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Medicine and Health

Peer review of “Expression of the pluripotency markers Oct3/4, Nanog and Sox2 in human breast cancer cell lines”

Professor Demetrios A. Spandidos, Ph.D.

Editor

Oncology Letters

Spandidos Publications

10 – Vriaxidos Street, Athens 116 34, Greece

 

Dear Dr. Spandidos,

I have conducted a detailed review of the publication entitled “Expression of the pluripotency markers Oct3/4, Nanog and Sox2 in human breast cancer cell lines” written by Gui-Qin Ling, Dong-Bo Chen, Bao-Qing Wang, and Lan-Sheng Zhang (2012). My comments, analysis, and recommendations will be summarized in the following paragraphs for your review.

General Evaluation

 The research study explores a new approach in evaluating breast cancer cell lines with the review of Oct3/4, Nanog, and Sox2, pluripotency markers (Ling et.al, 2012). The study determined that these transcription factors are relevant in breast cancer cell lines, but their validity beyond this basic association is unknown. The research protocol summarized in this study represents a step in the right direction towards the identification of transcription markers in human breast cancer cell lines but does not go far enough into detail with this premise. Therefore, the research article only serves as a starting point for future studies to further examine these specific transcription factors and their role in shaping diagnostic tools and treatments for patients. However, this research article does provide further evidence that various transcription factors may play an important role in breast cancer research and the potential development of new treatments for this patient population.

Major Comments 

The study conducted by Ling et.al (2012) addresses the importance of determining whether or not existing pluripotency-associated transcription factors are relevant to the creation of different types of cancer tumors across different patient populations.  Due to the complex nature of breast cancer, its many stages, and its ability to metastasize to other organs, this type of cancer mandates a greater level of genetic understanding in regards to its potential causes, prevalence, and impact on patients (Ling et.al, 2012). In order to address these issues more fully from a genetic perspective, the study of existing transcription factors Oct3/4, Nanog and Sox2 are warranted (Ling et.al, 2012). Human breast cancer cell lines may attributed to a number of genetic variations, including these transcription factors; however, these associations are not clearly understood and accepted in cancer research communities (Ling et.al, 2012). It is imperative to examine the role of these transcription factors to demonstrate whether or not their efficacy in the creation of breast cancer cell lines is warranted and worthy of further consideration (Ling et.al, 2012).

It is evident that gene transcription factors represent a means of cell development and variation that is unique to each individual cell and contribute to the overall genetic makeup of all living beings. However, the degree of specificity of these gene transcription factors and their role in shaping cell development is not clearly understood in many capacities. Cancer cell development is of particular relevance because of the commonality of different types of cancers and their relevance to society in the 21st Century. From a research-based perspective, cancer cell development is of critical importance because there must be methods in place to identify cancer cells and address treatment considerations before it is too late. At the same time, it is evident that with the appropriate research-based tools in place, the potential exists to eradicate cancer cell development before it even begins. The expression of various transcription factors is dependent on the type of research questions that are considered and the relevance of the protocol and design in addressing these research questions.

The nature of early-stage breast cancer is of particular importance to the researchers because it provides further evidence that at this stage, there is a greater potential to reduce the spread and limit the severity of the condition under certain circumstances (Ling et.al, 2012). Nonetheless, it is imperative to consider other factors which are representative of early-stage breast cancer, such as the potential for gene transcription factors to play a role in the development of breast cancer cell lines (Ling et.al, 2012). These factors also demonstrate that it is necessary for additional investigation into the elements which play into breast cancer cell development in female patients (Ling et.al, 2012). From the researcher’s perspective, it is necessary to develop a protocol that will examine the different causative factors of breast cancer, using a genetic approach to accomplish these objectives. It is possible that this type of investigation will shed new light on the scope and degree of influence of gene transcription factors in the formation of breast cancer cells and other factors associated with tumorigenesis (Ling et.al, 2012).

The study thoroughly examines the three transcription factors Oct3/4, Nanog, and Sox2, respectively and notes that they are derived from different genetic influences (Ling et.al, 2012). To be specific, the study notes that “Oct3/4, also known as OCT 3, OCT 4 and POU5F1, is one of the earliest transcription factors expressed in the embryo and is encoded by a homeobox-containing gene named Pou5f1 belonging to the family of Pit Oct Unc (POU) genes and recog­nized as fundamental in the maintenance of pluripotency and self-renewal in ESCs and in primordial germ cells” (Ling et.al, 2012, p. 1264). Nanog represents another form of genetic expression that is evident in some types of tumors (Ling et.al, 2012), but these tumor types are not addressed in detail in the study. Nanog and Oct3/4 have been identified as gene transcription factors in some forms of cancer, such as seminoma/germinoma as well as embryonal carcinoma in CNS and testicular forms (Ling et.al, 2012). Finally, Sox2 has also been identified in a number of tumors (Ling et.al, 2012), but the information regarding these tumor types is nonspecific. In order to conduct the study with the best possible sample, a heterogenous sample of human breast cancer cell lines was utilized to determine whether or not Oct3/4, Nanog, and/or Sox2 could be detected in these cell lines (Ling et.al, 2012).

The study method utilized the breast cancer cell lines known as MCF7, T-47D and MDA-MB-231, were cultured in standard media, and were grown under conditions that were optimal for growth (Ling et.al, 2012). The extraction of RNA was achieved using standard procedures to conduct a reverse transcription reaction and to analyze the transcription factors accordingly (Ling et.al, 2012).  In addition, staining was conducted using immunoflourescent techniques, with subsequent analysis to determine the impact of these transcription factors on the chosen breast cancer cell lines (Ling et.al, 2012). After the required tests were conducted, a statistical analysis was performed to determine standard deviations and statistically significant differences and was accomplished by using the SPSS 16 software program, with a P of less than 0.05 deemed statistically significant for further evaluation (Ling et.al, 2012).

In evaluating the study results, it is evident that MDA-MB-231 and MCF7 breast cancer cell lines had a greater prevalence of Oct3/4, while MCF7 and T-47D cells had a greater prevalence of Nanog (Ling et.al, 2012). Finally, Sox2 was detected more frequently in MCF7 cells (Ling et.al, 2012). These findings demonstrate that the researchers were valid in pursuing this evaluation because there was a consistency of all three gene transcription factors on the MCF7 cell lines, which represents a likelihood that these factors will lead to tumor development in this particular group of cell lines. In addition, the study noted that “The expression of various pluripotency marker genes was noted within the three breast cancer cell lines, suggesting that these pluripotency marker genes are reactivated during the process of breast cancer development” (Ling et.al, 2012, p. 1266). Finally, Nanog was also highly prevalent in three cell lines, which demonstrates its potential to be a key factor in the development of breast cancer cells in some patients (Ling et.al, 2012).

The broader discussion of this topic addresses a number of key challenges for oncology researchers in their efforts to ask the appropriate research questions and to draw viable conclusions to build upon in future studies. Based upon the study results, it is expected that there will be a common association between ESCs and cancer cells, depending upon whether or not they share common characteristics (Ling et.al, 2012). These factors are also representative of ESC differentiation and the development of tumors in some patients (Ling et.al, 2012). The study results demonstrate that Oct3/4 plays a unique role in the expression and renewal of stem cells (Ling et.al, 2012); therefore, it is not surprising that these factors also play a role in breast cancer cell development because some of their characteristics are very similar (Ling et.al, 2012).

Finally, the presence of Nanog as a primary gene transcription factor represents its ability to differentiate and to contribute to cancer cell development (Ling et.al, 2012). In particular, Nanog has been identified in osteosarcoma and is likely to be a contributing factor to breast cancer cell development (Ling et.al, 2012). Sox2 represents another gene transcription factor that is capable of differentiation and cancer cell growth, particularly in the following manner: In breast cancer, Sox-2 has also been shown to be a possible driver of the basal-like phenotype and play an early role in breast carcinogenesis (Ling et.al, 2012, p. 1267). These conditions represent a means of exploring the different dimensions of breast cancer cell development and if overlap and/or duplication of these transcription factors plays a greater role in breast cancer tumorigenesis (Ling et.al, 2012).

In general, the article provides a number of positive merits from a research perspective because it addresses the significance of three separate transcription factors and their potential role in breast cancer cell development in greater detail. This information is relevant in testing because it reflects a capacity to conduct tests for breast cancer of the genetic variation to determine if patients currently without breast cancer might have a predisposition to develop breast cancer at a later age. This study serves as an important opportunity for researchers in future studies to explore the different dimensions of breast cancer tumorigenesis and to determine the impact of Oct3/4, Nanog, and Sox2 on this type of cell development. These efforts are critical to the overall success of genetic diagnostic tools and the predisposition to develop breast cancer at some point in the adult life span.

Finally, the study provides a number of key points that are worthy of merit and of further consideration in future studies. This study was limited to some degree but it provides a strong basis to explore this topic further in future studies and to perhaps evaluate other transcription factors in a similar manner. These efforts are important contributors to the analysis of genetic transcription factors and their role in many forms of disease. It is evident that with the conclusions drawn in this research study, additional issues should be explored in future studies to capture a greater understanding of gene transcription factors and their role in the formation of different types of cancer cells. With this framework, it is likely that future studies will examine the applicability of these and other transcription factors on other forms of cancer which impact males and females throughout the life span. The reversal of these transcription processes is well beyond the scope of this study, but is worthy of further evaluation in other studies.

