Kinase-independent function of E-type cyclins in liver cancer Yan Genga,b, Wojciech Michowskia,b, Joel M. Chickc, Yaoyu E. Wangd, M. Emmanuelle Jecroisa,b, Katharine E. Sweeneya,b, Lijun Liua,b, Richard C. Hana,b, Nan Kea,b, Agnieszka Zagozdzona,b, Ewa Sicinskae, Roderick T. Bronsonf, Steven P. Gygic, and Piotr Sicinskia,b,1 aDepartment of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215; bDepartment of Genetics, Harvard Medical School, Boston, MA 02115; cDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; dCenter for Cancer Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02215; eDepartment of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA 02215; and fDepartment of Biomedical Sciences, Tufts University Veterinary School, North Grafton, MA 01536 Edited by Mariano Barbacid, Spanish National Cancer Research Centre, Madrid, Spain, and approved December 20, 2017 (received for review June 26, 2017) E-type cyclins (cyclins E1 and E2) are components of the core cell for 745,000 deaths annually (10). The 5-y survival rate for patients cycle machinery and are overexpressed in many human tumor with HCC is only ∼7%. E cyclins are overexpressed in the majority of types. E cyclins are thought to drive tumor cell proliferation by HCC cases, while nearly 20% of tumors display amplification of the activating the cyclin-dependent kinase 2 (CDK2). The cyclin E1 gene CCNE1 and CCNE2 genes (11–14).ThemajorriskfactorsforHCC represents the site of recurrent integration of the hepatitis B virus are chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) in- in the pathogenesis of hepatocellular carcinoma, and this event fections (15). The CCNE1 gene represents one of the most frequent is associated with strong up-regulation of cyclin E1 expression. sites of HBV integration in HCC, and this event is associated with Regardless of the underlying mechanism of tumorigenesis, the strong up-regulation of cyclin E1 expression (16). Moreover, the majority of liver cancers overexpress E-type cyclins. Here we used hepatitis C virus core protein was shown to promote cell proliferation conditional cyclin E knockout mice and a liver cancer model to by up-regulating cyclin E levels (17). Regardless of the pathogenesis, test the requirement for the function of E cyclins in liver HCC occurs more than five times more frequently in males than in tumorigenesis. We show that a ubiquitous, global shutdown of females, and this was attributed in part to up-regulation of cyclin E GENETICS E cyclins did not visibly affect postnatal development or physi- expression by testosterone (18). Collectively, these findings suggest an ology of adult mice. However, an acute ablation of E cyclins halted important role for cyclin E in pathogenesis of HCC. liver cancer progression. We demonstrated that also human liver In this study, we tested the requirement for E cyclins in pro- cancer cells critically depend on E cyclins for proliferation. In contrast, gression of HCC using mouse cancer models. We report that E we found that the function of the cyclin E catalytic partner, CDK2, is cyclins play an essential, rate-limiting role in liver cancer progres- dispensable in liver cancer cells. We observed that E cyclins drive sion, while being dispensable for proliferation of normal tissues. proliferation of tumor cells in a CDK2- and kinase-independent Unexpectedly, we found that the function of E cyclins in liver mechanism. Our study suggests that compounds which degrade or cancer cell proliferation is independent of the canonical role of E inhibit cyclin E might represent a highly selective therapeutic strategy cyclins as activators of CDK2. for patients with liver cancer, as these compounds would selectively cripple proliferation of tumor cells, while sparing normal tissues. Results Cyclin E Function Is Dispensable in Postnatal Animals. We and others cell cycle | E-type cyclins | cyclin-dependent kinase CDK2 | liver cancer previously reported that constitutive, germline ablation of cyclin − − − − E1 and E2 resulted in an embryonic lethality of cyclin E1 / E2 / -type cyclins (cyclins E1 and E2, collectively called “cyclin EE”) are components of the core cell cycle machinery. These Significance two cyclins are encoded by separate genes and are usually coexpressed in proliferating cells (1). E-type cyclins bind and Amplification of the cyclin E genes and overexpression of cyclin E activate their kinase partner, the cyclin-dependent kinase 2 proteins is very frequent in several human tumor types. In partic- (CDK2). Cyclin E–CDK2 complexes phosphorylate proteins ular, overexpression of E cyclins was implicated in the pathogen- governing cell cycle progression, including the retinoblastoma pro- esis of liver cancers. According to the current cell division models, E tein and retinoblastoma-related p107 and p130 proteins, as well as cyclins drive tumor cell proliferation by activating its kinase part- proteins involved in histone biosynthesis, centrosome