screening revealed 5 novel and 7 previously reported sequence variants in PANK2 . The variants identified were in the form of missense changes, small exonic deletions and intronic mutations with a probable splicing effect. The presenting features were dystonia and gait disturbance in early onset patients, whereas the presenting symptoms were variable for the late onset group. The progression rate of the disease was not uniform. Conclusion The current report is the first patient series of PKAN from Turkey that expands the clinical and genetic spectrum of the disease.

Soy consumption in human diet has been linked to decreased incidence of a variety of cancers, suggesting a potential role of soy products in cancer prevention and control. Furthermore, a substantial body of evidence in the literature suggests that soy supplementation may improve the efficacy and prevent the adverse effects of cancer chemotherapy and radiation therapy. Isoflavones constitute the predominant anticancer bioactive compounds in soy. Genistein, which is the most abundant and active isoflavone in soy, has a multitude of effects on cancer cells, including inhibition of NF-κB activation and DNA methylation, enhancement of histone acetylation, inhibition of cell growth and metastasis, and antiangiogenic, anti-inflammatory, and anti-oxidant effects. Isoflavones are orally bioavailable, easily metabolized, and usually considered safe. In this article, we review in vitro and in vivo evidence as well as the results of clinical and epidemiological studies on the effects of soy isoflavones, with a focus on sensitization of cancer cells to chemotherapy and radiation while at the same time protecting normal cells from the harmful effects of these treatments.

Purpose. In the present study, we aimed to investigate the effects of tomato powder (TP) on glucose and lipid metabolism, as well as oxidative stress and the NF-κB, mTOR, and Nrf2 pathways during the aging process in healthy rats. Methods and Results. Male Wistar rats were randomly assigned to four groups as follows: (i) Control group 1 (n=15, 3-week old): rats were fed standard diet for 7 weeks; (ii) TP group 1 (n=15, 3-week old): rats were fed standard diet supplemented with TP for 7 weeks; (iii) Control group 2 (n=15, 8-week old): rats were fed standard diet for 69 weeks; and (iv) TP group 2 (8-week old): rats were fed standard diet supplemented with TP for 69 weeks. TP supplementation significantly reduced the hyperglycemia, hypertriglyceridemia, and hypercholesterolemia and improved liver function and kidney function in 77-week old rats compared with the control animals (P<0.05). In addition, TP significantly decreased the serum and liver MDA levels (P<0.003 and P<0.001, respectively) while increasing the activities of liver SOD (P<0.001), CAT (P<0.008), and GPx (P<0.01) compared with the control groups in both 10-week-old and 77-week-old rats (P<0.05). Age-related increases in phosphorylation of NF-κBp65, mTOR, 4E-BP1, and P70S6K were observed in livers of 77-week-old rats compared to those of 10-week-old rats (P<0.001). TP supplementation decreased the expression of NF-κBp65 and activation of mTOR, 4E-BP1, and P70S6K in livers of 77-week-old rats compared to the control animals. Moreover, TP supplementation significantly elevated Nrf2 expression in livers of both 10-week-old and 77-week-old rats (P<0.05). Conclusion. TP ameliorates age-associated inflammation and oxidative stress through the inhibition of NF-κBp65, mTOR pathways, and Nrf2 activation may explain the observed improvement in glucose and lipid metabolism as well as the improved liver and kidney functions.

Understanding remains incomplete of the mechanisms underlying initiation and progression of prostate cancer, the most commonly diagnosed cancer in American men. The transcription factor SOX4 is overexpressed in many human cancers, including prostate cancer, suggesting it may participate in prostate tumorigenesis. In this study, we investigated this possibility by genetically deleting Sox4 in a mouse model of prostate cancer initiated by loss of the tumor suppressor Pten. We found that specific homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibited tumor progression initiated by homozygous loss of Pten. Mechanistically, Sox4 ablation reduced activation of AKT and β-catenin, leading to an attenuated invasive phenotype. Furthermore, SOX4 expression was induced by Pten loss as a result of the activation of PI3K–AKT–mTOR signaling, suggesting a positive feedback loop between SOX4 and PI3K–AKT–mTOR activity. Collectively, our findings establish that SOX4 is a critical component of the PTEN/PI3K/AKT pathway in prostate cancer, with potential implications for combination-targeted therapies against both primary and advanced prostate cancers. Cancer Res; 76(5); 1112–21. ©2015 AACR.

We investigated if previously demonstrated inhibition of fluciclovine ((18)F) in vitro could be replicated in a PC3-Luc xenograft mouse model. Following intratumoral injection of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), alpha-(methylamino)isobutyric acid (MeAIB) or saline, fluciclovine PET tumor-to-background activity was 43.6 (± 5.4)% and 25.3 (± 5.2)% lower in BCH (n = 6) and MeAIB (n = 5) injected PC3 Luc xenografts, respectively, compared to saline-injected controls (n = 2). Partial inhibition of fluciclovine uptake by BCH and MeAIB can be demonstrated in vivo similar to previous in vitro modeling.

Purpose In the present study, we aimed to investigate the effects of tomato powder (TP) on glucose and lipid metabolism, as well as oxidative stress and the NF-κB, mTOR, and Nrf2 pathways during the aging process in healthy rats. Methods and Results Male Wistar rats were randomly assigned to four groups as follows: (i) Control group 1 (n=15, 3-week old): rats were fed standard diet for 7 weeks; (ii) TP group 1 (n=15, 3-week old): rats were fed standard diet supplemented with TP for 7 weeks; (iii) Control group 2 (n=15, 8-week old): rats were fed standard diet for 69 weeks; and (iv) TP group 2 (8-week old): rats were fed standard diet supplemented with TP for 69 weeks. TP supplementation significantly reduced the hyperglycemia, hypertriglyceridemia, and hypercholesterolemia and improved liver function and kidney function in 77-week old rats compared with the control animals (P < 0.05). In addition, TP significantly decreased the serum and liver MDA levels (P < 0.003 and P < 0.001, respectively) while increasing the activities of liver SOD (P < 0.001), CAT (P < 0.008), and GPx (P < 0.01) compared with the control groups in both 10-week-old and 77-week-old rats (P < 0.05). Age-related increases in phosphorylation of NF-κBp65, mTOR, 4E-BP1, and P70S6K were observed in livers of 77-week-old rats compared to those of 10-week-old rats (P < 0.001). TP supplementation decreased the expression of NF-κBp65 and activation of mTOR, 4E-BP1, and P70S6K in livers of 77-week-old rats compared to the control animals. Moreover, TP supplementation significantly elevated Nrf2 expression in livers of both 10-week-old and 77-week-old rats (P < 0.05). Conclusion TP ameliorates age-associated inflammation and oxidative stress through the inhibition of NF-κBp65, mTOR pathways, and Nrf2 activation may explain the observed improvement in glucose and lipid metabolism as well as the improved liver and kidney functions.

Background Dietary intake of lycopene has been associated with a reduced risk of ovarian cancer, suggesting its chemopreventive potential against ovarian carcinogenesis. Lycopene's molecular mechanisms of action in ovarian cancer have not been fully understood. Therefore, in the present study, we investigated the effects of lycopene on the ovarian cancer formation using the laying hen model, a biologically relevant animal model of spontaneous ovarian carcinogenesis due to high incidence rates similar to humans. Methods In this study, a total of 150 laying hens at age of 102 weeks were randomized into groups of 50: a control group (0 mg of lycopene per kg of diet) and two treatment groups (200 mg or 400 mg of lycopene per kg of diet, or ~26 and 52 mg/d/hen, respectively). At the end of 12 months, blood, ovarian tissues and tumors were collected. Results We observed that lycopene supplementation significantly reduced the overall ovarian tumor incidence (P < 0.01) as well as the number and the size of the tumors (P < 0.004 and P < 0.005, respectively). Lycopene also significantly decreased the rate of adenocarcinoma, including serous and mucinous subtypes (P < 0.006). Moreover, we also found that the serum level of oxidative stress marker malondialdehyde was significantly lower in lycopene-fed hens compared to control birds (P < 0.001). Molecular analysis of the ovarian tumors revealed that lycopene reduced the expression of NF-κB while increasing the expression of nuclear factor erythroid 2 and its major target protein, heme oxygenase 1. In addition, lycopene supplementation decreased the expression of STAT3 by inducing the protein inhibitor of activated STAT3 expression in the ovarian tissues. Conclusions Taken together, our findings strongly support the potential of lycopene in the chemoprevention of ovarian cancer through antioxidant and anti-inflammatory mechanisms.

