bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3778: Protein targets for prostate cancer prevention by α-keto acid metabolites of organoselenium compounds
Authors: Sinha Raghu , Sinha Indu , Pinto John
Published: February 2011

Our previous studies show that human glutamine transaminase K (GTK) and L-amino acid oxidase convert Se-methylselenocysteine (MSC) and selenomethionine (SM) to methylselenopyruvate (MSP) and α-keto-γ-methylselenobutyrate (KMSB), respectively. Further more, both of these α-seleno keto acids inhibit histone deacetylase (HDAC) activity in cultured human prostate cancer cells in a dose-dependent manner (Cancer Prev. Res. 2:683, 2009). Since human prostate cancer cells have GTK activity and can metabolize MSC to MSP but not SM to KMSB, we studied whether administration of SM and KMSB to human prostate cancer cells each function as direct-acting metabolites of organoselenium compounds and individually can result in differential regulation of protein targets critical for growth regulation. Using a 4-plex-iTRAQ proteomic approach, we examined the potential effects of either SM or KMSB on several protein targets in LNCaP prostate cancer cells. Analyses revealed significant changes (p<0.05) in at least 68 proteins (1-60 peptide residue coverage) following 5 hr incubation of LNCaP cells with SM (200 μM), KMSB (50 μM) and Trichostatin A (TSA) (20 μM), an inhibitor of class I and II mammalian HDACs. The majority of observed protein changes were cytosolic (49%), nuclear (31%), then plasma membrane (4%) and the remainder was extracellular or unknown. Six proteins that included histone 1c, histone 2a, PARP1, and Epoxide hydrolase 1 microsomal (xenobiotic) were up regulated and two proteins, namely, ribosomal protein SA and hematological and neurological expressed-1 protein were down regulated in both KMSB and TSA-treated cells. A total of six proteins (including annexin VI and splicing factor 3a) were significantly altered by KMSB treatment alone while fifteen proteins (among which HSP90, 14-3-3ε, phosphoglycerate kinase 1, and eukaryotic translation initiation factor 4γ) were significantly changed by SM treatment alone. Collectively, our data reveal that several novel protein targets for prostate cancer prevention may be selective for either KMSB or SM alone. Several of these recognized targets will be investigated further to confirm the selectivity of seleno amino acids or seleno keto acids to function as direct-acting metabolites of naturally-occurring organoselenium compounds that can lead to de-repression of silenced tumor suppressor proteins and/or regulation of genes and signal factors.

The study was supported by NCI R01CA111842.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3778.
Abstract 3784: Caloric restriction inhibits 1, 2-dimethylhydrazine-induced aberrant crypt foci formation and induces differential expression of sirtuins in colonic mucosa of F344 rats
Authors: Tomita Mariko , Kanemoto Sayuri , Nakano Mayumi , Matsui Yuri , Morioka Takamitsu , Cui Changxu , Yoshimi Naoki
Published: February 2011

Caloric restriction (CR), a 20-40% lowering of caloric intake, is associated with longevity, and decreased incidences of age-related diseases including cancer. The sirtuins are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent enzymes and have been implicated as a key mediator for the beneficial effects of CR on aging and longevity. However, the underlying mechanisms by which CR decreases cancer risk have not yet been elucidated. The present study was conducted to determine whether CR would modify the growth of preneoplastic colonic aberrant crypt foci (ACF), and whether sirtuin expression levels would be correlated with ACF formation. Five-week-old male F344 rats were randomly divided into four experimental groups. Groups 1 and 2 were given subcutaneous injection of 1, 2-dimethylhydrazine (DMH, 40 mg/Kg body weight), once a week for 2 weeks to induce ACF. Rats in groups 2 and 4 were fed a CR diet (60% of the ad libitum intake), and those in groups 1 and 3 were fed CE-2 diet (CLEA, Tokyo, Japan) ad libitum. All animals were sacrificed at 4 weeks after the first DMH injection. Rat colons were removed and fixed in 10% formalin. ACF were counted after 2% methylene blue staining. The expressions of sirt1 and sirt4 in the colonic mucosa were evaluated by RT-PCR. The number of ACF in colonic mucosa was significantly decreased in DMH-treated rats with CR (group 2) as compared to in DMH-treated rats without CR (group 1). No ACF was found in DMH-untreated animals with or without CR (groups 3 and 4). The expression levels of sirt1 and sirt4 in colonic mucosa were increased by CR (group 1 vs. group 2 and group 3 vs. group 4). Interestingly, DMH treatment also increased the expression of sirt1 and sirt4 (group 1 vs. group 3), and DMH-treated rats with CR (group 2) showed the highest expression levels of these genes. These results suggest that sirt1 and sirt4 induced by CR might inhibit colonic mucosa from formation and development of ACF. Our observations warranted further investigation of association between expression of sirtuins and prevention of colon carcinogenesis. We are currently analyzing the expression of other sirtuins and will compare the expression of all sirtuin family and ACF formation in colonic mucosa of the rats with or without CR.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3784.
Abstract 3798: Epigallocatechin-3-gallate and atorvastatin induce translocation of eGFP-LC3 in immortalized baby mouse kidney cells suggesting of a link between autophagy and apoptosis
Authors: Yang Zhihong , Huang Bridget , Strohecker Anne M. , White Eileen , Yang Chung S.
Published: February 2011

