Pharmacological Dual Inhibition of Tumor and Tumor-Induced ......Small Molecule Therapeutics Pharmacological Dual Inhibition of Tumor and Tumor-Induced Functional Limitations in a

Small Molecule Therapeutics

Pharmacological Dual Inhibition of Tumor andTumor-Induced Functional Limitations in aTransgenic Model of Breast CancerRuizhong Wang1, Poornima Bhat-Nakshatri1, Maria B. Padua1, Mayuri S Prasad1,Manjushree Anjanappa1, Max Jacobson2, Courtney Finnearty2, Victoria Sefcsik2,Kyle McElyea2, Rachael Redmond2, George Sandusky2, Narsimha Penthala3,Peter A Crooks3, Jianguo Liu1, Teresa Zimmers1, and Harikrishna Nakshatri1,4,5

Abstract

Breast cancer progression is associated with systemic effects,including functional limitations and sarcopenia without theappearance of overt cachexia. Autocrine/paracrine actions ofcytokines/chemokines produced by cancer cells mediate cancerprogression and functional limitations. The cytokine-inducibletranscription factor NF-kB could be central to this process,as it displays oncogenic functions and is integral to the Pax7:MyoD:Pgc-1b:miR-486 myogenesis axis. We tested this possi-bility using the MMTV-PyMT transgenic mammary tumormodel and the NF-kB inhibitor dimethylaminoparthenolide(DMAPT). We observed deteriorating physical and functionalconditions in PyMTþ mice with disease progression. Comparedwith wild-type mice, tumor-bearing PyMTþ mice showeddecreased fat mass, impaired rotarod performance, andreduced grip strength as well as increased extracellular matrix(ECM) deposition in muscle. Contrary to acute cachexia mod-els described in the literature, mammary tumor progression

was associated with reduction in skeletal muscle stem/satellite-specific transcription factor Pax7. Additionally, we observedtumor-induced reduction in Pgc-1b in muscle, which controlsmitochondrial biogenesis. DMAPT treatment starting at 6 to 8weeks age prior to mammary tumor occurrence delayed mam-mary tumor onset and tumor growth rates without affectingmetastasis. DMAPT overcame cancer-induced functional lim-itations and improved survival, which was accompanied withrestoration of Pax7, Pgc-1b, and mitochondria levels andreduced ECM levels in skeletal muscles. In addition, DMAPTrestored circulating levels of 6 out of 13 cancer-associatedcytokines/chemokines changes to levels seen in healthy ani-mals. These results reveal a pharmacological approach forovercoming cancer-induced functional limitations, and theabove-noted cancer/drug-induced changes in muscle geneexpression could be utilized as biomarkers of functional lim-itations. Mol Cancer Ther; 16(12); 2747–58. �2017 AACR.

IntroductionBreast cancer is one of the most common cancers and a leading

cause of cancer-associated morbidity/mortality in women world-wide (1). It is becoming increasingly clear that breast cancer is asystemic disease affecting multiple organs. The systemic effects ofbreast cancer are manifested in three distinct forms: functionallimitation, sarcopenia, and cachexia (2–4). Functional limitation,which is defined as muscle weakness and body pain with noobvious physical signs, is observed in 39% of breast cancer

patients and is associated with increased risk of noncancer causeof death (2). Functional limitation is found in women <40 yearseven at the time of breast cancer diagnosis and before drug orsurgical intervention (2, 5). Sarcopenia (severe depletion ofskeletalmuscle, despite appearance of normalweight, overweight,or obesity) is observed in 25% of metastatic breast cancerpatients and is associated with increased toxicity to chemo-therapy (3). Additionally, 26% of breast cancer patients meetone of the four criteria of cachexia (4). Because onset ofcachexia in breast cancer patients is not as rapid and progressiveas in lung and pancreatic cancer, mechanistic studies on cachex-ia in breast cancer are limited. Acute cancer cachexia modelsused for mechanistic studies of cachexia in colon, pancreatic,and lung cancers may not accurately reflect systemic impact ofbreast cancer. Therefore, model systems need to be developedto understand the systemic effects of functional limitations andsarcopenia in breast cancer and for the development of ther-apeutic strategies to treat these effects.

