Short-term Assays Research Articles

Abstract 2841: Her2/neu expression as potential marker of regorafenib resistance in CRC

Abstract Nature Reviews Clinical Oncology, (2017). doi:10.1038/nrclinonc.2017.43 Jason J. Luke 0 0 2017-03-27T13:39:00Z 2017-11-29T14:35:00Z 1 312 1781 14 4 2089 14.0 Normal 0 false false false EN-US JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:EN-US;} Regorafenib is a multikinase inhibitor currently approved for the treatment of chemorefractory mCRC patients. No predictive biomarkers of efficacy have been identified. We recently reported that amplification or mutations in HER-2/neu might predict resistance to regorafenib in a small cohort of patients treated with Regorafenib. We therefore sought to better investigate the role of HER-2/neu in response to Regorafenib in preclinical and clinical models. Short-term proliferation assay with Regorafenib was performed in a panel of CRC cell lines. Basal protein expression of total and phospho Her2 were analized by WB analysis. RPPA data from MD Anderson cell lines project were used for external validation of total and phospho HER-2/neu. Sperman correlation was applied to correlate the expression levels of total and phospo HER2/neu and IC50 to Regorafenib. HT29 CRC cell line (HT29) was made resistant to Regorafenib (HT29R). WB analysis of basal levels of proteins of the EGFR and Her2 axis were investigated in both HT29 and HT29R. Moreover, HT29 and HT29R were treated with EGFR inhibitors, Her2/neu inhibitors, MAPK and PIk3Ca inhibitors. Apoptosis was perfromed by FACs analysis by using Annexin-V staining. We found a negative correlation between pHer2 expression and response to Regorafenib in both our data set of CRC cell lines and in the MD Anderson panel. Overexpression of all the epitelial markers of the EGFR and HER2/neu axis was observed in HT29R as compared to HT29. This translated to an increased sensitivity and apoptosis to Trastuzumab in HT29R as compared to HT29 (IC50 with 50 times fold change) and PIk3Ca inhibitor (IC50 with 5 times fold change). No differences of IC50 were observed with other inhibitors. Moreover, combined treatment with Trastuzumab and PIk3Ca inhibitor induced significantly early and late apoptosis in HT29R as compared to HT29. Our preliminary data indicate pHer2 levels to be predictive of resistance to Regorafenib in CRC. Functional work and IHC analysis of CRC patients treated with Regorafenib is ongoing and will be presented. Citation Format: Loredana Vecchione, Stefania Napolitano, Valentina Belli, Erika Martinelli, Nunzia Matrone, Christophe Hapke, Ulrich Keilholz, Josep Tabernero, Fortunato Ciardiello, Teresa Troiani. Her2/neu expression as potential marker of regorafenib resistance in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2841.

Abstract 1853: The Chk1 inhibitor, SRA737, synergizes with niraparib to kill cancer cells via multiple cell death pathways

Abstract Targeting the DNA damage response (DDR) network is a promising strategy for the development of new cancer therapies. Checkpoint kinase 1, Chk1, is a central mediator of the DDR network and the potent, selective oral Chk1 inhibitor, SRA737, is being investigated in clinical trials. A distinct class of DDR inhibitors targeting PARP (PARPi) are approved for the treatment of ovarian cancers; however, tumors with functional homologous recombination (HR) repair are less sensitive to their effects, thereby limiting the clinical potential of these agents. Several reports have described the synergistic combination of Chk1i and PARPi, although the mechanism of anti-tumor activity has not been well defined. We explored the efficacy and mechanism of cytotoxicity of SRA737 in combination with the PARPi, niraparib, in HR repair proficient tumor cell lines. In short-term cell viability assays, the combination of SRA737 and niraparib elicited greater tumor cell death than either agent alone, as early as 12 hours after exposure to drug. Combination indices determined from colony forming assays indicated synergistic activity (CI < 0.7) using clinically achievable concentrations of each agent. Quantitative immunofluorescence studies revealed activation of ATM and phosphorylation of H2AX within 4 hours of treatment, indicating induction of DNA double strand breaks and activation of DDR signaling. Concurrent changes in the phosphorylation of mTOR, AMPK and the downstream target ULK1 suggested an induction of autophagy. Consistent with this hypothesis, the single agents, as well as the combination, led to decreases in p62 and LAMP2 levels and simultaneous increases in ATG5 and Beclin1 expression, and ATG13 phosphorylation. Autophagic flux was confirmed in cells expressing an LC3-GFP-RFP reporter plasmid. Genetic knockdown of autophagy components resulted in partial rescue of cell viability, suggesting that autophagy-dependent cell death may represent a mechanism of cytotoxicity for this DDR combination. Given that rescue of cell death was incomplete following abrogation of autophagy, we additionally examined the involvement of apoptotic pathways. SRA737 and niraparib treatment resulted in reduced levels of anti-apoptotic proteins, BCL-XL and MCL-1, and increased levels of the pro-apoptotic protein, BIM. Moreover, knock down of pro-apoptotic proteins or over-expression of anti-apoptotic proteins partially rescued combination-induced lethality. Collectively these results argue that toxic autophagy, as well as the intrinsic and extrinsic apoptosis pathways, contribute to SRA737 and niraparib-induced tumor cell killing. The involvement of multiple mechanisms of cell death may decrease the likelihood of cancer cells to acquire resistance to these agents. These findings support further investigation of SRA737 in combination with PARPi, including niraparib, in HR repair proficient cancers. Citation Format: Laurence Booth, Jane Roberts, Andrew Poklepovic, Ryan J. Hansen, Bryan Strouse, Snezana Milutinovic, Christian Hassig, Paul Dent. The Chk1 inhibitor, SRA737, synergizes with niraparib to kill cancer cells via multiple cell death pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1853.