In this chapter, the role of lycopene in protecting the liver against oxidative stress-induced damage has been discussed. Lycopene, which is a very potent antioxidant, provides defense against cellular damage caused by reactive oxygen species (ROS). Both conjugated and nonconjugated double bonds of lycopene make it highly reactive against oxidative stress-induced damage. A lycopene-rich diet is recommended for patients with liver diseases because numerous epidemiological studies have validated the protective effects of lycopene on the liver. Lycopene has been reported to exert its effects through pleiotropic molecular mechanisms, including transcriptional regulation, cell cycle control, apoptosis, hormonal balance, inflammation, angiogenesis, and metastasis. Molecular studies have indicated that lycopene modulates cellular processes by acting on molecular targets, such as NF-κB (nuclear factor-kappa B), Nrf2 (nuclear factor erythroid 2-related factor 2), PI3K/AKT (phosphoinositide 3-kinase/AKT), VEGF (vascular endothelial growth factor), ROS-producing enzymes (CYP-P450 enzymes, NADPH oxidase, iNOS, COX-2, and 5-LOX), leading to inhibition of liver diseases, including nonalcoholic fatty liver disease (NAFLD) and liver cancer. Here, we summarize the recent data from in vitro and in vivo studies as well as clinical trials to assess the protective effects of lycopene on oxidative stress in liver.

Renal cell carcinoma (RCC) is the most frequent upper urinary tract cancer in humans and accounts for 80–85% of malignant renal tumors. Eker rat represents a unique animal model to study RCC since these rats develop spontaneous renal tumors and leiomyoma, which may be due to tuberous sclerosis 2 (TSC2) mutation resulting in the activation of the mammalian target of rapamycin (mTOR) pathway. This study examines the role of a lycopene-rich diet in the development of RCC in the TSC2 mutant Eker rat model. Ten-week old female Eker rats (n = 90) were assigned in equal numbers to receive 0,100 or 200 mg/kg of lycopene as part of their daily diet. After 18 months the rats were sacrificed and the kidneys were removed. Immunohistochemical staining with antibodies against mTOR, phospho-S6 and EGFR were performed, as well as hematoxylin–eosin staining for histologic examination of the tumors. Tumors were counted and measured in individual kidneys. Presence of tumor decreased from 94% in control animals to 65% in the experimental group, but the difference was not statistically significant (P< 0.12). However, mean numbers of renal carcinomas were statistically significantly decreased in the lycopene-treated rats (P< 0.008) when compared to untreated controls. In the lycopene group, tumor numbers decreased (P< 0.002) and the numbers tended to decrease linearly (P< 0.003) as supplemental lycopene increased from 0 to 200. Control rats fed only basal diet had a greater length of tumors (23.98 mm) than rats fed lycopene supplement groups (12.90 mm and 11.07 mm) (P

Non-alcoholic fatty liver disease (NAFLD) is a growing health problem around the world, especially in developed countries. NAFLD includes all cases of fatty liver disease from simple steatosis to cirrhosis, without excessive alcohol intake, use of steatogenic medication or hereditary disorders. Pathogenesis is associated with dietary high fat intake, decreased free fatty acid (FFA) oxidation, increased hepatic lipogenesis and lipolysis from the adipose tissue. These metabolic alterations contribute to the hepatic fat accumulation. Consequently, stimulated oxidative stress and inflammation play a major role in hepatocellular damage. Therefore, antioxidant and anti-inflammatory agents may have a role in the prevention of this disease. Carotenoids are potent antioxidant and anti-inflammatory micronutrients, which have been investigated in the prevention and treatment of NAFLD. The main sources of the carotenoids are fruits and vegetables. In this article we review the potential role and possible molecular mechanism of carotenoids in NAFLD.

