The use of umbilical cord blood transplant has been substantially limited by the finite number of hematopoietic stem and progenitor cells in a single umbilical cord blood unit. Small molecules that not only quantitatively but also qualitatively stimulate enhancement of hematopoietic stem cell (HSC) self-renewal ex vivo should facilitate the clinical use of HSC transplantation and gene therapy. Recent evidence has suggested that the cyclin-dependent kinase inhibitor, p18INK4C (p18), is a critical regulator of mice HSC self-renewal. The role of p18 in human HSCs and the effect of p18 inhibitor on human HSC expansion ex vivo need further studies. Here we report that knockdown of p18 allowed for an increase in long-term colony-forming cells in vitro. We then identified an optimized small molecule inhibitor of p18, 005A, to induce ex vivo expansion of HSCs that was capable of reconstituting human hematopoiesis for at least 4 months in immunocompromised mice, and hence, similarly reconstituted secondary recipients for at least 4 more months, indicating that cells exposed to 005A were still competent in secondary recipients. Mechanistic studies showed that 005A might delay cell division and activate both the Notch signaling pathway and expression of transcription factor HoxB4, leading to enhancement of the self-renewal of long-term engrafting HSCs and the pool of progenitor cells. Taken together, these observations support a role for p18 in human HSC maintenance and that the p18 inhibitor 005A can enhance the self-renewal of long-term HSCs.
<div>AbstractPurpose:<p>Although platinum compounds are the first-line treatment for ovarian cancer, the majority of patients relapse and develop resistance to treatment. However, the mechanism underlying resistance is unclear. The goal of our study is to decipher the mechanism by which a metabolic kinase, diacylglycerol kinase alpha (DGKA), confers platinum resistance in ovarian cancer.</p>Experimental Design:<p>Metabolic kinase RNAi synthetic lethal screening was used to identify a cisplatin resistance driver in ovarian cancer. DGKA variants were used to demonstrate the need for DGKA activity in cisplatin resistance. Phospho-proteomic and genomic screens were performed to identify downstream effectors of DGKA. Therapeutic efficacy of targeting DGKA was confirmed and clinical relevance of DGKA signaling was validated using ovarian cancer patient-derived tumors that had different responses to platinum-based therapy.</p>Results:<p>We found that platinum resistance was mediated by DGKA and its product, phosphatidic acid (PA), in ovarian cancer. Proteomic and genomic screens revealed that DGKA activates the transcription factor c-JUN and consequently enhances expression of a cell-cycle regulator, WEE1. Mechanistically, PA facilitates c-JUN N-terminal kinase recruitment to c-JUN and its nuclear localization, leading to c-JUN activation upon cisplatin exposure. Pharmacologic inhibition of DGKA sensitized ovarian cancer cells to cisplatin treatment and DGKA–c-JUN–WEE1 signaling positively correlated with platinum resistance in tumors derived from patients with ovarian cancer.</p>Conclusions:<p>Our study demonstrates how the DGKA-derived lipid messenger, PA, contributes to cisplatin resistance by intertwining with kinase and transcription networks, and provides preclinical evidence for targeting DGKA as a new strategy in ovarian cancer treatment to battle cisplatin resistance.</p></div>
Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. When diagnosed at an early, localized stage, the five year survival rate is approximately 90%; however, when patients are diagnosed at the metastatic stage, this drops to 12%. Metastasis, the spread of cancer cells from the primary tumor to a distant organ, is the primary cause of death in cancer patients. In CRC, metastasis is typically found in the liver, asymptomatic, and often detected at the final stage when therapeutic options are few. The genetic and cellular mechanisms driving the early stages of metastasis are still poorly understood. Recent studies have shown that prior to the arrival of cancer cells at the secondary organ, molecular signals from the tumor direct the recruitment of bone marrow derived cells to create a pre-metastatic niche where cancer cells can attach and develop into a metastatic lesion. Identifying and understanding these molecules can lead to the development of methods for early diagnosis or targets for intervention. Using an orthotopic model of CRC liver metastasis, we performed microarray analyses of the liver microenvironment in tumor bearing mice before and after the arrival of metastatic cells. We found that Lipocalin-2 (LCN2, 24p3, NGAL) was highly expressed in the liver and in circulation of mice bearing highly metastatic cells. LCN2 is a 25 kD secreted glycoprotein that binds small lipophilic molecules. Its expression is upregulated in a number of primary tumors of epithelial origin, but its role in metastasis remains unclear. It has contrasting roles in metastasis in breast cancer models and has been associated with poor prognosis in CRC patients; however its role in the tumor microenvironment has not been studied. Our studies thus far show that overexpression of Lcn2 in mouse colon cancer cells either had little impact on tumor growth and invasiveness, or it enhanced invasion in the presence of macrophages; however, it resulted in an overall reduction of circulating iron levels in sera from tumor bearing mice. In-vitro, iron also seems to have an effect in conjunction with Lcn2 on the invasiveness of the tumor cells. The results from this study will allow us to better describe the role of LCN2 in tumor and stromal cells in the early stages of liver metastasis. Citation Format: Daniel Hughes, Celestia Davis, Yu Zhang, John Bonaparte, Maria Pena. The effects of Lipocalin-2 in the early stages of colorectal cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A22.
