Aurora A kinase is a serine/threonine protein kinase responsible for regulating several mitotic processes including centrosome separation, spindle assembly, and chromosome segregation. Small molecule inhibitors of Aurora A kinase are being pursued as novel anticancer agents, some of which have entered clinical trials. Despite the progress in developing these agents, terminal outcomes associated with Aurora A inhibition are not fully understood. Although evidence exists that Aurora A inhibition leads to apoptosis, other therapeutically relevant cell fates have not been reported. Here, we used the small molecule inhibitor MLN8054 to show that inhibition of Aurora A induces tumor cell senescence both in vitro and in vivo. Treatment of human tumor cells grown in culture with MLN8054 showed a number of morphologic and biochemical changes associated with senescence. These include increased staining of senescence-associated β-galactosidase, increased nuclear and cell body size, vacuolated cellular morphology, upregulation/stabilization of p53, p21, and hypophosphorylated pRb. To determine if Aurora A inhibition induces senescence in vivo, HCT-116 xenograft-bearing animals were dosed orally with MLN8054 for 3 weeks. In the MLN8054-treated animals, increased senescence-associated β-galactosidase activity was detected in tissue sections starting on day 15. In addition, DNA and tubulin staining of tumor tissue showed a significant increase in nuclear and cell body area, consistent with a senescent phenotype. Taken together, this data shows that senescence is a terminal outcome of Aurora A inhibition and supports the evaluation of senescence biomarkers in clinic samples. Mol Cancer Res; 8(3); 373-84. ©2010 AACR.

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Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.

Protein supplements having either a high (soybean meal, SBM) or low (escape protein, EP) extent of ruminal N degradability with or without lasalocid (L) were evaluated in digestion and growth trials. The SBM supplement included soybean meal while EP was a combination of dehydrated alfalfa and distillers dried grains. Nitrogen digestibility of SBM supplements was consistently higher than EP supplements when evaluated with two lamb trials. Digestibility of N was improved 8% in trial one (64.9 vs 60.3%) and 27% in trial two (66.3 vs 52.3%) with SBM vs EP. The addition of L to the supplements improved N digestibility by 6% in trial one (64.5 vs 60.6%) and 13% in trial two (62.9 vs 55.7%). No interactions between protein source and L were measured in either trial. Dry matter digestibility was not changed by protein source or L in either trial. Rumen propionate was increased and acetate to propionate ratio decreased when L was fed. Plasma urea N was lower over a 24 h sampling period when lambs were fed EP supplements compared with SBM supplements (11.07 vs 16.44 mg/100 ml); however, L did not appear to consistently alter the values. When steers were supplemented with the same protein sources during a 105-d winter pasture trial daily gains were not affected (P greater than .10) by either protein source or L (.429, .495, .476 and .514 kg/d for SBM, SBM+L, EP and EP+L, respectively) although numerically there did not appear to be main effect improvements due to EP and L.(ABSTRACT TRUNCATED AT 250 WORDS)

Autophagy is a major cellular process for bulk degradation of proteins and organelles in order to maintain metabolic homeostasis, and it represents an emerging target area for cancer. Initially proposed to be a cancer-restricting process for tumor initiation, recent studies suggest that autophagy can also promote cell survival in established tumors. ATG7 is an essential autophagy gene that encodes the E1 enzyme necessary for the lipidation of the LC3 family of ubiquitin-like proteins and autophagosome formation. In this study we identified a rare case of a cancer cell line, H1650 lung adenocarcinoma, which has lost ATG7 expression due to a focal biallelic deletion within the ATG7 locus. These cells displayed no evidence of ATG7 pathway activity; however, reconstituting the cells with wild-type ATG7 restored both LC3 lipidation and downstream autophagic consumption of autophagy substrates such as the SQSTM1/p62 protein. We characterized several phenotypes reported to be influenced by autophagy, and observed an ATG7-dependent increase in cell growth and clearance of proteasome-inhibitor induced protein aggregates. Cellular changes in mitochondrial metabolism or response to nutrient starvation were unaffected by ATG7 expression. In addition, parental H1650 cells that lacked ATG7 were still able to consume autophagy substrates SQSTM1, NBR1 and TAX1BP1 via a bafilomycin A-sensitive pathway, suggesting that these proteins were not exclusively degraded by autophagy. Overall, these findings highlight a unique outlier instance of complete loss of ATG7-dependent autophagy in a cancer cell line. The H1650 cell line may be a useful system for future studies to further understand the role of autophagy in tumorigenesis and potential redundant pathways that allow cells to circumvent the loss of ATG7-dependent autophagy in cancer.

Immunohistochemistry-based biomarkers are commonly used to understand target inhibition in key cancer pathways in preclinical models and clinical studies. Automated slide-scanning and advanced high-throughput image analysis software technologies have evolved into a routine methodology for quantitative analysis of immunohistochemistry-based biomarkers. Alongside the traditional pathology H-score based on physical slides, the pathology world is welcoming digital pathology and advanced quantitative image analysis, which have enabled tissue- and cellular-level analysis. An automated workflow was implemented that includes automated staining, slide-scanning, and image analysis methodologies to explore biomarkers involved in 2 cancer targets: Aurora A and NEDD8-activating enzyme (NAE). The 2 workflows highlight the evolution of our immunohistochemistry laboratory and the different needs and requirements of each biological assay. Skin biopsies obtained from MLN8237 (Aurora A inhibitor) phase 1 clinical trials were evaluated for mitotic and apoptotic index, while mitotic index and defects in chromosome alignment and spindles were assessed in tumor biopsies to demonstrate Aurora A inhibition. Additionally, in both preclinical xenograft models and an acute myeloid leukemia phase 1 trial of the NAE inhibitor MLN4924, development of a novel image algorithm enabled measurement of downstream pathway modulation upon NAE inhibition. In the highlighted studies, developing a biomarker strategy based on automated image analysis solutions enabled project teams to confirm target and pathway inhibition and understand downstream outcomes of target inhibition with increased throughput and quantitative accuracy. These case studies demonstrate a strategy that combines a pathologist's expertise with automated image analysis to support oncology drug discovery and development programs.

One lamb and three calf trials were conducted to determine if interactions existed between the effects of dry matter (DM) content of corn silage at harvest and sodium bicarbonate supplementation on diet digestibility, nitrogen balance, rate and site of digestion and feedlot performance. Corn plants were harvested from the same field when DM content was approximately 31% (early; E) or 44% (late; L). Sodium bicarbonate (1.2% of DM intake) was added in a completely mixed ration. When lambs were offered diets ad libitum, (trial 1), intakes were greater (P less than .05) for L-silage diets, but apparent digestibilities were similar. Nitrogen balance was greater for sodium bicarbonate-supplemented diets, and was a reflection of greater (P less than .10) DM intakes for these diets. At similar diet DM intakes, N balance was greater for L-silage diets, with no effects measured due to sodium bicarbonate addition. When growing, abomasally cannulated heifers (trial 2) were offered diets 12 times per day, a significant interaction among treatments was measured for total tract and acid detergent fiber (ADF) digestion. Adding bicarbonate to the E-silage diet increased (P less than .05) digestion by 9.1 percentage units (66.4 vs 75.5%) but decreased digestibility of the L-silage diet by 4.2 percentage units (73 vs 68.8%). Grams of ADF apparently digested in the rumen followed the same pattern as for total tract digestion. In trial 3, ruminal rates of liquid and particulate passage and in situ rats of NDF digestion were determined using four rumen-cannulated heifers calves.(ABSTRACT TRUNCATED AT 250 WORDS)

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