The membrane-associated form of cyclin D1 enhances cellular invasion

The essential G1-cyclin, CCND1, is a collaborative nuclear oncogene that is frequently overexpressed in cancer. D-type cyclins bind and activate CDK4 and CDK6 thereby contributing to G1–S cell-cycle progression. In addition to the nucleus, herein cyclin D1 was also located in the cytoplasmic membrane. In contrast with the nuclear-localized form of cyclin D1 (cyclin D1NL), the cytoplasmic membrane-localized form of cyclin D1 (cyclin D1MEM) induced transwell migration and the velocity of cellular migration. The cyclin D1MEM was sufficient to induce G1–S cell-cycle progression, cellular proliferation, and colony formation. The cyclin D1MEM was sufficient to induce phosphorylation of the serine threonine kinase Akt (Ser473) and augmented extranuclear localized 17β-estradiol dendrimer conjugate (EDC)-mediated phosphorylation of Akt (Ser473). These studies suggest distinct subcellular compartments of cell cycle proteins may convey distinct functions.


Introduction
The cyclin D1 (CCND1) gene, encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein (pRB), in order to promote cell cycle progression [1][2][3] . Newly synthesized cyclin D1 associates with CDK4/6 to form the holoenzyme that phosphorylates pRB, releasing E2F family transcription factors and inducing a gene expression network contributing to G 1 /S entry. Early studies demonstrated that cyclin D1 functions as a nuclear collaborative oncogene 4 . In this regard a cyclin D1 cDNA clone contributed to cellular transformation by complementing a transformation defective adenovirus E1A oncogene 4 . The requirement for cyclin D1 in oncogenic transformation has been established through cyclin D1 anti-sense 5,6 and genetic deletion studies in the mouse [7][8][9] . Furthermore, cyclin D1 targeted to the mammary gland was sufficient for the induction of mammary tumorigenesis 10,11 . Clinical studies have shown a correlation between cyclin D1 expression and tumorigenesis and increased cyclin D1 expression is associated with tumor invasion and metastasis [12][13][14][15] .
A growing body of evidence provides support for an extranuclear function of cyclin D1. Cyclin D1 is actively synthesized and located exclusively in an extranuclear location in hibernating hematopoietic stem cells (HSC) 16 , in postmitotic neurons 17 , cardiomyocytes 18 , and hepatocytes 19 . The cytoplasmic sequestration of cyclin D1 is important to maintain the non-proliferative state as nuclear enforced expression using a nuclear-localized form of cyclin D1 forces the cell into a proliferative state 18 . Cyclin D1 has been identified in the cytoplasmic membrane [20][21][22] and shown to bind and regulate the function of several cytoplasmic membrane-associated proteins including PACSIN II (Protein kinase C and Casein kinase Substrate In Neurons protein 2) 23 also known as syndapin), Filamin A 24 and paxillin 21 .
The association of cyclin D1 with cytoplasmic membrane proteins 21,23,24 is consistent with prior studies demonstrating other components of the cell-cycle control apparatus are located in the cytoplasmic membrane including p27 Kip1 and p16 INK4a 20,25 . Although the physiological function of cytoplasmic membrane-associated cell-cycle components was previously not well understood, p16 INK4a and CDK6 colocalize in membrane ruffles of spreading cells and functioned upstream of αvβ3dependent activation of PKC to regulate matrixdependent cell migration 25 . Cyclin D1-deficient mouse embryo fibroblasts (MEFs) and mammary epithelial cells exhibit increased adhesion and decreased motility compared with wild-type MEFs [26][27][28] . Transduction of cyclin D1 −/− cells with a human or murine cyclin D1 cDNA, reversed this adhesive phenotype, promoting cell migration 26 . The induction of cell migration by cyclin D1 correlated with the reduction of Rho GTPase activity 26 . Mutational analysis demonstrated that cyclin D1 reduction of cellular adhesion and induction of cellular migration were independent of the pRB-and p160 coactivator-binding domains 26 . Cyclin E knockin of cyclin D1 −/− MEFs rescued the DNA synthetic defect of cyclin D1 −/− MEFs but did not rescue the migration defect 26 suggesting the pRB binding of cyclins and the promigratory function may be dissociable.
Although cyclin D1 binds cytoplasmic membraneassociated proteins and correlative studies have suggested that cyclin D1 may promote cellular migration, no studies have selectively uncoupled the functional activity of the nuclear vs. cytoplasmic cyclin D1 pools. The current studies were conducted in order to determine the function of cyclin D1 when localized to either the cellular membrane or the nucleus.

