Cyclin A1 and P450 aromatase promote metastatic homing and growth of stem-like prostate cancer cells in the bone marrow

Bone metastasis is a leading cause of morbidity and mortality in prostate cancer (PCa). While cancer stem-like cells have been implicated as a cell of origin for PCa metastases, the pathways which enable metastatic development at distal sites remain largely unknown. In this study, we illuminate pathways relevant to bone metastasis in this disease. We observed that cyclin A1 (CCNA1) protein expression was relatively higher in PCa metastatic lesions in lymph node, lung, and bone/bone marrow. In both primary and metastatic tissues, cyclin A1 expression was also correlated with aromatase (CYP19A1), a key enzyme that directly regulates the local balance of androgens to estrogens. Cyclin A1 overexpression in the stem-like ALDHhigh subpopulation of PC3M cells, one model of PCa, enabled bone marrow integration and metastatic growth. Further, cells obtained from bone marrow metastatic lesions displayed self-renewal capability in colony forming assays. In the bone marrow, Cyclin A1 and aromatase enhanced local bone marrow-releasing factors, including androgen receptor, estrogen and matrix metalloproteinase MMP9 and promoted hte metastatic growth of PCa cells. Moreover, ALDHhigh tumor cells expressing elevated levels of aromatase stimulated tumor/host estrogen production and acquired a growth advantage in the presence of host bone marrow cells. Overall, these findings suggest that local production of steroids and MMPs in the bone marrow may provide a suitable microenvironment for ALDHhigh PCa cells to establish metastatic growths, offering new approaches to therapeutically target bone metastases

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1(IS) and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.</p

Response mechanisms of normal hematopoietic cells and leukemic cells to genotoxic agents and novel therapeutic strategies

Hematopoiesis is initiated by a rare population of hematopoietic stem cells (HSC) in the bone marrow (BM). HSCs give rise to red blood cells and white blood cells of myeloid and lymphoid lineages. The red blood cells are responsible for transporting oxygen throughout the body, while the myeloid and lymphoid cells are essentially required for immune responses. The HSCs reside in special niches in the bone marrow, and they are able to respond efficiently to external stresses and injury such as blood loss, infection, or damage caused by cytotoxic agents. Tumor cells and its associated microenvironment acquire multiple alterations which render the tumor cells to respond poorly to treatment. My thesis work is focused on several specific studies: (i) Study the function of Heme oxygenase 1 (HO-1), a key factor that regulates supply and transport of oxygen. HO-1 with its associated carbon monoxide (CO) mediates response of cells to DNA damages caused by irradiation or chemical genotoxic stress. (ii) Study the function of a key cell cycle regulatory gene, cyclin A1 in the regulation of proliferation, differentiation and migration of HSC, and the role of cyclin A1 and MMP9 in mediating response of the HSC and their adjacent BM microenvironment to genotoxic stress caused by irradiation. (iii) Study the role of the key cell cycle regulator CDK1 in mediating treatment response of leukemic cells to all-trans retinoic acid (ATRA). (iv) Characterize the cellular mechanisms of several anticancer drugs, and our newly developed anticancer drug candidate and its selective target PIP5K1a for treatment of metastatic cancer. We have used several sets of knockout mouse models in which HO-1 or cyclin A1 are deleted, either in germ lines or in specific tissues. In addition, we have used tumor xenograft mouse models in which human prostate cancer were implanted and grown in mice. We have also used normal and cancer specimens from patients with leukemia or prostate cancer. A panel of non-malignant and malignant cell lines has been applied in our in vitro functional studies. Our results have shown that mice lacking HO-1 gene has elevated H2AX, and loss of ATM expression and function. Induction of HO-1 expression reduces DNA damages and activates ATM-supported DNA repair via homologous recombination. We have shown that mice lacking cyclin A1 function has increased proliferation of HSC and migratory ability, and also impaired vascular niches. Mice without cyclin A1 function suffer early deaths after irradiation treatment compared to that of the wild-type mice. MMP9 may mediate the response of the BM to irradiation treatment. We further show that CDK1 plays an important role in mediating treatment response to ATRA-induced differentiation and cell cycle arrest. Finally, we show that our newly developed anticancer drug candidate ISA-2011B is a specific PIP5K1α inhibitor. ISA-2011B inhibits growth of invasive prostate cancer cells through its inhibitory effect on PIP5K1α/PI3K/AKT and AR/cell cycle pathways. Taken together, my work provides novel insights into stem cell regeneration and cancer therapy

Induction of apoptosis by staurosporine involves the inhibition of expression of the major cell cycle proteins at the G(2)/m checkpoint accompanied by alterations in Erk and Akt kinase activities.

