|Ahead of print publication
Putative stemness markers octamer-binding transcription factor 4, sex-determining region Y-box 2, and NANOG in non-small cell lung carcinoma: A clinicopathological association
Vinal A Upadhyay1, Kanisha A Shah1, Dimple P Makwana1, Apexa P Raval1, Franky Dhaval Shah1, Rakesh M Rawal2
1 Department of Cancer Biology, Stem Cell Biology Laboratory, The Gujarat Cancer and Research Institute, Civil Hospital, Ahmedabad, Gujarat, India
2 Department of Life Sciences, Gujarat University, Ahmedabad, Gujarat, India
Franky Dhaval Shah,
Department of Cancer Biology, Stem Cell Biology Laboratory, The Gujarat Cancer and Research Institute, Civil Hospital Campus, Asarwa, Ahmedabad - 380 016, Gujarat
Source of Support: None, Conflict of Interest: None
Background: The promising improvement in the clinical outcome of lung cancer can be possibly achieved by identification of the molecular events that underlie its pathogenesis. Cancer stem cell (CSC) being one of the subsets of tumor majorly participates in drug resistance and treatment failure because of the moderate cell cycle, lower proliferation, and increased expression of DNA repair and anti-apoptosis genes. Although many putative CSC markers exist, a precise characterization for non-small cell lung cancer is of utmost importance due to increased mortality rate and lack of targeted therapies. Hence, the article focuses on the expression of stemness-associated markers, namely octamer-binding transcription factor 4 (OCT4), NANOG, and sex-determining region Y-box 2 (SOX2) in non-small cell lung cancer (NSCLC) patients.
Methods: The expression of OCT4, NANOG, and SOX2 were evaluated in 32 histopathologically confirmed NSCLC tissues using real-time polymerase chain reaction. The obtained expression was correlated with clinical and pathological manifestations using the statistical test such as Student's t-test and Pearson correlation in varied statistical software.
Results: Results showed a significantly higher expression of OCT4 and NANOG compared to SOX2 in the tumor tissues. When the expression of these markers was correlated with the clinical parameters, higher expression was seen in males, patients with age above 60 years, and in adenocarcinoma subtype. In correlation with the habit, higher expression of OCT4 and SOX2 was observed in habituated patients. Expression of NANOG and OCT4 was higher even in patients with poor differentiation.
Conclusion: The expression and prognostic significance of CSC markers obviously vary depending on histological NSCLC subtype. Importantly, our findings suggest that OCT4, SOX2, and NANOG network together may be promising for ongoing targeted therapies in specific NSCLC subgroups.
Keywords: Cancer stem cell markers, NANOG, non-small cell lung cancer, octamer-binding transcription factor 4, real-time polymerase chain reaction, sex-determining region Y-box 2
|How to cite this URL:|
Upadhyay VA, Shah KA, Makwana DP, Raval AP, Shah FD, Rawal RM. Putative stemness markers octamer-binding transcription factor 4, sex-determining region Y-box 2, and NANOG in non-small cell lung carcinoma: A clinicopathological association. J Can Res Ther [Epub ahead of print] [cited 2020 May 25]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=277419
| > Introduction|| |
Lung cancer remains one of the foremost causes of cancer-related death worldwide with 1825 estimated new cases and 1590 estimated deaths according to the GLOBOCAN 2012. Lung cancers are majorly bifurcated into (i) small cell and (ii) non-small cell lung cancers (NSCLC) where approximately 80% of cases are NSCLC. On the basis of tumor histology, they are further categorized into adenocarcinoma (AdC), squamous cell carcinoma (SqCC), and large cell carcinoma, with 80% of NSCLC being either AdC or SqCC. The overall prognosis of lung cancer is poor often due to its late presentation, high recurrence frequency, and lack of curative systemic therapy. The evolution of lung cancer being a multistep process involves various genetic, epigenetic, and environmental factor interacting in its microenvironment. This causes dysregulation of key oncogenes and tumor suppressor genes, thereby culminating inactivation of cancer-related signaling pathways.
Cancer stem cell (CSC) theory states that a subset of tumor cells has the ability to self-renew and generates tumor heterogeneity leading to tumor progression and invasion. These cells exhibit pluripotency and self-renewal properties and give rise to a heterogeneous population of tumor cells. Self-renewal being a hallmark of stem cell and cancer, stemness programming could play an important role in cancer. Hence, CSCs are important to study treatment resistance and relapse, thereby focusing on discovering new therapies and treatment regimen. Studies have supported the role of CSCs in tumor initiation of many solid tumors, such as lung as well as breast, prostate, pancreatic, and colon.
