Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling

SummaryMany signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming

Down-Regulation of NDRG1 Promotes Migration of Cancer Cells during Reoxygenation

One characteristic of tumor microenvironment is oxygen fluctuation, which results from hyper-proliferation and abnormal metabolism of tumor cells as well as disorganized neo-vasculature. Reoxygenation of tumors can induce oxidative stress, which leads to DNA damage and genomic instability. Although the cellular responses to hypoxia are well known, little is known about the dynamic response upon reoxygenation. In order to investigate the transcriptional responses of tumor adaptation to reoxygenation, breast cancer MCF-7 cells were cultured under 0.5% oxygen for 24 h followed by 24 h of reoxygenation in normoxia. Cells were harvested at 0, 1, 4, 8, 12, and 24 h during reoxygenation. The transcriptional profile of MCF-7 cells upon reoxygenation was examined using Illumina Human-6 v3 BeadChips. We identified 127 differentially expressed genes, of which 53.1% were up-regulated and 46.9% were down-regulated upon reoxygenation. Pathway analysis revealed that the HIF-1-alpha transcription factor network and validated targets of C-MYC transcriptional activation were significantly enriched in these differentially expressed genes. Among these genes, a subset of interest genes was further validated by quantitative reverse-transcription PCR. In particular, human N-MYC down-regulated gene 1 (NDRG1) was highly suppressed upon reoxygenation. NDRG1 is associated with a variety of stress and cell growth-regulatory conditions. To determine whether NDRG1 plays a role in reoxygenation, NDRG1 protein was overexpressed in MCF-7 cells. Upon reoxygenation, overexpression of NDRG1 significantly inhibited cell migration. Our results revealed the dynamic nature of gene expression in MCF-7 cells upon reoxygenation and demonstrated that NDRG1 is involved in tumor adaptation to reoxygenation

Cancer targeted gene therapy of BikDD inhibits orthotopic lung cancer growth and improves long-term survival

[[abstract]]Lung cancer is a leading cause of cancer death due to the high incidence of metastasis; therefore, novel and effective treatments are urgently needed. A current strategy is cancer-specific targeted gene therapy. Although many identified that cancer-specific promoters are highly specific, they tend to have low activity compared with the ubiquitous cytomegalovirus (CMV) promoter, limiting their application. We developed a targeted gene therapy expression system for lung cancer that is highly specific with strong activity. Our expression vector uses the survivin promoter, highly expressed in many cancers but not normal adult tissues. We enhanced the survivin promoter activity comparable to the CMV promoter in lung cancer cell lines using an established platform technology, whereas the survivin promoter remained weak in normal cells. In mouse models, the transgene was specifically expressed in the lung tumor tissue, compared with the CMV promoter that was expressed in both normal and tumor tissues. In addition, the therapeutic gene BikDD, a mutant form of pro-apoptotic Bcl2 interacting killer, induced cell killing in vitro, and inhibited cell growth and prolonged mouse survival in vivo. Importantly, there was virtually no toxicity when BikDD was expressed with our expression system. Thus, the current report provides a therapeutic efficacy and safe strategy worthy of development in clinical trials treating lung cance

Cancer-Targeted Bikdd Gene Therapy Elicits Protective Antitumor Immunity against Lung Cancer

Targeted cancer-specific gene therapy is a promising strategy for treating metastatic lung cancer, which is a leading cause of lung cancer- related deaths. Previously, we developed a cancer-targeted gene therapy expression system with high tumor specificity and strong activity that selectively induced lung cancer cell killing without affecting normal cells in immunocompromised mice. Here, we found this cancer-targeted gene therapy, SV-BikDD, composed of the survivin promoter in the VP16-GAL4-WPRE integrated systemic amplifier system to drive the apoptotic gene BikDD, not only caused cytotoxic effects in cancer cells but also elicited a cancer-specific cytotoxic T lymphocyte response to synergistically increase the therapeutic effect and further develop an effective systemic antitumoral immunity against rechallenges of tumorigenic dose of parental tumor cells inoculated at distant sites in immunocompetent mice. In addition, this cancer-targeted gene therapy does not elicit an immune response against normal tissues, but CMV- BikDD treatment does. The therapeutic vector could also induce proinflammatory cytokines to activate innate immunity and provide some benefits in antitumor gene therapy. Thus , this study provides a promising strategy with benefit of antitumoral immune response worthy of further development in clinical trials for treating lung cancer via cancer- targeted gene therapy

