Role of the tumor suppressor gene, p16INK4a, in regulating stem cell phenotypes in embryonic stem cells and human epithelial cells.
The roles of stem cells are to generate the organs of the body during development and to stand ready to repair those organs through repopulation after injury. In some cases these properties are not correctly regulated and cells with stem cell properties expand in number. Recent work is demonstrating that the genes that control stem cell properties are sometimes the same genes that are mutated in cancer. This means that a cell can simultaneously acquire stem cell properties and cancer properties. In order to effectively use stem cells for therapeutic purposes we need to understand the link between these two programs and devise ways to access one program without turning on the other. In other words, we would like to expand stem cell populations without them turning into cancer. Recent work in our laboratory has found that the reduction of a specific tumor suppressor gene, p16, not only removes an important barrier to cancer but also confers stem cell properties within the cell. Cells that have reduced p16 activity can turn on a program that increases and reduces expression of specific genes that control differentiation. In this proposal we will test whether the continued reduction of this tumor suppressor gene creates human embryonic stem cells (hESC) that are unable to differentiate. We hypothesize that the lack of p16 represses multi-lineage potential by activating an epigenetic program and silencing genes that drive differentiation. To test this hypothesis we will first determine if lack of p16 activity is necessary for hESCs to develop into different cell types. Second, we will determine if continued lack of p16 activity is sufficient to inhibit differentiation of hESCs. Finally, we will determine if transient lack of p16 activity is sufficient for a non-stem cell to exhibit properties of a stem cell after propagation in a stem cell niche. Since these types of events are potentially reversible, targeting such events may become clinically useful. These new observations identify novel opportunities. They provide potential markers for determining if someone is susceptible to cancer, as well as, providing potential targets for prevention and therapy. We hypothesize that these properties are critically relevant to the formation of cancer and will provide insights into the role of epigenetic modifications in disease processes and stem cell characteristics.
Statement of Benefit to California:
Stem cells hold great potential to help us in repairing injured body parts or replacing damaged organs. In order to realize this potential the rules that control stem cell behavior need to be understood. Recent work is demonstrating that the genes that control stem cell properties are sometimes the same genes that are mutated in cancer. In the proposed study we hypothesize that we may learn about a fundamental switch that not only controls stem cells but also controls the formation of a cancer cell. In understanding how this switch works we may be able to identify biomarkers that indicate when a normal looking cell will become a cancer cell or identify a drug that will allow us to stop the potential cancer cell from increasing in number. Since cancer is a common disease in California, any insights we can gain to battle this disease will benefit the citizens of our State. There is also another side to the insights that may arise from the work in this proposal. Currently we believe the roles of stem cells are to generate the organs of the body during development and to stand ready to repair those organs through repopulation after injury. We do not know how to encourage a stem cell to repair, for example, some heart tissue rather than some bone tissue. If we could understand the code that directs the stem cells to differentiate in the proper fashion into one tissue or another, we could use these cells for clinical benefit. The pathways we are studying in this proposal tell the stem cells which genes to silence and which to activate. This is the program that allows the one original cells of your body (the embryo) to diversify into the multitude of specialized cells that work together to make a functioning person (eye cells, skin cells, nerve cells, etc.). In order to effectively use stem cells for therapeutic purposes we need to understand how they code their decisions and whether they can be changed after they have been set. These insights would allow us to aid in maintaining the health of the citizens of California. Finally, if we do gain insight into the code that regulates the differences between cancer cells and stem cells, this information would be the basis of a new area of biotechnology. The generation of knowledge in this area would help in the development of companies, the recruitment of bright young minds and in the fiscal health of our State
