Summary of Research
Cyclin D1 causes breast cancer by overexpression of its normal mRNA and protein. Cyclin D1 was initially identified by translocations into the parathyroid hormone locus in parathyroid tumors in work first accomplished at MGH. Importantly, cyclin D1 was particularly interesting because it was the first cyclin linked by cytogenetic evidence to cancer. Given the cytogenetic evidence that cyclin D1 lay in a key site on the 11q13 amplicon, it became especially important to show that cyclin D1 could cause cancer. Our MMTV-cyclin D1 transgenic mice were the first clear demonstration that a cyclin could be an oncogene (1). Using an improved affinity-purified antibody, we demonstrated that additional mechanisms can increase cyclin D1 expression in breast cancer (2). Using modeling studies in animals as a key in vivo test for oncogenic function, we collaborated with Dr. Andy Arnold to demonstrate that cyclin D1 has an important role in vivo in parathyroid function (3).
Genetic interactions of cyclin D1. Cyclin D1 knockout studies show that it is not required for cell division, indicating that it is primarily a regulatory molecule. It is the downstream connector to cell division in many signaling pathways. In particular, erbB2 signals through cyclin D1 in mouse mammary tumors. We examined this phenomenon in depth comparing human and mouse tumors (4). We found that the p16INK4A tumor suppressor that blocks cdk4/6 blocks tumor formation in MMTV-erbB2 mice using an MMTV-p16 transgene. However, the situation is more complex in human tumors. We have shown a well-described dissociation between cyclin D1-expressing and erbB2-positive tumors, which is particularly noteworthy because cyclin D1 overexpression is narrowly confined to the ER positive subset of tumors. Importantly, our study suggested that erbB2 is more directly controlling the cell cycle through other mechanisms because its overexpression was non-redundant with cyclin D1 overexpression, loss of p16 or loss of pRb. To find translational correlates for our findings, we evaluated a therapeutic agent that blocks cdk4 activity, flavopiridol, and found that it was synergistic with trastuzumab in treatment of erbB2 positive breast cancers (5, 6).
Since cyclin D1 likely functions in a particular genetic context, we have tested a variety of other possible genetic interactions. First, cyclin D1 works downstream of NFkB, especially in mammary development (7). Second, the oncoprotein kinase chaperone CDC37 functions as an oncogene in mice and collaborates with cyclin D1 in transformation of multiple tissues (8). We have found no evidence that it collaborates with p53 loss in tumorigenesis (9).
Regulation of cyclin D1. The G1 cyclins are positioned in cell cycle control to monitor the cellular environment and determine the rate of passage through G1, after which cells become committed to completion of cell division. We have long been interested in connections between cell growth and cell division because cells must first grow before they can divide (10-12). We first became interested in this problem when we found that the c-myc oncogene works in part by controlling cell growth through regulation of the translation initiation factor eIF4E (13, 14). To better evaluate the specificity of the effects of an otherwise general translation initiation factor we became interested in its ability to specifically enhance cyclin D1 protein levels without affecting cyclin D1 mRNA levels (15). This translational control has since been confirmed in many other labs, and appears to be related to effects on nuclear-cytoplasmic export of the cyclin D1 mRNA (16). We have further evaluated the expression patterns of cyclin D1 during pregnancy, and found an interesting cessation of cyclin D1 expression during mammary involution that may be important to the terminal differentiation of mammary glands after a first pregnancy (17).
Functions of cyclin D1. Our study of cyclin D1 expression during mammary involution suggested that it may contribute to tumorigenesis by altering cell differentiation and demonstrated a significant function for p16INK4A in post-natal mammary differentiation (17). These regulatory mechanisms used during mammary involution offered a potential explanation for the protective effect of pregnancy against breast cancer. Our MMTV-cyclin D1 mice develop mammary tumors after a prolonged latency of nearly 18 months, but with an eventual penetrance of around 50%. The MMTV-transgene’s effects on involution may be especially important in our model because no tumors have developed if a mouse has not gone through at least one pregnancy. Interestingly, our model is somewhat unique for mouse transgenics because the cyclin D1 tumors are estrogen receptor positive (18), like their human counterparts. The long latency of MMTV-cyclin D1 induced tumor formation particularly implies that additional genetic events are required to collaborate in mammary carcinogenesis. We therefore used laser capture microdissection to attempt to define the genetic differences between non-invasive hyperplastic tissues driven by cyclin D1 expression and invasive tumor tissues in the MMTV-cyclin D1 mice (18). Importantly, we further tested the genes identified in mouse microarray studies in human breast cancers expressing cyclin D1 in LCM-isolated tissues from patient-matched normal, ductal carcinoma in situ, and invasive ductal carcinoma. We identified higher expression levels of immediate early response protein IEX-1, small stress protein 1 (HSPB8), and tumor necrosis factor-associated factor-interacting protein mRNAs in invasive lesions. These genes induced anchorage independence, increased cell proliferation, and protected against apoptosis, singly or in collaboration with erbB2. Importantly, they were all up-regulated by 17beta-estradiol and cyclin D1, and cyclin D1 overexpression increased p300/CBP binding to their promoters, supporting a novel model that cyclin D1-estrogen receptor (ER) coactivator interactions are important in ER-positive breast cancer. The heat shock protein B8 (HSPB8) is particularly associated with cyclin D1 and ER positive tumors, and we recently found that it can mediate cyclin D1’s enhancement of radiation sensitivity (19). We are actively exploring the possible contributions of additional cyclin D1- and ER-regulated genes to its oncogenic functions.
