nano-delivery of rad6/translesion synthesis inhibitor smi#9 for … · small molecule therapeutics...

Small Molecule Therapeutics

Nano-delivery of RAD6/Translesion SynthesisInhibitor SMI#9 for Triple-negative Breast CancerTherapyNadia Saadat1,2, Fangchao Liu3, Brittany Haynes1,2, Pratima Nangia-Makker1,2, Xun Bao1,2,Jing Li1,2, Lisa A. Polin1,2, Smiti Gupta4, Guangzhao Mao3, and Malathy P. Shekhar1,2,5

Abstract

The triple-negative breast cancer (TNBC) subtype, regard-less of their BRCA1 status, has the poorest outcome com-pared with other breast cancer subtypes, and currently thereare no approved targeted therapies for TNBC. We havepreviously demonstrated the importance of RAD6-mediatedtranslesion synthesis pathway in TNBC development/progression and chemoresistance, and the potential thera-peutic benefit of targetingRAD6with aRAD6-selective small-molecule inhibitor, SMI#9. To overcome SMI#9 solubilitylimitations, we recently developed a gold nanoparticle(GNP)-based platform for conjugation and intracellularrelease of SMI#9, and demonstrated its in vitro cytotoxicactivity toward TNBC cells. Here, we characterized the invivo pharmacokinetic and therapeutic properties of PEGy-lated GNP-conjugated SMI#9 in BRCA1 wild-type andBRCA1-mutant TNBC xenograft models, and investigatedthe impact of RAD6 inhibition on TNBC metabolism by

1H-NMR spectroscopy. GNP conjugation allowed thereleased SMI#9 to achieve higher systemic exposure andlonger retention as compared with the unconjugated drug.Systemically administered SMI#9-GNP inhibited the TNBCgrowth as effectively as intratumorally injected unconjugat-ed SMI#9. Inductively coupled mass spectrometry analysisshowed highest GNP concentrations in tumors and liver ofSMI#9-GNP and blank-GNP–treated mice; however, tumorgrowth inhibition occurred only in the SMI#9-GNP–treatedgroup. SMI#9-GNP was tolerated without overt signs oftoxicity. SMI#9-induced sensitization was associated withperturbation of a common set of glycolytic pathways inBRCA1 wild-type and BRCA1-mutant TNBC cells. These datareveal novel SMI#9 sensitive markers of metabolic vulnera-bility for TNBC management and suggest that nanotherapy-mediated RAD6 inhibition offers a promising strategy forTNBC treatment.Mol Cancer Ther; 17(12); 2586–97.�2018 AACR.

IntroductionTriple-negative breast cancers (TNBC) account for approxi-

mately 15% of all the breast cancers and because they lackestrogen receptor (ER), progesterone receptor (PGR), and Her2/neu amplification, they are not treatable with therapies targetingthese molecules. TNBCs present with high histologic grade andpatients with TNBC have poorer outcomes compared with otherbreast subtypes (1). The triple-negative phenotype is most com-monly observed in patients with BRCA1/BRCA2 mutations (2).

BRCA1-associated and sporadic TNBCs share features such ashigh-grade cytokeratin expression, p53 mutation, and aberrantDNA repair pathways (3). Because clinicopathologic features ofTNBCs overlap with BRCA-related breast cancers, BRCA1 is con-sidered an important player in TNBC biology. BRCA1 is requiredfor homologous recombination–mediated DNA repair. BRCA1-deficient cells are unable to repair lesions and resort to error-proneDNA repair that increase the risk of cancer progression andchemoresistance. Although patients with TNBC initially respondbetter to chemotherapy, patients with TNBC, regardless of theirBRCA1 status, have decreased progression-free and overall sur-vival rates (4). Thus, strategies that target TNBCswith andwithoutBRCA1 mutation constitute an important component of TNBCtreatment.

RAD6 is a fundamental component of the translesion synthesis(TLS) DNA repair pathway. It is critical for the continuation ofreplication on damaged DNA templates, thereby preventing rep-lication fork stalling and resultant cell-cycle arrest and ultimatelycell death (5). Because the RAD6 pathway is essential for cellsurvival in the face of a variety of genotoxic insults, it plays animportant role in DNA damage tolerance (5, 6). However,because the RAD6-mediated TLS process involves low-fidelity TLSpolymerases, it is potentially an error-prone process. Thus, itsusage needs to be tightly regulated because it is potentiallymutagenic. The E2 ubiquitin–conjugating (UBC) activity ofRAD6is essential for TLS (7). We have previously reported that RAD6B,a.k.a.UBE2B orHHR6B, is weakly expressed in normal breast cellsand overexpressed in metastatic and chemoresistant breast

1KarmanosCancer Institute, Detroit, Michigan. 2Department ofOncology,WayneState University School of Medicine, Detroit, Michigan. 3Department of ChemicalEngineering and Materials Science, Wayne State University College of Engi-neering, Detroit, Michigan. 4Department of Nutrition and Food Sciences, WayneState University College of Liberal Arts and Science, Detroit, Michigan. 5Depart-ment of Pathology, Wayne State University School of Medicine, Detroit,Michigan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

N. Saadat and F. Liu contributed equally to this article.

Corresponding Authors: Malathy P. Shekhar, Wayne State University,Room 1148, Elliman Building, 421 East Canfield Avenue, Detroit, MI 48201.Phone: 313-578-4326; Fax: 313-578-4659; E-mail: [email protected];and Guangzhao Mao, Phone: 313-577-3804; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-18-0364

�2018 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 17(12) December 20182586

on January 7, 2021. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst September 21, 2018; DOI: 10.1158/1535-7163.MCT-18-0364

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-18-0364&domain=pdf&date_stamp=2018-11-21

http://mct.aacrjournals.org/

carcinomas (8–10). Constitutive RAD6B overexpression in nor-mal human breast epithelial cells induces tumorigenesis andchemoresistance (8, 11, 12), whereas RAD6B depletion compro-mises TLS, rendering cells chemosensitive (5). We have reportedthe development of a RAD6-selective small-molecule inhibitorSMI#9 that inhibits UBC activity of RAD6 (13). SMI#9 treatmentsuppresses proliferation andmigration of breast cancer cells whilesparing normal breast cells (13). We recently demonstrated thatintratumorally administered SMI#9 restores cisplatin sensitivityand inhibits growth of cisplatin-resistant TNBC cells. We showedthat this inhibition results from SMI#9 inhibition of TLS-medi-ated repair of cisplatin-induced DNA damage (14).

SMI#9 has poor aqueous solubility that limits its therapeuticefficacy. To improve SMI#9 solubility and uptake, we previouslydeveloped SMI#9-tethered gold nanoparticles (GNP) using achemistry that allows its intracellular release, and showed thatthe SMI#9 released from the GNP conjugate behaves similarly tothe parent-free drug and inhibits TNBC cell proliferation withoutaffecting normal cells (15). GNPs have beenwidely used in cancerdiagnostics (16–18) and targeted delivery (19, 20) because oftheir versatile surface chemistry and biological inertness. In thisstudy, we analyzed the pharmacokinetic, biodistribution, andtherapeutic properties of SMI#9-GNP on BRCA1 wild-type andBRCA1-mutant TNBC growth. Pharmacokinetic analysis showedthat SMI#9 is slowly released from the GNP conjugate allowingfor a more stable build-up of the drug as compared with uncon-jugated SMI#9. SMI#9-GNPwaswell toleratedwithout overt signsof toxicity. Systemically administered SMI#9-GNP inhibitedgrowth of both BRCA1wild-type andmutant TNBCs as effectivelyas intratumorally injected unconjugated SMI#9. QuantitativeGNP biodistribution analysis showed that despite highest accu-mulation of GNPs in the tumors and livers of blank-GNP andSMI#9-GNP–treated mice, tumor inhibition occurred only inSMI#9-GNP–treated mice. Metabolic transformations typified byswitch to aerobic glycolysis and profound mitochondrial repro-gramming arewidely considered to support the cancer phenotype,and TNBCs show increased glycolysis compared with other breastcancer subtypes (21, 22). RAD6 is implicated in control ofmitochondrial turnover (23). To determine whether RAD6 inhi-bition impacts TNBCmetabolism, we performed 1H-NMR–basedmetabolomics analysis. Our results showed that SMI#9-GNPselectively decreased pathways regulating lactate, glutamate, andglycinemetabolism in both BRCA1wild-type andmutant TNBCs.These data reveal novel metabolic vulnerability impacted byRAD6 inhibition, further strengthening the therapeutic potentialof targeting RAD6 for treatment of TNBCs.

Materials and MethodsMaterials

Materials are described under Supplementary Data.

Synthesis and characterization of RAD6 inhibitor SMI#9conjugated GNPs

SMI#9 was synthesized as described previously (13). SMI#9was chemically modified to enable formation of a biodegradableester bond between the drug and mercaptosuccinic acid (MSA)-capped GNP carrier (15). Synthesis of MSA-capped GNP (blank-GNP) and MSA-capped GNP carrying SMI#9 (SMI#9-GNP) fol-lowed the same procedure as described previously (15). Thermo-gravimetric analysis (TGA) was performed to determine the

amount of SMI#9 in the nanoconjugate. To improve the biocom-patibility and colloidal stability, thiolated PEG (Molecular weight3,000) was added to MSA-capped GNP or SMI#9-GNP. Thenanoconjugates were characterized by atomic force microscopy(AFM), transmission electron microscopy (TEM), UV-vis, anddynamic light scattering (DLS) as described previously (15).

Cell lines and cultureMDA-MB-231 (BRCA1 wild-type) and MDA-MB-468 (BRCA1

wild-type) human TNBC cells were purchased from ATCC, andSUM1315 (BRCA1 mutant) TNBC cells were purchased fromAsterand. All lines were maintained in DMEM/F12 (Invitrogen)containing 5% FBS. Following authentication by cell bank (shorttandem repeat PCR) and Mycoplasma screening (MycoAlert,Lonza), several aliquots of cells were frozen and used within10 passages.

Clonogenic assayMDA-MB-231 or SUM1315 cells were treated overnight with

SMI#9-GNP (5 mmol/L SMI#9 equivalent dose) or the equivalentamount of blank-GNP. Cells were trypsinized and reseeded at 150cells/well in 24-well plates. Colony formation was assessed withcrystal violet staining and colony-forming efficiency wasexpressed relative to blank GNP–treated cells.

Cell survival assayTNBC cell responses to SMI#9-GNP and PEGylated SMI#9-

GNP were compared by MTT assay as described previously (15).SUM1315 (5 � 103) cells seeded in triplicates in 96-well plateswere treated for 72 hours with 0–8 mmol/L of SMI#9-GNP,PEGylated SMI#9-GNP, or blank GNP. Results are expressedrelative to control from three independent experiments.

Acridine orange/ethidium bromide stainingMDA-MB-231 or SUM1315 (1 � 104) cells seeded on cover-

slips were treated with SMI#9-GNP (5 mmol/L SMI#9 equiva-lent dose) or blank GNP in triplicates for 24 hours. Cultureswere stained with acridine orange/ethidium bromide (each25 mg/mL) and quantitated as described previously (13).

1H-NMR metabolomics analysisMDA-MB-231 or SUM1315 (1.2� 106) cells seeded in 60-mm

dishes were treated overnight with 0.5–5 mmol/L SMI#9-GNP,equivalent amounts of blank-GNP, or left untreated. Details ofmetabolite extraction are described under Supplementary Data.1H-NMRwas conducted using Agilent DD2-600MHz.Metabolitepeaks within 0–10 ppm spectral width were captured and pro-cessed simultaneously using ACD/Spec Manager 7.00 (AdvancedChemistry Development, Inc.) to avoid variations during proces-sing. The spectra were phased, base line corrected, and digitizedinto 1,000 bins by intelligent binning.

The table of integrals was imported into SIMCA Pþ software(Umetrics Academy) and multivariate data analysis (MVDA)performed. Pareto scaling was performed and spectral regionscorresponding to peaks from DSS, imidazole, water, andexchangeable protons were removed before MVDA. Principalcomponent analysis (PCA) and partial least squares discriminantanalysis (PLS-DA) score plots were generated to reveal metabo-lites perturbed by SMI#9-GNP. CHENOMX NMR suite (CHE-NOMX Inc.) was used to identify and quantify the metabolitesusing DSS as the reference. Metabolite concentrations (mmol/L)

Rad6 Inhibitor–conjugated Nanoparticle Anticancer Activity

www.aacrjournals.org Mol Cancer Ther; 17(12) December 2018 2587




were normalized to protein content and expressed from threeindependent experiments.

Metabolic pathway analysisMetabolic pathways perturbed by SMI#9-GNP in the TNBC

cells were identified by MetaboAnalyst 4.0 software, which isbased on KEGG (http://www.genome.jp/kegg) and the HumanMetabolome (http://www.hmdb.ca/) databases (24).

Pharmacokinetics analysisFemaleNcr nudemicewere intravenously injectedwith a single

dose of SMI#9 (5 mg/kg body weight in PBS/2.5% DMSO) orPEGylated SMI#9-GNP (SMI#9 equivalent dose 5 mg/kg bodyweight in PBS). Blood samples were collected prior to injectionand at defined time points postinjection, and SMI#9 concentra-tions in plasma were determined by LC/MS-MS (15).

Orthotopic tumor growth assays and biodistribution analysisA total of 1 � 106 MDA-MB-468 or SUM1315 cells were

suspended in 100 mL of Matrigel and injected into the bilateralinguinal mammary gland fat pads of female Ncr nude mice.When the tumors reached approximately 150 mm3, mice wererandomly assigned to vehicle, SMI#9, PEGylated SMI#9-GNP,or blank GNP groups (n ¼ 6 mice/group) and treated twiceweekly with intratumoral injections of SMI#9 (1.5 mg/kg bodyweight in PBS/0.5% DMSO) or vehicle, or intraperitonealinjections of PEGylated SMI#9-GNP (SMI#9 equivalent doseof 0.85 mg/kg body weight in PBS) or blank GNP. Bidimen-sional tumor size and body weights were measured twiceweekly. Mice were sacrificed at 37 or 44 days postimplantationof SUM1315 or MDA-MB-468 cells, respectively, 3 days afterthe last drug administration. Tumors were harvested, and themean tumor burden mass for each group was determined.Animals were evaluated for gross abnormalities of organs bynecropsy. Excised tumors and vital organs were fixed in buff-ered formalin and paraffin-embedded for histologic analysis.Portions of tumors were snap-frozen for LC/MS-MS analysis ofSMI#9. Organs and tumor fragments were processed for induc-tively coupled mass spectrometry analysis of gold. The in vivostudies were conducted in accordance with the InstitutionalAnimal Care and Use Committee (IACUC) guidelines of WayneState University (Detroit, MI).

LC/MS-MS analysis of SMI#9SMI#9 concentrations in the plasma and tumors were deter-

mined by LC/MS-MS as described previously (15). Briefly, tumors(50–100 mg) were homogenized, and plasma or tissue homo-genates were extracted with ethyl acetate. The organic phase wasdried down and reconstituted in the mobile phase [methanol/0.45% formic acid in water (60:30, v/v)]. LC/MS-MS was per-formed on a Waters Xevo TQ-XS LC/MS-MS system with a WatersXterra MS C18 column and isocratic elution with the mobilephase. SMI#9 was monitored at the positive electrospray ioniza-tion mode at m/z 366.69 > 150.1 mass transitions (15).

Inductively coupled plasmamass spectrometry analysis of goldOrgans and tumors were digested with aqua regia (hydrochlo-

ric acid: nitric acid, 3:1 v/v) and analyzed using an Agilent 7700�inductively coupled plasma mass spectrometry (ICP-MS)equipped with an Agilent ASX-500 series autosampler. Externalcalibration using standards with internal standard correction of

bismuth (Bi) was performed prior to the measurements. GNPbiodistribution data were expressed as a percentage of injecteddose of gold/gram dry tissue.

Statistical analysisResults are presented as the mean � SD or SEM, and were

analyzed by two-tailed Student t test and one-way ANOVA.Statistical significance was accepted at a value of P < 0.05.

ResultsGNP characterization

GNPs with a 5-nm core diameter were functionalized withmodified SMI#9 prior to PEGylation (15). GNPs were character-ized by AFM, TEM, and UV-vis as described previously (15) andthe results are summarized in Table S1 in Supplementary data.The resulting SMI#9-GNP conjugate had a hydrodynamic size of41 nm, and approximately 26% of the MSA-GNP reactive siteswere occupied by SMI#9.

SMI#9-GNP decreases clonogenic potential of TNBC cellsWe have previously reported that SMI#9 inhibits TNBC colony

formation (13). To determine whether SMI#9-GNP similarlyaffects clonogenic potentials of TNBC cells, MDA-MB-231 orSUM1315 TNBC cells were treated overnight with 5 mmol/LSMI#9-GNP or blank-GNP. SMI#9-GNP treatment significantlydecreased the colony-forming efficiencies of both lines comparedwith controls (P <.0.01; Fig. 1A and B). SMI#9-GNP–inducedapoptosis was detected by differential uptake of acridine orangeand ethidium bromide. Early apoptosis marked by intercalatedacridine orange within fragmented DNA (25) and late-stageapoptosis marked by apoptotic body separation and reddish-orange color due to acridine orange binding to fragmented DNA(26) were observed by 24 hours in >85% of TNBC cells treatedwith SMI#9-GNP (Fig. 1C and D, long arrow). GNP presence inthe apoptotic cells is indicated by a short arrow in Fig. 1C and D.Blank GNP–treated cells were minimally affected as >98% of thecells showed a green intact nuclear structure (Fig. 1C and D), thusverifying the nontoxicity ofGNPs and the anticancer activity of theconjugated SMI#9.

SMI#9-GNP perturbs common metabolic pathways in BRCA1wild-type and BRCA1-mutant TNBC cells

Because SMI#9-GNP treatment compromises mitochondrialmembrane potential (15), we performed 1H-NMR spectroscopyto determine whether SMI#9-GNP induces metabolic changes.Fourier-transformed spectra with the major metabolite peaksidentified from MDA-MB-231 and SUM1315 cells are shownin Fig. 1E and G. PCA plots (Fig. 1F and H) and the correspond-ing loading plots for SUM1315 and MDA-MB-231 (Supple-mentary Fig. S1A and S1B, respectively) show the regionsresponsible for spectra separation between blank GNP andSMI#9-GNP groups (Fig. 1F and H). Consistent with the differ-ences in responses to blank GNP versus SMI#9-GNP, PLS-DAscore plots showed clear spectral separations between blankGNP and SMI#9-GNP–treated cells (Supplementary Data,Fig. S2A and S2B). Spectral regions corresponding to lactate,glutamate, and glycine (quantified and confirmed by CHE-NOMX) were similarly affected by SMI#9-GNP treatment inSUM1315 (Fig. 2A) and MDA-MB-231 (Fig. 2B) cells. Phos-phocholine, Sn-glycero-3-phosphocholine, proline, creatinine,

Saadat et al.

Mol Cancer Ther; 17(12) December 2018 Molecular Cancer Therapeutics2588



http://www.genome.jp/kegg

http://www.hmdb.ca/


Figure 1.

