類別:

非小細胞肺癌之新藥開發與相關藥物作用標的之研究– 以GRC0321及Monocarboxylate transporter 4為例

Sun, 01 Jan 2017 00:00:00 GMT

標題: 非小細胞肺癌之新藥開發與相關藥物作用標的之研究– 以GRC0321及Monocarboxylate transporter 4為例; Identify Novel Drug and Related Therapeutic Target in Non-Small Cell Lung Cancer - in terms of GRC0321 and Monocarboxylate transporter 4 作者: Ting-Chun Kuo; 郭廷群摘要: 肺癌至今仍然是全世界致死率最高的癌症之一。儘管藥物或是手術方面於近年皆有長足的進步，但在有效降低肺癌致死率的部分仍不盡理想。歸納造成抗癌藥物無法展現有效的抗癌效果及提供安全的藥物劑量之眾多原因可發現，大多與癌症細胞快速產生抗藥性及抗癌藥物對人體正常細胞產生嚴重的副作用有關。但近年來隨著精準醫學此一觀念在抗癌藥物研究領域之蓬勃發展，越來越多的肺癌診斷標記蛋白及藥物標的在肺癌細胞中被發現，也再次為此領域注入了新的希望與發展方向。於此博士論文中，我們運用了隨機藥物篩選及機制導向藥物設計兩種策略成功的找到了兩個可做為肺癌藥物標的之蛋白，katanin蛋白及Monocarboxylate transporter 4 (MCT4)蛋白。同時我們亦針對其分別發展出能有效抑制肺癌細胞生長之抗癌藥物，GRC0321及MCT4中和抗體。此兩藥物不論是在細胞內或是動物體內之實驗模型中都展現了良好的抗癌效果及優異的安全性。在詳細的藥物抗癌機轉研究中，我們發現小分子藥物GRC0321是透過影響katanin蛋白來破壞癌症細胞微管的完整性。而後，此一現象將會進一步地造成癌症細胞G2/M細胞週期停滯，並導致癌症細胞走向由caspase-9 蛋白所主導的細胞凋亡途徑。關於MCT4中和抗體造成糖解作用依賴型肺癌細胞生長停滯的機轉部分，我們則發現此一抗體能有效的抑制癌症細胞將堆積於細胞內之多餘乳酸運送到細胞外之機制。此一抑制機制可以進一步的造成癌症細胞細胞內之pH值降低，並同時產生過量之活性氧化物質。而最後則一樣會造成癌症細胞走向由caspase-9 蛋白所主導的細胞凋亡途徑。此外，我們亦發現MCT4中和抗體在MCT4大量表性之肺癌細胞中能展現較好之抗癌能力。詳細研究後發現，因為此類肺癌細胞較依賴糖解作用來得到能量，且會透過Sp1蛋白參與之轉錄機制來調控MCT4蛋白之大量表現，且此現象即使在一般正常氧分壓環境仍可觀察到，因此其對MCT4中和抗體的敏感性較高。根據目前之實驗結果數據，此兩種藥物具有極大的潛能進行後之藥物發展。但較差的水溶解性及不適當的免疫球蛋白種型可能會是造成日後發展瓶頸的兩大原因。因此，如何針對此兩大問題進行適當的解決及改善勢必是影響此兩種藥物後續能否成功發展成為臨床抗癌藥物的關鍵。慶幸的是，目前在這些藥物開發的領域都有許多新穎的技術及方法被研發出來。因此我們深信，藉由不同領域人員及技術的合作幫忙，GRC0321及MCT4中和抗體必定能在將來發展成為臨床抗癌藥物，提供廣大的肺癌病人族群更多的治療選擇與幫助。; Lung cancer is the leading cause of the cancer-related death in the world. Though many improvements in medical and surgical therapies has been done, the advancement in lung cancer mortality remains unsatisfactory. Drug resistance and serious side effects are two important reasons to limit the efficacy and safety of the therapeutic agents targeting lung cancer cells. However, with the introduction of precision medicine, more and more promising diagnostic markers and therapeutic targets have been identified in lung cancer cells recently. In this dissertation, we adopted two strategies, inclusive of randomizing drug screening and mechanism-based drug design, and successfully identified katanin and Monocarboxylate transporter 4 (MCT4) with corresponding therapeutic agents, GRC0321 and MCT4 neutralizing antibody, for anti-lung cancer treatment. We demonstrated the excellent anti-cancer effects and broad therapeutic windows of these two drugs in the in vitro and in vivo models. Besides, the detail mechanisms of the drugs and the expression regulations of the drug targets in lung cancer cells were also investigated. GRC0321, a small compound, destructed the microtubule structure of lung cancer cells by targeting katanin protein and caused a caspase-9 mediated cancer cell apoptosis through prolonging G2/M cell cycle arrest. On the other hand, the MCT4 neutralizing antibody inhibited the proliferation of glycolysis-dependent subtype of lung cancer cells by blocking the export of excess lactate in the cancer cells. This blockage could reduce the intracellular pH level and increase reactive oxygen species (ROS) production and further activate caspase-9 mediated cancer cell apoptosis. The MCT4 neutralizing therapy revealed excellent effects especially on the MCT4-overexpressed lung cancer cells. To explore the detail mechanisms, we found that this MCT4-overexpressed lung cancer cells had higher dependency on the glycolysis and would express more MCT4 on their cell membranes through a Sp1-mediated transcriptional regulation even in a normoxia condition. Though all the wonderful experimental results of these two drugs make them deserve further development, poor solubility and improper immunoglobulin isotypes are the two major issues encountered and need to be solved in the near future. Fortunately, more and more progresses have been made in these fields which makes us strongly believe that the two drugs could be developed successfully and become clinical therapies to help the patients with lung cancer.