ADAM9 Up-Regulates N-Cadherin via miR-218 Suppression in Lung Adenocarcinoma Cells

Lung cancer is the leading cause of cancer death worldwide, and brain metastasis is a major cause of morbidity and mortality in lung cancer. CDH2 (N-cadherin, a mesenchymal marker of the epithelial-mesenchymal transition) and ADAM9 (a type I transmembrane protein) are related to lung cancer brain metastasis; however, it is unclear how they interact to mediate this metastasis. Because microRNAs regulate many biological functions and disease processes (e. g., cancer) by down-regulating their target genes, microRNA microarrays were used to identify ADAM9-regulated miRNAs that target CDH2 in aggressive lung cancer cells. Luciferase assays and western blot analysis showed that CDH2 is a target gene of miR-218. MiR-218 was generated from pri-mir-218-1, which is located in SLIT2, in non-invasive lung adenocarcinoma cells, whereas its expression was inhibited in aggressive lung adenocarcinoma. The down-regulation of ADAM9 up-regulated SLIT2 and miR-218, thus down-regulating CDH2 expression. This study revealed that ADAM9 activates CDH2 through the release of miR-218 inhibition on CDH2 in lung adenocarcinoma

Characterization of Metastasis Associated Genes and Development of Clinical Prognosis Assay for Lung Cancer

