The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that drives cell autonomous cell cycle progression and proliferation. Herein we show cyclin D1 abundance is increased >30-fold in the stromal fibroblasts of patients with invasive breast cancer, associated with poor outcome. Cyclin D1 transformed hTERT human fibroblast to a cancer-associated fibroblast phenotype. Stromal fibroblast expression of cyclin D1 (cyclin D1Stroma) in vivo, enhanced breast epithelial cancer tumor growth, restrained apoptosis, and increased autophagy. Cyclin D1Stroma had profound effects on the breast tumor microenvironment increasing the recruitment of F4/80+ and CD11b+ macrophages and increasing angiogenesis. Cyclin D1Stroma induced secretion of factors that promoted expansion of stem cells (breast stem-like cells, embryonic stem cells and bone marrow derived stem cells). Cyclin D1Stroma resulted in increased secretion of proinflammatory cytokines (CCL2, CCL7, CCL11, CXCL1, CXCL5, CXCL9, CXCL12), CSF (CSF1, GM-CSF1) and osteopontin (OPN) (30-fold). OPN was induced by cyclin D1 in fibroblasts, breast epithelial cells and in the murine transgenic mammary gland and OPN was sufficient to induce stem cell expansion. These results demonstrate that cyclin D1Stroma drives tumor microenvironment heterocellular signaling, promoting several key hallmarks of cancer.

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 D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) 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.

Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa.