과당에 의해 활성화되는 YWHAH에 대한 KHK-A의 인산화 기능이 유방암 전이를 촉진 시키는 기전에 관한 연구

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의과학과, 2021. 2. 박종완.Harmful effects of high fructose intake on health have been widely reported in epidemiological and laboratory studies. Although it has been reported that fructose promotes cancer development and progression, little is known about the underlying molecular mechanisms. Here, I found that fructose triggers breast cancer metastasis through the ketohexokinase-A signaling pathway. Molecular experiments showed that ketohexokinase-A, rather than ketohexokinase-C, is necessary and sufficient for fructose-induced cell invasion. An orthotopic xenograft experiment revealed that ketohexokinase-A-overexpressing breast cancer is highly metastatic in fructose-fed mice. Mechanistically, ketohexokinase-A moves from the cytoplasm to the nucleus during fructose stimulation, which is mediated by the nuclear importers LRRC59 and KPNB1. In the nucleus, ketohexokinase-A phosphorylates YWHAH at Ser25 and, in turn, YWHAH recruits SLUG to the CDH1 promoter, which weakens cell adhesion and triggers cell migration. This study provides a new insight into the effect of nutrition on breast cancer metastasis. High intake of fructose should be restricted in cancer patients to reduce the risk of metastasis. From a therapeutic perspective, the ketohexokinase-A signaling pathway could be a potential target to prevent cancer metastasis.현대 사회에서 식습관의 서구화로 인하여 과당의 섭취가 늘어났으며 그에 따라 비만, 당뇨, 지방간을 악화시킨다고 보고되어 있다. 이 외에도 과당의 섭취는 유방, 소장, 및 대장암을 포함한 다양한 암의 발병과 전이에 관련이 있다는 것이 역학적으로 보고되었다. 암 대사는 다량의 포도당을 소비하도록 Reprogramming이 일어나며, 포도당이 고갈되어 있는 암세포는 과당을 대체에너지원으로 이용한다. 췌장암과 대장암에서 과당 대사 경로의 활성화로 인해 암의 증식과 전이가 촉진된다는 것이 보고되어 있지만, 이 현상을 뒷받침하는 기전은 보고된 바가 없다. Ketohexokinase는 fructose를 fructose-1-phosphate로 전환하는 효소이다. KHK는 alternative splicing을 통해 조직 특이적으로 KHK-A와 KHK-C를 발현한다. 과당의 F-1-P 로의 분해는 간, 신장에서 KHK-C에 의해 일어나며, 반면 KHK-A는 과당에 대한 효소 활성도가 매우 낮아 과당의 대사에 기여하지 않는 것으로 여겨졌다. 그럼에도 불구하고 KHK-A는 대부분의 조직에서 발현하기 때문에 에너지 대사 이외의 생물학적 기능을 가지고 있다는 의견이 제기되었다. 유방암 세포는 정상 유방세포 대비, 과당 수용체인 GLUT5의 발현양이 높고, 과당 처리 시, 세포의 형태가 mesenchymal 하게 변한다는 것이 알려져 있다. 하지만 그 분자 메커니즘에 대해서는 알려진 바가 없다. 먼저 유방암 세포주를 포함한 암세포주에 과당 처리 후 이동능의 증가를 확인하였고, 간, 신장 유래 암세포를 제외하고는 모두 KHK-A가 우세하게 발현하는 것을 확인하였다. 따라서 유방암 세포주에서 Fructose에 의한 암 전이과정에 KHK-A가 관여되어 있을 것이라는 가설을 세웠다. 마우스 xenograft model을 이용하여 과당 공급 시 KHK-A를 발현하는 유방암 세포주가 폐로 전이됨을 확인하였고, 유방암 세포주에서 과당 존재 시, KHK-A가 공통적으로 결합하고 있는 단백질을 선정하였다. LRRC59는 과당이 존재할 때, KPNB1과 결합하여 KHK-A를 세포질에서 세포핵으로 이동시키며, 핵으로 들어간 KHK-A는 YWHAH (14-3-3 eta)의 25번 Serine잔기를 인산화 시키는 것을 확인하였다. 인산화된 YWHAH는 Slug와 결합하고 CDH1의 전사를 억제함으로써 유방암 세포주의 상피간엽이행을 유도하여, 결과적으로는 암 전이에 기여하는 것을 확인하였다. YWHAH의 인산화는 유방암 xenograft 모델에서 폐를 포함하여 뇌, 비장, 간 등 다양한 주요 장기로의 전이를 촉진하였다. 유방암 환자 조직에서 nuclear KHK-A와 YWHAH-pSer25의 발현이 서로 양의 상관관계를 보이며, 유방암 예후와 연관되어 있음을 보여줌으로써 상기 기전을 확인하였다. 그런데 왜 KHK-A는 과당은 인산화 시키지 못하는 것일까? 본 연구에서는 2016년도에 KHK-A의 기질로 보고된 PRPS1과의 비교연구를 통하여, KHK-A는 아미노산 DDMA로 이루어진 모티프를 인식하여 기질 단백질과 결합, 인산화 시킴을 확인하였다. 따라서 위의 물음에 대하여 KHK-A는 과당 인산화효소가 아닌 Serine/Threonine 인산화 효소이기 때문이다. 라는 해답을 제시하였다. 이러한 연구 결과는 KHK-A가 과당에 의한 암 전이 과정에서 KHK-A가 결정적인 스위치 조절자라는 새로운 기전을 규명하였으며, 나아가 KHK-A-YWHAH-CDH1 기전이 과당에 의한 암의 전이를 억제하는 새로운 치료 표적이 될 가능성을 제시하였다. 또한 우리가 간과하기 쉬운 식습관의 중요성을 다시금 일깨우는 메시지를 제공하고 있다. 그 메시지는 암환자가 영양보충을 하고자 과당을 많이 섭취하는 것은 좋지 못한 결정이라는 것이다.Contents Abstract………………………………………………………………………………ⅰ Contents……………………………………………………………………………ⅲ List of tables and figures…………………………………………………………ⅳ List of abbreviations ………………………………………………………………ⅸ INTRODUCTION…………………………………………………………………1 MATERIALS AND METHODS…………………………………………………5 RESULTS…………………………………………………………………………30 FIGURES…………………………………………………………………………46 DISCUSSION……………………………………………………………………132 REFERENCES…………………………………………………………………137 ABSTRACT IN KOREAN ……………………………………………………142Docto

