Background: Global loss of methylated cytosines in DNA, thought to predispose to chromosomal instability and aneuploidy, has been associated with an increased risk of colorectal neoplasia. Little is known about the relationships between global hypomethylation and lifestyle, demographics, dietary measures, and genetic factors. Methods: Our data were collected as part of a randomized clinical trial testing the efficacy of aspirin and folic acid for the prevention of colorectal adenomas. At a surveillance colonoscopy f3 years after the qualifying exam, we obtained two biopsies of the normal-appearing mucosa from the right colon and two biopsies from the left colon. Specimens were assayed for global hypomethylation using a pyrosequencing assay for LINE-1 (long interspersed nucleotide elements) repeats. Results: The analysis included data from 388 subjects. There was relatively little variability in LINE meth-

Gene-specific promoter methylation of several genes occurs in aging normal tissues and may predispose to tumorigenesis. In the present study, we investigate the association of blood folate levels and dietary and lifestyle factors with CpG island (CGI) methylation in normal colorectal mucosa. Subjects were enrolled in a multicenter chemoprevention trial of aspirin or folic acid for the prevention of large bowel adenomas. We collected 1,000 biopsy specimens from 389 patients, 501 samples from the right colon and 499 from the rectum at the follow-up colonoscopy. We measured DNA methylation of estrogen receptor alpha (ERa) and secreted frizzled related protein-1 (SFRP1), using bisulfite pyrosequencing. We used generalized estimating equations regression analysis to examine the association between methylation and selected variables. For both ERa and SFRP1, percentage methylation was significantly higher in the rectum than in the right colon (P ¼ 0.001). For each 10 years of age, we observed a 1.7% increase in methylation level for ERa and a 2.9% increase for SFRP1 (P < 0.0001). African Americans had a significantly lower level of ERa and SFRP1 methylation than Caucasians and Hispanics. Higher RBC folate levels were associated with higher levels of both ERa (P ¼ 0.03) and SFRP1 methylation (P ¼ 0.01). Our results suggest that CGI methylation in normal colorectal mucosa is related to advancing age, race, rectal location, and RBC folate levels. These data have important implications regarding the safety of supplementary folate administration in healthy adults, given the hypothesis that methylation in normal mucosa may predispose to colorectal neoplasia. Cancer Prev Res; 3(12); 1552-64. Ó2010 AACR.

Keywords: CDK, cyclin, paclitaxel, Smad3, triple negative breast cancer Abbreviations: BCSC, breast cancer stem cells; CDK, cyclin dependent kinase; CDKi, cyclin dependent kinase inhibitor; CK, cytokeratin; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; Pin1, peptidyL-prolyl cis-trans isomerase NIMA-interacting 1; PR, progesterone receptor; TNBC, triple negative breast cancerBreast cancer onset and disease progression have been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFb in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options.

Platelet derived growth factor B (PDGF-B) and its receptor, PDGFR-β, play a critical role in pericyte maturation; however, the mechanisms by which PDGF-B is up-regulated in the tumor microenvironment remain unclear. We previously demonstrated that up-regulating stromal-derived factor, SDF-1α, in vascular endothelial growth factor (VEGF165)-inhibited Ewing’s sarcoma tumors (TC/siVEGF7-1) induced PDGF-B mRNA expression, increased infiltration and differentiation of bone marrow cells (BMCs) into pericytes and, rescued tumor growth. The purpose of this study was to investigate the mechanism by which SDF-1α increased PDGF-B expression and the role of this pathway in BM-derived pericyte differentiation. We demonstrated that SDF-1α induced expression of PDGF-B mRNA and protein both in vitro and in vivo. In contrast, inhibiting SDF-1α down-regulated PDGF-B. We cloned the 2-kb pdgf-b promoter fragment and showed that SDF-1α activates PDGF-B via a transcriptional mechanism. Chromatin immunoprecipitation demonstrated that the ELK-1 transcription factor binds to the pdgf-b promoter in response to SDF-1α. We confirmed the correlation between the SDF-1α/PDGF-B pathway and the differentiation of PDGFR-β+ BMCs into mature pericytes using an in vitro assay. These findings demonstrate that SDF-1α regulates PDGF-B expression and that this regulation plays a critical role in the differentiation of PDGFR-β+ BMCs into mature pericytes.

Bone marrow cells (BMCs) are critical to the expansion of the tumor vessel network that supports Ewing’s sarcoma growth. BMCs migrate to the tumor and differentiate into endothelial cells and pericytes. We recently demonstrated that stromal derived growth factor 1α (SDF-1α) regulates platelet derived growth factor B (PDGF-B) and that this pathway plays a critical role in BM-derived pericyte differentiation in vitro. We investigated the role of SDF-1α/PDGF-B in the tumor microenvironment in vivo in promoting BM-derived pericyte differentiation in Ewing’s tumors. The CXCR4 antagonist, AMD 3100, was used to disrupt the SDF-1α/CXCR4 axis in vivo in two xenograft Ewing’s tumor models. BMCs from green fluorescent protein (GFP+) transgenic mice were transplanted into lethally irradiated nude mice in order to track BMC migration to the tumor site. Following BMC engraftment, tumor-bearing mice received daily subcutaneous injections of either PBS or AMD 3100 for 3 weeks. Tumors were resected, and tumor sections were analyzed by immunohistochemistry. AMD 3100 inhibited BMC differentiation into desmin+ and NG2+ pericytes, affected the morphology of the tumor vasculature, decreased perfusion, and increased tumor cell apoptosis. We observed smaller vessels with tiny lumens and a decrease in the microvessel density. AMD 3100 also inhibited PDGF-B protein expression in vitro and in vivo. SDF-1α in the tumor microenvironment plays a critical role in promoting pericyte formation and Ewing’s sarcoma tumor neovascularization by regulating PDGF-B expression. Interfering with this pathway affects tumor vascular morphology and expansion.

Beta2-glycoprotein 1 (beta2GP1), a 50 kDa serum glycoprotein that binds anionic phospholipid-containing membranes, plays a regulatory role in physiology and pathology. The protein is a member of the short consensus repeat (SCR) superfamily containing four typical repeating domains and an aberrant fifth domain constructed into an SCR-like core at the C-terminus. To investigate the contribution of the individual domains to the binding of beta2GP1, a series of sequential domain-deleted recombinant protein fragments were generated and assessed for their interaction with PS-containing vesicles. Spectral analyses of lipid binding-dependent alterations in tryptophan emission spectra revealed that the (single) tryptophan residues of the individual domains underwent binding-dependent conformational alterations. Depending on the ionic strength, some domains moved from polar to nonpolar environments, while others moved from less polar to more polar environments. Analysis of a series of acrylamide quenching and resonance energy transfer experiments indicated that the binding of N-terminal domain 1 to PS membranes exists in two, ionic strength-dependent, conformations. At low ionic strengths, domain 1 bound to the vesicles and induced their precipitation and/or aggregation. At physiologic ionic strengths, domain 1 detached from the membrane surface while the remaining domains maintained their association with the membrane. Under these conditions, membrane-bound conformationally altered domain 1 projects away from the membrane surface, enabling it to interact with other proteins and/or cell surface ligands or receptors.

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.

Background: Several neoadjuvant trials have been conducted directed at treating triple negative breast cancer (TNBC) patients with platinum agents with pathologic complete response (pCR) rates ranging from 16%-32%. Eribulin mesylate, a nontaxane microtubule dynamics inhibitor with a novel mechanism of action, has clinical activity as monotherapy in breast cancer and other solid tumors. A recent phase I trial found that the combination of eribulin mesylate with carboplatin was well tolerated and showed activity in advanced solid tumors. The recommended dose for future trials was eribulin mesylate 1.1 mg/m2 and carboplatin AUC6. We proposed a neoadjuvant phase II trial with the combination of carboplatin and eribulin in patients with TNBC. CDK inhibition has also recently shown efficacy in the treatment of TNBC in vivo. Thus we proposed correlative studies examining the impact of eribulin/carboplatin in conjunction with CDK inhibition. Methods: 30 patients were enrolled between November 2011 and February 2013. Patients received eribulin at 1.4 mg/m2 (intravenously over 2-5 minutes) on days 1 and 8 followed by carboplatin AUC=6 (intravenously over 30 minutes) on day 1 every 21 days for a total of 4 cycles. Definitive surgery was performed 3-8 weeks after completion of therapy, which concluded the duration of the study. Our primary endpoint was to determine the pCR in TNBC patients treated with the combination of carboplatin and eribulin. Secondary objectives included determination of the clinical response rate, toxicity evaluation, in addition to correlative markers including TLE3, Smad3, cyclins/CDKs, and PIN1. Additionally, we examined the effect of eribulin in combination with a CDK2 inhibitor on proliferation and the expression levels of phosphorylated Smad3 (p-Smad3-179) in vitro using TNBC MDA-MB-231 cells. Results: There was an initial safety run-in to evaluate the appropriate dose of eribulin in the study population. After the 10th patient, the study was temporarily suspended; toxicity was assessed for the first 10 patients (cycle 1 only) to assess whether eribulin at 1.4mg/m2 or a dose reduction to 1.1 mg/m2 would be required for the remaining patients. Of the first 10 patients, only 2 of 10 experienced grade 3 or 4 neutropenia, and 0 of 10 patients experienced grade 3 or 4 peripheral neuropathy. Therefore, the study was continued for the remaining 20 patients with eribulin dosed at 1.4 mg/m2 and carboplatin AUC=6. Thirty of the planned 30 patients have been enrolled to date. Of the 30 patients, 24 have completed therapy and 6 are currently on study. Of the 24 patients who have completed therapy, 11 have achieved a pCR (45.83%). All patients are scheduled to complete therapy by August 2013. Furthermore, the combination of eribulin and carboplatin was well tolerated with a predictable side effect profile. Of the 24 patients who have completed therapy, 8 (33%) required a dose reduction in eribulin for grade 3 or 4 neutropenia and 1 for neutropenic fever. There were no dose reductions for thrombocytopenia thus far. Combination studies with eribulin and CDK2 inhibition showed synergistic effects at concentrations of 3 μM and 1 nM for CDK2 inhibitor and eribulin respectively as tested by an MTS assay. Further, in comparison to the control, both treatments alone and in combination resulted in a decrease in the expression levels of p-Smad3-179 as examined using western blotting. Preliminary results using a transcription factor array showed that Eribulin treatment activates p53/73 and Smad3/4 transcription factors, with examination of additional factors in progress. Conclusion: The combination of carboplatin and eribulin in the neoadjuvant setting appears to be a safe and very promising treatment for TNBC. Also, preliminary in vitro data using the eribulin/CDK2 inhibitor combination suggests a potential therapeutic effect regulated, in part, by downstream TGFβ signaling factors. Statistical analysis re outcomes for the entire study population and correlative study results on pre- and post-treatment tissues are forthcoming. Citation Format: Sara Barnato Giordano, Shreyas Rao, Randala Hamdan, Jacqueline Jeruss, Kevin Bethke, Nora Hansen, Seema Khan, Jamie Von Roenn, Steven Rosen, William J. Gradishar, Kalliopi Siziopikou, Caitlin Meservey, Virginia Kaklamani. Neoadjuvant phase II trial with eribulin and carboplatin in patients with triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A065.