Abstract 5767: Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy

Abstract Background Photoimmunotherapy (PIT) is a new class of molecular targeted cancer theranostics, which employs monoclonal antibody (mAb) conjugated to a photosensitizer, IR700, that is activated by focal near-infrared (NIR) light irradiation leading to necrotic cell death by cell membrane rapture where mAb-IR700 conjugates binds to target membrane proteins specifically. We have previously demonstrated HER2-targeted PIT employing trastuzumab-IR700 conjugates for HER2 expressing cancers. However, some cancer cells were survived partly because of tumor heterogeneity and inhomogeneous micro-distribution of mAb-IR700 conjugates. In this study, we developed a new type of PIT agents targeting VEGFR-2 expressed on vascular endothelium in a tumor and evaluated the feasibility by comparing conventional PIT in vitro and in vivo. Methods HER2-positive human gastric cancer cells, NCI-N87, were used for the experiments. HER2-targeting trastuzumab and VEGFR-2-targeting ramucirumab were conjugated to IR700. Cells were treated with mAb-IR700 conjugates followed by NIR light irradiation after washing the cells. LIVE/DEAD assay was performed to assess short-term phototoxicity and trypan blue dye exclusion assay to assess long-term phototoxicity. Next, mouse tumor xenograft models were created for in vivo PIT. Tumor-bearing mice were randomized and treated with mAb-IR700 conjugates followed by NIR light irradiation under anesthesia. Antitumor effects were monitored by measuring tumor diameters with a caliper. Results PIT utilizing ramucirumab-IR700 conjugates did not induce phototoxic effect in vitro because of the absence of membranous expression of VEGFR-2 in NCI-N87 cells, while PIT utilizing trastuzumab-IR700 conjugates induced rapid phototoxic effect because of the strong membranous expression of HER2 in NCI-N87 cells. By contrast, antitumor effects were observed in NCI-N87 xenograft tumors in vivo utilizing both ramucirumab- and trastuzumab-IR700 conjugates followed by NIR light irradiation. Microscopic analysis of the harvested tumors showed decreasing tumor micro-vessel densities only when tumors were treated with PIT utilizing ramucirumab-IR700 conjugates but not trastuzumab-IR700, which represents a different mechanism than that of conventional PIT targeting antigens expressed on the tumor cell membrane. Conclusion We demonstrated a new mechanism of PIT utilizing ramucirumab-IR700 conjugates. As VEGFR-2 is upregulated in many types of solid cancers, this method may be considered as being applicable to various types of cancers in future clinical settings. Citation Format: Makoto Mitsunaga, Takashi Nishimura, Kimihiro Ito. Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5767.

Antigen-specific primed cytotoxic T cells eliminate tumour cells in vivo and prevent tumour development, regardless of the presence of anti-apoptotic mutations conferring drug resistance.