長鏈非編碼核糖核酸HOXB-AS3在急性骨髓性白血病及骨髓分化不良症候群患者之角色

Sun, 01 Jan 2023 00:00:00 GMT

標題: 長鏈非編碼核糖核酸HOXB-AS3在急性骨髓性白血病及骨髓分化不良症候群患者之角色; The role of long non-coding RNA HOXB-AS3 in patients with acute myeloid leukemia and myelodysplastic syndrome 作者: 黃懷萱; Huai-Hsuan Huang 摘要: 背景資料在細胞內的轉錄物（transcripts）裡，長鏈非編碼核糖核酸（Long non-coding RNAs，lncRNAs）佔了一大部分，也在造血作用（hematopoiesis）的過程中扮演重要角色。然而，長鏈非編碼核糖核酸對於血液惡性疾病，如：急性骨髓性白血病（acute myeloid leukemia，AML）和骨髓分化不良症候群（myelodysplastic syndrome，MDS），有何重要影響，目前所知有限。在這個研究裡，我們研究的主角是HOXB-AS3，一個在人類HOXB 群組基因裡的長鏈非編碼核糖核酸；研究其在骨髓性細胞株裡的特性，及在急性骨髓性白血病患者和骨髓分化不良症候群患者裡，他們的臨床表徵及預後。研究方法我們利用短髮夾核糖核酸（short hairpin RNA，shRNA）去抑制HOXB-AS3在細胞株裡的表現，並觀察細胞生長情形，再使用微陣列（microarray）的方式，來分析及找尋細胞內被影響的基因，接著利用定量即時聚合酶鏈鎖反應（quantitative polymerase chain reaction）的方式來驗證。另外，我們也利用溴化去氧尿嘧啶流式細胞儀分析（BrdU flow assay），顯示HOXB-AS3對細胞生長分裂的影響。更進一步地，我們回溯性研究HOXB-AS3，對於193位急性骨髓性白血病患者及157位骨髓分化不良症候群患者，其表現量與臨床表現及預後之影響。研究結果在急性骨髓性白血病細胞株OCI-AML3內，降低HOXB-AS3的表現，會抑制細胞的生長。在微陣列的分析中， HOXB-AS3不影響HOX基因的表現，反而會影響細胞周期及去氧核糖核酸複製相關的基因表現。在急性骨髓性白血病患者裡，HOXB-AS3高表現的患者，其預後較低表現患者差（高表現者的整體存活期中位數為17.7 個月，相較於低表現者為未達到，P < 0.0001；高表現者的無復發存活期中位數為12.9 個月，相較於低表現者為未達到，P = 0.0070）。在骨髓分化不良症候群患者裡，也可看到類似狀況（高表現者的整體存活期中位數為14.6個月，相較於低表現者為42.4個月，P = 0.0018）。我們的分析結果，使用美國癌症基因體圖譜計畫急性骨髓性白血病患者群（TCGA AML cohort）及本院另外一組骨髓分化不良症候患者群，作為驗證組，也能重現。在骨髓分化不良症候群患者的次族群分析裡，在國際預後評分系統（international prognostic scoring system ，IPSS）低風險及中低風險的患者中，HOXB-AS3高表現能預測患者有較差的預後（高表現者的整體存活期中位數為29.2個月，相較於低表現者為77.3個月，P = 0.0194）；然而在高風險患者中，則無太大差異。結論本研究發現，HOXB-AS3在骨髓惡性疾病裡所扮演的角色，也進一步展現出，HOXB-AS3的表現量，對於急性骨髓性白血病患者及骨髓化不良症候群患者，在預後上的影響。; Background Long non-coding RNAs (lncRNAs) represent the majority of cellular transcripts and play pivotal roles in hematopoiesis. However, their clinical relevance in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains largely unknown. Here, we investigated the functions of HOXB-AS3, a lncRNA located at human HOXB cluster, in the myeloid cells, and analyzed the prognostic significances