Team:SDU CHINA/description





Lung cancer is a leading cause of cancer-related death worldwide, and non-small cell lung cancer (NSCLC) accounts for about 80–85% of all patients with lung cancer [1-2]. In clinical treatment of NSCLC, chemotherapy and chemoradiotherapy have been used as the first-line therapies but suffer from lack of efficacy and also of several toxic adverse effects. Gradually,immunotherapy has moved into people’s field of vision, the success of immune checkpoint blockade therapy in NSCLC has recently gained widespread recognition [3-4]. Immune checkpoint blockade therapy by targeting the programmed death protein 1(PD-1)/programmed death ligand 1 (PD-L1) axis using antibodies has yielded promising clinical response in patients with NSCLC [5-6]. However, owing to the dynamic expression of PD-L1, degree of mutational/neoantigen load, intratumoral heterogeneity, and infiltrated immune cells of tumor microenvironment of NSCLC, the response rates to these agents are limited, despite several companies diagnostic assays by detecting PD-L1 in tumor cells have been introduced into clinical practice [1]. Therefore, more precise and effective therapy for NSCLC is expected.

Figure1: Incidence of all kinds of cancer, which shows that lung cancer is the biggest one.




overview of non-small cell lung cancer(NSCLC)


Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Histologically, NSCLC is divided into adenocarcinoma, squamous cell carcinoma (SCC) (see the image below), and large cell carcinoma. As a chronic and inflammatory promoting disease, tumor has the most complex mechanism and progression. Although chemotherapy and radiotherapy have developed quickly, as well as the molecular targeted drugs, the prognosis of patients with NSCLC is still not optimistic, which means the five years survival rate is almost 15% or below. We must develop new treatments to conquer the NSCLC.


Figure2: Micrograph of a squamous carcinoma, a type of non-small-cell lung carcinoma (NSCLC).



overview of PD-1/PD-L1


PD‑1, also known as CD279 and a member of the CD28 family, is a co‑inhibitory receptor that plays a crucial role in immune escape during tumor progression [7]. PD-1 is composed of an extracellular immunoglobulin-like binding domain, a transmembrane region and a cytoplasmic domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) [12]. These motifs are implicated in its immunosuppressive effects. The PD‑1 ligands such as PD‑L1 (CD274) and PD‑L2 (CD273) are B7 family members and are known to be overexpressed on the surface of tumor cells where they block cytotoxic T cells [7–9]. PD-L1 is widely expressed in a variety of immune cells, epithelial cells and tumor cells. At present, many studies have shown that a large number of human tumor cells express PD-L1 molecules, and the clinical pathological characteristics and prognosis are closely related to PD-L1 expression, which has being a new biological indicator for tumor detection and prognosis. Tumor cells bind to PD-1 on the surface of T cells and transmit negative regulatory signals through high expression of PD-L1 molecules, leading to tumor-specific T cell-induced apoptosis and immune incapacity [7–11]. Interfering with PD-L1 and PD-1 signal transduction either by antibody blockade or any other means enhances T cell functions by potentiating signal transduction from the TCR signalosome.


Figure 3:PD1-dependent inhibitory mechanisms.



(A)Direct inhibitory mechanisms over the TCR signalosome are shown. The figure represents PD1-dependent proximal inhibitory mechanisms, which depend on the recruitment of SHP1 and SHP2 phosphatases as shown. These phosphatases inhibit ZAP70 and PI3K activities (blue arrows). Downstream intracellular pathways are also terminated, as exemplified in the figure with ERK and PKCθ. (B) Indirect inhibitory mechanisms over TCR signaling and T cell proliferation are shown through regulation of CK2 expression and activities. On the left, the PI3K-dependent signaling pathway activating CDK2 and inhibiting SMAD3 is shown. Briefly, PIP3 activates AKT leading to production of the ubiquitin ligase SCF that degrades the CDK2 inhibitor Kip1. Activated CDK2 triggers cell cycle progression and inactivates SMAD3 by phosphorylation. These pathways are negatively regulated by the PTEN phosphatase that degrades PIP3 . During TCR activation CK2 phosphorylates PTEN with a concomitant decrease in its activities. When PD1 is engaged CK2 expression and activities decrease resulting in active PTEN that eliminates PIP3 shutting off AKT activation. (C) Regulation of TCR surface expression by PD1. PD1 engagement promotes expression of E3 ubiquitin ligases of the CBL family, as shown. As indicated, other ligases may be up-regulated as well. These ubiquitin ligases ubiquitylate TCR chains and PI3K, leading to the removal of TCRs from the T cell surface, possibly by endocytosis. Thus, T cells cannot respond to antigenic stimulation. (D) Metabolic control by PD1. Engaged PD1 alters T cell metabolism from glycolysis to β-oxidation by inhibition of ERK and PI3K-AKT activities. PD1- stimulated T cells would then metabolically resemble long-lived memory T cells.( www.impactjournals.com/oncotarget/)