Cyclin D1 is located at the cytoplasmic membrane
The endogenous cytoplasmic membrane-associated protein PACSIN II was shown to bind cyclin D1 in liver tissue 23 and cyclin D1 bound to PACSIN II and paxillin (Pxn) in 3T3 cells 21,23 . In order to characterize the function of membrane-associated cyclin D1, studies were conducted in the human diploid fibroblast cell line (MRC-5) and human breast cancer samples. Using immunohistochemistry, endogenous cyclin D1 was identified at the MRC-5 cellular leading edge, in proximity with PACSIN II (Fig. 1a, Fig. S1). Paxillin (Pxn) is a structural and regulatory component of FAs and is also found along the cell membrane. Cyclin D1 was identified co-staining with tyrosine phosphorylated paxillin (Paxillin-P Tyr118 Fig. 1b,  Fig. S2A). PACSIN II and tyrosine phosphorylated paxillin colocated at the leading edge Fig. 1c, Fig. S2B), consistent with prior studies conducted of the individual proteins in other cell types 21,23,24 .
Inflammatory breast cancer (IBC) is very aggressive breast cancer linked to poor prognosis. In order to assess  Fig. 1 Cyclin D1 is located in the cytoplasmic membrane. a The human diploid fibroblast cell line (MRC-5) was stained for cyclin D1, PACSIN II, and F-actin. Merged images demonstrate the presence of cyclin D1 at the membrane (arrow), shown at high magnification in the right-side panel. Size bar is 20 μm. b Cyclin D1 co-staining with tyrosine phosphorylated Paxillin (Y118) and F-actin. Focal contacts are identified by the tyrosine phosphorylated Paxillin. c Co-staining of PACSIN II with tyrosine phosphorylated Paxillin (Y118) with merged staining shown by yellow arrows.
the location of cyclin D1 in human breast cancer we compared membrane-associated cyclin D1 in patients with IBC and other breast cancers. Samples from 6 IBC patients and 17 non-IBC patients were stained for cyclin D1 and analyzed by a clinical pathologist (Fig. S3A, B). The subcellular distribution was assigned using the standard Aperio digital analysis algorithm for cellmembrane staining. The entire slide was scanned enabling analysis of >1000 cells per sample. Five of six IBCs stained for membrane associated cyclin D1, whereas only 2/17 non IBCs stained for membrane-associated cyclin D1. Cytoplasmic-membrane associated cyclin D1 was observed in 5 of 6 IBC patient samples and 2 of 17 non-IBC patient samples had membrane-associated cyclin D1 (Fig. S3). All 6 IBC and 16 of 17 non-IBC patients had nuclear localized cyclin D1. We next conducted immunofluorescent studies for cyclin D1 in non-IBC patients in order to provide more sensitive detection of membraneassociated cyclin D1. Costaining of cancer cells with a pan-cytokeratin antibody and underexposing the immunofluorescence (IF) signal provides an effective way of delineating the cellular boundaries, revealing tumor cores with clusters of cells displaying membrane-associated cyclin D1. In a tissue microarray of 50 ERα-positive breast cancers examined, membrane-associated cyclin D1 was detected in four cases ( Fig. S3C-F).
Cytoplasmic membrane-targeted cyclin D1 promotes transwell cellular migration and increases cellular migratory velocity Cyclin D1 is known to promote cellular migration of fibroblasts and mammary epithelial cells 26,28 . In order to further characterize the molecular mechanisms by which cyclin D1 governs the induction of cellular migration we conducted subfractionation of nuclear and cytoplasmic cellular fractions from cyclin D1 WT vs cyclin D1 −/− 3T3 cells (Fig. 2a). Western blot analysis demonstrated enrichment of histone H2A in the nuclear fraction, α-tubulin in the cytoplasmic fraction and Na + / K + -ATPase in the membrane-associated fraction as previously described 29 . Cyclin D1 was identified in each of the subcellular fractions, consistent with prior studies conducted by confocal microscopy 21,23 . In order to determine the function of the cytoplasmic membranelocalized fraction of cyclin D1, cyclin D1 −/− 3T3 were transduced with a cyclin D1 expression vector encoding either cyclin D1 WT , cyclin D1 NUC , or cyclin D1 MEM (Fig.  2b) and functional analysis were conducted. Cherry-lacR-NLS-CD1 NUC which encodes a nuclear localized form of cyclin D1, was previously well characterized 30,31 . Cyclin D1 was cloned at the C-terminus of the Cherry-lacR-NLS vector 32,33 . For cyclin D1 MEM the cyclin D1 cDNA was cloned in frame to pECFP-Mem (Clonetech), which encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin, also called GAP-43, and a cyan fluorescent variant of the enhanced green fluorescent protein. The neuromodulin fragment contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets ECFP to cellular membranes. Expression of ECFP-Mem in mammalian cells results in strong labeling of the plasma membrane and had been used to target proteins including ERα to the plasma membrane 34 . Using electroporation the transfection efficiency was >90%. Cyclin D1 WT enhanced transwell migration twofold (Fig. 2c, f), cyclin D1 NUC did not enhance transwell migration (Fig.  2d, f) and cyclin D1 MEM enhanced transwell migration threefold (Fig. 2e, f). Transwell migration assays were next conducted in MCF-7 cells that were serum starved to reduce endogenous cyclin D1. Compared with the respective vector control transwell migration was enhanced 6.6-fold by cyclin D1 WT , 2.2-fold by cyclin D1 NUC and 12.6-fold by cyclin D1 MEM (Fig. S4).
Cyclin D1-deficient fibroblasts show the same diameter size as wild-type cells, but attach and spread more rapidly after seeding on fibronectin-coated plates 26,28 . Herein, time lapse video microscopy demonstrated the induction of cellular velocity by cyclin D1 WT (Fig. 3a, d). Expression of Cyclin D1 MEM , but not cyclin D1 NUC , promoted cellular migratory velocity ( Fig. 3b-d).
The subcellular distribution of cyclin D1 MEM and cyclin D1 NUC , was further characterized using confocal microscopy ( Fig. S5). Transfected cells were examined by confocal microscopy and by Z series reconstruction with the nucleus stained with Hoechst 33342. The cells expressing membrane-associated cyclin D1 showed green fluorescence predominantly at the cellular membrane ( Fig. S5A, B), whereas the cyclin D1 NUC showed red fluorescence predominantly in the nucleus (Fig. S5C, D).

Cytoplasmic membrane-targeted cyclin D1 augments DNA synthesis and contact independent growth
Reintroduction of cyclin D1 into cyclin D1 −/− fibroblasts may enhance DNA synthesis associated with a reduction in the proportion of cells in the G 0 /G 1 phase of the cell cycle. In order to determine the capacity of membrane-targeted cyclin D1 to regular the cell-cycle distribution, fluorescence activated cell sorting (FACS) analysis was conducted. Comparison was made to the   (Fig. 5a). Colony formation as an assay of contact-independent growth, showed an increase in both colony number and colony size with either cyclin D1 WT , cyclin D1 NUC or cyclin D1 MEM (Fig. 5d-l) with a twofold increase in colony number and size with cyclin D1 MEM (Fig. 5j-l). In order to determine potential mechanisms by which cyclin D1 NUC and cyclin D1 MEM may induce proliferative signaling, we assessed the impact of signaling induced using downstream reporter target genes (Fig. S6). Consistent with prior studies, that cyclin D1 repressed the (AOX)3-LUC reporter gene 36 , herein both cyclin D1 NUC and cyclin D1 MEM repressed the (AOX)3-LUC reporter gene (Fig. S6). The immediate early gene c-Fos-LUC and cyclin D1-LUC were induced approximately twofold more by cyclin D1 MEM than by cyclin D1 NUC (Fig. S6). These studies show that cyclin D1 MEM activates immediate early gene c-Fos and cyclin D1 transcription and suggest that cyclin D1 MEM may promote distinct signaling pathways to augment cellular growth. Cytoplasmic membrane-targeted cyclin D1 augments estrogen-dependent Akt kinase activation via K112 The estrogen receptor α (ERα) is known to convey both genomic and extra genomic activities 37 . The extranuclear estrogen signaling pathway is thought to involve a membrane-associated ERα, which activates PI3-kinase and thereby Akt signaling 38 . Maximal activation of Akt requires phosphorylation on the carboxy-terminal site, S473, by mTORC2 39,40 . In recent studies, membraneassociated estrogen signaling was shown to occur via cyclin D1 32 . We investigated the impact of expressing cyclin D1 as either total, nuclear, or membrane-tethered forms of cyclin D1 (Fig. 6). The human breast cancer cell line (MCF-7) was transduced with expression vectors encoding cyclin D1 targeted to the nucleus (Cherry-CD1 NUC ), to the cytoplasmic membrane (PECFP-CD1 MEM ) or expressed in both cytoplasmic and nuclear compartments (MSCV-CD1 TOT ). Increased expression of cyclin D1 via an MSCV expression vector (cyclin D1 WT ), resulted in increased cyclin D1 abundance and increased phosphorylation of Akt1 at Ser473 compared with vector control (Fig. 6a, lanes 1 vs. 2). The ectopic expression of cyclin D1 MEM enhanced phosphorylation of Akt1 at Ser473 compared with vector control (Fig. 6a, lanes 3 vs.  4). Estradiol (E 2 ) increased phosphorylation of Akt1 at Ser473 compared with vehicle control (Fig. 6a, lanes 7 vs. 1). The ectopic expression of cyclin D1 MEM increased E 2induced phosphorylation of Akt1 at Ser473 compared with vehicle control (Fig. 6a, lanes 10 vs. 4). The extranuclear vs. nuclear E 2 -induced signaling pathways can be distinguished using 17β-estradiol linked to a dendrimer conjugate (EDC), which excludes estradiol from the nucleus 41,42 . In order to define the residues of cyclin D1 that participate in Akt activation, mutational analysis of cyclin D1 was conducted. Breast cancer epithelial cells (MCF-7 cells) were treated with either EDC or dendrimer control. Expression of a membrane-associated cyclin D1 under control of the MSCV promoter (cyclin D1 MEM ) induced phosphorylation of Akt1 at Serine473 (Fig. 6b, lanes 1 vs. 6). The addition of EDC to cyclin D1 MEM MCF-7 cells, augmented phosphorylation of Akt1 at Serine473 (Fig. 6b, lanes 6 vs. 7, 5 min S.E. (shorter exposure)). Mutation of cyclin D1 at K112 reduces CDK4/ 6 and p27 KIP1 binding 43,44 . Expression of a membranetethered mutant of cyclin D1 at K112 (cyclin D1 MEM-KE) ) demonstrated an approximately 90% reduction in EDCmediated induction of Akt1 Serine473 phosphorylation compared with empty vector control cells (Fig. 6b, lane 1 vs. 11; lanes 2 vs. 12).