Sox4 is an essential gene, and genetic deletion results in embryonic lethality. In an effort to develop mice with tissue-specific deletion, we bred conditional knockout mice bearing LoxP recombination sites flanking the Sox4 gene, with the LoxP sites located in the Sox4 5'UTR and 3'UTR.The number of mice homozygous for this LoxP-flanked conditional knockout allele was far below the expected number, suggesting embryonic lethality with reduced penetrance. From over 200 animals bred, only 11% were homozygous Sox4(flox/flox) mice, compared to the expected Mendelian ratio of 25% (p0.001). Moreover, there was a significant reduction in the number of female Sox4(flox/flox) mice (26%) relative to male Sox4(flox/flox) mice (p=0.0371). Reduced Sox4 expression in homozygous embryos was confirmed by in-situ hybridization and Quantitative real-time polymerase chain reaction (QPCR).LoxP sites in the 5' and 3' UTR of both alleles of Sox4 resulted in reduced, but variable expression of Sox4 message.

Background Triple-negative breast cancer (TNBC) is an aggressive clinical subtype of breast cancer that is characterized by the lack of estrogen receptor (ER) and progesterone receptor (PR) expression as well as human epidermal growth factor receptor 2 (HER2) overexpression. The TNBC subtype constitutes approximately 10%–20% of all breast cancers, but has no effective molecular targeted therapies. Previous meta-analysis of gene expression profiles of 587 TNBC cases from 21 studies demonstrated high expression of Wnt signaling pathway-associated genes in basal-like 2 and mesenchymal subtypes of TNBC. In this study, we investigated the potential of Wnt pathway inhibitors in effective treatment of TNBC. Methods Activation of Wnt pathway was assessed in four TNBC cell lines (BT-549, MDA-MB-231, HCC-1143 and HCC-1937), and the ER+ cell line MCF-7 using confocal microscopy and Western blot analysis of pathway components. Effectiveness of five different Wnt pathway inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4 and XAV-939) on cell proliferation and apoptosis were tested in vitro. The inhibitory effects of iCRT-3 on canonical Wnt signaling in TNBC was evaluated by quantitative real-time RT-PCR analysis of Axin2 and dual-luciferase reporter assays. The effects of shRNA knockdown of SOX4 in combination with iCRT-3 and/or genistein treatments on cell proliferation, migration and invasion on BT-549 cells were also evaluated. Results Immunofluorescence staining of β-catenin in TNBC cell lines showed both nuclear and cytoplasmic localization, indicating activation of Wnt pathway in TNBC cells. iCRT-3 was the most effective compound for inhibiting proliferation and antagonizing Wnt signaling in TNBC cells. In addition, treatment with iCRT-3 resulted in increased apoptosis in vitro. Knockdown of the Wnt pathway transcription factor, SOX4 in triple negative BT-549 cells resulted in decreased cell proliferation and migration, and combination treatment of iCRT-3 with SOX4 knockdown had a synergistic effect on inhibition of cell proliferation and induction of apoptosis. Conclusions These data suggest that targeting SOX4 and/or the Wnt pathway could have therapeutic benefit for TNBC patients.

