Citations

Abstract Introduction Cytidine drugs, such as gemcitabine, undergo rapid, inactivating catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine drug metabolism. However, p.o. THU is at most 20% bioavailable (Beumer et al.), which limits its preclinical evaluation and clinical use. Therefore, the more lipophilic prodrug triacetyl THU (taTHU) was developed. We characterized THU pharmacokinetics after administration of taTHU to mice. Methods Mice were dosed 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters and p.o. bioavailability were calculated non-compartmentally and compartmentally. We assessed taTHU inhibition of metabolism of gemcitabine by recombinant CD. ResultsTHU parameterstaTHU i.v. 150 mg/kgtaTHU p.o. 150 mg/kgCmax (µg/mL)10611.8Tmax (min)560Half-life (min)235122AUC0-inf (mg/mL*min)7.553.36Vd/F (L/kg)4.495.26Cl/F (mL/min/kg)13.229.8F-THUi.v. (%)68.030.3% dose in 0-16 h urine405.6% dose in 0-24 h urine5512 THU, after 150 mg/kg taTHU i.v., had a 235 min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. A multi-compartment model fit the data best. 150 mg/kg p.o. taTHU produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122 min half-life. Approximately 68% of i.v. taTHU was converted to THU. Approximately 30% of p.o. taTHU reached the systemic circulation as THU. After i.v dosing, renal excretion accounted for 40-55% of the taTHU dose, 26-35% as THU and an additional 14-19% as acetylated forms of THU. After p.o. dosing, the renal excretion percentages were 6-12%, 5.2-11%, and 0.42-0.71%, respectively. taTHU did not inhibit recombinant CD. Conclusion The availability of THU after p.o. taTHU in mice is 30%, as compared to the 20% achieved after p.o. THU. Clinical studies are warranted to evaluate availability of THU from taTHU in humans. Support: N01-CM07106, N01-CM52202, P30-CA47904 from the NCI and P41-EB001978. 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 4549.

Abstract Breast cancer is the most common form of cancer diagnosed and the second leading cause of cancer death among women in the United States. Cancer metastases are the major cause of morbidity and mortality among breast cancer patients. The central importance of the HER2/Neu/EGFR/RAS signaling pathway has been well established in the growth, invasion and metastasis of human breast cancer, even though oncogenic RAS mutations are rare in breast cancer, activation of the tumor-promoting ERBB/RAS pathway has been consistently documented in high-grade breast tumors. As such, novel approaches to inhibit activated ERBB/RAS signals constitute important measures to block tumor growth and metastasis in mammary tumors. In this study, instead of targeting an upstream signaling component such as HER2/EGFR/RAS, we targeted the most downstream signaling module identified in the RAS pathway - the SIAH proteolytic machinery. SIAH is an evolutionarily conserved RING E3 ligase that is a critical “gatekeeper” required for RAS signal transduction in human cancer cells. SIAH is specifically expressed in tumor cells in 120 human breast cancer patients examined so far. The increased SIAH expression is correlated with increased grades and aggressiveness of human breast cancer. We then asked whether inhibiting the gatekeeper function of SIAH would be effective in blocking mammary tumorigenesis and metastasis in human breast cancer. Our results indicated that SIAH2-deficiency successfully abolished tumor growth of three breast cancer cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468) in soft agar assays as well as in athymic nude mice. Importantly, by inhibiting SIAH2 function in highly aggressive human breast cancer cells, we are able to completely block tumorigenesis and metastasis of two highly invasive and metastatic human breast cancer cell lines (MDA-MB-231 and MDA-MB-435). These findings suggest that SIAH2 may be an attractive new therapeutic target for novel breast cancer therapy and developing anti-SIAH molecules will aid in expanding our arsenal of effective anticancer therapies. More effective breast cancer treatments may be obtained by multi-pronged synergistic inhibitions at upstream (HER2/EGFR membrane receptors), midstream (RAS/RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in the HER2/EGFR/RAS pathway in parallel. Thus, by attacking the growth-promoting ERBB/RAS pathway at multiple signaling modules simultaneously, we may be in a great position to halt the genesis, progression and metastasis of human breast cancer. If successful, this study has the promise to be translated to the cancer clinics to alleviate tumor burdens, inhibit cancer metastasis, improve the efficacy for the treatment of breast cancer, and in particularly help cancer patients with metastatic diseases. 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 4518.

Abstract Introduction: Despite significant progress over the last decade in the development of therapeutics for metastatic cancers including breast cancer (MBC), non small cell lung cancer (NSCLC), and renal cell carcinoma (RCC), most patients will ultimately succumb to their malignancy; more effective therapeutics are sorely needed. The microtubule (MT) is a well-validated target for anti-cancer therapeutics. Ixabepilone (Ixa), a MT stabilizer is approved for MBC and has demonstrated efficacy against other solid tumors. mTOR is another validated target for anti-cancer therapeutics. Temsirolimus (Tem) inhibits mTOR and is approved for advanced RCC. We tested the hypothesis that the addition of Ixa to Tem might augment its anti-tumor activity in vitro. Methods: The cell lines, A498 (RCC), MDA-MB-231 (MBC), and A549 (NSCLC) were treated with Tem alone, Ixa alone, or the combination. All cell lines were also treated with the combination of Tem and Ixa in a fixed ratio of 1:20; Tem doses ranged from 0.125 [nM] to 5 [nM] and Ixa doses ranged from 2.5 [nM] to 100 [nM]. The effects of the treatments were evaluated with clonogenic assays. Synergism was evaluated using CalucSyn 2.0 in accord with the isobologram method of Chou and Talalay. Results: Both Tem and Ixa inhibited cell growth in all tested cell lines as single agents in dose-dependent fashions. Moreover, the combination of Tem and Ixa synergistically enhanced cytotoxicity in all tested lines (see table below).Cell LineED50 [nM]CI (lowest value)A4980.449280.404A5490.285440.263MDA-MB-2310.483550.373 Conclusion: The combination of Ixa and Tem displayed promising activity in all tested cell lines. These findings support the further evaluation of this combination in a phase I clinical trial. 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 4480.

Abstract The number of non-coding transcripts (NCTs) exceeds the number of transcripts that code for protein. Their diverse and important functions are now being identified and utilized in the lab as well as in the clinic. Utilizing the Affymetrix Gene Chip Human Tiling 1.0R Array Set, we analyzed the transcription across the entire genome in both normal human bronchial epithelial cells (NHBE) alone and cells exposed to the tobacco carcinogen (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), NNK. We focused our efforts on the identification and function of novel long non-coding transcripts that had increased expression after exposure to NNK. We report on the identification of 12 long NNK-induced non-coding transcripts (NiTs) that are 300 nucleotides and larger in length. We first validated that these transcripts were indeed stress responsive using quantitative real-time RT-PCR (qPCR) and by Northern blot analysis revealed that these transcripts were between two and four kilobases in length. We also show using qPCR that these transcripts are more abundantly expressed in normal rapidly growing tissues or tissues that are under cellular stress. We show that there is altered expression of NiTs in a panel of lung cancer cell lines and many of these transcripts had increased expression in many breast cancer cell lines. We further analyzed one of these transcripts, NiT5 and identified it to be a 2.5 kb antisense, polyadenylated transcript by RLM-RACE and by Northern Blot analysis. Isolating nuclear and cytoplasmic fractions in BT474 cells, we identified Nit5 to be localized in the nucleus and found it to be transcribed by RNA polymerase III. NiT5 also has increased expression in breast, ovarian and cervical cancer-derived cell lines relative to normals from these three sites, but not increased expression in lung cancer cell lines. In summary, our results suggest that these novel long non-coding transcripts play an important role in stress also may play a role in 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 4083.

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer death in men in the United States. Definitive treatment consists of either radical prostatectomy or radiotherapy followed by androgen deprivation. Although majority of the prostate cancers usually responds to these therapies and can be cured, some of them will eventually relapse. It is believed that the relapsed cancers obtained new ability that facilitates their resistance to these therapeutic stresses. Increased expression of VEGF-C and its receptor Neuropilin-2 (NRP-2) in hormone-refractory and metastatic prostate cancers indicates that the VEGF-C/NRP-2 axis could play an important role in the progression of prostate cancer. Indeed, we observed VEGF-C/Neuropilin-2 axis promotes survival of prostate cancer cells during stress induced by hydrogen peroxide or radiation. Detail signaling events downstream of neuropilin-2 for this stress-resistant function have also been determined. Our findings therefore demonstrated a novel and distinct function of NRP-2 in protecting cancer cells from stress-induced cell death, thereby facilitating cancer recurrence 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 3950.

Abstract Crosstalk between signaling pathways is crucial to the initiation, progression and maintenance of the transformed phenotype. Therefore, characterization of these signaling interactions is fundamental for the understanding of the complex and varied network of events that leads to the development and spread of tumors. The Hedgehog (HH)-GLI pathway, an important carcinogenic cascade, is required for the growth and survival of tumors of diverse origin. Recent reports, mainly from animal models, suggest that key signaling interactions are required by HH-GLI signaling to modulate cancer-associated cellular functions. However, the molecular mechanisms underlying this phenomenon in cancer and non-cancer cells remain elusive. Our preliminary data show a crosstalk between the HH-GLI axis and TGF-beta pathway, a well-characterized tumor suppressor/oncogenic signaling pathway. Luciferase reporter and expression assays in combination with genetic and pharmacological manipulations of the TGFbeta pathway show that this cascade is able to activate GLI transcriptional activity, even in the absence of an active HH signaling. The TGFbeta pathway is required to maintain active GLI transcriptional complexes at GLI target genes. Further analysis of this molecular interaction suggests that TGFbeta pathway-mediated activation of GLI transcription factors requires an intact SMAD4, thus suggesting a potential mechanism mediating this functional interaction. The knowledge derived from this study provides both mechanistic insight into the contribution of this HH-GLI/TGF-beta pathway interaction to carcinogenesis and could serve as a foundation for development of new therapeutic approaches for tumors with active HH-GLI and TGF cascades. 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 3917.

Abstract Genome-wide association analyses have identified an ovarian cancer susceptibility locus near BNC2 at 9p22.2 (OR 0.82, p 5.1 × 10−19, Nat Genet 41:996-1000, 2009), based on 1,772 and 2,354 Stage I cases and controls, 4,803 and 5,237 Stage 2 cases and controls, and extensive Stage 3 analysis in independent samples. Here, we report on Stage 3 analysis of nine additional regions with Stage 2 results at p < 10−4. Stage 1 samples were genotyped using the Illumina 610Quad and imputed at up to 2 million SNPs; Stage 2 samples were genotyped on an Illumina iSelect of 22,790 SNPs. Stage 3 samples from 4,335 cases (including 2,197 serous subtype) and 5,951 controls (14 studies) were genotyped using Illumina or Sequenom at up to 32 SNPs on chromosomes 1, 2, 3, 7, 8, 11, and 17. No evidence of heterogeneity by study site was observed. Logistic regression revealed four regions with ordinal p ≤ 10−7 in all cases or serous subtype only. The most significant finding for all cases was for rs2072590 at 2q31.1 (all cases OR 1.15, p=8.1 × 10−13; serous only OR 1.19 p=9.0 × 10−13) which is located 3.8 kb 3-prime of HOXD3, a homeobox gene which also had an intronic SNP (rs2301301) with p=3.1 × 10−5 (all cases). Second, we observed a locus at 8q24.21 highlighted by rs10088218 (all cases OR 0.84, p=1.3 × 10−9; serous only OR 0.75 p=2.2 × 10−14). This locus resides on the opposite side of MYC and 1.3 Mb from the 8q24 regions harboring loci for other cancers, and is independent of the other cancer-associated variants (r2 < 0.02). Third, at 3q25.31, promising results were observed at rs2665390, a SNP intronic to TIPARP (TiPARP, TCDD-inducible poly(ADP-ribose) polymerase; all cases OR 1.19, p=8.7 × 10−7; serous only OR 1.26, p=3.2 × 10−8). Finally, at 17q21.32, rs9303542 was associated with risk particularly among serous cases (all cases OR 1.13, p=1.5 × 10−6; serous only OR 1.16 p=4.3 × 10−7); this SNP is intronic to SKAP1 (src kinase associated phosphoprotein 1). We evaluated the functional role of genes at these loci in ovarian cancer and normal ovarian epithelial cells and in an in vitro model of neoplastic initiation and progression from normal ovarian epithelial cells after CYMC and KRAS activation. Both BNC2 and TiPARP were down regulated in cancer versus normal cells (p=5 × 10−9 and p=3.4 × 10−5, respectively) suggesting that loss of function is required for cancer development. Over-expressing CMYC (at 8q24) induced neoplastic transformation of normal ovarian cells. Both BNC2 and TiPARP were down-regulated following CMYC over-expression and showed significant loss of function with further transformation after mutant KRAS activation (p=7.8 × 10−7 and p=6.3 × 10−4, respectively). These results emphasize the importance of genome-wide association studies and consortia for the identification of moderate-risk alleles in rare cancers. Importantly, our findings unravel the genetic components of this disease and suggest functionally-significant novel targets. 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 3860.

Abstract Purpose: This study uses spatially accurate image-guided tissue analysis to evaluate the correlation between Perfusion MRI (pMRI) and tissue microvascular density parameters as markers for tumor recurrence and treatment response in high-grade glioma (HGG). Methods: Following Institutional Review Board approval, we recruited previously treated (including chemo-radiation therapy) HGG patients undergoing surgical re-resection of new enhancing lesions on surveillance MRI. Preoperatively, we acquired pMRI and contrast-enhanced stereotactic T1-Weighted (T1W) MRI data sets. Intraoperatively, we recorded stereotactic locations of multiple biopsies based on T1W data sets that guided surgery. Following surgery, we coregistered pMRI and T1W data sets to calculate localized relative cerebral blood volume (rCBV) for each stereotactic specimen location. For each specimen, we performed 1) standard H&E staining to diagnose tumor recurrence or post-treatment radiation effect (PTRE); and 2) immunohistochemical analysis with CD34 to highlight tissue vessels. We analyzed CD-34 stained slides with Axiovision Automeasure 3.4 (Zeiss, Germany) to calculate both total microvessel number (MVN) and total microvessel area (MVA), each normalized to total slide specimen area (μm2). We calculated Pearson correlations to establish relationships between a) rCBV and MVN; and b) rCBV and MVA. We also performed t-test to compare MVN, MVA, and rCBV values between tumor and PTRE samples. A neuroradiologist performed all coregistration and pMRI calculations, and a neuropathologist analyzed all tissue specimens, without knowledge of corresponding data. A biostatistician performed all statistical comparisons. Results: In this preliminary study, we included 16 tissue specimens (from 8 subjects), each diagnosed as either tumor (n=7) or PTRE (n=9). We successfully calculated localized rCBV and determined both MVA and MVN for each specimen. The rCBV values showed highest correlation with total vessel area (MVA) (r=0.65, p=0.007) and slightly less correlation with vessel number (MVN) (r=0.52, p=0.04). Tumor showed significantly higher values than PTRE for all parameters: rCBV (1.87 + 0.82 versus 0.78 + 0.2, p=.002); MVA (0.22 + 0.03 versus 0.04 + 0.007, p=0.0001); and MVN (0.0068 + 0.001 versus. 0.0016 + 0.0006, p=0.0004). Conclusion: These preliminary results show the promise of Perfusion MRI to non-invasively estimate tissue microvessel density and distinguish tumor recurrence from treatment effects. The current study is ongoing to confirm results in a larger patient population. 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 3748.

