Expression And Significance Of OCT4 Pseudogenes In Glioma And Breast Carcinoma

Octamer-binding transcription factor 4 (OCT4) is a POU family transcription factor, notable for its maintaining the pluripotency and self-renewal of embryonic stem (ES) cells and for generating induced pluripotent stem (iPS) cells, as a uniquely essential reprogramming factor to date. Recently, it becomes a hot topic to study the expression and function of OCT4 in human tumours. Although OCT4 is putatively expressed in and associated with germ cell tumours with pluripotent potential, its expression in human somatic tumours remains controversial. Several studies showed that OCT4 is expressed in adult stem cells and somatic cancers, and OCT4-positive cells identified in cancers are likely to represent cancer stem cells (CSCs) and, further, OCT4 expression is required for maintaining the survival and self-renewal property of cancer stem-like cells. However, there is ample evidence suggesting that OCT4 is not expressed in somatic tumours and tumour cell lines. In view of this and to fully clarify the molecular mechanism of tumour biology, it would be vital to first determine whether OCT4 is expressed in human somatic cancers.In general, cancer cells, which are immortal, relatively undifferentiated, and invasive, are very similar to early embryonic cells. However, the cancer cell populations in most cancers are intrinsically heterogeneous, encompassing cells ranging from very immature to nearly differentiated. With stem cell theory being applied to cancer study, the undifferentiated cells in cancer may be considered to be CSCs, which have the exclusive ability to self-renew and give rise to nontumourigenic cancer cells. It is the small population of CSCs that drives tumour formation and growth. CSCs have been isolated from many somatic cancers including glioma and breast cancer and cultured in vitro. Recently, CSCs were proposed to be derived either from normal stem cells with dysregulated self-renewal or from restricted progenitors or differentiated cells regaining the capacity to self-renew. Therefore, it is likely that aberrant activation of the self-renewal pathway is a key event in carcinogenesis, and it becomes imperative and important to examine the expression and significance of genes involved in the regulation of stem cell self-renewal in cancers, including OCT4.The human OCT4 gene is located on chromosome 6 and comprises five exons and four introns. There are two isoforms generated by alternative splicing designated as OCT4A and OCT4B. They have identical POU DNA binding and C-terminal domains but differ in their N termini. OCT4A is localized in the nucleus, whereas OCT4B is mainly localized in the cytoplasm and, unlike OCT4A, it can not maintain the self-renewal of ES cells. OCT4 is commonly referred to as OCT4A and the function of OCT4 to maintain the self-renewal of ES cells should be attributed to OCT4A. In addition, there are six pseudogenes for human OCT4, which are highly homologous to human OCT4 gene. While OCT4B could be detected in various non-pluripotent somatic cancer cells, two OCT4 pseudogenes, OCT4-pg1 and OCT4-pg5, were found to be transcribed in somatic cancers and cancer cell lines. The existence and expression of both OCT4 alternative splicing variant and various pseudogenes are likely to yield undesirable false positive results. Therefore, when addressing OCT4 expression, it is necessary to distinguish the bona fide OCT4 from its alternative splicing variant and various pseudogenes.Although it was reported that the existence of OCT4 splicing variants and pseudogenes might have led to some confusion in cancer and stem cell research, the issue has not received much attention in many recent studies. In the present study, we used RT-PCR and sequencing analysis to detect OCT4 expression in two types of human somatic tumours, glioma and breast carcinoma, from which CSCs have already been isolated and cultured in vitro. Our results showed that three OCT4 pseudogenes, viz. OCT4-pg1, OCT4-pg3 and OCT4-pg4 rather than OCT4 gene, are expressed in these two human solid tumours. We further demonstrated their protein expression and explored the function and activity of their protein products.Part I Cloning and sequencing analysis of OCT4 pseudogenes from gliomas and breast carcinomasTo detect OCT4 expression in glioma and breast carcinoma, we collected 42 cases of gliomas and 45 cases of breast carcinomas. All tumour specimens were verified by pathological analysis. Gliomas were classified according to the WHO classification standard, whereas breast cancer subtypes were defined according to the status of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). The adult normal brain and breast tissues were obtained from surgical resections of four and five trauma patients, respectively. In RT-PCR assays, we used two pairs of primers to detect OCT4 mRNA expression. OCT4A-specific primer set is able to specifically amplify OCT4A, while OCT4-full length primers were designed to amplify the entire coding sequence of OCT4A. When using OCT4A-specific primers, except for seminoma, which served as a positive control,OCT4A mRNA (496bp) was not detected in any tumour and normal tissues. But, when using OCT4-full length primers, besides seminoma, the predicated 1086bp PCR products were detected in gliomas of different grades and breast cancers of various subtypes. In addition, we detected OCT4 mRNA expression in many human glioma cell lines and breast cancer cell lines. When using OCT4A-specific primers, except for human pluripotent embryonal carcinoma cells NTcra-2, which served as a positive control, OCT4A mRNA (496bp) was not detected in U251, U87, MCF7, MDA-MB-231, BT474 and SK-BR-3 cells. But, when using OCT4-full length primers, besides NTera-2 cells, the predicated 1086bp PCR products were detected in these tumour cell lines.Next, we used DNA cloning and sequencing analysis to detect OCT4 expression in glioma and breast carcinoma. First, all the 1086bp DNA fragments were extracted from the agarose gel, cloned into pGEM-T easy vector and amplified. Plasmids with an insert of correct size were sequenced. Sequencing results showed that while the transcripts of OCT4 gene were only found in samples of positive control, the seminoma and NTera-2 cells, transcripts of three OCT4 pseudogenes, OCT4-pg1, OCT4-pg3 and OCT4-pg4, which are mapped to chromosomal bands 8q24,12p13 and 1q22, respectively, were found in these cancer tissues and cancer cell lines. Our sequencing results confirmed that it was the transcripts of OCT4 pseudogenes that contributed to the RT-PCR products in the cancer tissues and cancer cell lines when using OCT4-full length primers for PCR amplification.PartⅡProtein expression and localization of OCT4 pseudogenesTheoretically, the transcripts from OCT4-pg1, OCT4-pg3 and OCT4-pg4 can translate into protein products of 359,186 and 286 amino acids, respectively. Although there are amino acid deletions or substitutions, all these three putative proteins contain N-terminal domains similar to that of human OCT4.In order to investigate the expression and subcellular localization of these OCT4 pseudogene products, we constructed their expression plasmids with HA tag at their C termini. We then transfected them into NIH 3T3 cells and carried out immunocytochemistry staining. Our results showed that OCT4-pg1 and OCT4-pg4, as well as OCT4, were mainly localized in the nuclei, whereas OCT4-pg3 was mainly localized in the cytoplasm. Besides OCT4, OCT4-pg1 and OCT4-pg3 could also be detected by two anti-OCT4 antibodies, ab18976 and sc-5279. By contrast, OCT4-pg4 could not be detected by these two antibodies. Similar results were obtained in U251 and MCF7 cells transfected with the plasmids, except that OCT4-pg3 was located in both the nucleus and cytoplasm. Furthermore, Western blotting showed that OCT4-pg1, OCT4-pg3, OCT4-pg4 and OCT4 migrated at 50 kDa,30 kDa,30 kDa, and 50 kDa, respectively. In agreement with the immunocytochemistry results, subcellular fractionation analysis confirmed that OCT4-pgl, OCT4-pg4 and OCT4 were localized in the nuclei, while OCT4-pg3 was localized in the cytoplasm of NIH 3T3 cells. Besides OCT4, OCT4-pg1 and OCT4-pg3 could also be stained by sc-5279 in Western blotting. The two antibodies, ab18976 and sc-5279, could not recognize OCT4-pg4, possibly due to amino acid deletion or substitution. We further used immunochemistry to detect the endogenous expression of OCT4 pseudogenes in cancer cells. As expected, the fluorescence signal was strongly detected in the nucleus of NTera-2 cells when using the polyclonal ab18976 antibody. As to U251 glioma cells and MCF7 breast cancer cells, the fluorescence signals were detected in both the nucleus and cytoplasm of almost all cells, indicating the expression of OCT4 pseudogenes in these cancer cells. In contrast to extensive and nuclear staining of OCT4 in seminoma, only a few cells in human glioma and breast cancer were immunostained by ab18976, with positive staining in both the nucleus and cytoplasm of some cells, indicating the expression of OCT4 pseudogenes in these cancer tissues. In addition, the cells that were positively stained by ab 18976 in glioma also expressed the cancer stem cell marker CD 133 or the astroglial marker GFAP, indicating that expression of OCT4 pseudogenes is not restricted to CSCs in gliomas.These results suggest that, when detecting OCT4 expression in cancers, the expression of OCT4 pseudogenes might lead to false detection for OCT4 in both immunochemistry and Western blotting. This might also contribute to the misinterpretation of OCT4 expression in cancers and stem cells.Part III Protein activity and function of OCT4 pseudogenesTo determine the biological activities of these OCT4 pseudogenes, U251, U87, MCF7 and MDA-MB-231 cells were transfected with expression plasmids of OCT4 or its pseudogenes. The cell viability was measured by MTT assay. There was a significant increase in cell viability in cancer cells transfected with OCT4 compared to the control group, but there was no significant difference between cell groups transfected with OCT4 pseudogenes and the mock vector. The colony formation assay showed that the colony formation rate of cancer cells transfected with OCT4 was significantly higher than that of the control group cells, while there was no significant difference between cell groups transfected with OCT4 pseudogenes and the mock vector. These results showed that the OCT4 pseudogenes could not increase cell viability in MTT analysis and did not promote colony formation rate of cancer cells, indicating that the OCT4 pseudogenes have no effect on the proliferation of cancer cells. Finally, the transcriptional activation potentials of OCT4 pseudogenes were detected by luciferase reporter assay. OCT4 strongly transactivated three OCT4 reporter constructs (6×W, PORE and MORE), but there was no significant difference between OCT4 pseudogenes and the mock vector, suggesting that OCT4-pgl, OCT4-pg3 and OCT4-pg4 have no significant transcriptional activity toward these three OCT4 luciferase reporter constructs.The high degree of homology between OCT4 and its pseudogenes suggests that they might have similar functions, but our results showed that OCT4-pg1, OCT4-pg3 and OCT4-pg4 did not possess OCT4-like activities. The functional implication of OCT4 pseudogenes expression in cancers and roles of these genes playing in carcinogenesis remain to be further investigated.In conclusion, we have demonstrated the expression of three OCT4 pseudogenes, OCT4-pgl, OCT4-pg3, and OCT4-pg4 with an apparent lack of OCT4 in human glioma and breast carcinoma, indicating that OCT4 may not be essential for somaic tumourigenesis. We further demonstrated their protein expression by using immunochemistry and Western blotting. Our results suggest that the protein expression of these pseudogenes might have produced the false detection of OCT4 in human somatic cancers. Remarkably, we have revealed that the OCT4 pseudogenes did not display OCT4-like activities. We suggest that, in order to obtain reliable results, combined approaches with appropriate controls must be followed to exclude possibility of false-positive results in investigations of OCT4 expression in cancers and stem cells.