SMI#9-GNP treatment inhibits colony formation and alters metabolite profiles in TNBC cells. Colony-forming potentials of MDA-MB-231 (A) and SUM1315 (B) cells.Results are mean � SD from three independent experiments. � , P < 0.001. Insets a and a' show typical colonies formed in blank-GNP and SMI#9-GNP(5 mmol/L)-treated cells, respectively. Apoptosis detection by acridine orange/ethidium bromide staining: MDA-MB-231 (C) and SUM1315 (D) cells. Long arrow,apoptotic bodies; short arrow, GNPs in apoptotic cells. Original magnification, � 20. Stacked plots of representative Fourier transformed 1H-NMR spectraof blank GNP and SMI#9-GNP–treated MDA-MB-231 (E) and SUM1315 (G) cells with major peaks labeled. PC, phosphatidyl-choline; GPC, Sn-glycerol-3-phosphocholine; FA, fatty acids; DSS, 4,4-Dimethyl-4-silapentane-1-sulfonic acid. PCA plots of MDA-MB-231 (F) and SUM1315 (H) cells treated with blank GNP (graycircles) and SMI#9-GNP (black circles).






and alloisoleucine were also decreased by SMI#9-GNP inSUM1315 cells (Fig. 2C) but not in MDA-MB-231 cells (Fig.2D). To identify the pathways perturbed by SMI#9, the met-abolic profiles were analyzed using MetaboAnalyst 4.0. Path-

ways regulating glycine, pyruvate, and alanine/aspartate/gluta-mate metabolism were found to be most impacted by SMI#9-GNP in both TNBC models. A summary of pathway analysis isshown in Fig. 3A and C, and the tabulated results are shown

Figure 2.

SMI#9-GNP decreases bioenergetics, glutamate, one-carbon, and phospholipid metabolism. SMI#9-GNP (1 mmol/L; gray bars) and blank GNP (black bars)-treatedSUM1315 (A and C) and MDA-MB-231 (B and D) cells. Values are mean � SE (mmol/L) normalized to protein (mg).

Saadat et al.





in Fig. 3B. Results for the individual cell models are shown inSupplementary Tables S2 and S3 (Supplementary Data). Thesedata show that SMI#9-GNP perturbs common metabolic path-ways in BRCA1 wild-type and BRCA1-mutant TNBC cells.

SMI#9 and SMI#9-GNP pharmacokineticsSMI#9 concentrations in plasma were measured by LC/MS-MS

at 5 minute to 24 hours after a single intravenous injection ofSMI#9 or PEGylated SMI#9-GNP. Following injection of the

unconjugated drug, SMI#9 achieved maximum plasma concen-tration (Cmax) of approximately 74 nmol/L at 5 minutes, and wasrapidly cleared by 30 minutes (Fig. 4A). In contrast, followinginjection of PEGylated SMI#9-GNP, SMI#9 was slowly releasedand achieved Cmax of approximately 1,200 nmol/L at 8 hours(Fig. 4B). These data confirm that SMI#9 is released from thePEGylated SMI#9-GNP conjugates and its slow release helpsachieve higher systemic exposure as compared with the uncon-jugated SMI#9.

Figure 3.

Metabolic pathways perturbed by SMI#9-GNP in TNBCcells.A, SMI#9-GNP–inducedmetabolite changes in themost relevant pathways. Size and color of the circlesindicate pathway impact and significance, respectively;red, most significant and white, least significant.Pathways with values >0.1 were considered to beperturbed. B, Summary of metabolic pathwaysperturbed/unperturbed by SMI#9-GNP. C, Metabolicscheme indicating SMI#9-GNP–induced perturbations.






In vivo therapeutic evaluation of SMI#9 and PEGylated SMI#9-GNP

Postsynthesis surface modification of SMI#9-GNP with PEGwas performed as PEG improves colloidal stability and preventsaggregation (27, 28). Furthermore, PEG reduces adsorption ofcellular proteins and increases the circulation time of nanopar-ticles. We first determined whether PEGylated SMI#9-GNP sim-ilarly sensitizes TNBC cells in vitro as non-PEGylated SMI#9-GNP.SUM1315 cells were treated with various concentrations of

SMI#9-GNP or PEGylated SMI#9-GNP (or blank-GNP) and theresponses measured by MTT assays. SUM1315 cells respondedsimilarly to both formulations with GI50 values (based uponSMI#9 concentration) of approximately 0.75–1 mmol/L, verifyingthat tethering of PEG did not significantly affect the activity of theSMI#9 payload (Fig. 4C). To compare the in vivo effects of SMI#9and PEGylated SMI#9-GNP on TNBC xenografts, MDA-MB-468or SUM1315 TNBC cells were orthotopically implanted in femalenude mice. When the tumors reached approximately 150 mm3,

Figure 4.

SMI#9-GNP inhibits TNBC growth. Pharmacokinetic analysis of unconjugated SMI#9 (A) and PEGylated SMI#9-GNP (B) administered intravenously at 5 mg/kgbody weight. C, In vitro activities of SMI#9-GNP and PEGylated SMI#9-GNP on SUM1315 cell proliferation. Results are mean � SD of triplicates from twoindependent experiments. Tumor volumes (mean � SEM; n ¼ 6 mice/group) of MDA-MB-468 (D) or SUM1315 (F) xenografts treated with unconjugated SMI#9(1.5 mg/kg body weight), PEGylated SMI#9-GNP (0.85 mg/kg body weight), or controls. P < 0.0001 by one-way ANOVA. Vertical scatter plots of excisedtumor mass of MDA-MB-468 (E) and SUM1315 (G) xenografts.

Saadat et al.





mice were randomly assigned for twice weekly treatments withvehicle, SMI#9, PEGylated SMI#9-GNP, or blank GNP. To facil-itate easy tumor access, unconjugated SMI#9 (or vehicle) wasadministered intratumorally. To assess tumor accessibility andbioavailability of conjugated SMI#9, PEGylated SMI#9-GNP(or blank GNP) was injected intraperitoneally. Vehicle, blankGNP, and unconjugated SMI#9-treated groups initially trackedsimilar growth, however, continued treatment with unconjugatedSMI#9 significantly retarded MDA-MB-468 and SUM1315 tumorgrowth (Fig. 4D and F; P < 0.0001). PEGylated SMI#9-GNPtreatment significantly retarded growth of MDA-MB-468 (P <0.0001; Fig. 4D) and SUM1315 (P ¼ 0.0038; Fig. 4F) tumors ascomparedwith blankGNP, andwas slightlymore efficacious thanunconjugated SMI#9 (P<0.01). Comparisonof the excised tumormasses upon sacrifice confirmed that both SMI#9 and PEGylatedSMI#9-GNP treatments significantly inhibitedMDA-MB-468 andSUM1315 tumor growth as compared with the correspondingcontrols (P � 0.05; Fig. 4E and G).