Bmi-1 is a polycomb family transcription factor that represses expression of the INK4A/ARF locus. In mouse models expression of Bmi-1 is necessary for the self-renewal of stem cell populations in various organs including the hematopoeitic and central nervous systems. Thus, loss of Bmi-1 causes stem cell depletion, and this can be rescued at least partially by concomitant loss of p16Ink4a and Arf. Recently published work from the applicants laboratory suggests that repression of p16 by Bmi-1 leads to upregulation of certain E2F target proteins that are involved in histone methylation, and consequently leads to an increase in DNA methylation. The applicants hypothesize that this represses the expression of genes that drive differentiation, and thus may help to promote self-renewal of uncommitted progentior cells. Here, the applicants propose to test the hypothesis that Bmi-1 expression is important for human ES cell self-renewal, and that this is mediated by by repression of p16Ink4a. SIGNIFICANCE AND INNOVATION: The proposed line of studies has a high degree of innovation as it ties together two seemingly disparate lines of investigation -- studies of carcinogenesis mediated by suppression of tumor suppressor gene expression and the pathways that maintain pluripotency vs. drive cells to differente. It appears that the investigator has found an important switch in the carcinogenic process -- namely p16, suppression of which results in activation of a program of epigenetic remodeling of chromatin and DNA methylation. The series of studies here is aimed to determine whether the same pathway may be involved in hES cells. The results of these studies could yield important insights into how one could control either the state of pluripotency or differentiation in ES cell cultures. Specific Aim 3 has potentially high significance -- if the approach proposed by the investigator were to reprogram a differentiated cell, namely human mammary epithelial cells (HMEC), into an undifferentiated ES-like cell via epigenetic alterations induced by reduced p16 expression -- this would open the door to a new method to derive autologous human adult stem cell populations. STRENGTHS: The investigators strong background and experience in studying the p16 tumor suppressor gene and the impact of its expression (up or down) on the tumorigenic phenotype of mammary epithelial cultures. The preliminary data from that system provide very convincing data to suppport the hypothesis and proposed studies of the current grant proposal -- namely that similar pathways may be at play in both tumor cells and in hES cells. Aims 1 and 2 are important experiments that are highly feasible and very likely to succeed. In Aim 1 the applicant will knockdown expression of Bmi-1 in human ES cells and then measure proliferation, cell death, and differentiation into embryoid bodies. In Aim 2 the applicant will determine whether stable suppression of p16 interferes with the differentiation of human ES cells. This will be evaluated by using microarrays to asses the patterns of gene expression in embryoid bodies exposed to differentiation medium in the presence of absence of p16 knockdown. The applicants hypothesis is that upregulation of p16 is necessary to permit the reprogramming of chromatin structure and activation of genes necessary for differentiation. The applicant has extensive experience studying the effects of cell cycle pathways in cellular differentiation and tumorigenesis, and I would therefore anticipate that she will make important and creative contributions tO understanding the roles of these pathways in ES cell biology. WEAKNESSES: The research methods are a little vague regarding the introduction of the siRNA or shRNA constructs against BMI1 or p16. In particular, it's not clear whether transient transfection methods or lentivirus vectors will be used (both are listed). If the former, the investigator should be aware that transient expression may not be sufficient to allow them to address the studies that are proposed. In that if they really want to induce differentiation into embryoid bodies and assess the impact of the down-modulation of either BMI1 or p16 on the ability or the ES cells to differentiate, then the knock-down should be present throughout the full course of that experiment. The transient expression therefore may not be sufficient, and they should really be prepared to use a lentivirus vector for both of the gene knock-downs they want to do. Aim 3 is both a weakness and a strength. Here the applicant proposes to test the idea that enforced reduction of p16 would be sufficient to convert a human mammary epithelial cell into a pluripotent stem cell if it were cultured in a stem cell niche. This is a potentially high impact experiment. However, success of this experiment requires that the epigenetic changes dictated by p16 be sufficient to reprogram a fully differentiation cell into a pluripotent stem cell. This seems highly unlikely. It further requires the undefined participation of placental feeder cells, that are supposed to represent a “validated stem cell niche”. I am not clear at all about what this means, other than that these cells have previously been shown to support proliferation of ES cells (but so can MEFs). Most mammary epithelial cells growing in vitro have already silenced p16 expression, and at least under standard culture condiitions show no evidence of "reprogramming". So, somehow the idea must be that the feeder cells would provide a critical signal, but I have no idea what this would be. The experimental design is also problematic as it requires not only knockdown of p16, but then spontaneous reversion of the knockdown so that it can be tested whether the cells have acquired the ability to differentiate into multiple cell types. Reversion is not documented and its frequency is unknown. It would have been much better to design an inducible knockdown vector. DISCUSSION: This is a strong proposal from a strong investigator. Aims 1 and 2 are very clear and are based on work in the mouse. Aim 3 is risky, but is of high significance and innovation.