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HSPC111 governs breast cancer growth by regulating ribosomal biogenesis

HSPC111Governs Breast Cancer Growth by Regulating Ribosomal Biogenesis

Changwen Zhang1,2,Chunyang Yin1,Lei Wang1,Shuping Zhang1,Yi Qian1,Juan Ma1,

Zhihong Zhang2,Yong Xu2,and Sijin Liu1

Abstract

Activation of c-Myc plays a decisive role in the development of many human cancers.As a transcription factor,c-Myc facilitates cell growth and proliferation by directly transcribing a multitude of targets,including rRNAs and ribosome proteins.However,how to elucidate the deregulation of rRNAs and ribosome proteins driven by c-Myc in cancer remains a signi?cant challenge and thus warrants close investigation.In this report,a crucial role for the HSPC111(NOP16)multiprotein complex in governing ribosomal biogenesis and tumor growth was determined.

It was discovered that enhanced HSPC111expression paralleled the upregulation of c-Myc and was directly regulated by c-Myc in breast cancer cells.Knockdown of HSPC111dramatically reduced the occurrence of tumorigenesis in vivo,and largely restrained tumor cell growth in vitro and in vivo.In stark contrast,HSPC111 overexpression signi?cantly promoted tumor cell growth.Biochemically,it was demonstrated that RNA 30-phosphate cyclase(RTCD1/RTCA)interacted with HSPC111,and RTCD1was involved in the HSPC111 multiprotein complex in regulating rRNA production and ribosomal biogenesis.Moreover,HSPC111and RTCD1synergistically modulated cell growth and cellular size through commanding rRNA synthesis and ribosome assembly coupled to protein production.Finally,overall survival analysis revealed that concomitant upregulation of HSPC111and RTCD1correlated with the worst prognosis in a breast cancer cohort.

Implications:Inhibition of HSPC111-dependent ribosomal biosynthesis and protein synthesis is a promising therapeutic strategy to diminish breast cancer tumor progression.Mol Cancer Res;12(4);583–94.ó2014AACR.

Introduction

Breast cancer is by far the most common cancer among women and the leading cause of cancer–related deaths world-wide.The primary tumor and metastases to distant organs often cause signi?cant morbidity and mortality.Numerous studies suggest that the oncogene c-Myc plays a crucial role in the development and progression of breast cancer.Along with its partner protein Max,c-Myc regulates an estimated10%to 15%of genes in the human genome,and globally reprograms cells and drives proliferation(1–3).Aberrant regulation and overexpression of c-Myc are observed in most tumor types,and the c-Myc signaling is believed to play a critical role in oncogenesis(4–7).A large body of studies have shown that c-Myc is pathologically ampli?ed and/or overexpressed in breast cancers(8,9),particularly in late-stage tumors(10–12).Its activation seems to be a prognostic marker in predicting recurrence and adverse outcomes in patients with breast cancer(5–7,12–16).

Enhanced rRNA synthesis and ribosomal biogenesis are commonly seen in human cancers coupled with resultant protein translation and cell-proliferation acceleration(17–19).The effect of c-Myc on drivingcell growth is in part due to its role in transcribing rDNA and advancing ribosomal biogenesis(20).The precise mechanisms responsible for c-Myc-controlled ribosomal biogenesis linked to protein synthesis are still largely unknown,such as the cross-talk and coordination among c-Myc–regulated ribosomal proteins, particularly in cancers.A c-Myc target,HSPC111,was recently reported to be overexpressed in breast cancers,and it might have a role in rRNA synthesis and ribosomal assembly (21,22).Moreover,the upregulation of HSPC111was associated with poor prognosis in patients with breast cancer (21).However,its biologic role in tumor development and progression has not been recognized,and the molecular basis underlying its involvement in rRNA synthesis and ribosomal biogenesis(such as its partners)has not been characterized. Here,we embarked on the biologic effects of the HSPC111multiprotein complex on ribosomal biogenesis

Authors'Af?liations:1State Key Laboratory of Environmental Chemistry

and Ecotoxicology,Research Center for Eco-Environmental Sciences,

Chinese Academy of Sciences,Beijing;and2Department of Urology,

Second Hospital of Tianjin Medical University,Tianjin Institute of Urology,

Tianjin,China

Note:Supplementary data for this article are available at Molecular Cancer

Research Online(https://www.wendangku.net/doc/ea9228245.html,/).

Corresponding Authors:Professor Sijin Liu,State Key Laboratory of

Environmental Chemistry and Ecotoxicology,Research Center for Eco-

Environmental Sciences,Chinese Academy of Sciences,Beijing100085,

China or Professor Yong Xu,Department of Urology,Second Hospital of

Tianjin Medical University,Tianjin Institute of Urology,Tianjin300211,

China.Phone:010********:FAX:010********:E-mail:

sjliu@https://www.wendangku.net/doc/ea9228245.html, or xuyong8816@https://www.wendangku.net/doc/ea9228245.html,

doi:10.1158/1541-7786.MCR-13-0168

ó2014American Association for Cancer Research.

https://www.wendangku.net/doc/ea9228245.html,583

and consequential tumor growth.We demonstrated that HSPC111knockdown largely inhibited cell growth of MDA-MB-231breast cancer cells in vitro and in vivo. HSPC111protein was identi?ed to be predominantly local-ized in nucleus,and was certi?ed to modulate ribosomal biosynthesis.In addition,HSPC111was testi?ed to interact with RNA30-phosphate cyclase(RTCD1),an enzyme catalyzing conversion of a30-phosphate group into the 20,30-cyclic phosphodiester at the30end of RNA(23, 24).Knocking down HSPC111or RTCD1,in particular knocking down both,largely hampered overall rRNA syn-thesis and consequential protein translation in cancer cells. Therefore,targeting HSPC111or its partners might repre-sent a novel approach for breast cancer therapeutics. Materials and Methods

Cell culture

The human breast cancer cell lines MDA-MB-231,MCF-7,and T47D were purchased from the Cell Resource Center Af?liated to the Chinese Academy of Medical Sciences.All cells were cultured in RPMI1640medium with10% newborn calf serum and penicillin–streptomycin(100 units/mL)at37 C with5%CO2.

Quantitative real-time PCR analysis

Total RNAs were performed from cells using TRizol (Invitrogen),and quantitative real-time polymerase chain reaction(qRT-PCR)analysis was assessed with a kit from Promega according to the manufacturer's instruction.A standard curve was constructed to calculate the relative content of interest mRNAs.glyceraldehyde-3-phosphate dehydrogenase(GAPDH)was used as an internal control. Primer sequences were listed in Supplementary Table S1.

Viral particle infection

MDA-MB-231cells were infected with lentiviral trans-duction particles of HSPC111short hairpin RNAs(shRNA) and nontarget shRNAs(scrambled control;Sigma). HSPC111shRNA transduction particles include5individ-ual constructs,which target5different regions of its mRNA. Stable transfectants were obtained15days after selection with puromycin(10m g/mL;Sigma).

siRNA molecule and plasmid transfection Prevalidated siRNA molecules were used to target RTCD1or HSPC111mRNAs.Following the instructions from the manufacturer,MDA-MB-231cells were trans-fected with siRNA molecules and nontarget siRNAs(scram-bled control)using siPORTTM NeoFXTM Transfection Agent(Ambion).HSPC111or c-Myc overexpression con-struct was transfected into MCF-7cells using Lipofectamine 2000(Invitrogen).Experiments were performed48hours after transfection.

Protein concentration determination and Coomassie Brilliant Blue staining

Cells(5.0?105)were harvested into lysis buffer(Solar-bio)after washing with cold PBS.Protein concentration determinations were performed with the Lowry protein assay (Solarbio)according to the instructions provided by the manufacturer.Equal amounts of protein(10m L/lane)were subjected to10%SDS-PAGE and processed for Coomassie Brilliant Blue staining as described previously(24).

Western blot analysis

Equal amounts of protein(30–50m g/lane)were subjected to10%SDS-PAGE and processed for Western blot analysis as described previously(25).Antibodies(Abs)used were anti-c-Myc(1:2,000;Sigma),anti-RTCD1(1:1,000; Abcam),anti-HSPC111(1:200)and anti-GAPDH (1:8,000;the latter2Abs from Santa Cruz Biotechnology).

Coimmunoprecipitation and mass spectrometry

Cells were lysed with the mammalian protein extraction reagent(Cwbio.Inc.),and the supernatants were incubated with2m g anti-HSPC111,or normal immunoglobulin G (IgG).Thereafter,immunoprecipites were collected with the Gamma-Bind A Sepharose beads(GE Healthcare).Immu-noprecipitated proteins were eluted with4?SDS-PAGE sample buffer by boiling for5minutes,and then assessed by Western blot analysis.The proteins within the puri?ed immunoprecipitates were also identi?ed by MALDI-TOF MS(Bruker Daltonics)following the standard procedures (26,27).

Immuno?uorescent staining

Cells were?xed by formaldehyde followed by remobili-zation with Triton X-100.Thereafter,cells were blocked for 1hour with1%Fetal calf serum(FCS)in PBS,and were incubated for2hours at room temperature with the anti-HSPC111rabbit antibody(1:200)and anti-RTCD1mouse antibody(1:200).Cells were then washed3times with PBS followed by incubation with dylight594-af?nipure goat anti-rabbit secondary antibody and dylight488-af?ni-pure goat anti-mouse IgG secondary antibody(EarthOx, LLC)for1hour.After washing with PBS,cells were examined under a confocal laser-scanning microscope.Cell nuclei were counter stained with40,6-diamidino-2-pheny-lindole(DAPI;Molecular Probes).

