bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3997: Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic aberrant crypt foci.
Authors: Drew David A. , Devers Thomas , Horelik Nicole , Yang Shi , O'Brien Michael , Wu Rong , Rosenberg Daniel W.
Published: August 2014

Oncogenic activation resulting in hyperproliferative lesions within the colonic mucosa has been identified in putative precancerous lesions, aberrant crypt foci (ACF). KRAS (codons 12 and 13) and BRAFV600E mutation status was determined in 172 ACF identified in colorectum of screening subjects by in situ high-definition, magnifying chromoendoscopy. Lesions were stratified according to histology: serrated hyperplastic versus distended (non-serrated) hyperplastic. Due to their limiting size, however, it was not technically feasible to examine downstream signaling consequences of these oncogenic mutations. We have combined ultraviolet-infrared (UV/IR) micro-dissection with an ultra-sensitive nanofluidic proteomic immunoassay (NIA) to enable accurate quantification of post-translational modifications to mitogen-activated protein kinase (MAPK) in total protein lysates isolated from hyperproliferative crypts and adjacent normal mucosa. Using this approach, levels of singly and dually (activated) phosphorylated isoforms of extracellular receptor kinase(ERK)-1 and -2 were quantified in samples containing as little as 16-ng of total protein recovered from <200 colon epithelial cells. ERK activation is responsible for the observed hyperplasia found in these early lesions, but its activation state is not directly dependent on KRAS and/or BRAF mutation status. This study describes the novel use of a sensitive nanofluidic platform to measure oncogene-driven proteomic changes in diminutive lesions procured at the time of endoscopy and highlights the advantage of this approach over classical immunohistochemistry-based analyses.

Citation Format: David A. Drew, Thomas Devers, Nicole Horelik, Shi Yang, Michael O'Brien, Rong Wu, Daniel W. Rosenberg. Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic aberrant crypt foci. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3997. doi:10.1158/1538-7445.AM2013-3997
Abstract 40: Mutation of EPCAM gene is less frequent in Korean and EPCAM immunohistochemistry can identify Lynch syndrome in patients who do not meet Bethesda criteria.
Authors: Kang So Young , Kang Shin Woo , Do In-Gu , Chang Dong Kyung , Kim Kyoung M.
Published: August 2014

Lynch syndrome is an inherited tumor predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes, mainly MLH1 and MSH2. Recently, germline deletions affecting the epithelial cell adhesion molecule (EPCAM) gene located upstream of MSH2 were identified as a novel mutational mechanism causing Lynch syndrome. However, most studies were conducted in western populations, where the incidence of CRC is high. To identify the incidence of germline deletions of EPCAM gene in Korea where the incidence of CRC is relatively low and socioeconomic condition is different, we conducted a comprehensive study in 4,993 patients diagnosed and treated with CRCs at a single institute between 2006 and 2011. In all tumor samples, microsatellite instability (MSI) test and immunohistochemistry (IHC) for MMR proteins were performed. In cases with loss of MLH1 protein, methylation of MLH1 by MethyLight was performed and cases with hypermethylation of MLH1 gene were excluded. Germline mutations of MLH1, MSH2 and MSH6 were performed in patients who fulfilled at least one of the revised Bethesda criteria. In 97 patients with loss of MSH2 protein, EPCAM IHC and multiplex ligation-dependent probe amplification for germline deletions of the EPCAM and MSH2 gene were performed. Loss of expression of MMR proteins and/or MSI-high was observed in 337 (6.7%) patients, 112 (2.2%) patients met revised Bethesda criteria, and 54 (1.1%) patients harbored germline mutations of MMR genes. Out of 97 MSH2-negative CRCs, 5 (5.2%) showed concomitant loss of EPCAM expression and EPCAM exon 9 deletionmutations. Out of 5 cases with EPCAM mutation and protein loss, four patients showed familial histories of CRCs. However, one patient showed no family history. In this patient, although multiple liver metastases were the first manifestation, he lived 6 more years after diagnosis. In this large single study cohort, we found that 5.2% of MSH2-negative CRCs were caused by EPCAM deletion mutations, which is lower than previously reported incidences in western populations. Moreover, we could find one candidate patient with de novo mutation of EPCAM gene, which has not been reported yet. Our results strongly support that in routine screening of patients with CRC for Lynch syndrome, a simple and sensitive EPCAM IHC should be included even in cases do not meet Bethesda criteria.

Citation Format: So Young Kang, Shin Woo Kang, In-Gu Do, Dong Kyung Chang, Kyoung M. Kim. Mutation of EPCAM gene is less frequent in Korean and EPCAM immunohistochemistry can identify Lynch syndrome in patients who do not meet Bethesda criteria. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 40. doi:10.1158/1538-7445.AM2013-40
Abstract 400: Coordinated loss of CD9 and IGSF8 expression on the prostate cancer cell surface contributes to metastasis and hormone responsiveness.
Authors: Li Na , Fazli Ladan , Li Estelle , Buttyan Ralph
Published: August 2014