in patients with AML and MDS. Methods We used shRNAs to downregulate lncRNA HOXB-AS3 in the cell lines, and then observed the cell growth. We investigated the downstream genes by microarray analysis, and validated the results with quantitative polymerase chain reaction. We also illustrated the effects of lncRNA HOXB-AS3 in the myeloid cell lines by BrdU proliferation flow assay. Further, we retrospectively analyzed lncRNA HOXB-AS3 expression in 193 patients with AML and 157 with MDS by microarray analysis and evaluated its clinical significance. Results Downregulation of lncRNA HOXB-AS3 suppressed cell proliferation in the myeloid cell line, OCI-AML3. In the microarray analysis, lncRNA HOXB-AS3 potentiated the expressions of several key factors in cell cycle progression and DNA replication without affecting the expressions of HOX genes. In AML, patients with higher HOXB-AS3 expression had shorter survival than those with lower HOXB-AS3 expression (median overall survival (OS), 17.7 months versus not reached, P<0.0001; median relapse-free survival, 12.9 months versus not reached, P=0.0070). In MDS, patients with higher HOXB-AS3 expression also had adverse prognosis compared with those with lower HOXB-AS3 expression (median OS, 14.6 months versus 42.4 months, P=0.0018). The prognostic significance of lncRNA HOXB-AS3 expression was validated in the TCGA AML cohort and another MDS cohort from our institute. The subgroup analyses in MDS patients showed that higher HOXB-AS3 expressions could predict poor prognosis only in low and low-intermediate IPSS risk groups (median OS, 29.2 months versus 77.3 months, P=0.0194), but not in high and intermediate-high IPSS risk groups. Conclusions This study uncovers a promoting role of lncRNA HOXB-AS3 in myeloid malignancies and identifies the prognostic value of lncRNA HOXB-AS3 expression in AML and MDS patients, particularly in the lower-risk group.