BACKGROUND: Staurosporine is a therapeutic agent that inhibits tumor cell growth by inducing cell death via intrinsic apoptotic pathways. Our previous studies in clinical settings have suggested that certain subpopulations of patients with acute myeloid leukemia (AML) had poor response to chemotherapy. MATERIALS AND METHODS: The effect of staurosporine on apoptosis and cell cycle distribution in human leukemic cell line U-937 cells was determined. U-937 cells were treated with staurosporine at 0.5 microM for 18 hours or 1 microM for 24 hours. Analyses of cell cycle distribution and apoptosis were performed using flow cytometric analysis. The effects of staurosporine on the targeted proteins were assessed by immunoblot analysis. RESULTS: A blockade of the cell cycle at the G(2)/M phase was observed in U-937 cells treated with staurosporine. A concomitant induction of apoptosis and activation of caspase-3 in U-937 cells was also achieved. Treatment of U-937 cells with staurosporine at 1 microM for 24 hours, compared with 0.5 microM for 18 hours, appeared to kill the leukemic more efficiently cells and this dose and duration may specifically target p27, Erk and Akt pathways that are important for cancer cell survival and resistance to treatment. We also show that the effects of stauroporine on cell cycle progression and apoptosis in U-937 cells are closely linked. CONCLUSION: Our results suggest that induction of apoptosis and inhibitory proliferation and survival pathways are important events induced by staurosporine. Understanding the conditions under which staurosporine shows high specificity and low toxicity in treatment of leukemic cells is of great importance for improving the efficacy of targeted therapeutics and overcoming resistance to chemotherapeutic agents

DNA Methylation in ATRA-treated Leukemia Cell Lines Lacking a PML-RAR Chromosome Translocation.

A deficient retinoic acid signaling has been suggested to be an important cause of the clinical inefficacy of all-trans retinoic acid (ATRA) therapy in non-promyelocytic (non-PML) forms of acute myeloid leukemia (AML). The general aim of the present work was to explore novel ways to take advantage of the anti-leukemic potential of ATRA, and, specifically, to search for a synergism between ATRA and epigenetic drugs. Because previous reports have found no major influence of ATRA on DNA methylation, we investigated whether ATRA-mediated differentiation of the U937 and HL-60 AML cell lines, both lacking a PML-retinoic acid receptor (RAR) fusion product, is accompanied by early-appearing and weak changes in CpG methylation. We report that in HL-60 cells, by using a highly quantitative analysis of a set of genes found to be abnormally expressed in AML, polymerase chain reaction (PCR)-amplified p16 gene promoter molecules (each with 15 CpG sites), exhibited a CpG methylation level of 0-4% in untreated cells, which increased to 4-21% after treatment with ATRA for seven days. In contrast to HL-60 cells, U937 cells exhibited a very high CpG methylation level in p16, and ATRA did not influence the promoter methylation of this gene. In the total CCGG sites of the genome, analysed using a methylation-sensitive restriction enzyme, CpG methylation was significantly lower in ATRA-treated HL-60 (p<0.01) and U937 cells (p<0.05) than in controls. Taken together, our findings show that ATRA can influence DNA methylation, and suggest that future research should investigate whether epigenetic modulation may evoke a clinical effect of ATRA in leukemia

Who Needs Cream and Sugar When There Is Eco-Labeling? Taste and Willingness to Pay for “Eco-Friendly” Coffee

