Phospho-histone H3 Positive Cells Research Articles

Abstract P5-02-11: Assessment of tumor proliferation by mitotic activity index, phosphohistone H3 and Ki67 in early stage ER+/Her2- breast cancer

Abstract Introduction: Determining expression of tumor proliferation by mitotic index or Ki67 expression is one of the strongest prognostic factors in breast cancer, but its reproducibility is not optimal. Recently, phosphohistone H3 (PHH3) was proposed as a novel marker specific for mitosis. For the present study, we hypothesized that counting PHH3 positive cells would be more reproducible than mitoses counting in H&E sections (MAI) and Ki67. Methods: Tumor samples of 159 early breast cancer patients were immunohistochemically stained for Ki67 and PHH3. MAI and PHH3 scores were assessed by three breast cancer dedicated pathologists. Ki67 scores were assessed separately by two breast cancer pathologists. We used for Ki67 a threshold of 20% and for PHH3 the threshold score was 13. Intraclass correlation coefficient (ICC) and Cohen’s κ statistics were used to assess inter-observer agreement. The impact of swapping mitotic index with PHH3 in histologic grading was assessed. Results: The ICCs of PHH3 as a measure for inter-observer agreement on a continuous scale were 0.78 (obs. 1 vs. 2), 0.76 (obs. 2 vs. 3) and 0.97 (obs. 1 vs. 3). The PHH3 kappa scores (κ 0.78, 0.80, 0.68, table 1) were contrasted by fair to moderate inter-observer variation for Ki67 and MAI: Ki67, k 0.55 (obs. 1 and 2) and MAI k 0.38 (obs. 1 vs. 2), k 0.26 (obs. 2 vs. 3), k 0.56 (obs. 1 vs. 3). When using PHH3 in the Nottingham grading score instead of MAI, variation in histologic grading decreased (MAI k 0.43, 0.35, 0.32 vs. PHH3 k 0.52, 0.48, 0.52), while the proportion of grade III tumors increased (table 2). Conclusion: PHH3 seems to outperform Ki67 and MAI as a reproducible measure for tumor proliferation in breast cancer. Variation in histologic grading might be improved by using PHH3, but the resulting increase of high-grade cancers remains to be addressed. Table 1 The inter-observer agreement of PHH3 scored by three different breast cancer pathologistsObs. 2Obs. 3Obs. 3Obs. 1PHH3<13PHH3 ≥13PHH3<13PHH3 ≥13Obs. 2PHH3<13PHH3 ≥13PHH3<13583905PHH3<13597PHH3 ≥138371054PHH3 ≥13931Kappa0.780.800.67Abbreviations: PHH3, phosphohistone H3, Obs, observer.The concordance between obs. 1 & 2, obs. 1 & 3 and obs. 2 & 3 was 90%, 91% and 85%, respectively. Table 2. The impact of swapping mitotic index with PHH3 in histologic gradingGrade I n(%)Grade II n(%)Grade III n(%)Observer 1H&E42(26)104(65)13(8)PHH330(19)77(48)52(33)Observer 2H&E35(33)62(58)9(8)PHH323(22)57(54)26(24)Observer 3H&E25(16)101(64)33(21)PHH320(13)90(56)49(31)Abbreviations: PHH3, phosphohistone H3, H&E, hematolylin and eosin Citation Format: Julia E.C. van Steenhoven, Anne Kuijer, A. M. van Leeuwen, Joost M. van Gorp, Paul J. van Diest, Thijs van Dalen. Assessment of tumor proliferation by mitotic activity index, phosphohistone H3 and Ki67 in early stage ER+/Her2- breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-11.

Abstract A49: Using IL10-/- mouse model to assess heavy-ion radiation exposure-associated colitis and colorectal cancer incidence