Abstract Human germinal center–associated lymphoma (HGAL) is an adaptor protein specifically expressed in germinal center lymphocytes. High expression of HGAL is a predictor of prolonged survival of diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma. Furthermore, HGAL expression is associated with early-stage DLBCL, thus potentially limiting lymphoma dissemination. In our previous studies, we demonstrated that HGAL regulates B-cell receptor signaling and cell motility in vitro and deciphered some molecular mechanisms underlying these effects. By using novel animal models for in vivo DLBCL dispersion, we demonstrate here that HGAL decreases lymphoma dissemination and prolongs survival. Furthermore, by using an unbiased proteomic approach, we demonstrate that HGAL may interact with multiple cytoskeletal proteins thereby implicating a multiplicity of effects in regulating lymphoma motility and spread. Specifically, we show that HGAL interacts with tubulin, and this interaction may also contribute to HGAL effects on cell motility. These findings recapitulate previous observations in humans, establish the role of HGAL in dissemination of lymphoma in vivo, and explain improved survival of patients with HGAL-expressing lymphomas.
Two novel compounds, 2‐(2‐hydroxyethylthio)‐5,8‐dimethoxy‐1,4‐naphthoquinone (HEDMNQ) and 2‐(6‐hydroxyhexylthio)‐5,8‐dimethoxy‐1,4‐naphthoquinone (HHDMNQ), were synthesized to investigate the kill effects and mechanism of 1,4‐naphthoquinone derivatives in lung cancer cells. The results of the CCK‐8 assay showed that HEDMNQ and HHDMNQ had significant cytotoxic effects on A549, NCI‐H23, and NCI‐H460 NSCLC cells. Flow cytometry and western blot results indicated that HHDMNQ induced A549 cell cycle arrest at the G2/M phase by decreasing the expression levels of cyclin‐dependent kinase 1/2 and cyclin B1. Fluorescence microscopy and flow cytometry results indicated that HHDMNQ could induce A549 cell apoptosis, and western blot analysis showed that HHDMNQ induced apoptosis through regulating the mitochondria pathway, as well as the MAPK, STAT3, and NF‐ κ B signalling pathways. Flow cytometry results showed that intracellular reactive oxygen species (ROS) levels were increased after HHDMNQ treatment, and western blot showed that ROS could modulate the intrinsic pathway and MAPK, STAT3, and NF‐ κ B signalling pathways. These effects were blocked by the ROS inhibitor N‐acetyl‐L‐cysteine in A549 cells. Our findings suggest that compared with HEDMNQ, HHDMNQ had the stronger ability to inhibit the cell viability of lung cancer cells and induce apoptosis by regulating the ROS‐mediated intrinsic pathway and MAPK/STAT3/NF‐ κ B signalling pathways. Thus, HHDMNQ might be a potential antitumour compound for treating lung cancer.
