Genome-wide Screening and Isolation of Differentially Methylated Genes in Non-small Cell Lung Cancer : 비소세포폐암에서 유전체 전체 검색을 통한 차등 메틸화되는 유전자 발굴

최준영 경북대학교 대학원 2008 국내박사

Lung cancer is a major public health problem in the world and remains a leading cause of cancer deaths but its impact in developed and developing Asian countries is also devastating (ACS, 2005; Jemal et al., 2007). Extensive prospective epidemiologic data clearly have estblished cigarette smoking as the major cause of lung cancer (Sasco et al., 2004). Despite an impressive body of research that has defined the major risk factors for the disease (Alberg & Samet, 2003), little has changed in the management of the disease in the last decade. Barriers to reducing the mortality of this disease include high percentage of patients presenting with advanced stage disease without effective treatment options, the lack of a proven screening method to identify patients at earlier and potentially more curable stages of disease, and the high risk of relapse and second primaries in patients who have undergone curative resection. Lung cancer is clinically subdivided into small cell lung cancer (SCLC, 15% of lung cancers), the most aggressive form of lung cancer, and non-small cell lung cancer (NSCLC), consisting of adenocarcinoma (AD), squamous cell carcinoma (SQ), large cell carcinoma (LC), and miscellaneous other types such as carcinoids, pleomorphic and mixed carcinomas and a range of neuroendocrine cancers (Travis, 2002). In United States, AD has now surpassed SQ as the most common histological subtype of lung cancer, and continues to increase worldwide, particularly in women (Travis, 1995). AD generally arises more peripherally in the lung. Its increased incidence is thought to be related at least in part to changes in the structure and composition of cigarettes (lower tar, lower nicotine, leading deeper inhalation) (Thun et al., 1997; Stellman et al., 1997). It is also the most common type of lung cancer in previous and never-smokers, and among young patients with AD, the percentage of women and non-smokers is usually high (Liu et al., 2000). Recently, certain human papilloma virus subtypes have been implicated in AD in non-smoking women in Asian countries (Chen et al., 2004). These observations point to the importance of gaining a better understanding in the etiology of lung AD, and in the molecular changes underlying this disease. Cancer has long been regarded as a genetic disease and mutations and deletions of many genes have been shown to be critical in the development of cancers. However, in recent years it has become apparent that epigenetic changes are as important as genetic alterations in the process of tumorigenesis (Fig. 1 and Table 1)(Laird & Jaenisch, 1996). As opposed to the irreversible nature of genetic events, which introduce changes in the primary DNA sequence, epigenetic silencing is now recognized as a 'third pathway' in Knudson's model of tumor-suppressor gene inactivation in cancer and can affect gene function without genetic changes (Fig. 2). Such epigenetic changes fall predominantly into two categories - covalent changes to DNA itself, which is essentially limited to DNA methylation at cytosine residues and secondly modifications of DNA associated with histones which can take the form of acetylation, methylation, phosphorylation or ubiquitination (Berger, 2002). DNA methylation is a postreplicative modification of DNA that occurs primarily on the 5-position of cytosine rings that are located in CpG dinucleotides. This is a normal and essential enzymatic modification that is required for proper development of the organism (Esteller, 2006). In cancer, the pattern of DNA methylation normally seen in healthy cells is altered; there is a global loss of methylation, associated with chromosomal stability, and local changes in methylation in clusters of CpG residues, called CpG islands (CGI) (Esteller, 2006; Jones & Baylin, 2002; Laird, 2003). The majority, 80%, of CpG dinucleotides are found in repetitive sequences, and in this context they are normally dispersed. Such dispersed CpG dinucleotides are observed also the bodies of genes (coding regions, introns) (Esteller & Herman, 2002). In contrast, up to 15% of CpG dinucleotides in mammals occur in CGI (Roberson & Wolffe, 2000; Fruhwald & Plass, 2002). Because ~40% of human genes carry promoter CGI (Lander et al., 2001), CGI hypo- and hypermethylation provides an enormous potential for epigenetic alterations in cancer. In addition, the functional importance of CGI was revealed by studies demonstrating that methylation of CGI within gene promoters leads to transcriptional repression of the associated gene in a variety of ways (Fig. 3) (Richardson et al., 2002). Studies of DNA methylation in lung cancer to date strongly suggest that analysis of DNA methylation profiles will be of great utility both understanding the molecular basis of lung cancer development and for developing tools fro accurate and early lung cancer diagnosis and prognostication (Tsou et al., 2002; Belinsky, 2004). In addition, the reversibility of DNA methylation suggests that clinical strategies might be developed to reactivate genes silenced by methylation (Yoo & Jones, 2006). Much current work still focuses on the important and gargantuan task of cancer-specific DNA methylation alterations in lung cancer, a task that is far from complete. DNA methylation-based technologies have promising future in both clinical diagnostics and therapeutics. DNA methylation markers have obvious applications in diagnostics, but can also contribute indirectly to the therapeutics as predictors of response to therapy. Since many of currently used targets of methylation are methylated at rather low frequencies in various cancer types even though the gene may frequently disrupted by other mechanisms (Esteller et al., 2001), it would be useful to develop additional markers that are methylated at high frequency in cancer being studied. The investigation of aberrant CGI methylation has mostly been conducted on the promoter regions of the specific target genes involved in growth regulation, DNA repair, and apoptosis using a candidate gene approaches. Assessment of the clinical potential of hypermethylation profiles and the identification of relevant marker genes, however, require means and methods to detect hypermethylation on a genome-wide level. Most of the genome-scanning techniques rely on methylation-sensitive restriction digestion, although their reliance on methylation-sensitive restriction enzymes renders them poorly suited for the routine analysis of formalin-fixed clinical specimens. The large-scale discovery of methylation markers for cancer has just begun, and it should be recognized that even the genome-scanning techniques are confined by the distribution of the relevant restriction enzyme recognition motifs the genome. Here, We have used a technique, called NotI-MseI methylation-sensitive amplified fragment length polymorphism (MSAFLP). NotI-MseI MS-AFLP is an efficient and sensitive method that permits the genome-wide evaluation of the spectrum of epigenetic alterations (and probably genetic alterations also), as well as the identification and cloning of DNA fragments that exhibit alterations. In this study, in order to identify novel CGIs that are hypo- or hypermethylated in human NSCLC, we analyzed genomic DNA from normal lung tissues and NSCLC cell lines using MS-AFLP. The methylation status of isolated putative CGIs was also validated in resected primary lung cancers tissues using methylation specific-PCR (MSP). Finally, the data was analyzed with reference to patients' clinicopathological features.