Abstract Underlying risk of γ-ray-induced colitis and colorectal cancer incidence is emphasized by the studies on atom bomb survivors and also in patients undergoing radiotherapy. However, uncertainties in colitis and CRC risk prediction after exposure to high-LET space (particle) radiation exposure has not been realized due to lack of reliable in vivo data. Here we report IL10-/- mice as a suitable mouse model for assessment of colitis and CRC risk after exposure to space radiation. Male mice (n=12 mice/group) were whole-body exposed to sham-radiation, γ-rays (137Cs-source, 2.0 Gy), and 28Si-ion (300 MeV/n; 70 keV/μm; 1.4 Gy). We used γ-ray equitoxic doses using relative biologic effectiveness (RBE) values determined earlier. Mice were sacrificed 90 days after irradiation, and colon swiss rolls were prepared and histopathologic assessment of colitis score and CRC grade was noted. Further, inflammation-associated gene-expression analysis was conducted in both radiation-induced tumor and adjoining normal tissue area. Immunohistochemistry (β-catenin, Cyclin-D1, and phospho-histone H3) was also performed to assess HZE-induced proliferative signaling. Relative to controls and γ-ray, colitis score, colon tumor incidence, size, and grade were significantly higher after space (28Si-ion) radiation exposure. Additionally, tumor incidence per unit of radiation (per cGy) was also higher after 28Si-ion radiation relative to γ radiation. Higher number of phospho-histone H3-positive cells in 28Si-ion exposed mouse colon demonstrated increased proliferative index resulting in increased intestinal tumors of larger size and grade was due to greater activation of β-catenin and its downstream effector cyclin D1. Inflammatory gene signature was more pronounced after 28Si-ion exposure. Significant upregulation of GATA4, ICAM1, ITGA2, and EGFR was exclusive to 28Si-ion exposed mouse colon; however, PTGES and TGFβ1 were upregulated in both γ and 28Si-ion exposed mice. Overall, our findings suggest that IL10-/- mice are a suitable model to assess IR-induced colitis and CRC risk and space radiation carries higher risk of colitis and CRC incidence, relative to γ-rays at comparable doses. This study has implications for risk prediction in astronauts planning for deep-space missions and also for cancer patients planning to undergo particle radiotherapy. Citation Format: Shubhankar Suman, Bo-Hyun Moon, Albert J. Fornace, Jr., Kamal Datta. Using IL10-/- mouse model to assess heavy-ion radiation exposure-associated colitis and colorectal cancer incidence [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A49.

Digital image analysis of Ki67 in hot spots is superior to both manual Ki67 and mitotic counts in breast cancer.

During pathological examination of breast tumours, proliferative activity is routinely evaluated by a count of mitoses. Adding immunohistochemical stains of Ki67 provides extra prognostic and predictive information. However, the currently used methods for these evaluations suffer from imperfect reproducibility. It is still unclear whether analysis of Ki67 should be performed in hot spots, in the tumour periphery, or as an average of the whole tumour section. The aim of this study was to compare the clinical relevance of mitoses, Ki67 and phosphohistone H3 in two cohorts of primary breast cancer specimens (total n = 294). Both manual and digital image analysis scores were evaluated for sensitivity and specificity for luminal B versus A subtype as defined by PAM50 gene expression assays, for high versus low transcriptomic grade, for axillary lymph node status, and for prognostic value in terms of prediction of overall and relapse-free survival. Digital image analysis of Ki67 outperformed the other markers, especially in hot spots. Tumours with high Ki67 expression and high numbers of phosphohistone H3-positive cells had significantly increased hazard ratios for all-cause mortality within 10 years from diagnosis. Replacing manual mitotic counts with digital image analysis of Ki67 in hot spots increased the differences in overall survival between the highest and lowest histological grades, and added significant prognostic information. Digital image analysis of Ki67 in hot spots is the marker of choice for routine analysis of proliferation in breast cancer.

Prolyl isomerase Pin1 is required sperm production by promoting mitosis progression of spermatogonial stem cells

A prolyl isomerase Pin1 deficient (Pin1−/−) male mice had severe testicular atrophy. We investigated the function of Pin1 in spermatogenesis by analyzing the Pin1−/− mice at reproductive age. Pin1−/− mice had lessαPLZF positive spermatogonia (undifferentiated spermatogonia) than wild type (WT). Nevertheless, the Pin1−/− testis contained approximately the same number of GFRα1 positive spermatogonia (SSCs in steady state) as the WT testis. Furthermore, degeneration of the spermatogenia appeared in seminiferous tubules of 10 months old Pin1−/− mouse testis, and abnormal shape GFRα1 positive spermatogonia were observed. In Pin1−/− spermatogonia, the ratio of the phospho-histone H3 positive cells (mitotic cells) in GFRα1-positive spermatogonia was higher than that of WT. These results suggest that Pin1 promotes the progression of the mitotic cell cycle of SSC in steady-state, which is required for the sperm production from SSCs.

Pathophysiological analyses of leptomeningeal heterotopia using gyrencephalic mammals

Leptomeningeal glioneuronal heterotopia (LGH) is a focal malformation of the cerebral cortex and frequently found in patients with thanatophoric dysplasia (TD). The pathophysiological mechanisms underlying LGH formation are still largely unclear because of difficulties in obtaining brain samples from human TD patients. Recently, we established a new animal model for analysing cortical malformations of human TD by utilizing our genetic manipulation technique for gyrencephalic carnivore ferrets. Here we investigated the pathophysiological mechanisms underlying the formation of LGH using our TD ferrets. We found that LGH was formed during corticogenesis in TD ferrets. Interestingly, we rarely found Ki-67-positive and phospho-histone H3-positive cells in LGH, suggesting that LGH formation does not involve cell proliferation. We uncovered that vimentin-positive radial glial fibers and doublecortin-positive migrating neurons were accumulated in LGH. This result may indicate that preferential cell migration into LGH underlies LGH formation. Our findings provide novel mechanistic insights into the pathogenesis of LGH in TD.