Graph Representation Learning in Biomedicine

Biomedical networks are universal descriptors of systems of interacting elements, from protein interactions to disease networks, all the way to healthcare systems and scientific knowledge. With the remarkable success of representation learning in providing powerful predictions and insights, we have witnessed a rapid expansion of representation learning techniques into modeling, analyzing, and learning with such networks. In this review, we put forward an observation that long-standing principles of networks in biology and medicine -- while often unspoken in machine learning research -- can provide the conceptual grounding for representation learning, explain its current successes and limitations, and inform future advances. We synthesize a spectrum of algorithmic approaches that, at their core, leverage graph topology to embed networks into compact vector spaces, and capture the breadth of ways in which representation learning is proving useful. Areas of profound impact include identifying variants underlying complex traits, disentangling behaviors of single cells and their effects on health, assisting in diagnosis and treatment of patients, and developing safe and effective medicines

Evaluation of novel positron emission tomography radiotracers in humans: tissue distribution kinetics and potential for cancer diagnosis and staging

Positron emission tomography (PET) imaging has emerged as an important decision-making tool in oncology with respect to diagnosis, staging, and assessment of treatment response. We proposed to investigate the ligand binding and retention kinetics of two novel PET/CT tracers in human tumours that do not normally exhibit high [18F]fluorodeoxyglucose ([18F]FDG) uptake, and a third tracer in the context of specific death mechanism. Biological validation of the imaging endpoint included histological correlation with PET/CT data and establishment of an optimum PET/CT methodologies for the probe for implementation into clinical practice. The internal dosimetry and receptor-mediated tumour localisation of the ‘click’ labelled [18F]fluoroethyl triazole octreotate analogue, [18F]FET-βAG-TOCA, in neuroendocrine tumours (NETs) were investigated for the first time in humans. The biomarker demonstrated favourable dosimetry, biodistribution and safety. The calculated effective dose over all subjects (mean ± SD) was 0.029 ± 0.004 mSv/MBq. Regarding staging, [18F]FET-βAG-TOCA PET/CT showed high tumoural uptake with high sensitivity (per lesion) compared with [68Ga]DOTATATE PET/CT (92.8% vs 87.5%). Tissue retention kinetics of the novel choline analogue, [18F]fluoromethyl-[1,2-2H4]- choline ([18F]D4-FCH) were investigated in the staging of muscle invasive bladder cancer (MIBC) and non-small cell lung cancer (NSCLC). The biomarker showed high contrast in lung cancer but poor contrast in bladder cancer. In lung tumours, [18F]D4-FCH uptake was quantitatively lower than [18F]FDG. Pharmacokinetic modelling revealed net tracer influx in tumour consistent with radiotracer phosphorylation via choline kinase, however choline kinase-alpha expression did not correlate with PET parameters. Beyond staging, we evaluated for the first time a caspase-3/7 imaging biomarker, [18F](S)- 1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluorophenoxymethyl)- 8 pyrrolidine-1-sulfonyl) ([18F]ICMT-11), for imaging apoptosis and/or necrosis in patients; [18F]FDG-PET is not a marker of caspase-3/7 activation. In breast cancer, lung cancer and lymphoma patients receiving first-line chemotherapy treatment, [18F]ICMT-11 and cytokeratin-18 analysis (blood) were performed. [18F]ICMT-11 showed low uptake pre- and post-chemotherapy in all tumours consistent with unremarkable changes in