Prostate cancer is the most common cancer and the second leading cause of cancer mortality in American men, underscoring the significance of unraveling the molecular mechanisms involved in the initiation and progression of the disease. The sex-determining region Y-box 4 (SOX4) gene is overexpressed in many types of human cancers, including prostate cancer, suggesting that SOX4 plays a fundamental role in tumorigenesis. In this study, we demonstrate that SOX4 is critical for PTEN-mediated prostate cancer progression in vivo. We show that homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibits tumor progression initiated by homozygous loss of the Pten tumor suppressor, demonstrating the key role of SOX4 in the development of prostate cancer. Homozygous deletion of Sox4 also reduces the activation of AKT and β-catenin in Pten-null mice, resulting in inhibition of an invasive cancer phenotype. We also show that SOX4 expression is induced by loss of PTEN, and that PI3K-AKT-mTOR signaling activity is critical for SOX4 expression, suggesting a positive feedback loop between SOX4 protein and PI3K-AKT-mTOR activity. Our findings indicate that SOX4 is a critical component of the PTEN-PI3K-AKT pathway in prostate cancer, suggesting that SOX4 may be a promising molecular target for novel combinatorial therapies for both primary and advanced prostate cancers. Citation Format: Birdal Bilir, Adeboye O. Osunkoya, W. Guy Wiles, Soma Sannigrahi, Veronique Lefebvre, Daniel Metzger, Demetri D. Spyropoulos, W. David Martin, Carlos S. Moreno. SOX4 is essential for PTEN-mediated prostate tumorigenesis in vivo. [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 2023.

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer death among men in the United States, with an estimated 220,800 new cases and 27,540 deaths in 2015. Epidemiological studies have shown that dietary compounds have significant effects on prostate carcinogenesis. Among dietary agents, genistein, the major isoflavone in soybean, is of particular interest because high consumption of soy products has been associated with a low incidence of prostate cancer, suggesting a preventive role of genistein in prostate cancer. In spite of numerous studies to understand the effects of genistein on prostate cancer, the mechanisms of action have not been fully elucidated. Here, we investigated the differences in methylation and gene expression levels of prostate specimens from a clinical trial of genistein supplementation prior to prostatectomy (Lazarevic B, et al. Nutr Cancer 2011). This study was a randomized, placebo-controlled, double-blind clinical trial on forty-seven Norwegian patients who received 30 mg genistein or placebo capsules daily for 3-6 weeks before prostatectomy. Whole genome methylation and expression profiling identified significantly differentially methylated sites and expressed sites between placebo and genistein groups. Differentially regulated genes were involved in developmental processes, stem cell markers, proliferation, and transcriptional regulation. These findings highlight the effects of genistein on global changes in DNA methylation and gene expression in prostate cancer, and provide additional insight into the multiple molecular pathways involved in prostate tumorigenesis. Citation Format: Birdal Bilir, Jeongseok Edward Lee, Nitya V. Sharma, Bato Lazarevic, Aud Svindland, Omer Kucuk, Carlos S. Moreno. Effects of genistein supplementation on genome-wide DNA methylation and gene expression in patients with localized prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A46.

Background Among American men, prostate cancer is the most common, non-cutaneous malignancy that accounted for an estimated 241,000 new cases and 34,000 deaths in 2011. Previous studies have suggested that Wnt pathway inhibitory genes are silenced by CpG hypermethylation, and other studies have suggested that genistein can demethylate hypermethylated DNA. Genistein is a soy isoflavone with diverse effects on cellular proliferation, survival, and gene expression that suggest it could be a potential therapeutic agent for prostate cancer. We undertook the present study to investigate the effects of genistein on the epigenome of prostate cancer cells and to discover novel combination approaches of other compounds with genistein that might be of translational utility. Here, we have investigated the effects of genistein on several prostate cancer cell lines, including the ARCaP-E/ARCaP-M model of the epithelial to mesenchymal transition (EMT), to analyze effects on their epigenetic state. In addition, we investigated the effects of combined treatment of genistein with the histone deacetylase inhibitor vorinostat on survival in prostate cancer cells. Methods Using whole genome expression profiling and whole genome methylation profiling, we have determined the genome-wide differences in genetic and epigenetic responses to genistein in prostate cancer cells before and after undergoing the EMT. Also, cells were treated with genistein, vorinostat, and combination treatment, where cell death and cell proliferation was determined. Results Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 μM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1). In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation. Conclusion Our results suggest that there are a number of pathways that are affected with genistein and vorinostat treatment such as Wnt, TNF, G2/M DNA damage checkpoint, and androgen signaling pathways. In addition, genistein cooperates with vorinostat to induce cell death in prostate cancer cell lines with a greater effect on early stage prostate cancer.

