Lung Cancer Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

Lung Cancer Signaling Pathway

Fig.1 Lung cancer signaling pathway. Targeted agents (listed in orange boxes) include those in clinical use (colored in green) and those in preclinical or early phase development (colored in red) for the treatment of advanced stage lung cancer.

An Introduction to Lung Cancer

Lung cancer (LC) has become the leading cause of cancer death in many industrialized countries claiming more than 160000 and 50000 lives annually in the United States and Japan, respectively. The majority of lung cancers, 85% of non-small-cell lung cancer and 98% of small cell lung carcinoma, arise in smokers. Carcinogens from tobacco smoke target both the central and peripheral compartments. Among the 20 carcinogens that are present in tobacco smoke and strongly associated with lung cancer development, the best known are polycyclic aromatic hydrocarbons and nicotine-derived nitroso-aminoketone, which lead to genetic mutations through DNA adduct formation. Adduct formation is caused by metabolic activation of these carcinogens by P450 cytochromes, enzymes encoded by the CYP family of genes, and glutathione S-transferases (GSTs). Significant advances in lung cancer biology may lead to customised therapy based on targeting specific genes and pathways. The main signalling pathways that could provide roadmaps for therapy include the following: RTK pathways, integrin-mediated pathways, growth inhibitory pathways. These networks result in, amongst others, proliferation, evasion of apoptosis and angiogenesis. The genetic and epigenetic pathways involved in lung tumorigenesis differ between smokers and nonsmokers, and are tools for cancer diagnosis, prognosis, clinical follow-up and targeted therapies.

1 Main Signaling Pathways in Lung Cancer Therapy

1.1 RTK signaling cascade

Whereas receptor tyrosine kinases （RTKs） activity in normal resting cells is tightly regulated, mutations or deregulated expression might cause them to function as potent oncogenes. Ligands, such as EGF, VEGF, HGF or others, bind to the homo- and heterodimer kinase domain , resulting in activation and receptor transphosphorylation. This creates docking sites for the adaptor proteins, Grb2 and Sos, which recruit Ras and phosphatidylinositol 3-kinase (PI3K), leading to the formation of two major signalling pathway branches, Ras/MAPK and PI3K/Akt and PLC-PKC pathways. The PI3K-AKT signaling phosphorylates and inhibits several apoptosis-inducing genes. NF-kB is convergently activated by several distinct stimuli including growth factors. So the activation of the RTK pathway can thus have noteworthy downstream effects on angiogenesis, evasion of apoptosis, cell growth.

1.2 Integrin-mediated signaling cascade

Integrins (ITGA/ITGB) are cell adhesion receptors that allow a cell to interact with extracellular matrix (ECM) in the local environment. The affinity with which integrins bind to ligands is highly dynamically regulated, between low affinity (suppressed) and high affinity (activated) states. Engagement and cross-linking integrins stimulates multiple intracellular signalling pathways, including those mediated by Ras, phosphoinositide 3-kinase (PI3K). NF-kB is activated by extracellular matrix attachment. These signalling cascades promote cell growth and mitogenesis.

1.3 Growth inhibitory signaling cascade

p53 is a sensor of cell stress, such as DNA damage and oncogenic stimuli, and functions as a transcription factor. Its target genes play roles in the control of the G1 arrest pathway, cyclin-dependent kinases (CDKs) are key regulatory enzymes, each consisting of a catalytic CDK subunit and an activating cyclin subunit. CDKs regulate the cell's progression through the phases of the cell cycle by modulating the activity of key substrates. Downstream targets of CDKs include transcription factor E2F and its regulator Rb. In addition，susceptibility to apoptosis (Bax/Bcl-2), and control of the tumour necrosis factor receptor-like apoptosis inducing ligand (TRAIL) receptor 5 (DR5), Fas. It also enters into a DNA repair protein complex with proliferation cell nuclear antigen (PCNA).

2 Lung cancer diagnosis

2.1 Molecular Markers for Lung Cancer

Molecular diagnostics-guided targeted therapies have become a standard treatment for patients with lung cancer. Here, we grouped molecular biomarkers into three categories-mutations, gene rearrangements, and amplifications.