肺癌細胞轉移的分子基礎研究有助於治療方式的改善及發現新治療標的分子。正確的預後評估及治療方式的選擇則決定癌症病人的存活率。因此，本論文的研究目標在於探討肺癌轉移相關基因及研發臨床預後的方法。 首先利用資料庫分析，並以一對具有不同轉移力的肺癌細胞株來探討癌轉移相關基因。經過篩選後，發現一個有趣的基因－人類第八號激肽釋放酶，它是屬於人類組織激肽釋放酶基因家族中的一員。人類第八號激肽釋放酶為絲胺酸蛋白酶，已知為卵巢癌病人預後良好的評估指標。然而，其生物意義卻不清楚。實驗結果顯示藉由轉殖於具有高侵襲力肺癌細胞株中人類第八號激肽釋放酶基因的過量表現，可抑制肺癌細胞的侵襲力。相反地，若利用具專一性的短髮夾核糖核酸來抑制細胞內生性人類第八號激肽釋放酶，則可增加肺癌細胞的侵襲力。根據原位分解技術及細胞附著力偵測的結果顯示，人類第八號激肽釋放酶的剪接產物可分解血清纖維結合蛋白，進而改變細胞外圍環境。去氧核糖核酸微陣列實驗及細胞內肌動蛋白纖維染色結果，顯示了人類第八號激肽釋放酶的剪接產物分解血清纖維結合蛋白後，抑制了整合素的訊息傳遞途徑，並藉由抑制F肌動蛋白的重排而妨礙肺癌細胞的移動能力。此外，以人類Alu序列為標的來偵測並定量小鼠動物實驗中鼠血液內人類肺癌細胞存在多寡的實驗顯示，在活體試驗中人類第八號激肽釋放酶可抑制腫瘤生長及轉移。近一步由臨床研究肺癌病人檢體發現，若肺癌早期病人(第一、二期)的癌組織中測得較高的人類第八號激肽釋放酶基因的表現，則病人有明顯較長的緩和期及較低的復發率。此發現可歸納出人類第八號激肽釋放酶的功能為阻礙腫瘤的轉移，並可利用此基因來當作非小細胞肺癌病人預後的指標。 臨床上最有效於提高療效的方法為早期診斷出癌轉移並施予有效的療程。因此，另一個計畫著重於早期檢測血液中癌細胞的研究。目前肺癌分期及病況評估主要是根據腫瘤影像法。然而此法受限於不夠靈敏，而無法正確地診斷出早期癌轉移的發生。此研究利用數個指標基因來偵測血液循環中的癌細胞，以增加目前肺癌分期及病況評估的正確度，並可用於快速評估藥效。我們利用基因庫來搜尋合適的基因用以偵測血液循環中的癌細胞，經實驗證明其中四個基因可當作標的基因。利用這四個標的基因來偵測五十四個非小細胞肺癌病人血液中癌細胞的存在與否，可達到百分之七十二檢出率。若利用即時定量聚合脢放大偵測法轉換成癌細胞載荷量來評估肺癌病人血液中癌細胞量與臨床結果的關聯性，則病人癌細胞載荷量越高者其治療效果較差且存活時間較短。治療效果不好的病人，其治療後仍可測到血液中癌細胞的存在，並有較短的存活期。藉由此四個標的基因及癌細胞載荷量來反映出肺癌病人血液中癌細胞量，可增強傳統肺癌分期法，進而提高檢測率及快速評估治療效果。此外也可輔佐醫師在治療肺癌病人上給予更適當的治療方式。Research investigations on the molecular basis of lung carcinoma metastasis are helpful to identify therapeutic targets for metastasis. An accurate prognosis and selection of therapeutic modality determines the survival of cancer patients. Therefore, this thesis aims to characterize metastasis associated genes and develop clinical prognosis assay for lung cancer. Firstly an in silico analysis approach was used to examine metastasis associated genes by a cell line model of human lung adenocarcinoma with different invasive abilities. After screening, one interesting gene was found, human kallikrein 8 (KLK8), a member of human tissue kallikrein gene family. The serine protease KLK8 protein (hK8) is known to be a favorable prognostic marker in ovarian cancer, but the biological basis of this is not understood. The experimental results showed that overexpressing the KLK8 gene in highly invasive lung cancer cell lines suppressed their invasiveness. This role in invasiveness was further confirmed by the fact that inhibition of endogenous KLK8 expression with a specific short hairpin RNA enhanced cancer cell invasiveness. In situ degradation and cell adhesion assays showed that proteins produced from KLK8 splice variants modify the extracellular microenvironment by cleaving fibronectin. DNA microarray experiments and cell staining for actin filaments revealed that the degradation of fibronectin by hK8 suppresses integrin signaling and retards cancer cell motility by inhibiting actin polymerization. In addition, studies in a mouse model coupled with detection of circulating tumor cells by quantitative PCR for the human Alu sequence demonstrated that KLK8 suppresses tumor growth and invasion in vivo. Furthermore, studies of clinical specimens from non-small cell lung cancer (NSCLC) patients showed a 52% recurrence rate for early-stage (stage I & II) patients with low KLK8 expression in their tumor cells and a 23% recurrence rate for patients with high KLK8 expression. Collectively, these findings show that KLK8 retards cancer metastasis and that further investigation of KLK8 as a prognostic marker for NSCLC is warranted. The most promising way to improve prognosis is by means of early metastasis detection. Thus, the other project in this thesis study is focused on detection of disseminated cancer cells of non-small cell lung cancer patients in their peripheral blood. Current lung cancer staging and prognosis methods are based on imaging methods which may not be sensitive enough for early and accurate detection of metastasis. A panel of markers was validated for circulating cancer cell detection to improve the accuracy of cancer staging, prognosis, and as a rapid assessment of therapeutic response. NCI-CGAP database was used to identify