Cytotoxic CD8+ T (Tc) cells are the main executors of transformed and cancer cells during cancer immunotherapy. The latest clinical results evidence a high efficacy of novel immunotherapy agents that modulate Tc cell activity against bad prognosis cancers. However, it has not been determined yet whether the efficacy of these treatments can be affected by selection of tumoural cells with mutations in the cell death machinery, known to promote drug resistance and cancer recurrence. Here, using a model of prophylactic tumour vaccination based on the LCMV-gp33 antigen and the mouse EL4 T lymphoma, we analysed the molecular mechanism employed by Tc cells to eliminate cancer cells in vivo and the impact of mutations in the apoptotic machinery on tumour development. First of all, we found that Tc cells, and perf and gzmB are required to efficiently eliminate EL4.gp33 cells after LCMV immunisation during short-term assays (1-4 h), and to prevent tumour development in the long term. Furthermore, we show that antigen-pulsed chemoresistant EL4 cells overexpressing Bcl-XL or a dominant negative form of caspase-3 are specifically eliminated from the peritoneum of infected animals, as fast as parental EL4 cells. Notably, antigen-specific Tc cells control the tumour growth of the mutated cells, as efficiently as in the case of parental cells. Altogether, expression of the anti-apoptotic mutations does not confer any advantage for tumour cells neither in the short-term survival nor in long-term tumour formation. Although the mechanism involved in the elimination of the apoptosis-resistant tumour cells is not completely elucidated, neither necroptosis nor pyroptosis seem to be involved. Our results provide the first experimental proof that chemoresistant cancer cells with mutations in the main cell death pathways are efficiently eliminated by Ag-specific Tc cells in vivo during immunotherapy and, thus, provide the molecular basis to treat chemoresistant cancer cells with CD8 Tc-based immunotherapy.

Effects of short- and long-term exposure of silver nanoparticles and silver ions to Nitrosomonas europaea biofilms and planktonic cells

The increasing use of silver nanoparticles (AgNPs) in consumer products, and their resulting influx into wastewater, may pose a threat to biological nutrient removal in wastewater treatment plants. Planktonic ammonia-oxidizing bacteria (AOB), which convert ammonia to nitrite in the first step of nitrification, are highly sensitive to AgNPs and their released silver ions (Ag+), but the sensitivity of AOB biofilms to AgNPs and Ag+ is less clear. This study demonstrated that biofilms of Nitrosomonas europaea, a model AOB, were more resistant to both short-term and long-term exposure to AgNP and Ag+ than planktonic cells. The increased resistance of N. europaea biofilms was attributed primarily to the increased biomass and slower growth rates present in the biofilm. Similar inhibition mechanisms were observed for AgNPs and Ag+ in both planktonic cells and biofilms with enzymatic inhibition observed at lower concentrations and cell lysis observed at higher concentrations. Long-term continuous exposure to AgNPs lowered the inhibitory concentration by 1–2 orders of magnitude below that required by short-term exposures. Although the total AgNP load was similar between the short and long-term exposure scenarios, the long-term exposure resulted in an order of magnitude more silver being associated in the biofilms and is the primary reason for the increased sensitivity observed. This suggests that short-term batch toxicity assays may greatly underestimate the sensitivity of biofilm treatment systems to long-term exposures of low concentrations of AgNPs and Ag+.

FTI-277 inhibits smooth muscle cell calcification by up-regulating PI3K/Akt signaling and inhibiting apoptosis.

BackgroundVascular calcification is associated with increased cardiovascular morbidity and mortality in patients with atherosclerosis, diabetes and chronic kidney disease. However, no viable treatments for this condition have been identified. This study aimed to determine whether farnesyl transferase inhibitors (FTIs) can reduce vascular calcification and the mechanism by which this reduction occurs.ResultsWe demonstrate that FTI-277 significantly inhibits phosphate-induced mineral deposition by vascular smooth muscle cells (VSMC) in vitro, prevents VSMC osteogenic differentiation, and increases mRNA expression of matrix Gla protein (MGP), an inhibitor of mineralization. FTI-277 increases Akt signaling in VSMC in short-term serum-stimulation assays and in long-term mineralization assays. In contrast, manumycin A has no effect on Akt signaling or mineralization. Co-incubation of VSMC with FTI-277 and SH6 (an Akt inhibitor) significantly reduces the inhibitory effect of FTI-277 on mineralization, demonstrating that FTI-277 inhibits calcification by activating Akt signaling. Over-expression of the constitutively active p110 sub-unit of PI3K in VSMC using adenovirus activates Akt, inhibits mineralization, suppresses VSMC differentiation and significantly enhances MGP mRNA expression. FTI-277 also inhibits phosphate-induced activation of caspase 3 and apoptosis of VSMC, and these effects are negated by co-incubation with SH6. Finally, using an ex vivo model of vascular calcification, we demonstrate that FTI-277 inhibits high phosphate-induced mineralization in aortic rings derived from rats with end-stage renal failure.ConclusionsTogether, these results demonstrate that FTI-277 inhibits VSMC mineral deposition by up-regulating PI3K/Akt signaling and preventing apoptosis, suggesting that targeting farnesylation, or Akt specifically, may have therapeutic potential for the prevention of vascular calcification.

