Knocking down cyclin D1b inhibits breast cancer cell growth and suppresses tumor development in a breast cancer model

Knocking down cyclin D1b inhibits breast cancer cellgrowth and suppresses tumor development in abreast cancer modelMin Wei,1 Li Zhu,2 Yafen Li,2 Weiguo Chen,2 Baosan Han,2 Zhiwei Wang,1 Jianrong He,2 Hongliang Yao,3

Zhongyin Yang,1 Qing Zhang,1 Bingya Liu,1 Qinlong Gu,1,4 Zhenggang Zhu1 and Kunwei Shen2

1Key Laboratory of Shanghai Gastric Neoplasms, Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai; 2ComprehensiveBreast Health Center in Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai; 3School of Science, Nanjing University of Science andTechnology, Nanjing, China

(Received January 17, 2011 ⁄ Revised April 9, 2011 ⁄ Accepted April 11, 2011 ⁄ Accepted manuscript online April 26, 2011 ⁄ Article first published online June 1, 2011)

4To whom correspondence should be addressed. E-mail: [email protected]

Cyclin D1 is aberrantly expressed in many types of cancers, includ-ing breast cancer. High levels of cyclin D1b, the truncated isoformof cyclin D1, have been reported to be associated with a poorprognosis for breast cancer patients. In the present study, we usedsiRNA to target cyclin D1b overexpression and assessed its abilityto suppress breast cancer growth in nude mice. Cyclin D1b siRNAeffectively inhibited overexpression of cyclin D1b. Depletion ofcyclin D1b promoted apoptosis of cyclin D1b-overexpressing cellsand blocked their proliferation and transformation phenotypes.Notably, cyclin D1b overexpression is correlated with triple-nega-tive basal-like breast cancers, which lack specific therapeutic tar-gets. Administration of cyclin D1b siRNA inhibited breast tumorgrowth in nude mice and cyclin D1b siRNA synergisticallyenhanced the cell killing effects of doxorubicin in cell culture, withthis combination significantly suppressing tumor growth in themouse model. In conclusion, the results indicate that cyclin D1b,which is overexpressed in breast cancer, may serve as a novel andeffective therapeutic target. More importantly, the present studyclearly demonstrated a very promising therapeutic potential forcyclin D1b siRNA in the treatment of cyclin D1b-overexpressingbreast cancers, including the very malignant triple-negative breastcancers. (Cancer Sci 2011; 102: 1537–1544)

C yclin D1 (CycD1), encoded by cyclin D1 ⁄ CCND1 andoverexpressed in a broad range of solid malignancies, is an

important cell cycle regulator that promotes G1 ⁄ S phase transi-tion by activation of Cdk4 and Cdk6 kinase activity.(1,2) Expres-sion of CycD1 in normal dividing cells is upregulated in the lateG1 phase by transcription activation through E2F family transac-tivators.(3–5) The CycD1 accumulated during the G1 ⁄ S transitionsimultaneously forms complexes with Cdk4 and Cdk6 and sub-sequently promotes initiation of DNA replication and centro-some duplication.(6–8) The abundant CycD1 eventually becomesphosphorylated and destroyed by ubiquitin-mediated proteolysis,which allows normal cell cycle progression. However, the levelof CycD1 and the activity of the CycD1–Cdk4 and CycD1–Cdk6 complexes can be regulated aberrantly, with excessiveCycD1–Cdk4 and CycD1–Cdk6 activity, in turn, driving cellsto replicate their DNA prematurely, resulting in genome insta-bility and tumorigenesis.(9,10) Emerging data from a number ofexperimental systems indicate that CycD1 also has multiple,kinase-independent cellular functions. First, CycD1 assists insequestering CDK inhibitors (e.g. p27kip1) and thus bolsteringlate G1 phase CDK activity.(11) Second, CycD1 is known to bindand modulate the function of several transcription factors thatare significant in human cancers.(12) Thus, CycD1 impinges onseveral distinct pathways that govern cancer cell proliferation.Although intragenic somatic mutation of CycD1 in human

doi: 10.1111/j.1349-7006.2011.01969.xªª 2011 Japanese Cancer Association

disease is rare, CycD1 gene translocation, amplification, and ⁄ oroverexpression are frequent events in selected tumor types. Inaddition, a polymorphism in the CycD1 locus that may affectsplicing has been implicated in increased cancer risk or pooroutcome.(13) Recent functional analyses of an established CycD1splice variant, namely cyclin D1b (CycD1b), revealed thatCycD1b harbors a stronger oncogenic potential than anotherfull-length variant of CycD1, referred to as cyclin D1a.(14,15)

However, the molecular mechanisms of CycD1b-driven tumori-genesis have not been fully elucidated. Importantly, total levelsof CycD1b in tumor tissues are inversely correlated with sur-vival in patients with breast cancer;(16,17) patients with tumorsthat have high levels of CycD1b at Stage I die within 5 years ofdiagnosis, whereas patients with low CycD1b expression intumors have a much longer survival. These observations indicatethat overexpression of CycD1b may be an important cause ofbreast cancer mortality and that CycD1b may be an importanttherapeutic target for the development of anticancer drugs.