Protein biomarker of cell proliferation determines survival to discharge in cases of equine large colon volvulus

Progenitor cells play critical roles in epithelial repair following ischaemic injury. Protein biomarkers have been used to identify intestinal progenitor cell subpopulations. This study aims to determine if a critical number of intestinal progenitor cells can predict tissue viability and survival to discharge of large colon volvulus (LCV) cases. The objectives were to 1) identify intestinal progenitor cell subpopulations using biomarkers: proliferating cell nuclear antigen (PCNA), sex determining region Y box 9 (SOX9), phospho-histone H3 (PHH3) and Ki-67, 2) define cut-off values for critical numbers of positive cells and 3) determine if survival to discharge is associated with cut-off values. Retrospective cohort study. Adult horses admitted to the Farm and Equine Veterinary Medical Center at NC State's Veterinary Hospital and Peterson and Smith Equine Hospital between 2006 and 2016 that underwent an exploratory coeliotomy with a diagnosis of LCV of ≥360 degrees, had pelvic flexure biopsy and that recovered from general anaesthesia were selected for inclusion in the study. Immunohistochemical analyses were performed and positive cells were counted. Optimal cut-off values were determined using receiver operator curves. A Fisher's exact test was used to associate cut-off values with survival to discharge. In this study, 23 cases of LCV ≥360° were included. Of 23 horses, 13 (57%) survived to discharge. A cut-off value of <2.1 PHH3 positive cells per crypt correctly predicted death with 100% sensitivity (95% CI; 69.15-100%) and 84.62% specificity (95% CI; 54.55-98.08%). LCV cases with <2.1 PHH3 positive cells per crypt were 96.6 times more likely to die (95% CI; 4.14-2255 and P<0.0001). Biomarkers PCNA, SOX9 and Ki-67 did not predict short-term survival. The population size was small. PHH3 immunohistochemical analysis may assist in more accurate prediction of survival to hospital discharge of LCV cases. The summary is available in Spanish - see Supporting Information.

Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway.

The Cancer Genome Atlas (TCGA) data indicate that high MDM2 expression correlates with all subtypes of breast cancer. Overexpression of MDM2 drives breast oncogenesis in the presence of wild-type or mutant p53 (mtp53). Importantly, estrogen-receptor positive (ER+) breast cancers overexpress MDM2 and estrogen mediates this expression. We previously demonstrated that this estrogen-MDM2 axis activates the proliferation of breast cancer cell lines T47D (mtp53 L194F) and MCF7 (wild-type p53) in a manner independent of increased degradation of wild-type p53 (ie, p53-independently). Herein we present data supporting the role of the estrogen-MDM2 axis in regulating cell proliferation and mammary tissue architecture of MCF7 and T47D cells in a p53-independent manner. Inducible shRNA mediated MDM2 knockdown inhibited colony formation in soft agar, decreased mass size and induced lumen formation in matrigel and also significantly reduced mitosis as seen by decreased phospho-histone H3 positive cells. The knockdown of MDM2 in both cell lines decreased Rb phosphorylation and the level of E2F1 protein. This signaling was through the estrogen receptor because fulvestrant (a selective estrogen receptor degrader) decreased MDM2 protein levels and decreased phosphorylation of Rb. Taken together these data indicate that in some ER+ breast cancers the estrogen-MDM2-Rb-E2F1 axis is a central hub for estrogen-mediated p53-independent signal transduction. This is the first indication that estrogen signaling utilizes the estrogen-MDM2 axis to provoke phosphorylation of Rb and increase E2F1 while promoting abnormal mammary architecture.

Postweaning development of porcine small intestinal morphology and epithelial cell proliferation1