Themain underlying pathophysiology of cancer-induced func-tional limitations/cachexia is thought to involve the release ofcytokines/chemokines such as tumor necrosis factor alpha(TNFa) and interleukin 1beta (IL-1b) from tumors that interferewith host immunity and skeletal muscle function (6). TNFa hasbeen shown to impair muscle oxidative phenotype and causemuscle wasting (7). Thus, muscle wasting in advanced breast

1Department of Surgery, Indiana University School of Medicine, Indianapolis,Indiana. 2Department of Pathology and Laboratory Medicine, Indiana UniversitySchool of Medicine, Indianapolis, Indiana. 3Department of PharmaceuticalSciences, College of Pharmacy, University of Arkansas for Medical Sciences,Little Rock, Arkansas. 4Department of Biochemistry and Molecular Biology,Indiana University School of Medicine, Indianapolis, Indiana. 5Richard L Roude-bush VA Medical Center, Indianapolis, Indiana.

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

Corresponding Author: Harikrishna Nakshatri, C218C, 980 West Walnut Street,Indianapolis, IN 46202. Phone: 317-278-2238; Fax: 317-274-0396; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-17-0717

�2017 American Association for Cancer Research.

MolecularCancerTherapeutics

www.aacrjournals.org 2747

on April 15, 2021. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 4, 2017; DOI: 10.1158/1535-7163.MCT-17-0717

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-17-0717&domain=pdf&date_stamp=2017-11-15

http://mct.aacrjournals.org/

cancer may be due to potent catabolic effects of inflammatorycytokines through their downstream signaling. Muscle loss incancer patients may be similar to age-associated muscle loss,which is accompanied with systemic inflammation and increasedlevels of cytokines including IL-1b, IL-6, IL-10, IL-13, TNFa, andgranulocyte–macrophage colony-stimulating factor (GM-CSF;ref. 8). NF-kB is a major signaling molecule downstream of, and,in some cases, upstream of these cytokines (9). NF-kB is not onlycritical in tumor initiation and progression as demonstrated by usand others (9, 10), but also regulates skeletal muscle mass andfunction by reducing protein synthesis or enhancing degradationof myogenic transcription factors such as MyoD (11). Activationof NF-kB through muscle-specific transgenic expression of acti-vated IkB kinase beta (IKKb) causes profoundmusclewasting thatresembles clinical cachexia (12). Therefore, NF-kB has beenproposed to be a potential target for the treatment of loss ofskeletal muscle mass in cancer cachexia (11, 13). However, mostof the studies that examined the role of NF-kB in cancer cachexiawere done using acute models of cachexia (�30 days) and it is yetto be ascertained whether these findings are relevant for breastcancer, which has a slower progression rate than other cancers,and usually does not display classic cachexia features.

Our previous study has shown that breast cancer patients withmetastasis have lower level of circulatingmyogenicmiR-486 (14).In line with human data, we observed that mice with mammarytumors have lower miR-486 concentrations in plasma and skel-etal muscles (14). Mechanistic studies showed that tumor-induced cytokines such as TNFa and IL-1 are responsible forlower miR-486 expression in muscle cells. miR-486 is an integralpart of amyogenesis signaling network that involves Pax7,MyoD,myostatin, and NF-kB (15–17), and MyoD actively induces itsexpression (17). In addition, miR-486 is essential for survival ofcardiomyocytes because it blocks PTEN to upregulate PI3K/AKT(18). Furthermore, loss of miR-486 is a major defect in musculardystrophy, and transgenic expression of miR-486 in muscle canrescue muscular dystrophy phenotype in animal models (19).Considering the intricate network in muscle that involvesboth NF-kB andmiR-486, this study aimed to determine whetherpharmacological inhibition of NF-kB could overcome the cancer-induced musculoskeletal defects and expression of which amongthe genes within the broader NF-kB–Pax7–MyoD–miR-486 sig-naling axis could be restored in muscle upon pharmacologicalinhibition.