Extended fish short term reproduction assays with the fathead minnow and Japanese medaka: No evidence of impaired fecundity from exposure to atrazine

Short-term reproduction assays were conducted with fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes) to evaluate responses from atrazine exposure at environmentally relevant concentrations and above. Breeding groups of fish with multiple males and females were exposed to atrazine under flow-through conditions. Fathead minnows were exposed to mean measured concentrations of 1.0, 10, 26, 52, and 105 μg atrazine/L for 28 days. Medaka were exposed to mean measured concentrations of 9.4, 48, 74, 97, and 244 μg atrazine/L for 28 or 29 days. Fish were evaluated for survival, fecundity, fertility, total length, wet weight, secondary sex characteristics, gonadosomatic index (GSI) (P. promelas only), plasma or hepatic vitellogenin (VTG), and histopathology of gonads. General observations of health and behaviour were also conducted. There were no statistically significant effects (i.e., p < 0.05) of atrazine on survival, size, reproduction, behaviour, GSI, VTG, or secondary sex characteristics in either species at any exposure level. In fathead minnows, there were no histopathological findings associated with atrazine exposure in male fish, but there was an increased proportion of Stage 4.0 ovaries accompanied by an increase in proportion of Grade 3 post-ovulatory follicles in females of the 105 μg/L treatment group. Without a concomitant increase in oocyte atresia, neither of these findings are considered adverse for the health of the fish. In medaka, there were no significant effects of atrazine exposure on histopathology in either sex. These data support current weight-of-evidence assessments that atrazine does not cause direct adverse effects on fish reproduction at environmentally realistic concentrations.

Transcriptional Responses Reveal Similarities Between Preclinical Rat Liver Testing Systems.

Toxicogenomics (TGx) is an important tool to gain an enhanced understanding of toxicity at the molecular level. Previously, we developed a pair ranking (PRank) method to assess in vitro to in vivo extrapolation (IVIVE) using toxicogenomic datasets from the Open Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System (TG-GATEs) database. With this method, we investiagted three important questions that were not addressed in our previous study: (1) is a 1-day in vivo short-term assay able to replace the 28-day standard and expensive toxicological assay? (2) are some biological processes more conservative across different preclinical testing systems than others? and (3) do these preclinical testing systems have the similar resolution in differentiating drugs by their therapeutic uses? For question 1, a high similarity was noted (PRank score = 0.90), indicating the potential utility of shorter term in vivo studies to predict outcome in longer term and more expensive in vivo model systems. There was a moderate similarity between rat primary hepatocytes and in vivo repeat-dose studies (PRank score = 0.71) but a low similarity (PRank score = 0.56) between rat primary hepatocytes and in vivo single dose studies. To address question 2, we limited the analysis to gene sets relevant to specific toxicogenomic pathways and we found that pathways such as lipid metabolism were consistently over-represented in all three assay systems. For question 3, all three preclinical assay systems could distinguish compounds from different therapeutic categories. This suggests that any noted differences in assay systems was biological process-dependent and furthermore that all three systems have utility in assessing drug responses within a certain drug class. In conclusion, this comparison of three commonly used rat TGx systems provides useful information in utility and application of TGx assays.

Integrated system with constructed wetlands for the treatment of domestic wastewaters generated at a rural property – Evaluation of general parameters ecotoxicity and cytogenetics

The present research investigated the efficiency of an integrated system for the treatment of wastewaters generated at a rural property located in the city of Vera Cruz do Sul, southern Brazil. The integrated system was composed of an anaerobic unit (UASB/AB), 4 subsurface constructed wetlands (SSFCWs), and two photoreactors (UV-254 nm). The evaluation of the performance was based on the reduction of the load parameters and mainly on the detoxification of the wastewaters. The ecotoxicological analysis included short-term assays with the microcrustacean Daphnia magna whereas the cytogenetic potential of the wastewaters was assessed by using the Allium cepa test system. On the one hand, the raw wastewaters presented a strong pollution potential due to high COD, BOD5, TKN, N-NH3 and total P values. On the other hand, the effluents treated by the integrated system showed high reductions of the abovementioned parameters and fully attend the Brazilian and International legal requirements. Furthermore, the treated wastewaters presented a complete absence of the acute ecotoxicity (100%) as well as significant reductions of the cytotoxicity, genotoxicity, and mutagenicity when compared to the raw wastewaters. Therefore, the investigated system can be considered an interesting low-cost sanitation alternative, especially for wastewaters produced in rural areas, where decentralized systems are more suitable. Besides, the results obtained in the present work demonstrate the importance of including toxicity analysis in the monitoring of waters and wastewaters in order to obtain a broader evaluation concerning the efficiency of different treatment systems.