Cytoplasmic membrane-targeted cyclin D1 augments EDCdependent Akt kinase activation at the cell membrane
We conducted IF to assess the relative abundance and subcellular distribution of Akt1 Serine 473 phosphorylation upon EDC treatment in cells transduced with the distinct located forms of cyclin D1. MCF-7 cells expressing the membrane-associated cyclin D1 (PECFP-CD1 MEM ) showed the characteristic enrichment of membranous GFP staining (Fig. 7a). Akt1 phosphorylated at Serine473 may be either nuclear or cytoplasmic, related to additional signaling partners 45 . In the vehicle treated cells, cyclin D1 MEM expression was associated with the induction of nuclear p-Ser473-Akt1. EDC treatment of vector control cells increased nuclear p-Ser473-Akt1. EDC treatment of cyclin D1 MEM transduced MCF-7 cells correlated with the induction of p-Ser473-Akt1, which was found to be in a cytoplasmic membranous distribution (Fig. 7a). In MCF-7 cells transduced with cyclin D1 NUC , cyclin D1-RFP was located primarily in the nucleus. Nuclear localized cyclin D1 (cherry-CD1 NUC ) did not induce p-Ser473-Akt1 significantly (Fig. 7b). EDC treatment of MCF-7 cells augmented phosphorylation p-Ser473-Akt1, which was primarily nuclear in distribution (Fig. 7b). Careful quantitation evidenced that cyclin D1 NUC did not augment EDC-induced nuclear Akt1 Serine473 phosphorylation (Fig. 7b, d). MCF-7 cells transduced with cyclin D1 TOT showed nuclear, cytoplasmic, and membraneassociated cyclin D1, and an enhancement of EDC induced Akt1 Serine473 phosphorylation. p-Ser473-Akt1 was located in both the nucleus and membrane (Fig. 7c, d). Thus, the cytoplasmic membrane localized cyclin D1 (PECFP-CD1 MEM ), but not the nuclear localized form (cherry-CD1 NUC ), augmented Akt1 phosphorylation at Serine473.