Epigallocatechin-3-gallate (EGCG), the major polyphenolic compound in green tea, and atorvastatin (ATST), a commonly used cholesterol-lowing medication, have been reported to have cancer preventive activities and to induce cancer cell apoptosis. However, the mechanisms of these activities are still unclear. Here we report that EGCG and ATST could induce cell autophagy, which may be linked to the induction of cell apoptosis. In immortalized baby mouse kidney (iBMK) cells that overexpress eGFP-tagged LC3, EGCG at 3, 6 and 12 μM exhibited a dose-dependent induction of the translocation of eGFP-LC3, an indicator of the formation of autophagosomes, as observed by fluorescent microscopy. ATST at 2 and 4 μM also induced the translocation of eGFP-LC3. Western-blot analysis showed that 25 μM EGCG and 4 μM ATST treatment for 24 h induced the appearance of LC3-II, a higher migrating band, indicating the occurrence of autophagy. Beclin +/− iBMK cells also displayed autophagy upon treatment with 25 μM EGCG for 24h, but to a less extent as compared to Beclin +/+ cells. The induction of the appearance of eGFP-LC3 puncta could not be eliminated by addition of superoxide dismutase plus catalase, enzymes that were used to inhibit the auto-oxidation of EGCG and the production of reactive oxygen species. The results suggest that the induction of autophagy is not caused by EGCG auto-oxidation produced oxidative stress. ATST inhibited the proliferation of iBMK cells in 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, but the inhibition was to a less extent in Beclin +/− cells as compared to Beclin +/+ cells. EGCG and ATST treatment resulted in an increase of cleaved caspase 3, a hallmark of cell apoptosis, and a decrease of anti-apoptotic Bcl-2. Beclin +/+ iBMK cells showed stronger and earlier induction of these apoptotic markers than Beclin +/− cells, indicating that induction of apoptotic marker is correlated to the ability of cells to produce autophagy. EGCG at 25 μM and ATST at 2 μM also induced the appearance of endogenous LC3-II in human colon cancer HCT116 cells. The induction of autophagy may be a key mechanism for EGCG and ATST to inhibit cancer growth and formation (This research is supported by NIH grants CA 120915 and CA 133021).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3798.
Abstract 380: Sunitinib treatment induces up-regulation of CXCR4
Authors: Kubacka Urszula , Nagengast Wouter B. , Kruizinga Roeliene C. , Lub-deHooge Marjolijn N. , Gietema Jourik A. , Oosting Sjoukje F. , Timmer-Boscha Hetty , Hospers Geke A. , den Dunnen Wilfred F. , de Vries Elisabeth G. , Huls Gerwin , Walenkamp Annemiek M.
Published: February 2011

Background: Chemokine receptors expressed on solid tumor cells and their corresponding ligands are an example of a network which is used by these cells to interact with their microenvironment to sustain survival, proliferation, invasive growth, angiogenesis, and to promote organ-specific localization of distant metastases. Indeed, CXCR4/CXCL12 signaling can induce angiogenesis and progression of tumors, for example by increasing the expression of vascular endothelial growth factor (VEGF). Inversely, it is hypothesized that anti-angiogenic treatment increases the expression of CXCR4/CXCL12 which could explain the in preclinical models observed invasive growth characteristics of tumors.

Methods: A human A2780 ovarian xenograft mouse model was used. When the tumor was established, mice were treated with vehicle or sunitinib (60 mg/kg i.p.) daily for 2 weeks. CXCR4 expression was assessed by immunohistochemistry at baseline and after 1 and 2 weeks of treatment and compared with markers like HIF1α, VEGF, Ki67, VEGFR2, mean vessel density (MVD) and blood vessel diameter. In addition human plasma VEGF levels were analyzed.

Results: Moderate cell membrane CXCR4 expression was seen in untreated tumors. One week of sunitinib treatment resulted in decrease angiogenice markers and tumor proliferation (Ki67) in both the tumor center and rim. Two weeks of sunitinib treatment resulted in up regulation of CXCR4 with a 2.2 fold increase (P = 0.046) particularly in the tumor rim. This coincided with normalization of blood vessel diameters, intense VEGF staining and increased tumor proliferation (Ki67) to baseline levels in the tumor rim. In contrast, an increase of HIF1α (P = 0.017) and decrease of angiogenic markers like VEGFR2 (P = 0.019) and MVD (P = 0.016) was still observed in the tumor center.

