bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3139: Chromosomal copy number alterations involved in Ras/PI3-kinase pathway in endometrial carcinomas
Authors: Ikeda Yuji , Oda Katsutoshi , Murayama-Hosokawa Satsuki , Nakagawa Shunsuke , Ishikawa Shumpei , Yamamoto Shogo , Shoji Keiko , Uehara Yuriko , Hiraike Osamu , Kawana Kei , Yano Tetsu , McCormick Frank , Kozuma Shiro , Taketani Yuji , Aburatani Hiroyuki
Published: February 2011

[Objectives]: Activation of Ras/PI3-kinase (PI3K) pathway plays a direct causal role in human cancer. Oncogenic mutant Ras remains in active state, and activates various Ras effectors, including PI3K pathway. Ras/PI3K pathway activating mutations are very frequent in endometrial cancer, as reported at 35-55% for PTEN, 25-36% for PIK3CA and 10-20% for K-Ras. NF1 is a negative regulator of Ras and its alterations have been reported in several types of tumors. Although LOH at PTEN locus (10q23.3) is reported at 15-30%, other chromosomal imbalances involved in the Ras/PI3K pathway are not well characterized in endometrial cancer. In this study, we focused on chromosomal copy number alterations (CNAs), including copy number neutral (CNN) LOH, involved in the Ras/PI3K pathway in endometrial carcinomas.

[Materials and Methods]: Under the approval of institutional ethical committees, we performed 250K or 50K Single Nucleotide Polymorphism (SNP) arrays in 31 endometrial carcinomas with paired DNA and examined allele-specific CNAs at K-Ras, NF1, PIK3CA and PTEN.

[Results]: We detected deletions of PTEN locus in eight out of 31 (26%) tumors; one with homozygous deletions (HD), four with CNN LOH and three with LOH without gain of the opposite allele. In addition to one HD (3%) and three LOH (10%) at NF1 locus, we found gains at the loci of PIK3CA (3q26.3) in four (13%) tumors and K-Ras (12p12.1) in four (13%) tumors. Taken together, we detected 12 (39%) tumors with one or more CNAs involved in the Ras/PI3K pathway. All the four tumors with NF1 deletions coexist with other CNAs involved in the Ras/PI3K pathway (Table). [Conclusion]: Our data unveiled CNN LOH of PTEN and homozygous deletions of PTEN and NF1, which had not been reported by conventional CGH analysis. Various types of CNAs might play roles in activation of the Ras/PI3K pathway and targeting the Ras-PI3K pathway might be effective in endometrial carcinomas with the Ras-PI3K related CNAs.Table.Coexistent CN alterations in the Ras/PI3K pathwayPTEN (Loss)PIK3CA (Gain)K-Ras (Gain)NF1 (Loss)LOH Gain LOH  LOH Gain LOHLOHGain HDLOHGainGainLOH

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 3139.
Abstract 314: Characterization of the Y83C Gab1 mutation in breast cancer
Authors: Holgado-Madruga Marina , Badal Shawn , Wong Albert J.
Published: February 2011

Following addition of various growth factors and cytokines, the Gab1 docking protein is tyrosine phosphorylated, which then activates different signaling pathways. A screening of human breast cancers revealed a mutation in the Pleckstrin homology domain of Gab1, at the 83rd amino acid changing the tyrosine to a cysteine. To explore the impact of this mutation in human breast cancer, the mutation was overexpressed in the MDA-MB 468 cell line. Upon addition of EGF there was a significant higher increase in tyrosine phosphorylation of the mutant versus the wild type that correlated with an increased association with Shc, Shp2, Grb2 and p85. Pull down experiments with the GST/RBD (Ras binding domain of Raf) fusion protein detected higher activity levels of Ras in the mutant form in comparison with the wild type. Soft agar colony formation assays indicated a 2-3 fold increase in the number of colonies in the Y83C mutation with respect to the wt Gab1. The information understood from this study can help to characterize a mutant of Gab1 and this plays an important role in the context of personalized medicine by elucidating a specific target against tumors which express the mutation.

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 314.
Abstract 3168: Knock down of m-calpain in a mouse breast carcinoma cell line reduced tumor growth in mouse engraftment studies and compromises the Akt pathway and in vitro cell migration
Authors: Ho Wai-chi , Greer Peter A.
Published: February 2011

Calpains are a family of calcium-dependent intracellular cysteine proteases consisting of fourteen members, with µ- and m-calpains (calpain 1 and 2) being ubiquitously expressed. Calpains play crucial roles in a various cellular functions including cell survival, movement and calcium homeostasis. Abnormal expression or activation of calpain is linked to pathological conditions such as neurodegenerative diseases, tumor growth and metastasis, platelet malfunction and muscular dysfunction. Studies have showed that calpain is involved in retraction of focal adhesions at the rear of migrating cells, indicating a major role of calpain in cell movement and cytoskeletal organization. In addition, calpain may be a key component in regulating cell survival and gene expression.