Studies on Lung Cancer Biomarkers
 by Proteogenomic Analysis

김용인 서울대학교 대학원 2019 국내박사

Biomarkers have been in high demand for disease diagnosis and therapeutics. Traditional hypothesis-based research has been challenging due to massive screening studies. Together with the emergence of omics technologies, currently, the paradigm for disease research has been moving toward evidence-based large-scale discovery studies. Proteins, as key effector molecules, can serve as ideal biomarkers for various diseases because they catalyze every biological function. Proteomics, which is represented by mass spectrometry (MS) technologies, stands as a solution for disease diagnosis and drug target discovery. CHAPTER I includes a portion of a report from of the human proteome project (HPP) related to chromosome 9 (Chr 9). To identify missing proteins (MPs) and their potential features in regard to proteogenomic view, both LC-MS/MS analysis and next-generation RNA sequencing (RNA-seq)-based tools were used for the clinical samples including adjacent non-tumor tissues. When the Chr 9 working group of the Chromosome-Centric Human Proteome Project (C-HPP) began this project, there were 170 remaining MPs encoded by Chr 9 (neXtProt 2013.09.26 rel.); currently, 133 MPs remain unidentified at present (neXtProt 2015.04.28 rel.). Proteome analysis in this study identified 19 missing proteins across all chromosomes and one MP (SPATA31A4) from Chr 9. RNA-seq analysis enable detection of RNA expression of 4 nonsynonymous (NS) SNPs (in CDH17, HIST1H1T, SAPCD2, and ZNF695) and 3 synonymous SNPs (in CDH17, CST1, and HNF1A) in all 5 tumor tissues but not in any of the adjacent normal tissues. By constructing a cancer patient sample-specific protein database based on individual RNA-seq data, and by searching the proteomics data from the same sample, 7 missense mutations in 5 genes (LTF, HDLBP, TF, HBD, and HLA-DRB5) were identified. Two of these mutations were found in tumor tissues but not in the paired normal tissues. Additionally, this study discovered peptides that were derived from the expression of a pseudogene (EEF1A1P5) in both tumor and normal tissues. In summary, this proteogenomic study of human primary lung tumor tissues enabled detection of additional missing proteins and revealed novel missense mutations and synonymous SNP signatures, some of which are predicted to be specific to lung cancer. CHAPTER II describes a study of the combination marker model using multiple reaction monitoring (MRM) quantitative data. Misdiagnosis of lung cancer remains a serious problem due to the difficulty of distinguishing lung cancer from other respiratory lung diseases. As a result, the development of serum-based differential diagnostic biomarkers is in high demand. In this study, 198 serum samples from non-cancer lung disease and lung cancer patients were analyzed using nLC-QqQ-MS to examine the diagnostic efficacy of seven lung cancer biomarker candidates. When the candidates were assessed individually, only SERPINEA4 showed statistically significant changes in the sera of cancer patient compared to those of control samples. The MRM results and clinical information were analyzed using logistic regression analysis to a select model for the best ‘meta-marker’, or combination of biomarkers for the differential diagnosis. Additionally, under consideration of statistical interaction, variables having a low significance as a single factor but statistically influencing the meta-marker model were selected. Using this probabilistic classification, the best meta-marker was determined to comprise two proteins SERPINA4 and PON1, with an age factor. This meta-marker showed an enhanced differential diagnostic capability (AUC=0.915) to distinguish the lung cancer from lung disease patient groups. These results suggest that a statistical model can determine optimal meta-markers, which may have better specificity and sensitivity than a single biomarker and may thus improve the differential diagnosis of lung cancer and lung disease patients. 정밀의료 패러다임의 등장 이후, 질환의 진단 및 치료를 위해서 바이오마커에 대한 수요는 높아지고있다. 가설기반연구는 전통적으로 당연하게 사용되오던 연구수행체계이지만, 바이오마커 발굴에서 필연적으로 마주치게되는 광범위한 스크리닝 작업에서는 효율성의 한계를 드러낸다. 오믹스기술의 등장과 함께 질환연구의 패러다임은 증거기반 대규모 타겟발굴방식으로 변화하고 있다. 단백질은 생체 기능조절에 직접적으로 관여하는 물질이기 때문에 바이오마커로 활용할 수 있는 가장 이상적인 물질로 여겨진다. 질량분석기를 이용한 단백체분석은 단백질을 직접 정성 및 정량할 수 있을 뿐만 아니라 매우 생산성이 높아 질환 바이오마커 발굴에 유용하다. 이 논문에서는 질량분석기를 이용하여 폐암 바이오마커의 발굴을 위한 고도화된 분석기법인 프로테오지노믹스 기법의 적용과, 스크리닝된 바이오마커후보 단백질의 정량검증 및 폐암 감별진단 조합마커의 생성연구에 대하여 알아본다. CHAPTER I에서는 인간염색체기반 단백체프로젝트 (C-HPP)의 일환으로 수행된 염색체9번에 대한 단백체연구가 포함되어있다. 미확인 단백질과 유전단백체에서 발견되지 않았던 시그니처를 밝혀내기 위해 LC-MS/MS분석과 RNA-seq 차세대염기분석기법을 적용하여 샘암종 폐암환자 5명의 정상-종양조직을 분석하였다. 염색체중심-인간단백체프로젝트의 2013년 리포트에서는 neXtProt 인간단백체 데이터베이스를 기준으로 염색체 9번에서 170개의 미확인 단백질이 있는 것으로 알려졌으며, 본 논문의 연구가 진행된 2015년에는 133개가 계속 미확인상태로 남아있었다. 본 논문의 단백체분석에서는 19개의 미확인 단백질을 동정할 수 있었으며, 그 중에서 염색체9번에 해당하는 단백질은 SPATA31A4 한 개 였다. RNA-seq분석으로는 샘종폐암조직 5개에서 공통적으로 검출되면서 정상조직에서는 검출되지 않는nonsynonymous SNP 5개 (CDH17, HIST1H1T, SAPCD2, ZNF695) 그리고 synonymous SNP 3개를 발굴할 수 있었다. 프로테오지노믹 분석을 위해서 각 시료별 RNA-seq데이터를 가공하여 맞춤형 데이터베이스를 구축하였다. 이렇게 생성된 시료맞춤형 데이터베이스를 단백체 질량분석데이터 검색에 활용하여 5개 유전자(LTF, HDLBP, TF, HBD, HLA-DRB5)에 해당하는 7개의 돌연변이를 검출하였다. 두 개의 돌연변이는 정상조직에서는 검출되지 않고 암조직에서만 검출되었다. 또한, 이 결과에서는 정상-암조직 모두에서 위유전자 (EEF1A1P5) 펩티드를 검출할 수 있었다. CHAPTER II에서는 다중반응검지법 (MRM) 을 이용한 단백질 바이오마커 검증과 조합마커 구성에 대한 연구를 서술하였다. 폐암과 다른 폐질환은 감별이 어렵기 때문에 폐암은 오진단 위험이 큰 질병이다. 따라서 혈청기반의 폐암감별진단 바이오마커개발의 필요성은 널리 인정되고있다. 이 단원에서는 폐암환자와 대조군폐질환 환자 198명의 혈청시료를 활용하여 일곱개의 폐암바이오마커 후보단백질을 나노유속 액체크로마토그래피-다중반응검지법으로 정량하였다. 후보단백질을 개별로 분석하였을 때에는 SERPINA4만이 통계적으로 유의성있게 혈중농도가 감소하는 것으로 나타났다. 다중반응검지법 전체데이터를 임상정보와 함께 로지스틱회귀모델에 적용하여 하나의 조합마커로 만들 수 있었다. 이 과정에서 개별마커로는 통계적인 유의성이 두드러지지 않지만 간섭효과를 만들어낼 수 있는 변수를 고려하여 모델링을 진행하였다. 최종적으로 SERPINA4, PON1, 나이를 조합하였을 때 가장 최적의 조합마커가 생성되었다. 이 조합마커는 AUC 0.915 의 감별진단 성능을 보여주었으며, 모델을 만드는데 사용되었던 시료와는 별개의 검증군에서도 성능은 유지되었다. 이와 같이 통계모델을 이용하여 생성한 조합마커는 개별 분자마커를 이용했을 경우보다 개선된 폐암 감별진단능력을 보여줄 수 있음을 제시한다.