Conclusion: This study shows that anti-angiogenic treatment with sunitinib up regulates CXCR4 expression, mainly in the rim of the tumor. CXCR4 is a potential target to block anti-angiogenic induced invasive growth/metastasis.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 380.
Abstract 3802: PPAR gamma mediated induction of transglutaminase in aerodigestive preneoplasia in vitro
Authors: Weidman Elizabeth K. , Wuertz Beverly R. , Gaffney Patrick M. , Ondrey Frank G.
Published: February 2011

Currently there are no validated biomarkers or therapies for human aerodigestive preneoplasia. There is increasing interest in the utilization of PPAR gamma mediated therapy for oral leukoplakia reversal based on preliminary clinical trial results. Transglutaminases (TGMs) are associated with squamous differentiation, and therefore might be hypothesized to be upregulated with PPAR gamma differentiation therapies. In the present study we sought to examine the feasibility of utilizing transglutaminases as a downstream readout for PPAR gamma activator treatment of oral leukoplakia. First we examined our Affymetrix database of human head and neck tumors for the presence or absence of transglutaminases 1-3 in 41 tumors compared to 13 normal oral mucosa samples. We discovered that TGM 1 and 3 were significantly upregulated in normal tissues compared to tumors (p<0.0001). Next, we examined whether PPAR gamma activators could activate TGM1 reporter genes in cell lines. We found that TGM1 luciferase reporters were significantly activated by ciglitazone (0-5uM) and pioglitazone (0-10 uM) in MSK leuk and HOK oral preneoplastic cell lines, as well as CA-9-22 oral cancer cells in several experiments. Next we examined pioglitazone activation of transglutaminases in these cell lines by real time PCR. We found that pioglitazone significantly activated TGM 1 and 2 in MSK leuk and CA-9-22 oral cancer cells on qRT-PCR. We conclude that the clinically relevant PPAR gamma activator, pioglitazone, can activate transglutaminases in aerodigetsive cancer cell lines and may be useful in upregulating differentiation in oral preneoplasia in clinical trials. We also conclude that transglutaminases may provide a family of proteins for PPAR gamma effects and should be included as exploratory biomarkers in leukoplakia clinical trials with PPAR gamma activators.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3802.
Abstract 3805: Furanodienone downregulates EGFR and Her-2 signaling in BT-474 breast cancer cells
Authors: Li Yingwei , Zhu Guoyuan , Dong Suisui , Shen Xiaoling , Yu Zhiling , Fong Wang-fun David
Published: February 2011

Epidermal growth factor receptor (EGFR) and Her-2 are members of the ErbB receptor family of type 1 tyrosine kinases. Also, ligand-induced EGFR /Her-2 heterodimerization triggers potent proliferative and survival signals. EGFR and Her2 frequently overexpress in breast cancer and their overexpression is correlated with a poor prognosis. Therefore dual EGFR/ Her-2 inhibition is an attractive therapeutic strategy for breast cancer treatment. Furanodienone is a compound isolated from Rhizoma Curcumae, which is commonly used in cancer treatment in China. In this study, we used the breast cancer cell BT-474 as the model to investigate the mechanism of furanodienone in breast cancer treatment. We found that furanodienone induced G1 arrest and effectively blocked EGF-stimulated cell cycle progression in BT-474 cells. Moreover, furanodienone decreased protein levels of EGFR, phosphorylation of EGFR and Her-2, so that it prevented the activation of downstream molecules like Akt, Gsk3β and c-Raf, which resulted in inhibiting cell cycle progression. These findings suggested that furanodienone induced growth inhibition in BT-474 human breast cancer cells through inhibiting EGFR /Her-2 signaling pathway. Furthermore, furanodienone and its related compounds provided insights into novel approaches for the prevention and treatment of human breast cancers, especially for those with EGFR /Her-2 overexpression.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3805.
Abstract 3814: Association of Toll-like receptor-4 (TLR4) expression with stage and grade in endometrial cancer
Authors: Papez Michael , Ko Emily , Kaufman David , Gehrig Paola , Young Steven , Bae-Jump Victoria L.
Published: February 2011

Objectives: Toll-like receptors (TLRs) are known to initiate immune responses upon binding bacterial glycolipids and have been implicated in the pathogenesis of a variety of human diseases including cancer. It is thought that cancers may sabotage TLR signaling pathways leading to enhanced tumor cell invasion and immune surveillance evasion. Upon glycolipid binding, TLR4 induces the production of proinflammatory cytokines through the adaptor protein, MyD88. Although TLR4 expression has been evaluated in normal endometrium, it has not been well-characterized in endometrial cancer. Thus, we sought to assess TLR4 and MyD88 expression in endometrial cancer specimens.

Methods: TLR4 and MyD88 expression was evaluated by immunohistochemistry in 82 formalin-fixed, paraffin-embedded endometrial cancer specimens and 14 specimens of benign endometrium. Tissue microarrays were constructed, and two cores were analyzed for each patient. Immunohistochemical staining was scored according to the intensity (1+ to 3+) and qualitative percent cells staining per core. A “histoscore” was calculated, based on multiplying these two determinants ranging from 0 to 300 (i.e. intensity x % labeled). Results were analyzed using the Student t-test and ANOVA.

