摘要:研究目的:探討腸道菌叢失衡及毒性因子對大腸直腸癌生成之影響,主要聚焦於貼附型細菌在腫瘤環境如上皮細胞與免疫細胞交互作用下引起之訊息傳遞途徑,並探索具潛力之抗生素介入治療。大腸直腸癌在癌症相關死亡率中排名第二,其中發炎性腸疾病患是癌症發生之高危險群。正常情況下腸道共生菌與上皮細胞並無接觸,然而在發炎性腸疾與大腸直腸癌病患檢體中發現有大量貼附侵襲型腸桿菌,暗示了菌叢失衡可能參與發炎性腸癌之生成過程。本人過去實驗證實宿主對細菌脂多醣(LPS)的異常辨識與腸道癌化有密切關係 (Kuo et al. 2015, Cell Death Diff. In press);惟腸菌物質何以大量暴露或貼附黏膜尚無法解釋,而相關致癌機轉的研究仍付之闕如。本計畫之實驗假設是腸道細菌毒性生成與宿主免疫異常的共存現象為導致大腸直腸癌化之重要原因。時至今日,特定細菌與毒性因子的致癌角色仍有待釐清。我們初步實驗結果顯示在發炎性腸癌小鼠模式中,腸道菌相組成有所改變,且口服給予抗生素可降低腫瘤數目。野生型小鼠的腫瘤生成相較於TLR4基因突變型小鼠為高;而腫瘤發生率與腸桿菌量、細菌貼附性及脂多醣受體表現量有正相關。本篇研究目標為:(1) 利用發炎性腸癌小鼠模式評估微生物菌相改變時程,並找出以抗生素治療腸癌之適當時機點;(2) 探討在腫瘤進程及抗生素治療後變化的細菌毒性特質,如貼附性、基因毒性或訊息途徑擬態現象;(3) 建構具有或刪除毒性因子之候選細菌,以植入無菌小鼠、大腸類器官、人類細胞株的方式證實其對致癌作用之影響。本計畫將利用化學物誘導發炎性腸癌生成,並探究以抗生素重整腸道菌叢治療癌症之潛力。預計以先進之bacterial 16S rDNA sequencing和high through-put illumina-based RNA sequencing技術來分析致癌過程和抗生素治療後之腸道菌相變化,並配合PCR和fluoresence in situ hybridization技術以偵測腸道黏膜細菌之毒性因子。經由比較野生型和基因突變型小鼠之間的菌相差異及細菌貼附性,以及與人類貼附侵襲型細菌毒性因子作比對,初步選擇可能有致癌性的細菌;再利用人類腸癌細胞株,或上皮-免疫細胞共同培養方式作攻毒試驗,篩選出有加速細胞分裂能力或誘發DNA傷害之候選細菌。最後透過phage -red/Flp recombinase method刪除細菌毒性基因後,將原生型和基因刪除之候選細菌植入於尖端技術之大腸類器官培養和無菌小鼠模式,以確認細菌毒性因子之致癌性。本研究成果冀望能為腸癌病患帶來突破性之療法。
Abstract: Objective: To investigate the profile of bacterial dysbiosis and virulence factors on colorectal cancer (CRC) development, focusing on adherent bacterial-derived signaling in tumor environment, i.e. epithelial and immune cell interaction, and explores potential antibiotic intervention. CRC is the second leading cause of cancer-related mortality, of which patients with inflammatory bowel disease (IBD) are of higher risk. Gut commensals are normally not in contact with epithelium in healthy subjects, whereas large amounts of adherent-invasive enterobacteriaceae are observed in IBD and CRC patients, suggesting that microbiota dysbiosis may be involved in colitis-associated colon tumorigenesis. Previous studies from our laboratory have demonstrated that aberrant recognition of bacterial lipopolysaccharide (LPS) is associated with CRC growth (Kuo et al., 2015. Cell Death Diff. In press). However, it remains unclear why enteric bacteria are capable of maximizing exposure and adherence to gut mucosa, and its involvement in carcinogenesis has not yet been explored. Our working hypothesis of a two-hit theory is that co-existing bacterial virulent changes and abnormal host immune signaling promote colon tumor development. To date, the roles of specific bacteria and virulence factors on carcinogenesis remain poorly understood. Pilot studies showed that microbiota composition was altered in CRC mice and oral antibiotics decreased colon tumor numbers. Wild-type mice showed higher tumor burden compared to TLR4-mutant mice; tumor susceptibility was positively correlated with abundance of enterobacteriaceae, bacterial adhesion, and LPS receptor expression. The aims of the project are to (1) evaluate time-dependent microbiota changes and explore the appropriate timing for antibiotic treatment to modulate carcinogenesis using colitis-associated CRC mouse models; (2) investigate bacterial virulence properties (e.g. adherence, genotoxicity, or signaling mimicry) during cancer progression and after antibiotic pressure; (3) construct virulence factors in candidate bacteria to validate the impact on carcinogenesis using gnotobiotic mouse models, colonic organoid cultures, and human cell lines. We plan to explore potential antibiotic use to inhibit cancer development by altering gut microbiota in mouse models of chemical-induced CRC. We will employ bacterial 16S rDNA sequencing and high through-put illumina-based RNA sequencing to evaluate microbiota profiles at various stages during cancer development and after antibiotic treatment. Moreover, PCR-based analysis and fluoresence in situ hybridization will be conducted to examine virulence factors on adherent mucosa-associated bacteria. By comparing the discrepancy of microbiota profile and bacterial adherence in wild-type and mutant mice, and by cross-validating with pathogenicity islands identified in human adherent/invasive enterobacteriaceae, a pool of bacteria with potential pro-tumorigenic properties will be chosen. Candidate bacteria that accelerate cell division and/or induce DNA damage in human colon carcinoma cell cultures, or epithelial-immune cell co-cultures will be further selected. Lastly, candidate bacteria and those with virulent gene deletion by using phage -red and Flp recombinase method will be inoculated into state-of-the-art colonic organoid cultures and gnotobiotic mouse models to validate the pro-tumorigenic properties. This research project will lead to novel strategies for development of advanced colon cancer therapy.