비소세포폐암의 진단을 위한 TTF-1과 Napsin A 면역화학염색의 유용성

안종범 건국대학교 농축대학원 2022 국내석사

Lung cancer is a collective term for all cancers that occur in the respiratory epithelium, such as alveoli and bronchial tubes. In Korea, the incidence of lung cancer is not high. However, it is the cancer with the highest mortality rate. Lung cancer can be broadly divided into small cell lung cancer and non-small cell lung cancer. Among them, non-small cell lung cancer has a very high therapeutic effect due to the development of targeted therapies according to molecular biological characteristics, and thus targeted treatment is currently possible. Targeted therapy for non-small cell lung cancer patients is possible if there is an epidermal growth factor receptor gene mutation or ALK gene translocation, and it appears particularly high in adenocarcinoma. And because adenocarcinoma has a very high response rate to targeted therapies, the diagnosis of adenocarcinoma among non-sofeso-pulmonary cancers is very important. Among non-small cell lung cancers, if it is not confirmed by general tissue staining to diagnose adenocarcinoma, immunochemical staining is performed as an auxiliary method for histological differentiation. TTF-1 (Thyroid transcription factor-1) and Napsin A are used as markers for immunochemical staining of adenocarcinoma. TTF-1 is an important item in the immunochemical staining method for the diagnosis of adenocarcinoma of non-small cell lung cancer. However, if it is necessary to differentiate it from metastatic thyroid cancer or cancer metastasized in the digestive tract, it should be differentiated using other antibodies. Napsin A is overexpressed in normal tissues of the lung and kidney, but underexpressed in the spleen, and is characterized in that it is expressed in adenocarcinoma of the lung. In particular, since it shows very high specificity in adenocarcinoma of the lung, it is useful in distinguishing primary lung adenocarcinoma from adenocarcinoma of other organs. In this study, immunohistochemical staining was used to diagnose primary lung adenocarcinoma with lung tissue obtained through postoperative biopsy, and among them, the usefulness of TTF-1 and Napsin A immunochemical staining was tested. Among the 78 cases confirmed as primary non-small cell lung cancer, 46 cases of adenocarcinoma and 19 cases of squamous cell carcinoma were performed. Of the 46 adenocarcinoma cases, 38 cases (84%) were TTF-1 positive and 40 cases (88%) were Napsin A positive. Of the 19 squamous cell carcinoma cases, 6 cases (29%) were TTF-1 positive. Two cases (8%) were positive. The positive incidence rate of immunohistochemical staining for diagnosing adenocarcinoma was confirmed in order of Napsin A and TTF-1. Therefore, if the diagnosis is made using TTF-1, a useful marker for diagnosing primary lung adenocarcinoma, and Napsin A, a useful marker for differentiating from squamous cell carcinoma, it can be confirmed that it is a useful test method for diagnosing primary lung adenocarcinoma and adenocarcinoma of other organs. did In the future, if additional research is conducted on the usefulness of immunochemical staining in lung cancer diagnosis such as bronchial aspiration through cytology or ultrasound endoscopy and fine needle aspiration cytology for samples suspected of lung cancer, it will be meaningful as a comparison and analysis with existing studies. 폐암은 폐포, 기관지등 호흡상피에서 발생하는 모든 암을 통칭한다. 한국에서는 폐암 발생률은 높지 않다. 그러나 사망률이 가장 높은 암종이다. 폐암은 크게 소세포폐암과 비소세포폐암으로 나눌 수 있다. 그중에서 비소세포폐암은 분자생물학적 특징에 따라 표적치료제의 발전으로 인해 치료 효과가 매우 높아서 현재 표적치료가 가능한 실정이다. 비소세포폐암 환자의 표적치료는 상피세포성장인자 수용체 유전자 돌연변이나 ALK 유전자 전위가 있는 경우 가능하고 선암인 경우에 특히 높은 빈도로 나타난다. 그리고 선암은 표적치료제에 대한 반응률이 매우 높아서 비소페소폐암 중 선암의 진단은 매우 중요하다. 이 중 비소세포폐암 중 선암을 진단하기 위해 일반조직염색으로 확진되지 않는 경우 조직학적 감별을 위해서 보조적인 방법으로 면역화학염색법을 시행한다. TTF-1(Thyroid transcription factor-1)과 Napsin A는 선암의 면역화학염색법의 마커로 사용된다. TTF-1은 비소세포성폐암의 선암의 진단을 위한 면역화학염색법에서 중요한 항목이며 약75%정도 양성을 보이며 분화가 좋을수록 그 비율이 높아진다. 그러나 전이성 갑상선 암이나 소화기에서 전이하는 암과 감별해야 하는 경우는 다른 항체를 이용하여 감별해야 한다. Napsin A는 폐와 신장의 정상조직에서 과발현 양상을 보이나 비장에서는 저발현 양상을 보이며 폐의 선암에서 발현되는 것이 특징이다. 특히 폐의 선암에서 매우 높은 특이성을 나타내기 때문에 원발성 폐 선암과 다른 장기의 선암을 구분하는데 유용하다. 본 연구는 수술 후 생검을 통해 얻은 폐 조직으로 원발성 폐 선암을 진단하기 위하여 면역조직화학염색법을 이용, 그중에서 TTF-1과 Napsin A 면역화학염색의 유용성에 대해서 실험하였다. 원발성 비소세포폐암으로 확진된 78증례 중 선암 46건 그리고 편평상피세포암 19건을 실시하였다. 그 중 선암 46건 중 TTF-1 양성은 38건(84%), Napsin A 양성은 40건(88%)이었으며 편평상피세포암 19건 중 TTF-1 양성은 6건(29%)이었으며 Napsin A 양성은 2건(8%)이였다. 선암을 진단하기 위한 면역조직화학염색의 양성 발생율은 Napsin A, TTF-1 순으로 확인되었다. 따라서 원발성 폐 선암을 진단하는데 유용한 표지자인 TTF-1과 편평상피세포암과 감별하는데 유용한 표지자인 Napsin A를 활용하여 진단을 하면 원발성 폐 선암과 다른 장기의 선암 진단 시에 유용한 검사 방법이라는 것을 확인 가능하였다. 향후 폐암이 의심되는 검체에 세포검사나 초음파내시경을 통한 기관지 흡인술과 세침흡인 세포검사 등의 폐암진단에서도 면역화학염색의 유용성에 대해 추가적으로 연구한다면 기존 연구와의 비교, 분석으로 의미가 있을 것이라고 사료된다.

The lung cancer specificity of membrane ATPase : membrane ATPase의 폐암 특이성

Jun-won Jang 한양대학교 2016 국내석사

Lung cancer is the fatal cancer with the highest morality worldwide. Though there are many diagnostic, these are not clear solution for low sensitivity to early stage lung cancer.Lung cancer is difficult to be diagnosed due to its symptomless and non-destruction inspection can hardly search tumor until the last stage of lung cancer. Physical, chemical, biological approach has been performed in various fields, one of which is utilize biological property of lung cancer. Cell membrane proteins are specific for their function and microenvironment, some of which were exposed to circulation system. They are biomarker candidates of lung cancer to tell lung cancer to normal lung tissue and they may suppress the survival rate of lung cancer. Cell membrane proteins were investigated which express on the surface of four typical lung cancer cells, A549(Adenocarcinoma), H146(Small cell lung carcinoma), NCI-H460(Non-small cell lung carcinoma) and SK-MES-1 (Squamous cell lung carcinoma) using bioinformatic database and 16 proteins were verified as candidate biomarkers, one of which was investigated how much it is suitable for diagnostics and therapeutics. Its specific expression on the membrane of 4 typical human lung cancer cell was confirmed through western blot, immunocytochemistry and flow cytometry. This was identified that Sodium/Potassium-Transporting ATPase Subunit Alpha-1 was expressed on the surface of human lung cancer, showing possible candidate target[1-3]. Its specificity on the surface of lung cancer tissue was also examined in subcutaneous xenograft nude mice. Anti-ATPase antibody restrained population growth of human lung cancer cell lines in complement-dependent cytotoxicity assay. Anti-tumor effect was verified by anti-ATPase antibody injection into subcutaneous tumor-bearing mice model. This suggested that ATPase is one of the human lung cancer biomarker for inspection or treatment.

Aquilaria crassna Extract Triggers ROS-mediated Apoptosis in Human Small Cell Lung Cancer NCI-H889 Cells

Hwang, You Lim 부산대학교 2021 국내석사

Lung cancer causes the most common cancer death worldwide including Korea, despite declining the smoking rate for a decade. It is divided into two subtypes, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). In addition, there is another form of NSCLC called neuroendocrine non-small cell lung cancer (NE-NSCLC) resembled to SCLC by expressing NE markers. SCLC and NE-NSCLC have more aggressive cancer characteristics than NSCLC, whereas those are suitable for chemotherapy in the beginning. Unfortunately, chemotherapy has many side-effects and develops drug resistance. To overcome these disadvantages, natural products has been used as an alternative because of their peculiar features which are low toxicity and high efficacy at low doses. Aquilaria crassna was chosen as a candidate for lung cancer agent, which has been used as a traditional herb to treat allergy, diabetes, and neurological disorder. This study was to investigate anti-cancer activities on human lung cancer cells and, in addition, general biological activities of wood chips. A. crassna was extracted with methanol (MeOH) and partitioned into ethyl acetate (EtOAc), n-butanol (BuOH) and water layers, depending on their polarity. EtOAc layer of MeOH extract from A. crassna (ACM/E) showed biological activities which are associated with several diseases. The inhibition of α-glucosidase (at 50 µg/ml) and PTP1B (at 100 µg/ml) was involved in anti-diabetes. Also, anti-obesity, whitening, anti-gout and anti-inflammation of ACM/E were represented through inhibition of lipase (at 150 µg/ml), tyrosinase (at 150 µg/ml), xanthine oxidase (at 100 µg/ml) and nitric oxide (at 30 µg/ml), respectively. Interestingly, A. crassna was not well studied for anti-cancer activity, so far. Therefore, we testified whether ACM/E induces cell cytotoxicity on several lung cancer cell lines (A549, NCI-H889 and HCC1833). Their cell viability was reduced and their morphology was changed to spherical shape in a dose-dependent manner. Also, ACM/E increased ratio of apoptotic cells, cleavage of apoptotic-related proteins (caspase-3 and PARP) and intracellular reactive oxygen species (ROS) generation in lung cancer cells. Especially, apoptotic cell death of ACM/E treated-NCI-H889 and HCC1833 increased higher than A549 in a dose-dependent manner. That was consistent with the previous reports that SCLC and NE-NSCLC is more susceptible to cytotoxicity by drug treatment than NSCLC. To determine whether ACM/E-induced apoptosis is mediated with ROS, NCI-H889 cells that were the most sensitive to ACM/E, was chosen and tested. Pre-treatment with N-acetyl-L-cysteine (NAC), ROS inhibitor, recovered ACM/E-induced production of ROS, leading to restoring dysfunction of mitochondrial membrane potential (MMP) and apoptosis in NCI-H889 cells. Taken together, it suggested that ACM/E from A. crassna induces ROS-mediated apoptosis in NCI-H889 SCLC cells and may be a possibility for novel therapeutic agent for intractable lung cancer.