Results: TLR4 and MyD88 staining was localized in the cytoplasm of both the benign and malignant endometrium. TLR4 and MyD88 expression was highest in the atrophic endometrium (mean 276 & 251, respectively) and lowest in the secretory endometrium (mean 182 & 136, respectively) (p=0.0182 for TLR4; p=0.0043 for MyD88). Expression was intermediary in the proliferative endometrium (mean 214 for TLR4; p=0.0278) (mean 210 for MyD88; p=0.0520). Of the endometrial cancer specimens, 87% were of endometrioid histology, and 13% were of serous histology. 77% of these tumors were from women with early stage cancers (stage 1/2), and 24% were from patients with advanced stage disease (stage 3/4). 89% of the endometrial cancers expressed TLR4, and 91% expressed MyD88. Decreased TLR4 expression correlated with higher stage (mean 205 for stage 1/2 and 169 for stage 3/4; p=0.0163) and grade (mean 214 for G1 and 176 for G3; p=0.0165). TLR4 and MyD88 expression did not significantly differ between endometrioid and serous histologies.

Conclusions: TLR4 and MyD88 are expressed in endometrial cancers, and decreased TLR4 expression was associated with advanced stage and higher grade. Thus, the TLR4 pathway may represent a novel therapeutic target in endometrial cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3814.
Abstract 3819: Activation of toll-like receptor 5 on breast cancer cells by flagellin suppresses tumor development and growth
Authors: Zhenyu Cai , Shi Zhongcheng , Amir Sanchez , Zhang Dekai , McKeehan Wallace L
Published: February 2011

Toll-like receptors (TLRs) play key roles in both the innate and adaptive immune systems, particularly in inflammatory responses against pathogen infection. Recent evidence showed that functional TLRs are also expressed on a wide variety of tumor cells; however, their activation exhibits either antitumor or pro-tumor effects. In order to investigate the function of TLR signaling pathways in breast cancers, we analyzed the expression of TLRs and their functional status in breast cancer cells by RT-PCR and NF-κB reporter gene assays in response to synthetic ligands for these receptors. We found that only TLR5 signaling is highly responsive in breast cancer cells. The phosphorylation levels of IκB, ERK, and JNK after flagellin (an agonist of TLR5) stimulation were increased in tumor cells. Activation of TLR5 signaling in tumor cells by flagellin induces the secretion of various cytokines and chemokines including interleukin-6, interleukin-8, and TNF-α. Moreover, flagellin/TLR5 signaling in MCF-7 cells inhibits an anchorage-independent growth, a hallmark of tumorigenic transformation. In addition, the secretion of soluble factors induced by flagellin contributed to the growth-inhibitive activity in an autocrine fashion. The inhibitive activity was further confirmed in mouse xenografts of human breast cancer model. Administration of flagellin inhibits tumor growth and therefore prolongs the survival of tumor-bearing mice. These findings indicate that TLR5 activation by flagellin mediates innate immunity to elicit potent antitumor activity in breast cancers, which may serve as a novel therapeutic target for human breast cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3819.
Abstract 3820: Inhibition of M1 macrophages may aid the conversion process to M2, which may aid tumor survival and growth
Authors: Enz Ryley A. , Knechtel Charles , Enz Amanda Dee , Swanson Bryan , Robison Richard , O'Neill Kim
Published: February 2011

Macrophages perform a variety of functions, from phagocytosis to the repair and remodeling of damaged tissues. Two phenotypes have been proposed: M1 and M2. The M1 macrophage performs phagocytosis of tumor cells and releases pro-inflammatory cytokines. The M2 macrophage acts as a support cell by promoting the healing of damaged cells by angiogenesis and tissue remodeling. The transition from M1 to M2 may be facilitated by tumor environments. To further study the relationship between cancer and macrophages we developed a live-cell migration assay, which measures cancer's ability to recruit macrophages. We hypothesize that cancer cell lines have varied abilities to recruit macrophages and convert them into M2, and that this ability corresponds to the aggressiveness of the cell line. Macrophages were derived from the human mononuclear phagocyte cell line U937, after addition of phorbol 12-myristate 13-acetate (PMA). These U937 cells were incubated with three adenocarcinomas of varying aggressivity. The adenocarcinomas were MDA-MB-435, MDA-MB-231, and MCF7. The U937 cells were separated from the three cell lines by 3.0 µm trans-permeable polycarbonate membranes (Corning Inc.) which allowed the U937 cells to migrate to the cell lines. After 1 hour non-migratory U937 cells were isolated and counted by means of a hemocytometer. The control without cancer caused 73.86% of U937 cells to remain, MDA-MB-435 69.28% (N=24, P=0.29), MCF7 61.09% (N=24, P<0.01), and MDA-MB-231 55.83% (N=24, P<0.01). This data was then compared with expression levels of macrophage Migration Inhibition Factor (MIF) analyzed from Real Time PCR data. These samples consisted of monocytes, from human blood, which were co-incubated with the cell lines for 48 hours. The cells were separated by 0.4 µm trans-permeable polycarbonate membranes (Corning Inc.) to prevent cells from coming into contact and allowing signal exchange. The RT-PCR data correlated with the migration data. The amount of MIF increased 7.56 fold, (N=9) in MDA-MB-435 when compared to controls, 3.13 fold for MCF7 (N=9), and 1.33 fold for MDA- MB-231 (N=9). This data suggests that the more aggressive the cancer the more MIF emitted and the greater the potential to inhibit macrophages. Previous studies have shown Interleukin-10, (IL-10), IL-6, IL-4, and prostaglandin F2 (PGF2) to be released from tumors and inhibit the cytotoxic activity of macrophages. This exposure to IL-10 has been suggested to convert M1 macrophages to a M2 phenotype. The migration inhibition observed from this study might be the initial step in the conversion process from M1 to M2.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3820.
Abstract 3824: Induction of potent antitumor immunity by intratumoral injection of mT-bet transduced dendritic cells
Authors: Qu Yanyan , Lipscomb Michael W. , Taylor Jennifer L. , Storkus Walter J.
Published: February 2011