We have examined the role of m-calpain in tumor growth. Mouse mammary carcinoma cells AC2M2 were transduced with a lentiviral vector expressing shRNA directed against m-calpain or a control shRNA. A mouse xenograft experiment was then carried out by injecting control cells or m-calpain knockdown cells into the mammary fat pads of nude mice. Preliminary results showed that mice injected with m-calpain knockdown cells grow smaller tumors. In vitro transwell migration assay showed that the m-calpain knockdown cells migrated slower than the control cells. Furthermore, biochemical studies demonstrated that these AC2M2 m-calpain knockdown cells have reduced activation of key the survival mediator Akt, although there was no significant difference in the growth rate between the knockdown and the control cells. Other biochemical studies implicate the transcription factor Foxo, a downstream target of Akt, in mediating the m-calpain-regulated tumor progression through activation of transcription of genes that are important in cell survival such as p27 Kip1 and Bim. In summary, our current data suggests that m-calpain mediates tumor growth through regulation of Akt signaling. This in turn regulates Foxo in the activation of gene transcription during cell survival signaling.

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 3168.
Abstract 3182: Decreased expression of PTEN enhances CXCR4-mediated proliferation and tumorigenesis in prostate cancer cells
Authors: Chetram Mahandranauth A. , Hinton Cimona V.
Published: February 2011

The progression of human prostate cancer is a result of metastasis from the primary tumor to vital organs. Metastasis is a complex process that involves invasion, intravasation, extravasation, and metastatic colonization. To date, the biology underlying the various mechanisms of metastasis has not been fully elucidated. Chemokines are pro-inflammatory molecules that bind to chemokine receptors, which are members of the G-protein coupled receptor (GPCR) family. The interaction between chemokines and their receptors results in a diverse array of biological and biochemical functions, such as chemotaxis, hematopoiesis and angiogenesis. Likewise, neoplastic cells employ chemokines and their receptors to promote metastasis, and encourage cell survival. It has been observed that the CXCR4 chemokine receptor is overexpressed on the cell surface of prostate cancer cells, which directed metastatic cells to tissues where its CXCL12 ligand is overexpressed, such as the bones and lungs. The Phosphate and Tensin homolog deleted on chromosome 10 (PTEN) is the second most mutated tumor suppressor in human cancer, and has been shown to be mutated in metastatic prostate cancer cells. Gao et al demonstrated that reconstituted PTEN in lymphocytes down-regulated CXCR4-mediated chemotaxis towards its ligand, CXCL12. Additionally, progenitor PTEN-deficient smooth muscle cells (SMC) increased migration towards CXCL12-producing SMC, suggesting that an alteration in PTEN expression negatively regulated CXCR4-mediated events. We analyzed prostate cancer cells for the expression of CXCR4 by flow cytometry, and observed that CXCR4 was expressed in human metastatic prostate cancer cell lines, PC3 and LnCaP. We analyzed for the expression of PTEN, and observed no expression of PTEN in PC3 and LnCaP cells at the protein level by western blot, nor at the mRNA level in PC3 cells by PCR. Furthermore, when reconstituted with PTEN, PC3 cells demonstrated a mesenchymal to epithelial-like phenotypic change in morphology. Additionally, we observed that reconstituted PTEN inhibited CXCR4-meditated proliferation via an MTT assay. Therefore, we hypothesize that the absence of PTEN expression correlates with an up-regulation of CXCR4-mediated proliferation and tumorigenesis in prostate cancer cells. In summary, our results showed that PTEN induced an epithelial-like morphological change, and inhibited CXCR4-mediated proliferation in metastatic prostate cancer cell lines. In future studies, we will investigate if the reconstituted expression of functional PTEN regulates CXCR4-mediated metastatic events in prostate 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 3182.
Abstract 3201: Identification of the type 2 cystatins (SA/SN) as a novel biomarkers for stress-induced senescence in human non-small cell lung cancer lines
Authors: Cheng Guanjun , Wang Liang-Chuan S. , Crisanti Cecilia , Sun Jing , Kapoor Veena , Albelda Steven M.
Published: February 2011

Purpose: Stress-induced cellular senescence (SIS) is becoming increasingly well recognized as an important process in premalignant neoplastic lesions and the response to chemotherapeutic drugs and radiation. In addition, senescent cells may release growth factors, cytokines, proteases, and other factors that affect the tumor microenvironment. The standard maker of SIS (staining for β-galactoside [β-gal] at pH 6) cannot be used in fixed tissues. New and more consistent biomarkers of SIS that could be used in tissue sections are thus needed to improve early detection of premalignant lesison, to design and monitor cancer therapeutic approaches, and to gain additional mechanistic understanding.

Experimental Procedures: Doxorubicin (Dox) was employed to induce senescence in A549 and L55 human non-small cell lung cancer lines. Gene expression profiles were compared in control vs Dox-treated A549 cells. Validation was carried out by real time quantitative PCR (qPCR). Type 2 Cystatin (SA/SN) protein expression levels were evaluated by immunoblotting and immunohistochemistry (IHC) using an antibody (Santa Cruz) that recognized both proteins.

