Citations

Since the microbiological assay of the antibiotic content of serum generally requires 18 to 24 hr of incubation, results of such procedures may not become available in time to make appropriate adjustments in subsequent dosages of antibiotic. A 4-hr bioassay for determining concentrations of gentamicin in serum has been developed in which Staphylococcus aureus ATCC 6538P is used as the test organism. Poured plates have yielded satisfactory results after storage at 4 C for 5 days. Results of the 4-hr procedure agree closely with those of a conventional 18-hr disc-plate assay performed with the same test organism.

Abstract BACKGROUND. The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. We determined whether mTOR inhibition can induce cytoprotective autophagy resulting in anti-cancer drug resistance, and whether inhibition of autophagy using the lysomotrophic agent, chloroquine, can restore chemosensitivity. EXPERIMENTAL METHODS. Inhibition of mTOR was accomplished in human colon cancer cells (HT-29, DLD1) using lentiviral shRNA or by AZD8055, a TORC1 and TORC2 kinase inhibitor. These cells were then incubated with 5-FU (0-100 µM) or CPT-11 (0-20 µM) alone and in combination with chloroquine. LC3I/II expression, phospho-histone H2A.X (a DNA damage marker), and caspase cleavage were analyzed by Western blotting using highly specific antibodies, and clonogenic survival assays were also performed. RESULTS. AZD8055 was shown to attenuate caspase-3 and -9 cleavage and to suppress the expression of phospho-histone H2A.X induced by 5-FU or CPT-11 in colon cancer cells. AZD8055 was also found to antagonize the ability of 5-FU to inhibit clonogenic survival. Similarly, shRNA knockdown of mTOR attenuated 5-FU-induced caspase cleavage and phospho-histone H2A.X expression. Inhibition of mTOR by AZD8055 or shRNA enhanced the conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form, consistent with induction of autophagy. Inhibition of autophagy by chloroquine was shown to attenuate the ability of AZD8055 to suppress 5-FU-induced caspase cleavage. Consistent with this result, chloroquine sensitized DLD1 cells to AZD8055 and 5-FU in a clonogenic survival assay. CONCLUSION. Inhibition of mTOR can protect colon cancer cells from DNA damage induced by cytotoxic chemotherapy. This cytoprotection was due, in part, to induction of pro-survival autophagy since chloroquine was able to partially restore chemosensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2011-5485

Abstract Metastatic malignant melanoma is an incurable disease with average 5-year survival rates for patients with disseminated disease ranging from 5-18%. Despite the recent development of promising targeted and immunological approaches for treatment of melanoma patients, there remains an urgent need to improve the effectiveness of conventional chemotherapeutics such as TMZ treatment for those patients ineligible or unresponsive to these approaches. We hypothesized that genetic alterations or contexts of vulnerability (COVs) exist in melanoma cells that mediate sensitivity to TMZ, and thus developed RNA-interference-based (RNAi) TMZ chemosensitization assays in melanoma cell lines in order to better understand how to pharmacologically augment TMZ response. First, we extensively developed RNAi sensitization assay in 17 melanoma cell lines followed by screening siRNA kinome library in these cell lines. This screening result, combined with our previous melanoma genomic data (gene expression, aCGH, SNP, and gene mutations), would prioritize the critical essential genes in melanoma. In addition, we profiled 17 melanoma cell lines for TMZ sensitivity and subsequently optimized RNAi screening parameters for detection of TMZ sensitization in one sensitive (UACC903) and four resistant (UACC647, UACC1308, UACC91 and UACC1940) cell lines. We then performed high-throughput RNAi screens with a 7,000 gene siRNA library targeting the druggable genome in these five cell lines. The data from the screens were rigorously evaluated and top TMZ-sensitizing hits were identified and confirmed. Based on these data, we prioritized the ATR/ATM/Chk1 pathway for further evaluation. We tested two Chk1 inhibitors for synergistic effects when combined with TMZ. Notably, both Chk1 inhibitors demonstrated good synergistic effects in the TMZ-resistant cell lines, with moderate/marginal effects in the sensitive line UACC903. These data are consistent with previous reports that inhibition of Chk1 can sensitize TMZ-induced cytotoxicity in glioma cells, and suggest that targeting this pathway in melanoma may augment TMZ response in melanoma. We are presently following up these results across a larger panel of melanoma cell lines, as well as evaluating pharmacological inhibitors of additional TMZ-sensitizing siRNA hits and pathways with the hope that these results may serve as a starting point for the development of novel combination therapies for melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5457. doi:10.1158/1538-7445.AM2011-5457

Abstract Catabolism of fluoropyrimidine drugs such as 5-fluorouracil (5-FU) and its prodrug capecitabine depends on an exclusive enzymatic step, dihydropyrimidine dehydrogenase (DPD), which results in drug inactivation. DPD exhibits wide interindividual variation, with partial to complete DPD deficiency occurring in approximately 3 to 5% of the general population. Patients with less pronounced, but decreased DPD activity may also be unable to efficiently inactivate 5-FU, such that this may contribute to 5-FU toxicity. Though numerous tests have been developed to phenotypically assess the DPD enzyme, they remain labor intensive and time consuming with difficulty in defining threshold values for separating DPD deficient patients from those with normal DPD enzyme activity. To date, attempts to develop genotypic assays have been complicated by the size and complex structure of the gene encoding the DPD enzyme (DPYD). Numerous sequence variants have been reported with varying frequencies across different populations. Unfortunately many DPYD variants have not been examined to determine whether they are associated with loss of function in the DPD enzyme. Current genotypic methods are limited due to the inability to rapidly assess multiple variants in a large number of samples at one time. In the current study, we have developed a rapid, high-throughput genotypic method using 6 multiplexed PCR assays covering 115 DPYD variants analyzed with the Sequenom matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Selected variants included previously identified and functionally characterized SNPs as well as newly identified mutations within the coding region from a previous mutation discovery analysis in Coriell cell lines and patient samples. Due to the potential importance of variants in the untranslated regions (UTRs), we have included known variants from the 5′- and 3′- UTRs (9 and 16 respectively) within our plexes. A total of 95 DNA samples derived from 4 colorectal cancer cell lines, 76 Coriell cell lines (40 Caucasian and 36 African American), and 7 Caucasian patients with clinically observed 5-FU toxicity and/or decreased DPD activity were utilized for initial assay testing. To assess “calling” efficiency and presence of false positives, 8 Coriell DNA samples plated in duplicate and 1 negative control were included. Our results showed that identified variants were 100% in agreement across duplicated samples, and only four variants within the 6 different plexes were removed due to PCR/extension failure. In summary, this method provides a comprehensive and rapid analysis of 111 DPYD variants within 6 different plexes with both sensitive and accurate detection suitable for high-throughput patient screening. Functional studies in a large patient population are underway (CA116964). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2011-5451

Abstract Mesenchymal Stem Cells (MSCs) have the ability to migrate specifically to tumours in vivo, and coupled with their capacity to bypass immune surveillance, are potentially attractive vehicles for tumor-targeted delivery of therapeutic agents. The aim of this study was to determine the potential of MSC-mediated expression of the sodium iodide symporter (NIS) for in vivo imaging and therapy of breast tumours. Expression of NIS allows cells to concentrate radionuclides including technetium-99m (Tc-99m) and 131-Iodine (131-I). This could potentially support imaging of NIS-expressing MSCs following engraftment, and 131-I therapy of surrounding tumour tissue based on the bystander effect of the radionuclide. Methods: Tumor bearing animals (MDA-MB-231 flank) were given an intravenous or intratumoral injection of NIS expressing human MSCs (MSC-NIS), followed by imaging 3, 7, 10 or 14 days later. Imaging was performed using a Bazooka SPECT γ-camera, following intraperitoneal injection of 2mCi/74MBq Tc-99m. Following imaging, animals were sacrificed and organs harvested for analysis of hNIS expression by RQ-PCR. A second group of animals received an intraperitoneal injection of 1mCi/37MBq 131-I or saline (controls) 14 days following intratumoral or intravenous injection of MSC-NIS, and tumour volume was tracked for 8 weeks. Results: Bazooka SPECT imaging of animals revealed uptake of tracer (Tc-99m) in the mouse thyroid/salivary glands and stomach, representing native NIS expression. Following injection of MSC-NIS, an image of animal intestines was also observed at D3, with a weak image of the tumour also visible. By Day14, uptake of tracer was visible at the site of the tumour with no significant image of non-target tissue observed. Expression of hNIS in harvested organs supported the imaging data, with hNIS detected in the intestines, heart, lungs and tumour at early timepoints. While NIS expression depleted in non-target tissues by D7, gene expression persisted at the tumour site. Based on imaging/biodistribution data, animals were given a therapeutic dose of 131-I 14 days following MSC-NIS injection to avoid toxicity to non-target organs. No adverse effect of injection of MSC-NIS or radionuclide was observed. Tracking of tumour volume revealed a significant reduction in tumour growth in animals that had received an intravenous injection of 131-I 14days following MSC-NIS injection (Mean ± SEM, 236 ± 62mm3 versus 665 ± 204 mm3 in controls). Following intratumoral injection of MSC-NIS + 131-I, although tumour size was reduced compared to controls (472 ± 80 mm3), the difference in volume was not significant. Conclusion: The ability to non-invasively track MSC migration and transgene expression in real time prior to therapy is a major advantage to this strategy. This promising data supports the viability of this approach as a novel therapy for metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5392. doi:10.1158/1538-7445.AM2011-5392