The tetraspanin web is a complex multi-molecular cell structure that tethers cell surface signalling proteins (i.e. integrins) to their intermediate intracellular signalling partners (i.e. small GTPases) and anchors actin filaments. Loss of certain components of the web (CD9, CD82 or CD316) are associated with increased invasive and metastatic behaviors of prostate cancer (PCa) cells and here we demonstrate that two of these tetraspanin web components, CD9 and CD316, also play a role in regulating androgen signaling in PCa cells. Additionally, we utilized human prostate cancer tissue microarrays (TMAs) containing 261 prostate/PCa specimens of differing stages, grades and states of progression (including 60 castration resistant tumors [CRPCs]) to study the relationship between CD9 and CD316 expressions and progression to CRPC. Using androgen-dependent LNCaP cells as a model, we found that androgen deprivation (AD) suppressed expression of CD9 protein and mRNA without affecting CD316 expression. FACS analysis showed that the presence of CD9 on the cell surface was coordinately reduced by AD as was cell surface CD316 which was relocalized to the cytoplasm. This effect was reversed by addition of R1881. Knockdown of CD9 or CD316 in LNCaP cells using siRNA reduced expression of androgen receptor (AR) mRNA but increased expression of PSA, an AR target gene. CD9 or CD316 knockdown also increased LNCaP growth in fetal bovine serum (FBS) containing medium but not in charcoal-stripped FBS (CS-FBS) whereas dual-knockdown further increased growth in FBS as well as in CS-FBS. TMAs immunostained for CD9 or CD316 were assessed for correlation to clinicopathological parameters. For untreated, primary tumors, CD9 and CD316 expression were inversely correlated with Gleason scores (R2=0.9612). CD9 expression was diminished during progression to metastatic (p<0.0038, compared to non-metastatic) and further in CRPC (p<0.040, compared to metastatic non-treated) disease. Conversely, CD316 expression was increased in primary (non-metastatic) as well as in metastatic untreated disease but then reduced (p<0.046) during progression to metastatic CRPC. We propose that androgen deprivation (first line therapy for advanced PCa) may potentially trigger loss of both CD9 and IGSF8 expression on the cell surface that contributes to metastatic behaviors as well as to responsiveness to androgen ablation therapies. This work is supported by the Cancer Research Society (Canada).

Citation Format: Na Li, Ladan Fazli, Estelle Li, Ralph Buttyan. Coordinated loss of CD9 and IGSF8 expression on the prostate cancer cell surface contributes to metastasis and hormone responsiveness. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2013-400
Abstract 4004: Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces metallothionein gene expression in human colon cancer.
Authors: Shinagawa Kei , Kitadai Yasuhiko , Yuge Ryo , Onoyama Mieko , Ohnishi Mayu , Tanaka Shinji , Yasui Wataru , Chayama Kazuaki
Published: August 2014

Bone marrow-derived mesenchymal stem cells (MSCs) are reported to contribute to the formation of tumor stroma. We reported recently that in an orthotopic nude mouse model of human colon cancer, MSCs migrated to tumor stroma, where they differentiated into carcinoma-associated fibroblast (CAF)-like cells and promoted tumor growth and metastasis. Furthermore, the migratory and invasive abilities of KM12SM colon cancer cells were shown by Transwell invasion assay to be enhanced by indirect co-culture with MSCs. However, the molecular mechanisms of this phenomenon have not been elucidated. We performed microarray analysis of gene expression in KM12SM cells and found that expression of metallothionein (MT) mRNA increased markedly by indirect co-culture of KM12SM cells with MSCs. Additionally, in an orthotopic nude mouse model of colon cancer, MT mRNA expression levels were shown to be higher in the tumors produced by KM12SM cells mixed with MSCs than in the tumors produced by KM12SM cells alone. MTs comprise a family of low-molecular-weight, cysteine-rich intracellular proteins that are associated with protection against DNA damage, oxidative stress, and apoptosis. The potential role of MTs in carcinogenesis remains unknown. We examined MT expression in surgical specimens of human colon cancer and colon adenoma by immunohistochemistry. A total of 101 tumors were analyzed. MT immunoreactivity was detected in tumor cells and in atypical stromal cells. Atypical stromal cells with strong MT expression were found in invasive cancers, at the invasive edge, but not in mucosal cancers or adenomas. Levels of MT expression in tumor cells correlated inversely with disease stage. These findings suggest that the interaction between MSCs recruited into tumor stroma and the tumor cells of invasive colon cancers induces and depends on MT expression. Further studies are needed to clarify the specific effects of MTs on tumor cells and stromal cells.

Citation Format: Kei Shinagawa, Yasuhiko Kitadai, Ryo Yuge, Mieko Onoyama, Mayu Ohnishi, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces metallothionein gene expression in human colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4004. doi:10.1158/1538-7445.AM2013-4004
Abstract 4013: S100A4 promotes cell migration and growth through focal adhesion kinase and Src mediated dual signaling pathways in pancreatic cancer cells.
Authors: Cai Guoqiang , Kerr Elizabeth H. , Hu Meng , Yang Youfeng , Buchsbaum Donald J. , Grizzle William E. , Ding Qiang
Published: August 2014

BACKGROUD: Pancreatic cancer is one of the deadliest types of cancer. Activation and expression of focal adhesion kinase (FAK) and Src kinase is abnormally upregulated in pancreatic cancers. The results of clinical trials targeting either Src or FAK by inhibitors are largely unknown. S100A4 is a calcium-binding protein and aberrant S100A4 expression is an independent biomarker for poor outcomes of pancreatic cancer; however, the involved mechanism is unclear.