Mutations including EGFR, KRAS, BRAF, HER2, and MET. Activating somatic mutations including point substitution, small insertion, and in-frame deletion are major oncogenic drivers in lung cancer. The discovery of activating EGFR mutations and their close relation with the response to EGFR TKIs opened the new era of precision medicine. A variety of methods can be used for detecting mutations including direct sequencing, real-time polymerase chain reaction (PCR), and commercial kits. Gene rearrangements including ALK, ROS1, RET, NTRK1, NRG1, and FGFR, The presence of an ALK rearrangement and ROS1 proto-oncogene receptor tyrosine kinase (ROS1) rearrangement in lung cancer has become the best predictor of response to crizotinib. Several methods are currently available for assessing gene rearrangement, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), reverse-transcriptase polymerase chain reaction (RT-PCR), and NGS. Amplifications including FGFR1, EGFR, MET, and HER2, gene amplification is an important mechanism for oncogene activation. HER2-targeting therapy has been implemented in breast and gastric cancer on the basis of molecular testing for HER2. Amplifications of several genes have been studied in lung cancer. Several methods can be used for detection of gene amplifications. FISH using a locus-specific intensifier (LSI) gene and a chromosome-specific centromere (CEP) probe is a standard method for the detection of gene amplifications. Although the FISH assay requires technical expertise and experience for interpretation, this method has the advantage of being able to evaluate gene amplification by selecting only cancer cells.

2.2 Protein Markers for Lung Cancer

Chest radiography and computer tomography are the most commonly used methods for lung cancer diagnosis. However, as they can only identify visible and irreversible changes in lung, there is a need for additional methods for early diagnosis. In order to overcome this challenge, it is essential to discover novel, highly sensitive, and specific biomarker. Therefore, accurate identification of the histological type of cancer and respective protein biomarkers is crucial for adequate therapy. Proteins are the most suitable biomarkers for lung cancer diagnosis because of their involvement in cellular processes. Unfortunately, a uniform serum biomarker composition capable of distinguishing lung cancer types is yet to be discovered. Here, we discuss the recent advances in conventional and prospective aptamer based strategies for biomarker discovery.

Panels of various biomarkers have been recently applied and are becoming more popular as this technique improves early lung cancer detection. We suggest the use of a panel consisting of eight tumor-associated biomarkers-CEA, CYFRA21-1, ProGRP, CEA, PSF3, MUC, SCCA, and SST-allow us to differentiate between each histological type of lung cancer and to define the metastasis rate. In addition to conventional biomarker discovery methods, aptamer-based detection of several lung cancer biomarkers, such as LMN and VIM, DEF, and TUB, could be helpful for accurate diagnostics. An important clinical method often used for diagnosis of tumor biomarkers is immunohistochemistry; usually, hematoxilin and eosin staining is sufficient for diagnosis. Immunostaining is based on histological identification of tumor biomarkers and abnormal blood vessels by specific agents such as antibodies, but this method has some limitations such as: relatively high cost, difficulties in quantifying results. Cancer-related proteins can be detected using various sensors, most of them rely on antibody-antigen interaction in a sandwich-like system that requires two different types of antibodies for target identification.

3 Targeted therapy for lung cancer

The numerous molecular mechanisms implicated in the pathogenesis of lung cancer present exciting avenues for target-specific approaches to therapy. Major components of cell signaling pathways, such as the receptor tyrosine kinases (RTKs), protein kinase C (PKC), and the ras/mitogen-activated protein kinase (MAPK) systems, are altered in lung cancer cells by oncogenes through overexpression or mutation, leading to dysregulated cell signaling and cell proliferation. Here, we summarize potentially viable targets and new agents that have been developed and employed in recent, ongoing and future clinical trials to attempt to improve clinical outcomes in this disease (Table1-11).

3.1 Lung cancer therapy for RTK pathway

RTKs comprise the largest family of dominant oncogenes.Targeting EGFR: Cetuximab and Panitumumab competitively inhibit activation of the EGFRTK, causing cell cycle arrest in G1. ZD1839, CI-1033, and OSI774, as EGFR tyrosine kinase inhibitors, are undergoing various phases of clinical testing. Targeting HER-2: Trastuzumab cause growth arrest in the G0-G1 phase of the cell cycle. Antibodies elicited by the chimeric peptide MVF-HER-2 had antitumor activity in vitro and prevented tumor development in vivo. Targeting VEGFR: Bevacizumab is a mAB targeting the VEGF-A receptor. DC101 is a mAB that specifically binds murine VEGFR-2. Cediranib is highly potent inhibitor of VEGFR-1, -2 and -3 tyrosine kinases. Aflibercept binding to VEGFA and B, thereby preventing binding to their cell receptors. It has demonstrated inhibition of tumor growth and metastasis in vivo. Targeting c-Met: Cabozantinib, a multi-targeted c-Met inhibitor; Capmatinib is a selective c-Met inhibitor. Targeting PKC: many kinase inhibitors that act by blocking the catalytic site are not highly specific and may act as inhibitors of PKC, such as Staurosporine, Bryostatin. Targeting ras/MAPK pathway: ISIS 2503 reduces H-ras mRNA expression through Rnase H-mediated cleavage of the hybridized H-ras mRNA. BAY 43-9006 is a potent oral selective inhibitor of Raf-1 and the first in its class to enter clinical trials. PD-184352 is an active difluorobenzamide that nanomolar inhibition of MEK activation. CCI-779 inhibits mTOR and abrogate the uncontrolled proliferation of malignant cells.

