Naoko Kobayashi

Pten Loss and RAS/MAPK Activation Cooperate to Promote EMT and Metastasis Initiated from Prostate Cancer Stem/Progenitor Cells

PTEN loss or PI3K/AKT signaling pathway activation correlates with human prostate cancer progression and metastasis. However, in preclinical murine models, deletion of Pten alone fails to mimic the significant metastatic burden that frequently accompanies the end stage of human disease. To identify additional pathway alterations that cooperate with PTEN loss in prostate cancer progression, we surveyed human prostate cancer tissue microarrays and found that the RAS/MAPK pathway is significantly elevated in both primary and metastatic lesions. In an attempt to model this event, we crossed conditional activatable K-ras(G12D/WT) mice with the prostate conditional Pten deletion model. Although RAS activation alone cannot initiate prostate cancer development, it significantly accelerated progression caused by PTEN loss, accompanied by epithelial-to-mesenchymal transition (EMT) and macrometastasis with 100% penetrance. A novel stem/progenitor subpopulation with mesenchymal characteristics was isolated from the compound mutant prostates, which was highly metastatic upon orthotopic transplantation. Importantly, inhibition of RAS/MAPK signaling by PD325901, a mitogen-activated protein (MAP)-extracellular signal-regulated (ER) kinase (MEK) inhibitor, significantly reduced the metastatic progression initiated from transplanted stem/progenitor cells. Collectively, our findings indicate that activation of RAS/MAPK signaling serves as a potentiating second hit to alteration of the PTEN/PI3K/AKT axis, and cotargeting both the pathways is highly effective in preventing the development of metastatic prostate cancers.

Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae

The stress-responsive DDR2 gene (previously called DDRA2) of Saccharomyces cerevisiae is transcribed at elevated levels following stress caused by heat shock or DNA damage. Previously, we identified a 51-bp promoter fragment, oligo31/32, which conferred heat shock inducibility on the heterologous CYC1-lacZ reporter gene in S. cerevisiae (N. Kobayashi and K. McEntee, Proc. Natl. Acad. Sci. USA 87:6550-6554, 1990). Using a series of synthetic oligonucleotides, we have identified a pentanucleotide, CCCCT (C4T), as an essential component of this stress response sequence. This element is not a binding site for the well-characterized heat shock transcription factor which recognizes a distinct cis-acting heat shock element in the promoters of many heat shock genes. Here we demonstrate the ability of oligonucleotides containing the C4T sequence to confer heat shock inducibility on the reporter gene and show that the presence of two such elements produces more than additive effects on induction. Gel retardation experiments have been used to demonstrate specific complex formation between C4T-containing fragments and one or more yeast proteins. Formation of these complexes was not competed by fragments containing mutations in the C4T sequence nor by heat shock element-containing competitor DNAs. Fragments containing the C4T element bound to a single 140-kDa polypeptide, distinct from heat shock transcription factors in yeast crude extracts. These experiments identify key cis- and trans-acting components of a novel heat shock stress response pathway in S. cerevisiae.

A class of activation domains interacts directly with TFIIA and stimulates TFIIA-TFIID-promoter complex assembly

TFIIA is a general transcription factor that interacts with the TFIID-promoter complex required for transcription initiation by RNA polymerase II. Two lines of evidence suggest that TFIIA is directly involved in the mechanism by which some activators stimulate transcription. First, binding of TFIIA to a TFIID-promoter complex is a rate-limiting step that is enhanced by transcriptional activators GAL4-AH and Zta. Second, recombinant TFIIA greatly enhances activator-dependent transcription. In this study, we found that the activation domains of Zta and VP16 bind directly to TFIIA. Both Zta and VP16 stimulated rapid assembly of a stable TFIID-TFIIA complex on promoter DNA. Analysis of deletion derivatives of the VP16 activation domain indicated that the ability to bind to TFIIA correlates with the ability to enhance TFIID-TFIIA-promoter ternary complex assembly. Thus, we propose that a class of activators stimulate transcription initiation through direct interactions with both TFIIA and TFIID, which stimulate the assembly of an activated TFIIA-TFIID-promoter complex.

