Austin J Cancer Clin Res 2015; 2(7): 1062.
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
Senchenko VN1*, Dmitriev AA1, Krasnov GS1, Kudryavtseva AV1, Dardyk MV2, Beniaminov AD1 and Kondratieva TT2
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Russia
2N.N. Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences, Russia
*Corresponding author: Senchenko VN, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova 32, 119991 Moscow, Russia
Received: June 01, 2015; Accepted: August 05, 2015; Published: August 08, 2015
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 p16INK4A–Cdk/cyclin D1–Rb and p53/ p21Waf1 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
Multidisciplinary approach for a non-small cell lung cancer (NSCLC) diagnostics allows to choose the most adequate treatment strategy . Particular relevance acquired differential diagnosis of lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) that requires specific markers for each histological type . In case of ADC, targeted therapy has a great success and is one of the perspective treatment directions . However, targeted therapy, aimed at individual targets, is often accompanied by the rapid development of drug resistance . 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 . 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 .
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 p16INK4A– Cdk/cyclin D1–Rb and p53/p21Waf1 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.
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  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 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.
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.
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/p21Waf1, 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):
Expression ratio, n-fold
Negative apoptosis regulators
G2 and S phase-expressed protein 1 (B99 homolog)
Baculoviral IAP repeat-containing protein 5
(Apoptosis inhibitor survivin)
G1/S-specific cyclin-D1 (PRAD1 oncogene)
Cyclin-dependent kinases (CDK)
Cell division control protein 2 homolog (CDK1)
Cell division protein kinase 7
APC complex subunits and activators
Anaphase-promoting complex subunit 4 (APC4)
Cell division cycle protein 20 homolog (p55CDC)
M-phase inducer phosphatase 1
M-phase inducer phosphatase 3
Cyclin-dependent kinase 5 activator 1 precursor
(CDK5 activator 1)
Cyclin-dependent kinase inhibitor 1 (p21)
Checkpoint system elements
Serine-protein kinase ATM
Serine/threonine-protein kinase Chk1
Proliferating cell nuclear antigen (PCNA)
Cell cycle checkpoint protein RAD17 (hRad17)
DNA repair protein RAD51 homolog 1 (hRAD51)
Mitotic spindle assembly checkpoint protein
MAD2A (MAD2-like 1)
Transcription factor E2F1 (E2F-1)
Transcription factor E2F2 (E2F-2)
Transcription factor E2F3 (E2F-3)
Transcription factor E2F5 (E2F-5)
DNA replication licensing factor MCM2
(Minichromosome maintenance protein 2 homolog)
Serine/threonine-protein kinase PLK1
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.
1. Over-expression of many genes from the panel was stronger in lung ADC than SCC and more pronounced in metastatic tumors.
2. For 19 genes we showed 3-18-fold up-regulation in the metastatic ADC compared to metastasis-free. Six genes were downregulated in the metastatic SCC compared to non-metastatic one. The expression level was 3-6-fold higher in both metastatic ADC and SCC compared to non-metastatic for only two genes – survivin (BIRC5) and cyclin – B2 (CCNB2). (Potential markers of metastatic activity of ADC and/or SCC).
3. Six genes – CCND1 and CCND2 (cyclins D1 and D2), CDK7 (cyclin-dependent kinase), RAD17 (element checkpoints), E2F5 (transcriptional factor) and CUL3 (cullin) showed alterations in expression specific to NSCLC subtype. The CCND2 mRNA level was 3-fold higher in SCC compared to ADC. The expression levels of other genes were in a 3-9 times higher in ADC compared to SCC. (Potential markers of discrimination between ADC and SCC).
4. The highest mRNA level was observed for the following genes: CDC2 (cyclin-dependent kinase, CDK), CCNA2 and CCNE1 (cyclins A2 and E1), CDC20 (activator anaphase-promoting complex, APC) and CDC25C (activators CDK), RAD51 and MAD2L1 (elements mechanism checkpoints), BIRC5 and GTSE1 (inhibitor of apoptosis), PLK1 (APC activator) in all samples regardless NSCLC subtype and the presence of metastases. (Potential targets for combined therapy of NSCLC – ADC and SCC).
The next efforts in this research direction will help to develop a new optimal gene panel for searching of general and specific targets for the treatment of NSCLC subtypes. These problems are particularly acute in connection with the relevance of accurate ADC and SCC detection because erroneous diagnosis and choice of therapy can cost a patient’s life [15,16].
The expression analysis of the genes participating in a cell cycle progression revealed evident dysfunctions of Rb/E2F and other pathway checkpoints in NSCLC while the essential impact comes from the RB1 and CTDSPL silencing. The summary of currently available data includes: a) an inhibition of RB1 gene expression at the transcriptional level and, probably, via microRNA interference takes place in different cancers ; b) the dramatic increase of expression of cyclins E and others cyclins, also known to be up-regulated in various types of cancer  can accelerate Rb phosphorylation by Cyclin-CDK complexes; c) p21, an inhibitor of these complexes, may be under-expressed ; d) transfection of tumor cells with CTDSPL can lead to the content decrease of inactive phosphorylated form of Rb ; and e) CTDSPL down-regulation can decline miR-26amediated suppression of CDK6, cyclins D2, E1, E2 expression .
Pronounced character of expression abnormalities of two tumor suppressors – CTDSPL and its target Rb, along with various cell cycle control genes in NSCLC supports the new continuum model of tumor suppression , in which authors integrated “two-hit” model of carcinogenesis with a hypothesis of subtle dosage effects of tumor suppressors. It underlines the importance of TSG expression attenuation. Our results propose that a decline of tumor suppression is resulted from a gradual falling of TSG expression even without dramatic loss of alleles: dozens of slight aberrations in various genes may lead to significant tumor promotion effect.
We showed expression deregulation of the majority of cell cycle regulation genes involved in associated with down-regulation of two tumor suppressor genes RB1 and CTDSPL. Six genes (CCND1, CCND2, CDK7, RAD17, E2F5, and CUL3) showed subtype-specific alterations of their expression and were suggested potential markers of NSCSL subtype – ADC or SCC. Two genes BIRC5 and CCNB2 revealed metastasis-specific expression up-regulation and can be speculated as markers of NSCLC aggressiveness. Many genes (survivin, cyclins, Ser/Thr-protein kinases, etc.) with expression gains (up to 100-fold) could be potential targets for future biotherapy approach of NSCLC – especially four genes (BIRC5, CCNE1, CDC25C, and RAD51) that had high expression level in all studied tumors independently of particular NSCLC subtype, and presence of metastases.
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Citation: Senchenko VN, Dmitriev AA, Krasnov GS, Kudryavtseva AV, Dardyk MV, Beniaminov AD, et al. 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. Austin J Cancer Clin Res 2015; 2(7): 1062.