Identification of a new immune escape related gene in pancreatic cancer

Abstract

[한글]췌장암은 면역학적으로 가장 면역원성이 낮은 암 종 중의 하나이다. 현재 환자의 면역체계를 활성화하여 면역치료법을 통해 췌장암을 치료하려는 시도가 활발히 연구되고 있으며, 아울러 췌장암의 연구에서 확보할 수 있는 면역 억제 현상과 관련된 인자들이 규명이 될 수 있다면 향후 종양면역학에 커다란 기여를 할 수 있음과 아울러 새로운 치료법 개발에 커다란 도움이 될 수 있을 것이다. 종양환자들에 있어 면역세포들의 양적, 질적인 억제현상이 유도되어있다는 사실은 이미 밝혀져 있으나, 아직도 구체적인 mechanism의 규명과 함께 관여하는 인자들의 동정은 충분하지 못한 상황이다.본 연구는 췌장암에 있어서 면역 회피를 위해 췌장암 세포들이 이용하는 체액성 인자를 (1) 유전체 정보로부터 동정하고, (2) 동정된 인자들의 존재와 함량을 환자의 체액과 조직 등에서 확인 (validation)하며, (3) 체외 배양 조건에서 선발된 인자들이 기본적인 면역세포의 기능에 억제효과를 발휘하는지를 확인하여 종양에 의한 면역억제의 원인들을 규명하는 것을 목적으로 한다. 이러한 정보는 궁극적으로 췌장암에 있어 새로운 치료법을 제시하는 동시에, 여러 종양과 면역 억제의 mechanism을 규명하여 질병 치료에 응용할 수 있게 한다.인체 암 조직으로부터 얻어진 mRNA를 Affymetrix사의 U133 human chip을 이용하여 microarray 실험을 수행하였다. 그 결과 Microarray chip data의 유전자들 중, 정상인보다 췌장암 환자에서 'FC (fold change) >2, p-value<0.05' 조건의 유전자들을 선택하고, 그들 중 GO (gene othology) term에 따른 gene grouping을 거쳐 secretory protein, growth factor, receptor, ligand, extra-cellular matrix와 관련된 유전자들을 선정하여, 췌장암 환자에서 발현이 증가한 유전자들을 선정하였다. 이들 중 췌장암, immune system에 관한 연구 보고가 없는 유전자들만 1차 선발하여, 최종 4개 이내의 유전자를 선정하였다.최종 선택된 gene들이 췌장암 세포주와 환자 혈청에서의 발현여부를 확인하기 위해, 각 유전자를 췌장암 세포주(Mia PaCa-2, CFPAC-1, PANC-1, AsPC-1, HPAC, Capan-1, Capan-2, BxPC-3)에 적용하여, RT-PCR을 수행한 결과, 대부분의 췌장암 세포주에서 발현함을 확인하였다.선택된 NMU, TFF2 유전자들이 갖는 면역기능을 조사하기 위해, 이 재조합 단백질들을 처리하여 실험관에서 대표적인 immune cell인 강력한 전문항원제시세포인 수지상세포 (dendritic cells) 에 미치는 영향을 분석하였다. 그 결과, 수지상세포의 migration 유도됨을 보였다. 따라서 본 연구는 면역관련 유전자 (NMU, TFF2) 들이 갖는 면역기능을 이용하여 이들이 췌장암의 면역 억제 유전자로써 역할을 할 수 있는지 검증할 수 있었다. 또한 상기의 인자들 이외에도 추가적인 면역억제 능력을 갖는 인자들의 동정은 향후의 면역치료법이나 환자의 면역결핍 증상을 완화시키는데 정보를 제공할 것이다.

[영문]In pancreatic cancer, soluble factors produced by and for the protection of the tumor environment have been detected and are often distributed to the patient’s circulatory system where they may cause a more generalized immunosuppression. Tumors exploit several strategies to evade immune recognition, including the production of a large number of immunosuppressive factors, which lead to reduced numbers and impaired functions of dendritic cells (DCs) in the vicinity of tumors.In this study, several immune related genes were used to obtain gene expression profiles of pancreatic cancer for immune escape, and validation of gene expression in pancreatic cancer cell lines and patient sera. The inhibitory function of immune cells in vitro was then confirmed and the selection genes controlling susceptibility to induced immunosuppression were identified.We used Affymetrix GeneChip arrays to identify genes differentially expressed in pancreatic cancer. Samples were hybridized to the complete Affymetrix U133A oligonucleotide microarray for simultaneous analysis of 45,000 fragments corresponding to 33,000 known genes and 6,000 ESTs. Genes with a 2-fold greater increase in expression in the 8 pancreatic adenocarcinoma tissues compared to 17 normal tissues were identified. We primarily selected fragments that were expressed at least 2-fold more in pancreatic cancer samples compared to normal tissues, of which 4 corresponded to known gene fragments ranged from less than p value <0.05 (modified Welch t test). Next, we selected genes related to secretory proteins, growth factors, receptor, ligands, and extra-matrix proteins. For each of the 225 genes identified, a search was performed by means of the online NCBI database PubMed using search parameters of the gene name together with the terms “pancreas cancer” or “immune.” Of the 225 genes analyzed, most of the genes and gene products were reported to be associated with pancreatic cancers. Many of them were typically associated with the general phenomenon of inflammation and pancreatitis. A few assessed good geness were primarily reported to be associated with immune suppression. Finally, four genes that were not reported to be associated with either pancreatic cancer or the immune system were selected for the further study. Candidate genes were selected for verification of expression in samples of pancreatic cancer cell lines. These four genes were expressed in approximately 50% of pancreatic cancer cells, in support of their initial identification as differentially expressed genes by Affymetrix GeneChip.In addition, this study has provided evidence that the selected genes are involved in immune function. We used recombinant protein to study these genes in DC. We demonstrated that Neuromedin U (NMU) and Trefoil Factor 2 (TFF2) interfere with the differentiation of monocytes into DC in vitro, resulting in the down-regulation of CD40, CD80, and HLA-DR. These results suggested that NMU and TFF2 had little effect on the expression of cell surface molecules in antigen presenting cells (APC). Moreover, we found that DC treated with NMU and IL-10 had a higher capacity than control DC for uptake of FITC-dextran. These results suggested that NMU-treated DC generated in the presence of the absence of IL-10 had a similar degree of endocytic activity. In addition, we found that in our cultures, migration of immature DC was induced by both NMU and TFF2.In conclusion, we provide evidence here that NMU and TFF2 modulate DC function. The identification of signal-transduction events that participate in the modulation of DC function via NMU and TFF2 would further contribute to the elucidation of the mechanisms underlying the complex anti-inflammatory effects of NMU, and TFF2 and would enable the construction of a theoretical framework for its eventual therapeutic use. The results described here suggest that the presence of NMU and TFF2 from the onset of the tumor can promote progressive induction of immune silencing in synergy with other described immunosuppressive factors.