Minor Comments

            The research article does provide a brief summary of the incidence of breast cancer throughout the world, with approximately 690,000 new cases per region ( Ling et.al, 2012). In addition, the author points out that cases involving breast cancer are more common in developed countries than those currently under development, and that the process of early detection has been quite useful in curbing the advancement of the disease in many specific cases, many early-stage cases are subject to a recurrent nature and are difficult to treat from a clinical perspective (Ling et.al, 2012). With the incidence of breast cancer quite high in many countries throughout the world, and in particular, developed countries, one might question if there are environmental factors or other considerations that are leading to increased numbers of cases in these developed countries (Ling et.al, 2012).These elements are critical to the successful understanding of gene transcription factors and their impact on cancer cell development and tumorigenesis in otherwise healthy patients. In addition, this information is likely to be beneficial in the examination of transcription factors in patients who have already been diagnosed with cancer in one form or another. It is important for the researchers to consider the impact of this type of evaluation on the incidence of breast cancer because of its significance to society and the loss of productivity and of life. Mortality rates for some forms of breast cancer are very high and warrant further attention. Therefore, it is the responsibility of researchers to take the data that is received and to make sense of it in the context of other types of cancer. Since cancer is such a critical factor in the health and wellbeing of millions of people throughout the world, this type of research is necessary on an ongoing basis.

Changes Required for Publication and Suggestions for Improvement

The research study by Ling et.al (2012) represents a challenge to these and other researchers to expand upon existing research questions and protocols in order to develop new methods and testing approaches for improved evaluation of the causes of breast cancer and other forms. In some ways, this research study provides significant insight into the work that is performed by three separate gene transcription factors. However, it is also important to recognize that the future of breast cancer research requires scientists to build upon existing frameworks such as the framework derived in this protocol and to determine how to best move forward to achieve greater results in future studies. These developments are particularly relevant because they reflect upon the challenging nature of breast cancer research and the creation of protocols which examine the specific genetic codes and frameworks which lead to various markers for breast cancer. These efforts are conducive to the creation of a research environment that supports expanded genetic research and testing in recognizing the risk factors associated with breast cancer in females. With this framework in mind, it is possible to develop new strategic approaches to accommodate patients who already have breast cancer so that new treatments are derived to treat their cases accordingly. At the same time, it is important to distinguish between these roles and the creation of breast cancer genetic testing mechanisms which will draw accurate conclusions regarding the potential risks of developing breast cancer in the adult years when a diagnosis is not imminent.

The development of advanced research methods to examine transcription factors in the development of breast cancer is quite promising and should continue to be examined in future studies. This study only begins to shed some light on this topic and its value to the study of all types of cancers. The researchers performed the study well and addressed its most relevant points, thereby leading to greater insight regarding genetic factors associated with breast cancer. However, this study only scratched the surface of what is necessary to fully develop a genetic testing and evaluation framework for breast cancer, as well as for other forms of cancer. It is important to recognize the value of gene transcription mechanisms as a means of promoting effective outcomes in the treatment of one or more forms of cancers. Perhaps most importantly, this type of research protocol could be a highly effective tool in conducting pre-genetic screening mechanisms to detect the potential to form breast cancer cells or other forms of cancer cells. These elements support the development of new ideas and approaches to support the possible reversal of genetic transcription factors which serve as precursors to breast cancer cells and tumorigenesis.

With respect to the study and its research questions and methods, this study provides a valid starting point in the development of other studies of a more advanced or specific nature which attempt to answer questions regarding breast cancer cell development in females. This information serves as a guide in facilitating the development of other studies with similar objectives and also serves to promote effective outcomes in treating breast cancer and other types of cancer patients. The nature of genetic testing includes the development of new ideas and approaches to determine how genetic material is formed and modified to create the formation of potential cancer cells in patients. The specificity of genetic material is difficult to measure without advanced testing procedures and equipment to achieve the desired research objectives. Therefore, it is important for researchers to take steps forward by using this type of research as a guide. The research article in question demonstrates that pleuripotent genetic factors play a role in the formation of precursor cancer cells and tumorigenesis. It is important for researchers to recognize this relationship and how to cultivate it in other studies that capture an extension of the research provided in this article.

Finally, it is important to demonstrate that there are a number of challenges that this article presents because it only emphasizes three transcription factors. However, the combinations of transcription factors and subsequent traits are so significant that duplication of this study with other transcription factors may be very difficult. Therefore, its duplicity is called into question. In addition, the ability of this research study to be duplicated in other types of cancer cell lines is uncertain, although practical knowledge would indicate that this is possible. In general, the article only emphasizes a very small segment with three transcription factors and a small number of breast cancer cell lines; therefore, additional research in this area is required to ensure that patients who face the possibility of breast cancer or who already have the disease are provided with many options to treat their conditions rather than a single option or no hope at all. The study is advanced in some ways, but weak in others and requires further reflection and analysis from other researchers in order to accomplish the objectives that were sought at the beginning.

References

Ling, G.Q, Chen, D.B., Wang, B.Q., and Zhang, L.S. (2012). Expression of the plurpotency markers Oct3/4, Nanog and Sox2 in human breast cancer cell lines. Oncology Letters, 4, 1264-1268.