METHODS: The effects of inhibitors on FAK and Src activation, and on cell migration and growth, were examined in MiaPaca2 and S2VP10 cell lines. To determine whether FAK and Src depend on each other or independently regulate pancreatic cancer cell phenotypes, the effects of FAK or Src mutant expression were determined. The role of S100A4 on FAK and Src activation was evaluated through loss/gain approaches. The association of S100A4 expression with FAK and Src activation was determined by immunostaining on human pancreatic cancer tissue sections.

RESULTS: FAK and Src kinase inhibitors blocked activation in a dose dependent manner in both MiaPaca2 and S2VP10 cells. However, it required more inhibitor to inhibit either FAK or Src in S2VP10 cells. Blocking FAK (or Src) does not completely inhibit Src (or FAK) activation. This is confirmed by the finding that FAK mutant expression completely inhibited FAK activation, but not Src activation. Similarly, Src mutant expression did not inhibit FAK activation. Downregulation of S100A4 expression significantly decreased FAK and Src activation. Importantly, increased S100A4 expression is associated with increased FAK and Src activation in pancreatic cancer.

CONCLUSIONS: These data support that the Src-FAK axis forms a dual signaling pathway, and each of them contributes independently to the aggressive pancreatic cancer cell phenotype. Also, these data support that S100A4 promotes the Src-FAK dual signaling pathway which possibly leads to a feed-forward loop facilitating pancreatic tumor progression.

Citation Format: Guoqiang Cai, Elizabeth H. Kerr, Meng Hu, Youfeng Yang, Donald J. Buchsbaum, William E. Grizzle, Qiang Ding. S100A4 promotes cell migration and growth through focal adhesion kinase and Src mediated dual signaling pathways in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4013. doi:10.1158/1538-7445.AM2013-4013
Abstract 4089: Differential regulation of androgen receptor by PIM-1 kinases via phosphorylation-dependent recruitment of distinct ubiquitin E3 ligases.
Authors: Linn Douglas , Yang Xi , Xie Yingqiu , Alfano Alan , Deshmukh Dhanraj , Wang Xin , Shimelis Hermela , Chen Hegang , Li Wei , Xu Kexin , Chen Mingyuan , Qiu Yun
Published: August 2014

Androgen receptor (AR) plays a pivotal role in prostate cancer. Regulation of AR transcriptional activity by post-translational modifications, such as phosphorylation by multiple kinases, is well documented. Here, we report that two PIM-1 kinase isoforms which are upregulated during prostate cancer progression, namely PIM-1S and PIM-1L, modulate AR stability and transcriptional activity through differentially phosphorylating AR at Serine 213 (S213) and Threonine 850 (T850). Although both kinases are capable of interacting with and phosphorylating AR at S213, only PIM-1L could phosphorylate T850. We also showed that PIM-1S induced S213 phosphorylation destabilizes AR by recruiting the ubiquitin E3 ligase Mdm2 and promotes AR degradation in a cell cycle dependent manner, while PIM-1L-induced T850 phosphorylation stabilizes AR by recruiting the ubiquitin E3 ligase RNF6 and promotes AR-mediated transcription under low-androgen conditions. Furthermore, both PIM-1 isoforms could promote prostate cancer cell growth under low-androgen conditions. Our data suggest that these kinases regulate AR stability and transcriptional activity through recruitment of different functional partners in a phosphorylation-dependent manner. As AR turnover has been previously shown to be critical for cell cycle progression in prostate cancer cells, PIM-1 kinase isoforms may promote prostate cancer cell growth, at least in part, through modulating AR activity via distinct mechanisms.

Citation Format: Douglas Linn, Xi Yang, Yingqiu Xie, Alan Alfano, Dhanraj Deshmukh, Xin Wang, Hermela Shimelis, Hegang Chen, Wei Li, Kexin Xu, Mingyuan Chen, Yun Qiu. Differential regulation of androgen receptor by PIM-1 kinases via phosphorylation-dependent recruitment of distinct ubiquitin E3 ligases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4089. doi:10.1158/1538-7445.AM2013-4089
Abstract 409: In vitro and in vivo characterization of protein biomarkers of metastasis in melanoma.
Authors: Lie Wen-Rong , Becker-Hapak Michelle , Chan Megan , Graham James , Mistry Jehangir , Linette Gerald , Carreno Beatriz
Published: August 2014