potential marker genes for the detection of circulating cancer cells in peripheral blood. A panel of 4 marker genes was identified and experimentally validated. With these marker genes, the results achieved an overall positive detection rate of 72% for circulating cancer cells in the peripheral blood of 54 NSCLC patients. Nested real-time quantitative PCR (qPCR) and a scoring method using cancer cell load, Lc, were employed to correlate the amount of circulating cancer cells with clinical outcomes in NSCLC patients. Patients who had higher Lc values had worse outcomes and shorter survival times. Patients with poor therapeutic response were revealed by positive detection of circulating cancer cells after therapy. The results correlated well with the patients’ survival time. Circulating cancer cell detection by a panel of markers and the Lc scoring method can supplement the current TNM staging method for improved prognosis and for rapid assessment of therapeutic response. Together, they may facilitate the design of better therapeutic strategies for the treatment of NSCLC patients.中文摘要…………………………………………………………………………… 1 ABSTRACT…………………………………..…………………………..………… 3 1. Metastasis in Lung Cancer – from Molecular Study to Clinical Prognosis 1.1 Human lung cancer……………………….……….………………………………5 1.2 The basic biology of metastasis …………………………………………………. 6 1.3 Molecules involved in metastasis…………………………….…………………… 7 1.3.1 Changes in cell-cell and cell-matrix adhesion…………………….…………… 8 1.3.2 Metastasis-promoting and metastasis-suppressor genes…………………….… 9 1.4 Clinical tools in the detection of lung cancer metastasis……………………..… 10 1.5 The clinical problem: predicting metastatic propensity…………………………. 10 2. Identify Metastasis Associated Genes in Lung Cancer 2.1 Molecular signature of metastasis and its implication…….…………..…..…… 13 2.2 Transcript-specific expression………….………………..….……………….…. 14 2.3 Strategy to identify the metastasis associated genes in the lung cancer model… 15 2.4 Materials and methods 2.4.1 RT-PCR analysis…………….……………………………………………. 15 2.5 Identification of metastasis associated genes in the lung cancer model…….…. 15 3. Characterization of the Role of Kallikrein 8 in Metastasis 3.1 Kallikreins in cancer progression……………………………………………….…. 17 3.2 Kallikrein 8 as a biomarker for cancer prognosis………………………..….…. 18 3.3 Rationale for characterizing the association of KLK8 in metastasis……………. 18 3.4 Materials and methods 3.4.1 Cell lines ………………………………………..………………..………. 18 3.4.2 RT-PCR analysis of KLK8 expression in cancer cell lines ……….……… 19 3.4.3 KLK8 gene transcripts construction and retroviral infection ……………. 19 3.4.4 Detection of proteins generated by the KLK8 splice variants …………….. 20 3.4.5 Lentiviral short hairpin RNA (shRNA)-mediated knockdown of KLK8 in CL1-0 cells……………………………………………………………..… 20 3.4.6 in situ fibronectin (FN) degradation and cell adhesion assays…………… 21 3.4.7 Microarray gene expression profile analysis……………………………… 22 3.4.8 qPCR detections……………………………………………..……..……. 22 3.4.9 Immunofluorescence imaging of F-actin and filopodia …………………. 24 3.4.10 Analysis of tumor growth rate affected by hK8 expression……..……… 24 3.4.11 Monitoring tumor growth by magnetic resonance imaging (MRI)….…. 24 3.4.12 Mouse tumor cell invasion model…………..………………………..…. 25 3.4.13 Lung cancer patients and tissue specimens……………………..……….. 25 3.4.14 Statistical analysis………………………………………….……………. 26 3.5 Results 3.5.1 High expression of KLK8 transcripts correlates with low invasiveness of cancer cell lines………….…………………………………………..… 26 3.5.2 KLK8 is overexpressed in weakly invasive lung cancer cells……..…….… 27 3.5.3 Overexpression of KLK8 decreases the invasiveness of lung cancer cells …………………………………………………..………………… 28 3.5.4 hK8 degrades FN and decreases cell adherence………………..…………. 29 3.5.5 Gene expression profiling in KLK8-transfected cells…..………………… 29 3.5.6 KLK8 overexpression suppresses tumor growth and cancer cell invasion in vivo………………..