CSCs can be specifically differentiated from other cancer cells on the basis of specific surface markers. Lung CSCs have characteristic surface markers, for example, CD133, CD44, ABCG2, and ALDH1, which confer drug resistance and stemness as opposed to somatic lung cancer cells. Recent reports also suggest that NANOG, octamer-binding transcription factor 4 (OCT4), and sex determining region Y-box 2 (SOX2) are potential diagnostic and prognostic markers in lung carcinoma. Recent studies have demonstrated that CSCs are commonly activated tumor oncogene that further activates epidermal growth factor receptor signaling pathways in human cancers, altogether indicating its complex biological role in cell proliferation and differentiation. NANOG, OCT4, and SOX2 together make a strong transcription regulatory circuit that facilitates cell pluripotency and helps to regulate the self-renewal of embryonic stem cells [Figure 1]. However, each of these genes is involved in the regulation and pathogenesis of lung carcinoma and is interconnected. These stemness markers have also been collectively showed to its effect on A549 cell lines by converting these epithelial cells to mesenchymal phenotype and causing epithelial–mesenchymal transition transdifferentiation. NANOG, OCT4, and SOX2 together or individually, lead to tumorigenicity, tumor metastasis, and distant metastasis after chemo or radiotherapy in different types of cancer.,
The aim of this study was to address the potential role of OCT4, NANOG, and SOX2 markers in NSCLC patients. Thus, we assessed the expression of these markers in 32 histopathologically confirmed cases of NSCLC and in healthy lung tissue using quantitative real-time polymerase chain reaction (qRT-PCR). The study also correlated the relative expression of NANOG, OCT4, and SOX2 with clinicopathological parameters (age, gender, habit, as well as the association with histological subtypes and histological grade). In addition, intercorrelation among the markers and pathway prediction was also carried out. These results led us to conclude that the CSC theory in the future may play an important role in the diagnosis and treatment of cancer. Although the clinical impact of these markers is unclear, they may have a prognostic or predictive value in NSCLC.
| > Methods|| |
Patients and tissue specimens
Fresh tissue samples were collected from the radiology clinic during computed tomography-guided lung biopsy. A total number of 32 histopathologically confirmed NSCLC tissue samples were analyzed in the present study. The obtained tissues were further sectioned into two mirror-image specimens. One of the sections was evaluated by the pathology department, in order to confirm the presence or absence of malignancy. The other tissue specimens were immediately frozen in liquid nitrogen after the biopsy and stored at −80°C until further use. None of the patients received either chemotherapy or radiotherapy. Seropositive for HIV/HBsAg/HCV was excluded from the study. RNA extracted from normal lung (Agilent Technologies, USA) was used as control for the study. The study was approved by the Institutional Review Board and Ethics Committee. Informed consents were obtained from individual cases before sampling.
Extraction of total RNA and c-DNA synthesis
Total RNA was extracted from 30 mg tumor tissue using RNeasy tissue kit (Qiagen 74106) based on the principal of silica gel membrane technology by selective binding, stepwise washing, and elution of RNA following manufacturer's instructions. A digestion step on the spin column for DNA was performed using RNase-free DNase set (Qiagen 79254). The concentration of the isolated RNA was quantified with Qubit 3.0 Fluorometer (Invitrogen by Life Technologies, CA, USA). The extracted RNA was then stored at −80°C until further analysis. Complementary DNA synthesis from the isolated RNA was performed by reverse transcription using kit (Bio-Rad iScript™ cDNA Synthesis kit, cat #170-8891). The cyclic conditions were 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min.
Real-time polymerase chain reaction (quantitative real-time polymerase chain reaction)
SYBR green-based qRT-PCR assays were performed for the gene expression analysis using gene-specific primers for OCT4, SOX2, NANOG, and β-actin as housekeeping gene. All the primers were custom ordered using the sequences as shown in [Table 1]. A total of 100 ng of the transcribed cDNA was taken as input for qRT-PCR reaction. The reaction mixtures of 20 μl consisted of 10-μl Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies, USA), 0.5 μl (200 nM) each of the forward primer and reverse primer, and 2-μl cDNA. β-actin gene expression was measured as endogenous control.
Amplification and detection was performed using AriaMx real-time PCR System (Agilent Technologies, USA) at the following cycling conditions: 1 cycle of 3 min at 95°C for the initial denaturation step and 40 cycles of 5 s at 95°C for the denaturation step, 20 s at 60°C for the annealing and extension step. Melting curve analysis was performed following the amplification. The generation of PCR product was tested using the ΔΔCT method and β-actin was taken as the endogenous control. All experiments were performed in triplicate independently and average Ct value was calculated for the quantification analysis. The obtained results were correlated with the results of normal lung sample.