The RNAi system is used to regulate gene. The functionalmediator of RNAi is a 21-nucleotide (nt) siRNA generated bythe cleavage of double-stranded (ds) RNA via a complex con-sisting of Dicer, TAR RNA-binding protein (TRBP), and proteinactivator of protein kinase PKR (PACT).(18–20) In fact, in recentyears, chemically synthesized siRNA oligonucleotides havebeen demonstrated to be superior agents to conventional anti-sense oligonucleotide approach that can effectively knock downgene expression by sequence-specific degradation of comple-mentary mRNA in cell culture.(21) Compared with conventionalantisense oligonucleotide approaches, siRNA-mediated geneknock down is much more specific and potent. The selection oftargeting sequences for siRNA is less restricted, so the rate ofproducing effective duplexes is higher.(22,23) In addition, siRNAis a double-stranded RNA, which is more resistant to nucleasedegradation and it therefore has prolonged stability in in vivostudies.(24,25) These unique properties make siRNAs potentialtreatments for cancer by targeting mutation- or overexpression-activated oncogenes in cancers. Numerous studies have shownthat siRNA can effectively suppress oncogene expression in can-cer cells and some siRNA cancer therapies are, indeed, in thepreclinical or early stages of clinic trials.(26–28) In the presentstudy, to investigate whether CycD1b can serve as a noveltherapeutic target and whether an siRNA-based approach caneffectively treat CycD1b-overexpressing breast cancer, weused CycD1b siRNA to target CycD1b overexpression andassessed its ability to suppress breast cancer growth in nudemice. The results clearly demonstrated a very promisingtherapeutic potential for CycD1b siRNA in the treatment of

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CycD1b-overexpressing breast cancer, including the highlymalignant triple-negative breast cancer.

Materials and Methods

Cell culture. All cell lines used were obtained from the Amer-ican Type Culture Collection (Rockville, MD, USA) and werecultured at 37�C in a 5% CO2 incubator. The immortalized nor-mal human mammary epithelial cell line MCF-10A was culturedas described previously.(29) Breast cancer cell lines, includingthe basal-type cell lines MDA-MB436 and MDA-MB157 (estro-gen receptor [ER] and progesterone receptor [PR] negative) andthe luminal-type cell lines SK-BR3 (ER and PR negative andoverexpressing human epidermal growth factor receptor 2[HER2 ]), MDA-MB453 (ER and PR negative), and T47D (ERand PR positive), were cultured in DMEM supplemented with10% FBS, 1% L-glutamine, and 1% penicillin ⁄ streptomycin(Sigma, St Louis, MO, USA). For MDA-MB436, 10 mg ⁄ mLinsulin was also added to the culture medium.

Transfection with siRNA oligonucleotides. The siRNA oligo-nucleotides for CycD1b and luciferase (Luc) were synthesizedby Qiagen (Valencia, CA, USA). The breast cancer cells(1 · 105 ⁄ well) in six-well plates were transfected with siRNAoligonucleotides (0.3 lg ⁄ well) using Oligofectamine reagent(Invitrogen, Carlsbad, CA, USA) according to the manufac-turer’s instructions.

Western blotting analysis. Western blotting assays were per-formed as what previously described.(17,30) Forty-eight hoursafter transfection, cells were lysed into mammalian cell lysisbuffer (Pierce, Rockford, IL, USA) supplemented with Halt Pro-tease Inhibitor Cocktail (Thermo, MA, USA). After quantifiedas previously described,(17,30) lysates (50 lg) were analyzed byimmunoblotting. The anti-CycD1b polyclonal antibody used inthe present study was obtained from Santa Cruz Biotechnology(Santa Cruz, CA, USA), whereas the anti-GADPH antibody wasfrom Sigma. The density of each protein band in each samplewas determined by densitometry and standardized against GAD-PH as an internal control. The standardized density of CycD1bin the SK-BR3 cells was arbitrarily set at 100%, with relativelevels of CycD1b in other samples calculated by comparing theGADPH-normalized densities against that of the SK-BR3 cells.The data are shown as the mean ± SD of three independentexperiments.