duplication, ner, the cyclin-dependent kinase 2 (CDK2). In this study, we dem- and firing of DNA replication origins (2). These functions of cyclin onstrate that E cyclins are dispensable for adult mouse physiology, E–CDK2 kinase promote progression of cells through the G1 phase but essential for liver cancer progression. Surprisingly, we found of the cell cycle and entry into DNA synthesis (S phase) (2). that the function of E cyclins in liver tumorigenesis is kinase in- Consistent with their growth-promoting roles, amplification of dependent. Our study suggests that agents targeting E cyclins the cyclin E1 and E2 genes (CCNE1 and CCNE2, respectively) would be efficacious in liver cancer treatment, as they would halt and pathological overexpression of their proteins have been proliferation of tumor cells while sparing normal tissues. documented in many cancer types (3–7). Indeed, the CCNE1 gene located on chromosome 19q12 represents one of the most fre- Author contributions: Y.G. and P.S. designed research; Y.G., W.M., J.M.C., M.E.J., K.E.S., L.L., R.C.H., N.K., A.Z., and E.S. performed research; W.M., J.M.C., and S.P.G. contributed quently amplified loci across all human tumor types (8). Over- new reagents/analytic tools; Y.G., Y.E.W., R.T.B., and P.S. analyzed data; and Y.G. and P.S. expression of E cyclins was shown to generally correlate with poor wrote the paper. clinical outcome (3, 4). In the case of HER2-positive breast can- Conflict of interest statement: P.S. is a consultant and a recipient of a research grant cers, cyclin E amplification/overexpression represents a molecular from Novartis. mechanism of resistance to trastuzumab (Herceptin) treatment This article is a PNAS Direct Submission. (9). It is currently assumed that overexpressed E cyclins drive the Published under the PNAS license. tumorigenic process through hyperactivation of the CDK2 kinase. 1To whom correspondence should be addressed. Email: [email protected]. Hepatocellular carcinoma (HCC) represents the second most This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. common cause of death from cancer worldwide, and it is responsible 1073/pnas.1711477115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1711477115 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 mice due to placental and heart abnormalities (19, 20). To circum- S1 D and E). Importantly, efficient deletion of cyclin E1 was vent this lethality, and to study the requirement of cyclin E function maintained after 1 y even in highly proliferative organs such as at later stages of development, we generated a conditional cyclin intestine or spleens (Fig. S1B, see 1 y). We concluded that E E1 knockout (E1F/F) mouse strain (21). We interbred cyclin E1F/F cyclins are largely dispensable for proliferation of cells in post- − − − − and cyclin E2 / mice giving rise to cyclin E1F/FE2 / animals, which natal animals. Despite the efficient deletion of cyclin E1 after will be further referred to as “conditional cyclin E knockout” mice. Cre activation, we detected low levels of nondeleted floxed cyclin In the first set of experiments, we crossed conditional cyclin E E alleles in many organs (Fig. S1B). Hence, we cannot conclude knockout mice with Esr1-Cre animals (22), which ubiquitously that all cells within a given compartment do not require cyclin E. express tamoxifen-inducible Cre recombinase. Administration of tamoxifen to Esr1-Cre mice activates Cre, leading to a global Requirement for Cyclin E Function in Liver Cancer Progression. We deletion of the “floxed” sequences (22). We administered ta- next asked what the impact would be of cyclin E shutdown on − − moxifen to pregnant females bearing cyclin E1F/FE2 / embryos liver tumorigenesis. To efficiently delete both E cyclins in livers, at day 17.5 of gestation, and continued treatment of postnatal we turned to Mx1-Cre mice, which express inducible Cre animals with tamoxifen to ensure ubiquitous deletion of E cyclins recombinase. Administration of a double-stranded RNA analog (Fig. S1A). We then verified an efficient deletion of cyclin E1 in polyI–polyC to Mx1-Cre animals activates Cre, leading to nearly most organs analyzed (Fig. S1B, see P36) and observed the an- 100% deletion of the floxed sequences in liver and in the bone imals for 1 y. We found that an acute and global shutdown of E marrow cells (Fig. S2 A and B) (23). First, we administered − − cyclins did not compromise the animals’ viability and had no polyI–polyC to cyclin E1F/FE2 / /Mx1-Cre animals and de- outward effect on their health, except for slightly reduced body termined that ablation of both E cyclins had no effect on he- weights of cyclin E-deleted animals (Fig. S1C). Cyclin E-deleted matological parameters (Fig. S2 C and D). These findings are animals displayed normal biochemical parameters in the pe- consistent with the observation that bone marrow cells express ripheral blood and normal parameters of liver function (Fig.