To evaluate the effects of unconjugated and GNP-conjugatedSMI#9 on tumor morphology, the tumors from control andtreated groups were analyzed by hematoxylin and eosin(H&E) staining. Compared with the robust tumors in controlmice, SMI#9 and PEGylated SMI#9-GNP–treated MDA-MB-468(Fig. 5A) and SUM1315 (Fig. 5B) tumors were sparsely populatedand contained several apoptotic cells marked by nuclear pyknosisand apoptotic bodies (arrows in insets of Fig. 5A and B) as scoredby H&E diagnostic criteria for apoptosis (29). These data areconsistent with the data in Fig. 1C and D that showed robustapoptosis inductionby SMI#9-GNP. Todeterminewhether PEGy-lated SMI#9-GNP treatment affected vital organs, H&E-stainedtissues were scored for organ damage. Tissues harvested fromuntreated nontumor-bearing normal mice were used as referencefor detecting GNP-induced alterations. No differences in tissuecolor and appearance were observed after GNP treatments. H&E-stained tissues showed normal morphologies for lung, liver,spleen, kidney, heart, and small intestines in blank-GNP andSMI#9-GNP–treated mice with no evidence of atrophy or inflam-mation, and were indistinguishable from normal control mice(Fig. 5C). Mice treated with unconjugated SMI#9, PEGylatedSMI#9-GNP, or blank-GNP show similar body weights as thevehicle control group (Fig. 5D). Taken together, these data con-firm that blank- or SMI#9-GNP treatments are tolerated well withnegligible histopathologic changes and organ toxicity.

Biodistribution analysis of SMI#9 and GNPTo verify whether the tumor growth inhibition in SMI#9-

GNP–treated mice resulted from SMI#9 release from the nano-conjugate, we quantitated SMI#9 levels in tumor fragmentscollected at sacrifice by LC/MS-MS. Compared with the controlgroups, measurable amounts of SMI#9 were detected inSUM1315 and MDA-MB-468 tumors treated with SMI#9 orSMI#9-GNP (Fig. 6A and B). However, there was variability inthe detected levels, which could have potentially resulted fromtumor tissue samplings as tissue fragments rather than wholetumors were used for analysis or from loss of SMI#9 resultingfrom therapy-induced tumor cell death in the responsivetumors.

Next, we determined the biodistribution and retention ofGNPsin tumor and organ tissues collected at sacrifice (Fig. 6C) by ICP-MS. The injected dosage of gold was calculated on the basis of theTGA data: weight ratio of gold to SMI#9 equivalent to 1:0.083 in

the nanoconjugate (15). Thus, a SMI#9 dosage of 0.85 mg/kgbodyweight corresponds to golddosageof 10.24mg.On thebasisof 0.02 kg average weight of mouse, the gold dosage per injectionis 0.2mg and the total gold injected is 1.8mg after nine injections.Among the tissues analyzed, liver and tumors showed the highestaccumulation of gold ranging from 0.19% to 0.4%; however,therewereno significant differences in gold content between liversand tumors of blank GNP and SMI#9-GNP groups (Fig. 6C).Despite multiple injections, this accumulation was comparablewith those reported for a single injection (30). GNP bioaccumu-lation in liver and spleen are regulated by the reticuloendothelialsystem (RES; ref. 31). The low levels of GNP accumulation in thespleen potentially reflect the low GNP doses administered in ourstudy and/or the time intervals between the injections. Further-more, because the tissue samples were collected 3 days after thelast GNP injection, larger increases in gold depositions wereperhaps not observed because of active renal clearance. The GNPsused here are 5-nm particles with a hydrodynamic size of 41 nmfollowing SMI#9 conjugation. Because the kidney filtrationthreshold is 6–8 nm (32), GNPs that were stripped off theirSMI#9 cargo would be small enough to be cleared by the kidneysleaving behind the nonbiodegraded SMI#9-GNPs. Becausetumors contained higher levels of gold, it is likely that enhancedpermeability and retention (EPR) effects from leaky vasculature inthe tumors contributed to accumulation of gold in the tumors(ref. 33; Fig. 6C).

DiscussionIn this article, we evaluated the toxicity, biodistribution, and

therapeutic activity of systemically administered PEGylatedSMI#9-conjugated GNPs to assess their utility as a drug deliveryplatform for treatment of BRCA1 wild-type and BRCA1-mutantTNBCs. We demonstrate that the tethered SMI#9 prodrug isreleased from the GNP conjugate and inhibits growth of bothBRCA1 wild-type and mutant TNBCs. We also demonstrate thatPEGylated SMI#9-GNP inhibits TNBC growth as effectively asintratumorally administered unconjugated SMI#9. Intratumoralinjection of SMI#9, a clinically unsuitable mode of drug admin-istration, was necessary because of its poor solubility and poorpharmacokinetic properties.Delivery of SMI#9 as a prodrug–GNPconjugate not only greatly improved its circulation stability anddrug exposure, but also allowed for a clinically acceptable mode,that is, systemic administration of the drug because of improvedsolubility.

Surface functionalization of GNP with PEG is commonly usedto prevent rapid clearance by the RES. PEGylation preventsnonspecific GNP interactions with serum proteins improvingGNP circulation time (27, 28). Solid tumors are characterizedby defective vasculature and impaired lymphatic drainage/recov-ery system that promote EPR effect (33, 34). Consequently, tumoraccumulation of GNPs increases with increased blood circulationtime and a leaky vasculature. MDA-MB-468 and SUM1315 TNBCgrowth is strongly inhibited by systemically administered PEGy-lated SMI#9-GNP compared with blank-GNP. This is supportedby our data from ICP-MS analysis that showed high accumulationof gold in the tumor tissues, and further corroborated by LC/MS-MS analysis that showed SMI#9 in the residual tumors of SMI#9-GNP–treated mice. SMI#9 presence in the tumors establishesSMI#9-GNP tumor uptake, lysosomal processing of the prodrug(SMI#9)–GNP conjugate, and the hydrolytic release of the






tethered SMI#9 (15). Our data confirm that SMI#9 released fromthe PEGylated–GNP conjugate is therapeutically active as itpotently inhibits tumor growth with similar efficacy as the intra-tumorally injected unconjugated SMI#9.

Perrault and colleagues (35) tested GNPs with size range of10–100 nm and found that although GNPs with hydrodynamicdiameter of 60–100 nm with PEGylation efficiently utilize EPReffect for enhanced tumor accumulation, these larger GNPs pen-etrated weakly into the tumors and localized in the perivascularregions. GNPs are generally considered nontoxic when the size islarger than2nm,whereas ultrasmall nanoparticles are found tobetoxic (36). GNPs of larger size (35–50 nm) were found to entercells more efficiently without toxicity than small nanoparticles(1.4 nm; refs. 37–39). Consistentwith these data, our results show

that 41-nm SMI#9-GNPs are taken up efficiently by tumor cellsand display no apparent toxicity. Quantitative analysis of GNPbiodistribution by ICP-MS analysis showed higher accumulationof gold in tumors and liver as compared with other organs ofSMI#9-GNP and blank GNP–treated mice. These data are inagreement with other studies that showedmaximal accumulationof nanoparticles in the liver regardless of their size, shape, dosage,or composition (31, 40, 41). However, our data suggest that theGNPs are actively cleared because the amounts of gold depositedin the liver and other tissues despite repeated injections resemblethose in mice receiving a single dose. Because the GNPs used inour study have a 5-nm core diameter and a hydrodynamic size of41 nm for the SMI#9 nanoconjugate, our data suggest that themajority of GNPs are stripped off their SMI#9 payload rendering

Figure 5.