Apoptosis and cell cycle analysis. Standard FACS analysiswas used to determine the apoptosis of cells or the phase distri-bution of cells in the cell cycle, as described previously.(31,32)

Colony formation assay in soft agar. The standard colony for-mation assay was performed as described previously.(31) Briefly,breast cancer cells were transfected without (mock) or withsiRNA oligonucleotides targeting CycD1b or Luc. Two daysafter transfection, cells (1 · 103 cells ⁄ well) were plated in 24-well plates in culture medium containing 0.35% agar overlyinga 0.7% agar bottom layer and cultured at 37�C with 5% CO2.Two to three weeks later, the top layer of the culture was stainedwith p-iodonitrotetrazolium violet (1 mg ⁄ mL) and colonies>100 lm in diameter were counted. To monitor the cell viabilityof each group, cells (1 · 103 cells ⁄ well) were plated in 10-cmculture plates with normal complete medium and, 2–3weekslater, colonies were stained with 1% crystal violet and counted.Finally, the number of colonies in soft agar for the Luc orCycD1b groups was standardized against that for the controlgroup (mock cell; set at 100%).

In vivo assays. In in vivo experiments, 1 · 106 MDA-MB436, SK-BR3, or T47D cells, mixed with Matrigel Matrix(BD Biosciences, San Jose, CA, USA), were injected into thesecond pair of mammary glands of nude mice (two injectionsites per mouse; 45 mice for each cell line). Two weeks later,nude mice with a tumor burden were randomly divided into

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three groups (n = 15 for each group) and then treated without(mock) or with the an siRNA complex (Luc siRNA or CycD1bsiRNA) by intratumoral injection. Each complex contained10 lg siRNA (for mock injections, mice were injected with PBSinstead of siRNA) and 7.5 lL Oligofectamine (Invitrogen) inPBS, which was mixed according to the manufacturer’s instruc-tions. Mice were treated weekly for 4 weeks and were killedeither 1 week after the last treatment (for tumor size compari-sons) or 2 days after the last treatment (for anti-CycD1b immu-nohistochemistry or TUNEL assays). Tumor size was measuredjust before each treatment or after mice had been killed and thetumor volume was determined as described previously.(30)

To assess the effectiveness of combination treatment withCycD1b siRNA and doxorubicin (Dox), MDA-MB436 cellswere injected into the second pair of mammary glands of thenude mice as described above (n = 70). Beginning on Day 14after tumor cell injection, mice with a tumor burden were ran-domly divided into five groups (n = 14 per group): mock (lipo-some alone), Luc siRNA, CycD1b siRNA, Dox (alone), andDox+CycD1b siRNA. For the mock, Luc siRNA, and CycD1bsiRNA groups, mice were treated with repeated weekly intratu-moral injections for 4 weeks of liposome alone, Luc siRNA(10 lg), or CycD1b siRNA (10 lg), respectively. Mice in theDox group were injected weekly with Dox (2 mg ⁄ kg, i.p.) for4 weeks. Mice in the Dox+CycD1b siRNA group were treatedfor 4 weeks with weekly injections of Dox (2 mg ⁄ kg, i.p.), fol-lowed the following day with CycD1b siRNA treatment. Allthese experiments were repeated three times.

All animal protocols performed in the present study wereapproved by the Institutional Animal Care and Use Committeeof Shanghai Jiaotong University. Nude mice, aged 4–5 weeks,were used for the in vivo studies.

Immunohistochemistry. Tumors were dissected 2 days afterthe last mock or siRNA treatment and then sectioned, deparaffi-nized, rehydrated, and stained with anti-CycD1b antibody (SantaCruz Biotechnology) according to manufacturer’s instructionsand then assayed using the Pierce BCA Protein Assay kit(Thermo Scientific, MA, USA). For each treatment group, atleast two tumor samples and two slides per sample were ana-lyzed. For each slide examined, 1000 cells were counted fromfive fields at ·200 magnification and the number of CycD1b-positive cells as a percentage of total cells determined.

TUNEL assay. Apoptotic cells were identified using the TUN-EL labeling kits from Roche (Indianapolis, IN, USA), accordingto the manufacturer’s instructions. Brown-stained (apoptotic)cells were counted under the microscope at ·200 magnification.The apoptotic index (AI) was defined as the percentage of browncells among total cells for each sample. For each cell line ana-lyzed, 200 cells were counted; for each tumor sample analyzed,1000 cells were counted from each fields. Experiments wererepeated three times.