SPC에 의해 유도되는 상피 간엽 천이과정에 미치는 Ethacrynic acid의 억제 효과에 관한 연구

유루 동국대학교 2019 국내석사

Lung cancer is a fatal disease that accounts for 14% of new cancer cases. Metastasis is the primary cause of death in patients with lung cancer. Epithelial-mesenchymal transition (EMT) contributes to lung cancer invasion and metastasis. Therefore, it is crucial to find appropriate therapy for lung cancer and EMT. Ethacrynic acid (ECA) is a diuretic that inhibits cellular ion flux that leads to an increase in intracellular Na concentrations. We studied the effects of ethacrynic acid (ECA) on sphingosylphosphorylcholine (SPC)-induced EMT in A549 lung cancer cells by RT-PCR, Western blot, and RNA sequencing. We found that ECA inhibits SPC-induced EMT in A549 lung cancer cells. ECA inhibited SPC-induced migration and invasion in A549 lung cancer cells. We identified norrin (NDP) and Wnt2 as ECA responsive genes in A549 lung cancer cells through transcriptome analysis by RNA seq. NDP and WNT2 were validated by RT-PCR. NDP is found to be involved in SPC-induced WNT activation by NDP si-RNA. These results suggested that ECA suppresses SPC-induced EMT of A549 lung cancer cells via downregulation of NDP expression and ECA might be used as an anti-metastatic drug for lung cancer.

Phospholipase D6 acts as a potential therapeutic target against lung cancer

강래희 Graduate School, Yonsei University 2023 국내석사

Lung cancer has the second-worst prognosis of all cancers, with the greatest fatality rate among newly diagnosed patients. Targeted therapy and immunotherapy are currently the leading lung cancer treatments. Targeted therapy is frequently utilized in patients who have a bad prognosis, in particular. The prognosis for lung cancer is still poor, thus discovering a novel target protein for the disease is crucial to overcome lung cancer. Previous studies have shown that oxidative phosphorylation (OXPHOS) increased lung cancer metabolism. Increased OXPHOS enhances growth and metastasis. Since OXPHOS is regulated by different mitochondrial cycles including mitochondrial dynamics, we determined whether mitochondrial fusion increases OXPHOS promoting tumorigenesis. The significance of the PLD6's role in mitochondrial fusion in lung cancer was therefore anticipated. In this study, it was demonstrated that Phospholipase D6 (PLD6) was overexpressed in lung cancer. In addition, PLD6 induced mitochondrial fusion and OXPHOS, which in turn stimulate cancer cell growth. Furthermore, it was discovered that EGFR, which is hyperactive in lung cancer, phosphorylates the tyrosine residue of PLD6 to increase its activity. These findings help us understanding the function of PLD6 and suggest the possibility of PLD6 as a target protein for lung cancer. 폐암은 신규 환자 진단 2위를 차지하며, 암 환자 사망률 1위를 가지는 예후가 좋지 않은 암이다. 현재 폐암 치료의 주요 치료는 표적 치료와 면역 치료가 주를 이루며, 특히 표적 치료는 예후가 좋지 않은 환자에게 많이 사용된다. 하지만 예후는 여전히 좋지 않아 폐암의 새로운 표적 단백질을 찾는 것이 폐암의 치료에 매우 중요한 과제이다. 따라서 본 연구는 폐암에서의 새로운 표적 치료 단백질을 찾는 것을 목표로 하였다. 선행 연구에 따르면 폐암의 대사에서 OXPHOS가 증가되어 있어 있음을 확인하였고, 이는 페암의 성장과 전이를 증진시킨다. 미토콘드리아에서 일어나는 OXPHOS는 미토콘드리아의 역학에 의해 조절되며, 미토콘드리아의 웅합이 증가하면 OXPHOS가 증가된다고 연구되었기에, 미토콘드리아 융합에 관여하는 단백질인 PLD6의 폐암에서의 기능을 연구하고자 하였다. 또한 PLD6의 암에서의 기능은 많이 밝혀지지 않았기 때문에 새로운 폐암의 표적 단백질을 제시할 수 있을 것으로 예상하였다 본 연구를 통해 페암에서 PLD6는 과발현 되어 있으며, 이는 미토콘드리아의 융합을 촉진하여 OXPHOS를 증진시킴으로서 폐암을 진행을 촉진한다는 것을 규명하였다. 이는 폐암에서 과활성화된 EGFR에 의해 타이로신 잔기가 인산화됨으로서 활성이 더욱 증가됨을 알 수 있었다. 이로써 이 논문은 폐암에서의 PLD6의 기능을 이해하고, 새로운 페암의 치료 단백질로서 PLD6의 가능성을 제시한다.