Cell-proliferation assay

Cell proliferation was determined by cell number count-ing,the MTT proliferation assay,and the BrdUrd incorpo-ration assay(Roche).Brie?y,cells were serum starved overnight and then seeded at a concentration of5.0?104cells per well in100m L culture medium with1%FCS. Cell culture continued for48hours,and then cell growth was assessed following the instructions provided by the manufacturers.

Metabolic labeling of nascent pre-rRNAs with32P in MDA-MB-231cells

MDA-MB-231cells were transfected with HSPC111or RTCD1siRNA molecules,or nontarget siRNAs as described above for48hours.The culture medium was replaced with phosphate-free medium supplemented with

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[32P]Orthophosphate (Perkin-Elmer Life Sciences)at a ?nal concentration of 100m Ci/mL,and cells were incubated for 1.5hours.Thereafter,the culture medium was replaced with nonradioactive medium,and cells were incubated for anoth-er 2hours.Total RNAs were extracted using TRizol from the same number of cells (5.0?105)for each group,and ?nally dissolved in 30m L ddH 2O.An equal volume of total RNAs (20m L)from each sample was separated with 1%agarose formaldehyde gel,and the radioactive bands were detected by autoradiography.

Animal experiments

All animal care and surgical procedures were approved by the Animal Ethics Committee at RCEES,Chinese Academy of Sciences.Six-week-old immunode ?cient (BALB/c nude)female mice were maintained under aseptic sterile condi-tions.Surgeries were performed under sterile conditions and mice received antibiotics (Gentamycin)in drinking water up to 2weeks following surgical procedures.MDA-MB-231cells (4.0?106)were orthotopically inoculated into the bilateral mammary fat pads of nude mice.The cells were injected in 1:2diluted matrigel (BD Biosciences):sterile PBS using a Hamilton syringe.Tumor size was closely monitored with a vernier caliper,and calculated according to the formula p /6?L ?W 2along time course.Mice were sacri ?ced when tumors reached a size of 1.0cm 3.Survival analysis

A publicly-available dataset was used to evaluate the role of HSPC111and RTCD1in affecting the outcome of patients with breast cancer.This dataset was generated using the Affymetrix oligonucleotide microarray U133a GeneChip,

representing 22,000transcripts,from 286lymph node –negative patients who had not received adjuvant systemic treatment (28).The normalized mRNA expression data and clinical information were obtained from the NCBI/Gen-Bank GEO database (accession number GSE2034).The 6-year survival rates (for 72months)were estimated with the Kaplan –Meier method,and the threshold was determined as previously described,that is the expression value 330for HSPC111and 1085for RTCD1(28).

Quanti ?cation of Western blots and statistical analysis The intensities of autoradiogram were quanti ?ed with Image J (NIH,https://www.wendangku.net/doc/ea9228245.html,),and quanti ?ed data of each protein were normalized with those of GAPDH.Two-tailed Student t test was used to analyze experimental data between 2groups.Data were shown in means ?SE.P <0.05was considered statistically signi ?cant.

Results

A contributive role of HSPC111in tumor growth

As a crucial transcriptional factor,c-Myc facilitates tumor cell growth through directly transcribing a variety of targets (4–7).A previous study suggested that HSPC111is a target of c-Myc (25);however,its role in tumorigenesis has not been characterized yet.In this study,to elucidate the role of HSPC111in breast cancer development and progression,we ?rst assessed the expression of c-Myc and HSPC111in breast cancer cells.The mRNA level of c-Myc in malignant cell line MDA-MB-231was considerably increased com-pared with that in nonmalignant cell lines,T47D and MCF-7(P <0.05,Fig.1A).HSPC111's promoter has a c-Myc binding site,and its expression is directly transcribed

Figure 1.Increased c-Myc and HSPC111expression in malignant breast cancer cells.A,relative expression of c-Myc and HSPC111in malignant cell line MDA-MB-231compared with that in

nonmalignant cell lines,T47D and MCF-7,assessed by qRT-PCR analysis (n ?5–6).B,Western blot analyses of c-Myc and HSPC111in MDA-MB-231cells,MCF-7

cells,and T47D cells.C,Western blot analysis of c-Myc and

HSPC111protein concentrations in MCF-7cells upon transfection with c-Myc plasmid with or

without combination of HSPC111siRNAs for 48hours.D,MCF-7cells were transfected with c-Myc plasmid with or without

combination of HSPC111siRNAs for 48hours,and then cell

growth was assessed with the MTT assay and cell number counting (n ?6).

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c-Myc(25).Consistent with the alteration to c-Myc,the mRNA level of HSPC111was also markedly increased in MDA-MB-231cells compared with T47D and MCF-7cells (P<0.05,Fig.1A).Similar results were observed for c-Myc and HSPC111at the protein level evidenced by the Western blot analysis(Fig.1B),in parallel to the previous?ndings (25).