We used MMTV-PyMT transgenic mice (called PyMTþ micehereafter) as an animal model of mammary tumors in order tocharacterize tumor progression and functional limitations (20).Genome-wide comparative analyses of multiple transgenic mam-mary tumormodels have suggestedPyMTþmodel to represent theluminal B–intrinsic subtype of breast cancer (21). We useddimethylaminoparthenolide (DMAPT), awater-soluble analogueof parthenolide, which we have previously shown to have potentNF-kB–inhibitory activity (22) and has been shown to overcomethe adverse effects of persistent activation of NF-kB in BRCA1-deficient mammary luminal progenitor cells in vivo (23). Wefound that mammary tumor development in PyMTþ mice wasaccompanied with functional limitations such as reduced gripstrength, impaired rotarod balance, causing altered body com-position as well as molecular changes in muscle, includingreduced expression of miR-486 and MyoD as we reported previ-ously (14). Unlike in acute models of cachexia, which involvespersistent expression of self-renewing factor Pax7 and block in

myogenic differentiation (24), we observed lower Pax7 expres-sion in muscle of PyMTþ mice. We also observed reduced expres-sion of peroxisome-activated receptor coactivator 1-beta (Pgc-1b)in skeletal muscle of PyMTþ mice. MyoD, in combination withRelB, an alternative NF-kB, has been shown to maintain theexpression of Pgc-1b in muscle and Pgc-1b is required for oxida-tive phosphorylation andmitochondrial biogenesis (25).DMAPTtreatment ameliorates many of the functional limitations as wellas themolecular changes inmuscle, including restoration of Pax7,MyoD, and Pgc-1b expression in muscle. These results have thepotential for translation into a clinical strategy for targeting bothtumors and functional limitations via inhibition of NF-kB toimprove outcomes in breast cancer patients.

Materials and MethodsTransgenic mice, functional limitation studies, and drugtreatment

National Institutes of Health regulations concerning the useand care of experimental animalswere followedwhile conductinganimal studies and the Indiana University School of Medicineanimal use committee approved this study. Male PyMTþ mice ona FVB/N background were purchased from The Jackson Labora-tory, and randomly bred with nontransgenic FVB/N females toobtain females heterozygous for the PyMT oncogene. Tumorprogression was monitored periodically. Tumor volume wascalculated as (length) � (width)2/2. Echo-MRI was used todetermine body compositions. Grip strength was measured bylayingmice on awiremesh connected to a testmeter (Bioseb), andpulling the tail directly back parallel to the mesh surface, thenrecording the amount of force. Three trials of each test wereperformed with minimum 5-minute intervals, and the averagevalue from the three recordings is presented. For rotarod perfor-mance (Harvard Apparatus), the period of timemice were able toremain on a rotating rod at 10 rpm was recorded 3 times atminimum 5-minute intervals and the average from the threerecordings is presented. For the drug intervention study, DMAPT(100mg/kg) or vehicle (2.5%mannitol) was orally administratedto mice from Monday to Friday, starting 6 to 8 weeks post birthuntil the end of the experiment. Age-matched normal femalemicewere used as controls. Structure of DMAPT has been describedpreviously (26). Blood and tissues were collected at the time ofsacrifice as per the recommendation of the attending veterinarianfor miRNA, mRNA, and protein preparation or for histologicalanalysis. Drug-treated animals were sacrificed at two differenttime points; one time point set at the same age as the control micewith tumors and the second set as recommended by the attendingveterinarian to measure biochemical parameters.

