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INT6 is a Potential Breast Tumor Suppressor Gene that Regulates the Proteasome
Background – Mouse mammary tumor virus (MMTV) has been used as an insertional mutagen to identify genes that are pivotal for breast tumorigenesis. “INT” genes are loci where MMTV integration can induce tumor formation. All well-characterized INT genes encode proteins with tremendous relevance to oncogenesis, e.g., aromatase (essential for estrogen synthesis), FGF, Notch, and Wnt. In mouse breast tumors, MMTV insertions in INT6 create C-terminally truncated proteins (Int6?C) that are dominant-negative, and in human breast tumors, INT6 expression is frequently diminished. Moreover, MMTV insertion in INT6 leads to mammary tumors in a feral strain of mice with no known tumor propensity and no insertion into another INT loci, so that INT6 inactivation by itself appears to be a potent promoter of tumorigenesis. Nevertheless, the role of INT6 in breast tumor formation is poorly defined. We have characterized the INT6 homolog yin6 in the fission yeast genetic model system. We discovered that Yin6 binds and positively controls assembly of the proteasome, which functions to degrade mitotic regulatory proteins that are marked by polyubiquitination. Yin6 inactivation weakens the proteasome and causes accumulation of mitotic regulatory proteins, some of which, e.g., securin, an oncoprotein that is overexpressed in breast cancer, lead to chromosome instability. Human and yeast Int6 are 43% identical in primary sequence, and full-length human Int6, but not Int6 deltaC, can fully substitute for yeast Yin6 to mediate proteasome function and chromosome segregation, indicating that Int6 functions are highly conserved across species.
Objective and Hypothesis – We hypothesize that INT6 is a tumor suppressor gene for breast cancer by regulating the proteasome. Int6 inactivation can promote tumorigenesis by allowing accumulation of mitotic regulators, thus globally disrupting cell functions (genetic stability and cell division). Our objective is to test this idea in human mammary epithelial cells (HMEC’s) and breast tumors.
Specific Aims – We plan to determine whether weakening Int6 functions can influence (1) cellular transformation and chromosome stability, and (2) proteasome functions. To further examine the direct relevance of Int6 regulation of the proteasome to breast cancer, we will (3) determine if its C-terminal domain, which is essential for its activity and its proteasome regulation in yeast, is important for Int6 function in HMEC’s, and whether this domain is mutated in human breast tumors. Finally, we plan to (4) further delineate how Int6 may regulate the proteasome by identifying Int6 binding proteins.
Study Design – We will first weaken Int6 functions by using the RNAi technology to reduce its expression. Additionally, we will interfere with endogenous Int6 function by expressing Int6 deltaC, since this is thought to function in a dominant-negative manner. We will examine INT6-deficient cells to determine whether they are transformed and whether they show chromosomal instability and abnormal mitosis. Additionally, we will examine whether Int6 associates with the proteasome in normal cells; in parallel, we will analyze INT6-deficient cells for proteasome dysfunction by monitoring accumulation of polyubiquitinated proteins and mitotic regulators and the integrity of the proteasome. C-terminal truncation of Int6 leads to breast cancer formation in mice, and we have identified a specific conserved amino acid residue in the C-terminus that is essential for its function in yeast. Thus, we will express these mutated Int6 proteins in HMEC’s to determine if they induce cell transformation and proteasome abnormalities. In addition, we will screen breast tumor DNA for mutations altering the C-terminus. Finally, we will identify Int6-binding proteins by Int6 pull-down and high resolution mass spectroscopy.
Potential Outcomes and Benefits – Confirmation of our model would identify a novel breast tumor suppressor, and this would allow INT6 inactivation to be monitored as a risk factor for breast cancer. The proteasome is already emerging as a target for cancer treatment (e.g., PS-341 is now in clinical trials). Establishing a direct link between the proteasome and genetic stability via Int6 could provide a new angle for understanding tumor suppression mechanisms and targets for cancer prevention/treatment.
Conversion of a normal cell into a malignant one is usually a slow process because it requires numerous alterations, and each of these changes is in itself a rare event. Consequently, the chance of developing a full-blown tumor within a lifetime is greatly increased if the rate of such changes is increased. Inactivation of tumor suppressors frequently causes a reduction of genetic stability and an accelerated global change of cell growth and behavior, which facilitate tumorigenesis.
We originally set out to study the regulation of cell growth using yeast, because the mechanisms governing cell division are very similar between yeast and mammals. In fact, the current understanding of how cell division is regulated in human cells was established based on pioneering work in yeast, for which a Nobel prize was awarded in 2001 to scientists at the Imperial Cancer Research Fund and the Fred Hutchison Cancer Center.
During the course of studying growth regulation in yeast, we discovered a gene that is the yeast version of a mammalian gene, INT6, which has been directly implicated in breast cancer. INT6 belongs to a group of INT genes that were first discovered as integration sites for the Mouse Mammary Tumor Virus (MMTV) in mouse breast tumors. MMTV integration can alter gene functions by reprogramming the level of gene expression, or by disrupting the gene sequence leading to the production of shortened proteins, or both. Studies of other INT genes have consistently provided valuable insights into cancer biology (e.g. one encodes the aromatase, a rate-limiting enzyme for estrogen synthesis). However, very little is known about the function of INT6, even though a reduction in INT6 expression is often found in human breast tumors. Moreover, MMTV insertion in INT6 leads to mammary tumors in a wild strain of mice with no known tumor propensity and no insertion into another INT loci, so that INT6 inactivation by itself appears to be a potent promoter of tumorigenesis.
Our data indicate that a key function of INT6 is to activate the proteasome, which in turn regulates the level of numerous regulatory proteins central for cell growth control. If the proteasome is inactivated, these regulatory proteins accumulate to dangerously high levels, thus disrupting how cells normally divide. For example, our data suggest that the securin protein (an oncoprotein in humans that is overexpressed in breast tumors) accumulates as a result of INT6 inactivation, and this in turn causes chromosome missegregation. Chromosome abnormalities are hallmarks of human tumors, and this supports the idea that chromosome abnormalities and the associated genetic instability can allow mutations favorable for tumor formation to occur efficiently. Based on these results, we hypothesize that INT6 is a tumor suppressor gene for the development of breast cancer. INT6 inactivation may impede proteasome functions, thus globally disrupting cell activities such as maintenance of chromosome stability, and promoting tumorigenesis. The goal of this proposal is to test this hypothesis in human mammary cells and breast tumors. Specifically, we plan to inactivate INT6 in normal human mammary cells and then determine whether growth regulation, genetic stability, and proteasome function are affected. We also plan to examine whether mutations inactivating INT6 occur frequently in human breast tumors. Breast cancer is among the most complex of cancers, and so far there is no single molecular marker that can be monitored to cover the general population. For example, mutations in BRCA1 and BRCA2, the two best documented breast tumor suppressor genes, are found in only 5-10% of all breast tumors. If INT6 can be proven to be a novel tumor suppressor, its functionalities can be monitored as an additional risk factor. A good understanding of tumor biology can lead to rational therapeutics. Indeed, drugs interfering with proteasome functions (e.g., PS-341) have already entered clinical trials; thus, it could be possible to target the Int6 proteasome interaction to prevent or treat breast cancer.