Participants tasted two cups of coffee, decided which they preferred, and then rated each coffee. They were told (in lure) that one of the cups contained “eco-friendly” coffee while the other did not, although the two cups contained identical coffee. In Experiments 1 and 3, but not in Experiment 2, the participants were also told which cup contained which type of coffee before they tasted. The participants preferred the taste of, and were willing to pay more for, the “eco-friendly” coffee, at least those who scored high on a questionnaire on attitudes toward sustainable consumer behavior (Experiment 1). High sustainability consumers were also willing to pay more for “eco-friendly” coffee, even when they were told, after their decision, that they preferred the non-labeled alternative (Experiment 2). Moreover, the eco-label effect does not appear to be a consequence of social desirability, as participants were just as biased when reporting the taste estimates and willingness to pay anonymously (Experiment 3). Eco labels not only promote a willingness to pay more for the product but also lead to a more favorable perceptual experience of it.</p

Who Needs Cream and Sugar When There Is Eco-Labeling? Taste and Willingness to Pay for "Eco-Friendly" Coffee

Participants tasted two cups of coffee, decided which they preferred, and then rated each coffee. They were told (in lure) that one of the cups contained "eco-friendly" coffee while the other did not, although the two cups contained identical coffee. In Experiments 1 and 3, but not in Experiment 2, the participants were also told which cup contained which type of coffee before they tasted. The participants preferred the taste of, and were willing to pay more for, the "eco-friendly" coffee, at least those who scored high on a questionnaire on attitudes toward sustainable consumer behavior (Experiment 1). High sustainability consumers were also willing to pay more for "eco-friendly" coffee, even when they were told, after their decision, that they preferred the non-labeled alternative (Experiment 2). Moreover, the eco-label effect does not appear to be a consequence of social desirability, as participants were just as biased when reporting the taste estimates and willingness to pay anonymously (Experiment 3). Eco labels not only promote a willingness to pay more for the product but also lead to a more favorable perceptual experience of it.Funding Agencies|University of Gavle, Sweden||</p

Heme detoxification by heme oxygenase-1 reinstates proliferative and immune balances upon genotoxic tissue injury

Phenotypic changes of myeloid cells are critical to the regulation of premature aging, development of cancer, and responses to infection. Heme metabolism has a fundamental role in the regulation of myeloid cell function and activity. Here, we show that deletion of heme oxygenase-1 (HO-1), an enzyme that removes heme, results in an impaired DNA damage response (DDR), reduced cell proliferation, and increased cellular senescence. We detected increased levels of p16INK4a, H2AXγ, and senescence-associated-β-galactosidase (SA-β-Gal) in cells and tissues isolated from HO-1-deficient mice. Importantly, deficiency of HO-1 in residential macrophages in chimeric mice results in elevated DNA damage and senescence upon radiation-induced injury. Mechanistically, we found that mammalian target of rapamycin (mTOR)/S6 protein signaling is critical for heme and HO-1-regulated phenotype of macrophages. Collectively, our data indicate that HO-1, by detoxifying heme, blocks p16INK4a expression in macrophages, preventing DNA damage and cellular senescence

Cyclin A1 regulates the interactions between mouse hematopoietic stem and progenitor cells and their niches.

It remains poorly understood how the hematopoietic stem/progenitor cells (HSPC) are attracted to their niches and the functional consequences of such interaction. In the present study, we show that the cell cycle regulator cyclin A1 in association with vascular endothelial growth factor receptor 1 (VEGFR1), is required for HSPC and their niches to maintain their function and proper interaction. In the absence of cyclin A1, the HSPC in the BM are increased in their frequency and display an increased migratory and homing ability. Concomitantly, the ability of the endosteal and central BM niche zones to attract and home the wild-type HSPC is significantly reduced in cyclin A1-null mice as compared to the wild-type controls. The impaired proliferation and homing of HSPC in the BM of cyclin A1-null mice are attributed to the increased density of microvessels in the endosteal and central BM niche zones, which is associated with the increased VEGFR1 expression. Thus, modulation of cyclin A1 and VEGFR1 in HSPC and their niches may provide new insights into therapeutic approaches

The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer.

Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy