Streptococcus thermophilus inhibits colorectal tumorigenesis through secreting β-galactosidase

Ph.D.Background & Aims: A lot of colorectal cancer (CRC)-enriched bacteria have been shown to promote intestinal tumorigenesis. However, the possible tumor-suppressive effect of under-represented probiotics is still elusive. Our shotgun metagenomics-based meta-analysis of fecal samples revealed that Streptococcus thermophilus, was depleted in patients with CRC. Here, we aim to investigate if this bacterium could act as a prophylactic for CRC prevention and its possible mechanisms involved in this tumor-suppressive process.Methods: Stool samples were collected from 78 CRC patients and 78 normal subjects and the abundance of S. thermophilus in those stool samples were assessed by quantitative PCR. The anti-tumor effects of S. thermophilus were assessed in cultured colonic epithelial cells and in two murine models of intestinal tumorigenesis. The tumor-suppressive protein produced by S. thermophilus was identified by mass spectrometry. The mutant strain of S. thermophilus was constructed by homologous recombination. The contribution of S. thermophilus on gut microbiota compositation was assessed by shotgun metagenomic sequences. The associated molecular mechanism was studied by RNA-sequencing. The activation of oxidative phosphorylation was validated by the Seahorse experiment.Results: S. thermophilus was significantly depleted in stool samples of CRC patients as compared with healthy individuals. Oral gavage of S. thermophilus significantly reduced tumor number and tumor load in both Apcᵐⁱⁿ/⁺ and azoxymethane (a colon carcinogen)-injected mouse. Co-incubation with S. thermophilus or its conditioned medium decreased the viability of cancerous but not normal colonic epithelial cells. β-galactosidase secreted by S. thermophilus inhibited the proliferation, lowered the colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft. The mutant S. thermophilus without functional β-galactosidase lost its tumor-suppressive effect. In addition, S. thermophilus modulated the gut microbiota by enriching the abundance of Bifidobacterium and Lactobacillus via β-galactosidase. β-galactosidase-dependent production of galactose partially mediated the anticancer effects of S. thermophilus through regulating the Hippo signaling and reversing the Warburg effect.Conclusion: S. thermophilus is a novel prophylactic for CRC prevention in mice. The tumor-suppressive effect of S. thermophilus is mediated by the secretion of β-galactosidase. This probiotic might be used as a prophylactic for preventing CRC in humans with minimal side effects.研究背景及目的：許多在大腸癌中高豐度表達的細菌已證實與大腸癌的發生發展密切相關，然而有關大腸癌中低豐度益生菌的抗癌作用尚無報導。通過對大腸癌患者糞便的全基因組檢測，我們發現相較于正常人，嗜熱鏈鏈球菌 S.thermophilus 的豐度在大腸癌患者中明顯減低。本研究旨在探索這種大腸癌低豐度的益生菌是否可以用作大腸癌的預防藥用於腸癌的預防，以及其潛在的抗腫瘤機制。方法：從 78 例 CRC 患者和 78 例正常人中收集糞便樣本，並通過 q-PCR 評估糞便樣本中嗜熱鏈球菌的豐度。通過結腸上皮細胞與嗜熱鏈球菌共培養並同時將嗜熱鏈球菌灌胃給兩種 腸癌模型小鼠中評估嗜熱鏈球菌的抗腫瘤作用。通過質譜鑒定嗜熱鏈球菌產生的腫瘤抑制蛋白。並通過同源重組構建嗜熱鏈球菌的突變株。通過 RNA 測序探索嗜熱鏈球菌抑癌的相關分子機制。通過 線粒體壓力 實驗證實嗜熱鏈球菌導致的氧化磷酸化啟動。結果：與健康個體相比，大腸癌患者糞便樣品中嗜熱鏈球菌豐度顯著減少。嗜熱鏈球菌的灌胃處理顯著減少 Apc ᵐⁱⁿ/⁺ 和乙氧基甲烷 （結腸致癌物）的小鼠 的腫瘤數量和腫瘤負荷。與嗜熱鏈球菌的共培養顯著降低了癌性結腸上皮細胞的增殖能力，但對正常結腸上皮細胞無顯著影響。嗜熱鏈球菌分泌的β-半乳糖苷酶 可顯著抑制結腸上皮癌細胞的增殖，降低克隆形成，誘導細胞週期停滯，並癌細胞凋亡，同時阻止裸鼠移植 瘤的生長。無法產生功能性β-半乳糖苷 酶 的突變嗜熱鏈球菌失去了其腫瘤抑制作用。 此外 β-半乳糖苷酶的產生可顯著增加雙歧桿菌和乳酸菌的豐度。β-半乳糖苷酶 依賴性半乳糖的產生可通過調節 Hippo信號通路和逆轉 Warburg 效應來部分介導嗜熱鏈球菌的抗癌作用。結論：嗜熱鏈球菌是一種可用於預防小鼠大腸癌的新手段。嗜熱鏈球菌的腫瘤抑制作用是由其分泌的β-半乳糖苷酶所介導的。這種益生菌可以以很小的副作用用於大腸癌的預防。Li, Qing.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 115-123).Abstracts also in Chinese.Title from PDF title page (viewed on February 8, 2022 )