Nearly one out of every two Californians born today will develop cancer at some point in their lives, and it is likely that one in five persons will die of the disease. We propose to study the mechanisms of action of the RB gene, which is mutated in a broad range of human cancers, including pediatric cancers of the eye and the bone, and adult tumors such as lung, breast, prostate and liver cancers. RB normally acts as a tumor suppressor. When RB is mutated, cells lose the ability to sense when to cycle or not and they divide too much, thereby initiating cancer. Because RB is mutated in so many human cancers, therapies that could re-introduce RB function in cancer cells would benefit a great number of cancer patients. A key question is to determine in which cell type loss of RB function is most detrimental. Knowing the answer to this question would help to diagnose cancer early and target specific cells within tumors, making treatment more effective. Recent evidence suggests that loss of RB may initiate cancer in stem cells . Because human embryonic stem cells (hESCs) give rise to any other stem cells, we will study the role of RB in hESCs. The results of these experiments will thus be applicable to a broad range of human cancers. Specifically, we will use novel tools that will allow us to precisely alter RB levels in hESCs. We will then study the consequences of these manipulations for the proliferation of these cells; lower levels of RB may promote proliferation, while higher levels of RB may slow proliferation and push these embryonic stem cells to become more mature. We will then investigate the molecular mechanisms underlying these observations, beginning with the cellular pathway leading to retinal development because of RB’s involvement in retinal cancer. Because RB is usually deleted in cancer cells, there is no simple way to re-express RB function in these cells. However, two genes related to RB, p107 and p130, are rarely deleted in cancers and can compensate for loss of RB in mouse cells. Therefore, we will also study the role of p107 and p130 in hESCs, to determine if the functions of these two genes also overlap with RB function in these human cells and their progeny. If this is the case, knowing how to control the expression of p107 and p130 in hESCs may result in the development of a novel therapeutic strategy against human cancers associated with loss of RB. A better knowledge of the cells from which cancer arises and of the molecular mechanisms by which cancer initiates will lead in the future to the development of novel means to diagnose cancer earlier, thereby increasing the chances of a successful therapy. In addition, because of the central role of RB family members in multiple cellular functions, these experiments in hESCs may provide novel insight into the basic biology of these stem cells, which will eventually allow us to manipulate these cells more efficiently to treat a broad range of human diseases.
Statement of Benefit to California:
Despite significant decreases in the incidence and mortality rates of cancers in California over the past decade, nearly one out of every two Californians born today will still develop cancer at some point in their lives, and it is likely that one in five persons will die of the disease. Overall, in 2007, more than 50,000 people will die of cancer in California. These statistics underscore the need for the development of novel approaches to detect and treat human cancers. Stem cells hold the promise of treatments and cures for human diseases that affect millions of people. In particular, recent models suggest that cancer may arise from mutant stem cells whose progeny form the bulk of the tumor. Thus, in the future, one anti-cancer strategy may be to replace mutant stem cells in patients with normal stem cells. Another approach may be to repair the defects in these mutant stem cells. However, these approaches will only be possible when the mechanisms controlling the proliferation of these stem cells and their capacity to produce their functional progeny are better understood under normal and pathological conditions. We propose to study the mode of action of a key cancer gene, the RB gene, in human embryonic stem cells (hESCs). RB is inactivated in a broad range of human tumors, including adult lung, brain, breast, and prostate cancers, as well as pediatric eye and bone tumors. Thus, RB is a major target for the development of therapeutic strategies that may benefit a wide range of cancer patients. However, the mechanisms by which RB mutation triggers cancer are still poorly understood, hampering the development of such anti-cancer strategies. We believe that by studying RB function in hESCs, we will gain novel insights into both the mechanisms of action of RB and the biology of these stem cells. Exploring the effects of altering RB levels in hESCs will increase our knowledge of RB's mode of action and will eventually provide new ways to treat human cancers. In addition, these experiments may identify novel means of manipulating hESCs to control the fate of these cells when transplanted into patients. Because hESCs have the capacity to form any type of cell in the human body, these experiments will be relevant to the numerous cancer types associated with loss of RB function and may be ultimately translated into novel anti-cancer strategies. In addition, the results of these experiments may lead to novel avenues of research and may lay the groundwork for the development of therapies against diseases occurring in organs in which RB plays a central role, such as the eyes and the bones. Thus, the proposed research may benefit a broad range of patients, from young children to senior citizens, in California and elsewhere.