Sample processing miRNA and total RNA extractionMouse blood was collected in a K2 EDTA coated tube, which

was centrifuged at 2000 rpm for 15 minutes at 4�C. Immediatelyfollowing centrifugation, supernatant was transferred into a cleanEppendorf tube for storage at�80�C. AQiagenmiRNeasy serum/plasma kit was used to isolate miRNA from 200 mL of storedplasma (Qiagen #217184). Tibialis anterior and gastrocnemiusmuscles from the hind limb of the mouse were harvested, snap-frozen, and stored at �80�C. A Qiagen miRNeasy Mini Kit(#217004) or Qiagen RNeasy Mini Kit (#74106) was used toisolate miRNA or total RNA for RNA amplification from �30 mgof stored muscle. First, 700 mL QIAzol Lysis reagent/or RLT lysisbuffer was added to the tube containing dissectedmuscle, and the

Wang et al.

Mol Cancer Ther; 16(12) December 2017 Molecular Cancer Therapeutics2748




mixture was then homogenized using a Qiagen TissueLyser LTunit with metal beads at a speed of 50 Hz for 3minutes, followedby 10 seconds of sonication following the Qiagen-recommendedprotocol.

Quantitative reverse transcription PCRFive microliters of miRNAs extracted from plasma (20 ng/mL)

was used for cDNA synthesis using a Taqman miRNA ReverseTranscription Kit (Applied Biosystems). Total RNA frommuscletissues (200 ng/mL) was reverse transcribed into cDNAsusing Bio-RAD iScript cDNA synthesis kit in a final volumeof 20 mL. Quantitative PCR (qPCR) was performed using Taq-man universal PCR mix (Applied Biosystems) and specificprimers. miRNA primers for U6 (#001973), miR-202(#002579), miR-486 (#001278), miR-214 (#002293), miR-16 (#000391), miR-1(002222), miR-146a (000468), miR206(#000510), and mRNA primers for Hsp90ab (#Mm00833431-g1), Pax7 (#Mm01354484-m1), MyoD (#Mm00440387-m1),Pgc-1b(#Mm00504730-m1), Acvr2b (#Mm00431664-m1),Dmpk(#Mm00446261-m1), Lmna (#Mm00497783-m1),Prkag1(#Mm00450298), Hoxa9 (#Mm00439364), Mt1(#Mm00496660-g1), and Mt2 (#mm00809556-s1) were pur-chased from Applied Biosystems. Pgc-1b primers amplify exons11 and 12 and measure only the "A" isoform of Pgc-1b. Eachamplification reaction was performed in duplicate in a finalvolume of 20 mL with 4 mL of cDNA. The expression levels ofmiR-486 were normalized to miR-202 (mouse sera) and U6(skeletal muscle) using the 2�DDCt method as described in aprevious study (14).

Histological and immunohistochemical analysisTissues were immersed into 10% buffer formalin and stored in

a cold room. For H&E staining, formalin-fixed tissues were trans-ferred to the pathology lab, paraffin-embedded, sliced, stainedand analyzed by a pathologist. For immunofluorescence, forma-lin-fixed tissues were transferred to 20% sucrose in PBS overweekend, frozen, sliced with cryostat at 10 micron, mounted onpositively charged slides, then washed with PBS three times, andincubated with Cox IV antibody (Abcam #ab16056; 1:1000). Thenext day, sections were washed three times with PBS and subse-quently incubated with secondary antibody conjugated with afluorescent dyes. After a final wash with PBS, sections were sealedby mounting media with DAPI, and viewed under fluorescentmicroscope. For ECMhistochemical examination, formalin-fixed,frozen sliced sections were washed with PBS three times and thenincubated with Texas-red-conjugated-wheat germ agglutinin(WGA; Invitrogen, #W21405, 1:1000). WGA staining was quan-tified to measure ECM area by using ImageJ software. The areaoccupied by WGA was expressed relative to the total area of themuscle cross-section.

Western blottingFreshly harvested or frozen mouse muscles (�30 mg tissue)

were lysed in RIPA buffer with protease/phosphatase inhibitors(Sigma) and then homogenized usingQiagen TissueLyser LTwithmetal beads at a speed of 50 Hz for 3 minutes, followed by 10seconds of sonication. Thirty micrograms of proteins were usedfor Western blotting. Antibodies including mouse monoclonalPax7 (Developmental Studies Hybridoma Bank, AB 528428) andMyoD (#sc-760, Santa Cruz Biotechnology) were used for West-ern blot analyses as per instructions from the manufacturers.

Cytokine measurementsCirculating cytokines were measured using 100 mL of plasma

and a MILLIPLEX MAP Mouse Cytokine/Chemokine MagneticBead Panel (MCYTMAG-70K-PX32, Millipore) that measures32 cytokines/chemokines from limited samples. Tgfb1, 2, and3 were measured separately. Each group contained plasma sam-ples from six animals. Those samples with no detectable valueswere given a score of zero for the analyses.

Statistical analysisMultiple group datawere compared using ANOVA. A P value of

<0.05 was considered statistically significant.

ResultsTumor progression in PyMTþ transgenic mice is accompaniedwith functional limitations

PyMTþ transgenic mice develop mammary tumors at 100%penetrance (20). Palpable tumors in females were evident as earlyas 60 days with a slow progression (by volume) for the first fewdays followed by rapid tumor growth requiring animal sacrificedue to humane reasons within 2 to 3 weeks following tumoroccurrence. During an observation period from 8 to 14 weeks ofage, we noted increased body weight in PyMTþ mice comparedwith control mice (Fig. 1A), which was significant by 12 weeks.While PyMTþmice weighed 26� 0.7 grams, the weight of controlmice at the same age was 23� 0.4 grams. By 14 weeks, differencesbecame even more significant with tumor-bearing mice reaching30� 1.1 grams, whereas control mice remained at 23� 0.4 grams(Fig. 1A). This weight gain in PyMTþ mice is likely due to rapidlygrowing tumors.

We used echo-MRI to determine body composition. PyMTþ

mice had significant loss of body fat with progressive diseasecompared with control mice (Fig. 1B). For instance, the percent-age of body fat was 15% � 1.3% at the age of 8 weeks in PyMTþ

mice and significantly reduced to 10%� 0.5% at 14 weeks of age(Fig. 1B). In contrast to body fat, body lean mass increased withgrowth of tumors, and was particularly obvious at 14 weeks (Fig.1C). Parallel to the changes in body lean mass, the percentage ofbody free water and body total water increased in PyMTþ mice(Fig. 1D and E). This water retentionmay also have contributed toincreased body weight in PyMTþ mice and could be the result oftumor-induced impairment in lymphatic drainage. In line withbody composition changes, we found that PyMTþ mice hadreduced grip strength (Fig. 1F). Eight-week old PyMTþ mice had191 � 6.0 grams gripping force, which was further reduced to162� 6.6 grams and 162� 2.7 grams gripping force by 12 and 14weeks, respectively. The gripping force in FVB/N wild-type miceremained �200 grams during this observation period (Fig. 1F).

In order to test whether heart function was affected by mam-mary tumor burdens, we used the ECGenie device to acquire ECGsignals from live mice. Surprisingly, tumor burden did not sig-nificantly change heart beating intervals and heart beating rate(Supplementary Fig. S1). One reason for the lack of effects oncardiac function could be the rapid tumor growth forcing animalsacrifice before any cardiac effects of tumor burden were notice-able with available measurement tools.

DMAPT inhibits mammary tumors in PyMTþ miceTo investigate whether NF-kB is the central node in cancer-

induced systemic effects, we treated PyMTþ mice with the NF-kBinhibitor DMAPT. DMAPT is a water-soluble analogue of

Targeting Systemic Effects in Breast Cancer

www.aacrjournals.org Mol Cancer Ther; 16(12) December 2017 2749




parthenolide, an active ingredient of the herb feverfew (22, 27). Aswithmany targeted therapies,DMAPTdisplaysNF-kB–dependentandNF-kB–independent activities; however, itsmajor therapeuticeffects are likely through NF-kB inhibition (22). Treatment withDMAPT (100mg/kg, 5 times/week, orally), starting at 6 to 8weeksof age, prior to visual appearance of mammary tumors, delayedmammary tumor visual onset and slowed tumor growth ratescompared with vehicle treatment (Supplementary Table S1). At10 weeks, 60% of PyMTþ mice with vehicle treatment haddeveloped mammary tumors, while only 10% of PyMTþ micewith DMAPT treatment had mammary tumors. By 12 weeks,tumor burden was 3.6 � 1.2 cm3 in mice treated with vehicle,while it was 0.7� 0.3 cm3 in mice with DMAPT treatment. Thesedifferences in tumor growth persisted until the termination of thestudy. Tumor burden was 10.6 � 1.6 cm3 in mice treated withvehicle treatment versus 6.3 � 1.3 cm3 in mice treated withDMAPT by 14 weeks (Supplementary Table S1). Tumors ofDMAPT-treated animals compared with tumors from vehicle-treated animals contained �40% lower levels of transcripts forIL-6 (P ¼ 0.006), a NF-kB target gene, suggesting that the drug istargeting NF-kB in tumors. These differences in tumor growth/characteristics also translated into time of sacrifice of animals asrecommended by the attending veterinarian. By this measure-ment, overall survival of animals in theDMAPT-treated groupwassignificantly longer compared with control mice (Fig. 2A). For the

purpose of tissue collection, we repeated the experiment withcontrol and drug-treated groups and collected tissues at the sameage point. Lungs from nontransgenic, PyMTþ, and PyMTþ þDMAPT mice that were harvested at the same age as well asanother group of control, untreated and drug-treated PyMTþ

animals, which were allowed to survive until recommended foreuthanasia by the attending veterinarian, were examined formetastasis. Surprisingly, despite reduced tumor growth andimproved survival, DMAPT-treated animals showed similarmetastasis burden as untreated PyMTþ mice (Fig. 2B). Note thatall FVB/N background wild-type mice did not develop mammarytumors during the entire period of these observations (Supple-mentary Table S1).

DMAPT restores functional performance in PyMTþ miceWe next examined functional limitations including grip

strength and rotarod performance.Weobserved that both vehicle-andDMAPT-treatedmice had significantly lower gripping force tothe metal wires of the test meter at 12 and 14 weeks of agecompared with control mice. However, gripping force ofDMAPT-treated mice was significantly better than vehicle-treatedmice (Fig. 3A). For instance, vehicle-treated mice had a grippingforce of 159�3.9 g,whilemice that receivedDMAPThadgrippingforce of 193 � 3.9 g. In addition, PyMTþ mice that receivedDMAPT treatment had better rotarod performance. In the rotarod

Figure 1.

Characterization of systemic effects ofmammary tumors in PyMTþ mice.A, Body weight. B, Percentage of fatover total body weight. C, Percentageof lean mass over total body weight.D, Percentage of body free water overtotal body weight. E, Percentage oftotal body water over total bodyweight.F,Grip strength. � , Significancebetween PyMTþ and wild-typegroups, P < 0.05. #, Significancebetween baseline (8 weeks) andexperimental data from the samegroup; P < 0.05. n¼ 5 (wild-type) and8 (PyMTþ) mice/group.

Wang et al.





test, FVB/N wild-type mice rotarod performance was 113 � 21seconds,while PyMTþmicewith vehicle could only hold on to therotating rod for 11.6 � 2.7 seconds. DMAPT-treated mice stayedon the rod for 37 � 9.7 seconds (Fig. 3B).

To examine whether DMAPT ameliorates alterations of bodycomposition associated with mammary tumors in PyMTþ mice,we monitored body weight, body fat, body lean mass, body freewater, and body total water every other week utilizing echo-MRIanalysis (Fig. 3C–F). Both vehicle- and DMAPT-treated miceexhibited a similar trend of changes in body composition in thethree categories of body fat, body leanmass and total body water,but not in body free water (Fig. 3C–F). There was a significantincrease of body free water in mice treated with vehicle, but inmice treated with DMAPT body free water was significantly lowerand similar to levels in nontransgenic wild-type mice (Fig. 3E). Inaddition, DMAPT-treated mice gained less weight when com-pared with vehicle-treated mice (Fig. 3G).

Restoration of muscle function by DMAPT is accompaniedwith reversal of several cancer-induced molecular changes inskeletal muscle

To associate the beneficial effects of DMAPT with specificchanges in molecular parameters, we measured the levels ofskeletal-muscle enriched microRNAs in the circulation and inmuscle. Consistent with our previous report (14), plasma miR-486 levels were lower in tumor-bearing mice compared withcontrol mice (Fig. 4A). To our surprise, DMAPT treatment didnot significantly restore circulating miR-486 levels despiteimproving muscle function. However, we found elevated levelsof circulating miR-146a in animals treated with DMAPT. miR-146a, althoughNF-kB inducible, plays amajor role in attenuatinginflammation by targeting molecules in NF-kB–regulatory net-work, including cytokine receptors, thus reducing cytokine actionand NF-kB activity (28).

Wenext examinedmiR-486 levels inmuscles.miR-486 levels inthe muscle of PyMTþ mice were lower compared with controlmice (Fig. 4B), similar to our previous observations (14). As withcirculating miR-486, DMAPT treatment did not restore musclemiR-486 (Fig. 4B). Thus, it appears that DMAPT restores musclefunction that is independent of miR-486. miR-206 is anothermuscle-specific microRNA and inflammatory cytokines reduce its

expression to promote muscle degeneration, inflammatorymyopathies, and dermatomyositis (15, 29, 30). In addition,miR-206, secreted by myogenic progenitor cells, controls extra-cellular matrix (ECM) deposition (31). We measured miR-206levels in muscle to further document the effects of cancer pro-gression onmuscle. Indeed, miR-206 levels in muscle were lowerin PyMTþ mice compared with control mice (Fig. 4B). Amongothermuscle-relatedmicroRNAs, we observed downregulation ofmiR-214 but not miR-1 in skeletal muscle of PyMTþ mice com-pared with control mice (Fig. 4B). Surprisingly, DMAPT failed tochange the expression levels of any of thesemicroRNAs.We notedthat although circulating levels of miR-146a were elevated inDMAPT-treated animals, the levels did not change in muscle,suggesting thatmuscle is not the source of circulatingmiR-146a inDMAPT-treated animals.

DMAPT restores loss of Pax7, MyoD, Hoxa9, and Pgc-1b inskeletal muscles of PyMTþ mice

To determine whether enhanced functional performance uponDMAPT treatment can be correlated with the expression levels ofother NF-kB targets, we first measured Pax7 protein levels inskeletal muscles. We found lower levels of Pax7 protein inmuscleof PyMTþ mice compared with wild-type mice, and DMAPTrestored Pax7 levels in tumor-bearing mice to levels similar towild-type mice (Fig. 5A). Consistent with protein analysis, qRT-PCR showed lower levels of Pax7 mRNA in PyMTþ mice and thereverse in DMAPT-treated mice (Fig. 5B).

We also observed lower MyoD protein level in skeletal musclesof PyMTþ mice compared with control mice, although thesedifferences did not reach statistical significance (P ¼ 0.13). How-ever, MyoD protein levels in DMAPT treated animals were sig-nificantly higher than in untreated animals, suggesting thatNF-kBinhibition can lead to elevated MyoD levels in muscle (P ¼0.01; Fig. 5A). As with protein levels, MyoD mRNA levels werelower in the muscle of PyMTþ mice (Fig. 5B) but DMAPT treat-ment only marginally increased MyoD mRNA levels. Thus, itappears that DMAPT likely reduces MyoD protein turnover.

To identify additional targets of DMAPT, we performedMyogenesis and Myopathy PCR array (Qiagen) covering >90muscle-enriched genes using skeletal muscle RNA from control,PyMTþ, and PyMTþþDMAPT mice (n ¼ 5/group). Compared

Figure 2.