Results: Dox induced senescence of A549 and L55 cells as evidenced by cellular enlargement, cessation of proliferation, and positive staining for ß-gal at pH 6.0. Microarray screening revealed marked upregulation of the genes for Cystatin SA and SN, that was confirmed by qRT-PCR. High protein expression levels of type 2 cystatin (SA/SN) was demonstrated in Dox-treated cells by immunoblotting and IHC. IHC was successful in ethanol-fixed and formalin-fixed paraffin embedded slides.

Conclusions: The cystatin superfamily is a well-established family of cysteine protease inhibitors that have been suggested to affect cell differentiation, growth, and migration through protease inhibition and by direct receptor mediated effects. The role of cystatin SA/SN in SIS is under study. However, our results identify two Type 2 Cystatins (SA/SN) that appear to be promising biomarkers for SIS in human non-small cell lung cancer. Cystatin staining could be utilized in fixed tissue sections to screen for oncogene-induced premalignant lesions and for studying the effects of chemotherapeutic drugs and radiation in lung cancer or bronchial biopsy samples.

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 3201.
Abstract 3214: C-strand fill-in at human telomeres requires lagging strand synthesis machinery and is essential for maintaining functional telomeres
Authors: Chai Weihang , Huang Chenhui
Published: February 2011

Telomeric single-stranded G-overhangs are required for protecting chromosome ends and for telomerase elongation of telomeres. However, the molecular mechanism for G-overhang generation at human telomeres is unclear. We report here that G-overhangs undergo cell cycle-regulated dynamic changes independent of telomerase activity. G-overhangs are lengthened during S phase due to extensive C-strand resection, and shortened at G2 phase as a result of C-strand fill-in. This delayed C-strand fill-in is mediated by lagging-strand polymerases and regulated by cyclin-dependent kinase 1. Inhibiting C-strand fill-in induces telomere DNA damage response and a G2 arrest, suggesting a critical role of proper G-overhang generation in maintaining genome stability. In addition, the last nucleotide at C-strand remains precisely defined throughout the cell cycle. Our results indicate that both C-strand resection and fill-in are strictly regulated during the cell cycle to ensure proper G-overhang generation, and unveil an essential role of C-stand fill-in in maintaining functional telomeres.

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 3214.
Abstract 3227: Lung-targeted expression of B-RAF V600E in alveolar type II cells causes emphysema and delayed adenoma in mice
Authors: Zanucco Emanuele , Potapenko Tamara , Götz Rudolf , Carraretto Irene , Ceteci Fatih , Rapp Ulf R.
Published: February 2011

Classically oncogenes are found associated with neoplastic diseases. More recently a connection with developmental syndromes has been reported. Here we describe a link between the most prevalent mutant form of B-RAF oncogene (B-RAF V600E) with Chronic Obstructive Pulmonary Disease (COPD) that often precede lung cancer in men. By use of targeted expression of this mutant B-RAF in type II pneumocytes of mice we have developed an animal model for these devastating diseases that are epidemic worldwide. Constitutive expression of B-RAF V600E led to emphysema as early as late embryonic development that is persistent throughout adult life when it is often accompanied by chronic inflammation. Infiltrating immune cells include localized macrophages, mast cells, and leukocytes as in the case of human COPD. Inflammatory cell infiltration does not precede emphysema formation but is a prelude to tumor development. Consistently, ablation of RAG1 alleles did not eliminate emphysema or late tumor development. The oncogene involved in tumor formation is also B-RAF V600E because the most frequent mutations of spontaneous lung adenomas in mice were absent. These data support a model where the regenerative process triggered by continuous emphysema formation is the driving force that converts B-RAF V600E from a latent to an active oncogene in this target cell type. Our data suggest that B-RAF V600E is not a complete oncogene in type II cells and requires promoting conditions that can be provided by emphysema.

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 3227.
Abstract 3233: Inhibition of aldose reductase prevents lung cancer cell growth in vitro and in vivo
Authors: Ramana Kota V. , Tammali Ravinder , Singhal Sharad S. , Awasthi Sanjay , Srivastava Satish K.
Published: February 2011

Lung cancer is the second most common cancer and the leading cause of cancer death among both men and women. Although, tobacco smoking is the dominant etiologic factor responsible for the vast majority of lung cancers, other factors such as asbestos, pollutants, polycyclic aromatic hydrocarbons, nutritional and dietary factors which can cause pulmonary inflammation can also drive carcinogenesis by altering cellular and molecular targets and pathways that are crucial to normal tissue homeostasis. However, the molecular signaling events specific to the pulmonary inflammation and their effects on pulmonary cells leading to carcinogenesis are poorly understood. Hence, we investigated the role of polyol pathway enzyme aldose reductase (AR), an oxidative stress response protein that catalyzes the reduction of lipid peroxidation -derived lipid aldehydes and their glutathione conjugates, in the mediation of carcinogenic signals leading to lung cancer. We examined the effect of pharmacological and siRNA -mediated inhibition of AR on in vitro human lung carcinoma cell line A549 growth as well as tumor growth in nude mice xenografts. We have also examined the molecular mechanisms by which AR inhibition prevents carcinogenic signaling events that cause lung cancer cell growth. Treatment of A549 cells with AR inhibitors prevented the activation of NF-kB and expression of NF-kB- dependent inflammatory markers such as iNOS and Cox-2. AR inhibition also prevented the proliferation of cultured human lung cancer cells. Further, AR inhibition significantly arrested the tumor growth in nude mice bearing lung adenocarcinoma xenografts. Similar results were observed in AR-siRNA treated nude mice xenografts. Immunohistochemical analysis of nude mice tumor cross sections indicated that AR inhibition prevents activation of iNOS, Cox-2, AR and NF-kB. Our studies thus strongly suggest that AR may be a critical regulator of signaling pathway(s) that cause lung cancer. Hence, disrupting these signals by AR inhibition could be a novel preventive approach in lung cancer 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 3233.
Abstract 3239: Synergy of PIM1 kinase with c-MYC in prostate cancer
Authors: Wang Jie , Kim Jongchan , Roh Meejeon , Franco Omar E. , Martinez Erin , Hayward Simon W. , Wills Marcia L. , Abdulkadir Sarki A.
Published: February 2011

The oncogenes PIM1 and c-MYC are frequently up-regulated in human prostate tumors. We found that PIM1 and c-MYC were co-expressed in human prostate cancer and were associated with higher Gleason scores. We next used a model of prostate regeneration coupled with lentiviral-mediated gene transfer to directly show that Pim1 dramatically enhanced c-MYC tumorigenicity in naïve adult mouse prostatic epithelium in a kinase-dependent manner. Microarray analysis revealed that many signaling pathways including MAPK, focal adhesion calcium signaling pathway were activated in MYC/Pim1 tumor. Knockdown of Pim1 in MYC/Pim1 tumor derived cell line reduced ERK and Akt phosphorylation. We further showed that targeting Pim1 by shRNA inhibited tumorigenicity in vitro and in vivo. Pim1 kinase inhibitor quercetagetin also inhibited cell growth in vitro. These results directly demonstrate that MYC and PIM1 cooperate in promoting tumor development. PIM1 can be considered as a drug target for human prostate cancer treatment.

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 3239.
Abstract 3243: Differential effects of caloric and carbohydrate restriction upon prostate cancer tumor growth in a mouse model
Authors: Thomas Jean A. , Masko Elizabeth M. , Antonelli Jodi A. , Pooulton Susan H. , Phillips Tameika E. , Coburn Tonisha , Freedland Steve J.
Published: February 2011

Background: Caloric restriction (CR) has been shown to be anti_cancer. However, whether these effects are the result of generalized reduction in overall caloric intake or reduced intake of macronutrients (i.e. carbohydrates) are unknown. We sought to investigate the differential effects of caloric and carbohydrate restriction upon prostate cancer tumor growth in a xenograft model.

Materials and Methods: A total of 100 male SCID mice were injected subcutaneously with the LAPC-4 cell line. Mice were randomized to 1 of 4 diets: ad libitum Western diet (WD, 49% carbohydrates, 35% fat, and 16% protein), pair-fed No Carbohydrate Ketogenic Diet (NCKD; 1% carbohydrates, 83% fat, 16% protein), Western diet calorically restricted (WD-CR), and NCKD calorically restricted (NCKD-CR). Calorically restricted arms were fed 75% of the calories of their reference diet group. Tumor volumes and body weights were measured twice weekly. Mice were sacrificed when tumor volumes reached 1,000 mm3.

Results: Relative to WD, there was a suggestion of smaller body weights in both NCKD-CR and WD-CR arms (rank-sum, p≤0.06). There was trend for heavier mice in the NCKD group (rank-sum, p=0.09). By day 50, median tumor volumes for NCKD, WD-CR, and NCKD-CR were 45, 51, and 62% smaller than WD (rank-sum, all p≤0.001). At any time point, there were no significant differences in tumor volume among the NCKD, NCKD-CR, and WD-CR arms. However, there was a suggestion of smaller tumors in the NCKD-CR arm by day 42 relative to NCKD and WD-CR (p<0.11). Diet was significantly associated with mouse survival (log_rank, p=0.004). In 2_way comparisons, NCKD, NCKD-CR, and WD-CR had significantly prolonged survival relative to WD (log_rank, all p≤0.02). However, no difference in survival was seen among any caloric or carbohydrate restricted arms (log rank, p≤0.16). A total of 34 mice died due to infection prior to their tumors reaching 1,000mm3 and therefore were not included in the survival analyse (WD-CR, n=14; NCKD, n=5; NCKD-CR, n =15).

Conclusions: Both CR and carbohydrate restriction without weight loss are effective in delaying tumor growth and improving mouse survival. We found no differences between caloric and carbohydrate restricted arms, though this may relate to loss of study power due to mouse infections. CR may further weaken the immune system of immunocompromised mice subjecting them to increased risk of infections. A confirmatory study is underway in a high-level barrier facility.

