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.
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
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. Our laboratory has found that repression of the tumor suppressor p16 in human mammary epithelial cells (HMECs) endows them with specific properties that are only found in classical stem cells and tumor cells. Indeed, repression of p16INK4a in HMECs enables them to grow in culture for a long time, something that HMECs expressing p16INK4a cannot achieve. Importantly, we have previously shown that repression of p16INK4a is accompanied by the acquisition of pre-malignant features.
Thanks to the support of this CIRM grant, we have now established that a sub-population of these cells display stem cell properties. This means that these cells can self-renew but also differentiate in different breast cell types. Unexpectedly, these cells can also give rise to non-breast cells, such as brain cells, when grown in the appropriate cell culture conditions, making this unique cell model a powerful tool for cancer AND regenerative medicine research. Knowing that these cells can generate cells of different tissue types, we can now dissect the rules that dictate those different cell fates. We are also testing whether these exciting findings obtained in cell culture dishes (in vitro) can be confirmed in a mouse model (in vivo). In other words, can these cells generate a functional mammary gland? Other studies, beyond the scope of this application could also test whether these cells could rescue spinal injury.
So why do we bother using breast cells to generate brain cells (or other types of cells)? The answer is that we believe that the sub-population of cells we have identified in breast likely exists as a stem cell pool in any tissue (with some tissue-specific variations of course). If this hypothesis is confirmed, these cells could turn out to represent a major advancement in regenerative medicine. Another major advantage of these naturally occurring stem cells, compared to the widely used embryonic stem cell lines, is that they are directly isolated from fresh breast tissue without introducing artifacts that may result from establishment in long-term cell culture systems. Their properties are an accurate reflection of a fully functional stem cell pool actually existing physiologically in our body.
Understanding how stem cells code their decisions and whether cell fate can be changed after it has been set is key to the effective use of stem cells for therapeutic purposes. Gaining such insights will greatly improve our ability to manage wound repair and organ replacement. This should also help us characterize fundamental switches that control stem cells as well as control the formation of cancer cells since some of the genes that control stem cell properties are mutated in cancer. A mechanistic understanding of how these switches work may help us prevent adverse events that may result from the use of stem cells during regenerative medicine. Thus, we hope to contribute in improving the health of the citizens of California.
An important feature of adult stem cells is the ability to bypass negative growth signals and participate in wound healing. Based on this premise, we identified a small subpopulation of human breast epithelial cells that is capable of bypassing negative growth signals. We identified a differential expression of genes that allowed for the rapid isolation of this novel somatic cell population from fresh disease-free human breast tissue. Importantly, this cell population is characterized by the over-expression of Bmi-1, a protein that plays an essential role in the self-renewal of stem cells and represses the cell cycle inhibitor, p16. This population of cells is therefore poised to express pluripotency markers at a level similar to that measured in human embryonic stem cells. It has the ability to self-renew and can express phenotypes of any of the three mammary lineages in vitro using cell culture differentiation assays. Importantly, these cells are also functional in vivo as observed after implantation in mice. Indeed, these human cells can differentiate into functional mammary outgrowths of human origin in the host mouse as we could document secretion of human milk in mice transplanted with these human somatic cells. We are currently investigating whether these cells can also differentiate into other lineages (tissue types) when cultured in the appropriate conditions. Our preliminary studies support that these cells will hold great promise in regenerative medicine and cell replacement therapy and may help overcome some of the important ethical and technical roadblocks related to the use of human embryonic stem cells.