藉由系統性分析配對的原發性和復發性 IDH 野生型膠質母細胞瘤間的基因體和轉錄體差異尋找癌症復發相關的生物標記

Wed, 01 Jan 2025 00:00:00 GMT

標題: 藉由系統性分析配對的原發性和復發性 IDH 野生型膠質母細胞瘤間的基因體和轉錄體差異尋找癌症復發相關的生物標記; Systematic Analysis of Genomic and Transcriptomic Differences between Matched Initial and Recurrent IDH Wild-Type Glioblastomas Identifies Recurrence-Associated Biomarker 作者: 何偉民; Wei-Min Ho 摘要: 神經膠質母細胞瘤 (Glioblastoma multiforme，GBM) 是成人中最常見且侵襲性最強的惡性腦部腫瘤，對現有的治療方式仍具高度抗性，且在初次治療後常迅速復發。儘管針對GBM復發所涉及的基因組與轉錄組變化已進行了廣泛的研究，成對的原發及復發 GBM 之間的演化動態仍未完全理解。其中一個主要挑戰是找出與復發時間 (Relapse time/Time to relapse，RT/TTR) 相關的分子標誌，並建立一個能準確預測原發GBM患者RT的穩定預後模型。在本研究中，我們整合了多個患者匹配的GBM資料集的RNA測序和基因組數據 (Genomic and transcriptomic datasets)，特別關注於異檸檬酸脫氫酶野生型 (IDH wild-type) 的腫瘤。我們探討了RT與原發及復發 GBM之間分子異質性之間的關聯，著重於基因表現模式、腫瘤突變負荷 (Tumor mutational burden，TMB) 和腫瘤微環境。我們的研究結果顯示，RT與原發及復發 GBM之間轉錄組和基因組的差異程度呈正相關。此外，相較於RT較長的患者，RT較短的患者顯示出非間質型向間質型轉變(Non-mesenchymal-to-mesenchymal transitions)的比例較高，以及更高的間質型亞型表現。我們還觀察到原發及復發 GBM在基因表現模式和TMB上的高度相關性，且這些匹配的原發及復發 GBM基因表現一致性與RT呈負相關。基於這些觀察結果，我們鑑定出55個與RT相關的基因，並透過單變量和多變量Cox回歸分析建立了一個包含7個關鍵基因（ZSCAN10、SIGLEC14、GHRHR、TBX15、TAS2R1、CDKL1、CD101）的預後模型。該模型產生的風險分數在訓練集及兩個獨立驗證集中與RT呈顯著負相關。此外，該模型有效地將IDH wild-type GBM患者分成兩組，並顯示出顯著不同的無進展生存率 (Progression-free survival，PFS) 結果，且在所有數據集中對1年、2年及3年的PFS率預測表現出色。我們的研究結果為GBM復發時的分子機制提供了新的見解，並提出了潛在的治療靶點以供未來研發策略的參考。; Glioblastoma multiforme (GBM) represents the most pervasive and formidable malignant brain tumor in adults, characterized by its resistance to current therapeutic modalities and a propensity for rapid recurrence post-initial treatment. Despite substantial efforts to elucidate the genomic and transcriptomic landscapes underpinning GBM recurrence, the evolutionary dynamics linking initial/primary and recurrent (I-R/P-R) GBMs remain inadequately defined. A critical challenge lies in discerning molecular determinants predictive of relapse time/time to relapse (RT/TTR) and formulating a reliable prognostic framework to estimate RT in patients with initial/primary GBM (iGBM/pGBM). In this investigation, we leveraged RNA sequencing and genomic datasets from longitudinally collected, patient-matched GBM cohorts, with a specific focus on isocitrate dehydrogenase wild-type (IDH-wildtype) tumors. Our analysis interrogated the interplay between RT and molecular heterogeneity within matched I-R GBMs, emphasizing gene activity dynamics, tumor mutational burden (TMB), and the composition of the tumor microenvironment. Notably, our findings demonstrated a direct correlation between RT and the extent of transcriptomic and genomic divergence observed between initial and recurrent GBMs. Patients with shorter RTs exhibited elevated frequencies of non-mesenchymal-to-mesenchymal transitions and a predominance of mesenchymal subtypes, as opposed to those with prolonged RTs. Moreover, we identified a strong concordance in gene activity patterns and TMB between matched I-R GBMs, which inversely correlated with RT. From these analyses, we pinpointed 55 RT-associated genes and built a prognostic model incorporating seven pivotal genes (ZSCAN10, SIGLEC14, GHRHR, TBX15, TAS2R1, CDKL1, and CD101) through univariate and multivariate Cox regression methodologies. The model's risk scores demonstrated a significant inverse correlation with RT in the training cohort, validated across two independent datasets. Furthermore, the model stratified IDH-wildtype GBM patients into two distinct prognostic subgroups with significantly different progression-free survival (PFS) trajectories, while achieving robust predictive accuracy for one-, two-, and three-year PFS across all cohorts. These findings illuminate the molecular underpinnings of GBM progression and recurrence, offering a deeper understanding of its evolutionary biology. The study also highlights potential molecular targets for therapeutic intervention, paving the way for more personalized and effective strategies in GBM management.