Interaction between cancer and bacteria based on analysis of lung microbiome

최보윤 중앙대학교 대학원 2022 국내석사

폐 마이크로바이옴에 관한 연구와 폐암과 관련된 박테리아에 관한 연구는 최근 들어 끊임없이 계속되어지고 있지만, 여전히 많은 것이 알려지지 않았다. 폐암 환자들에게서 얻어진 조직을 무작위로 216개 조직을 선택하였다: 인접 정상 조직(Normal; n = 54), 표피생장인자수용체 (EGFR) 유전자 변이가 있는 선암 (AC, EGFR+; n = 54), EGFR유전자 변이가 없는 선암 (AC, EGFR-; n = 54), 편평상피세포암 (SCC; n = 54). 각 조직들은 gDNA 추출 과정을 거쳐 암과 관련된 폐 마이크로바이옴을 분석하기 위해 MiSeq platform으로 시퀀싱 되었다. QIIME2 pipeline을 사용하여 미생물 군집 분석이 수행되었다. 그 미생물 군집 분석 결과를 토대로, 조직에서 직접 Haemophilus influenza와 Stenotrophomonas maltophilia를 키웠으며, 폐암조직에서 분리된 H. influenzae와 S. maltophilia를 Oxford nanopore 시퀀싱을 이용하여 유전체 시퀀싱 후 분석을 진행하였다. 그 결과, 우리는 암 그룹이 정상 그룹보다 유의미하게 높은 박테리아 다양성을 가지는 것을 알 수 있었다. 편평상피세포암 그룹에서 다른 그룹들에 비해 H. influenzae가 많이 발견되었다. 또한 암의 재발 여부에 따라 미생물 군집을 비교하였을 때, 정상 그룹과 재발 그룹, 비재발 그룹의 모든 그룹 사이에 유의미한 차이가 있었다. 재발 그룹에서 Stenotrophomonas가 가장 많이 발견되었으며, Stenotrophomonas는 폐암의 재발과 연관성이 있을 수 있다는 가정을 할 수 있었다. 유전체 분석을 통해 폐암에서 분리된 H. influenzae의 전체 유전체를 확인하고 그들에게만 존재하는 유전자인 lsg B 유전자를 찾았으며, 그 유전자는 sialyltransferase였다. 따라서 우리는 선행 연구들을 참고하여 sialic acid가 풍부한 상태인 암 환경에서 H. influenzae가 더 잘 서식할 수 있으며, lsg B 유전자에 의해 더 강화된 독성이 암의 전이와 진행에 영향을 미칠 수 있다고 예상할 수 있었다. Research on the correlation between lung cancer and the microbiome has been continuously ongoing in recent years. However, most of them are still in question. Therefore, we randomly selected 216 tissues from lung cancer patients: adjacent normal tissue (Normal; n = 54), adenocarcinoma with epidermal growth factor receptor (EGFR) gene mutation (AC, EGFR+; n = 54), adenocarcinoma without EGFR gene mutation (AC, EGFR-; n = 54), and squamous cell carcinoma (SCC; n = 54). The gDNA extraction process was performed, and the sequencing was conducted using the MiSeq platform to analyze the cancer-related lung microbiomes. The microbial analysis was performed through the QIIME2 pipeline. Based on the results of microbial analysis , Haemophilus influenzae and Stenotrophomonas maltophilia were cultured directly from tissues, and H. influenzae and S. maltophilia isolated from lung cancer tissues were analyzed using whole-genome sequencing with the Oxford nanopore sequencing . As a result, we found that the cancer group had significantly higher bacterial diversity than the normal group. H. influenzae was detected in the SCC group. Also, when the microbial community was compared according to lung cancer recurrence, there was a significant difference between all groups; Normal, the group with lung cancer recurrence, and the group without lung cancer recurrence. Stenotrophomonas was detected the most in the group with lung cancer recurrence, and it could be predicted that Stenotrophomonas may be associated with the recurrence of lung cancer. Through whole-genome analysis, the genome of H. influenzae isolated from lung cancer was identified, and the lsgB gene, the gene that exists only in them, was found, and that gene was sialyltransferase. Therefore, referring to previous studies, we predicted that H. influenzae could have a better chance to be colonized in a cancer environment that is rich in sialic acid, and thus, increasing the toxicity by the lsgB gene could affect the metastasis and progression of lung cancer.

Longitudinal transition patterns of symptom clusters and health outcomes in patients with lung cancer surgery

김예솔 Graduate School, Yonsei University 2024 국내박사

Association between air pollution and recurrence of lung cancer after surgery in female never-smokers