SYNOPSIS: This project will look at roles for the RB gene in hESC growth and differentiation. In particular, RB and its family members p107 and p130 will be studied with regard to mechanisms of cell cycle progression in hESCs by way of: (1) Controlling the expression of RB family genes in hESCs; (2) Investigating how RB family members control cell cycle progression in hESCs; and (3) Determining how changes in RB family members affect the differentiation of hESCs. SIGNIFICANCE AND INNOVATION: This proposal is both innovative and significant because it will use different hESC lines for the first time to study a well known tumor suppressor gene (RB) that could lead to the development of better therapies for human cancers known to have lost RB, and to potentially gain insights into the control of the inherent tumorigenicity of hESCs which is a major problem in transplantation paradigms. It is also innovative and significant given the state of the field regarding the cell cycle and pluripotency of hES cells. STRENGTHS: As pointed out by the PI, “…Nothing is known about the role of RB family proteins in early human development or in hESCs…” Since RB normally acts as a transcriptional regulator and its loss can result in many cellular defects including hyperproliferation, lack of differentiation, and genomic instability (hallmarks of oncogenic transformation), its relevance toward an understanding of developmental biology and tumor biology, in ES cell studies as proposed here, is tremendous. Cell cycle progression in hESCs is understudied, and the present model is extremely well-suited for gleaning important normal and abnormal cell cycle events, related to RB expression, in different hES cell lines. Previous studies of hES cell cycle mechanisms have focused on only two lines that could skew our understanding of hES cell cycle regulation; the present proposal will examine several lines. Not only will this proposal examine consequences of altering RB family function in hES cells by way of looking at cell cycle control (e.g. G2/M transition), but also self renewal abilities (Oct 3/4 and Nanog expression) AND fate choice and differentiation by way of looking at ectoderm, mesoderm, and endoderm markers and potential altered differentiation into retinal cells (Reh collaboration). The application, from a gifted young PI, is extremely well-written, planned, and justified. The investigators have extensive experience in understanding Rb/pocket proteins in a number of other cellular contexts. Moreover, the proposal appears highly collaborative bringing together groups with distinct, but complementary strengths. The rational for CIRM support is strong, e.g. use of several independent hESC lines (including non-NIH approved lines), and the need to develop novel tools before going on to study something not known to be involved in hES cell development (therefore risky). WEAKNESSES: The PI has no experience at all on working with hESCs; however, a letter of support from Dr. Baker, who has apparently derived novel hESC lines (although all publications listed are on Xenopus), does help the cause, but the PI will be relying heavily on the collaboration with Dr. Baker. The mouse feeder layer could add problems in interpretation amongst other things to these hESC studies. The PI acknowledges this and rather boldly proposed to employ new feeder-free technologies (without details or experience). One other concern was the fact that there was no mention of the phenotype of the triple Rb knockout mouse ESCs (previously published) and whether the investigators expect a similar or different phenotype in hESCs. DISCUSSION: This proposal aims to study Rb in hESCs, specifically to look at a role for Rb in hESC growth and differentiation, in cell cycle progression control, and to assess changes in Rb and resultant differentiation effects in hESCs with the goal of finding better therapies for cancers and to provide insight on tumorigenicity. Studying Rb function in hESCs has significant potential to impact better therapies for types of cancer that have lost Rb and so the project is extremely innovative. The PI proposes a well-planned and unique cell cycle examination in several hESC lines. One strength is that nobody knows anything about the role of Rb in hESCs or a role for Rb during development. In addition, the cell cycle progression in hESCs is understudied. Weakness: the PI has no experience with hESCs; mouse feeders could significantly complicate this project. The secondary reviewer felt that understanding cell cycle in hESCs is a hot topic in the field. Because the Rb/pocket proteins are at the heart of the G2/G0 transition, they may play an important role in tumorigenicity. This study may shed light on how the Rb proteins contribute to tumorigenicity. The applicant is a leader in the Rb field and the lack of experience may not be overly problematic. A minor criticism - the applicant neglected to mention a study of the Rb protein in mESC. There were only minor technical issues highlighted during discussion. The PI has no experience with hESCs, and despite the collaboration letter from Dr. Baker, one reviewer recommended that he take a good hESC course.