Abstract In collaboration with NCI, we are developing endoxifen (END), the active metabolite of tamoxifen (TAM), as a drug for the treatment of estrogen receptor positive breast cancer. Tamoxifen treated women with reduced or absent CYP2D6 enzyme activity have significantly lower END plasma concentrations. In a retrospective analysis of two different prospective tamoxifen trials (NCCTG and ABCSG), we have demonstrated that genetic or drug-induced reductions in CYP2D6 metabolism are associated with a higher risk of breast cancer recurrence in Tam treated ER positive breast cancer. To test the hypothesis that clinically relevant concentrations and exposures of END are achieved following oral administration of END, we characterized the pharmacokinetics of i.v. and oral END and oral TAM in female mice. Endoxifen was synthesized and provided by NCI/DTP. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. Following i.v. administration of a 1 mg/kg dose, peak plasma concentration, terminal elimination half-life and plasma clearance values were 0.26 µM, 6.5 h and 11.8 L/h/kg, respectively. Oral pharmacokinetics and bioavailability were determined following doses of 50 and 200 mg/kg. Peak plasma concentrations of 0.76 µM were achieved with a 50 mg/kg dose and plasma concentrations above 0.1 µM were maintained for longer than 8 h. Peak plasma concentrations of 8 µM were achieved with a 200 mg/kg dose and plasma concentrations above 2 µM were maintained for 24 h. Bioavailability was 50% and greater than 100% following the 50 mg/kg and 200 mg/kg doses, respectively. Based on pharmacokinetic data for oral END and TAM normalized to a 50 mg/kg oral dose, END plasma concentrations and total exposure were 20-fold greater following oral END as compared to oral TAM. In conclusion, END has high oral bioavailability in mice and substantial plasma concentrations are achieved and maintained after a single oral END dose. Compared to an equivalent oral dose of Tam, substantially higher END concentrations are achieved following oral END. These data suggest that the primary administration of End may overcome the limitations related to human CYP2D6 metabolism, and support the ongoing development of endoxifen as a primary therapy for ER breast cancer. Supported by the Mayo Comprehensive Cancer Center Grant (CA15083) and the Mayo Clinic Breast Cancer SPORE (CA 116201; MMA, JMR, MPG). 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 3603.

Abstract Background Radiation therapy is used to treat half of the all cancer patients. Response to radiation therapy varies widely, in terms of both response and toxicity related events. Previous studies suggested almost 80% of the variation in normal tissue response to radiation treatment was likely to be due to genetic factors. Therefore, we performed a genome-wide association study (GWAS) to identify biomarkers that might help predict radiation response using a the “Human Variation Panel” lymphoblastoid cell lines (LCLs) that consists 287 ethnically defined cell lines. Method Basal gene expression level, 1.3 million genome-wide SNP data were obtained for 287 human LCLs using Affymetrix U133 plus 2.0 GeneChip, Illumina HumanHap 550K, 510S BeadChips and Affymetix SNP Array 6.0. Radiation cytotoxicity assay was also performed to obtain the area under radiation dose response curve (AUC), a summary measure for cytotoxicity for the same cell lines. Functional validation of candidate genes, selected based on the integrated analysis of SNP, expression and AUC data, was performed in multiple cancer cell lines using specific siRNAs followed by MTS and colony formation assays. Results 27 loci, containing at least 2 SNPs of p values <10−4 within 50kb were associated with radiation AUC. 270 expression probe sets were associated with radiation AUC (p< 10−3). The integrated analysis of 27 loci, 54,000 basal expression probe sets and radiation AUC identified 50 unique SNPs associated with AUC were also associated with gene expression of 39 unique annotated genes. In addition, these 39 genes were associated with radiation AUC with p values <10−3. Based on the GWAS and integrated analysis of SNP, expression array and radiation AUC data, 22 genes were selected for functional validation studies using siRNA knock down followed by MTS and colony formation assays in multiple tumor cell lines. C13orf34 (Bora), MAD2L1, PLK4, TPD52 significantly altered the radiation sensitivity in at least 2 cancer cell lines for both MTS and colony formation assays (p<0.05). Knock down of DEPDC1B, a gene containing 2 SNPs at the 3′-end that was significantly related with Bora gene expression level (p<10−4) significantly desensitized cancer cells to radiation treatment. Both microarray data and QRT-PCR analysis indicated that expression levels for those two genes were significantly correlated. Conclusion Our results using the lymphoblastoid cell lines might help identify novel biomarkers that contribute to variation in response to radiation therapy and help understand the mechanisms underlying the association. 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 3602.