Strong Down-Regulation of Tumor Suppressor Genes RB1 and CTDSPL is Associated with Aberrant Expression of Cell Cycle Regulation Genes in Non-Small Cell Lung Cancer

Abstract

The search of specific molecular markers for differential diagnostics and new targets for combined targeted therapy or gene therapy is one of the key goals of molecular oncology. The first step in this direction is a comparative expression analysis of genes involved in a complex network of interactions of cell cycle control molecules in tumors. In the present study, advanced quantitative expression analysis of 84 genes (Human Cell Cycle Regulation Panel, Roche) revealed dysfunctions of p16^INK4A–Cdk/cyclin D1–Rb and p53/ p21^Waf1 pathways associated with strong down-regulation of two tumorsuppressor genes, RB1 and CTDSPL, in non-small cell lung cancer (NSCLC). Rb protein, a key regulator of cell cycle, can be activated by small CTD-serine phosphatase CTDSPL/RBSP3. The analysis revealed that over-expression of many genes from the panel was stronger in lung adenocarcinoma (ADC) than squamous cell carcinoma (SCC) and more pronounced in metastatic tumors. A number of genes showed expression alterations which were specific to NSCLC histological type or metastases presence. These genes could be potential NSCLC biomarkers. Twenty five genes (survivin, cyclins, Ser/Thrprotein kinases, transcription factors, phosphatases, etc.) with the strongest expression gains (up to 100-fold) could be potential targets for future biotherapy approach of NSCLC (both ADC and SCC). Our data concerning incremental deregulation of RB1, CTDSPL and other cell cycle control genes are in flow with the continuum model for tumor suppression.

Keywords: Non-small cell lung cancer; Gene expression; Tumor suppressor gene; Cell cycle control gene; Biomarker; Target for biotherapy

Introduction

Multidisciplinary approach for a non-small cell lung cancer (NSCLC) diagnostics allows to choose the most adequate treatment strategy [1]. Particular relevance acquired differential diagnosis of lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) that requires specific markers for each histological type [2]. In case of ADC, targeted therapy has a great success and is one of the perspective treatment directions [3]. However, targeted therapy, aimed at individual targets, is often accompanied by the rapid development of drug resistance [3]. A new generation of anti-cancer drugs for combined targeted therapy and gene therapy involves the search for not only individual, but a number of targets at once for simultaneous blocking or restoring of their activity. In this regard, a concurrent expression analysis of many genes in a complex network of interactions of cell cycle control elements is currently relevant.

Deregulation of cell cycle control is recognized as the hallmark of carcinogenesis, including NSCLC. Numerous works were focused on aberrations of gene expression caused by genetic and epigenetic mechanisms for one or more of the key elements of cell cycle regulation in primary lung tumors [4,5]. However, only a few studies compared multiple expression markers among cell control elements and their value for a clinical use simultaneously.

The Rb protein encoded by RB1 gene (retinoblastoma 1, RB, pRb; 13q14.2) is a key component of the p16ink4A –Cdk/cyclin–Rb pathway responsible for cell division arrest at the G1/S checkpoint [6]. The lack of Rb activity correlated with lower survival of patients with various types of lung cancer [7,8]. The CTDSPL gene (also known as RBSP3; 3р21.3) belongs to the gene family of small CTD (carboxyterminal domain) serine phosphatases that preferentially catalyzes serine-5 dephosphorylation in the consensus repeat sequence of the RNA polymerase II (Pol II) largest subunit and other proteins. In case of Pol II, this modification leads to the negative regulation of transcriptional activity [9-11]. Another function of CTDSPL is believed to be an activation of the phosphorylated precursor of Rb by dephosphorylation of serine 807/811 [11].

In the present study, advanced quantitative expression analysis revealed strong down-regulation of two tumor suppressor genes (TSGs), RB1 and CTDSPL, associated with aberrant expression of 84 genes (Human Cell Cycle Regulation Panel, Roche) from p16^INK4A– Cdk/cyclin D1–Rb and p53/p21^Waf1 pathways in non-small cell lung cancer (ADC and SCC). Obtained data allowed to suggest novel potential differential diagnostics markers and targets for future biotherapy approach of NSCLC.

Materials and Methods

Tissue samples

Primary NSCLC (ADC and SCC) and the adjacent morphologically normal tissues were obtained from patients after surgical resection of tumors prior radiation or chemotherapy and characterized according to the International TNM Classification system [12] at the N.N.

