Human esophageal cancer occurs worldwide with a variable geographic distribution and ranks eighth in order of occurrence and sixth as the leading cause of cancer mortality, affecting men more than women.1
At diagnosis, nearly 50% of cases have cancer that extends beyond the primary locoregional confines and 75% of patients requiring surgery have proximal lymph node metastasis. At this stage, therapeutic modalities are limited in their success. Detecting cancers in early stages even in the premalignant state, means that current or future treatment modalities might have a higher likelihood of a true cure. In spite of the use of modern surgical techniques combined with various adjuvant treatment modalities, such as radiotherapy and chemotherapy, the overall 5-year survival rate of esophageal cancer patients still remains less than 10%. Hence, early detection of esophageal cancer lacks a specific symptom, a specific biomarker and accurate and reliable diagnostic, non-invasive modalities. Traditional methods of treating cancer include use of invasive or mildly invasive diagnostic tests, biopsy, and histological examination and so on. The invasive, unpleasant and inconvenient nature of the current diagnostic procedures limits the application of till date proven tumor markers. Hence, there is a pressing need for establishment of novel non-invasive biomarkers for early tumor diagnosis and monitoring response to therapy and prognosis.
Stem cell related genes, Oct 3/4, Sox2, Nanog and Bmi-1 are the factors responsible for the maintenance of the proliferation and pluripotent character in the Embryonic Stem Cells. Cancer cells, especially in poorly differentiated or undifferentiated tumors, have been characterized by many phenotypic traits similar to undifferentiated embryonic cells, indicating that stem cell related genes viz. Oct3/4, Sox2, Nanog and Bmi-1 may be expressed in solid tumors.
Oct-4, belongs to the POU (Pit-Oct-Unc) transcription factor family.2
The POU family of transcription factors can activate the expression of their target genes through binding an octameric sequence motif of an AGTCAAAT which is a consensus sequence.3a
This gene encodes a transcription factor containing a POU homeodomain. Oct-4 plays a key role in the maintenance of proliferation potential of embryonic stem cells as well as the human adult stem cells.4
Moreover, Pesce and Scholar, 1990 have shown Oct-4 to be downregulated in all the differentiated somatic cell types in vitro and in vivo.3b
Sox (SRY box) genes have been identified through their homology to the high mobility group (HMG) box (79 amino acids) of sex-determining factor SRY. The Sox2, a member of the Sox gene family which is an intronless gene encodes a group of transcription factors that are characterized by a highly conserved high-mobility group (HMG) domain.5
These genes are found throughout the animal kingdom, are expressed in a restricted spatial-temporal pattern, and play critical roles in stem cell biology, organogenesis, and animal development.6,7
For example, overexpression of Sox2 in mouse neural stem cells blocks their differentiation. Recently, SOX transcription factors have been found to be associated with human cancers.8
Nanog is another transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells. In humans, this protein plays a critical role in regulating the cell fate of the pluripotent inner cell mass during embryonic development.9
In vitro, Nanog mRNA is enriched in pluripotent cell lines such as embryonic stem, embryonic germ, and embryonic carcinoma cells but not in adult tissues. On differentiation of these pluripotent cells, Nanog expression is down-regulated. Takahashi et al 2007 reported that Nanog, works in concert with Oct-4 and Sox-2 to maintain pluripotency.10
Bmi-1 polycomb ring finger oncogene, also known as Bmi-1, is a protein which in humans is encoded by the BMI1 gene.11
The mRNA transcribed from Bmi-1 gene is linear and 3251 bp in length and Bmi1 has a RING finger at the N-terminus and a central helix-turn-helix domain.12
Bmi-1 has been reported to regulate p16 and p19, the cell cycle inhibitor genes and hence act as the oncogene rendering abnormal proliferative ability. Bmi-1 is necessary for efficient self-renewing cell divisions of adult hematopoietic stem cells as well as adult peripheral and central nervous system neural stem cells and is a downstream target in the Hedgehog (Hh) pathway.
Previous studies have demonstrated that many cancers express these genes and that their expression appears to be important for cancer cell survival. The Oct3/4 and Sox2 genes have also been shown to be expressed in human tumors including pancreatic, breast, esophageal and gastric carcinomas.13
Recent studies have shown the overexpression of Bmi-1 in many somatic solid tumors such as colon carcinoma,14
non small lung cancer,15
head and neck cancer17
and gastric carcinoma.18
He et al 2009 have shown its overexpression in ESCC.19
Although NANOG is generally found to be expressed in germ cell tumors20
as well as in somatic tumors like breast21,22
however, there are reports of its expression in esophageal cancer. Recently, microarray analysis of “Tip”-Side Population cells of esophageal cancer cell lines showing stem cell like characteristics revealed differential expression of several important stem cell– related genes viz.
Oct-4, Sox-2, Bmi-1, and ZFX.27
Therefore, it might be expected that cancer cells will express genes in common with the very early embryonic cells, especially genes specifically associated with deprogramming, return to the undifferentiated and proliferative stem cell state, and the maintenance of that state. Moreover, the identification of circulating levels of these genes in sera of ESCC patients may be instrumental in development of these makers as a diagnostic tool for the early diagnosis of esophageal cancer. Thus, keeping in view the above mentioned facts the aim of the present study is to analyze the expression of stem cell markers viz.
Oct-4, Sox-2, Bmi-1 and Nanog in tumor and sera in the ESCCs and evaluate their potential as minimally invasive blood based markers. To the best of our knowledge, this is the first study analyzing the levels of circulating mRNAs of these genes in sear of esophageal squamous cell carcinoma patients. It is also the first report showing the expression of Nanog in ESCCs.