Pelizaeus-Merzbacher disease is an early onset dysmyelinating leukodystrophy. About 80% of PMD cases have been associated with duplications and mutations of the proteolipid protein 1 (PLP1) gene. Pelizaeus-Merzbacher-like disease is a genetically heterogeneous autosomal recessive disease and rarely caused by mutations in gap junction protein α12 (GJA12/GJC2) gene. The molecular basis of the disease was investigated in a cohort of 19 Turkish families. This study identified novel chromosomal rearrangements proximal and distal to, and exclusive of the PLP1 gene, showed equal frequencies of PLP1 and GJA12/GJC2 mutations at least in our cohort, and suggested further genetic heterogeneity.

Early onset hereditary motor and sensory neuropathies are rare disorders encompassing congenital hypomyelinating neuropathy with disease onset in the direct post-natal period and Dejerine-Sottas neuropathy starting in infancy. The clinical spectrum, however, reaches beyond the boundaries of these two historically defined disease entities. De novo dominant mutations in PMP22, MPZ and EGR2 are known to be a typical cause of very early onset hereditary neuropathies. In addition, mutations in several other dominant and recessive genes for Charcot-Marie-Tooth disease may lead to similar phenotypes. To estimate mutation frequencies and to gain detailed insights into the genetic and phenotypic heterogeneity of early onset hereditary neuropathies, we selected a heterogeneous cohort of 77 unrelated patients who presented with symptoms of peripheral neuropathy within the first year of life. The majority of these patients were isolated in their family. We performed systematic mutation screening by means of direct sequencing of the coding regions of 11 genes: MFN2, PMP22, MPZ, EGR2, GDAP1, NEFL, FGD4, MTMR2, PRX, SBF2 and SH3TC2. In addition, screening for the Charcot-Marie-Tooth type 1A duplication on chromosome 17p11.2-12 was performed. In 35 patients (45%), mutations were identified. Mutations in MPZ, PMP22 and EGR2 were found most frequently in patients presenting with early hypotonia and breathing difficulties. The recessive genes FGD4, PRX, MTMR2, SBF2, SH3TC2 and GDAP1 were mutated in patients presenting with early foot deformities and variable delay in motor milestones after an uneventful neonatal period. Several patients displaying congenital foot deformities but an otherwise normal early development carried the Charcot-Marie-Tooth type 1A duplication. This study clearly illustrates the genetic heterogeneity underlying hereditary neuropathies with infantile onset., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Summary Autosomal recessive demyelinating Charcot–Marie– Tooth disease (CMT4), Dejerine–Sottas disease and congenital hypomyelinating neuropathy are variants of hereditary demyelinating neuropathy of infancy, a genetically heterogeneous group of disorders. To explore the spectrum of early-onset demyelinating neuropathies further, we studied the clinicopathological and genetic aspects of 20 patients born to unaffected parents. In 19 families out of 20, consanguinity between the parents or presence of an affected sib suggested autosomal recessive transmission. Screening of various genes known to be involved in CMT4 revealed six mutations of which five are novel. Four of these novel mutations occurred in the homozygous state and include: one in GDAP1, one in MTMR2, one in PRX and one in KIAA1985. One patient was heterozygous for a novel MTMR2 mutation and still another was homozygous for the founder mutation, R148X, in NDRG1. All patients tested negative for mutations in EGR2. Histopathological examination of nerve biopsy specimens showed a severe, chronic demyelinating neuropathy, with onion bulb formation, extensive demyelination of isolated fibres and axon loss. We did not discern a specific pattern of histopathology that could be correlated to mutations in a particular gene.