Abstract The mammalian target of rapamycin (mTOR) consists of two distinct complexes, mTORC1 and mTORC2. mTOR plays a pivotal role in Hepatocellular Carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 only moderately enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity. This limited effect may be attributable to the increase of AKT activation through S6K1-IRS1-PI3K negative feedback loop triggered by selective mTORC1 inhibition. While as AKT is the downstream effector of mTORC2, we hypothesize that dual targeting of mTORC1/C2 would be more efficient in suppressing AKT and in enhancing anti-tumor activity of HDACi. In this study, the interactions between mTOR kinase inhibitors (mTORKis) and HDACis were examined in vitro using established HCC cell lines and in vivo using patient-derived HCC tumor xenografts on SCID mice. Our results demonstrate that inhibition of both mTORC1/2 not only effectively blocked mTORC1 signaling, but also abrogated negative-feedback activation of AKT caused by mTORC1 inhibition. Furthermore, inactivation of mTORC1/C2 by mTORKis substantially enhanced HDACi-induced anti-tumor activity. Either dysfunction of mTORC2 by knockdown of Rictor (a component of mTORC2), or diminution of AKT1 significantly lowered the threshold of HDACi-induced apoptosis. Lastly, in vivo study indicated that the combination of AZD8055 and Vorinostat yielded much greater tumor-growth inhibition compared to either agent alone in primary HCC xenografts without obvious adverse effects. Conclusion: Dual mTORC1/2 inhibition is more efficient than selective mTORC1 inhibition in enhancing anti-tumor activity of HDACi due to abrogation of negative-feedback activation of AKT induced by selective mTORC1 inhibition. Our findings suggest that combining regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5363. doi:10.1158/1538-7445.AM2011-5363

Abstract Breast cancer recurrence is believed to be caused by a potently tumorigenic sub-population of cancer cells possessing stem cell attributes of resistance to chemotherapy, radiation and other forms of treatment. Recently, we and others have reported the generation of breast cancer stem cell (BCSC) characteristics by inducers of epithelial to mesenchymal transition (EMT). Both EMT and acquisition of BCSC properties are therefore important steps in cancer progression, although the physiological process by which BCSCs arise in vivo remains unclear. We show here that exposure to TGFβ and TNFα induces a stable BCSC phenotype, characterized by self-renewal, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. We further found that the TGFβ/TNFα-derived cells showed downregulated expression of Claudins 3, 4 and 7 and the luminal marker KRT18, demonstrating a shift to the claudin low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlation with poor prognosis. Furthermore, pathway analyses of the generated BCSCs showed deregulation of a number of breast cancer-associated genes and pathways including inflammatory cytokine and receptors, chemokines, homeobox, hedgehog, notch and metastases genes. These results confirm the role of EMT and BCSCs in resistance of breast cancer to conventional chemotherapy and identify novel target genes and pathways for validation as potential therapeutic targets. Our demonstration that stable BCSCs can be produced by specific microenvironmental signals also provides a valuable tool for studying cancer stem cells, as well as a means for screening potential cancer drugs for efficacy of targeting cancer stem cells to prevent recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2011-3341