Metastasis is a major cause of death in cancer patients. We report here the use of a multiplex proteomic analysis approach to in vitro and in vivo identify biomarkers involved in melanoma invasion/metastasis. An in vitro quantitative profile of the secretome (>200 proteins) was done on non-metastatic melanoma cell lines (A375, LH, M14, DM.6, Lox, and WUmel19) and their metastatic RhoC-expressing counterparts (A375-RhoC, LH-RhoC, M14-RhoC, DM.6-RhoC, Lox-RhoC, and WUmel19-RhoC). A heat map cluster analysis was performed to characterize protein expression levels on metastatic vs. the non-metastatic melanoma cells and identify secreted markers with significantly altered expression that may be involved in tumor invasion, angiogenesis, and metastasis. An in vivo characterization, using an animal model of human melanoma pulmonary metastasis, has identified differential expression of tissue and circulating protein biomarkers in tumor and sera, respectively. Moreover, use of species-specific assays has allowed us to dissect the contribution of stroma (mouse) and tumor (human). Increased expression of a panel of markers, including IL-8, PLGF, FGF-2, follistatin, MMP-1, MMP-9, TIMP-1, TIMP-2, GDF15, NSE, OPN, and MIF was detected in vivo in both tumor and sera, indicating their roles in inducing angiogenesis, tumor invasion and metastasis. Cell signaling pathway multiplex analyses revealed the activation of PI3K/mTOR pathway in the tumor. Elevated levels of phosphorylated targets of PI3K/mTOR pathway, p70S6K (pT412), GSK3α (pS21), GSK3β (pS9), RPS6 (pS235/236), TSC2 (pS939) and mTOR (pS2448) were detected in the pulmonary tumor metastasis. Hence this human melanoma pulmonary metastasis model may prove to be a useful animal model for studying the actions of PI3K/mTOR inhibitors in melanoma metastasis. Altogether, these in vitro and in vivo studies using multiplex proteomic analysis may assist in the discovery of biomarkers and therapeutic targets for human melanoma interventions.

Citation Format: Wen-Rong Lie, Michelle Becker-Hapak, Megan Chan, James Graham, Jehangir Mistry, Gerald Linette, Beatriz Carreno. In vitro and in vivo characterization of protein biomarkers of metastasis in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 409. doi:10.1158/1538-7445.AM2013-409
Abstract 4318: Development of a novel SurrobodyTM that simultaneously activates both death receptors DR4 and DR5 and induces cancer cell death with high potency.
Authors: Milutinovic Snezana , Zhou Sihong , Wimer Carina , Diaz Paul W. , Kashyap Arun K. , Bhatt Ramesh R. , Reed John C.
Published: August 2014

Death receptors of the Tumor Necrosis Factor (TNF) family are found on surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors-4 and -5 (DR4 and DR5) is tumor necrosis factor-related apoptosis-inducing ligand, TRAIL (Apo2L). TRAIL is expressed by many cell types, including Natural Killer (NK) cells that are involved in the immune surveillance and elimination of cells in vivo. Given that most normal untransformed cells are not susceptible to TRAIL-induced apoptosis, activators of death receptors have emerged as promising cancer therapeutic agents.

One strategy to stimulate death receptors in cancer patients has been to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. An alternative strategy to evade decoy receptor sequestration and to have improved pharmacokinetic properties is to generate DR4 or DR5 agonist antibodies. To this end, several agonistic monoclonal antibodies have been reported that overcome the limitations of short half-life, but exhibited the limitation of activating only one of the two death receptors. The SurrobodyTM binding protein is a novel therapeutic protein structure based upon the pre-B cell antigen receptor common chain that is capable of being engineered to bind a diversity of targets with high affinity and specificity. We exploited a previously discovered SurrobodyTM that potently activates both DR4 and DR5 in vitro and in vivo. Here we show experimental results for a dual agonist SurrobodyTM that offers a powerful and likely more reliable strategy for cancer therapeutics based on simultaneous stimulation of both TRAIL receptors, DR4 and DR5.

Citation Format: Snezana Milutinovic, Sihong Zhou, Carina Wimer, Paul W. Diaz, Arun K. Kashyap, Ramesh R. Bhatt, John C. Reed. Development of a novel SurrobodyTM that simultaneously activates both death receptors DR4 and DR5 and induces cancer cell death with high potency. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4318. doi:10.1158/1538-7445.AM2013-4318
Abstract 4343: Therapeutic efficacy of different double arms polyethyleneglycol-modified liposome containing doxorubicin on pulmonary metastasis.
Authors: Sugiyama Ikumi , Sadzuka Yasuyuki
Published: August 2014

[Purpose] Metastasis is responsible for most mortality in cancer therapy. Important things of metastatic treatment are not only prevention but also therapy of metastatic tumor. It was indicated that usability of liposomal doxorubicin (DOX) as drug delivery system formulation which were modified with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine dipolyethyleneglycol(different double arms PEG; DDA-PEG) (DDA-LDOX). DDA-PEG had two different PEG lengths, 2000 and 500, in one molecule. In this study, the effect of DDA-LDOX was examined on pulmonary metastasis compared with 1-monomethoxy-PEG(2000)-2,3-distearoylglycerol (PEG2000-DSG) modified liposomal DOX (2000-LDOX).[Methods] The liposomal DOX was prepared by L-α-distearoylphosphatidylcholine, cholesterol, L-α-distearoylphosphatidyl-DL-glycerol and DOX (100:100:60:18 μmol). Liposomal DOX was added 7.5 μmol DDA-PEG or 15 μmol PEG2000-DSG. In anti-metastasis experiment, BDF1 mice were transplanted B16F10 melanoma cells by intravenous inoculation and each liposome (2.5 mg/kg, DOX) was injected at 20, 23 and 26 day after tumor transplantation. After 48 hr from last administration, the mice were dissected and score of pulmonary metastasis was recorded (0∼10, 0: normal). Removed tissues were determined DOX concentration by fluorescence method. In vitro, it was examined DOX uptake into B16F10 melanoma cells from each liposome. [Results and Discussion] Pulmonary metastasis scores of control and 2000-LDOX were 6.5 and 2.6, respectively. In contrast, that of DDA-LDOX was 1.0. Relative pulmonary weight (%) was correlation with metastatic score (R2=0.954) and supported pulmonary metastasis score. DOX concentration of DDA-LDOX was twice higher than that of 2000-LDOX in the lung, namely, it was proved that DDA-LDOX was easy to accumulate in the lung. Especially, it was suggested that DDA-LDOX accumulated into pulmonary metastatic site because DOX concentrations of heart, spleen and kidney were equal level in other group. In the examination of DOX uptake into tumor cells, DOX level in DDA-LDOX group was significantly higher than that in 2000-LDOX group. The data had supported high accumulation into pulmonary metastasis in vivo. In conclusion, it was suggested that DDA-LDOX was useful drug carrier for suppressed pulmonary metastasis since it could have target ability to pulmonary metastasis.

Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. Therapeutic efficacy of different double arms polyethyleneglycol-modified liposome containing doxorubicin on pulmonary metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4343. doi:10.1158/1538-7445.AM2013-4343
Abstract 4392: Targeting HADC-2: Chemopreventive effect of OSU-HDAC42 on rat colon carcinogenesis and APCmin/+ Mice intestinal tumorigenesis.
Authors: Ravillah Durgadevi , Qian Li , Brewer Misty , Zhang Yuting , Biddick Laura , Madka Venkateshwar , Patlolla Jagan M.R. , Mohammed Altaf , Steele Vernon , Rao Chintalapally V.
Published: August 2014

Epigenetic modulators particularly histone deacetylases (HDACs) are valid targets cancer for prevention and Therapy. Recent studies from our laboratory and others suggest that HDAC-2 over-expression is associated with colon tumor progression and potential target for colon cancer prevention. The present study explored the chemopreventive effects of novel HDAC-2 inhibitor, OSU-HDAC42, in a mouse model of familial adenomatous polyposis (FAP). We used a rat colon carcinogenesis model assay, inhibition of aberrant crypt foci (ACF) development, as measure of the dose-response and chemopreventive effects. Colonic ACF were induced by azoxymethane (AOM) (15 mg/kg BW once weekly for 2 weeks by s.c. injection at the age of 8 and 9 weeks). One week after AOM-treatment groups of rats were fed AIN-76A diet containing 0, 75, 150 and 300 ppm OSU-HDAC42 for eight-weeks, and then colonic ACF were evaluated. Six week-old male C57Bl/6J-APCmin/+ and wild type mice were fed AIN-76A diet containing 150 ppm OSU-HADC42 or 300 ppm SAHA for 80 days. At ∼120 days of age, all mice were killed, and the intestines were evaluated for polyps. Dietary administration of OSU-HDAC42 produced a dose-dependent inhibition of AOM-induced colonic ACF formation (by 13-50%, p<0.05 -0.001) and reduced the number of foci with ≥4 crypts/focus (by 25-57%, P<0.05-0.0001) in F344 rats. 150 ppm OSU-HDAC42 significantly inhibited development of small intestinal (>46%, p<0.001) and colon tumors (>26%, P≥0.05) in APCmin/+ mice. Whereas 300 ppm SAHA showed limited non-significant inhibition of intestinal polyp formation in APCmin/+ mice. Further, OSU-HDAC42 showed inhibition in small intestinal polyps size measuring 1-2-mm (P<0.001). Importantly, mice fed with 150ppm OSU-HDAC42 showed significant reduction in HDAC-2, PCNA, CyclinA, CDK2, CDC25C expression pattern and increased p53 expression levels. These observations demonstrate that novel HDAC inhibitor, OSU-HDAC42 potential chemopreventive against the chemically-induced and polyposis models of colon tumorigenesis. {This work is supported by NCI-N01-CN53300}

Citation Format: Durgadevi Ravillah, Li Qian, Misty Brewer, Yuting Zhang, Laura Biddick, Venkateshwar Madka, Jagan M.R. Patlolla, Altaf Mohammed, Vernon Steele, Chintalapally V. Rao. Targeting HADC-2: Chemopreventive effect of OSU-HDAC42 on rat colon carcinogenesis and APCmin/+ Mice intestinal tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4392. doi:10.1158/1538-7445.AM2013-4392
Abstract 44: Tissue image analysis tool to quantify HER2 RNA and protein expression by colorimetric in situ hybridization and immunohistochemistry for breast cancer diagnosis.
Authors: Peljto Mirza , Krueger Joseph , Hagendorn Erik , Young David , Potts Steve , Nguyen Quan , Maqsodi Botoul , Yaoi Takuro , McMaster Gary
Published: August 2014