The qRT-PCR results were analyzed using ΔΔCt method using Ct values of test and control gene. Correlation study among these genes was carried out using Pearson test and significance was obtained by performing Student's t-test using MedCalc and GraphPad Prism 7 (California, USA). All statistical tests were performed at the 5% level of significance and P < 0.05 was considered statistically significant.
| > Results|| |
Altogether, a total of 32 patients with lung cancer diagnosed with NSCLC were enrolled in this study. The cohort predominantly comprised of males (78.2%) and older patients with median age above 60 years (65.7%). Histologically proven AdC (62.5%) patients with poor differentiation of the tumor tissues (28.2%) dominated the cohort. Most of the patients were either habituated (62.5%) to smoking or had exposure to smoke in one or other forms such as cooking on chulla, exposed to factory or industrial smoke as depicted in [Table 2].
Expression of stem cell markers in non-small cell lung cancer patients
Expression levels of NANOG, OCT4, and SOX2 were investigated in the tumor and healthy lung control. β-actin gene was used as an internal control and its expression level was correspondingly detected in the respective samples (β-actin is the housekeeping gene whose expression has been negated both normal lung and tumor tissue). The fold change expression was used to study the regulation mechanism of these markers.
Increased expression of all the three markers was noted in the tumor tissue as compared to healthy lung tissue suggestive of its role in tumor formation [Figure 2]a, [Figure 2]b, [Figure 2]c.
|Figure 2: mRNA expression of cancer stem cell markers in normal lung tissues and tumor tissues (a) NANOG, (b) octamer-binding transcription factor 4, (c) sex-determining region Y-box 2, and (d) relative expression of stem cell markers in non-small cell lung cancer patients|
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The internal correlation among these markers was studied to map out the relative association among them. The fold change was graphed negating the expression of the healthy lung tissue as depicted in [Figure 2]d. The expression of NANOG and OCT4 was comparatively higher as compared to the SOX2 expression. Further, Pearson correlation among the expression of these markers showed a significant correlation solely between NANOG and OCT4 (P < 0.0001, r = 0.9744).
Correlation studies of the cancer stem cell markers and clinicopathological parameters
The expression of these markers was then correlated with various clinicopathological parameters to study its regulation mechanism with the changing conditions.
NANOG, OCT4, and SOX2 showed higher expression in patients with age above 60 years [Figure 3]a. In case of gender correlation, higher expression of all the three genes was seen in females as compared to males; however, only SOX2 (P = 0.0001) expression was statistically significant [Figure 3]b. Higher expression of NANOG and OCT4 (P = 0.0001) was seen in tobacco habituates/smoke-exposed patients, whereas SOX2 (P = 0.02) expression markedly decreased as shown in [Figure 3]c.
|Figure 3: Expression correlation of markers with clinical characteristics (a) age, (b) gender, and (c) habit|
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Expression correlation exhibited higher levels of these stemness markers in AdC patients as compared to SqCC patients. All three markers were differently expressed in lung ADC and SqCC. The overexpression was significant in case of OCT4 and SOX2 expression (P = 0.0001) [Figure 4]a. NANOG, OCT4, and SOX2 gene have been reported to be amplified in human lung SqCCs, but the role is still unclear lung AdCs.
|Figure 4: Expression correlation of markers with (a) histological subtypes and (b) histological grade of patients|
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Histological grading of these patients showed that most of them were poorly and moderately differentiated. Elevated expression of NANOG and OCT4 was seen in poorly differentiated patients in contrast to the moderately differentiated ones. Moreover, there were patients with unknown histological grade that showed a higher expression of SOX2 [Figure 4]b.
| > Discussion|| |
The expression pattern of SOX2, NANOG, and OCT4 in 32 human NSCLC cancerous and normal human lung tissues was investigated in this study. Results indicate upregulation of OCT4, NANOG, and SOX2 in NSCLC tissues as compared to normal human adult lung tissue with higher expression of NANOG and OCT4 as compared to SOX2. Subsequently, OCT4 and SOX but not NANOG expression levels were significantly associated with histological subtype of the tumors. Besides, NANOG, SOX2, or OCT4 expression did not significantly correlated to age and histological grade of the patients enrolled in the study. In fact, a significant expression of OCT4 and SOX2 was observed in habituated patients as compared to NANOG which did not show any significance. SOX2 as a sole marker associated with the gender and showed significance, whereas OCT4 and NANOG were not significantly expressed.