Effect of Altering Dietary ω-6/ω-3 Fatty Acid Ratios on Prostate Cancer Membrane Composition, Cyclooxygenase-2, and Prostaglandin E2

Purpose: To determine whether altering the dietary content of ω-6 (n-6) and ω-3 (n-3) polyunsaturated fatty acids affects the growth of androgen-sensitive prostate cancer xenografts, tumor membrane fatty acid composition, and tumor cyclooxygenase-2 and prostaglandin E2 (PGE2) levels. Experimental Design: Individually caged male severe combined immunodeficiency mice were fed isocaloric 20% kcal fat diets with the fat derived either primarily from n-6 fatty acids (n-6 group) or with the fat consisting of n-6 and n-3 fatty acids in a ratio of 1:1 (n-3 group), and injected s.c. with Los Angeles Prostate Cancer 4 (LAPC-4) cells. Tumor volumes and mouse weights were measured weekly, caloric intake was measured 3 days per week, and tumors and serum were harvested at 8 weeks postinjection. Results: Tumor growth rates, final tumor volumes, and serum prostate-specific antigen levels were reduced in the n-3 group relative to the n-6 group. The n-3 group tumors had decreased proliferation (Ki67 staining) and increased apoptosis (terminal nucleotidyl transferase–mediated nick end labeling staining). In vitro proliferation of LAPC-4 cells in medium containing n-3 group serum was reduced by 22% relative to LAPC-4 cells cultured in medium containing serum from the n-6 group. The n-6/n-3 fatty acid ratios in serum and tumor membranes were lower in the n-3 group relative to the n-6 group. In addition, n-3 group tumors had decreased cyclooxygenase-2 protein and mRNA levels, an 83% reduction in PGE2 levels, and decreased vascular endothelial growth factor expression. Conclusion: These results provide a sound basis for clinical trials evaluating the effect of dietary n-3 fatty acids from fish oil on tumor PGE2 and membrane fatty acid composition, and serum and tumor biomarkers of progression in men with prostate cancer.

Effect of Low-Fat Diet on Development of Prostate Cancer and Akt Phosphorylation in the Hi-Myc Transgenic Mouse Model

This study evaluated the effect of dietary fat on prostate cancer development by using the Hi-Myc mouse transgenic prostate cancer model. Hi-Myc mice develop murine prostatic intraepithelial neoplasia (mPIN) as early as 2 to 4 weeks and invasive adenocarcinoma between 6 and 9 months due to the overexpression of human c-Myc in the mouse prostate. Three-week-old male Hi-Myc mice were placed on high-fat (HF; 42% Kcal) or low-fat (LF; 12% Kcal) diets, and equal caloric intake was maintained until euthanasia at 7 months. The number of mice that developed invasive adenocarcinoma at 7 months was 27% less in the LF diet group (12/28) compared with the HF diet group (23/33, P < 0.05). Epithelial cells in mPIN lesions in the LF group had a significantly lower proliferative index compared with epithelial cells in the HF group (21.7% versus 28.9%, P < 0.05). During the mPIN phase of carcinogenesis (4 months), the LF group had higher serum insulin-like growth factor (IGF) binding protein-1 levels (21.0 +/- 8.9 ng/mL versus 3.2 +/- 0.8 ng/mL, P < 0.05) relative to the HF group. Akt (Ser(473)) phosphorylation, Akt kinase activity, and phosphorylation of downstream targets of Akt in prostates were significantly reduced in the LF diet group compared with the HF group. We conclude that dietary fat reduction delays transition from mPIN to invasive cancer in this Myc-driven transgenic mouse model, possibly through suppression of the IGF-Akt pathway and decreased proliferation of mPIN epithelial cells.

Growth Inhibitory Effect of Low Fat Diet on Prostate Cancer Cells: Results of a Prospective, Randomized Dietary Intervention Trial in Men With Prostate Cancer