Early cancer diagnosis and risk assessment are powerful drivers in discovery of oncology biomarkers for diagnostic tests and simultaneous improvement of already identified biomarkers. Molecular tools such as RNA Colorimetric in situ hybridization (CISH) and immunohistochemistry (IHC) combined with conventional histopathology promise to increase the sensitivity and specificity of cancer staging and overall survival prediction. Changes in oncogene and proto-oncogene expression are a hallmark of cancer. For this reason, simultaneous quantification of mRNA and protein expression using RNA CISH and IHC combined with image-analysis is of interest. One such proto-oncogene is HER2, which is encoded by ERBB2 gene located at the long arm of human chromosome 17 (17q12). Here, we combine RNA CISH and IHC with image-analysis to develop a reliable quantitative HER2 assay that enables quantification of HER2 mRNA and protein expression in the context of human breast cancer. Since HER2 is only over-expressed in a subset of human breast cancers, we initially focused our analysis by screening tissue microarrays (TMAs) using RNA CISH and IHC. TMAs containing 20 normal and 20 breast tumor tissues were tested and whole-slide imaging was used to obtain CISH and IHC signals. HER2 mRNA and protein expression were analyzed, quantified, and compared both at tissue- and individual cell-levels using proprietary image-analysis based tools. Moreover, we compared heterogeneity of RNA and protein expression and further extended our studies to several other cancer-related genes. Finally, since oncogene expression plays a role in cancer pathologies, successful quantification of oncogene in situ expression in human cancer tissues may prove to be beneficial to further understanding of cancer pathology, for drug screening and development of biomarkers and ultimately diagnostic tests.

Citation Format: Mirza Peljto, Joseph Krueger, Erik Hagendorn, David Young, Steve Potts, Quan Nguyen, Botoul Maqsodi, Takuro Yaoi, Gary McMaster. Tissue image analysis tool to quantify HER2 RNA and protein expression by colorimetric in situ hybridization and immunohistochemistry for breast cancer diagnosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 44. doi:10.1158/1538-7445.AM2013-44
Abstract 4404: Multicellular tumor spheroid models to evaluate drugs targeting cell cycle checkpoints in 3D.
Authors: Ducommun Bernard , Lobjois Valérie , Laurent Jennifer , Frongia Céline , Mondesert Odile , Cazales Martine
Published: August 2014

MultiCellular Tumor Spheroid (MCTS) mimic the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. We report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization, the dynamics of cell cycle checkpoints and the effects of anticancer drugs in 3D. Spheroids expressing fluorescent reporters were engineered and used to explore cell cycle distribution and regionalization during growth. In these models we monitored cell cycle arrest in response to checkpoint activation after growth factor starvation, DNA injury or treatment with chemotherapeutic agents. The kinetics and regionalized aspects of the response were investigated both on fixed sections and on live spheroids using light sheet microscopy. All together, our data demonstrate the power of the combination of spheroids made of genetically modified cells expressing cell cycle reporters with both classical antibody-based detection and the use of innovative 3D imaging strategy. This study paves the way for the investigation of the molecular aspects of checkpoint response in 3D models and the dynamic studies of the 3D response to novel antiproliferative agents.

Citation Format: Bernard Ducommun, Valérie Lobjois, Jennifer Laurent, Céline Frongia, Odile Mondesert, Martine Cazales. Multicellular tumor spheroid models to evaluate drugs targeting cell cycle checkpoints in 3D. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2013-4404
Abstract 4501: Diverse microtubule stabilizers cause differential expression of mitotic kinases leading to distinct centrosomal and mitotic defects.
Authors: Rohena Cristina C. , Peng Jiangnan , Johnson Tyler A. , Crews Phillip , Mooberry Susan L.
Published: August 2014

Microtubule stabilizers are some of the most useful and successful drugs used to treat adult solid tumors. However, the molecular events responsible for their antimitotic actions are not yet fully understood. In this study we evaluated the mitotic defects and signaling events initiated by three structurally and biologically different microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel. These microtubule stabilizers cause the formation of aberrant, but morphologically distinct mitotic spindles leading to the hypothesis that they initiate distinct mitotic signaling events. Each microtubule stabilizer caused different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ caused centrosome separation failure to a much greater extent than paclitaxel or laulimalide. Taccalonolide AJ is also unique in that it was the only stabilizer to cause centrosome disjunction failure. These observations were consistent with the different defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer, which suggests a common mechanism of aster formation. However, taccalonolide AJ was the only stabilizer evaluated that caused pericentrin accumulation on every spindle aster. Pericentrin's localization to every spindle aster could facilitate the maintenance and stability of the highly focused asters formed by taccalonolide AJ. Laulimalide and paclitaxel caused completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Elucidating how chemically distinct microtubule stabilizers disturb mitotic signaling could identify key proteins involved in controlling sensitivity and resistance to the antimitotic actions of these agents.

Citation Format: Cristina C. Rohena, Jiangnan Peng, Tyler A. Johnson, Phillip Crews, Susan L. Mooberry. Diverse microtubule stabilizers cause differential expression of mitotic kinases leading to distinct centrosomal and mitotic defects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2013-4501
Abstract 4506: Photocaged molecules in targeted drug delivery.
Authors: Mitra Deboleena , Dcona Martin Michael , Hartman Matthew C.T.
Published: August 2014

We have developed a new targeted therapy that uses light to deliver cytotoxic agents into cancer cells. This strategy is based on the concept of photocaging in which a biologically active molecule is made inactive by the attachment of a light-cleavable blocking group. Upon exposure to UV radiation the photocleavable entity is removed and the biologically active molecule is released. Using this concept we have designed a prodrug that consists of a cell impermeable hydrophilic photocage attached to doxorubicin. The hydrophilic moiety makes the entire prodrug cell impermeable in the dark. Upon irradiation with non-toxic doses of UV light, the photosensitive group is removed and cytotoxic doxorubicin is released into the cells. In the presence of light the IC50 has been found to be 1.6 ±1.0 μM in JH-Eso-Ad1 cells. The FACS studies further confirmed that the cytotoxicity was due to light triggered release of free doxorubicin from the cell impermeable prodrug. This concept has been further developed by attaching a receptor binding molecule to the photocaged drug to enhance specificity. Specifically, we have attached folic acid and the PSMA binding ligand, DUPA to the photocaged doxorubicin. These targeting groups enable cancer cell selective drug delivery with light.