Histomorphometry and cell proliferation are markers of intestinal development, which we investigated in pigs from weaning to slaughter at 5 different small intestinal sites. Furthermore, the impact of enteral deoxynivalenol (DON) exposure on intestinal morphology and proliferation was studied. Postweaning pigs of 4 categories (1, 4, 8, and 12 wk after weaning) were slaughtered (4–6 pigs/category) and intestinal wall was collected from the duodenum, proximal jejunum, mid jejunum, and proximal and terminal ileum. Villus height and crypt depths were measured on histological specimens, and cell proliferation was determined by immunohistochemistry (5′-bromo-2′-deoxyuridine–positive or phosphohistone H3–positive cells) in the crypt area. All pigs were fed either a control diet or a DON-contaminated diet (approximately 3 mg/kg feed), and data were analyzed for the effect of gut axis, age, and diet. Villus height was only marginally affected by intestinal site, age, or diet, but crypt depth and cell proliferation showed a distinct pattern along the small intestinal axis. In pigs, irrespective of age or diet, the duodenum exhibited the deepest crypts (P < 0.05) but the lowest count of proliferating cell, albeit the latter was not always statistically significant. Moreover, there was a strong age effect on crypt depth at the duodenal and upper jejunal level but not in the lower intestinal sections: crypt depth increased dramatically (P < 0.01) between 1 (294 μm), 4 (505 μm), and 8 wk after weaning (508 μm). There was no difference between the 4-wk-after-weaning animals and older animals. We could not confirm a positive relationship between crypt depth and proliferating cell count. The presence of dietary DON did not significantly modify the development of the small intestine with respect to villus height, crypt depth, or epithelial cell proliferation.

Effect of methotrexate on rostral migratory stream in newborn rats.

Two-day-old rats were treated with subcutaneous injections of methotrexate (MTX) 5 mg/kg and 150 mg/kg, and their rostral migratory streams (RMS) were examined time-dependently. MTX treatment increased pyknotic and TUNEL-positive cells and decreased mitotic and phospho-Histone H3-positive cells at almost all time points in the vertical arm, elbow and horizontal arm regions of the RMS. There were more TUNEL-positive cells ratio in the MTX 150 mg/kg group than in the MTX 5 mg/kg group. Treatment with MTX 150 mg/kg decreased the cellularity in the vertical arm region on Postnatal day (PD) 4, but that with the MTX 5 mg/kg did not. TUNEL-positive cells ratio was the highest in the vertical arm region, followed by elbow and horizontal regions in both MTX-treated groups. TUNEL-positive cells ratio in the vertical arm and elbow regions reached their peaks on PD 4 in both MTX-treated groups, and both MTX-treatments significantly decreased Phospho-Histone H3-positive cells ratio on PDs 2.5 and 3 in the vertical arm, elbow and horizontal arm regions. The phospho-Histone H3-positive cells ratio in the vertical arm region recovered on PD4 in the MTX 150 mg/kg group. These findings suggested that RMS required a great amount of folic acid on PD 2 and that the folic acid-requirement differed depending on the anatomical region of the RMS. To our knowledge, this is the first report demonstrating the effect of MTX on the RMS and the necessity of the folic acid metabolism on RMS development in newborn rats.

Effect of methotrexate on cerebellar development in infant rats.

Six-day-old rats were treated intraperitoneal injections with methotrexate 1 mg/kg, and the cerebellum was examined. Both the length and width of the vermis decreased in the methotrexate-treated group instead of the control from 4 day after treatment (DAT) onward. A significant reduction in the width of the external granular layer was detected on 2 and 3 DAT in the methotrexate group. By 4 DAT, the width of the external granular layer of the methotrexate group was indistinguishable from the control, and by 8 DAT, it was greater than that of the control. The molecular layer of methotrexate group on 8 and 15 DAT was thinner than that of the control. On 1 DAT, in the methotrexate group, there were many TUNEL and cleaved caspase-3-positive granular cells throughout the external granular layer, and they decreased time-dependently. On 1 DAT, in the methotrexate group, phospho-histone H3-positive cells in the external granular layer were fewer than in the control and tended to increase on 2–4 DAT. The p21-positive-rate of the external granule cells in the MTX group was higher than in the control on 1–4 DAT. These results suggested that methotrexate exposure on postnatal day 6 induces a delay, slowing in the migration of external granular cells to the inner granular layer, attributed to decrease or inhibition in the production of external granular cells that had arisen from apoptosis and the decrease in cell proliferative activity, resulting in cerebellar hypoplasia.

Abstract 2252: Gadd45b deficiency impairs G2/M cell cycle progression leading to premature senescence