References:
1. Wang TC, Cardiff RD, Zukerberg L, Lees E, Arnold A, Schmidt EV. Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature 1994;369(6482):p669-71.
2. Zukerberg LR, Yang WI, Gadd M, et al. Cyclin D1 (PRAD1) protein expression in breast cancer: approximately one-third of infiltrating mammary carcinomas show overexpression of the cyclin D1 oncogene. Modern Pathology 1995;8(5):560-7.
3. Imanishi Y, Hosokawa Y, Yoshimoto K, et al. Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice. J Clin Invest 2001;107(9):1093-102.
4. Yang C, Ionescu-Tiba V, Burns K, et al. The role of the cyclin D1-dependent kinases in ErbB2-mediated breast cancer. Am J Pathol 2004;164(3):1031-8.
5. Nahta R, Trent S, Yang C, Schmidt EV. Epidermal growth factor receptor expression is a candidate target of the synergistic combination of trastuzumab and flavopiridol in breast cancer. Cancer Res 2003;63(13):3626-31.
6. Nahta R, Iglehart JD, Kempkes B, Schmidt EV. Rate-limiting Effects of Cyclin D1 in Transformation by ErbB2 Predicts Synergy between Herceptin and Flavopiridol. Cancer Res 2002;62(8):2267-71.
7. Cao Y, Bonizzi G, Seagroves TN, et al. IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 2001;107(6):763-75.
8. Stepanova L, Finegold M, DeMayo F, Schmidt EV, Harper JW. The oncoprotein kinase chaperone CDC37 functions as an oncogene in mice and collaborates with both c-myc and cyclin D1 in transformation of multiple tissues. Mol Cell Biol 2000;20(12):4462-73.
9. Hosokawa Y, Papanikolaou A, Cardiff RD, et al. In vivo analysis of mammary and non-mammary tumorigenesis in MMTV-cyclin D1 transgenic mice deficient in p53. Transgenic Res 2001;10(5):471-8.
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11. Schmidt EV. The role of c-myc in cellular growth control. Oncogene 1999;18(19):2988-96.
12. Polymenis M, Schmidt EV. Coordination of cell growth with cell division. Curr Opin Genet Dev 1999;9(1):76-80.
13. Jones RM, Branda J, Johnston KA, et al. An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc. Mol Cell Biol 1996;16(9):4754-64.
14. Rosenwald IB, Rhoads DB, Callanan LD, Isselbacher KJ, Schmidt EV. Increased expression of eukaryotic translation initiation factors eIF-4E and eIF-2 alpha in response to growth induction by c-myc. Proc Natl Acad Sci U S A 1993;90(13):6175-8.
15. Rosenwald IB, Lazaris-Karatzas A, Sonenberg N, Schmidt EV. Elevated levels of cyclin D1 protein in response to increased expression of eukaryotic initiation factor 4E. Molecular & Cellular Biology 1993;13(12):7358-63.
16. Rosenwald IB, Kaspar R, Rousseau D, et al. Eukaryotic translation initiation factor 4E regulates expression of cyclin D1 at transcriptional and post-transcriptional levels. J Biol Chem 1995;270(36):21176-80.
17. Gadd M, Pisc C, Branda J, et al. Regulation of cyclin D1 and p16(INK4A) is critical for growth arrest during mammary involution. Cancer Res 2001;61(24):8811-9.
18. Yang C, Trent S, Ionescu-Tiba V, et al. Identification of cyclin D1- and estrogen-regulated genes contributing to breast carcinogenesis and progression. Cancer Res 2006;66(24):11649-58.
19. Trent S, Yang C, Li C, Lynch M, Schmidt EV. HSPB8, a CDK-independent cyclin D1 target gene, contributes to its effects on radiation sensitivity. Cancer Research 2007;Revised manuscript under review.
