Citations

Abstract Metastatic solid tumors are aggressive and mostly drug resistant, leading to few treatment options and poor prognosis as seen with clear cell renal cell carcinoma (ccRCC) and triple-negative breast cancer (TNBC). Therefore, the identification of new therapeutic regimes for the treatment of metastatic disease is desirable. ccRCC and TNBC cell lines were treated with the HDAC inhibitor romidepsin and the methyltransferase inhibitor decitabine, two epigenetic modifying drugs approved by the U.S. Food and Drug Administration for the treatment of various hematologic malignancies. Cell proliferation analysis, flow cytometry, quantitative PCR, and immunoblotting techniques were used to evaluate the antitumor synergy of this drug combination and identify the reexpression of epigenetically silenced tumor suppressor genes. Combinatorial treatment of metastatic TNBC and stage IV ccRCC cell lines with romidepsin/decitabine leads to synergistic inhibition of cell growth and induction of apoptosis above levels of individual drug treatments alone. Synergistic reexpression of the tumor suppressor gene secreted frizzled-related protein one (sFRP1) was observed in combinatorial drug-treated groups. Silencing sFRP1 (short hairpin RNA) before combinatorial drug treatment showed that sFRP1 mediates the growth inhibitory and apoptotic activity of combined romidepsin/decitabine. Furthermore, addition of recombinant sFRP1 to ccRCC or TNBC cells inhibits cell growth in a dose-dependent manner through the induction of apoptosis, identifying that epigenetic silencing of sFRP1 contributes to renal and breast cancer cell survival. Combinatorial treatment with romidepsin and decitabine in drug resistant tumors is a promising treatment strategy. Moreover, recombinant sFRP1 may be a novel therapeutic strategy for cancers with suppressed sFRP1 expression. Mol Cancer Ther; 11(10); 2105–15. ©2012 AACR.

Abstract The terminology for human papillomavirus (HPV)–associated squamous lesions of the lower anogenital tract has a long history marked by disparate diagnostic terms derived from multiple specialties. It often does not reflect current knowledge of HPV biology and pathogenesis. A consensus process was convened to recommend terminology unified across lower anogenital sites. The goal was to create a histopathologic nomenclature system that reflects current knowledge of HPV biology, optimally uses available biomarkers, and facilitates clear communication across different medical specialties. The Lower Anogenital Squamous Terminology (LAST) Project was cosponsored by the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology and included 5 working groups; 3 work groups performed comprehensive literature reviews and developed draft recommendations. Another work group provided the historical background and the fifth will continue to foster implementation of the LAST recommendations. After an open comment period, the draft recommendations were presented at a consensus conference attended by LAST work group members, advisors, and representatives from 35 stakeholder organizations including professional societies and government agencies. Recommendations were finalized and voted on at the consensus meeting. The final, approved recommendations standardize biologically relevant histopathologic terminology for HPV-associated squamous intraepithelial lesions and superficially invasive squamous carcinomas across all lower anogenital tract sites and detail the appropriate use of specific biomarkers to clarify histologic interpretations and enhance diagnostic accuracy. A plan for disseminating and monitoring recommendation implementation in the practicing community was also developed. The implemented recommendations will facilitate communication between pathologists and their clinical colleagues and improve accuracy of histologic diagnosis with the ultimate goal of providing optimal patient care.

Abstract The Forkhead transcription factor, FoxO3a, is a known suppressor of primary tumor growth and is negatively regulated by growth factors via phosphorylation and mislocalization from the nucleus to the cytoplasm. Here we show for the first time that FoxO3a is nuclear in anaplastic thyroid carcinoma (ATC) and it transcriptionally upregulates cyclin A thereby promoting tumor growth in human ATC cells. Silencing FoxO3a in ATC cell lines leads to down-regulation of cyclin A mRNA and protein while over-expression of FoxO3a leads to upregulation of cyclin A protein. Transient co-transfection of cyclin A promoter linked to luciferase with FoxO3a leads to enhanced luciferase activity, which can be ablated when mutating the FoxO3a site on the cyclin A promoter. This combined data implicates an entirely novel function for FoxO3a in modulating cancer progression by promoting cell cycle progression and tumor growth. ATC is a poorly differentiated carcinoma with average survival upon diagnosis of 3–4 months with 99% lethality, notably the most aggressive and deadliest cancers known. This data also suggest that any therapies under development to inactivate FOXO3a may be effective at blocking tumor expansion. 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 1038. doi:10.1158/1538-7445.AM2011-1038

Abstract Renal cell carcinoma (RCC) accounts for 3% of all malignancies reported each year making it the sixth leading cause of cancer death in the United States. Within this year more than 50,000 individuals will be diagnosed with RCC and around 12,000 will die from this disease. Out of these RCCs the most frequently diagnosed form is clear cell renal cell carcinoma (ccRCC) which accounts for approximately 80% of all RCCs. We have previously identified through genomic profiling that loss of the type II and III TGFβ receptors in the progression of this disease lead to loss of TGFβ responsiveness and suggests a role for dysregulated TGFβ signaling in RCC progression and metastasis. Restoration of the TGFβ receptor type III (TβRIII) leads to apoptosis in RCC cell lines (Margulis et al. 2007) suggesting that re-expression of TβRIII is an attractive method for intervention of ccRCC. Recently we identified that loss of TβRIII is an early event in ccRCC and that this loss is not through epigenetic silencing but due to the loss of transcriptional regulation. The transcription factor GATA3 was identified to be a positive regulator of TβRIII expression through its actions on the proximal promoter of TβRIII. Through deletion analysis we have now identified a region within the proximal promoter that contains a number of putative transcription factor binding sites that are capable of negatively regulating the expression of TβRIII. We have identified that one of these transcription factors is over-expressed in ccRCC patient matched renal tissue samples and that silencing of this transcription factor through lentiviral shRNA, leads to a three fold induction of endogenous TβRIII in ccRCC cell lines. Furthermore, loss of this transcription factor in ccRCC cell lines leads to decreased cell proliferation when compared to non-target infected controls. Further evaluation of these putative negatively regulating transcription factors and their actions upon the proximal promoter of TβRIII are being analyzed utilizing EMSA and ChIP assays. These investigations will identify novel molecular targets that through their dysregulation will have the potential to up-regulate TβRIII in ccRCC inducing apoptosis within these cells. Together these findings will allow for a deeper molecular understanding of the events taking place in ccRCC. 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 2173. doi:10.1158/1538-7445.AM2011-2173

Abstract Bladder cancer is the 5th most commonly diagnosed cancer in the United States. Because superficial bladder cancer (SBC) recurs in about 60% of cases, standard treatment involves tumor resection followed by intravesical therapies to prevent recurrence, as well as lifelong periodic cystoscopy. There is an urgent need for better intravesical therapies to prevent both the recurrence and progression of SBC. Recent evidence has demonstrated that glycogen synthase kinase – 3 beta (GSK3β) plays an important role in pancreatic, ovarian and colon cancers; however, to date the possible role of this important kinase has not been examined in bladder cancer. We propose that GSK3β plays a cancer promoting role in bladder cancer and that inhibition of GSK3β will reduce bladder cancer growth. The GSK3β inhibitor, AR-A014418, has been demonstrated to reduce tumor growth in the previously cited cancers. AR-A014418 reduced cell proliferation of three bladder cancer cell lines (HT1376, T24 and UM-UC-3) with EC50 values of 17.5μM for HT1376, 8μM for T24, and 10μM for UM-UC-3 cells. The effect of AR-A014418 on downstream targets in these cell lines was evaluated by Western blots which indicated an increase in apoptosis in these cells in response to drug treatment as demonstrated by the presence by both PARP and caspase 3 cleavage. The NFkB downstream target XIAP also exhibited reduced protein levels after drug treatment. The HT1376 cell line was transduced with luciferase containing lentivirus particles to generate a luciferase expressing cell line, HT1376-LUC to use for both ectopic and orthotopic models of bladder cancer growth. The efficacy of AR-A014418 in reducing bladder cancer tumor growth was tested in an ectopic mouse model. HT1376-LUC cells mixed with matrigel were transplanted into the right flank of 50 female nude mice. After one week, mice containing tumors with an average volume of 52 mm3 were randomized into four treatment groups of 10 mice each. Mice were treated twice weekly via IP injection with placebo, 1, 5 or 7mg/kg AR-A014418. Tumor sizes were measured and imaged for luciferase activity once weekly. Tumor growth was inhibited by both 5mg/kg and 7mg/kg doses by 19 days of treatment (p < 0.05, Student's T test). Together, the in vitro and in vivo data suggest that GSK3β is a novel target for bladder cancer treatment. Future studies using an orthotopic model of bladder cancer are planned. 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 1671. doi:10.1158/1538-7445.AM2011-1671