Synergistic effects of CycD1b siRNA and Dox treatmentof cultured cells. SK-BR3 and MDA-MB436 cells (1 · 104

cells ⁄ well) were transfected with different concentrations ofCycD1b siRNA (0, 5 lg, 10 lg, 20 lg, 50 lg, 100 lg/well).Then, 24 h after transfection, cells were split into 96-well cultureplates and treated with different concentrations of Dox for 8 days(0, 2 ng, 5 ng, 20 ng, 50 ng, 100 ng ⁄ well). The number of viablecells was determined using the MTT assay, as described previ-ously.(33) The synergistic inhibitory effects of CycD1b siRNAand Dox were determined using combination indices of IC80,IC90, and IC95 using the combination index (CI) isobologrammethod developed by Chou,(34,35) where a CI of 1 indicates anadditive effect, a CI > 1 indicates synergism, and a CI < 1indicates antagonism.

Statistical analysis. Statistical analyses were performed usingtwo-tailed Student’s t-test. Data are presented as the mean ± SDand differences were considered significant at P = 0.05.


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Fig. 2. Apoptosis and decreased cell proliferation induced bydepletion of cyclin D1b (CycD1b) in CycD1b-overexpressing breastcancer cells (SK-BR3, MDA-MB157, and MDA-MB436). (a,b)Downregulation of CycD1b promotes apoptosis of SK-BR3, MDA-MB436 and MDA-MB157 cells (a), but not of T47D and MDA-MB453cells (b), in which CycD1b is low. Apoptosis was determined by flowcytometry 48 h after transfection of mock (n), CycD1b (h), orluciferase (Luc [control]; ) siRNA. (c,d) Apoptosis was confirmed bythe TUNEL assay 48 h after Luc or CycD1b siRNA transfection. (c)Apoptotic cells (brown) were detected in the MDA-MB436 cell line,but not in the T47D cell line. (d) Percentage of apoptotic cells in theMDA-MB436 and T47D cell lines following Luc (h) or CycD1b (n)siRNA transfection. (e) Cell cycle distribution, as determined by flowcytometry, in the five breast cancer cell lines (n = 3 for each group).(h), G1 ⁄ G0 phase; ( ), S phase; (n), G2 ⁄ M phase. The S phasepopulation was decreased by CycD1b siRNA in CycD1b-overexpressingcells, but not in cells in which CycD1b expression was low.

Results

CycD1b overexpression is suppressed in breast cancer cells bysiRNA targeting. To determine whether CycD1b can serve as anovel therapeutic target for breast cancer, we first used siRNAoligonucleotides to deplete CycD1b expression in breast cancercells. The siRNA oligonucleotides were transfected into threeCycD1b-overexpressing breast cancer cell lines (SK-BR3,MDA-MB157, and MDA-MB436) and two cell lines in whichCycD1b expression is low (T47D and MDA-MB453). Proteinlevels of the full-length CycD1b (50 kDa) were reduced by upto 96% by CycD1b siRNA in all five cell lines (Fig. 1). Theinhibitory effect of the CycD1b siRNA was shown to be specificbecause a control siRNA oligonucleotide targeting fireflyluciferase mRNA (Luc) had no effect on CycD1b expression(Fig. 1). Moreover, siRNA oligonucleotides did not producenon-specific downregulation of gene expression, as demon-strated by the GADPH control (Fig. 1). These data indicate thatCycD1b siRNA can effectively suppress the overexpression ofCycD1b.

Induction of apoptosis and cell cycle arrest in the G1 phasefollowing siRNA-mediated depletion of CycD1b overexpression.Because CycD1b and its functional complex CycD1b–Cdk2play pivotal roles in the G1 ⁄ S phase transition, we examinedchanges in the cell cycle distribution and apoptosis in CycD1b-depleted cells. To determine whether depletion of CycD1bpromotes tumor cell death, flow cytometry was performed aftersiRNA transfection. Cells were analyzed at different time pointsafter transfection (72 and 96 h) and significant sub-G1 (apopto-tic) populations were observed at 96 h in CycD1b-overexpress-ing cells (SK-BR3, MDA-MB157, and MDA-MB436).Approximately 14% of these cells underwent apoptosis aftertransfection of CycD1b siRNA (Fig. 2a). In contrast, only 4% ofthe same cell lines underwent apoptosis in the mock or LucsiRNA-treated groups (Fig. 2b). The CycD1b-overexpressingcells shrank, became rounder, and detached from the plates3 days after transfection of CycD1b siRNA, whereas controlsiRNA-treated cells remained attached on the plates and exhib-ited normal morphology, also suggesting that apoptosis hadoccurred. In addition, we confirmed CycD1b siRNA-inducedapoptosis in CycD1b-overexpressing cells using the TUNELassay (Fig. 2c). In contrast with CycD1b-overexpressing cells,we did not observe significant apoptosis in cells in whichCycD1b expression was low (MDA-MB453 and T47D) 96 hafter transfection (Fig. 2b,c), even though CycD1b protein levelsin these cells were suppressed by CycD1b siRNA treatment(Fig. 1). These data indicate that depletion of CycD1b specifi-cally triggers apoptosis in CycD1b-overexpressing cells.

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Fig. 1. Cyclin D1b (CycD1b) siRNA is able to suppress CycD1b overexpreMDA-MB436, MDA-MB453, and T47D) were transfected with siRNA oligonprotein lysates of the transfected cells were subjected to western blottingRelative levels of CycD1b in each cell line was determined after transfeMaterials and Methods.

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Furthermore, 48 h after CycD1b siRNA transfection, weobserved increased G0 ⁄ G1 and decreased S phase populations inall three CycD1b-overexpressing cell lines tested, but not in the

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ssion in breast cancer cell lines. Five cell lines (SK-BR3, MDA-MB157,ucleotides targeting either CycD1b or luciferase (Luc; control). (a) Theanalysis using anti-CycD1b and anti-GADPH antibodies (top panel). (b)

ction of mock (n), CycD1b ( ), or Luc (h) siRNA, as described in the

Cancer Sci | August 2011 | vol. 102 | no. 8 | 1539ªª 2011 Japanese Cancer Association

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Fig. 3. Inhibition of cell transformation by cyclinD1b (CycD1b) siRNA in vitro. (a) Growth curves forbreast cancer (SK-BR3, MDA-MB157, MDA-MB436,MDA-MB453, and T47D) and normal epithelial(MCF-10A) cells following CycD1b siRNAtransfection. Data are the mean ± SD (n = 3). (b)Suppression of colony formation in soft agar byCycD1b siRNA. SK-BR3 and T47D cells weretransfected with mock ( ), CycD1b ( ), orluciferase (Luc; ) siRNA and then seeded in0.35% agar containing DMEM and 10% FBS. Cellviability was determined by the colony formationassay, with the number of colonies following Luc orCycD1b siRNA transfection given as a percentage ofthat following mock siRNA transfection (set at100%). Data are the mean ± SD (n = 3). *P = 0.001compared with mock transfection.

cells in which CycD1b expression was low (MDA-MB453 orT47D; Fig. 2e). Together, these results indicate that CycD1bsiRNA has a specific effect on CycD1b-overexpressing breastcancer cells by promoting apoptosis as well as inhibiting G1 ⁄ Sphase transition.

Depletion of CycD1b inhibits proliferation and transformationin CycD1b-overexpressing breast cancer cells. To determinewhether CycD1b siRNA actually affects proliferation, weexamined the growth curves of three CycD1b siRNA-treatedgroups. Growth was significantly suppressed in the CycD1b-overexpressing cell lines and the two cells lines in whichCycD1b expression was low following CycD1b siRNA treat-ment. As shown in Fig. 3(a), CycD1b siRNA significantlydecreased cell numbers in all three CycD1b-overexpressing celllines (SK-BR3, MDA-MB157, and MDA-MB436) comparedwith control. However, for the two cells lines in which CycD1bexpression was low (T47D and MDA-MB453) or normal breastepithelial cells (MCF10A), cell number was not significantlyaffected after CycD1b siRNA transfection (Fig. 3a). In addi-tion, CycD1b depletion inhibited the transformation phenotypeof CycD1b-overexpressing cells. As shown in Fig. 3(b),CycD1b siRNA significantly reduced the ability of SK-BR3cells to grow on soft agar, a well known assay that measurescell transformation. This suppression of transformation was alsoobserved for the MDA-MB157 and MDA-MB436 cell lines fol-lowing depletion of CycD1b expression (data not shown). Incontrast, we did not observe significant inhibitory effects on thetransformation of T47D (Fig. 3b) or MDA-MB453 cells (datanot shown) following CycD1b siRNA targeting. Together, theseresults show that CycD1b siRNA can significantly inhibit pro-liferation and transformation of CycD1b-overexpressing breastcancer cells.

CycD1b siRNA treatment suppresses tumor growth in vivo. Todetermine whether CycD1b siRNA could suppress breast cancergrowth in vivo, we established breast tumors in nude mice andthen treated the mice with CycD1b siRNA. Because MDA-MB436 cells can grow easily in nude mice, we selected thisCycD1b-overexpressing cell line to establish the tumor model.

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As shown in Fig. 4(a), tumor growth in the CycD1b siRNA-trea-ted group was significantly inhibited compared with the controlgroup. To further assess whether CycD1b siRNA was able tosuppress the tumor growth, mice were not treated with thesiRNA until the tumor burden at each site was >50 mm, ataround 4 weeks after tumor cell injection. After 4 weeks treat-ment, tumor progression in the CycD1b siRNA-treated groupwas significantly suppressed (Fig. 4b), which clearly demon-strated the inhibitory effect of CycD1b siRNA on establishedtumors. In addition, western blotting and CycD1b immunohisto-chemistry showed that CycD1b protein levels were decreased inmost of the CycD1b siRNA-treated tumors (Fig. 4c,d). Indeed,considerably fewer cells overexpressing CycD1b were seen intumors treated with CycD1b siRNA compared with those treatedwith PBS or control siRNA (Luc siRNA; Fig. 4d). Furthermore,the TUNEL assay revealed that there were more CycD1b apop-totic cells in the CycD1b siRNA-treated tumors (Fig. 4e), indi-cating that CycD1b depletion was able to induce apoptosisin vivo, leading to tumor suppression. We also tested the inhibi-tory efficacy of CycD1b siRNA using the tumorigenicity assayon another CycD1b-overexpressing cell line, namely SK-BR3cells, as well as on T47D cells, a cell line in which CycD1bexpression is low. Treatment with CycD1b siRNA markedlyinhibited the tumor growth of SK-BR3 cells, but only exerted amild suppressive effect on the growth of T47D cells in mice(Fig. 4f). Together, these results show that treatment withCycD1b siRNA can inhibit the growth of CycD1b-overexpress-ing breast tumor cells in vivo, indicating that CycD1b siRNAmay serve as a novel therapeutic agent for the treatment ofbreast cancer in which CycD1b is overexpressed.

Synergistic inhibitory effects are achieved in vivo with acombination of CycD1b siRNA and Dox. To broaden any poten-tial clinical applications, we evaluated any synergistic effectson breast cancer cell growth of a combination of CycD1b siRNAand Dox (50 ng ⁄ mL), a chemotherapeutic drug commonlyused to treat breast cancer patients.(36,37) In this series ofexperiments, SK-BR3 and MDA-MB436 cells were treatedwith CycD1b siRNA or Dox alone or in combination. The


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Fig. 4. Tumor growth in vivo is inhibited by cyclin D1b (CycD1b) siRNA. (a) Growth of MDA-MB436 tumor xenografts is suppressed by CycD1bsiRNA treatment. The red arrows indicated the days when mice were administered with breast cancer cells. Mice were killed on Day 40 aftertumor cell injection and tumor samples were collected. Representative tumor samples from each group are shown on the right panel. Tumorsfrom CycD1b siRNA-treated groups were much than those treated with mock or luciferase (Luc) siRNA. Data are the mean ± SD (n = 3). *P = 0.05compared with mock transfection. ( ), mock; ( ), Luc siRNA; ( ), CycD1b siRNA. (b) Growth inhibition of established tumor xenografts byCycD1b siRNA. MDA-MB436 cells were injected into two mammary glands of each nude mouse. When the tumors reached approximately50 mm3 (around 4 weeks after tumor cell injection), mice were randomly treated with weekly intratumoral injections of mock or with Luc orCycD1b siRNA (10 mg). Mice were killed on Day 56 after cell injection and tumor samples were collected. Representative tumor samples fromeach group are shown on the right panel. Data are the mean ± SD (n = 3). *P = 0.05 compared with mock transfection. ( ), mock; ( ), LucsiRNA; ( ), CycD1b siRNA. (c) Assessment of reduced CycD1b protein levels in CycD1b siRNA-treated tumors. MDA-MB436 tumors were collected2 days after the last treatment with mock (M), Luc, or CycD1b siRNA and analyzed for CycD1b protein levels by western blotting. (d)Immunostaining confirmation of reduced CycD1b expression following CycD1b siRNA treatment. MDA-MB436 tumors were sectioned andanalyzed for CycD1b expression using anti-CycD1b immunohistochemical staining (left panels). Data are summarized in the right-hand graph.Data are the mean ± SD (n = 3). *P = 0.01 compared with mock transfection. (e) Induction of apoptosis in CycD1b siRNA-treated tumors, asdetermined by TUNEL assay, with data summarized in the graph. Apoptotic cells stained brown. Data are the mean ± SD (n = 3). *P = 0.01compared with mock transfection. (f) Tumor growth of SK-BR3, but not T47D, cells was significantly inhibited by CycD1b siRNA treatment inmice. From Day 14 after implantation of SK-BR3 or T47D cells, mice were treated weekly with indicated siRNA (10 lg) or liposome alone (mock).( ), mock; ( ), Luc siRNA; ( ), CycD1b siRNA. Data are the mean ± SD (n = 3). *P = 0.05 compared with mock transfection.