Blokhin Russian Cancer Research Center (Moscow, Russia). The clinical diagnosis was confirmed by pathomorphological examination at the Department of Tumor Pathologic Anatomy, Research Institute for Clinical Oncology (Moscow, Russia). Approval of the use of all human tissues was obtained from the Ethics Committee of N.N. Blokhin Russian Cancer Research Center. The patients gave written informed consent that is available upon request. The study was done in accordance with the principles outlined in the Declaration of Helsinki.

RNA Isolation and Reverse transcription

RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The quantity and quality of the RNA was determined using NanoDrop ND-1000 (NanoDrop Technologies Inc., USA). The reverse transcription reaction was carried out using M-MuLV Reverse Transcriptase (Fermentas, Lithuania) according to the manufacturer’s instructions.

Quantitative PCR

The preliminary expression analysis of two TSGs, CTDSPL and RB1, was done in 14 ADC and 16 SCC samples. QPCR was performed with commercial primers and TaqMan probes using a 7500 Real- Time PCR System (Applied Biosystems, USA). Then simultaneous expression profiling of 84 genes involved in a cell cycle regulation was performed in ADC and SCC samples with the use of a LightCycler 480 PCR System (Roche, Switzerland) and “Human Cell Cycle Regulation Panel” (Roche) according to the manufacturer’s protocol. These quantitative data were analyzed using three reference genes GAPDH, HPRT1 and YWHAZ and the relative quantification or ΔΔCt-method. All calculations were performed using our program ATG (“Analysis of Transcription of Genes”) [13,14]. At least 2-fold mRNA level changes were considered as significant because of mRNA level variability of the reference genes.

Results and Discussion

In the present study we compared expression profiles of two tumor suppressor genes, RB1 and CTDSPL, in the same primary lung tumors (NSCLC: ADC and SCC) to find mutually interdependent changes and their association with tumor characteristics. At the beginning, we revealed noticeable mRNA level decrease of both RB1 and CTDSPL in the majority of NSCLC samples (data not shown). The down-regulation was more pronounced in tumors with metastasis to the regional lymph nodes compared to metastasis-free tumors. We chose four samples for further comparison: moderately differentiated (T₁N₀M₀) and well differentiated (T₂N₃M₀) ADC (Figure 1A and 1B respectively); moderately differentiated (T₂N₀M₀) and poorly differentiated (T₂N₂M₀) SCC (Figure 1C and 1D respectively). These two pairs of ADC and SCC samples were characterized by the strongest decrease of the mRNA level in samples with metastases compared to those without metastasis: for CTDSPL – 3.4-fold vs. 2.1 (ADC) and 94-fold vs. 7.5 (SCC); for RB1 – 16-fold vs. 2.1 (ADC) and 19-fold vs. 2.7 (SCC). Then expression profiling was performed to reveal the association between the down-regulation of RB1 and CTDSPL and aberrations of 84 genes encoding cell cycle control elements of two basic signaling pathways, Rb (Rb, cyclin D1, p16INK4A) and p53/p21^Waf1, in the same pairs of ADC and SCC samples. In general, detected expression profiles abnormalities of examined cell cycle regulation genes (Figure 2) were reflected in different degrees of cell differentiation and severity of the cell nuclei atypia (Figure 1) in two major histological types of NSCLC.

The comparison of expression data revealed the following important features (Figure 2, Table 1):

  

    
Gene 
      
symbol 
    
Expression ratio, n-fold 
    
Gene name 
  
  

    
ADC
      
III/I 
    
SCC
      
III/I 
    
ADC/
      
SCC
  
  

    
Negative apoptosis regulators 
  
  

    
GTSE1 
    
8 ↑ 
    

    
 
    
G2 and S phase-expressed protein 1 (B99    homolog)
  
  

    
BIRC5 
    
5 ↑ 
    
3 ↑ 
    
 
    
Baculoviral IAP repeat-containing protein 5
      
(Apoptosis inhibitor survivin)
  
  

    
Cyclins 
  
  

    
CCNA2 
    
5 ↑ 
    

    
 
    
Cyclin-A2 (Cyclin-A)
  
  

    
CCNB1 
    
18 ↑ 
    

    
 
    
G2/mitotic-specific cyclin-B1
  
  