Abstract Gadd45a, Gadd45b, and Gadd45g encode for small (18 kD), evolutionarily conserved proteins that play a pivotal role in diverse cellular functions such as cell cycle arrest, DNA repair, and are regulated by the nature of the stress stimulus encountered, its magnitude, and the cell type. Here, we show that Gadd45b negatively regulates stress-induced senescence. By using the 3T3 growth protocol to study tissue culture-induced senescence; we observed that Gadd45b KO MEFs display reduced growth rates at early passages (by passage 5) compared to wild type (WT) MEFs. Notably, this is in contrast with Gadd45a KO MEFs that show enhanced growth rate and escape senescence. Furthermore, senescence associated (SA) βgal staining showed increased blue stained cells at earlier passages in Gadd45b KO MEFs compared to WT MEFs. When MEFs were cultured at 3% oxygen, Gadd45b KO MEFs at 3% oxygen showed increased growth rate compared to MEFs at 21% oxygen, but growth was still retarded compared to WT MEFs at 21%, indicating that Gadd45b KO MEFs have increased sensitivity to oxidative stress leading to increased senescence. Additionally, we also observed that Gadd45b levels in WT MEFs increased with increasing passage number. Propidium Iodide staining and FACS analysis showed that Gadd45b KO MEFs arrest at the G2M phase of cell cycle. At passage 7, the majority (75%) of Gadd45b KO MEFs were observed to arrest at the G2M phase, whereas only 15% of WT MEFs arrested at G2M. This finding is in striking contrast to other senescent MEFs, which normally arrest at G1. Furthermore, FACS analysis of phospho-histone H3 (ser10) stained cells (mitotic) showed that, Gadd45b KO MEFs have less phospho-histone H3-positive cells than WT MEFs indicating that Gadd45b KO MEFs are arrested in G2 phase rather than M phase. Recent data also indicate that Gadd45b KO MEFs show increased phospho-histone H2Ax and phospho-p53 staining compared to WT MEFs, suggesting that loss of Gadd45b leads to increased DNA damage signaling. Interestingly, other stressors such as sub-lethal H2O2 as well as UV irradiation, known to increase oxidative stress, were observed to trigger increased premature senescence in Gadd45b KO MEFs compared to WT MEFs. Notably, staining embryos for SA βgal, we show that embryos from Gadd45b KO mice exhibit increased SA βgal staining compared to WT embryos, thus providing in vivo evidence for increased senescence in Gadd45b KO mice. Taken together, these data provide the impetus to further elucidate the role of Gadd45b in G2 arrest and premature senescence. Note: This abstract was not presented at the meeting. Citation Format: Andrew Magimaidas, Barbara Hoffman, Dan Liebermann. Gadd45b deficiency impairs G2/M cell cycle progression leading to premature senescence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2252. doi:10.1158/1538-7445.AM2014-2252

Evaluation of repeated dose micronucleus assays of the liver using N-nitrosopyrrolidine: A report of the collaborative study by CSGMT/JEMS.MMS

The repeated dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens, and can be integrated into a general toxicological study. To assess the performance of the assay, N-nitrosopyrrolidine (NPYR), a genotoxic hepatocarcinogen, was tested in 14- or 28-day RDLMN assays. NPYR was orally administered to rats at a daily dose of 25, 50 or 100mg/kg. One day after the last administration, a portion of the liver was removed and hepatocyte micronucleus (MN) specimens were prepared by the new method recently established by Narumi et al. In addition, a bone marrow MN assay and a histopathological examination of the liver were conducted. The detection of Phospho-Histone H3 was performed by immunohistochemistry to evaluate the proliferation rate of hepatocytes. The results showed significant increase in the number of micronucleated hepatocytes and Phospho-Histone H3-positive cells from the lowest dose in both 14- and 28-day RDLMN assays. On the other hand, the bone marrow MN assay yielded a negative result, which was in accordance with the existing report of the bone marrow MN assay using mice. Upon histopathological examination, inflammatory lesions and hypertrophy were noted, which may explain the increase in the hepatocyte proliferation and the enhancement of MN induction by NPYR. Our findings indicate that the RDLMN assay could be a useful tool for comprehensive risk assessment of carcinogenicity by providing information on both genotoxicity and histopathology when integrated into a general repeat dosing toxicity assay.

Abstract P2-09-04: Development of novel combination therapy of IGF-1R/InsR and MEK inhibitors in triple-negative breast cancer (TNBC)

