Stromal cyclin D1 promotes prostate cancer stemness via the transcriptional regulatory E domain

• Introduction. Prostate cancer “stemness” increases with tumor progression, predicts poor prognosis and contributes to therapy resistance. The cyclin D1 gene (CCND1), which encodes the regulatory subunit of a holoenzyme that phosphorylates RB and conveys transcriptional properties, is expressed in prostate cancer and augments growth of some castrate resistant prostate cancers. Clinical trials are targeting the kinase activity of cyclin D1 in prostate cancer. The functional significance of the cyclin D1 E domain remained to be further defined. We investigated a potential kinase-independent function of cyclin D1 in prostate cancer. • Methods . Analysis of patient gene expression, tumor histology, gene knockout transgenic mice, tissue culture stem cell assays, proteomic analysis. • Findings . We show cyclin D1 expression is increased in PCa tumor stroma compared with adjacent tissue. Using single cell sequencing cyclin D1 we identified cyclin D1 in cancer associated fibroblasts. In vivo, cell markers of PCa stemness including Trop2 ICD activity. Deletion of both epithelial cell and stromal cyclin D1 further reduced markers of PCa stem cells suggesting a role for extra epithelial stromal cell cyclin D1 in the induction of PCa stemness. In vitro, cyclin D1 rescue of cyclin D1 deficient fibroblasts demonstrated cyclin D1 governed a secretome that augmented prostate cancer stemness, assessed by prostate cancer sphere size and number, and expression of pro-stemness chemokine receptors (CXCR2, CX3CR1). Mutational analysis showed the cyclin D1 heterotypic induction of stemness and chemokine receptor expression required an intrinsically disordered acidic rich “E domain” (AA 272-280) within the cyclin D1 carboxyl terminus. Integrated proteomics and ChIP Seq identified transcriptional targets governing cancer stemness and pro-tumorigenic inflammation. • Conclusions. Cyclin D1 augments prostate cancer stemness through heterotypic functions via an intrinsically disordered carboxyl terminal domain.