To further delineate the functional link between c-Myc and HSPC111,in other words,to?gure out the regulation of c-Myc on HSPC111and the dependence of c-Myc–driven cell growth on HSPC111as well,we elevated c-Myc level through forced expression with combination of HSPC111knockdown in MCF-7cells.As shown in Fig. 1C,the protein concentration of c-Myc was increased by 34%for cells upon forced expression,and,as a result, HSPC111was greatly induced by more than3-fold(lane 3)compared with the control,supporting the regulation of HSPC111by c-Myc(21,25).Consequentially,cell growth was signi?cantly promoted by more than2-fold compared with the control,evidenced by the MTT assay and direct cell number counting(Fig.1D,P<0.05).To further con?rm the regulation of HSPC111by c-Myc,we induced exogenous c-Myc expression using a conditionally regulated system by fusing c-Myc and the estrogen recep-tor hormone-binding domain.Upon induction of tamox-ifen(at1m mol/L)in MCF-7cells for24hours,c-Myc expression was signi?cantly induced by>3-fold,and,as a result,HSPC111expression was elevated by>5-fold with consequential great increase(approximately40%)of cell proliferation(Supplementary Fig.S1and data not shown). However,for cells transfected with vehicle control con-struct upon induction of tamoxifen,endogenous c-Myc expression was not signi?cantly affected(data not shown). These results suggested that the upregulation of HSPC111 was because of the induction of exogenous c-Myc rather than endogenous c-Myc for cells transfected with MYC-ER construct.These results together con?rmed the reg-ulation of HSPC111by c-Myc.In support of our?nding, Butt and colleagues recently demonstrated that HSPC111 was a target of c-Myc through luciferase reporter assay, electrophoretic mobility shift assay,and chromatin immu-noprecipitation assay(21).

When simultaneously overexpressing c-Myc and knock-ing down HSPC111(with more than60%reduction,lane 2in Fig.1C),c-Myc–derived cell growth was greatly restrained by approximately45%compared with the cells with c-Myc overexpression only,as re?ected by the MTT assay and cell number counting(Fig.1D,P<0.05).It should be noted that cell growth was still signi?cantly simulated in cells with c-Myc overexpression and HSPC111knockdown,compared with the control(Fig. 1D,P<0.05).These?ndings indicated that the ability of c-Myc in driving cell growth partially but not exclusively relied on HSPC111.These data together stressed the functional link between c-Myc and HSPC111in cancers, and implied that upregulation of HSPC111driven by c-Myc could be contributive to development and malig-nancy of breast cancers.

We thus reduced the endogenous expression of HSPC111 in malignant MDA-MB-231cells through infection of lentivirus-mediated shRNA constructs.We obtained stable transfectants with HSPC111shRNA expression after selec-tion.As shown in Fig.2A,The mRNA level of HSPC111 was reduced63%upon HSPC111-speci?c shRNAs,and similar results were also observed in the protein level. Consequentially,the cell proliferation of MDA-MB-231 was signi?cantly restrained by45%characterized with the BrdUrd incorporation assay(P<0.05,Fig.2B),and similar results were also re?ected by the MTT assay(P<0.05,Fig. 2B).To decipher the role of HSPC111in tumorigenesis and tumor progression in vivo,we implanted MDA-MB-231 cells with HSPC111knockdown into mammary fat pads of nude mice,and tumor formation and growth were closely monitored.We?rst compared the tumor take rate between the2groups.In the scrambled control group,100%mice (10of10)harbored bilateral tumors(for a total of20 tumors),whereas only50%mice(5of10)developed bilateral tumors and one mouse had a unilateral tumor(for a total of11tumors)in the HSPC111-downregulation group.Thus,the tumorigenesis was greatly suppressed by 45%in the HSPC111-downregulation group in comparison to the scrambled control group(55%vs.100%).As shown in Fig.2C,the tumor growth for cells with HSPC111 downregulation was substantially repressed along time course compared with the scrambled control from day24 to day38(P<0.001).The representative tumor image was shown in Fig.2C,and the?nal tumor weight was reduced by 86%in mice implanted with HSPC111-shRNA cells com-pared with the vehicle control(Fig.2D,P<0.05).

To con?rm the role of HSPC111in promoting cell proliferation,we thereafter enforced HSPC111expression in nonmalignant MCF-7cells.As shown in Fig.2E,the concentration of HSPC111protein was induced by>3-fold in cells upon HSPC111overexpression compared with the vector control.And cell growth was resultantly provoked by approximately110%evidenced by the MTT assay and by about70%re?ected by cell number counting,compared with the vector control(Fig.2F,P<0.05).These results demonstrated that HSPC111played a vital role in modu-lating tumor formation and progression,that is upregulation or downregulation of HSPC111could signi?cantly promote or impede tumor cell growth.