M30/M60 cytokeratin-18 cleavage products in the breast cohort suggesting a lack of predominantly apoptotic cell death mechanism in responding patients. In lung cancer, multi-parametric [18F]ICMT-11 PET/CT, diffusion weighted (DW-MRI) and dynamic contrast enhanced-MRI (DCE-MRI) showed that PET changes were concordant with cell death in the absence of significant perfusion changes. Thus, tumour response could occur in the absence of predominant chemotherapy-induced caspase-3/7 activation measured non-invasively across entire tumour lesions. In conclusion, the optimal clinical context whereby the [18F]ICMT-11 PET endpoint critically determines the outcome of therapy remains to be established.Open Acces

Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer

Introduction: The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets. Methods: Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription. Results: TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species. Conclusions: TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer

Background Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells. Results We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID /APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling- based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC- related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background. Conclusions The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue

Treatment time and circadian genotype interact to influence radiotherapy side-effects. A prospective European validation study using the REQUITE cohort

Breast cancer; Circadian rhythm; RadiotherapyCáncer de mama; Ritmo circadiano; RadioterapiaCàncer de mama; Ritme circadià; RadioteràpiaBackground Circadian rhythm impacts broad biological processes, including response to cancer treatment. Evidence conflicts on whether treatment time affects risk of radiotherapy side-effects, likely because of differing time analyses and target tissues. We previously showed interactive effects of time and genotypes of circadian genes on late toxicity after breast radiotherapy and aimed to validate those results in a multi-centre cohort. Methods Clinical and genotype data from 1690 REQUITE breast cancer patients were used with erythema (acute; n=340) and breast atrophy (two years post-radiotherapy; n=514) as primary endpoints. Local datetimes per fraction were converted into solar times as predictors. Genetic chronotype markers were included in logistic regressions to identify primary endpoint predictors. Findings Significant predictors for erythema included BMI, radiation dose and PER3 genotype (OR 1.27(95%CI 1.03-1.56); P < 0.03). Effect of treatment time effect on acute toxicity was inconclusive, with no interaction between time and genotype. For late toxicity (breast atrophy), predictors included BMI, radiation dose, surgery type, treatment time and SNPs in CLOCK (OR 0.62 (95%CI 0.4-0.9); P < 0.01), PER3 (OR 0.65 (95%CI 0.44-0.97); P < 0.04) and RASD1 (OR 0.56 (95%CI 0.35-0.89); P < 0.02). There was a statistically significant interaction between time and genotypes of circadian rhythm genes (CLOCK OR 1.13 (95%CI 1.03-1.23), P < 0.01; PER3 OR 1.1 (95%CI 1.01-1.2), P < 0.04; RASD1 OR 1.15 (95%CI 1.04-1.28), P < 0.008), with peak time for toxicity determined by genotype. Interpretation Late atrophy can be mitigated by selecting optimal treatment time according to circadian genotypes (e.g. treat PER3 rs2087947C/C genotypes in mornings; T/T in afternoons). We predict triple-homozygous patients (14%) reduce chance of atrophy from 70% to 33% by treating in mornings as opposed to mid-afternoon. Future clinical trials could stratify patients treated at optimal times compared to those scheduled normally.EU-FP7

Conduits of Intratumor Heterogeneity: Centrosome Amplification, Centrosome Clustering and Mitotic Frequency

Tumor initiation and progression is dependent on the acquisition and accumulation of multiple driver mutations that acti­vate and fuel oncogenic pathways and deactivate tumor suppressor networks. This complex continuum of non-stochastic genetic changes in accompaniment with error-prone mitoses largely explains why tumors are a mosaic of different cells. Contrary to the long-held notion that tumors are dominated by genetically-identical cells, tumors often contain many different subsets of cells that are remarkably diverse and distinct. The extent of this intratumor heterogeneity has bewildered cancer biologists’ and clinicians alike, as this partly illuminates why most cancer treatments fail. Unsurprisingly, there is no “wonder” drug yet available which can target all the different sub-populations including rare clones, and conquer the war on cancer. Breast tumors harbor ginormous extent of intratumoral heterogeneity, both within primary and metastatic lesions. This revelation essentially calls into question mega clinical endeavors such as the Human Genome Project that have sequenced a single biopsy from a large tumor mass thus precluding realization of the fact that a single tumor mass comprises of cells that present a variety of flavors in genotypic compositions. It is also becoming recognized that intratumor clonal heterogeneity underlies therapeutic resistance. Thus to comprehend the clinical behavior and therapeutic management of tumors, it is imperative to recognize and understand how intratumor heterogeneity arises. To this end, my research proposes to study two main features/cellular traits of tumors that can be quantitatively evaluated as “surrogates” to represent tumor heterogeneity at various stages of the disease: (a) centrosome amplification and clustering, and (b) mitotic frequency. This study aims at interrogating how a collaborative interplay of these “vehicles” support the tumor’s evolutionary agenda, and how we can glean prognostic and predictive information from an accurate determination of these cellular traits

Characterization of Human Prostate Cancer Using Sodium Magnetic Resonance Imaging

Overtreatment of prostate cancer is a significant problem in the health care of men. Development of non-invasive imaging tools for improved characterization of prostate lesions has the potential to reduce overtreatment. In this thesis work, we will evaluate the ability of tissue sodium concentration obtained from sodium magnetic resonance imaging (sodium-MRI) to characterize in vivo prostate lesions. Imaging data, including multi-parametric magnetic resonance imaging (mpMRI) and sodium-MRI, were obtained from a cohort of men with biopsy-proven prostate cancer and compared to digitized whole-mount histopathology after prostatectomy. Histopathology was independently graded for Gleason score to be used as the ground truth of tumour aggression. These imaging data were all accurately co-registered, allowing for direct comparison of imaging contrast to Gleason score. The results of this thesis work suggest that tissue sodium concentration assessed by sodium-MRI has utility as a part of a “non-invasive imaging-assay” to accurately characterize prostate cancer lesions. Sodium-MRI can provide clinically useful, complementary information to mpMRI; ultimately leading to better characterization of prostate lesions throughout the whole prostate. This has potential to improve patient outcomes of men with low-risk disease who do opt for active surveillance instead of treatment