Abstract P4-03-15: Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer

Abstract Background: Afatinib is an irreversible pan-HER inhibitor approved for non-small cell lung cancer. We have previously shown that afatinib inhibits growth of HER2-positive breast cancer cells and enhances response to trastuzumab. However, we have also shown that long-term exposure to tyrosine kinase inhibitors leads to the development of acquired resistance. To determine if acquired afatinib resistance develops in HER2-positive breast cancer cells, we exposed a HER2-positive breast cancer cell line to afatinib for 6 months and investigated alterations in the cells following long-term exposure. Methods: SKBR3 cells were treated with 150 nM afatinib twice-weekly for 6 months. Growth response to drug inhibitors was assessed by acid phosphatase assay. Drug sensitivity was examined in four HER2-positive cell lines (SKBR3, EFM192A, BT474 and HCC1954) and three acquired trastuzumab resistant cell lines (SKBR3-T, BT474-T and EFM192A-T). Reverse phase protein array (RPPA) was used to determine alterations in key signaling pathways. Src, p-Src, EGFR, p-EGFR, ERK1/2, p-ERK 1/2 levels were examined by Western blotting. To examine the prevention of the development of afatinib resistance, cells were treated twice weekly with afatinib, dasatinib, or the combination and stained with crystal violet when confluent. Results: Following 6 months of afatinib treatment, the SKBR3-A cells were more resistant to afatinib compared to parental cells (IC50 SKBR3-A 284 ± 28.2 nM vs SKBR3-Par 10.9 ± 3.4 nM). Furthermore, the resistant cells were cross-resistant to lapatinib, neratinib and trastuzumab. RPPA interrogation of the SKBR3-A cells showed alterations in several pathways, including significantly increased levels of p-Src (Y416). SKBR3-A cells were more sensitive to Src inhibition with dasatinib compared to SKBR3-Par cells and the combination of afatinib and dasatinib was highly synergistic in SKBR3-A cells (CI value = 0.09 ± 0.06). The combination of afatinib and dasatinib was also synergistic in the trastuzumab resistant SKBR3-T cells (Table). Afatinib and dasatinib inhibited EGFR and Src activation and ERK 1/2 signalling in SKBR3-A cells. Short-term resistance assays showed that the addition of dasatinib to afatinib blocks the emergence of resistant cells in three of four HER2 positive cell lines tested and two of the three acquired trastuzumab resistant cell lines tetsed. Conclusion: HER2-positive breast cancer cells that are highly sensitive to afatinib can develop acquired resistance to afatinib within six months. Src is a potential target to prevent the development of afatinib resistance and thus combined treatment with afatinib and dasatinib may be beneficial in patients with HER2-positive breast cancer. Percentage growth in HER2-positive cells treated with afatinib and/or dasatinib for 5 days. 20 nM Afatinib40 nM Dasatinib20 nM Afatinib + 40 nM DasatinibSKBR325.2 ± 6.1109.9 ± 6.222.6 ± 6.2SKBR3-A62.1 ± 0.787.3 ± 11.036.3 ± 0.3*SKBR3-T42.6 ± 4.9107.4 ± 9.729.3 ± 3.5*BT47418.2 ± 3.079.5 ± 5.213.3 ± 1.1BT474-T6.9 ± 2.795.3 ± 2.06.2 ± 2.3EFM192A39.1 ± 4.7105.7 ± 3.135.3 ± 2.7EFM192A-T30.4 ± 5.299.8 ± 2.528.5 ± 4.4HCC195461.8 ± 8.180.5 ± 8.219.4 ± 3.6** indicates enhanced anti-proliferative response for the combination compared to the single agents. Citation Format: Conlon N, Canonici A, Morgan C, Cremona M, Hennessey BT, Eustace A, O'Brien N, Slamon D, Crown J, O'Donovan N. Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-15.

Quantitative analysis of the complete larval settlement process confirms Crisp's model of surface selectivity by barnacles.

For barnacle cypris larvae at the point of settlement, selection of an appropriate surface is critical. Since post-settlement relocation is usually impossible, barnacles have evolved finely tuned surface-sensing capabilities to identify suitable substrata, and a temporary adhesion system for extensive surface exploration. The pattern of exploratory behaviour appears complex and may last for several hours, imposing significant barriers to quantitative measurement. Here, we employ a novel tracking system that enables simultaneous analysis of the larval body movement of multiple individuals over their entire planktonic phase. For the first time, to our knowledge, we describe quantitatively the complete settlement process of cyprids as they explore and select surfaces for attachment. We confirm the 'classic' behaviours of wide searching, close searching and inspection that comprise a model originally proposed by Prof. Dennis Crisp FRS. Moreover, a short-term assay of cyprid body movement has identified inspection behaviour as the best indicator of propensity to settle, with more inspection-related movements occurring in conditions that also promote higher settlement. More than half a century after the model was first proposed by Crisp, there exists a precise method for quantifying cyprid settlement behaviour in wide-ranging investigations of barnacle ecology and applied studies of fouling management.

Temperature effects on a marine herbivore depend strongly on diet across multiple generations.

Increasing sea surface temperatures are predicted to alter marine plant-herbivore interactions and, thus, the structure and function of algal and seagrass communities. Given the fundamental role of host plant quality in determining herbivore fitness, predicting the effects of increased temperatures requires an understanding of how temperature may interact with diet quality. We used an herbivorous marine amphipod, Sunamphitoe parmerong, to test how temperature and diet interact to alter herbivore growth, feeding rates, survival, and fecundity in short- and long-term assays. In short-term thermal stress assays, S. parmerong was tolerant to the range of temperatures that it currently experiences in nature (20-26°C), with mortality at temperatures >27°C. In longer term experiments, two generations of S. parmerong were reared in nine combinations of temperature (ambient, +2, +4°C) and diet (two high- and one low-quality algal species) treatments. Temperature and diet interacted to determine total numbers of amphipods in the F1 generation and the potential F2 population size (sum of brooded eggs and newly hatched juveniles). The size and development rate of F1 individuals were affected by diet, but not temperature. Consumption rates per capita were highest at intermediate temperatures but could not explain the observed differences in survival. Our results show that predicting the effects of increasing temperature on marine herbivores will be complicated by variation in host plant quality, and that climate-driven changes to plant availability will affect herbivore performance, and thus the strength of plant-herbivore interactions.

Chemotaxis to Atypical Chemoattractants by Soil Bacteria.

Although the mechanism of bacterial chemotaxis has been extensively studied in enteric bacteria, the hunt for novel and atypical chemoeffectors (in enterics and distantly-related species alike) has necessitated the modification of classic chemotaxis assays to deal with recalcitrant and potentially toxic chemicals. Here, we describe detailed protocols for the quantitative and qualitative assessment of chemotaxis responses that are categorized into short-term direct population response assays and long-term metabolism-based assays that can be used to identify novel chemoeffector molecules and the specific chemoreceptors involved. We emphasize the importance of behavior-based assays to verify the biochemical and physiological relevance of newly identified chemoeffector-receptor pairs.

Abstract B210: BET inhibitors show superior ability to suppress the progression of tumors containing the Pro47Ser single-nucleotide polymorphism of p53

Abstract The tumor suppressor TP53 is the most frequently mutated gene in human cancer and serves to restrict both tumor initiation and progression. Single-nucleotide polymorphisms (SNPs) in the p53 pathway can have a marked impact on p53 tumor suppressor function, and several have been associated with increased cancer risk. We have reported that a naturally occurring SNP affects codon 47 of TP53 (rs1800371, G/A). In most individuals, codon 47 encodes a proline (P47). However, approximately 2.5% of African-Americans express a p53 allele with a serine residue (S47) at this position. In order to compare the ability of the WT and S47 variants of p53 to suppress tumor development, we isolated mouse embryonic fibroblasts (MEFs) from WT and S47 mice and transformed them with the oncogenes E1A and Ras, in order to generate a facile model that would allow us to study responses to therapy. WT and S47 cells grew similarly in 10% serum. However, transformed S47 cells grew significantly better under nutrient-deprived conditions. S47 transformed cells also grew with increased dynamics in a xenograft mouse model and in a tail vein model of metastasis. These data support the premise that the S47 variant of p53 is significantly impaired as a tumor suppressor. We found that, for the majority of conventional genotoxic drugs, S47 tumor cells are resistant, compared to tumor cells with WT p53. In order to identify novel compounds that preferentially target S47 tumor cells, we performed a drug screen on WT and S47 transformed MEFs, using a library of over 200 conventional and experimental chemotherapeutic drugs. We found that S47 cells are far more sensitive to the class of bromodomain inhibitors, specifically BET inhibitors. Using short-term (96 hrs, Alamar Blue) cell viability assays, we found that S47 transformed cells were approximately 5-fold more sensitive to OTX-015, a BET inhibitor currently in clinical trials. We also found that S47 cells were significantly impaired in their ability to form colonies when pretreated for 24 hours with OTX-015. These data highlight the relevance of targeted therapy for patients harboring the S47 variant of p53, and suggest that BET inhibitors may preferentially target S47 tumors. Citation Format: Thibaut Barnoud, Subhasree Basu, Che-Pei Kung, Matthew Reiss, Rugang Zhang, Maureen Murphy. BET inhibitors show superior ability to suppress the progression of tumors containing the Pro47Ser single-nucleotide polymorphism of p53 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B210.

Abstract A117: Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells

Abstract ATM is a master regulator of the DNA damage response (DDR) elicited by double-strand breaks (DSB). After DNA DSB, ATM controls cell fate via cell cycle arrest, apoptosis, and DNA damage repair. Numerous tumor types have ATM gene loss-of-function mutations, resulting in genomic instability. Most knowledge on the effect of ATM loss on DDR and tumor therapeutic response is from ataxia-telangiectasia (A-T) patients and ATM knockout models. A distinct and more deleterious phenotype has been proposed for kinase-dead (KD) ATM mutants that retain ATM protein expression. Inhibition of ATM activity does not fully mimic ATM loss; therefore, KD ATM mutants may be better predictors of response to ATM inhibitors and chronic effects of ATM inhibition. Using an inducible lentiviral CRISPR/Cas9 genome-editing system, we generated in-frame ATM mutated clonal lines derived from a bladder cancer PDX model (BLX1), via targeting of ATM exon 57, which maps to the second exon in the kinase domain. CRISPR-edited clones consistently exhibited reduced phosphorylation of Kap1 (S824) but not CHK2, p53 or NBS1 when challenged with DSB-inducing agents such as ionizing radiation (IR) and etoposide. Similar results were obtained in unedited cells treated with the ATM inhibitor KU55933, confirming that changes observed are ATM-dependent and suggesting that reduced Kap1 phosphorylation is a robust readout of compromised ATM activity in response to DSB-inducing therapies. Radiosensitivity is a hallmark of the A-T syndrome and the CRISPR-edited clones had increased sensitivity to DSB-inducing agents including IR, bleomycin, etoposide, and doxorubicin but not to S-phase drugs gemcitabine and camptothecin in a 10-day colony formation assay. ATM deficiency increased the dependency of cancer cells on ATM-independent repair mechanisms. Indeed, short-term (4-day) proliferation assays demonstrated the synthetic lethality of KD ATM with ATR inhibitors (AZD6738, VE821), Chk1 inhibitors (MK8776, LY2606368), as well as high concentrations of PARP inhibitors (Olaparib, BMN673) but not the DNAPK inhibitor (NU7441). KD ATM clones did not have increased response to cisplatin as a single agent except at high concentrations (&amp;gt;10 µM); the combination of low-concentration cisplatin (&amp;lt;1 µM) and an ATR inhibitor had a greater effect in KD ATM clones compared to unedited cells, demonstrating increased dependence on ATR. In summary, CRISPR-Cas9 editing of the ATM kinase domain was a useful tool for characterizing ATM loss-of-function effects, identifying targets to resensitize ATM mutants and elucidating compensatory mechanisms driven by chronic ATM loss. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. El Touny, John Connelly, Curtis Hose, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A117.

Androgen receptor (AR) degradation enhancer ASC-J9® in an FDA-approved formulated solution suppresses castration resistant prostate cancer cell growth

ASC-J9® is a recently-developed androgen receptor (AR)-degradation enhancer that effectively suppresses castration resistant prostate cancer (PCa) cell proliferation and invasion. The optimal half maximum inhibitory concentrations (IC50) of ASC-J9® at various PCa cell confluences (20%, 50%, and 100%) were assessed via both short-term MTT growth assays and long-term clonogenic proliferation assays. Our results indicate that the IC50 values for ASC-J9® increased with increasing cell confluency. The IC50 values were significantly decreased in PCa AR-positive cells compared to PCa AR-negative cells or in normal prostate cells. This suggests that ASC-J9® may function mainly via targeting the AR-positive PCa cells with limited unwanted side-effects to suppress the surrounding normal prostate cells. Mechanism dissection indicated that ASC-J9® might function via altering the apoptosis signals to suppress the PCa AR-negative PC-3 cells. Preclinical studies using multiple in vitro PCa cell lines and an in vivo mouse model with xenografted castration-resistant PCa CWR22Rv1 cells demonstrated that ASC-J9® has similar AR degradation effects when dissolved in FDA-approved solvents, including DMSO, PEG-400:Tween-80 (95:5), DMA:Labrasol:Tween-80 (10:45:45), and DMA:Labrasol:Tween-20 (10:45:45). Together, results from preclinical studies suggest a potential new therapy with AR-degradation enhancer ASC-J9® may potentially be ready to be used in human clinical trials in order to better suppress PCa at later castration resistant stages.

