探討攜有pks island大腸桿菌感染的大腸癌細胞其細胞適應性的腫瘤突變負荷量

Abstract

腫瘤突變負荷量(Tumor mutation burden, TMB)代表腫瘤細胞的DNA中帶有的突變數，這些突變造成新生抗原(Neoantigen)的產生，在評估癌症的治療預後及研究上都扮演重要的角色。大腸直腸癌是世界上最盛行的癌症之一，近年來有許多研究指出腸道內的菌相組成(Gut microbiota)對於大腸直腸癌的發生和發展有其重要性。大腸桿菌(Escherichia coli, E. coli)是人體腸內菌的一員，其中某些菌株帶有一大段稱為pks island的基因，已知具有此基因的大腸桿菌(pks+ E. coli)感染真核細胞會造成細胞DNA斷裂，影響細胞週期並進一步造成基因不穩定(Genomic instability)及癌化(Tumorigenesis)的現象。在針對腸道發炎或易發生大腸癌的動物實驗中發現感染pks+ E. coli會誘發腫瘤的產生。臨床研究也顯示在慢性腸道發炎疾病患者的腸道菌相中有較高比例的pks+ E. coli組成，而在大腸直腸癌中pks+ E. coli比例則較健康人高出約三倍。大腸直腸癌的發生是由一連串基因突變造成的基因不穩定現象而導致，其中約有15%的大腸直腸癌帶有錯配修復(Mismatch repair)的缺失，其導致的微衛星不穩定(Microsatellite instability)現象易導致癌細胞基因的二次突變，若因此造成致癌基因(Oncogene)或抑癌基因(Tumor suppressor gene)的表現量改變則將使情況更為棘手。本篇欲研究細胞的錯配修復能力對於pks+ E. coli感染後所造成的DNA損傷及修復，以至於其引發的癌化現象上是否有所差異，藉以探討錯配修復在pks+ E. coli作用中所扮演的角色。我們利用回補染色體的大腸直腸癌細胞HCT116作為主要實驗材料：錯配修復能力缺失的HCT116 + chr. 2細胞及錯配修復能力正常的HCT116 + chr. 3細胞。透過單細胞電泳彗星實驗(Comet assay)觀察到在受pks+ E. coli感染後，相較於HCT116 + chr. 3細胞，HCT116 + chr. 2細胞中有較多且較嚴重的DNA損傷，以西方墨點法(Western blotting)和免疫螢光染色法(Immunofluorescence assay)偵測代表DNA雙股斷裂的H2AX蛋白也可見相同趨勢。我們也發現在感染pks+ E. coli後，HCT116 + chr. 2細胞的存活能力會下降約三成，其發展出腫瘤生成特性的能力也較明顯。此外，我們透過RNA干擾技術(RNA interference)抑制HCT116 + chr. 3細胞的MLH1蛋白表現，結果顯示在MLH1受抑制後，經pks+ E. coli感染會產生更嚴重的DNA損傷，亦使得細胞存活能力下降及腫瘤生成特性增加。綜上所述，我們認為細胞的錯配修復能力在pks+ E. coli的作用中有所參與並值得更進一步深入探討。

Tumor mutation burden (TMB) affects cancer initiation, progression, and development significantly. It also affects the generation of neoantigens which will dictate therapeutic prognosis and therapy resistance. Colorectal cancer (CRC) is one of the most prevalent cancer types in the world. Various risk factors are considered to be responsible for this high incidence, for example, recent studies have indicated that dysfunction in the microbiota-host interaction might play a critical role in CRC progression. Among microbiota, Escherichia coli (E. coli) has caught our attention. There are four subgroups of E. coli, namely A, B1, B2, and D. Subgroup A and B1 are the two major composing of normal flora in colon while the toxic strains mostly belong to subgroup B2 those harboring a high ratio of a 54 kb genomic pks island and correlated with a higher tumorigenic propensity. CRC genomic instability can be classified into microsatellite instability (MIN) which mostly results from defects in DNA mismatch repair (MMR) and chromosomal instability (CIN) which is due to chromosome breaks. Both forms of genomic instability are enabling factors for tumorigenesis and increase mutation burden, thus giving rise to cancer development. Interestingly, the pks island encodes a polyketide-peptide genotoxin named colibactin that has been proven to induce DNA break and lead to CIN. Among all the factors of MIN and CIN, we are particularly interested in how MMR and pks island would mediate the progression of tumorigenesis. Toward this, we employed paired colon cancer cell lines, including colorectal cancer cell HCT116 with extra chromosome 2 or 3 (HCT116 + chr. 2 without MLH1 expression, MMR-; HCT116 + chr. 3 with MLH1 expression, MMR+). We performed comet assay to identify the DNA damage induced by pks+ E. coli. Initial DNA break showed no significant difference between cells infected with pks+ and pks- E. coli for 4 h. Furthermore, after recovering for 12, 20, and 40 h, MMR+ HCT116 + chr. 3 showed less DNA damage than MMR- HCT116 + chr. 2. The expression of DNA double-stranded break marker H2AX protein was confirmed in accordance with the comet assay by western blotting and immunofluorescence assay. Clonogenic assay and anchorage-independent soft agar assay were used to evaluate the cell viability and tumor-promoting propensity respectively. Cell viability decreased in MMR- HCT116 + chr. 2 and there were more anchorage-independent colonies formed after infected with pks+ E. coli. We further knockdown the expression of MLH1 protein in MMR+ HCT116 + chr. 3 and observed increased DNA damage, decreased cell viability, and increased anchorage-independent growth ability. Taken together, MMR proficiency of cells might interact with the presence of pks island to regulate tumorigenesis and worth further investigation.