T-bet is a T-box transcription factor (Tbx21) previously identified as a master regulator of Type-1 polarization in T cells. We have previously demonstrated that ectopic expression of hT-bet converts dendritic cells (DC) into Type 1-biasing APCs. In the current study, we investigated whether mouse DC overexpressing mT-bet promote superior Type 1 anti-tumor T cell responses when applied as an intratumoral therapy. CD11c+ DCs were then purified and transduced with recombinant adenovirus to express high levels of mT-bet. DC. mT-bet stimulated stronger Tc1 and Th1 T cell responses when compared with control DC and retained the capacity to kill and then uptake tumor cells/debris, allowing for the cross-priming of specific T cell responses in vitro. Surprisingly, in contrast to human DC. T-bet, CD40L-activated DC. mT-bet produced elevated levels (vs. control DC) of IL-12p70/TNF-α. DC. mT-bet exerted potent anti-tumor activity in the (day 7 established) s.c. CMS4 sarcoma after intratumoral administration. These anti-tumor effects were mediated primarily by Tc1 cells, however, both CD4+ T cells and NK cells appeared to also be involved in therapeutic response. In situ imaging suggested that DC. mT-bet treatment inhibited the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells and tumor-associated vascular structures. Collectively, these findings suggest that DC. mT-bet-based therapy has the potential to promote superior Type-1 anti-tumor immunity in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3824.
Abstract 3828: Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate
Authors: Lee Ji Hee , Chung Ik-Joo , Umeyama Akemi , Lee Je-Jung , Takei Masao , Jung Young Do , Bae Woo Kyun
Published: February 2011

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DCs are used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferons (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to URC alone, or in combination with TNF-α, IFN-α and IFN-γ. All three cytokines induced phenotypically mature DC. The expression levels of CD38 and CCR7 on URC-primed DC were enhanced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. DC matured with URC plus IFN-γ produce high levels of IL-12p70 upon CD40 triggering. IFN-γ secretion from naïve T cells co-cultured with URC-primed DC also augmented by IFN-γ. In addition, DC maturated with URC plus IFN-γ has an intermediate migratory capacity towards CCL19 and CCL21. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α and IFN-α. These data support that DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3828.
Abstract 3830: Inhibition of IL-4 function decreases fibrosis and lymphatic dysfunction in a mouse model of lymphedema
Authors: Avraham Tomer , Daluvoy Sanjay V. , Kueberuwa Essie , Zampell Jamie C. , Yan Alan , Mehrara Babak J.
Published: February 2011

Background: Lymphedema, commonly encountered in cancer survivors, is characterized by chronic swelling and fibrosis. Previous studies have shown that fibrotic disorders involving other organ systems are dependent on T-helper 2 (Th-2) chronic inflammatory changes. The purpose of these experiments was therefore to evaluate the role of Th-2 cells in the pathological changes associated with lymphedema.

Methods: We developed a surgical mouse model that results in tail lymphedema for prolonged periods (>9 weeks) and is histologically is similar to clinical lymphedema. Using this model in wild-type and athymic nude mice we evaluated the role of T-cells in general on the development of lymphedema and fibrosis. Furthermore, using a specific monoclonal neutralizing antibody against IL-4, a molecule necessary for Th-2 differentiation, we evaluated the role of Th2 cells in the initiation and maintenance of chronic lymphedema.