Effect of Cinnamaldehyde Addition on Zebrafish (Danio rerio) Eggs Against Streptococcus agalactiae Infection

In this study, the effect of cinnamaldehyde (CA) towards Streptococcus agalactiae on zebrafish egg were investigated. To obtain the optimal concentration, CA and S. agalactiae were tested by short-term effects assay. Fresh zebrafish egg, CA and S. agalactiae were observed in different groups, single and mix treatment. The result showed that the larval survival rate (%) of single CA concentration (2.5 and 5 µg mL -1 ) were not significantly different compared to the control, with the value 94 ± 2.8% and 93 ± 3.3% respectively. Meanwhile, the single S. agalactiae concentration (10 9 and 10 10 CFU mL -1 ) were significantly different in comparison to the control with the value 73 ± 2.1% and 6 ± 2.5%. Mix groups, larval survival rate (%) value were significantly different with the best combination value was 17 ± 4.7% at 5 µg mL -1 of CA and 10 9 CFU mL -1 of S. agalactiae . Even though CA may potential for zebrafish egg shelter against S. agalactiae infection. But a mixture (CA and S. agalactiae ) treatments were not recommended for this study because the low value was shown compared to the single treatments. This possibility may occur because of the debris formation of two substances which leads to the poor environment and causes to low fish larval survival rate (%) value. For further study, the water quality assay is needed.

Influence of sulfate supply on selenium uptake dynamics and expression of sulfate/selenate transporters in selenium hyperaccumulator and nonhyperaccumulator Brassicaceae.

Stanleya pinnata not only hyperaccumulates selenium (Se) to 0.5% of its dry weight, but also exhibits higher tissue Se-to-sulfur (S) ratios than other species and its surroundings. To investigate the mechanisms underlying this Se enrichment, we compared S.pinnata with the nonhyperaccumulators S.elata and Brassica juncea for selenate uptake in long- (9d) and short-term (1h) assays, using different concentrations of selenate and competitor sulfate. Different sulfate pre-treatments (0, 0.5, 5mM, 3d) were also tested for effects on selenate uptake and sulfate transporters' expression. Relative to nonhyperaccumulators, S.pinnata showed higher rates of root and shoot Se accumulation and less competitive inhibition by sulfate or by high-S pretreatment. The selenate uptake rate for S.pinnata (1h) was three- to four-fold higher than for nonhyperaccumulators, and not significantly affected by 100-fold excess sulfate, which reduced selenate uptake by 100% in S.elata and 40% in B.juncea. Real-time reverse transcription PCR indicated constitutive upregulation in S.pinnata of sulfate transporters SULTR1;2 (root influx) and SULTR2;1 (translocation), but reduced SULTR1;1 expression (root influx). In S.pinnata, selenate uptake and translocation rates are constitutively elevated and relatively sulfate-independent. Underlying mechanisms likely include overexpression of SULTR1;2 and SULTR2;1, which may additionally have evolved enhanced specificity for selenate over sulfate.

Assessment of the hazard of nine (doped) lanthanides-based ceramic oxides to four aquatic species

The risk of environmental pollution with rare earth oxides rises in line with increasing application of these compounds in different sectors. However, data on potential environmental hazard of lanthanides is scarce and concerns mostly Ce and Gd. In this work, the aquatic toxicity of eight doped lanthanide-based ceramic oxides (Ce0.9Gd0.1O2, LaFeO3, Gd0.97CoO3, LaCoO3, (La0.5Sr0.5)0.99MnO3, Ce0.8Pr0.2O2, (La0.6Sr0.4)0.95CoO3, LaNiO4) and one non-doped oxide (CeO2) with primary size from 23 to 590nm were evaluated in four short-term laboratory assays with freshwater crustaceans and duckweeds. Results showed no acute toxicity (EC50>100mg/L) or very low acute toxicity for most studied oxides. Observed toxicity was probably due to bioavailable fraction of dopant metals (Ni and Co) but in the case of aquatic plants, decrease of nutrient availability (complexing of phosphorus by lanthanides) was also presumed. Studied oxides/metals accumulated in the aquatic plant tissue and in the gut of crustaceans and thus may be further transferred via the aquatic food chain. Accumulation of metals in the duckweed Lemna minor may be recommended as a cost-effective screening bioassay for assessment of potential hazard of poorly soluble oxides to aquatic ecosystems.

Identification of a Human Airway Epithelial Cell Subpopulation with Altered Biophysical, Molecular, and Metastatic Properties.

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of "metastatic inefficiency." Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514-24. ©2017 AACRSee related editorial by Hynds and Janes, p. 491.