Citation Format: Deboleena Mitra, Martin Michael Dcona, Matthew C.T. Hartman. Photocaged molecules in targeted drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4506. doi:10.1158/1538-7445.AM2013-4506
Abstract 4542: Towards the identification of PKR-like endoplasmic reticulum kinase (PERK) inhibitors.
Authors: Park Jihyun , Wang Qiantao , Ren Pengyu , Dalby Kevin N.
Published: August 2014

During tumorgenesis cancer cells must tolerate cellular stresses such as DNA damage, hypoxia, and oxidative stress. In order to adapt to and overcome such stresses, tumor cells remodel transcriptional and translational programs by activating pro-survival signaling pathways. PERK (PKR-like endoplasmic reticulum kinase) is one of the key components of the unfolded protein response (UPR). Many studies report that the UPR is activated in a variety of tumor types. Previous studies demonstrated that cells with a compromised PERK-eIF2α-ATF4 signaling pathway are sensitive to hypoxic stress in vitro and form tumors that grow more slowly in vivo. Together, these data suggest a substantial role for PERK and the UPR in tumor cell survival and adaptation to stress. Thus, selective inhibitors of PERK activity may be useful therapeutic candidates for the treatment of cancer. Using a high-throughput screening (HTS) approach a number of lead compounds were identified with demonstrated ability to inhibit PERK kinase autophosphorylation and activity in cells. Using a publically available structure of Apo PERK a PERK-ATP complex structure was generated by means of molecular dynamics (MD) simulation. Evidence is presented to support the notion that the simulation-generated structure represents a useful model for computer-aided lead optimization.

Citation Format: Jihyun Park, Qiantao Wang, Pengyu Ren, Kevin N. Dalby. Towards the identification of PKR-like endoplasmic reticulum kinase (PERK) inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4542. doi:10.1158/1538-7445.AM2013-4542
Abstract 4578: Deep targeted sequencing of 12 breast cancer loci in 4,700 women across four different ethnicities.
Authors: Lindström Sara , Chapman Brad , Chen Gary , Chen Constance , Ablorh Akweley , Le Marchand Loic , Henderson Brian , Hofmann Oliver , Mirel Daniel , Haiman Christopher , Kraft Peter
Published: August 2014

Genome-wide association studies (GWAS) have identified multiple genetic loci associated with breast cancer risk. However, the underlying genetic structure in these regions is not fully understood and it is likely that the index GWAS signal originates from one or more yet unidentified causal variants within the region. We used next-generation sequencing to characterize 12 GWAS-discovered breast cancer loci in a total of 2,300 breast cancer cases and 2,300 controls across four ethnic populations. Study subjects were women from the Nurses’ Health Study (NHS) and the Multiethnic Cohort (MEC). Region intervals spanned between 46kb and 973kb and we hybrid-captured and sequenced a total of 5.5Mb. On average, we were able to capture 82% of the non-repetitive sequence in the targeted regions, and the average fraction of captured bases sequenced with a depth %gt20x was more than 95%. After single nucleotide variant (SNV) calling and quality control, a total of 138,898 SNVs remained for analysis. 87.4% of those had a minor allele frequency less than 0.5% and 50.2% were private mutations. Across all regions, a total of 81 SNVs showed evidence for association with breast cancer (P<0.001) but we did not find unequivocal association evidence supporting a causal role for any individual SNV. However, in two of the regions (2q35 and 11q13), we were able to refine the GWAS signal and identify a high posterior probability (>90%) set including less than 15 SNVs. We also conducted burden tests of rare variants (MAF<0.005) but did not identify any significant associations after adjusting for number of tests. However, rare variants in FGFR2 and a second gene at 10q26 were nominally associated with breast cancer (P<0.05). We are currently imputing existing breast cancer GWAS using this sequencing data as a reference panel which will triple our current sample size. These results stress the importance of large study samples when attempting to refine GWAS signals and illustrate the challenges facing post-GWAS fine-mapping studies.

Citation Format: Sara Lindström, Brad Chapman, Gary Chen, Constance Chen, Akweley Ablorh, Loic Le Marchand, Brian Henderson, Oliver Hofmann, Daniel Mirel, Christopher Haiman, Peter Kraft. Deep targeted sequencing of 12 breast cancer loci in 4,700 women across four different ethnicities. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4578. doi:10.1158/1538-7445.AM2013-4578
Abstract 4589: Overexpression of RAD51 promotes brain metastases from breast cancer.
Authors: Woditschka Stephan , Palmieri Diane , Duchnowska Renata , Jassem Jacek , Badve Sunil , Sledge George W. , Steeg Patricia S.
Published: August 2014

The DNA double-strand break (DSB) repair gene RAD51 was found to be overexpressed (1.5-fold, p=0.001) at the mRNA level in resected brain metastases compared to primary breast tumors from the same patients. RAD51 was also overexpressed (1.4-fold, p=0.008) specifically in brain metastases from breast cancer compared to systemic (bone/lung) metastases. We therefore hypothesize that RAD51 promotes brain metastasis development in breast cancer patients and investigated RAD51 as functional driver of brain metastases, independent of its recognized role in resistance to DNA-damaging chemotherapy.

RAD51 was overexpressed at physiologically relevant levels in brain-metastatic variants of human MDA-MB-231 breast carcinoma cells (231-BR) and 4T1 murine mammary carcinoma cells (4T1-BR). In vitro, RAD51 overexpression results in a 35-40% (p<0.0001) reduction Gamma-H2AX foci during S-phase, a DNA DSB marker. Furthermore, using a Homologous Recombination (HR) assay we found that the frequency of HR was significantly increased (1.7-fold, p=0.002) in RAD51 overexpressers compared to vector controls. No effects on proliferation, motility or invasion were observed with RAD51 overexpression.

In intracardiac-injected mice, overexpression of RAD51 increased formation of large brain metastases, the equivalent of clinically detectable lesions, (3.3-fold, p=0.005; 3.3-fold, p=0.02) and micro-metastases (3.1-fold, p=0.01; 2.0-fold, p=0.04), compared to vector controls in 231-BR and 4T1-BR models, respectively. ShRNA-mediated RAD51 knockdown in 4T1-BR cells had the opposite effect, reducing large brain metastases (2.5-fold, p=0.001) and micro-metastases (2.5-fold, p=0.01). RAD51 modulation had no effect on lung metastasis development in tail-vein injected mouse models of 231-BR or 4T1-BR cells.

To distinguish between roles for RAD51 in initiation vs. outgrowth of brain metastasis, we quantified iron-labeled 231-BR cells in the brain microenvironment at 3, 7 and 12 days post-injection. On day 12, we observed significantly more RAD51-overexpressing single cells compared to vector controls, with a clear trend discernable by day 7 (fold-changes over vector: 1.1-fold, p=0.8; 2.4-fold, p=0.07 and 2.8-fold, p=0.001 for days 3, 7 and 12, respectively).

Together these data suggest that RAD51 drives initiation of brain metastases from breast cancer.

Citation Format: Stephan Woditschka, Diane Palmieri, Renata Duchnowska, Jacek Jassem, Sunil Badve, George W. Sledge, Patricia S. Steeg. Overexpression of RAD51 promotes brain metastases from breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4589. doi:10.1158/1538-7445.AM2013-4589
Abstract 460: Delicate balance among three types of T cells in concurrent regulation of tumor immunity.
Authors: Izhak Liat , Ambrosino Elena , O'Konek Jessica J. , Kato Shingo , Parish Stanley T. , Xia Zheng , Venzon David , Berzofsky Jay A. , Terabe Masaki
Published: August 2014

Various immunosuppressive cells, such as Tregs and type II NKT cells, mediate the immune regulation of cancer. Many studies have demonstrated the importance of regulatory cells; however, it is still not clear why different suppressive cells play a dominant role in different models. Here, we examined the relative role of two suppressors - Tregs and type II NKT cells – in a subcutaneous CT26 tumor model in three strains of mice: wild-type, NKT cell-deficient CD1dKO mice and Jα18KO, which lack type I NKT cells but still retain type II NKT cells. Tumors grew in all three strains, but Treg blockade by anti-CD25 mAb (PC61) led to tumor rejection in WT and CD1dKO but not in Jα18KO mice, suggesting that in Jα18KO mice, Tregs are not necessary for immune suppression. We hypothesized that cross regulation between type I and type II NKT cells in wild-type mice leaves Treg cells as primary suppressors, whereas in mice lacking type I NKT cells, unopposed type II NKT cells can suppress tumor immunity even when Tregs are blocked. We confirmed this hypothesis in PC61-treated Jα18KO mice by blocking type II NKT cells using anti-CD1d mAb (1B1) or by adoptive transfer of type I NKT cells combined with Treg blockade. These results support our hypothesis that it is necessary to block both suppressors, type II NKT cells and Tregs, in order to achieve protection. Also, activation of type II NKT cells by sulfatide in PC61-treated wild-type mice abrogated the protective effect of Treg blockade, indicating that shifting the balance between the two types of NKT cells toward immunosuppressive type II NKT cell dominance suppresses tumor immunity even in the absence of Tregs. We conclude that both Tregs and type II NKT cells can concurrently suppress tumor immunity and the balance between these is controlled in part by a third T cell, the type I NKT cell, regulating the regulators. Finally as cancer patients often have deficient type I NKT cell function somewhat like the Jα18KO mice, managing this delicate balance among three T cells may be critical for the success of immunotherapy of human cancer.

Citation Format: Liat Izhak, Elena Ambrosino, Jessica J. O'Konek, Shingo Kato, Stanley T. Parish, Zheng Xia, David Venzon, Jay A. Berzofsky, Masaki Terabe. Delicate balance among three types of T cells in concurrent regulation of tumor immunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 460. doi:10.1158/1538-7445.AM2013-460