The involvement of RTCD1in the HSPC111 multiprotein complex

To interpret the mechanism by which HSPC111reg-ulates tumor growth,we assessed HSPC111's location and potential interacting protein partners.The Western blot analysis demonstrated that HSPC111protein was pre-dominantly localized in nucleus,but not in cytoplasm (Fig.3A),consistent with the previous observation(21). Because of the principal function of nucleus is rRNA transcription and ribosome assembly,these above?ndings suggested a potential role of HSPC111in regulating ribosomal biosynthesis and nucleolar integrity.To this end,we thus surveyed the potential binding partners of

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HSPC111.Immunoprecipitation was thus carried out in the cellular extracts with the HSPC111antibody,and the immunoprecipitates were puri ?ed by TCA-Acetone fol-lowed by the mass spectrometry analysis.Based on the mass spectrometry analysis,a few candidate binding part-ners were determined by selecting the ones precipitated by the HSPC111Ab after deduction by those precipitated by normal IgG.Among them,RTCD1was assumed to be the one with great possibility of interacting with HSPC111.RTCD1was previously identi ?ed in the HeLa cell extract,and it is an enzyme that catalyzes conversion of a 30-phosphate group into the 20,30-cyclic phosphodiester at the 30end of RNAs (also named RNA 2,3-cyclic phos-phate)in an ATP-dependent manner (23,24).RNA 2,3-cyclic phosphate ends are crucially important in ribosome assembly through regulating RNA-protein binding and RNA stability (27,29–31).Previous studies have validated that RTCD1-mediated conversion of 2,3-cyclic phosphate ends on rRNAs is indispensable to 18S rRNA biogenesis (32,33).Because HSPC111is assumed to play an impor-tant role in ribosomal biosynthesis (21),there is likely a physical and functional overlapping between HSPC111and RTCD1.We therefore assessed the physical interac-tion between HSPC111and RTCD1through Co-IP.We ?rst used the HSPC111Ab to precipitate its binding proteins in MDA-MB-231cell extracts.As shown in Fig.3B (left),RTCD1was recognized to be coprecipitated with HSPC111.Correspondingly,we used the RTCD1Ab to precipitate its binding proteins to verify the inter-action between RTCD1and HSPC111.As presented in Fig.3B (right),HSPC111was identi ?ed to bind with RTCD1.Furthermore,immunohistochemical analysis was used to look into the localization of HSPC111and RTCD1.As shown in Fig.3C,HSPC111and RTCD1were found to overlap in nucleus for both MDA-MB-231and MCF-7cells.These results demonstrated

Figure 2.Tumor growth was modulated by deregulation of HSPC111.A,qRT-PCR and Western blot analyses for the mRNA and protein levels of HSPC111in MDA-MB-231cells infected with HSPC111-shRNA viral constructs or scrambled constructs.B,cell proliferation assessed with the BrdUrd

incorporation assay and the MTT method (n ?6).C,the growth curves and the representative image of tumors from the 2groups of mice.D,the ?nal weight of tumors from the mice implanted with HSPC111-knockdown cells or the control mice.E,Western blot analysis of HSPC111in MCF-7cells transfected with HSPC111plasmid for 48hours.F,MCF-7cells were transfected with HSPC111plasmid for 48hours,and then cell growth was determined by the MTT assay and cell number counting (n ?6).

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interaction between HSCP111and RTCD1,and a poten-tially important role of RTCD1in HSPC111multipro-tein complex –elicited biologic functions.

Diminished rRNA synthesis in cells with HSPC111and RTCD1reduction

As a master transcriptional factor,c-Myc controls prolif-eration by directly transcribing a variety of targets,including a number of rRNAs and ribosome proteins (34–36).Upon forced expression of c-Myc (as described in Fig.1C),the concentrations of 18S and 28S rRNAs were signi ?cantly elevated in MCF-7cells by 3.6-and 4.4-fold,respectively,compared with the vector control cells (Fig.4A).Meanwhile,HSPC111downregulation (as described in Fig.1C)could partially undermine c-Myc –simulated rRNA synthesis (Fig.4A),in agreement with the results of cell growth (Fig.1D).These ?ndings supported our hypothesis of a crucial role of HSPC111(i.e.,the HSPC111-RTCD1complex)in ribo-somal biogenesis.