combination of CycD1b siRNA and Dox exhibited a muchstronger cell-killing effect than CycD1b siRNA or Dox alone inboth cell lines (Fig. 5a). Alone, 50 ng ⁄ mL Dox had no apparent

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effect on the expression of CycD1b in SK-BR3 and MDA-MB436 cells (Fig. 5b). To determine whether this inhibitoryeffect was synergistic, we calculated the CI using the CI-isobo


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Fig. 5. Enhanced inhibitory effects followingcombination treatment with cyclin D1b (cycD1b)siRNA plus doxorubicin (Dox). (a) Sensitization ofbreast cancer cells to Dox by cycD1b siRNAtreatment. Cell viability was determined by theMTT assay after 8 days of the indicated treatment.Data are the mean ± SD (n = 3). *P = 0.05compared with mock transfection. (b) Western blotanalysis of the effects of Dox (50 ng ⁄ mL) on CycD1blevels in SK-BR3 and MDA-MB436 cells. (c)Synergistic cytotoxicity following combinationtreatment with cycD1b siRNA and Dox. SK-BR3 andMDA-MB436 cells were treated with differentconcentration of siRNA (0–0.1 mg ⁄ well in six-wellplates) and split into 96-well plates. After 8 daysincubation with different concentrations of Dox (0–100 ng ⁄ mL), cytotoxicity was determined using theMTT assay. The combination index (CI) wascalculated as described in the Materials andMethods. Data show the CI value at 80%, 90%, and95% cells killed. Data are the mean ± SD (n = 3). (d)Effects of CycD1b siRNA in combination with Doxon breast tumor growth in mice. As described inthe Materials and Methods, mice were treated for4 weeks from Day 14 after MDA-MB436 cellinjection with weekly injections of CycD1b siRNA(10 mg ⁄ injection; ), luciferase siRNA ( ),liposome alone (mock; ), Dox alone (2 mg ⁄ kg,i.p.; ), or CycD1b siRNA + Dox ( ). The effectsof treatment on tumor growth were determined bymeasuring tumor volume. Data are the mean ± SD.*P = 0.05, **P = 0.01 compared with mock afterfour administrations.

logram method developed by Chou.(34,35) As shown inFig. 5(c), CI values at IC90 and IC95 were <1 for MDA-MB436and SK-BR3 cells, indicating that there was a synergistic inhib-itory effect on these cancer cells of the combination of CycD1bsiRNA and Dox.

We also investigated the therapeutic effects of this com-bined treatment in mice. Approximately 14 days after inocula-tion of MDA-MB436 cells, mice were divided into five groups(n = 14 in each group). The different groups of mice weretreated with liposome alone (mock), Luc siRNA, CycD1b siR-NA, Dox alone, or a combination of Dox+CycD1b siRNA, asdescribed in the Materials and Methods. Treatment with acombination of CycD1b siRNA and Dox was able to suppressthe tumor growth of MDA-MB436 cells to a much greaterdegree than CycD1b siRNA oligonucleotides or Dox alone(Fig. 5d). These findings indicate that combined treatment withCycD1b siRNA plus Dox may provide a novel therapeuticoption for breast cancer in which there is CycD1b overexpres-sion.

Discussion

In many types of cancer, CycD1 is aberrantly expressed.(15,38–40)

Indeed, high levels of the two isoforms of CycD1 are associatedwith a poor prognosis for breast cancer patients.(16,41,42) Byusing CycD1b siRNA to target CycD1b overexpression andassessing breast cancer growth in nude mice, we have shownthat CycD1b siRNA effectively inhibits CycD1b overexpres-sion. Tumorigenesis results from a disturbances of cell cycleprogression and ⁄ or programmed apoptosis, which are perceivedas reciprocal processes. Depletion of CycD1b promoted apopto-

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sis of CycD1b-overexpressing cells and blocked their prolifera-tion and transformation phenotype. We also demonstrated thatCycD1b siRNA inhibited breast tumor growth in nude mice.Furthermore, we found that CycD1b siRNA synergisticallyenhanced the cell-killing effects of Dox in cell culture and thiscombination greatly suppressed tumor growth in mice. Thus, thepresent study clearly demonstrates the therapeutic potential ofCycD1b siRNA for the treatment of CycD1b-overexpressingbreast cancer, including triple-negative breast cancer. Ourresults also indicate that CycD1b, which is overexpressed inbreast cancer, may serve as a novel and effective therapeutictarget.