SMI#9-GNP treatment induces cytotoxicity in MDA-MB-468 and SUM1315 tumors but not organ toxicity. H&E analysis of MDA-MB-468 (A) and SUM1315 (B) tumors.Insets show enlarged images. Long arrows in insets indicate apoptotic cells (ref. 29); short arrow in SMI#9-treated tumors (B, inset) indicates multinucleatedgiant cells. Magnification,� 400. C, Comparison of histologies of H&E-stained organs from normal micewith those collected at sacrifice from PEGylated SMI#9-GNPor blank GNP–treated SUM1315 tumor-bearing mice. Magnification, � 100 (spleen, small intestine); � 200 (lung, liver, heart); � 400 (kidney). D, Body weightassessment in control (vehicle, blank GNP) and treated (unconjugated and conjugated SMI#9) mice. Data are mean � SEM (n ¼ 6 mice/group), P < 0.0001 byone-way ANOVA.

Saadat et al.





them small enough for renal clearance (32). These data areconsistent with those reported by Perrault and colleagues whoshowed rapid clearance of PEGylated 20-nm and 40-nm GNPsfrom circulation without accumulation in the spleen and liver(35). Regardless of the coating layer, nanoparticles always form a"protein corona" when encountering physiologic conditions(42, 43). This corona phenomenon potentially balances out anydifferences between blank GNP and SMI#9-conjugated GNPduring circulation and may explain the similar patterns of bio-distribution data. It is noteworthy that despite high accumulationof gold in the tumors of blank GNP–treated mice, tumor growthwas unaffected in these mice providing further support for SMI#9therapeutic activity and the nontoxicity of GNPs. Although tumorgrowth was robustly inhibited by SMI#9-GNP, the treatment didnot affect body weights or induce overt toxicity as determined bynecropsy and histopathologic analysis of the major vital organs.

TNBC cells are characterized by high glycolytic flux anddecreased mitochondrial respiration (44), and are primed toswitch to a glycolytic program regardless of the oxygen statusunlike nontransformed cells (44, 45). A siRNA screen showedthat TNBC cells are dependent on lactate dehydrogenase activityfor elevated glycolysis as compared with non-TNBC cells (46).Using 1H-NMR–based spectroscopy, we analyzed the impact ofRAD6 inhibition on metabolic profiles of BRCA1 wild-type andBRCA1-mutant TNBC cells. Using unsupervised analysis, fourdiscriminating metabolites (lactate, glycine, alanine, and gluta-mate) were found to be perturbed by SMI#9-GNP in both BRCA1wild-type and BRCA1-mutant TNBC cells. It is likely that a greaternumber of metabolites would have been identified by massspectrometry because of the sensitivity limitations of NMR spec-troscopy. It is noteworthy, however, that against the backdrop ofmyriads ofmetabolic changes, NMR spectroscopy helped identify

Figure 6.

SMI#9 and gold biodistribution analysis. LC/MS-MS analysis of SMI#9 tumor levels and corresponding vertical scatter plots of SUM1315 (A) or MDA-MB-468 (B)excised tumor mass at sacrifice. C, ICP-MS analysis of accumulated gold in the indicated tissue. Scale bars, mean � SEM. Data were analyzed by one-wayANOVA and two-tailed Student t test; NS, no significant difference.






quantitative and reproducible changes in key metabolic determi-nants perturbed by the RAD6 inhibitor. TNBC cells are charac-terized by high levels of lactate, glutamate, and glycine and havebeen correlated with high glycolytic activity and tumor aggres-siveness (47). Glycine is involved in the synthesis of proteins,nucleotides, and one-carbon metabolism, and high levels ofglycine have been shown to correlate with poor prognosis inbreast cancer (48). Compared with ER/PGR-positive tumors,TNBCs contain lower levels of glutamine and higher levels ofglutamate, which might result from increased glutaminolysis(49). Aberrant phosphatidyl-choline metabolism associated withincreases in phosphocholine has been reported in breast cancer(50). However, although SMI#9-GNP treatment inhibited tumorgrowth in both MDA-MB-468 and SUM1315 TNBC models, itdecreased phospholipid levels only in SUM1315 cells. These datasuggest that the SMI#9-GNP–inhibitory effects may not be uni-versally linked with phosphatidyl-choline metabolism.

The UBC activity of RAD6, also known as UBE2A, is implicatedin maintaining healthy mitochondria. RAD6, in combinationwith Parkin E3 ubiquitin ligase, regulates mitochondrial proteinubiquitination to facilitate autophagic clearance of dysfunctionalmitochondria (23). Consistent with these data, RAD6-deficienthumanandDrosophila cells showdefectivemitochondria turnover(23).Wehave shownpreviously that SMI#9 inboth free andGNP-conjugated forms induces accumulation of dysfunctional mito-chondria corroborating the role of Rad6 in mitochondrial func-tion (15). Interestingly, loss of mitochondrial function in SMI#9-treated TNBC cells did not result in concomitant increase inmetabolites associated with Warburg effect but rather causedreductions in glycolytic mediators. These data suggest that theincrease in glycolytic flux seen in TNBC cells may not be directlyrelated to mitochondrial function. Because RAD6 inhibition canconcurrently perturb mitochondrial function and glycolyticpotential besides its traditional role in TLS, our data suggest thattargeting Rad6 could offer a promising approach for TNBCtreatment.

In summary, we demonstrated the therapeutic utility of a GNP-based platform for delivering Rad6 inhibitor for treating BRCA1wild-type and BRCA1-mutant TNBCs, a breast cancer subtypewith poor prognosis and no targeted therapies. SMI#9 delivery asa prodrug–GNP conjugate not only improved its pharmacoki-netic properties but also allowed for a clinically acceptable

(systemic) mode of drug administration that inhibited TNBCgrowth. There was no evidence of blank or SMI#9-GNP–inducedtoxicity as measured by their impact on animal body weight andorgan histopathology. SMI#9-GNP–induced sensitization ofBRCA1 wild-type and BRCA1-mutant TNBCs was associated withperturbation of a core set of metabolic pathways revealing mar-kers of metabolic vulnerabilities that can be targeted for TNBCmanagement.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: N. Saadat, F. Liu, L.A. Polin, M.P. ShekharDevelopment of methodology: N. Saadat, F. Liu, X. Bao, J. Li, L.A. Polin,S. Gupta, G. Mao, M.P. ShekharAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Saadat, F. Liu, P. Nangia-Makker, X. Bao,L.A. Polin, G. Mao, M.P. ShekharAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Saadat, F. Liu, B. Haynes, X. Bao, L.A. Polin,S. Gupta, G. Mao, M.P. ShekharWriting, review, and/or revision of the manuscript: N. Saadat, F. Liu,B. Haynes, X. Bao, J. Li, L.A. Polin, S. Gupta, G. Mao, M.P. ShekharAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N. Saadat, L.A. Polin, G. Mao, M.P. ShekharStudy supervision: S. Gupta, G. Mao, M.P. Shekhar

AcknowledgmentsThisworkwas been supported byNCI (grant number R21CA178117, toM.P.

Shekhar), National Science Foundation (grant number CHE1404285, toG. Mao), NIH (grant number R01HD031550, to G. Mao), and MolecularTherapeutics Program of Karmanos Cancer Institute (to M.P. Shekhar andG. Mao). The Pharmacology Core and Animal Model and Therapy EvaluationCore facilities are supported by NIH Center grant P30 CA022453 to theKarmanos Cancer Institute at Wayne State University. B. Haynes was supportedby Initiative for Maximizing Student Diversity award from NIH to Wayne StateUniversity (R25 GM058905) and Ruth L. Kirschstein National Research ServiceAward T32-CA009531 training grant from NIH.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 9, 2018; revised August 2, 2018; accepted September 18, 2018;published first September 21, 2018.

References1. Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, et al.

Triple-negative breast cancer: clinical features and patterns of recurrence.Clin Cancer Res 2007;13:4429–34.

2. Lips EH, Mulder L, Oonk A, van der Kolk LE, Hogervorst FB, Imholz AL,et al. Triple-negative breast cancer: BRCAness and concordance of clinicalfeatures with BRCA1-mutation carriers. Br J Cancer 2013;108:2172–7.

3. Atchley DP, Albarracin CT, Lopez A, Valero V, Amos CI, Gonzalez-AnguloAM, et al. Clinical and pathological characteristics of patients with BRCA-positive and BRCA-negative breast cancer. J Clin Oncol 2008;6:4282–8.

4. Anders C, Carey L. Biology, metastatic patterns, and treatment of patientswith triple-negative breast cancer. Clin Breast Cancer 2009;9:S73–S81.

5. Yeeles JT, Poli J, Marians KJ, Pasero P. Rescuing stalled or damagedreplication forks. Cold Spring Harb Perspect Biol 2013;5:a012815.

6. Branzei D, Szakal B. DNA damage tolerance by recombination: molecularpathways and DNA structures. DNA Repair 2016;44:68–75.

7. Sung P, Prakash S, Prakash L. Stable ester conjugate between the Saccha-romyces cerevisiae RAD6 protein and ubiquitin has no biological activity.J Mol Biol 1991;221:745–9.

8. Shekhar MP, Lyakhovich A, Visscher DW, Heng H, Kondrat N. Rad6overexpression induces multinucleation, centrosome amplification,abnormal mitosis, aneuploidy, and transformation. Cancer Res 2002;62:2115–24.

9. ShekharMP, Biernat LA, Pernick N, Tait L, Abrams J, Visscher DW.Utility ofDNA postreplication repair protein Rad6B in neoadjuvant chemotherapyresponse. Med Oncol 2010;27:466–73.

10. Gerard B, Sanders MA, Visscher DW, Tait L, Shekhar MP. Lysine 394 is anovel Rad6B-induced ubiquitination site on beta-catenin. Biochim Bio-phys Acta 2012;1823:686–96.

11. Lyakhovich A, Shekhar MP. RAD6B overexpression confers chemoresis-tance: RAD6expressionduring cell cycle and its redistribution to chromatinduring DNA damage-induced response. Oncogene 2004;23:3097–106.

12. Lyakhovich A, Shekhar MP. Supramolecular complex formation betweenRad6 and proteins of the p53 pathway during DNA damage-inducedresponse. Mol Cell Biol 2003;23:2463–75.

13. SandersMA, BrahemiG,Nangia-Makker P, Balan V,MorelliM, KothayerH,et al. Novel inhibitors of Rad6 ubiquitin conjugating enzyme: design,

Saadat et al.





synthesis, identification, and functional characterization. Mol Cancer Ther2013;12:373–83.

14. Sanders MA, Haynes B, Nangia-Makker P, Polin LA, Shekhar MP. Phar-macological targeting of Rad6 enzyme-mediated translesion synthesisovercomes resistance to platinum-based drugs. J Biol Chem 2017;292:10347–63.

15. Haynes B, Zhang Y, Liu F, Li J, Petit S, Xun B, et al. Gold nanoparticleconjugated Rad6 inhibitor induces cell death in triple negative breastcancer cells by inducing mitochondrial dysfunction and PARP-1 hyper-activation: synthesis and characterization. Nanomedicine 2016;12:745–57.

16. Bardhan R, Lal S, Joshi A, Halas NJ. Theranostic nanoshells: fromprobe design to imaging and treatment of cancer. Acc Chem Res2011;44:936–46.

17. Lin D, Feng SY, Pan JJ, Chen YP, Lin JQ, Chen GN. Colorectal cancerdetection by gold nanoparticle based surface-enhanced Raman spec-troscopy of blood serum and statistical analysis. Opt Express 2011;19:13565–77.

18. Hainfeld JF, O'Connor MJ, Dilmanian FA, Slatkin DN, Adams DJ,Smilowitz HM. Micro-CT enables microlocalisation and quantificationof Her2-targeted gold nanoparticles within tumour regions. Br J Radiol2011;84:526–33.

19. Brown SD, Nativo P, Smith JA, Sterling D, Edwards PR, Venugopal B,et al. Gold nanoparticles for the improved anticancer drug deliveryof the active component of oxaliplatin. J Amer Chem Soc 2010;132:4678–84.

20. Zhang Y, Walker JB, Minic Z, Liu F, Goshgarian H, Mao G. Transporterprotein and drug-conjugated gold nanoparticles capable of bypassing theblood-brain barrier. Sci Rep 2016;6:25794.

21. Sciacovelli M, Gaude E, Hilvo M, Frezza C. The metabolic alterations ofcancer cells. Methods Enzymol 2014;542:1–23.

22. Timmerman LA, Holton T, Yuneva M, Louie RJ, Padr�o M, Daemen A,et al. Glutamine sensitivity analysis identifies the xCT antiporter as acommon triple-negative breast tumor therapeutic target. Cancer Cell2013;24:450–65.

23. Haddad DM, Vilain S, Vos M, Esposito G, Matta S, Kalscheuer VM, et al.Mutations in the intellectual disability gene Ube2a cause neuronaldysfunction and impair parkin-dependent mitophagy. Mol Cell2013;50:831–43.

24. Xia J, Sinelnikov I, Han B, Wishart DS. MetaboAnalyst 3.0 – makingmetabolomics more meaningful. Nucl Acids Res 2015;43:W251–7.

25. Abdel Wahab SI, Abdul AB, Alzubairi AS, Elhassan MM, Mohan S. In vitromorphological assessment of apoptosis induced by zerumbone on (HeLa).J Biomed Biotechnol 2009:2009:769568.

26. RenvoizeC, Biola A, PallardyM, Breard J. Apoptosis: identification of dyingcells. Cell Biol Toxicol 1998;14:111–20.

27. Gao J, Huang X, Liu H, Zan F, Ren J. PEG colloidal stability of goldnanoparticles modified with thiol compounds: bioconjugation and appli-cation in cancer cell imaging. Langmuir 2012;28:4464–71.

28. Manson J, Kumar D, Meenan BJ, Dixon D. Polyethylene glycol functio-nalized gold nanoparticles: the influence of capping density on stability invarious media. Gold Bull 2011;44:99–105.

29. Elmore SA, Dixon D, Hailey JR, Harada T, Herbert RA, Maronpot RR, et al.Recommendations from the INHAND apoptosis/necrosis working group.Toxicol Pathol 2016;44:173–88.

30. De JongWH,HagensWI, Krystet P, BurgerMC, SipsAJ,GeertsmaREParticlesize-dependent organ distribution of gold nanoparticles after intravenousadministration. Biomaterials 2008;29:1912–9.

31. Sadauskas E, Wallin H, Stoltenberg M, Vogel U, Doering P, Larsen A, et al.Kupffer cells are central in the removal of nanoparticles from the organism.Part Fibre Toxicol 2007;4:10.

32. Ohlson M, Sorensson J, Haraldsson B. A gel-membrane model of glomer-ular charge and size selectivity in series. Am J Physiol Renal Physiol2001;280:F396–405.

33. Maeda H. The enhanced permeability and retention (EPR) effect in tumorvasculature: the key role of tumor-selectivemacromolecular drug targeting.Adv Enz Reg 2001;41:189–207.

34. Modi S, Jain JP, Domb AJ, Kumar N. Exploiting EPR in polymer drugconjugate delivery for tumor targeting. Curr Pharm Design 2006;12:4785–96.

35. Perrault SD,Walkey C, Jennings T, Fischer HC, ChanWC.Mediating tumortargeting efficiency of nanoparticles through design. Nano Lett 2009;9:1909–15.

36. Senut MC, Zhang Y, Liu F, Sen A, Ruden DM, Mao G. Size-dependenttoxicity of gold nanoparticles on human embryonic stem cells and theirneural derivatives. Small 2016;12:631–46.

37. Chithrani BD, Ghazani AA, Chan WC. Determining the size and shapedependence of gold nanoparticle uptake into mammalian cells. Nano Lett2006;6:662–8.

38. Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Gold nanopar-ticles are taken upby human cells but donot cause acute cytotoxicity. Small2005;1:325–7.

39. Shukla R, Bansal V, Chaudhary M, Basu A, Bhonde RR, Sastry M. Biocom-patibility of goldnanoparticles and their endocytotic fate inside the cellularcompartment: a microscopic overview. Langmuir 2005;21:10644–54.

40. Sadauskas E, Danscher G, Stoltenberg M, Vogel U, Larsen A, Wallin H.Protracted elimination of gold nanoparticles from mouse liver. Nanome-dicine 2009;5:162–9.

41. Zhang G, Yang Z, Lu W, Zhang R, Huang Q, Tian M, et al. Influence ofanchoring ligands and particle size on the colloidal stability and in vivobiodistribution of polyethylene glycol-coated gold nanoparticles in tumor-xenografted mice. Biomaterials 2009;30:1928–36.

42. Herrwerth S, Eck W, Reinhardt S, Grunze M. Factors that determine theprotein resistance of oligoether self-assembled monolayers – internalhydrophilicity, terminal hydrophilicity, and lateral packing density. J AmerChem Soc 2003;125:9359–66.

43. Heuberger M, Drobek T, Spencer ND. Interaction forces and morphol-ogy of a protein-resistant poly(ethylene glycol) layer. Biophys J 2005;88:495–504.

44. Pelicano H, Zhang W, Liu J, Hammoudi N, Dai J, Xu RH, et al. Mitochon-drial dysfunction in some triple negative breast cancer cell lines: role ofmTOR pathway and therapeutic potential. Breast Cancer Res 2014;16:434.

45. Diers AR, Vayali PK,Oliva CR, Griguer CE,Darley-Usmar V, Hurst DR, et al.Mitochondrial bioenergetics ofmetastatic breast cancer cells in response todynamic changes in oxygen tension effects of HIFa. PLoS One 2013;8:e68348.

46. McClelandML, Adler AS, Shang Y, Hunsaker T, Truong T, Peterson D, et al.An integrated genomic screen identifies LDHBas an essential gene for triplenegative breast cancer. Cancer Res 2012;72:5812–23.

47. Cao MD, Lamichhane S, Lundgren S, Bofin A, Fjøsne H, Giskeødega�rd GF,

et al. Metabolic characterization of triple negative breast cancer. BMCCancer 2014;14:941.

48. Sitter B, Bathen TF, Singstad TE, Fjosne HE, Lundgren S, Halgunset J, et al.Quantification of metabolites in breast cancer patients with differentclinical prognosis using HR MAS MR spectroscopy. NMR Biomed 2010;23:424–31.

49. Wise DR, Thompson CB. Glutamine addiction: a new therapeutic target incancer. Trends Biochem Sci 2010;35:427–33.

50. Aboagye EO, Bhujwalla ZM. Malignant transformation alters membranecholine phospholipid metabolism of human mammary epithelial cells.Cancer Res 1999;59:80–4.






2018;17:2586-2597. Published OnlineFirst September 21, 2018.Mol Cancer Ther Nadia Saadat, Fangchao Liu, Brittany Haynes, et al. Triple-negative Breast Cancer Therapy

/Translesion Synthesis Inhibitor SMI#9 forRAD6Nano-delivery of

Updated version

10.1158/1535-7163.MCT-18-0364doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2018/09/21/1535-7163.MCT-18-0364.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/17/12/2586.full#ref-list-1

This article cites 50 articles, 9 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/17/12/2586To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-18-0364

http://mct.aacrjournals.org/content/suppl/2018/09/21/1535-7163.MCT-18-0364.DC1

http://mct.aacrjournals.org/content/17/12/2586.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/17/12/2586


nano-delivery of rad6/translesion synthesis inhibitor smi#9 for … · small molecule therapeutics...

Documents