Abstract Recent studies found that TNBC had a highly activated profile in the insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (InsR) pathway. Patients in whom the IGF-1R/InsR pathway was activated had a worse prognosis than did those in whom the pathway was not activated. We also previously found that the tumor-initiating cells in mouse TNBCs had a highly activated IGF-1R pathway. On the basis of these findings, we tested an IGF-1R/InsR dual kinase inhibitor, KW-2450, in TNBC. Results: To investigate the antitumor effects of KW-2450 in TNBC, we first confirmed the high IGF-1R and low InsR expression in TNBC cell lines (e.g., SUM149, MDA-MB-231, MDA-MB-468). An in vitro growth inhibition assay revealed that KW-2450 inhibited cell growth (all IC50 &amp;lt;0.5 μM), and an agar assay for colony formation confirmed this antitumor effect in TNBC cells. We also tested KW-2450's inhibitory effect against cancer stem cell (CSC) activity. With KW-2450 treatment of SUM149 and MDA-MB-231 cells, proportions of CSCs, profiled as CD44+CD24−, were significantly reduced in a dose-dependent manner. Indeed, KW-2450 dose-dependently inhibited mammosphere-forming, a hallmark of CSC activity. Cell cycle analysis revealed that KW-2450 induced mitotic accumulation and apoptosis in TNBC cells. Interestingly, MDA-MB-468 cells were the most susceptible to death, and their sensitivity to KW-2450 was associated with the high activation level of the mitotic checkpoint, the levels of which were determined by accumulation of cyclin B1 (on Western blot) and of phospho-histone H3–positive cells, a mitosis marker (on FACS). In contrast, SUM149 and MDA-MB-231 cells, which are relatively unsusceptible to death from KW-2450, exited from mitosis (as indicated by an accumulation of 8N; octaploidy) without significant cell death. This variable sensitivity to KW-2450 was also observed in in vivo studies. We confirmed that MDA-MB-468 tumors that were treated with 80 mg/kg of KW-2450 in vivo were stable and had many more mitotic cells than did those treated with vehicle control, which suggests that mitotic accumulation is a key process for this antitumor effect. Since it is known that any of the MAPKs (e.g., JNKs, p38 kinase, ERKs) become activated at the mitotic phase in mammalian cells, we next investigated whether the activation levels of MAPKs play critical roles in either mitotic progression or mitotic death in TNBC. Western blot analysis revealed that KW-2450 activated ERKs, but not JNKs or p38 kinase, in KW-2450–insensitive MDA-MB-231 and SUM149 cells, suggesting that ERK activation may promote mitotic progression but not mitotic death. Indeed, the combination of a MEK inhibitor (AZD6244), which inhibits ERK activation, and KW-2450 significantly reduced the 8N fraction and increased cell death in SUM149 and MDA-MB-231 cells. Conclusion: KW-2450 had significant antitumor effects in vitro and in vivo in TNBC cells. KW-2450–induced cell death, accompanied by mitotic accumulation, depended on mitotic checkpoint activity. TNBC cells refractory to KW-2450 were sensitized to KW-2450 by the addition of a MEK inhibitor. This novel combination therapy targeting IGF-1R/InsR and MEK in TNBCs, whose mitotic checkpoints are commonly abrogated, needs to be developed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-04.

Irradiation to the young mouse brain impaired white matter growth more in females than in males

Modern therapy cures 80% of all children with brains tumors, but may also cause long-lasting side effects, so called late effects. Radiotherapy is particularly prone to cause severe late effects, such as intellectual impairment. The extent and nature of the resulting cognitive deficits may be influenced by age, treatment and gender, where girls suffer more severe late effects than boys. The reason for this difference between boys and girls is unknown, but very few experimental studies have addressed this issue. Our aim was to investigate the effects of ionizing radiation on the corpus callosum (CC) in both male and female mice. We found that a single dose of 8 Gray (Gy) to the brains of postnatal day 14 mice induced apoptosis in the CC and reduced the number of proliferating cells by one third, as judged by the number of phospho-histone H3 positive cells 6 h after irradiation (IR). BrdU incorporation was reduced (62% and 42% lower in females and males, respectively) and the number of oligodendrocytes (Olig2+ cells) was lower (43% and 21% fewer in females and males, respectively) 4 months after IR, so the lack of developing and differentiated cells was more pronounced in females. The number of microglia was unchanged in females but increased in males at this late time point. The density of microvessel profiles was unchanged by IR. This single, moderate dose of 8 Gy impaired the brain growth to some extent (8.1% and 0.4% lower brain/body weight ratio in females and males, respectively) but the CC growth was even more impaired (31% and 19% smaller in females and males, respectively) 4 months after IR compared with non-irradiated mice. In conclusion, this is the first study to our knowledge demonstrating that IR to the young rodent brain affects white matter development more in females than in males.

A phase I, dose-escalation study of the Eg5-inhibitor EMD 534085 in patients with advanced solid tumors or lymphoma