    
CCNB2 
    
6 ↑ 
    
3 ↑ 
    
 
    
G2/mitotic-specific cyclin-B2
  
  

    
CCND1
    
7 ↑ 
    
 
    
9 ↑ 
    
G1/S-specific cyclin-D1 (PRAD1 oncogene)
  
  

    
CCND2
    
 
    
 
    
3 ↓ 
    
G1/S-specific cyclin-D2
  
  

    
CCNE1 
    
5 ↑ 
    

    
 
    
G1/S-specific cyclin-E1
  
  

    
CCNF 
    
3 ↑ 
    

    
 
    
G2/mitotic-specific cyclin-F
  
  

    
CCNT1
    

    
3 ↓ 
    
 
    
Cyclin-T1 (CycT1)
  
  

    
CCNT2
    

    
4 ↓ 
    
 
    
Cyclin-T2 (CycT2)
  
  

    
Cyclin-dependent kinases (CDK) 
  
  

    
CDC2 
    
7 ↑ 
    

    
 
    
Cell division control protein 2 homolog (CDK1)
  
  

    
CDK7
    
 
    
 
    
3 ↑ 
    
Cell division protein kinase 7
  
  

    
APC complex subunits and activators 
  
  

    
ANAPC4 
    

    
4 ↓ 
    
 
    
Anaphase-promoting complex subunit 4 (APC4)
  
  

    
CDC20 
    
9 ↑ 
    

    
 
    
Cell division cycle protein 20 homolog    (p55CDC)
  
  

    
CDK activators 
  
  

    
CDC25A 
    
9 ↑ 
    

    
 
    
M-phase inducer phosphatase 1
  
  

    
CDC25C 
    
5 ↑ 
    

    
 
    
M-phase inducer phosphatase 3
  
  

    
CDK5R1 
    
5 ↓ 
    

    
 
    
Cyclin-dependent kinase 5 activator 1    precursor
      
(CDK5 activator 1)
  
  

    
CDK inhibitors 
  
  

    
CDKN1A
    

    
6 ↓ 
    
 
    
Cyclin-dependent kinase inhibitor 1 (p21)
  
  

    
Checkpoint system elements 
  
  

    
ATM 
    

    
6 ↓ 
    
 
    
Serine-protein kinase ATM
  
  

    
CHEK1 
    
6 ↑ 
    

    
 
    
Serine/threonine-protein kinase Chk1
  
  

    
PCNA 
    
5 ↑ 
    

    
 
    
Proliferating cell nuclear antigen (PCNA)
  
  

    
RAD17
    
 
    
 
    
4 ↑ 
    
Cell cycle checkpoint protein RAD17 (hRad17)
  
  

    
RAD51 
    
3 ↑ 
    

    
 
    
DNA repair protein RAD51 homolog 1 (hRAD51)
  
  

    
MAD2L1 
    
6 ↑ 
    

    
 
    
Mitotic spindle assembly checkpoint protein
      
MAD2A (MAD2-like 1)
  
  

    
Transcription factors 
  
  

    
E2F1 
    
11 ↑ 
    

    
 
    
Transcription factor E2F1 (E2F-1)
  
  

    
E2F2 
    
4 ↑ 
    

    
 
    
Transcription factor E2F2 (E2F-2)
  
  

    
E2F3
    

    
3 ↓ 
    
 
    
Transcription factor E2F3 (E2F-3)
  
  

    
E2F5
    
 
    
 
    
3 ↑ 
    
Transcription factor E2F5 (E2F-5)
  
  

    
Miscellaneous genes 
  
  

    
MCM2 
    
7 ↑ 
    

    
 
    
DNA replication licensing factor MCM2
      
(Minichromosome maintenance protein 2 homolog)
  
  

    
PLK1 
    

    
4 ↑ 
    
 
    
Serine/threonine-protein kinase PLK1
  
  

    
CUL3
    
 
    
 
    
4 ↑ 
    
Cullin-3 (CUL-3)
  
  

    
Note: Here genes with 3-fold (and more) mRNA    level changes are represented (ADC and SCC stage III vs. stage I; ADC vs. SCC); ↑ – increase; ↓ – decrease. 
      
 
  

Table 1:  The most considerable relative mRNA level changes (increase and decrease, n-fold) of the cell cycle regulation genes in ADC and SCC.