High levels of CycD1 are correlated with triple-negativebreast cancers.(43,44) Indeed, we found that the overexpression ofone isoform of CycD1, namely CycD1b, is correlated with tri-ple-negative (ER, PR, and HER2 negative) basal-like breast can-cers, which lack specific therapeutic targets. In the presentstudy, two of three CycD1b-overexpressing cell lines usedare triple-negative cancer cells (MDA-MB436 and MDA-MB157).(45,46) Of note, CycD1b siRNA alone or in combinationwith Dox effectively inhibited the growth of these cancer cellsboth in vitro and in vivo in mice. Thus, this combination therapymay be an effective treatment option for triple-negative breastcancers because, as yet, there are no specific treatment guide-lines for these types of cancers, which appear to be very meta-static and have a poor prognosis.

Our results demonstrate that CycD1b siRNA exerts robustantitumor activity by promoting apoptosis and inhibitingDNA replication. The induced apoptosis is only observed inCycD1b-overexpressing cells (SK-BR3, MDA-MB157, andMDA-MB436) and not in cells in which CycD1b expression is


low (MDA-MB453 and T47D). This specificity of action shouldincrease the therapeutic index of siRNA-based therapies forCycD1b-overexpressing cancers. It is unclear how CycD1bdepletion triggers apoptosis in CycD1b-overexpressing cells.However, it is well known that tumor cells are highly dependenton activated oncogenes for their survival and ⁄ or proliferation, aphenomenon called ‘‘oncogene addiction’’. Inactivation of theseactivated oncogenes results in apoptosis and an antitumor action.The most convincing evidence for ‘‘oncogene addiction’’ comesfrom the examples of therapeutic efficacy of antibodies or drugsthat target biomarkers in human cancers, such as the antibodytrastuzumab (Herceptin, Genentech Inc., South San Francisco,CA, USA), targeting the receptor tyrosine kinase HER2 ⁄ NEU inhuman breast cancer. The results of the experiments in the pres-ent study support the concept of ‘‘CycD1b addiction’’ inCycD1b-overexpressing cancers; that is, these cancers rely heav-ily on the activity of CycD1b for continued cell proliferationand survival. Importantly, in addition to promoting apoptosis,depletion of CycD1b by its siRNA sensitizes CycD1b-overex-pressing breast cancer cells to the cell-killing effect of Dox.Doxorubicin is a chemotherapeutic drug that targets S-phasecells by inhibiting topoisomerase II.(47) Flow cytometry datafrom the present study demonstrate that depletion of CycD1bdoes not lead to accumulation of cells in the S phase, suggestingthat sensitization to Dox is effected via mechanisms other thanS phase accumulation. Interestingly, it has been shown thatcell survival pathways, such as NF-jB, Akt, and the Bcl-2 fam-ily, can be activated to antagonize the cytotoxic effects ofDox.(48–51) Blockade of these pathways sensitizes breast cancercells to Dox.(52) Therefore, it will be interesting to investigate infuture studies whether CycD1b overexpression may enhancesurvival pathways and therefore increase resistance to Dox.

Wei et al.

An effective delivery system is required for the practicalapplication of siRNA in vivo therapy. In the present study,CycD1b siRNA was delivered by intratumoral injections ofliposomes and this route of administration was found to beeffective for the inhibition of CycD1b expression in vivo, result-ing in tumor suppression. However, the CycD1b siRNA used inthe present study was not chemically modified and so has rela-tively limited in vivo stability and membrane permeability. Toapply CycD1b siRNA therapy in the clinical setting, it would beof considerable benefit if the siRNA could be modified toreduce its sensitivity to nucleases and to increase its cellularuptake. Various approaches have recently been used to chemi-cally modify siRNA to increase its nuclease resistance andintracellular uptake.(53–56) It will be interesting to test the effectsof these chemical modifications on the CycD1b siRNA, particu-larly with respect to improvements in therapeutic efficacy, espe-cially for systemic treatment in mice. In addition to chemicalmodifications of siRNA, novel delivery systems can be used toimprove the stability and efficacy of CycD1b siRNA,(57–59) suchas carrying siRNA by targeted nanoparticles, and to thus furtherimprove the therapeutic efficacy of CycD1b siRNA.

Acknowledgments

This work was supported by grants from the Science and TechnologyCommission of Shanghai Municipality (J-50208) and Shanghai NaturalScience Funding (11ZR1422900).

Disclosure Statement

The authors have no conflict of interest.

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Knocking down cyclin D1b inhibits breast cancer cell growth and suppresses tumor development in a breast cancer model