The kinesin spindle protein Eg5 is involved in mitosis, and its inhibition promotes mitotic arrest. EMD 534085, a potent, reversible Eg5 inhibitor, demonstrated significant preclinical antitumor activity. This first-in-man, single-center, open-label, phase I dose-escalation study (3 + 3 design) investigated EMD 534085 safety, pharmacokinetics and antitumor activity in refractory solid tumors, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. EMD 534085 (starting dose 2 mg/m²/day) was administered intravenously every 3 weeks. Doses were escalated in 100% steps in successive cohorts of 3 patients until grade 2 toxicity occurred, followed by 50% until the first dose-limiting toxicity (DLT) arose. If <2 of 6 patients experienced a DLT, doses were further increased by 25%. Dose-escalation was stopped if a DLT occurred in ≥2 of 6 patients. Forty-four patients received EMD 534085. Median treatment duration was 43 days (range, 21-337). Thirty-eight patients (86%) received ≥2 cycles. DLTs were grade 4 neutropenia (1 patient each at 108 and 135 mg/m²/day), and grade 3 acute coronary syndrome with troponin I elevation (1 patient at 135 mg/m²/day). The maximum tolerated dose (MTD) was 108 mg/m²/day. The most common treatment-related adverse events were asthenia (50%) and neutropenia (32%). EMD 534085 appeared to have linear pharmacokinetics. Increase in phospho-histone H3 positive cells in paired pre- and on-treatment biopsies showed evidence of target modulation. No complete or partial responses were observed. Best response was stable disease in 23 patients (52%). EMD 534085 appeared to be well tolerated; MTD was 108 mg/m²/day. Preliminary antitumor results suggested limited activity in monotherapy.

Abstract 2772: Inhibition of EGFR activity reduces AML cell survival via inhibition of downstream Akt and Erk signaling pathways.

Abstract Epidermal growth factor receptor (EGFR) inhibitor erlotinib, has been shown to induce complete remission of acute myeloid leukemia (AML) in two patients with concurrent lung cancer, suggesting a prominent role for EGFR in AML. However, EGFR expression analysis in AML is poorly defined and the role of EGFR in AML is still unclear. In this study, we determined the EGFR protein expression in different human AML cell lines and studied the impact of EGFR inhibition by canertinib on the AML cell survival, proliferation, apoptosis and their downstream signaling routes. Using flowcytometry, we have shown that total EGFR as well as the phosphorylated residue Tyr1068 is expressed in five out of the eight AML cell lines (range of 4.9-46.6% for total and phosphorylated EGFR). EGFR phosphorylation was found to be induced by EGF stimulation, which could be prevented by canertinib treatment, showing functional activity and specificity of the EGFR phosphorylation site. Canertinib was shown to decrease the AML cell survival sufficiently (at a median IC50 of 6.2 μM, range: 0.9 - 15.2 μM canertinib in n=8 AML cell lines) as measured by WST-1 assays. The lower cell survival was the net result of lower numbers of mitotic phospho-histone H3 positive cells and an increase in the number of Annexin V positive cells. Immunoblot analysis revealed that canertinib treatment (5uM) reduced the phosphorylation of Akt and Erk intracellular proteins downstream of the EGFR signaling pathway in AML cells. Long-term canertinib treatment (5uM) showed that downstream targets were re-activated after 24 hours, which suggest kinomic reprogramming by alternative escape mechanisms compensating for the loss of EGFR activity. These findings suggest encouraging possibilities for targeting EGFR as a therapeutic strategy in AML. Future studies are necessary to determine the exact mechanism through which EGFR activity might contribute to AML progression, relapses as well as to interact with possible escape mechanisms of AML cells. Citation Format: Ellen R. Scheepers, Kim R. Kampen, Eveline SJM de Bont. Inhibition of EGFR activity reduces AML cell survival via inhibition of downstream Akt and Erk signaling pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2772. doi:10.1158/1538-7445.AM2013-2772

Effect of methotrexate on rat placenta development

The sequential changes in the placenta from rats exposed to methotrexate were examined histopathologically. Methotrexate was intraperitoneally administered at 0.2mg/kg/day during gestation days (GDs) 11-12 or GDs 13-14, and the placentas were sampled on GDs 13, 15, 17 and 21. The fetal mortality rates tended to increase throughout the experimental period, and fetal weights were significantly decreased on GD13 in the GD11,12-treated group. A significant reduction in placental weights was detected on GDs 13 and 15 in the GD11,12-treated group. Histopathologically, in the GD11,12-treated group, a significant thinning of the basal zone was detected throughout the experimental period, whereas the thickness of the labyrinth zone decreased significantly during GDs 13-17. The severity of thinning of the basal zone was higher than that of the labyrinth zone. In addition, a marked decrease in glycogen cell-islands in the basal zone was detected on GD 15. A significant decrease in Phospho-Histone H3-positive cells and a significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were detected on GD 13 in the basal zone of the GD11,12-treated group. In the GD13,14-treated group, there were no other significant changes in placentas and fetuses throughout the experimental period other than thinning of the labyrinth zone on GD 17. These results suggest that component cells of the basal and labyrinth zones during GDs 11-12 are more highly sensitive for methotrexate than those during GDs 13-14 and methotrexate affects the component cells of basal zone more strongly than those of the labyrinth zone.

