ANKHD1, a novel component of the Hippo signaling pathway, promotes YAP1 activation and cell cycle progression in prostate cancer cells

ANKHD1 is a multiple ankyrin repeat containing protein, recently identified as a novel member of the Hippo signaling pathway. the present study aimed to investigate the role of ANKHDI in DU145 and LNCaP prostate cancer cells. ANKHD1 and YAP1 were found to be highly expressed in prostate cancer cells, and ANKHD1 silencing decreased cell growth, delayed cell cycle progression at the S phase, and reduced tumor xenograft growth. Moreover, ANKHD1 knockdown down-regulated YAP1 expression and activation, and reduced the expression of CCNA2, a YAP1 target gene. These findings indicate that ANKHD1 is a positive regulator of YAP1 and promotes cell growth and cell cycle progression through Cyclin A upregulation. (C) 2014 Elsevier Inc. All rights reserved.Univ Estadual Campinas, Hematol & Hemotherapy Ctr, Hemoctr, Inst Nacl Ciencia & Tecnol Sangue,UNICAMP, BR-13083878 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUniv Estadual Campinas, Integrated Ctr Childhood Oncohematol Invest, BR-13083878 Campinas, SP, BrazilUniv São Paulo, Dept Internal Med, Ribeirao Preto Med Sch, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilWeb of Scienc

Plasma Hsp90 Level as a Marker of Early Acute Lymphoblastic Leukemia Engraftment and Progression in Mice

<div><p>Current monitoring of acute lymphoblastic leukemia (ALL) in living mice is based on FACS analysis of blood hCD45+ cells. In this work, we evaluated the use of human IGFBP2, B2M or Hsp90 as soluble markers of leukemia. ELISA for B2M and IGFBP2 resulted in high background levels in healthy animals, precluding its use. Conversely, plasma levels of Hsp90 showed low background and linear correlation to FACS results. In another experiment, we compared Hsp90 levels with percentage of hCD45+ cells in blood, bone marrow, liver and spleen of animals weekly sacrificed. Hsp90 levels proved to be a superior method for the earlier detection of ALL engraftment and correlated linearly to ALL burden and progression in all compartments, even at minimal residual disease levels. Importantly, the Hsp90/hCD45+ ratio was not altered when animals were treated with dexamethasone or a PI3K inhibitor, indicating that chemotherapy does not directly interfere with leukemia production of Hsp90. In conclusion, plasma Hsp90 was validated as a soluble biomarker of ALL, useful for earlier detection of leukemia engraftment, monitoring leukemia kinetics at residual disease levels, and pre-clinical or mouse avatar evaluations of anti-leukemic drugs.</p></div

Correlation between plasma Hsp90 level and percentage of ALL cells in the different tissues analyzed.

<p>One representative case of three BCP-ALL or T-ALL analyzed is shown. For complete data refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.s002" target="_blank">S2 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.s003" target="_blank">S3 Fig</a> ELISA Hsp90 and flow cytometry hCD45+ data were transformed to log10 and analyzed by Pearson’s correlation. Correlations between ALL in peripheral blood and in the different tissues are shown for comparisons. Dotted line represents cut-off values for ALL detection by flow cytometry (0.5%) or Hsp90 levels (0.1 ng/mL). Data points correspond to individual samples. Numbers near each data point represent time point of sampling (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.g002" target="_blank">Fig 2</a>). PB; peripheral blood. BM; bone marrow. Circles, BCP-ALL. Triangles, T-ALL.</p

Analysis of three potential leukemia plasma biomarkers.

<p>(A) Peripheral blood B2M, IGFBP-2 and Hsp90 levels (ELISA) in NOD/SCID mice transplanted with a primary human T-ALL or healthy controls. Animals were divided into groups according to the percentage of ALL cells (hCD45+) in peripheral blood by FACS. Data points correspond to individual samples, and horizontal bars correspond to median. *P<0.05; Mann-Whitney U test. (B) Correlation between Hsp90 levels and % ALL cells in peripheral blood. Data points correspond to individual samples. Data were transformed to log10 and analyzed by Pearson’s correlation. (C) Cut-off value of Hsp90 (dotted line) as determined by adding 2 times SD to mean. Animals transplanted with different BCP-ALL (n = 3) and T-ALL (n = 2), sacrificed at the earliest time point (time point 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.g002" target="_blank">Fig 2</a>) were included in the analysis. Leukemia engraftment was confirmed by FACS analysis. Data points correspond to individual samples. PB, peripheral blood; SD, standard deviation.</p

ELISA of plasma Hsp90 levels for earlier engraftment confirmation.

<p>(A) Experimental workflow. Cryopreserved primary ALL cells were thawed and injected into 3 mice for expansion. Fresh xenograph ALL cells were then injected into 6 to 12 secondary animals for experiments. For RS4;11 and TALL-1, cultured cells were directly injected in animals for the experiments. Animals were monitored weekly and sacrificed as indicated. (B) Kinetic of Hsp90 plasma levels over time, following ALL injection. Data points represent mean of 3 animals. The cut-off Hsp90 value (0.1 ng/mL) is indicated. Asterisks represent starting week of sequential sacrifice of animals (Time Point 1). Some groups did not have enough animals for the experiment to be carried out until the third week of sacrifice. (C) Percentage of ALL cells (hCD45+) in bone marrow (BM) and peripheral blood (PB) at the different time points. Data points represent mean of 3 animals. Dotted line represents the usual cut-off (0.5%) for ALL detection by flow cytometry in blood samples. Different ALL cells are represented by colors. Circles; BCP-ALL. Triangles, T-ALL.</p

Chemotherapy effects on biomarkers levels.

<p>Plasma biomarkers levels (ELISA) and matched percentage of ALL cells (hCD45+) in peripheral blood (PB) from animals under chemotherapy treatment. Samples were collected for analysis at the beginning (C1 = day 5), middle (C2 = day 19) and end (C3 = day 33) of treatment. Colored bars represent mean ± SD of biomarker levels (left Y axis) for individual mice samples analyzed in duplicate. Different colors represent different treatments. The PI3K inhibitor used was AS605240. Black bars represent percentages of ALL cells in peripheral blood (right Y axis). The dotted line in the Hsp90 graphic represents the ELISA cut-off value. Red arrows highlight lower than expected ELISA values, when compared to levels found in untreated animals with similar percentage of ALL cells.</p

Immune response resetting in ongoing sepsis

Cure of severe infections, sepsis, and septic shock with antimicrobial drugs is a challenge because morbidity and mortality in these conditions are essentially caused by improper immune response. We have tested the hypothesis that repeated reactivation of established memory to pathogens may reset unfavorable immune responses. We have chosen for this purpose a highly stringent mouse model of polymicrobial sepsis by cecum ligation and puncture. Five weeks after priming with a diverse Ag pool, high-grade sepsis was induced in C57BL/6j mice that was lethal in 24 h if left untreated. Antimicrobial drug (imipenem) alone rescued 9.7% of the animals from death, but >5-fold higher cure rate could be achieved by combining imipenem and two rechallenges with the Ag pool (p < 0.0001). Antigenic stimulation fine-tuned the immune response in sepsis by contracting the total CD3(+) T cell compartment in the spleen and disengaging the hyperactivation state in the memory T subsets, most notably CD8(+) T cells, while preserving the recovery of naive subsets. Quantitative proteomics/lipidomics analyses revealed that the combined treatment reverted the molecular signature of sepsis for cytokine storm, and deregulated inflammatory reaction and proapoptotic environment, as well as the lysophosphatidylcholine/phosphatidylcholine ratio. Our results showed the feasibility of resetting uncontrolled hyperinflammatory reactions into ordered hypoinflammatory responses by memory reactivation, thereby reducing morbidity and mortality in antibiotic-treated sepsis. This beneficial effect was not dependent on the generation of a pathogen-driven immune response itself but rather on the reactivation of memory to a diverse Ag pool that modulates the ongoing response.203512981312FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2014/10068-

The Wnt signaling pathway regulates Nalm-16 b-cell precursor acute lymphoblastic leukemic cell line survival and etoposide resistance

10 p. : il.B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common malignancy in children. The Wnt signaling pathway has been found to be extensively involved in cancer onset and progression but its role in BCP-ALL remains controversial.Weevaluate the role of the Wnt pathway in maintenance of BCP-ALL cells and resistance to chemotherapy. Gene expression profile revealed that BCP-ALL cells are potentially sensitive to modulation of Wnt pathway. Nalm-16 and Nalm-6 cell lines displayed low levels of canonical activation, as reflected by the virtually complete absence of total b-catenin in Nalm-6 and the b-catenin cell membrane distribution in Nalm-16 cell line. Canonical activation with Wnt3a induced nuclear b-catenin translocation and led to BCP-ALL cell death. Lithium chloride (LiCl) also induced a cytotoxic effect on leukemic cells. In contrast, both Wnt5a and Dkk-1 increased Nalm-16 cell survival. Also, Wnt3a enhanced the in vitro sensitivity of Nalm-16 to etoposide (VP-16) while treatment with canonical antagonists protected leukemic cells from chemotherapy-induced cell death. Overall, our results suggest that canonical activation of the Wnt pathway may exerts a tumor suppressive effect, thus its inhibition may support BCP-ALL cell survival