Results: Lymphedema in wild-type mice resulted in a chronic inflammation consisting predominantly of CD-4+cells with a mixed Th1-Th2+ differentiation. Interestingly, application of this model to athymic nude mice resulted in markedly decreased tail edema and fibrosis, demonstrating that these outcomes are T-cell dependent. While inhibition of Th2 differentiation immediately after surgery did not prevent initiation of lymphedema, it did result in early resolution, with a marked reduction in fibrosis and improved lymphatic function 6 weeks post-operatively. Consistent with this, inhibition of Th2 cell function in mice with established lymphedema resulted in resolution of lymphedema and fibrosis with just 3 weekly treatments. In contrast, lymphedema was sustained in animals treated with isotype control.

Conclusions: We have shown that Th2 differentiation is necessary for maintenance of the pathologic changes associated with lymphedema. Inhibition of Th2 differentiation completely reversed the fibrosis and lymphatic dysfunction in animals with established lymphedema, with resultant resolution of swelling. These results indicate that similar approaches may be clinically useful in patients with chronic lymphedema.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3830.
Abstract 3831: Sodium butyrate induces differential expression of Th17 and inflammatory cytokines during apoptosis in human U937 leukemia cells
Authors: Pulliam Stephanie , Copper Roland S. , Adunyah Samuel Evans
Published: February 2011

Sodium butyrate (NaB) is a sodium salt of butyric acid. It is a strong HDAC1 inhibitor, which has been implicated in many studies as a potential therapy for various forms of cancer including prostate cancer. Also, it has been suggested as a potential therapy for hemoglobin disorders including sickle cell diseases and leukemia because of its ability to promote both total globin and gamma globin synthesis during erythroid differentiation. Previously NaB has been shown to active apoptosis in several cancer cell lines including U937 and K562 leukemia cell lines. However, it is not known whether it has the capability to modulate cytokines expression in cancer cells and alter the cancer microenvironment, which was the purpose of this study. We exposed U937 leukemia cells to NaB at 5 mM or 10 mM for 24 or 48 hours, conditions under which NaB is known to induce apoptosis in these cells. The culture media of the cells were harvested and analyzed by Human Multi-Analyte Cytokine ELISArray method and by protein/antibody cytokine arrays for levels of TH17-type and Inflammatory cytokines. We also monitored cells for caspase 3 activation and cell viability as evidence of apoptosis. Our results indicate that NaB induces differential expression of several TH17-type and Inflammatory cytokines from 2 to 10-fold within 24 hours. The ranking order of induction of TH17-type cytokines by NaB was IL-17A>IL-5>IL-4>G-CSF>IL-10>TNF>IL-12>IFNγ while induction of inflammatory cytokines was of the ranking order: IL-1A>IL-8>IL-10. Also within 24 hours, NaB treatment caused at least 2-fold activation of caspase 3 and reduced the cell viability by 60 %. These observations indicate that NaB induces differential expression of both TH17 and Inflammatory cytokines during induction of apoptosis. The data also suggest that while promoting cancer cell death by apoptosis, NaB also has the potential to influence cancer microenvironment by inducing differential expression and secretion of cytokines. Such an effect could influence the biology of both the normal and cancer cells and could alter the status of the patient who is receiving NaB therapy. Further studies are in progress to determine whether NaB has similar effects in human monocytes. Supported by NCI U54 cancer grants # 5 U54 CA091408-09.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3831.
Abstract 3835: Impaired pSTAT3 and pSTAT5 induction by IL-2 does not affect IFNγ production in NK cells of breast cancer patients
Authors: Konjevic Gordana M. , Radenkovic Sandra S. , Srdic Tatjana , Jelic Svetislav , Stamatovic Ljiljana , Inic Momcilo
Published: February 2011

Although pSTAT3 and pSTAT5 in tumors have oncogenic potential, STAT dysregulation has not been extensively investigated in peripheral blood lymphocytes (PBL) of breast cancer patients. Alterations in JAK-STAT pathway in cancer patients contribute to immunosuppression and inadequate antitumor immune response. In this study baseline level and cytokine-mediated activation of pSTAT3 and pSTAT5 were investigated in PBL of breast cancer patients and healthy controls after in vitro treatment with IL-2 for 24h at 37C. Induction of pSTATs was assessed in cellular lysates by Western blotting using anti-pSTAT antibodies. PBL of breast cancer patients and controls were analyzed for intracellular IFNγ, CD3 and CD16 receptor expression and NK cell activity using Flow cytometry and 51Cr-release assay, respectively. The induction of pSTAT3 and pSTAT5 in PBL by IL-2 is significant for controls and patients, although the effect in patients is lower. Moreover, PBL and CD3-CD16+ NK cells of breast cancer patients compared to controls have significantly lower baseline level of IFNγ. IL-2 significantly increases the production of IFNγ in PBL inspite of impaired induction of pSTAT3 and pSTAT5 in breast cancer patients. Evaluation of NK cell subset shows highly significant induction of IFNγ by IL-2 in breast cancer patients and in healthy controls. Inspite of this, we found that NK cell activity of breast cancer patients is significantly lower compared to healthy controls suggesting that NK cell activity is more dependent on STATs inducibility than IFNγ production in these patients. These results provide evidence that evaluation of STAT expression and cytokine induction in PBL of breast cancer patients could be of diagnostic and therapeutic significance during non-specific active immunotherapy.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3835.
Abstract 3838: Akt isoform-specific signaling in breast cancer cell invasive migration
Authors: Chin Yuet Ming Rebecca , Toker Alex
Published: February 2011