The immediate activation of Akt1 by insulin requires cyclin D1
Recent studies identified a dichromic fluorescent (DCF) dye substrate for cellular Akt1 activity 46 . The diserine DCF substrate was shown to serve as a specific substrate for Akt1, which can be used to quantitatively assess the enzyme's activity in real time 46 . Insulin activation of cellular Akt phosphorylates a single serine residue of the diserine DCF substrate in a time dependent manner, resulting in a spectral shift that can be used to assess longitudinally the stimulation and reversibility of Akt1 activity. The dichromic dye LS456 is phosphorylated by Akt1, but not a variety of other kinases (including PKA, PKC, RSK1, P70S6K, and PI3K) 46 . The binding of insulin to its cell surface receptor stimulates phosphoinositide-3 kinase (PI3K), which then induces the second messenger, phosphotidylinositol-3, 4, 5-triphosphate (PIP3). PIP3 activates Akt and additional downstream effectors. As LS456 was shown to serve as a specific substrate for Akt1 in response to 150 nM insulin, we examined the kinetics of insulin-mediated activation of LS456 in cyclin D1 −/− MEF compared with wild-type MEFs. Insulin stimulation of Akt1 activity assessed by LS456 was delayed with reduced induction in cyclin D1 −/− cells compared with the cyclin D1 WT rescued cells (Fig. S7).

Cyclin D1 restrains RhoA activity via K112
In the current studies, cytoplasmic membrane-tethered cyclin D1 augmented cellular migratory velocity and estrogen-dependent induction of Akt1 Ser473 phosphorylation. In prior studies cyclin D1 rescue of cyclin D1 −/− MEFs reduced RhoA activity 26 . Although these prior studies suggested that cyclin D1 may augment cellular migration by restraining RhoA activity, Rac1 and Cdc42 can also participate in cellular migration 47 . In order to examine the functional interactions with cyclin D1 and Rho GTPases we deployed the FRET based fluorescent probes for RhoA, Rac, and Cdc42 (Fig. 8a). pRaichu-RhoA consists of a truncated RhoA (aa 1-189), the RhoAbinding domain (RBD) and the FRET pair of CFP and YFP. When RhoA binds to GTP, and thereby the RBD, RhoA recruits CFP in close proximity to YFP, thereby increasing the FRET activity between CFP and YFP. We examined the functional interaction between cyclin D1 and RhoA using FRET. The image from a typical FRET experiment was shown in Fig. 8b   co-transfected with pRaichu-RhoA and either cyclin D1 WT , cyclin D1 KE , or their corresponding vector control. Spectral images in 10 channels from 470 to 566 nm with excitation at 458 nm were simultaneously recorded. YFP was inactivated by photobleaching with a 514 nm laser at 100% power output (Fig. 8b). The emission spectra within the ROI increased in the CFP signal at 481 nm after photobleaching with YFP which has an emission peak at 534 nm (Fig. 8c). FRET efficiency was used to quantitatively compare the difference in RhoA activity among the

cells. (FRET efficiency was defined as (F
where F B is the intensity of the donor (CFP) after photobleaching and F 0 is the intensity of the donor before photobleaching, see "Methods"). FRET efficiency was reduced 40% by cyclin D1 WT but was not significantly reduced by expression of the cyclin D1 KE (Fig. 8d). Similar analysis of FRET for the related Rho family members, Rac1 and Cdc42, failed to elicit changes in FRET efficiency upon re-expression of cyclin D1 wild type. By using FRET, we extend prior studies demonstrating cyclin D1 reduces Rho GTPase activity 26 , to define the interaction of cyclin D1 occurs with RhoA, not Rac or Cdc42, and demonstrate the residue K112 of cyclin D1 is required for interaction with RhoA.

Discussion
The well characterized nuclear functions of cyclin D1 include firstly, serving as the regulatory subunit of a holoenzyme that phosphorylates the pRB protein, and secondly serving as part of a transcriptional regulatory complex that drives proliferative gene expression 48 . Consistent with previous studies, that either identified cyclin D1 associated with the cytoplasmic membrane or cytoplasmic membrane proteins [20][21][22][23][24] , the current studies identified cyclin D1 colocalized with PACSIN II and paxillin PTyr118 at the cytoplasmic membrane. The current studies extend our understanding of cyclin D1 through characterizing the function and signaling pathways regulated by cyclin D1 at the cytoplasmic membrane vs. the nucleus. Firstly, herein membrane-associated cyclin D1 augmented transwell migration and enhanced the velocity of cellular migration. In contrast, the nuclearlocalized form of cyclin D1 neither enhanced cellular migratory velocity nor induced transwell migration in 3T3 cells. These studies are consistent with previous findings that cyclin D1 promotes migration 21,26,28,43,44 , but extend these findings by demonstrating that it is the membraneassociated form of cyclin D1 that mediates this function. Secondly, these studies show both nuclear and membrane-associated cyclin D1 augment cellular DNA synthesis, cellular proliferation, and contact-independent growth. Thirdly, these studies demonstrate that cyclin D1 tethered to the cytoplasmic membrane induces Akt signaling, characterized by the induction of Akt1 Ser473 phosphorylation. Furthermore, membrane-associated cyclin D1 augmented a physiological function of estrogen, to induce Akt1 Ser473 phosphorylation. Fourthly, as activity of Rho GTPase at the cellular membrane may inhibit cellular adhesion and migration and restrain Akt activity 49 , we examined and defined a role for cyclin D1 to inhibit Rho activity. Collectively these studies define a novel function for cytoplasmic membrane associated cyclin D1 that may augment aberrant growth control and cellular invasion.
Prior studies had shown the induction of cellular migration by cyclin D1 21,26,28,43,44 . Cyclin D1 −/− cells show a more spread morphology than the corresponding wild type and display an increased number of focal adhesions (FAs) with higher levels of tyrosinephosphorylated paxillin 21,26,28,43,44 . Herein, using cyclin D1 −/− cells, we demonstrated the membrane-associated pool of cyclin D1 is sufficient to augment transwell migration. We identified cyclin D1 at the plasma membrane in inflammatory breast cancer, and cyclin D1 colocalized to the cytoplasmic membrane with PACSIN II and Paxillin (Y118) in MRC-5 cells. Cyclin D1 was previously shown by mass spectrometry to bind the membrane-associated proteins PACSIN II 23 , Filamin A 24 , Paxillin 21 , and several additional proteins 50 . PACSIN II is involved in cell spreading 51 , as well as endocytosis of cell-surface receptors like the EGF receptor 52 and in caveolae-mediated endocytosis 53,54 . In view of clinical analyses showing a correlation between total cyclin D1 expression and tumor invasiveness and metastasis [12][13][14][15] , our studies suggest further studies assessing membraneassociated cyclin D1 may be warranted.
Herein, cytoplasmic membrane-associated cyclin D1 augmented phosphorylation of Akt1 at Ser473. Akt, also known as Protein Kinase B, promotes cellular survival, proliferation, growth, and migration 55 . Akt hyperactivation contributes to human cancer correlating with poor prognosis and therapy resistance and genetic deletion demonstrated Akt1 is required for ErbB2-induced breast cancer progression and tumor metastases in vivo 56 . Herein the acute nongenomic E 2 activation of Akt1, was augmented by the membrane-associated cyclin D1 pool. Estradiol acutely activates Akt 57,58 in part through the association of ERα at the plasma membrane associated with the p85 regulatory subunit of PI3-kinase and other proteins including the scaffold protein caveolin-1, G proteins, Src kinase, Ras, and Shc 57,59-61 . ERα regulates nuclear gene expression via genomic and extranuclear non-genomic signals 37,59 . Extranuclear pools of ERα reside in the plasma membranes 62 and the ability to distinguish nuclear from extranuclear ERα signaling has been enabled through the generation of a 17β-estradiol dendrimer conjugate (EDC) which is localized to the extranuclear compartment 41,42 . Herein, using nuclear excluded E 2 dendrimers, cyclin D1 was shown to participate in the acute non-genomic E 2 response. Genetic deletion studies in the mouse demonstrated E 2 -dependent induction of genes governing growth factors, growth factor receptor and promigratory processes in the mammary gland requires cyclin D1 63 . The biological effects of estrogen, are critically dependent upon cyclin D1 in vivo 63,64 , with the current studies suggesting an important component is mediated via membraneassociated cyclin D1.
RhoA, Rac1, and Cdc42 are the best characterized members of the Rho GTPase branch of the Ras superfamily and are known to regulate cellular morphology and migration 47 . In the current studies, cyclin D1 restrained RhoA activity, requiring K112. Cyclin D1 −/− cells have increased RhoA activity, increased ROCK II kinase and increased LIM kinase activation (threonine 505/508). LIM kinase phosphorylation at threonine 505/508 in turn phosphorylates the actin-depolymerizing protein cofilin at serine 3 and MLC2 at Thr18/Ser19 26 . Herein, FRET analysis evidenced cyclin D1 restrained Rho GTPase activity. In contrast, neither Rac-GTPase nor Cdc42 activity was influenced by cyclin D1. The reduction in RhoA GTPase FRET by cyclin D1 was abolished by mutation of cyclin D1 residue K112. Cyclin D1 participates in multiple functions via K112 including CDK4/6mediated pRB phosphorylation 65 and binding to p27 KIP144 . Rho GTPase is an important modulator of ERα activity 66,67 , and E 2 enhances ERα association with the p85 subunit of PI3 kinase thereby inducing Akt phosphorylation 57 . An increase in ERα/PI3K interactions in patient-derived xenografts (PDXs) correlates with acquired resistance to tamoxifen 68 . RhoA represses Akt Ser473 phosphorylation 49 and the repression of RhoA activity by cyclin D1 may have contributed to the induction of pAkt1-Ser473. The role of cyclin D1 in restraining RhoA, thereby inducing ERα activity and tamoxifen resistance, warrants further investigation.
Several lines of evidence support the importance of cyclin D1 nuclear location in aberrant growth including elegant studies showing that a mutant of cyclin D1 (D1T286A), that is defective in phosphorylation-mediated nuclear export, induces cell transformation in cell culture assays and triggers B-cell lymphoma in a mouse model of mantle cell lymphoma 69,70 . Furthermore, transgenic mice that overexpress the identical mutant cyclin D1 driven by the MMTV promoter (MMTV-D1T286A) developed mammary adenocarcinoma with a shorter latency relative to mice over-expressing the wild-type cyclin D1 (MMTV-D1) 71 . That said, the current studies suggest that in addition to the nuclear function of cyclin D1, a membrane-associated pool of cyclin D1 contributes to cellular migration, induction of Akt1 activity and the induction of a signaling pathway, defined through transcriptional reporters, that activates the immediate early gene c-Fos and cyclin D1. c-Fos is a target of Akt1 induction and Fos family members induce cell-cycle entry though the induction of cyclin D1 72 , suggesting a mechanism by which membrane associated cyclin D1 may augment cellular growth. The major adjuvant therapy for the~70% of ERα expressing human breast cancer involves anti-estrogen therapy. The ERα/PI3K/Akt complex pathway is hyperactivated in aggressive breast tumors 73 . The non-genomic actions of E 2 /ERα, mediated via cytoplasmic membrane-associated cyclin D1, may provide an important additional target 58 . As membrane-associated cyclin D1 augments activity of the ERα/PI3K/Akt complex pathway, the cytoplasmic membrane pool of cyclin D1 may be a new target for ERα expressing breast cancer treatments 74,75 .

