Chromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1 -/-mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNAbound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1-rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland-targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability. IntroductionChromosomal instability (CIN) in tumors (1-3) is characterized by an elevated rate of gain or loss of whole chromosomes (i.e., aneuploidy) and/or as structural chromosomal aberrations (i.e., translocations, deletions, and duplications). Aneuploidy is one of the most striking differences between cancer and normal cells. The molecular mechanisms inducing CIN as well as the timing of CIN in tumor progression, invasion, and metastasis is poorly understood (4, 5). Cell cycle-associated factors have been implicated in CIN, including cyclin E (6). The relative enrichment of a molecular genetic signature of CIN-related genes has been used to quantitate a CIN score (7); this signature includes AURKB (a component of the chromosomal passenger complex [CPC]), TOP2A, CENPP, MLF1IP (a component of the CENPA-NAC kinetochore complex protein), ZW10 (a kinetochore-associated mitotic checkpoint protein), and CKAP2 (a mitotic spindle-associated protein) (3) as well as the retinoblastoma (pRb) protein. Supernumerary centrosomes increase the frequency of dual attachment of 1 sister kinetochore to 2 spindle poles. Cyclin E activity promotes centrosome duplication during S phase onset. Loss of pRb can also alter centrosome number and formation of micronuclei, leading to

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Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1−/− mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1WT or cyclin D1KE in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1KE induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1WT and cyclin D1KE to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

Polyhomeotic-like 3 (PHC3) is a ubiquitously expressed member of the polycomb gene family and part of the human polycomb complex hPRC-H. We found that in normal cells PHC3 associated with both hPRC-H complex components and with the transcription factor E2F6. In differentiating and confluent cells, PHC3 and E2F6 showed nuclear colocalization in a punctate pattern that resembled the binding of polycomb bodies to heterochromatin. This punctate pattern was not seen in proliferating cells suggesting that PHC3 may be part of an E2F6-polycomb complex that has been shown to occupy and silence target promoters in G 0 . Previous loss of heterozygosity (LoH) analyses had shown that the region containing PHC3 underwent frequent LoH in primary human osteosarcoma tumors. When we examined normal bone and human osteosarcoma tumors, we found loss of PHC3 expression in 36 of 56 osteosarcoma tumors. Sequence analysis revealed that PHC3 was mutated in nine of 15 primary osteosarcoma tumors. These findings suggest that loss of PHC3 may favor tumorigenesis by potentially disrupting the ability of cells to remain in G 0 .

Secretory phospholipase A2 (sPLA2-IIA) has been shown to attenuate intestinal tumorigenesis in Apc(Min) mice, demonstrating that it is a tumor modifier. To further explore the actions of sPLA2-IIA in tumorigenesis, sPLA2-IIA was overexpressed in two cell lines where it is normally absent, the murine colon tumor cell line AJ02nm0, and human colon carcinoma cell line HCT-116. Two allelic variants of sPLA2-IIA were tested in this study; sPLA2-IIA(AKR) and sPLA2-IIA(SWR), which are derived from AKR/J and SWR/J mice, respectively, and differ by a single amino acid at position 63 in the calcium- and receptor-binding domain. There was no change in cell-doubling time for either allele when compared to vector controls. Furthermore, sodium butyrate and arachidonic acid (AA)-induced cell death were unchanged in control and transfected cells. Addition of the sPLA2 substrate, palmitoyl-arachidonoyl-phosphatidic acid (PAPA), to AJ02nm0 cells resulted in a modest (12%-24%), but significant (P < 0.01), inhibition of growth that was dependent on sPLA2-IIA expression. However, when AJ02nm0 and HCT-116 cells were injected subcutaneously (sc) into nude mice, Pla2g2a expression resulted in a 2.5-fold increase in tumor size. In addition, sPLA2-IIA expressing HCT-116 tumors were found to be more infiltrative than controls. We conclude that the ability of sPLA2-IIA to slow tumor cell growth is dependent upon the availability of substrate, and that in some instances sPLA2-IIA may actually enhance tumor growth. Mechanisms that may account for differences between the tumor explant model versus the Apc(Min) model of intestinal cancer are discussed.