Abstract Certain regions of China have high rates of esophageal squamous cell carcinoma (ESCC). Previous studies of human papillomavirus (HPV), a proposed causal factor, have produced highly variable results. We attempted to evaluate HPV and ESCC more definitively using extreme care to prevent DNA contamination. We collected tissue and serum in China from 272 histopathologically‐confirmed ESCC cases with rigorous attention to good molecular biology technique. We tested for HPV DNA in fresh‐frozen tumor tissue using PCR with PGMY L1 consensus primers and HPV16 and 18 type‐specific E6 and E7 primers, and in formalin‐fixed paraffin‐embedded tumor tissue using SPF 10 L1 primers. In HPV‐positive cases, we evaluated p16 INK4a overexpression and HPV E6/E7 seropositivity as evidence of carcinogenic HPV activity. β‐globin, and thus DNA, was adequate in 98.2% of the frozen tumor tissues (267/272). Of these, 99.6% (95% confidence interval (CI) = 97.9–100.0%) were negative for HPV DNA by PGMY, and 100% (95% CI = 98.6–100%) were negative by HPV16/18 E6/E7 PCR. In the corresponding formalin‐fixed paraffin‐embedded tumor specimens, 99.3% (95% CI = 97.3–99.9%) were HPV negative by SPF 10 . By PGMY, 1 case tested weakly positive for HPV89, a noncancer causing HPV type. By SPF 10 , 2 cases tested weakly positive: 1 for HPV16 and 1 for HPV31. No HPV DNA‐positive case had evidence of HPV oncogene activity as measured by p16 INK4a overexpression or E6/E7 seropositivity. This study provides the most definitive evidence to date that HPV is not involved in ESCC carcinogenesis in China. HPV DNA contamination cannot be ruled out as an explanation for high HPV prevalence in ESCC tissue studies with less stringent tissue procurement and processing protocols.
Objective: Analysis of the molecular characteristics of eosinophilia. Methods: Targeting sequence to 24 patients with chronic eosinophilic leukemia (CEL) with rearrangement of PDGFRA, PDGFRB, or FGFR1 and 62 patients with hyper-eosinophilic syndrome (HES). Mutation annotation and analysis of amino acid mutation using authoritative databases to speculate on possible pathogenic mutation. Results: Thirty-seven kinds of clonal variant were detected from 17 patients with CEL, no recurrent mutation site and hot spot region were found. No pathogenic mutation was detected in 19 patients with PDGFRA rearrangement, but pathogenic mutations of ASXL1, RUNX1 and NRAS were detected from 2 patients with FGFR1 rearrangement who progressed to acute myeloid leukemia and 1 patient with PDGFRB rearrangement who progressed to T lymphoblastic lymphoma, respectively. One hundred and two kinds of clonal abnormalities were detected in 49 patients with HES. The main hot spot mutation regions included: CEBPA Exon1, TET2 Exon3, ASXL1 Exon12, IDH1 Y208C, and FGFR3 L164V. CRRLF2 P224L and PDGFRB R370C point mutations were detected separately in 2 patients with HES who treated with imatinib monotherapy and achieved hematologic remission. Conclusion: The pathogenesis of CEL with PDGFRA, PDGFRB or FGFR1 rearrangement is usually single, and the progression of the disease may involve other driver mutation. A variety of genes with hot mutation regions may be involved in the pathogenesis of HES, and some mutation sites are sensitive to tyrosine kinase inhibitors.目的： 分析嗜酸性粒细胞增多症的分子学特征。 方法： 对24例伴PDGFRA、PDGFRB或FGFR1重排的慢性嗜酸性粒细胞白血病（CEL）及62例高嗜酸性粒细胞综合征（HES）患者进行靶向测序，利用权威数据库进行变异注释及氨基酸突变分析，推测可能致病性突变。 结果： 17例（71%）CEL患者检出37种克隆性异常，但未发现重现性突变位点及热点突变区域，19例伴PDGFRA重排患者未检出致病性突变，2例进展为急性髓系白血病的伴FGFR1重排患者及1例进展为T淋巴母细胞淋巴瘤的伴PDGFRB重排患者分别检出ASXL1、RUNX1和NRAS的致病性突变。49例（71%）HES患者检出102种克隆性异常，主要热点突变区域包括：CEBPA Exon1、TET2 Exon3、ASXL1 Exon12、IDH1 Y208C和FGFR3 L164V。其中2例经伊马替尼单药治疗获得血液学缓解的HES患者分别检出CRRLF2 P224L和PDGFRB R370C点突变。 结论： 伴PDGFRA、PDGFRB或FGFR1重排的CEL致病因素单一，疾病进展可能有其他突变参与。HES可能由具有热点突变区域的多种基因参与致病，部分突变位点对酪氨酸激酶抑制剂治疗敏感。.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)