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 3243.
Abstract 3247: Establishment of a castration-resistant prostate cancer model, JDCaP-hr: Implications for prostate cancer recurrence
Authors: Nakata Daisuke , Masaki Tsuneo , Tanaka Akira , Kakoi Yuichi , Nakayama Yusuke , Kusaka Masami , Watanabe Tatsuya
Published: February 2011

There are few in vivo models of androgen-dependent prostate cancer (ADPC), and limited availability of the models makes it difficult to evaluate anticancer agents for early stage prostate cancer and to investigate the recurrence of ADPC. We previously reported JDCaP xenograft model, an ADPC model with wild type androgen receptor (AR) and TMPRSS2-ERG gene fusion, derived from skin metastatic lesions of a prostate cancer patient. JDCaP xenografts completely regressed after surgical or medical castration; however, some of them relapsed several months after castration. The relapsed JDCaP subline, designated JDCaP-hr (hormone refractory), was established by serial passage in castrated nude mice. The AR antagonist bicalutamide did not show any effect on tumor growth of JDCaP-hr, while testosterone treatment induced tumor regression of JDCaP-hr, indicating that ligand binding to AR is not necessary, rather cytotoxic, for the growth of JDCaP-hr. Direct sequencing analysis of full length AR revealed that JDCaP-hr retained wild type AR. AR mRNA expression in JDCaP-hr was approximately 7-fold higher than in JDCaP, and interestingly, truncated AR protein with molecular weight of approximately 75 kDa in addition to full length AR (110 kDa) was expressed in JDCaP-hr. Microarray analysis revealed that ubiquitin-dependent protein catabolism pathway was markedly upregulated in JDCaP-hr compared with JDCaP. JDCaP-hr cells proliferated in vitro in steroid-free medium under co-culture condition with mouse stromal cells, and secreted prostate-specific antigen into the conditioned medium. Taken together, JDCaP/JDCaP-hr is a valuable model which simulates the recurrence of ADPC to castration-resistant prostate cancer (CRPC) with common clinical features such as AR overexpression. This model provides not only useful in vivo and in vitro systems to evaluate anticancer agents for ADPC and CRPC but also intriguing implications for the recurrence of ADPC.

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 3247.
Abstract 3248: PSMA specific imaging of prostate cancer
Authors: Reddy Joseph A. , Xu Le-Cun , Dorton Ryan , Bloomfield Alicia , Nelson Melissa , Klein Patrick , Kularatne Sumith , Low Philip , Leamon Christopher
Published: February 2011

A peptide derivative of a prostate specific membrane antigen (PSMA) targeting ligand [Glu-C(O)-Glu] was designed to efficiently coordinate 99mTc. This chelate, referred to as EC0652, was found to bind cultured PSMA-positive LNCaP tumor cells in a concentration-dependent manner with very high affinity (Kd ∼ 20 nM). Its binding was reduced > 10 fold in the presence of the ligand competitor PMPA (a high affinity PSMA binder). In addition, this molecule did not bind to panel of PSMA negative cells (4T1, A549, IGR-OV1, J6456, KB, L1210 and M109), thereby demonstrating the agent's PSMA-specific binding property. Following intravenous injection into nu/nu mice, 99mTc-EC0652 was rapidly cleared from circulation (and non-targeted tissues), and only 1.3% of the injected dose remained in the blood after 1 hour. Data from quantitative biodistribution studies confirmed that 99mTc-EC0652 predominantly accumulates in PSMA-positive subcutaneous LNCaP tumor and kidney (PSMA/glutamate carboxypeptidase positive) tissues. Net uptake in the tumor and kidney remained steady throughout the 8 h experiment resulting in a continued increase in tumor to background tissue radioactivity with time. Radioactive uptake in both of these tissues was reduced > 10 fold when this radiodiagnostic imaging agent was co-dosed with PMPA. The safety of decayed 99mTc-EC0652 was evaluated as a single intravenous dose in an acute toxicology study in male mice. All doses tested were well tolerated with no demonstrated toxicity. The study established a no observed adverse effect level (NOAEL) of 14.0 mg/kg, a dose which provides an appropriate safety margin relative to the FDA designated Phase 0 dose. Equipped with these encouraging results, preparations have begun for a Phase 0 clinical trial to evaluate 99mTc-EC0652 distribution in patients with metastatic prostate cancers.

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 3248.
Abstract 3255: Relevance of mutated PIK3CA to normal mammary gland development and malignancy
Authors: Merino Vanessa F. , Park Sunju , Liang Xiaohui , Chun Yong Soon , Sukumar Saraswati
Published: February 2011

In the current study we generated transgenic mice with mammary gland specific- overexpression of PIK3CA wild type and mutant protein with the aim to characterize the effects of activation of PI3K signaling on normal mammary gland and tumor development. PIK3CA encodes p110α, the predominant isoform of the catalytic subunit of class 1A phosphatidylinositol 3-kinase (PI3K), a lipid phosphokinase. The family of PI3Ks provides signaling for diverse cellular functions, including proliferation, metabolism, migration, translation, apoptosis avoidance, and angiogenesis. Disruption of this tightly regulated pathway by gene loss (PTEN), mutation (PIK3CA, AKT1, or less commonly PIK3R1), or amplification (PIK3CA), is one of the most common alterations in human cancers. PIK3CA mutations result in constitutive activation of p110α, increasing lipid kinase activity and resulting in an increase in activated AKT. The majority of mutations in breast cancer occur at three hotspots: E542K and E545K at exon 9, which encode the helical domain, and H1047R at exon 20, which encodes the kinase domain. Previously, it was described that human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. In a mouse model in which PI3K is activated by forced recruitment of p110α to the membrane, increased ductal branching, alveolar hyperplasia, intraductal neoplasia and a low frequency of mammary tumors were observed. To date, no in vivo studies have been reported to determine if PIK3CA mutations play a causative role in breast tumorigenesis. By performing whole mount analysis of mammary gland at 8 weeks of age, we have shown that mice harboring E545K mutation showed increased ductal branching, while expression of H1047R (high copy numbers) results in duct enlargement and defects in duct elongation. Two mice harboring low and high copy numbers of H1047R mutation developed mammary tumors at 14 and 16 months of age respectively. Histological analysis revealed that both were carcinomas with tubular, papillary and comedo features. In conclusion, transgenic mice with mammary gland specific- overexpression of PIK3CA wild type and hot spot mutations were generated. In transgenic mouse studies, we show that PI3K signaling activation appears to alter normal mammary gland development and initiate mammary tumor formation.

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 3255.
Abstract 3259: HIV protease inhibitors induce apoptotic and non-apoptotic cell death in ovarian cancer cells in vitro and in vivo
Authors: Vandeven Natalie A. , McLean Karen , Sorenson Dorothy R. , Liu J. Rebecca
Published: February 2011

Introduction: HIV patients taking antiretroviral protease inhibitors have a lower incidence of HIV associated malignancies, leading to the hypothesis that these drugs have antineoplastic activity. Given the need for novel treatment approaches in ovarian cancer, we investigated the antineoplastic effects of the HIV protease inhibitors saquinavir and nelfinavir in vitro and in vivo. Methods: A panel of ovarian cancer cell lines was treated with saquinavir and nelfinavir. The effect of protease inhibitors on cell growth, viability, apoptotic and non-apoptotic cell death was determined. Stimulation of endoplasmic reticulum stress (ERS) response was assessed by immunoblotting for ERS regulators GRP78 and ATF6. Induction of autophagy was assessed using transmission electron microscopy (TEM), and confocal microscopy was performed to demonstrate changes in green fluorescent protein-labeled LC3 expression patterns. A xenograph model of ovarian cancer was used to assess the antineoplastic effect of nelfinavir in vivo. Results: HIV protease inhibitors induced cell death in chemosensitive and chemoresistant ovarian cancer cells in a time- and dose-dependent manner. Treatment with protease inhibitors resulted in caspase-dependent apoptosis and caspase-independent cell death characterized by induction of ERS and autophagy. Cellular morphology assessed by TEM revealed apoptotic, autophagic, and necrotic cell death. Preliminary results indicate that nelfinavir administer intraperitoneally inhibits tumor growth in vivo without inducing weight loss. In contrast, the dose of intraperitoneal cisplatin required to attain a similar antineoplastic effect resulted in significant weight loss and morbidity. Conclusions: Because protease inhibitors can induce both apoptotic and non-apoptotic cell death, these drugs may circumvent chemoresistance due to alterations in apoptotic response. Saquinavir and Nelfinavir are FDA-approved agents for the treatment of HIV, and our data suggest that these drugs may also have clinical application in the treatment of ovarian 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 3259.
Abstract 327: Genomic profiling of sentinel lymph node breast cancer metastasis
Authors: Torresan Clarissa , Santos Savana C. , Camps Jordi , Pereira Silma F. , Ribeiro Enilze M. , Cavalli Iglenir J. , Haddad Bassem R. , Cavalli Luciane R.
Published: February 2011