缺乏氧化壓力傳遞者NPGPx在小鼠與人類引起肥胖

Tue, 01 Jan 2013 00:00:00 GMT

標題: 缺乏氧化壓力傳遞者NPGPx在小鼠與人類引起肥胖; Deficiency of NPGPx, an oxidative stress transducer, causes obesity in mice and human 作者: Yi-Cheng Chang; 張以承摘要: 流行病學顯示氧化壓力與肥胖緊密相關, 傳統上認為氧化壓力上升是肥胖所造成的結果, 但最近的證據顯示, 氧化壓力也會促進脂肪組織形成。NPGPx (non-selenocysteine-containing phospholipid hydroperoxide glutathione peroxidase or glutathione peroxidase 7, GPx7)是一個演化中保守的氧化壓力感受與傳遞者。我們之前的研究發現, 缺乏NPGPx會增加氧化壓力。在本研究中, 我們發現NPGPx在脂肪前趨細胞表現量豐富, 而減少其NPGPx的表現, 會促進脂肪前趨細胞分化為脂肪細胞。此促進脂肪分化的作用, 是藉由氧化壓力增加造成的CCAAT/enhancer-binding protein beta (C/EBPβ)的活化所達成, 抗氧化劑N-acetylcysteine可以壓制這些作用。我們也發現, 缺乏NPGPx的小鼠脂肪組織增加且脂肪細胞肥大,以N-acetylcysteine餵食NPGPx缺乏的小鼠, 可以防止這些表現型產生。我們進一步發現, 人類NPGPx基因的單一核苷酸多型性, 在數個人類族群中, 與身體質量指數有相關,也與人類脂肪組織中NPGPx訊息核醣核苷核酸的表現量有相關。總和來說, 本研究顯示NPGPx會經由調控氧化壓力, 影響脂肪細胞分化, 造成肥胖, 這結果揭示了氧化壓力在引發肥胖的角色, 以及抗氧化劑預防肥胖的可能療效。; Elevated oxidative stress is closely associated with obesity. Emerging evidence shows that instead of being a consequence of obesity, oxidative stress may also contribute to fat formation. NPGPx (non-selenocysteine-containing phospholipid hydroperoxide glutathione peroxidase or glutathione peroxidase 7, GPx7) is an evolutionally conserved oxidative stress sensor/transducer. Deficiency of NPGPx causes accumulation of reactive oxygen species (ROS). In this study, we showed that NPGPx was highly expressed in preadipocytes of adipose tissue. Deficiency of NPGPx in preadipocytes facilitated its differentiation into adipocytes via ROS-dependent activation of CCAAT/enhancer-binding protein beta (C/EBPβ) pathway. This enhanced adipogenesis was suppressed by antioxidant N-acetylcysteine (NAC). Consistently, NPGPx-deficient mice exhibited markedly increased fat mass and adipocyte hypertrophy, while treatment with NAC ablated these phenotypes. Furthermore, single nucleotide polymorphisms in human NPGPx gene, which correlated with lower NPGPx expression level in adipose tissue, were associated with higher body mass index in several independent human populations. These results indicate that NPGPx modulates fat accumulation in mice and human via regulating ROS and highlight the importance of targeting redox homeostasis for treating obesity.