Regulation of cytosolic Ca2+ concentration ([Ca2+]cyt) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca2+]cyt, helping prevent Ca2+ overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca2+ buffering by mitochondria and mitochondrial Ca2+ transport mechanisms in the regulation of [Ca2+]cyt in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca2+]cyt and mitochondrial Ca2+ concentration ([Ca2+]mito) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca2+]cyt and [Ca2+]mito, with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na+/Ca2+ exchanger (1 μM CGP-37157) increased [Ca2+]mito responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca2+]mito responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca2+]cyt. TNF-α and IL-13 both increased [Ca2+]cyt, which was associated with decreased [Ca2+]mito in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca2+]cyt and [Ca2+]mito were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca2+]cyt after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca2+]mito in different parts of ASM cells may serve to locally regulate Ca2+ fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca2+]cyt is increased.

RATIONALE Sphingolipids are important components of cell membranes that serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. De novo ceramide biosynthesis depends on fatty acid availability, but whether muscle uses circulating free fatty acids or pre‐existing intracellular stores is unknown. Our goal was to develop a method to detect the incorporation of intravenously infused [U‐ 13 C]palmitate into intramyocellular ceramides. METHODS We used liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipid species and 13 C‐isotopic enrichment of 16:0‐ceramide. Chromatographic separation was performed using ultra‐performance liquid chromatography. The analysis was performed on a triple quadrupole mass spectrometer using a positive ion electrospray ionization source with selected reaction monitoring (SRM). RESULTS The sphingolipids ions, except enriched ceramide, were monitored as [M+2+H] + . The [ 13 C 16 ]16:0‐ceramide was monitored as [M+16+H] + . By monitoring two different transitions of the [ 13 C 16 ]16:0‐ceramide (554/536 and 554/264) we could indirectly measure enrichment of the palmitate that is not a part of the sphingoid base. Concentration and enrichment could be measured using 20 mg of muscle obtained from volunteers receiving a low dose [U‐ 13 C]palmitate infusion. CONCLUSIONS LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo . Copyright © 2012 John Wiley & Sons, Ltd.

Abstract The 19p13.1 breast cancer susceptibility locus is a modifier of breast cancer risk in BRCA1 mutation carriers and is also associated with the risk of ovarian cancer. Here, we investigated 19p13.1 variation and risk of breast cancer subtypes, defined by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) status, using 48,869 breast cancer cases and 49,787 controls from the Breast Cancer Association Consortium (BCAC). Variants from 19p13.1 were not associated with breast cancer overall or with ER-positive breast cancer but were significantly associated with ER-negative breast cancer risk [rs8170 OR, 1.10; 95% confidence interval (CI), 1.05–1.15; P = 3.49 × 10−5] and triple-negative (ER-, PR-, and HER2-negative) breast cancer (rs8170: OR, 1.22; 95% CI, 1.13–1.31; P = 2.22 × 10−7). However, rs8170 was no longer associated with ER-negative breast cancer risk when triple-negative cases were excluded (OR, 0.98; 95% CI, 0.89–1.07; P = 0.62). In addition, a combined analysis of triple-negative cases from BCAC and the Triple Negative Breast Cancer Consortium (TNBCC; N = 3,566) identified a genome-wide significant association between rs8170 and triple-negative breast cancer risk (OR, 1.25; 95% CI, 1.18–1.33; P = 3.31 × 10−13]. Thus, 19p13.1 is the first triple-negative–specific breast cancer risk locus and the first locus specific to a histologic subtype defined by ER, PR, and HER2 to be identified. These findings provide convincing evidence that genetic susceptibility to breast cancer varies by tumor subtype and that triple-negative tumors and other subtypes likely arise through distinct etiologic pathways. Cancer Res; 72(7); 1795–803. ©2012 AACR.