PURPOSE A high fat Western diet and sedentary lifestyle may predispose men to prostate cancer through changes in serum hormones and growth factors. We evaluated the effect of a low fat diet on serum factors affecting prostate cancer cell growth by performing a prospective, randomized dietary intervention trial in men with prostate cancer. MATERIALS AND METHODS We randomized 18 men with prostate cancer who did not receive prior therapy to a low fat (15% kcal), high fiber, soy protein supplemented diet or a Western (40% kcal fat) diet for 4 weeks. Fasting serum was collected at baseline and after the intervention to measure prostate specific antigen, sex hormones, insulin, insulin-like growth factor I and II, insulin-like growth factor binding proteins, lipids and fatty acids. LNCaP cells (ATCC(R)) were cultured in medium containing pre-intervention and post-intervention human serum to assess the in vitro effect of the diet on prostate cancer cell proliferation. RESULTS Subjects in each group were highly compliant with the dietary intervention. Serum from men in the low fat group significantly decreased the growth of LNCaP cells relative to Western diet serum (p = 0.03). There were no significant between group changes in serum prostate specific antigen, sex hormones, insulin, insulin-like growth factor I and II, and insulin-like growth factor binding proteins. Serum triglyceride and linoleic acid (omega-6) levels were decreased in the low fat group (p = 0.034 and 0.005, respectively). Correlation analysis revealed that decreased omega-6 and increased omega-3 fatty acid correlated with decreased serum stimulated LNCaP cell growth (r = 0.64, p = 0.004 and r = -0.49, p = 0.04, respectively). CONCLUSIONS In this prospective, randomized dietary intervention trial a low fat diet resulted in changes in serum fatty acid levels that were associated with decreased human LNCaP cancer cell growth. Further prospective trials are indicated to evaluate the potential of low fat diets for prostate cancer prevention and treatment.

Phase II Prospective Randomized Trial of a Low-Fat Diet with Fish Oil Supplementation in Men Undergoing Radical Prostatectomy

Preclinical studies suggest lowering dietary fat and decreasing the ratio of omega-6 to omega-3 polyunsaturated fatty acids decreases the risk of prostate cancer development and progression. We conducted a phase II randomized trial to test the effect of decreasing dietary fat combined with decreasing the dietary omega-6:omega-3 ratio on biomarkers related to prostate cancer development and progression. Patients undergoing radical prostatectomy were randomly assigned to receive a low-fat diet with 5 grams of fish oil daily (dietary omega-6:omega-3 ratio of 2:1) or a control Western diet (omega-6:omega-3 ratio of 15:1) for four to six weeks prior to surgery. The primary endpoint was change in serum insulin-like growth factor I (IGF-1) between arms. Secondary endpoints were serum IGFBP-1, prostate prostaglandin E2 levels, omega-6:omega-3 fatty acid ratios, COX-2, and markers of proliferation and apoptosis. Fifty-five patients were randomized and 48 completed the trial. There was no treatment difference in the primary outcome. Positive secondary outcomes in the low-fat fish oil versus Western group were reduced benign and malignant prostate tissue omega-6:omega-3 ratios, reduced proliferation (Ki-67 index), and reduced proliferation in an ex vivo bioassay when patient sera was applied to prostate cancer cells in vitro. In summary, four to six weeks of a low-fat diet and fish oil capsules to achieve an omega-6:omega-3 fatty acid ratio of 2:1 had no effect on serum IGF-1 levels, though in secondary analyses, the intervention resulted in decreased prostate cancer proliferation and decreased prostate tissue omega-6:omega-3 ratios. These results support further studies evaluating reduction of dietary fat with fish oil supplementation on modulating prostate cancer biology. Cancer Prev Res; 4(12); 2062–71. ©2011 AACR.

DA-complex assembly activity required for VP16C transcriptional activation

ABSTRACT One class of transcriptional activation domains stimulates the concerted binding of TFIIA and TFIID to promoter DNA. To test whether this DA-complex assembly activity contributes significantly to the overall mechanism of activation in vivo, we analyzed mutants of the 38-amino-acid residue VP16C activation subdomain from herpes simplex virus. An excellent correlation was observed between the in vivo activation function of these mutants and their in vitro DA-complex assembly activity. Mutants severely defective for in vivo activation also showed reduced in vitro binding to native TFIIA. No significant correlation between in vivo activation function and in vitro binding to human TATA binding protein, human TFIIB, or Drosophila melanogaster TAFII40 was observed for this set of VP16C mutants. These results argue that the ability of VP16C to increase the rate and extent of DA-complex assembly makes a significant contribution to the overall mechanism of transcriptional activation in vivo.