MEK1 inactivates Myt1 to regulate Golgi membrane fragmentation and mitotic entry in mammalian cells

The pericentriolar stacks of Golgi cisternae are separated from each other in G2 and fragmented extensively during mitosis. MEK1 is required for Golgi fragmentation in G2 and for the entry of cells into mitosis. We now report that Myt1 mediates MEK1's effects on the Golgi complex. Knockdown of Myt1 by siRNA increased the efficiency of Golgi complex fragmentation by mitotic cytosol in permeabilized and intact HeLa cells. Myt1 knockdown eliminated the requirement of MEK1 in Golgi fragmentation and alleviated the delay in mitotic entry due to MEK1 inhibition. The phosphorylation of Myt1 by MEK1 requires another kinase but is independent of RSK, Plk, and CDK1. Altogether our findings reveal that Myt1 is inactivated by MEK1 mediated phosphorylation to fragment the Golgi complex in G2 and for the entry of cells into mitosis. It is known that Myt1 inactivation is required for CDK1 activation. Myt1 therefore is an important link by which MEK1 dependent fragmentation of the Golgi complex in G2 is connected to the CDK1 mediated breakdown of Golgi into tubules and vesicles in mitosis.

Effect of estrogen on rat placental development depending on gestation stage

We examined the sequential histopathological changes in the placenta from rats exposed to estrogen. 17 β-estrogiol-3-benzoate was intraperitoneally administered at 100 μg/animal/day during GD 6 to GD 8 (GD6-8 treated group), GD 9 to GD 11 (GD9-11 treated group) and GD 12 to GD 14 (GD12-14 treated group), and the placentas were sampled on GDs 11, 13, 15, 17, and 21. Fetal mortality rates were increased up to approximately 50% in the GD6-8 and 9-11 treated groups, but there was no change of fetal weight on GD 21. An increase in placental weight and a reduction in fetal/placental weight ratio were detected during GD 17 to GD 21 in the GD6-8 treated group. Histopathologically, hypoplasia of metrial gland was detected with defective development of spiral arteries in the GD6-8 and GD9-11 treated groups. A decrease in the thickness of metrial gland was observed from GD 11 onwards in the GD6-8 treated group and from GD 13 onwards in the GD9-11 treated group. The endovascular trophoblasts invaded into the spiral arteries in the deep part of metrial gland in these treated groups. The number of phospho-histone H3 positive cells was decreased on GD 11 or GD 13 in these groups. In the decidua basalis, transitory necrosis was observed with hemorrhage on GD 13 in the GD6-8 and GD9-11 treated groups. In the labyrinth zone, cystic dilatation of the sinusoid was observed with congestion in the GD6-8 treated group, resulting in an increased placental weight. Therefore, we consider that estrogen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland, and leads to metrial gland hypoplasia with less development of the spiral arteries. The reduced utero-placental blood flow is supposed to be one of the important factors for poor reproductive performance.

Inhibition of Aurora-B kinase activity confers antitumor efficacy in preclinical mouse models of early and advanced gastrointestinal neoplasia

The Aurora family of kinases, play a fundamental role in cell division and are overexpressed in several cancers including colon. The activity of barasertib-hQPA, a selective inhibitor of Aurora-B kinase (ABK) was investigated in a range of preclinical models of gastrointestinal cancer. Treatment with barasertib-hQPA produced anti-proliferative and cytotoxic effects across a panel of human colorectal cancer (CRC) cell lines in vitro. Prodrug, barasertib [48-h subcutaneous (s.c.) infusion; 150 mg/kg/day] inhibited the growth of SW620, Colo205, HCT116 human colorectal tumor xenografts in nude mice significantly (Student's t-test, P<0.05, n=10-12 per group). Flow cytometric analysis of single cells from disaggregated barasertib-treated SW620 tumors revealed a decrease in phosphorylated histone H3 (phH3) and an increase in tumor cells with ≥4N DNA content P<0.05). The activity of barasertib was then examined in ApcMin/+ mice, a spontaneous model of early intestinal neoplasia. Macroscopic evaluation of the small intestine revealed that barasertib treatment [25 mg/kg intra-peritoneal (i.p.) Q1Dx4 each week for 3 weeks] of 8-week old ApcMin/+ mice produced a 39% reduction in macroadenoma number (P=0.02) and a 43% reduction in overall adenoma burden (P=0.02) compared with vehicle-treated controls. Quantification of microscopic adenomas revealed a >64% reduction in the number of adenomas spanning more than one villus. Histological analysis of these adenomas revealed a number of distinct changes in barasertib-treated ApcMin/+ mice, including a 94% reduction in the proportion of phospho-histone H3-positive cells (P<0.001) and a 53% reduction in the number of cells per adenoma (P=0.001). These results provide a scientific rationale for investigating ABK inhibitors as a treatment for intestinal cancer.