…………………………………………………… 31 3.5.7 Early-stage NSCLC patients with high KLK8 expression have a longer remission time and a lower rate of recurrence…..…….……………….. 32 3.6 Discussion ………………………………………………………………………. 33 4. Development of Clinical Prognosis Assay for Non-Small Cell Lung Cancer Patients 4.1 Metastasis in early stage NSCLC － an increasing problem………………… 37 4.2 Early detection of metastasis…………………………………………..……….. 37 4.3 Strategy of detecting circulating tumor cells in blood……..…..……………… 38 4.4 Materials and methods 4.4.1 Patients and specimens …………………………………………………… 39 4.4.2 Identification of candidate marker genes ……..……..………………….. 40 4.4.3 Sample collection and RNA preparation ……………….………………… 40 4.4.4 Nested RT-PCR assay …………………………………………………….. 40 4.4.5 Semi-quantification of the nested PCR results ……………….…………. 41 4.4.6 Statistical analysis ……………………………………………………….. 42 4.5 Results 4.5.1 Marker genes for detecting circulating NSCLC cells …………………….. 42 4.5.2 Enhancement of positive detection rate with multiple genes.….…..……. 43 4.5.3 Circulating cancer cell load and patient outcome ……………….….…….. 44 4.5.4 Assessment of therapy efficacy …………..………………………..……… 45 4.6 Discussion ………………………………………………………………………. 46 FIGURES 1. Differential transcript-specific expression profiles in the lung cancer model……. 51 2. KLK8 expression profiles in different cancer cell types with different degrees of invasiveness………………………………………………………………..…… 52 3. Multiple splice variants of KLK8 are overexpressed in weakly invasive lung cancer cells, CL1-0……………………………………………………………..………. 53 4. Overexpression of KLK8 in lung cancer cell line, CL1-5………………………. 54 5. KLK8 was associated with cancer cell invasion…………………………………. 55 6. hK8 degrades FN and decreases cell adherence…………………………………. 56 7. Gene expression profiling in KLK8-transfected cells…………………..……….. 57 8. Analysis of tumor growth rate affected by hK8 expression in an animal model…58 9. Spin echo T2-weighted images and maps of permeability factor (K) of MRI detection in an animal model…………………………………………………… 59 10. Tumor mass analysis on the 14th day after subcutaneous implantation of tumor cells……………………………………………………………………..……… 60 11. Detection of disseminated tumor cells in an animal model…………………….. 61 12. Expression of KLK8 in tumor tissue specimens from 88 NSCLC patients…….. 62 13. The flowchart of screening a panel of marker genes for detecting circulating cancer cells………………………………………………………………..……. 63 14. Determination of the positive detection threshold for TRIM28 marker gene with residual expression in leukocytes………………………………………………. 64 15. Analysis of positive detection rates with the multi-marker gene panel………… 65 16. Lc value characterization……………………………………………………….. 66 17. Survival analysis of late stage patients with high (³1) or low (<1) Lc values…. 67 18. Assessment of therapy efficacy with circulating tumor cell detection for six different NSCLC patients…………………………………………….………… 68 TABLES 1. Clinicopathologic characteristics and their correlation with KLK8 expression of NSCLC patients……………………………………………………………..…. 69 2. DNA sequences of the PCR primer pairs for detecting the marker genes……….. 70 3. List of a panel of 19 marker genes……………………………………………….. 71 4. List of a panel of four marker genes………………………………………………. 72 5. Clinicopathologic characteristics and their correlation with Lc value of NSCLC patients………………………………………………………………………….. 73 APPENDIX 1. AS primers of 190 genes…………………………………………………………. 74 2. List of 34 genes with single transcript difference in cancer model……………… 81 3. List of 18 genes with multiple isoforms difference in cancer model…………….. 82 4. Genes whose expression profiles are concordant with the expected profile…….. 83 5. Pathways involving the 448 genes of the most concordant cluster……………… 96 6. DGED list from lung cancer library vs. leukocyte library……………………….. 98 REFERENCE………………………………………………………………………10