The CSCs theory might be one of the ideal models to understand the genesis and the development of lung cancer tumor which may provide the possibility of a better therapeutic outcome or even prevention to disease. This crucial transcription factors SOX2, OCT4, and NANOG maintain embryonic stem cell differentiation and pluripotency and have also been recognized as having “stemness” characteristics in cancer cells. It has been reported earlier that these CSC markers may lead to immortality, self-renewal, and invasive properties of cancer cells., Supporting this hypothesis, it was suggested that knocking down SOX2, NANOG, and OCT4 in tumor-initiating cells would lead to the loss of the self-renewal, proliferation, and tumorigenic capacities of the cells resulting in CSC-like cell apoptosis of these tumor cells.,
AdC accounts for approximately 80% of the most frequent and aggressive types of NSCLC and recent studies have highlighted the fact that stem cells might be involved in their development and maintenance. Giangreco et al., in 2002, observed that bronchoalveolar stem cells (BASCs), that are capable of multipotent differentiation and self-renewal in both lung cancer and normal lung, are involved in the process, and lead to the development of AdC from normal cells of the distal lung. Therefore, these stem cells can be considered as putative cells of origin for this subtype of lung cancer. Recent reports have stated that OCT4 is exhibited by these BASCs. Our study suggests that not only OCT4 but SOX2 may also be playing an impotent role in the pathogenesis of lung carcinoma by activating other downstream signaling pathways and leading to uncontrolled proliferation and differentiation. No relevant studies have been reported yet for AdC, but our research revealed significantly higher expression of SOX2 and OCT4 in AdC as compared to SCC. This signifies the hypothesis that these two transcription factors OCT4 and SOX2 play an important role in the AdC neoplastic processes of lung cancer. Moreover, further research needs to be carried out to understand the mechanism involved in this disease.
OCT4 is one of the most important genes that control the process of self-renewal and differentiation of embryonic stem cells as well as in the tumor cells. OCT4 has been known to be expressed in different cancers such as Germ cell Tumours (GCTS), tumor cell lines of colon, liver, prostate bladder, and breast., Recent evidence suggested that OCT4 plays a crucial role in the identification of new prognostic factor for recurrence and classification of patients for appropriate treatment. NANOG is another key factor in maintaining the pluripotency of stem cells regulates self-renewal and is essential for the reprogramming of the differentiated cells. Similar to OCT4, NANOG expression is also observed in human tumors and embryonic stem cell as well as carcinomas. NANOG is associated with REX1 which governs the self-renewal of the stem cells. SOX2 is another genetic marker that has been conferred with stemness property and plays a key role in maintaining neural stem cells proliferation and differentiation. Increased SOX2 expression has been observed in patients with ovarian carcinoma, melanoma, bladder, and brain and liver cancers. SOX2 expression has been reported in embryonic stem cells and it is one of the most important transcription factors that are involved in the regulation of embryonic stem cell self-renewal.
Recent studies have shown that OCT4 expression regulates NANOG expression through binding sites in the promoter of NANOG. It is known that SOX2 is involved as a cofactor in OCT4 target genes transcription. OCT4 interacts with SOX2 and regulates downstream target molecules and the binding site for OCT4-SOX2 complex exists in NANOG promoter. REX1 gene is required for stem cell self-renewal and is activated by NANOG. Moreover, REX1 is downstream target gene of OCT4-SOX2 complex., Schonhals et al. showed coexpression of OCT4 and SOX2 in tumor tissues such as brain, bladder, and lung. Different studies have demonstrated that these genes OCT4, NANOG, and SOX2 are simultaneously expressed in embryonic cancer cells, gastric cancer, colon cancer, and testicular cancer.,, Recently, researchers have observed that SOX2 mRNA expression in whole blood alone might be a promising noninvasive marker for molecular screening of SCLC and important prognostic marker in advanced chemotherapy-treated SCLC patients, altogether indicating important role of cancer stem-like cell (CSC) regulators in cancer metastasis. However, the function of SOX2, OCT4, and NANOG in NSCLC cells together are not completely understood and remains ambiguous. Taken together, these molecular markers form a strong transcriptional circuit [Figure 5] for pluripotency and self-renewal of stem cells. Each of these three genes involved in the regulation of other genes and forms a new class of molecular markers for the diagnosis of cancer. However, the combinatorial analysis of SOX2, NANOG, and OCT4 expression in NSCLCs is more helpful than the single-marker analysis.
|Figure 5: Predicted pathway that depicts the core transcriptional circuit in non-small cell lung carcinoma|
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Moreover, not many studies have worked on correlating the expression of OCT4, SOX2, and NANOG with other clinical parameters. Much of the studies have dedicatedly worked in SqCCs and NSCLC has been less explored.
Hence, the articles concurrently summarize that there is collectively increased expression of these stemness markers with significant correlation between NANOG and OCT4. It has a major incidence found in adult patients and that possessing strong habit history. Results have shown a significant expression in AdCs as compared to the SqCC showing its at par contribution in the aggressive state of the disease.
| > Conclusion|| |
Conclusively, the present study indicated that OCT4, SOX2, and NANOG can be potential markers in predicting the prognosis of NSCLC. To confirm the significance of these factors in the diagnosis and therapy of NSCLC, further experiments pertaining to the knockdown of these transcription factor needs to be carried out. Moreover, these genes potentially could be used as a marker for tumor diagnosis or prognosis of NSCLC.
Financial support and sponsorship
This study was financially supported by the Gujarat Cancer Society and the Gujarat Cancer and Research Institute.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]