Materials and methods
A detailed description is provided in the Supplementary Materials.

Transwell migration
The assessment of transwell migration 77 , migratory velocity, and migratory distance 26 were conducted as previously described.

Fluorescence resonance energy transfer (FRET) imaging
HEK293T cells, co-transfected with 3×FLAG vector, cyclin D1 wild-type or cyclin D1 KE mutant and FRET reporters (pRaichu-RhoA, pRaichu-Cdc42 or pRaichu-Rac1 78,79 ), were cultured in a four-well chamber and imaged using a Zeiss laser-scanning microscope, LSM510META, with a 40× oil immersion Doc Plan-Neofluar lens objective (numerical aperture of 1.3). To detect FRET between CFP and YFP, we used time-lapse and lambda stack acquisition linked with the photobleaching command 80 .
Immunostaining IF staining and confocal microscopy of cultured cells was conducted as described previously 77 . Chromogen immunostaining of human breast cancer samples was conducted on the breast tissue with the Ventana Benchmark autostainer using deintified archival tissue which are exempt from review by the Thomas Jefferson University Institutional Review Board. Fluorescence-based immunohistochemistry for cyclin D1 multiplexed with pancytokeratin and DAPI counterstain was performed as previously described 81-83 on a tissue microarray containing cores of 50 de-identified ER-positive breast cancer specimens provided by the Medical College of Wisconsin Tissue Bank under IRB-approved protocol.

Live cell Akt activity monitoring
Live cell imaging studies were conducted as described 46 .