Akt/PKB (protein kinase B) is a proto-oncogene that plays critical roles in cell survival, proliferation and metabolism. There are three mammalian Akt isoforms: Akt1, Akt2 and Akt3. They share a high degree of amino acid similarity and are activated by similar mechanisms. The current paradigm is that all three Akt isoforms promote cancer cell survival and growth. Interestingly, recent studies from our laboratory have shown that Akt1 inhibits breast cancer cell migration and invasion. Conversely, Akt2 has been shown to promote breast cancer cell migration. We therefore seek to test the hypothesis that Akt isoforms regulate breast cancer cell invasive migration in a distinct manner, and investigate the mechanistic basis for this selectivity. Current experiments are undertaken to identify novel substrates of Akt1 and Akt2, which modulate cell motility, with a combination of shRNA and phospho-proteomic approaches. shRNAs specific to Akt1 or Akt2 have been generated and delivered into human breast cancer cells by lentiviral vectors. Using an antibody that recognizes phosphorylated serine/threonine residues contained within a consensus Akt motif (RXRXXS/T), we showed that palladin, a component of actin-containing microfilaments, is phosphorylated at serine 507 by Akt1 but not Akt2. Downregulation of palladin using RNA interference enhances migration and invasion of breast cancer cells and induces abnormal branching morphogenesis in 3D cultures, indicative of an anti-migratory function for palladin. Importantly, palladin is required for Akt1-mediated inhibition of breast cancer cell migration, whereby the migratory effect induced by palladin silencing is reversed by wild-type but not a S507A mutant. Finally, palladin phosphorylation by Akt1 is functionally required for bundling F-actin and maintaining an organized actin cytoskeleton network in cells. These findings identify palladin as an Akt1-specific substrate that regulates cell motility and further provide a molecular mechanism that accounts for the functional distinction between Akt isoforms in breast cancer cell migration.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3838.
Abstract 3840: Bone morphogenetic proteins require the type II TGFβ receptor for growth arrest in mammary tumor cells
Authors: Owens Philip , Gorska Agniesszka E. , Aakre Mary E. , Moses Harold L.
Published: February 2011

Bone Morphogenetic Proteins (BMPs) are members of the Transforming Growth Factor β (TGFβ) superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth arrest in cancers as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers. We sought to understand how a potential tumor suppressor could be overexpressed in human breast cancers. We analyzed several tumor cell lines and normal mammary epithelia and found that BMPs are sufficient to induce growth arrest in normal and transformed mammary epithelia. Surprisingly, when we treated cells derived from the PyVmT mouse model lacking the type II TGFβ receptor with BMP-4, these cells failed to undergo growth arrest. We determined by flow cytometry that BMP treated cells did not undergo apoptosis. Cell cycle analysis demonstrated that cells with functional TGFβ signaling were arrested in G1 and cells lacking TGFβ signaling capabilities remained in a typical distribution of the cell cycle. We also found that Smads which mediate both BMP and TGFβ signaling were not only still present, but also responsive to BMP stimulation resulting in BMP Smad1/5/8 phosphorylation. Furthermore, we assessed the ability for synergy between BMP-4 and TGFβ1 and found that BMP-4 could induce an increased growth response in the presence of maximal TGFβ inhibition. We conclude that the dual functions of TGFβ as a tumor suppressor and promoter are connected to a BMP mediated tumor suppressive mechanism that shares common and distinct pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3840.
Abstract 3841: An integrin αvβ3/c-Src oncogenic unit promotes anchorage independence and tumor progression
Authors: Desgrosellier Jay S. , Seguin Laetitia , Barnes Leo A. , Shields David J. , Huang Miller , Lau Steven K. , Prevost Nicolas , Tarin David , Shattil Sanford J. , Cheresh David A.
Published: February 2011

Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin αvβ3 is associated with progression of a variety of human tumors. We recently described an adhesion-independent role for integrin αvβ3 in pancreatic cancer and other carcinomas resulting in increased anchorage-independent tumor growth in vitro and lymph node metastases in vivo. These effects required recruitment of c-Src to the β3 integrin cytoplasmic tail, leading to c-Src activation. Pharmacological blockade of c-Src kinase activity with Dasatinib or genetic suppression of endogenous c-Src not only inhibited anchorage-independent growth, but also suppressed metastasis in vivo. We now show that αvβ3 exerts these effects through ligand-independent clustering of the integrin on the surface of non-adherent tumor cells. Self-assembly/clustering appears to be a unique property of αvβ3 relative to other integrins and is dependent on c-Src recruitment to αvβ3. These data describe a c-Src-dependent self-assembly property of αvβ3 leading to increased anchorage-independence and malignancy of tumor cells expressing this integrin. These findings also underscore the potential benefits of using Src inhibitors, such as Dasatinib, for treating patients with αvβ3-expressing solid tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3841.
Abstract 3847: Metastasizing host stromal cells potentiate colonization of metastatic tumors
Authors: Duyverman Annique M. , Duda Dan G. , Kohno Mitsutomo , Steller Ernst JE , Fukumura Dai , Jain Rakesh K.
Published: February 2011