김보미 서울대학교 대학원 2021 국내석사

Background: Lung cancer is a common cancer all over the world, and it is the third most common cancer in Korea. While groundbreaking anticancer drugs and radiotherapy have been developed, surgery is still known as the most appropriate treatment in early-stage lung cancer. However, recurrence after surgery is the most common event of treatment failures among lung cancer patients, thereby shortening the survival period. Although stage at diagnosis and vascular lymphatic metastases have been estimated to be risk factors for recurrence, causes for recurrence are still unclear. Smoking is the highest risk factor for lung cancer, but never-smokers account for more than half among female lung cancer patients. Many studies have investigated air pollution as one of the risk factors in lung cancer incidence, but there is little research on the association between air pollution and lung cancer recurrence. Objectives: Under the hypothesis that air pollution will have an effect on the recurrence of lung cancer after surgery with a similar mechanism to the effect on lung cancer incidence, this study aims to investigate the association between air pollution and lung cancer recurrence after surgery in female never-smokers. Methods: This study selected 132 female never-smoker lung cancer patients who had surgery from February 2013 and January 2017 at Seoul National University Hospital and collected clinical information, including stage at diagnosis, primary tumor size, lymph node invasion, EGFR (Epidermal growth factor receptor) mutation, family history of lung cancer, symptoms related to lung cancer at diagnosis, history of hypertension, history of type 2 diabetes mellitus and secondhand smoking. The air pollution data were extracted according to each subject’s residence and were merged after calculating the average concentrations at each time-interval (3, 6, 12, 24 and 36 months intervals from surgery until recurrence). Statistical analysis was performed using multiple logistic regression models after adjusting for the aforementioned clinical variables. Results: Estimated odds ratios for the recurrence of lung cancer with increase of 1 ppb in SO2 was identified as 1.73 (95% CI: 1.07-2.80) and 2.14 (95% CI: 1.31-3.49) during 24 and 36 months from surgery until recurrence in fully adjusted models. Estimated odds ratios for the recurrence of lung cancer with increase of 10 ppb in NO2 and 10 μg/m^3 in PM2.5 were respectively 2.02 (95% CI: 1.01-4.04) and 3.35 (1.02-10.99) during 36 months from surgery until recurrence. In the case of increase of 10 ppb in O3, adjusted odds ratios for the recurrence were 0.32 (0.11-0.94) and 0.34 (0.12-0.98) during 12 and 36 months from surgery until recurrence. These results were identically indicated in two-pollutant models. In subgroup analyses, only O3 and PM10 had interactions with EGFR mutation but the patterns of associations between each air pollutant and recurrence of lung cancer differed by the presence of EGFR mutation. Conclusion: This study identified that air pollution can be an associated risk factor for the recurrence of lung cancer in female never-smokers and the association was founded to be more relevant with longer exposure periods. However, this study showed that O3 was negatively associated with the recurrence of lung cancer. It was also shown that the association between air pollution and the recurrence of lung cancer was different according to the presence or absence of EGFR mutation. Subsequent studies should further investigate uncertain effect of ozone on lung cancer recurrence. The results of this study will serve as the base for further studies concerning risk factors of lung cancer recurrence in female never-smokers in the interest of public health. 연구배경: 폐암은 전 세계적으로 흔한 암으로, 우리나라에서는 세 번째로 흔한 암이다. 획기적인 항암제와 방사선 치료법이 개발되어왔으나, 여전히 초기 폐암에서는 수술적 치료가 가장 적절한 치료로 알려져 있다. 그러나 수술 후 재발은 많은 폐암 환자들이 경험하는 치료 실패이자 생존기간을 단축시키는 원인으로, 진단 당시 암 병기, 혈관림프절 전이 여부가 재발 위험요인으로 추정되지만 그 원인은 아직까지 명확하게 밝혀진 바가 없다. 폐암의 가장 큰 위험요인으로 흡연이 꼽히지만, 여성 폐암에서 비흡연인 경우는 절반 이상을 차지한다. 대기오염은 폐암의 위험요인으로 많은 연구가 진행되어 왔으나 수술 후 재발과의 연관성에 대한 연구는 거의 전무하다. 연구목적: 본 연구는 대기오염이 폐암 유발에 미치는 영향과 유사한 메커니즘으로 수술 후 재발에도 영향이 있을 것이라는 가설 아래, 한국에서 대기오염과 비흡연 여성 폐암 환자의 수술 후 재발 간의 연관성에 대해 확인하고자 한다. 연구방법: 본 연구는 2013년 2월부터 2017년 1월까지 서울대학교병원에서 폐암 진단 후 수술을 받은 비흡연 여성 폐암환자 132명을 대상으로, 진단 당시 암 병기, 암 크기, 림프절 전이 여부, EGFR 변이 여부, 폐암 가족력, 진단 당시 암 관련 증상 여부, 고혈압 및 당뇨 병력, 간접흡연 노출 여부 등 임상 관련 특성에 관한 정보를 수집하였다. 대기오염자료는 대상자 거주지 별 대기오염 자료를 추출하여, 수술 후 재발까지 3, 6, 12, 24, 36개월 간격의 각 평균 농도를 산출하여 병합하였다. 통계분석은 다중 로지스틱 회귀분석을 이용하여, 임상관련 특성을 보정한 후 폐암 재발과의 연관성을 확인하였다. 연구결과: SO2 평균 농도의 1 ppb 단위 증가에 따른 폐암 재발 오즈비는 수술 후 재발까지 24개월 및 36개월 동안 1.73 (95% CI: 1.07-2.80)와 2.14 (95% CI: 1.31-3.49)로 나타났다. NO2의 10 ppb 단위 증가에 따른 폐암 재발 오즈비와 PM2.5의 10μg/m^3 단위 증가에 따른 폐암 재발 오즈비는 수술 후 재발까지 36개월 동안 각각 2.02 (95% CI: 1.01-4.04) 및 3.35 (1.02-10.99)로 관찰되었다. O3의 경우, 수술 후 재발까지 12개월 및 36개월 동안 10 ppb 증가 당 폐암 재발 오즈비는 0.32 (0.11-0.94), 0.34 (0.12-0.98)로 나타났다. 이러한 결과는 두 오염 물질 모형 (two-pollutant model)에서도 동일하게 나타났다. 하위그룹분석에서 유일하게 O3와 PM10이 EGFR 변이 간에 교호작용이 있는 것으로 관찰되었으나, 그 연관성이 상반된 것으로 나타났다. 결 론: 본 연구를 통해 비흡연 여성 폐암의 재발에 대기오염이 영향을 미치며, 대기오염에 대한 장기 노출이 보다 더 관련이 있는 것을 확인하였다. 본 연구에서는 오존과 수술 후 재발 간 음의 연관성으로 확인되었다. 또한 EGFR 변이 여부에 따른 대기오염과 폐암 재발 간의 연관성이 서로 다른 것으로 나타났다. 수술 후 폐암 재발에 대한 오존의 불확실한 영향에 대하여서는 후속 연구에서 추가로 시행되어야 할 것이다. 본 연구 결과는 보건학적 관심이 필요한 비흡연 여성 폐암 환자의 재발 위험 요인에 대한 연구의 기반이 될 것이다.