Table 1 Clinical trials of EGFR mAB Cetuximab

Nct id

Status

Lead sponsor

Study first posted

Table 2 Clinical trials of EGFR mAB Panitumumab

Nct id	Status	Lead sponsor	Study first posted
NCT00979212	Active, not recruiting	Radiation Therapy Oncology Group	September 17, 2009
NCT03582124	Recruiting	Eben Rosenthal	July 10, 2018
NCT01061788	Active, not recruiting	Jeffrey Clarke	February 3, 2010
NCT01416688	Active, not recruiting	Southwest Oncology Group	August 15, 2011
NCT01726309	Recruiting	Cancer Trials Ireland	November 14, 2012

Table 3 Clinical trials of EGFR inhibitor ZD1839

Nct id

Status

Lead sponsor

Study first posted

According to statistics, a total of 71 ZD1839 projects targeting lung cancer EGFR are currently in clinical stage, of which 33 are recruiting and 38 are not recruiting.

Table 4 Clinical trials of EGFR inhibitor OSI774

Nct id

Status

Lead sponsor

Study first posted

According to statistics, a total of 104 OSI774 projects targeting lung cancer EGFR are currently in clinical stage, of which 49 are recruiting and 55 are not recruiting.

Table 5 Clinical trials of HER2 mAB Trastuzumab

Nct id

Status

Lead sponsor

Study first posted

Table 6 Clinical trials of VEGFR mAB Bevacizumab

Nct id

Status

Lead sponsor

Study first posted

According to statistics, a total of 58 Bevacizumab projects targeting lung cancer VEGFR are currently in clinical stage, of which 26 are recruiting and 32 are not recruiting.

Table 7 Clinical trials of VEGFR inhibitor Cediranib

Table 8 Clinical trials of c-Met inhibitor Cabozantinib

Nct id

Status

Lead sponsor

Study first posted

Table 9 Clinical trials of c-Met inhibitor Capmatinib

Nct id

Status

Lead sponsor

Study first posted

Table 10 Clinical trials of Raf inhibitor BAY 43-9006

Table 11 Clinical trials of mTOR inhibitor CCI-779

Nct id

Status

Lead sponsor

Study first posted

According to statistics, a total of 14 CCI-779 projects targeting lung cancer VEGFR are currently in clinical stage, of which 7 are recruiting and 7 are not recruiting.

3.2 Lung cancer therapy for integrin-mediated pathway

Matrix metalloproteinases (MMPs) are a family of enzymes responsible for remodeling of the extracellular matrix in processes of growth and morphogenesis for which they have been implicated in metastasis and angiogenesis. Targeting EMC: BMS-275291 is an orally bioavailable, nonpeptidic MMPI that exhibits antiangiogenic effects. Tetracycline inhibit collagenase activity through zincing chelation at the MMP active site, downregulation, inhibition of oxidative activation. Targeting integrins: LM609 monoclonal antibody, directed against integrin,  intervene the angiogenic process.

3.3 Lung cancer therapy for growth inhibitory pathway

The human p53 protein is a tumor suppressor nuclear phosphoprotein, once p53 genes are deleted or mutated, cells become susceptible to DNA damage and dysregulated cell growth. Targeting p53: ONYX-015 is an attenuated, replicationselective adenovirus that replicate in tumor cells lacking functional p53, with consequent tumor cell lysis. Targeting CDK: Flavopiridol exhibits potent antiproliferative and antitumor activity by selectively inhibiting the activity of cdk1, cdk2, cdk4, and cdk7 with subsequent cell cycle arrest at the G1/S and G2/M boundaries as well as downregulation of cyclin D1.