HDAC inhibition impedes epithelial–mesenchymal plasticity and suppresses metastatic, castration-resistant prostate cancer

PI3K (phosphoinositide 3-kinase)/AKT and RAS/MAPK (mitogen-activated protein kinase) pathway coactivation in the prostate epithelium promotes both epithelial–mesenchymal transition (EMT) and metastatic castration-resistant prostate cancer (mCRPC), which is currently incurable. To study the dynamic regulation of the EMT process, we developed novel genetically defined cellular and in vivo model systems from which epithelial, EMT and mesenchymal-like tumor cells with Pten deletion and Kras activation can be isolated. When cultured individually, each population has the capacity to regenerate all three tumor cell populations, indicative of epithelial–mesenchymal plasticity. Despite harboring the same genetic alterations, mesenchymal-like tumor cells are resistant to PI3K and MAPK pathway inhibitors, suggesting that epigenetic mechanisms may regulate the EMT process, as well as dictate the heterogeneous responses of cancer cells to therapy. Among differentially expressed epigenetic regulators, the chromatin remodeling protein HMGA2 is significantly upregulated in EMT and mesenchymal-like tumors cells, as well as in human mCRPC. Knockdown of HMGA2, or suppressing HMGA2 expression with the histone deacetylase inhibitor LBH589, inhibits epithelial–mesenchymal plasticity and stemness activities in vitro and markedly reduces tumor growth and metastasis in vivo through successful targeting of EMT and mesenchymal-like tumor cells. Importantly, LBH589 treatment in combination with castration prevents mCRPC development and significantly prolongs survival following castration by enhancing p53 and androgen receptor acetylation and in turn sensitizing castration-resistant mesenchymal-like tumor cells to androgen deprivation therapy. Taken together, these findings demonstrate that cellular plasticity is regulated epigenetically, and that mesenchymal-like tumor cell populations in mCRPC that are resistant to conventional and targeted therapies can be effectively treated with the epigenetic inhibitor LBH589.

MP64-16 HYPOFRACTIONATED STEREOTACTIC RADIOTHERAPY INCREASES BOTH TUMOR-INFILTRATING LYMPHOCYTES AND SUPPRESSIVE IMMUNE CELLS IN PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Introduction: We reported functional driver genes and network interaction of race specific and differentially expressed genes by use of bioinformatics and ingenuity pathway analysis among a large population of African American men (AAM) and European American men (EAM) (Powell, I et al 2013, Cancer Epid Bio Prev.). When unraveling the network we identified multiple biological signaling pathways, parts of which reported elsewhere, but when put together, they activate directly or indirectly the androgen receptor and other pathways associated with invasiveness and metastasis. The drivers of these pathways are pro-inflammatory cytokines. We will present these signaling pathways from our interactive network. METHODS: Method: In our initial microarray analysis from formalin fixed radical prostatectomy specimens, we examined 227 genes from 517 genes associated with PCa showing significantly greater expression in PCa from AAM and EAM, and a subset of these genes were identified by bioinformatics and the network from Ingenuity Pathway analysis to be functionally interrelated and driver genes. RESULTS: Results: Pro-Inflammatory cytokines TNF (tumor necrosis factor) IL1B, IL6 and IL8 from our gene profile activated several intermediaries that interact and activate transcription factors in the nucleus, NFkB and TGFB included in our network . These factors bind to the promoter region of the androgen receptor and promote PCa cell proliferation (Zhang 2009,Cao 2015). Inflammatory cytokines TNF and IL1B up-regulate MMP9 (Matrix metalloproteinase 9) in the microenvironment which down regulates cell adhesion molecule E-Cadherin and allows tumors cells to become invasive (Zhang 1998, Canbay, 2015 ) Beta Catenin that is released into the cytoplasm after down regulation of E-Cadherin by MMP9 is translocated to the nucleus by IL8 (WNT pathway) (Tung-Yuan Lai, 2010). TNF and IL1B up regulate CXCR4 which facilitates movement of prostate cancer cells within the prostate and from the prostate to bone on lipid rafts (Han, 2001). These pro-inflammatory cytokines are more expressed among AAM. CONCLUSIONS: Conclusion: These pro-inflammatory cytokines, the same cytokines identified in several other cancers and associated with aggressiveness and disease progression, are associated with PCa aggressiveness, disease progression and castrate resistant prostate cancer. These interactive pathways may become targets for therapy.