To further test our hypothesis,we looked into the ef ?cacy of overall RNA synthesis affected by the HSPC111–RTCD1complex.We reduced the endoge-nous expression of HSPC111and RTCD1in MDA-MB-231cells through transfection of siRNA molecules.As shown in Fig.4B,the protein level of HSPC111and RTCD1was reduced >50%and >60%,respectively.We then surveyed the concentrations of nascent RNAs labeled with 32P from cells.As shown in Fig.4C,the concentra-tions of 28S rRNA and 18S rRNA were greatly reduced in cells transfected with HSPC111-speci ?c siRNAs or RTCD1-speci ?c siRNAs compared with those in the parental control and scrambled control cells (P <0.05).Moreover,a more pronounced decline in the 28S rRNA and 18S rRNA concentrations was observed in cells upon binal HSPC111and RTCD1downregulation,compared with the single HSPC111-or RTCD1-knockdown cells (Fig.4C,P <0.05).Similar results were demonstrated for the levels of 18S and 28S rRNA validated by the qRT-PCR analysis (Fig.4D,P <0.05).In contrast,when we elevated the HSPC111level in MCF-7cells,the levels of 18S and 28S rRNAs were markedly increased by 3.8-and 3.4-fold,respectively,compared with the control (Fig.4E,P <0.05).These results con ?rmed a crucial role of HSPC111in directing rRNA synthesis and assembly,in agreement with the ?nding from a previous study (21).These results also implied an important role of RTCD1in modulating rRNA synthesis,consistent with previous studies documenting the involvement of RTCD1in 18S rRNA biogenesis (37,38).The further reduction of rRNA concentrations in cells with synergistic HSPC111

and

Figure 3.The interaction between HSPC111and RTCD1.A,Western blot analysis for the distribution of HSPC111in cytoplasm and nucleus.B,the Co-IP assessment of the interaction between HSPC111and RTCD1.MDA-MB-231cell extracts were immunoprecipitated with the anti-RTCD1Ab or

anti-HSPC111Ab,and coimmunoprecipitated proteins were then determined with anti-HSPC111Ab or the anti-RTCD1Ab through Western blot analysis,respectively.The blue arrow indicates RTCD1(left)or HSPC111(right)in the IP assessment,and the red arrow indicates the existence of HSPC111

(left)and RTCD1(right)in the coimmunoprecipitated proteins.C,confocal images indicative of colocalization of HSPC111and RTCD1in MDA-MB-231and MCF-7cells.Cells were immunostained with antibodies against HSPC111(red,detected with a dylight 594-af ?nipure goat anti-rabbit secondary

antibody)or RTCD1(green,detected with a dylight 488-af ?nipure goat anti-mouse secondary antibody),and were also counter stained with DAPI for nuclei (blue).The original magni ?cation,?400.

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RTCD1downregulation veri ?ed their interactive depen-dence in commanding rRNA synthesis and assembly.Attenuated protein synthesis upon HSPC111and RTCD1reduction

Elevated rRNA concentrations are commonly seen in human cancers associated with enhanced protein synthesis and accelerated cell proliferation (19,39).For example,increased rRNA transcription and ribosomal biogenesis were observed in cancers subject to c-Myc activation (17,18).To this end,we therefore examined the protein concentrations and cell proliferation in MDA-MB-231cells with HSPC111/RTCD1knockdown.As shown in Fig.5A,a signi ?cant decline in protein translation as evidenced by the total protein concentrations was observed in HSPC111-low cells or RTCD1-low cells compared with those in

the

Figure 4.HSPC111and RTCD1modulate rRNA synthesis.A,the concentrations of 18S and 28S rRNAs in MCF-7cells transfected with c-Myc plasmid with or without combination of HSPC111siRNAs for 48hours (n ?6).B,the protein levels of HSPC111and RTCD1in MDA-MB-231cells transfected with HSPC111or RTCD1siRNAs for 48hours,determined by

Western blot.C,autoradiogram of RNA pulse-labeled with [32

P]orthophosphate in HSPC111-low and/or RTCD1-low cells.MDA-MB-231cells were transfected with HSPC111and/or RTCD1siRNAs for 48hours.The arrows indicate the positions of 28S rRNA and 18S rRNA,https://www.wendangku.net/doc/ea9228245.html,ne 1,the parental control;lane 2,the scrambled control;lane 3,

HSPC111-siRNA;lane 4,RTCD1-siRNA;lane 5,Binal HSPC111and RTCD1-siRNA.The band

intensities for the overall RNAs after normalization to the cell numbers were shown in the bar graph.D,the RNA levels of 18S and 28S rRNAs in MDA-MB-231cells transfected with HSPC111

and/or RTCD1siRNAs for 48hours (n ?6).E,the concentrations of 18S and 28S rRNAs in MCF-7cells transfected with HSPC111plasmid for 48hours (n ?6).

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parental control and scrambled control cells (P <0.05).And the total protein concentrations were further reduced in binal-knockdown (HSPC111-low and RTCD1-low)cells,compared with the individual HSPC111-knockdown or RTCD1-knockdown (P <0.05).These results were similar to the studies demonstrating diminished protein translation upon c-Myc loss (20).The molecular bases determining cellular shape and size largely rely on the concentrations of cytosolic macromolecules,such as ribosomes and proteins.Because of reduction in rRNA and protein concentrations upon HSPC111and/or RTCD1downregulation,the mor-phology was greatly altered in MDA-MB-231cells

with

Figure 5.HSPC111and RTCD1modulate protein synthesis,cellular morphology,and cell

growth.A,the total cellular protein concentrations of MDA-MB-231cells transfected with HSPC111and/or RTCD1siRNAs.The graph shows quanti ?ed data of the total cellular proteins determined by the Lowry protein assay after

normalization to the cell numbers (n ?6).B,the images showed the representative morphologic alterations for cells transfected with HSPC111and/or RTCD1siRNA molecules for 48hours.The original magni ?cation,?100.The cellular size was assessed

with a software Image-Pro Plus,and the quanti ?ed data for the cellular size were shown in the left panel (n ?20).C and D,cell growth upon HSPC111and/or RTCD1downregulation.Cells were

transfected with HSPC111and/or RTCD1siRNA molecules for 48hours,and then cell growth was assessed with cell number

counting (C)and the MTT method (D;n ?6).

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condensed cellular body (Fig.5B).The corresponding cellular size was decreased by approximately 22%in HSPC111-low cells and 24%in RTCD1-low cells,com-pared with the parental control and scrambled control,respectively (P <0.05),and a further reduction was observed in the double-knockdown cells by 36%(Fig.5B,P <0.05,),in agreement with the results of attenuation of RNA syn-thesis and protein translation (Fig.4).A similar phenotype was observed to cells upon ribosomal protein S6(rpS6)de ?ciency,as the rpS6P à/àb cells had smaller size with diminished insulin production (40).

Impeded cell growth in response to HSPC111and RTCD1reduction

Ribosomal biogenesis and protein synthesis fundamen-tally determine cell growth (19,41,42).Cell growth was remarkably restrained by approximately 25%in MDA-MB-231cells with HSPC111knockdown characterized by cell number counting and the MTT assay,compared with the parental control and scrambled control (P <0.05,Fig.5C and D).A similar suppression on cell growth was observed in cells upon RTCD1knockdown with $30%decline,re ?ected by cell number counting and the MTT assay,compared with the parental control and scrambled control (P <0.05,Fig.5C and D).Moreover,a further inhibition on cell growth was validated in concomitant-knockdown cells with >35%reduction,compared with the single HSPC111or RTCD1knockdown (P <0.05,Fig.5C and D).These combined results are in agreement with the notion that c-

Myc –induced cell growth is dependent on c-Myc's ability in promoting protein synthesis (20,43).However,the FACS analysis showed that HSPC111/RTCD1single or double knockdown did not trigger cell death via either apoptosis or necrosis in MDA-MB-231cells using FITC-Annexin V and PI staining (data not shown).Thus,these data together highlighted the crucial role of HSPC111in promoting cell growth through regulating the HSPC111–RTCD1multi-protein complex –conducted ribosome production and pro-tein synthesis.

High expression of HSPC111and RTCD1correlated to poor survival

To recognize the signi ?cance of the HSPC111–RTCD1multiprotein complex in tumor development under the clinical setting,we characterized the association of deregu-lated HSPC111and RTCD1expression with survival in patients with breast cancer.We delineated the Kaplan –Meier survival curves of the relationship between HSPC111and/or RTCD1expression and patient survival in a breast cancer cohort database (28).As shown in Fig.6A,the 6-year (for 72months)survival curves indicated that upregulated HSPC111or RTCD1expression correlated to poor prog-nosis in patients with breast cancer.More strikingly,con-comitant upregulation of HSPC111and RTCD1(HSPC111high /RTCD1high )was associated to the worst survival,and concomitant downregulation of HSPC111and RTCD1(HSPC111low /RTCD1low )correlated to the best survival,compared with the rest with

different

Figure 6.High expression of HSPC111and RTCD1correlated to poor survival in a breast cancer cohort.Kaplan –Meier survival curves depicting the relationship between HSPC111and/or RTCD1mRNA expression and survival rates in a publicly available breast cancer cohort.A,the survival curves related to HSPC111and RTCD1mRNA levels,respectively.B,the survival curves describing the synergistic

upregulation or downregulation of HSPC111and RTCD1mRNA expression.

HSPC111Governs Ribosomal Biogenesis

https://www.wendangku.net/doc/ea9228245.html, Mol Cancer Res;12(4)April 2014591

expression levels of HSPC111and RTCD1(P <0.001,Fig.6B).These results suggested that abnormal expression of HSPC111or RTCD1was signi ?cantly involved in tumor development,and the synergistic upregulation of HSPC111and RTCD1exerted more robust impact on tumor progres-sion than one individual gene upregulation only.Discussion

Protein translation is concertedly ruled,and deregula-tion at any step of translational control might predispose cells to transformation or deteriorate tumor progression (20,42).The oncogene c-Myc enhances protein transla-tion through transactivating diverse targets,such as initi-ation factors,ribosomal proteins,and rRNAs.Activation of c-Myc is found in a variety of cancers,including breast cancer,and its activation seems to be a surrogate marker for cancers (44–49).The role of c-Myc in driving protein synthesis is in part attributed to promoting ribosome production through transcribing a number of ribosomal proteins (34–36).HSPC111,as a target of c-Myc,was previously characterized as a ribosomal protein residing in a large RNA-dependent nucleolar complex (25).In this study,to the best of our knowledge,we for the ?rst time demonstrated that HSPC111played a critical role regu-lating cell growth by involving in rRNA synthesis and ribosomal biogenesis,and the biologic function of HSPC111essentially relied on its direct binding partner RTCD1.

