雖然PDL1在腫瘤免疫逃避中扮演著重要的角色，但PDL1在肺腺癌癌化過程中的角色仍然不清楚。我們的研究中發現一部份的肺腺癌會高度表達PDL1，且此類癌細胞生長會受到PDL1抑制。當抑制PDL1時，則會促進細胞週期進行和增進其抵抗失巢凋亡的能力。DNA微陣列分析指出在肺腺癌中，TGFBI為PDL1訊息的下游標的。
此外，PDL1和TGFBI的表現在肺腺癌中有密切的關聯性，且皆能預測較高的存活率。抑制TGFBI能降低細胞非貼附性生長能力，顯示PDL1-TGFBI訊息路徑可對抗癌化過程。我們發現PDL1和TGFBI會透過SMAD2的路徑被TGF-β所調控。同時，抑制PDL1和TGFBI會促進CITED2表現，然而TGF-β刺激會降低CITED2表現。抑制CITED2表現會導致G1/S週期停滯和阻擋細胞非貼附性生長。綜合上述結果，在肺腺癌中PDL1-TGFBI-CITED2訊息路徑可當成預測癌症惡化的指標。

Although the expression of PDL1 in tumor has been known to play a critical role in immune escape, the role of PDL1 in the oncogenesis of lung adenocarcinoma is not clear. Here, we report that PDL1 was highly expressed in a subset of lung adenocarcinoma, the proliferation of which was suppressed by PDL1 expression. We found that knockdown of PDL1 promoted cell cycle progression and anoikis resistance. Gene expression profiling analysis revealed that TGFBI was a downstream target regulated by PDL1 signaling in lung cancer cells. Correlation analysis showed that PDL1 in lung adenocarcinoma was associated with the expression of TGFBI, which predicted a good survival outcome in patients. TGFBI-silencing attenuated anchorage-independent growth in lung cancer, suggesting an anti-oncogenic role of PDL1-TGFBI signaling. We found that both PDL1 and TGFBI were induced by TGF-β through a SMAD2 dependent pathway. Q-PCR analysis revealed that CITED2 was enhanced by PDL1- or TGFBI- silencing while TGF-β stimulation downregulated CITED2 in lung cancer cells. Knockdown of CITED2 caused G1/S cell cycle arrest and inhibited anchorage-independent cell growth. Thus, our findings reveal a new role of PDL1-TGFBI-CITED2 signaling in lung adenocarcinoma with the potential to serve as predictive biomarkers in cancer progression.

摘要 1
Abstract 2
致謝 3
目錄 4
Introduction 6
Lung cancer 6
Anoikis 6
Immunotherapy and immune suppression 7
Clinical applications of PD1/PDL1 blockade 8
Transforming growth factor-beta signaling pathway 8
The CITED2 involved TGF-β regulated cell quiescence 9
The candidate biomarker in PD1/PDL1 axis 10
Materials and Methods 12
Cell culture 12
Lentiviral infection 12
Quantitative Real-time PCR 13
Protein extraction and western blotting 13
Soft agar assay 14
Clonogenic assay 14
Cell-Cycle Analysis 15
TCGA database statistics 15
Tomida database statistics 15
Statistical analysis 16
Table 1: Q-PCR primers 17
Table 2: lentiviral shRNA sequence 18
Results 19
Correlation of PDL1 with better survival outcomes in patients with lung adenocarcinoma. 19
PDL1 regulates cell growth and anoikis in lung cancer cells. 19
TGFBI, regulated by PDL1, inhibits cell growth in lung cancer cells. 20
PDL1-TGFBI axis is regulated by TGF-β through TGFBR-SMAD2 signaling in lung adenocarcinoma. 21
CITED2, downregulated by TGF-β-mediated PDL1-TGFBI signaling axis, is required for cell growth and anoikis resistance in lung cancer cells. 22
Discussion 24
TGF-β-mediated upregulation of PDL1 24
TGFBI mediates PDL1-induced growth inhibitory effect 25
CITED2 functions as a molecular switch of PDL1-TGFBI mediated growth control 26
Figures 27
Supplemental Figures 38
References 42

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