Abstract Microarray gene expression profiling has facilitated identification of molecular subtypes which can be associated with disease outcome and treatment response. However current platforms lack disease focus, potentially missing vital information contained in patient tissue samples. Moreover, a wide range of FFPE sample cohorts are available but their use is limited by the mRNA degradation inherent to the sample fixation process. By combining the Breast Cancer DSA® with powerful data analysis methodology, we reproduced the five major molecular subtypes in breast cancer based on a cohort of 107 FFPE samples. Furthermore, we showed that the established subtypes do not extend to BRCA1 and BRCA2 mutant tumours, necessitating expansion of the original breast cancer subtypes definition. Following RMA pre-processing of the 107 raw profiles, the data was transformed to overcome the association between profile quality and expression variability. If left untreated, this technical variability, often observed with FFPE tissues, can impair the study of inherent biological variations. Using the intrinsic list of 277 genes previously identified as differentiating breast cancer subtypes in fresh frozen samples [Sorlie et al., 2003], initially the sporadic samples were examined using hierarchical agglomerative clustering. All five original subtypes were identified, verifying the ability to detect subtypes in FFPE samples using the breast cancer DSA®. When the intrinsic list was applied to all 107 samples including BRCA1 and BRCA2 mutant samples, the subtypes were still distinguishable, although 31% of the mutant tumors were unclassified suggesting that specific biological processes defining BRCA1/BRCA2 mutant tumours are not captured by the original breast cancer molecular classification. We have successfully overcome the limitation imposed by FFPE sample microarray profiling by using the Breast Cancer DSA® and advanced data manipulation and exploration to reproduce molecular subtypes previously identified using fresh frozen samples. Further investigation to extend the previously identifed subtypes will progress the investigation of the heterogeneous biology underlying breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2011-326

Abstract Pancreatic cancer is the most lethal type of cancer due to its high proliferation, metastasis and resistance to chemotherapy. In pancreatic cancer, pancreatic ductal adenocacinoma (PDAC) is the most common form. Current opinion is that PDAC originates from acinar or centroacinar cells though a process called “acinar-to-ductal metaplasia” (ADM). During ADM, acinar cells undergo a transformation to ductal PanIN (pancreatic intraepithelial neoplasia) cells, which is believed as the first step in PDAC formation. Transforming growth factor α (TGFα) is produced by pancreatic cancer cells and promotes tumor growth. In an ex vivo explant culture system, TGFα transforms murine pancreatic acinar cells into ductal cells. Here we describe a cell signaling pathway that TGFα utilizes to induce acinar-to-ductal transformation. We show that K-ras is downstream of TGFα and is sufficient to drive ADM. We further show the mechanisms that K-ras uses to induce cell structural rearrangement of acinar cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1123. doi:10.1158/1538-7445.AM2011-1123

Abstract DNA methylation profiles could signify the risk of prostate cancer and predict the progression of cancer before the appearance of clinical symptoms. Specific CpG sites methylation of genes identified in our study could be used to improve the sensitivity and specificity for early diagnosis and to predict the risk of prostate cancer recurrence. To identify methylation changes associated with prostate cancer, we measured gene methylation using the Infinium Human Methylation27 bead chips. Tissue DNA isolation and bisulfite conversion was done using ZR Genomic DNA kit and EZ methylation kit. The methylation27 microarrays was used to analyze methylation of 27,578 CpG sites of 14,495 genes in 198 tumor tissues and 40 matched normal tissues. Methylation data was processed using the Genome Studio software, differentially methylated genes identified using one-way analysis of variance, and correction for multiple testing done using the false discovery rate q-value. To identify putative diagnostic and prognostic biomarkers we performed four subgroup comparisons: tumor vs. matched normal, recurrent tumors vs. non-recurrent tumors, clinical recurrence vs. biochemical recurrence, and systemic recurrence vs. local recurrence. Clinicopathological and molecular features including patient preoperative PSA levels, Gelason score, TNM stage, GPSM, recurrence type, were also evaluated for subgroup discrimination. Discriminative subgroup candidate methlyation markers were selected using a p value <0.05 and a mean difference between two contrast groups >0.05. Between the tumor and matched normal tissue samples, 164 CpG sites representing 147 genes were identified as significantly differentially methylated (p<9.99E-25, fold change>2.0). Between recurrent tumor and non-recurrent tumor tissues there were 78 CpG sites representing 75 genes that were significantly differentially methylated (p <0.002, fold change >1.5). Between clinically recurrent and biochemically recurrent tumors, 16 CpG sites representing 16 genes were significantly differentially methylated (p <0.05, fold change >1.5). Between systemic recurrent and local recurrent tumors there were 73 CpG sites representing 68 genes that were significantly differentially methylated (p <0.01, fold change >1.5) Our analysis revealed differential methylation of the genes, implicating their role in prostate cancer development and progression. We demonstrated that the hypermethylation of genes could be used as a sensitive molecular tool in detection of prostate tumorigenesis and prediction of tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 109. doi:10.1158/1538-7445.AM2011-109