Ribosomal biogenesis involving the mRNA-to-protein translational machinery is necessary for cell growth and proliferation.Increased ribosome production coupled to elevated protein translation facilitates cell growth and pro-liferation,and crucially contributes to tumor progression (19,42).Studies have demonstrated that elevated rRNA synthesis is closely associated with cell transformation and cell proliferation,and a few rRNAs are overexpressed in cancers,such as prostate cancer,gastric cancer,and breast cancer (22,37).Deregulated control on ribosomal biogen-esis linked to enhanced translational capacity signi ?cantly contributes to tumorigenesis and tumor aggressivity,as a previous study demonstrated that augmented overall ribo-somal production and translational capacity were closely associated with tumor progression in breast cancers (38).In contrast,attenuation on ribosomal biogenesis could repress cell growth and proliferation (38,50).We here deciphered the function of HSPC111in governing rRNA synthesis and ribosomal assembly,and also delineated a synergistic inter-play between HSPC111and RTCD1in rRNA metabolism and ribosomal biogenesis.Cell proliferation and cellular size were remarkably attenuated in cells with HSPC111or RTCD1downregulation,likely because of the reduction of overall rRNA synthesis and ribosome production in these cells.A further decline of cell proliferation and cellular size was demonstrated in cells with synergistic HSPC111and RTCD1downregulation,supporting a role of their inter-active dependence in commanding rRNA synthesis and ribosome assembly.

A previous study suggested that overexpressed HSPC111was signi ?cantly associated with poor prognostic outcome in patients with breast cancer (21).Similar to HSPC111,we here demonstrated that increased expression of RTCD1was also associated with poor overall survival in patients with breast cancer.Noticeably,concomitant upregulation for both HSPC111and RTCD1(HSPC111high /RTCD1high )corre-lated to the worst prognosis in the breast cancer cohort used in this study.These combined data highlighted the crucial role of HSPC111–RTCD1in driving cell growth through reg-ulating rRNA synthesis,and also pinpointed a promising direction for cancer therapeutic development by suppressing HSPC111/RTCD1-mediated ribosomal biogenesis.

To summarize,our data suggest that the upregulation of HSPC111contributes to breast cancer development,and knocking down the endogenous HSPC111could

greatly

Figure 7.A schematic diagram for the role of the HSPC111–RTCD1multiprotein complex in regulating tumor growth.

Zhang et al.

Mol Cancer Res;12(4)April 2014Molecular Cancer Research

592

restrain tumor progression.Importantly,we veri?ed that RTCD1is a binding partner of HSPC111in the HSPC111 multiprotein complex formation,and these proteins together synergistically regulate rRNA synthesis and ribosomal assem-bly,as knocking down HSPC111or RTCD1,particularly when knocking down both,could profoundly diminish cancer cell growth by repressing ribosomal biogenesis and protein synthesis.Moreover,the synergistic upregulation of HSPC111 and RTCD1indicated the worst prognosis in a breast cancer cohort.These combined data indicate that interference on the HSPC111–RTCD1multiprotein complex might exert inhi-bition on ribosomal biogenesis-driven cell growth.A schematic diagram depicting the possible mechanism responsible for the role of the HSPC111–RTCD1multiprotein complex in regulating tumor growth is illustrated in Fig.7.Therefore, our study has implications for the development of therapeutics by targeting HSPC111-mediated ribosomal biosynthesis for the treatment of breast cancers.

Disclosure of Potential Con?icts of Interest No potential con?icts of interest were disclosed.Authors'Contributions

Conception and design:Z.Zhang,Y.Xu,S.Liu

Development of methodology:C.Zhang,C.Yin,L.Wang,J.Ma Acquisition of data(provided animals,acquired and managed patients,provided facilities,etc.):C.Zhang,L.Wang,S.Liu

Analysis and interpretation of data(e.g.,statistical analysis,biostatistics,compu-tational analysis):C.Zhang,C.Yin,L.Wang,S.Zhang,Y.Xu,S.Liu Writing,review,and/or revision of the manuscript:C.Zhang,L.Wang,Z.Zhang, Y.Xu,S.Liu

Administrative,technical,or material support(i.e.,reporting or organizing data, constructing databases):C.Zhang,Y.Qian

Study supervision:S.Liu

Acknowledgments

We thank lab members for great assistance with experiments and reagents. Grant Support

This work was supported by a grant from the national"973"program(grant No. 2014CB932000),and grants from the National Natural Science Foundation of China (grant Nos.21377159and81172451).

The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solely to indicate this fact. Received April9,2013;revised December13,2013;accepted December26,2013; published OnlineFirst January14,2014.

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