Our project

Although the antibodies of PD-1/PD-L1 have been applied into the research of NSCLC which showed promising, antibody immunotherapy did not increase the five years survival rate. This is possible due the unchanged genetic materials. In our project, gene edition is used as a new way of immunotherapy to eradicate the immune escape occurred in NSCLC. By constructing plasmid with Crispr-Cas 9 system targeting the gene coding for PD-1 ligand(PD-L1), PD-L1 expression in situ will be expected to be inhibited, and therefore the immune system supervision on tumor cells is hoped to be restored. To ensure the safety of our design and prevent gene pollution, another plasmid with Crsipr-Cas 9 system to cut off the housekeeping gene of two plasmids will be also constructed. The promoter does not start the Cas 9 expression in nontumorous cells, in which two plasmids would commit suicide to ensure biosafety. Longer overall survival of NSCLC, and more efficient, affordable way to conquer the NSCLC is expected to be achieved by our project.


Figure 4:Mechanisms of CRISPR/Cas9 Genome Editing..



REFERENCES:

[1]Liu X, Cho WC. Precision medicine in immune checkpoint blockade therapy for non-small cell lung cancer. Clinical and Translational Medicine. 2017;6:7. doi:10.1186/s40169-017-0136-7.

[2]Ferlay J, Shin H-R, Bray F, Forman D, Mathers C, Parkin DM.Estimates of worldwide burden of cancer in 2008: GLOBOCAN2008. Int J Cancer 2010;127:2893e2917.

[3]Liu X, Cho WC. Precision medicine in immune checkpoint blockade therapy for non-small cell lung cancer. Clinical and Translational Medicine. 2017;6:7. doi:10.1186/s40169-017-0136-7.

[4]Yang J, Chen J, Wei J, Liu X, Cho WC (2016) Immune checkpoint blockade as a potential therapeutic target in non-small cell lung cancer. Expert Opin Biol Ther 16(10):1209–1223.

[5]Rebelatto MC, Midha A, Mistry A, et al. Development of a programmed cell death ligand-1 immunohistochemical assay validated for analysis of non-small cell lung cancer and head and neck squamous cell carcinoma. Diagnostic Pathology. 2016;11:95. doi:10.1186/s13000- 016- 0545-8.

[6]Inoue Y, Yoshimura K, Mori K, et al. Clinical significance of PD-L1 and PD-L2 copy number gains in non-small-cell lung cancer. Oncotarget. 2016;7(22):32113-32128. doi:10.18632/ oncotarget. 8528.

[7]Dong H, Strome SE, Salomao DR, Tamura H, Hirano F,Flies DB, Roche PC, Lu J, Zhu G, Tamada K, Lennon VA,Celis E, Chen L. Tumor‑associated B7‑H1 promotes T‑cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002; 8:793–800.

[8]Zou W Chen L. Inhibitory B7‑family molecules in the tumour microenvironment. Nat Rev Immunol. 2008;8:467–477.

[9]Rosenwald A, Wright G, Leroy K, Yu X, Gaulard P, Gascoyne RD, Chan WC, Zhao T, Haioun C, Greiner TC, Weisenburger DD, Lynch JC, Vose J, et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med.2003; 198:851–862.

[10]Chinai JM, Janakiram M, Chen F, Chen W, Kaplan M,Zang X. New immunotherapies targeting the PD‑1 pathway. Trends Pharmacol Sci. 2015; 36:587–595.

[11]Inoue Y, Yoshimura K, Mori K, et al. Clinical significance of PD-L1 and PD-L2 copy number gains in non-small-cell lung cancer. Oncotarget. 2016;7(22):32113-32128. doi:10.18632/ oncotarget.8528.

[12]Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol. 2007; 8:239–45.