The sentinel lymph node (SLN) is the first node in the axilla to harbor malignant cells in breast tumors with metastasis, and its positivity is an indication for axillary lymph node dissection. Despite significant improvement in the methods for detection of tumor cells at this site, there are a still significant number of false negative cases and the inability to detect malignant cells in lower abundance, such as in the case of micrometastasis. We propose to use methods based on the genomic alterations, which present higher sensitivity and accuracy, and can be used to augment the current methods to identify the epithelial malignant cells at the SLN. In this study, we performed CGH and array-CGH analysis in microdissected tumors cells from the SLN metastatic lesions to identify DNA copy number changes. We compared the changes observed in the SLN lesion to the ones present in the corresponding primary tumors (PT) from the same patients. Here we present the results from the initial analysis of 10 FFPE (formalin-fixed paraffin-embedded) breast cancer samples (5 pairs of SLN and PT). DNA copy number changes were detected by both methods in all the cases analyzed. A remarkable similarity in the DNA profiles between the SLN metastatic lesions and the corresponding PT were observed, despite the high level of heterogeneity observed among the cases. CGH analysis revealed most frequently gains of chromosomal regions (at both lesions) involving 1p, 1q, 6q, 9q, 11p, 11q, 13q, 16p, 17q, 19p, 19q and Xq. Array-CGH analysis detected both amplifications and deletions, affecting mostly the chromosome regions 1p (genes affected KLHL17, NOC2L, PPIAL4), 9q (ANKRD20A, COL5A), 11p (PSMD13, SIRT3), 15q (MAN2C1), 19p (MUC16, AZU1) and 20q regions (PSMA7). In addition, several genes of the HIST, FLJ, SLC and TRIM family as well as the homeobox families HOX and NK were involved in the alterations. FISH analysis is being conducted to confirm the array-CGH findings as well as the analysis of additional matching samples. The assessment of copy number changes in the SLN metastasis (and in its correspondent PT) using genomic profiling is a very sensitive method and will lead to the identification of genomic alterations that can reliably characterize breast tumor cells at the SLN lesions. Once these alterations are validated in larger series of patients, they can be used as molecular markers that can improve the predictive value of the current established methods of SLN evaluation in the identification of patients at higher risk of developing axillary lymph node metastasis.

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 327.
Abstract 3277: The Fps/Fes protein-tyrosine kinase serves a stromal tissue-specific supporting role in an orthotopic mouse model of breast cancer
Authors: Zhang Shengnan , Elliott Bruce B. , Greer Peter A.
Published: February 2011

Fes (also known as Fps) is a cytoplasmic protein tyrosine kinase that is highly expressed in breast epithelial cells during lactation, as well as in vascular endothelial and myeloid cells. Retrovirally encoded oncogenic alleles of Fps/Fes are associated with tumors in chickens and cats and cause tumors in transgenic mice; however, a role for Fes in human cancer has not been established.

We hypothesized that Fes promotes breast tumorigenesis through tumor cell intrinsic functions as well as through functions in stromal tissues including vascular endothelial cells and macrophages. To test the potential tumor cell intrinsic role, AC2M2 cells, a highly metastatic mouse mammary carcinoma cell line, were transduced with expression plasmids encoding wild-type, kinase-inactivated or activated Fes. These cells were then engrafted into the mammary fat pads of nude mice and tumorigenesis was assessed by ultrasound (longitudinal tumor volume measurement), Doppler (tumor-associated vascular flow) and biophotonic imaging of GFP-expressing tumor cells (lung metastasis). There was no obvious difference in tumor growth and angiogenesis at the orthotopic site between each cell line and no apparent difference in lung metastasis. To test the potential stromal role of Fes in breast tumorigenesis, parental AC2M2 cells were injected into the mammary fat pads of wild type and Fes-null nude mice, and tumorigenesis and metastasis were assessed as described above. Wild type mice developed tumors significantly faster than Fes-null mice and there was earlier tumor neovascularization and more recruitment of macrophages. There was also a significantly higher degree of lung metastasis in wild type vs Fes-null mice.

These observations are consistent with a tumor promoting role for Fes acting at the level of the vascular endothelium and macrophages. This might include promoting early tumor neovasularization through direct roles in vascular endothelial cells as well as indirect roles associated with macrophages. The increased metastatic potential also suggests roles in both these cell lineages. This provides genetic evidence that the Fes protein-tyrosine kinase represents a potential novel therapeutic target in breast 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 3277.
Abstract 3278: Mitochondria derived from breast cancer MDA-MB-231 cells confer oncogenic properties in the normal breast epithelial nuclear background
Authors: Ma Yewei , Wong Lee-Jun C.
Published: February 2011

Previous reports showing somatic mitochondrial DNA alterations, the reduction of respiratory chain function, and the essential roles of mitochondria in apoptosis and autophagy, have implicated that mitochondria are vital in cancer development. To study the contribution of cancer mitochondria to tumor properties, the major effects from the nucleus must be removed. We developed an in vitro transmitochondrial cybrid system, in which, mitochondria derived from normal breast epithelial cells (MCF-10A) or breast cancer cells (MDA-MB-231) were fused with ρ0 nuclear donor cells derived from immortalized normal breast epithelial A1N4 cells. Studies of these cybrids showed reduced respiratory chain activities, ATP synthesis rates, and oxygen consumption, in 231/A1N4 cybrids compared to 10A/A1N4 cybrids. In addition, higher production of ROS and lactate were also noted. These results suggested that cancer mitochondria had reduced oxidative energy production function. With the same normal breast epithelial nuclear background, cybrids containing cancer mitochondria exhibited higher expression of oncogenes and growth factors than cybrids containing mitochondria derived from non-cancer epithelial cells (MCF-10A). The 231/A1N4 cybrid cells lost growth contact inhibition, had higher ability to form colonies, and were more resistant to serum starvation compared to 10A/A1N4 cybrids, supporting the hypothesis that mitochondria derived from cancer cells contribute to tumor properties even in the normal nuclear background. These studies provide useful insights to the cross-talk between the mitochondrial and nuclear genomes of altered mitochondria to normal 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 3278.
Abstract 3280: Generation of orthotopic mouse models of breast cancer
Authors: Palafox Marta , Pellegrini Pasquale , Barbera Laura , Urruticoechea Ander , Climent Josefina , Soler Maria Teresa , Gonzalez-Suarez Eva
Published: February 2011

The current systemic treatments for breast cancer include chemotherapy, and targeted approaches such as anti-hormonal and anti-Her2 treatments. These treatments are effective only in a percentage of cases and even those initially responsive tumors eventually acquire resistance. It is critical to define the mechanisms of resistance and to identify the population of resistant cells to target them efficiently. We have generated mouse models for the study of breast cancer tumors based on the orthotopic implantation of fresh primary human tumor pieces and cancer cells isolated from pleural effusions in immunodeficient mice. A total of 55 fresh tumor pieces and tumor cells isolated from 5 pleural effusions have been implanted into the cleared fat pad of nude, NOD/SCID and SCID/beige mice. Slow release estrogen pellets have been implanted in the intercapsular region at the same time as tumors. Nine out of the 60 samples implanted gave rise to palpable lesions, 5 derived from fresh tumor pieces and 4 from tumor cells isolated from pleural effusions and are being maintained by serial passages in mice. Currently we have five well-established tumors, 2 TNBC (triple negative breast cancer), 2 luminal tumors and one Her2 positive tumor. Morphologically tumors growing in mice resemble human tumors. Latency to tumor development and growth rates are being evaluated in different serial passages and host mice. Tumors at different passages are being characterized at the histological level and for the expression of ER, PR, Her2, EGFR, cytokeratins and p53, and compared with the primary human tumor. Once tumors are established and characterized we will test their sensitivity to drugs currently used in the clinic. Orthotopic tumor xenografts will allow gaining insight into the molecular mechanisms involved in the resistance. Genetic and epigenetic analysis will allow to identify the chromosomal regions related with resistance. Similarly, differential gene expression profiling will be determined by microarray analysis.

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 3280.
Abstract 3283: Developmental and metabolic deregulation in 14-3-3zeta knockout mouse
Authors: Yang Jun , Li Ping , Yu Dihua
Published: February 2011

14-3-3s are a family of proteins conserved throughout eukaryotics. By binding to specific phosphorylated serine/threonine containing motifs in the target proteins, 14-3-3s modulates their molecular structure, stability, subcellular localization and/or enzymatic activity. Our lab recently found that 14-3-3ζ (zeta) is overexpressed in ∼45% breast cancers and 14-3-3ζ overexpression predicts poor patient survival. Additionally, 14-3-3ζ prevents anoikis by suppressing p53 and promotes epithelial mesenchymal transition by activating TGB-β signaling pathway, which plays important roles during tumor formation and progression. These led us to we hypothesizing that 14-3-3ζ plays important role in breast cancer.

In our initial attempt to systematically study the role of 14-3-3ζ in breast cancer in vivo, we have recently generated 14-3-3ζ conventional knockout mouse by Gene Trap. We obtained the ES cell line RRR334 (which has no functional 14-3-3ζ based on the 5’ RACE data of the Baygenomics) from the Mutant Mouse Regional Resource Center. We confirmed silencing of 14-3-3ζ in the clone by RT-PCR. The integration site of the Gene Trap vector was located about 5kb downstream of exon 3 inside the intron 3 and PCR genotyping primers were designed accordingly. The ES cells were injected into C57Bl/6 albino mouse blastocysts. Chimera mice with high ES cell contributions based on their fur color were mated with C57Bl/6 albino mice to determine germ line transmission. When we mated the founder 14-3-3ζ heterozygous (+/−) male with 14-3-3ζ +/− female, only ∼ 5% of the offspring's were 14-3-3ζ homozygous (−/−) mice, indicating 14-3-3ζ −/− mice are embryonic lethal. This lethality hinders our study of the role of 14-3-3ζ in tumorigenesis and progression. To overcome this, we outbred to CD-1 mouse strain and the resulting mice are viable and fertile. Interestingly, the 14-3-3ζ knockout mice are smaller than their littermates. This growth retardation did not seem to be mediated by deregulated growth hormone level. The 14-3-3ζ null mice also had lower blood glucose level and their livers and brain had a reduced glucose uptake. Our preliminary studies indicated that HIF1-α, a well known oncogene and a master regulator of metabolism (e.g., glycolysis), is downregulated in 14-3-3ζ null mice. We are currently further characterizing the phenotypes of 14-3-3ζ knockout mouse and also studying the molecular mechanism of how loss of 14-3-3ζ leads to growth retardation and how 14-3-3ζ regulates glucose metabolism.

We are also studying whether loss of 14-3-3ζ has any impact on tumor formation and metastasis.

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 3283.