Metastatic cancer cells only grow in the secondary sites with a permissive microenvironment. Here, we show that the metastatic cells can also carry their own “soil”, including activated fibroblasts from the primary site. Moreover, we found that these non-malignant carryover host-derived cells can potentiate metastatic cell colonization in the lungs and increase cancer's metastatic efficiency.

Materials and Methods: Using the isolated renal perfusion model we were able to collect fragments shed by the primary tumor and assess their viability. Next, we adjusted the parabiosis model to transplant a 2 cm Actb-GFP+ skin graft onto a wild type mouse. Tumors were allowed to grow in the graft and metastasize to the lung. GFP+ cells carried over by the tumor to the lung in the lung were characterized using immunohistochemistry. In a spontaneous metastases model we used two lung tumor cell lines (LLC1 and LA-P0297) in combination with human carcinoma associated fibroblasts to study the modulation of the metastatic efficiency by stromal cells.

Results: We show here that, in addition to single cells (81%), tumors shed fragments consisting of both tumor and host stromal cells. These heterogeneous fragments are more viable in the circulation compared to tumor cells alone (p < 0.05) and their stromal component proliferates in the metastic site for up to seven days. Next, we assessed the nature of these host stromal cells using immunohistochemistry and indentified 80% of them being of fibroblast origin. When we selectively depleted the host stromal cells in the tumor microenvironment the spontaneous formation of metastases was significantly reduced three weeks after the primary tumor resection as compared to control mice (p < 0.05). Also, in mice with metastases, survival was significantly decreased after co-implantation of CAFs with LLC1 compared to mice implanted with cancer cells alone. This effect could be reversed by selective depletion of the “circulating” CAFs after primary tumor removal (p < 0.01).

Discussion: We found that tumor-associated stromal cells can escape primary tumors along with cancer cells, survive in blood circulation and in metastatic nodules. Pre-existence of a tissue-like structure in the form of heterotypic cell fragments may increase the viability of cancer cells in blood circulation and promote metastasis efficiency at the secondary site. How this mechanism contributes to the organo-tropism of certain metastatic tumors is not known. But demonstration of the involvement of some of the same tumor “soil” in both local and distant tumor invasion has important conceptual and clinical implications for colonization during metastasis progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3847.
Abstract 3851: Protein switches that activate a prodrug in the presence of HIF-1a
Authors: Wright Chapman M. , Eshleman James , Ostermeier Marc
Published: February 2011

We have engineered yeast cytosine deaminase (CD) to be activated by the tumor marker, hypoxia-inducible factor 1-alpha (HIF-1a) - turning on its ability to convert 5-fluorcytosine (5FC) into the anticancer drug 5-fluoruracil (5FU). HIF-1a is overexpressed in many types of cancer but is virtually undetectable in normal tissue due to rapid degradation. We sought a “tumor-sensing” protein whose prodrug converting activity is activated in the presence of HIF-1a. Such a protein could be used in a therapeutic treatment similar to gene-directed enzyme prodrug therapy (GDEPT). However, our approach would have an advantage over GDEPT because the “suicide” gene could be delivered using very efficient methods for gene delivery, whether or not they were selective, since the selectivity occurs at the protein level and not the gene delivery level.

To create our desired protein, we constructed a library of fusion proteins between CD and the CH1 domain of p300 - a domain known to interact with HIF-1a. This library was subjected to a two-tiered genetic selection in E. coli designed to identify genes that conferred increased sensitivity to 5FC when HIF-1a was co-expressed. We identified two library members (#3 and #59), which conferred to E. coli sensitivity to 5FC equivalent to wildtype CD but only in the presence of co-expressed HIF-1a. In the absence of HIF-1a, #3 conferred a 5-fold higher LC50 to 5FC and #59 did not confer any sensitivity to 5FC. Both proteins were purified and characterized in vitro for their activity in the presence and absence of HIF-1a. Protein #3 exhibited a 4-fold increase in its CD activity in the presence of HIF-1a, whereas protein #59's activity did not have a significant dependence on HIF-1a. Currently, the two genes are being examined in human cancer cell lines for their ability to confer sensitivity to 5FC in the presence of HIF-1a.

Using advanced protein engineering techniques, we have created two proteins that confer sensitivity to 5FC in the presence of the tumor-marker, HIF-1a. We believe that these genes have therapeutic potential for the treatment of cancers that exhibit elevated HIF-1a levels. Our strategy circumvents a limitation of GDEPT by not requiring selective delivery of the gene to cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3851.