The effect of DMAPT on survival andmetastasis. A, Oral administration of DMAPT(100 mg/kg, M-F/week, starting at 6–8weeks of age) improved survival. B, Left,representative lung metastasis; right,average lung metastasis area per mouse. n¼5 mice/group. Note that this analysisincluded two sets of mice; lungs from age-matched untreated and DMAPT-treatedanimals and from animals collected at theend of the terminal experiments. In both sets,there were no statistical differences in therate of metastasis between groups, althoughthere was a trend of lower metastasis in theDMAPT-treated group.






with control mice, only six genes in this panel showed signif-icantly lower expression in muscle of PyMTþ mice and noneshowed elevated expression (AcvR2b, Dmpk, Il-1b, Lmna,Nos2, and Prkag1; Supplementary Table S2). In DMAPT-treatedmice, only Dmpk mRNA levels remained lower compared withcontrol animals. In validation experiments, while AcvR2b,Dmpk, Lmna, and Prkag1 mRNA levels remained low in theskeletal muscle of tumor-bearing animals, DMAPT failed toimprove their expression levels. Thus, among myogenesis-asso-ciated genes, DMAPT was able to restore the expression of onlyPax7 and MyoD.

We examined the expression levels of four other genes associ-ated with NF-kB signaling and are also recently been linked tometabolism, differentiation, and aging of muscle. These includePgc-1b, which is induced by MyoD and alternate NF-kB, and is anegative regulator of canonical NF-kB signaling in skeletalmuscle(25, 32), Hoxa9, which is typically elevated in aging satellite cellsof muscle and its expression is repressed by NF-kB (33, 34),and metallothioneins 1 and 2 (Mt1 and Mt2), which controlskeletal muscle mass and strength (35). Pgc-1b and Hoxa9 butMt1 and Mt2 mRNA levels were lower in the skeletal muscle oftumor-bearingmice,whichwere reverseduponDMAPT treatment

Figure 3.

Oral administration of DMAPT (100mg/kg, M-F/week) modulates bodycomposition and functionalperformance in PyMTþ mice.A, DMAPT restored grip strength.B, DMAPT partially amelioratedtumor-induced decline in rotarodperformance. C,DMAPT treatment didnot alter tumor-induced loss of fat. D,DMAPT had minimal effect on tumor-induced changes in body lean mass. E,DMAPT reduced percentage of bodyfree water. F, DMAPT did notsignificantly change total body waterratio. G, DMAPT slowed down bodyweight gain. � , Significance betweenexperimental groups and wild-typegroup, P < 0.05. #, Significancebetween the PyMTþ group withvehicle and with DMAPT treatments atthe same age-testing point, P < 0.05.n ¼ 10 mice/group.

Wang et al.





(Fig. 5B). Thus, DMAPT restored the expression of four proventargets of NF-kB inmuscle (i.e., Pax7,MyoD, Pgc-1b, andHoxa9).

DMAPT reverses cancer-induced loss in muscle mitochondriaPgc-1b is necessary for oxidative phosphorylation and mito-

chondria biogenesis (25). Because Pgc-1b levels were lower inskeletal muscle of PyMTþ mice, we next determined mitochon-dria content by staining for Cox IV, which is a mitochondria-enriched protein. Skeletal muscle of PyMTþ mice containedlower levels of Cox IV compared with control mice and DMAPTtreatment restored Cox IV levels (Fig. 6A). Thus, cancer-inducedloss of Pgc-1b has an effect on mitochondria biogenesis inmuscle, which may be a contributing factor for weakenedmuscle function.

DMAPT attenuates ECM deposition in skeletalmuscles of PyMTþ mice

The ECMprovides an appropriate and permissive environmentfor muscle development and functioning (36). Dynamic remo-deling of the ECM is directed by the activity of matrix metallo-

proteinases such as MMP9, which is a known transcriptionaltarget of NF-kB (37). Moreover, enhanced satellite cell to myo-genic progenitor cell differentiation leads to increased ECMaccumulation in muscle (31). Because muscle of tumor-bearingmice expressed low levels of the two satellite-specific transcriptionfactors Pax7 andHoxa9, which could be due to enhanced satellitecell to progenitor cell differentiation, we determined ECM com-position in muscle. PyMTþ mice had twice the level of ECMcompared with control mice, and this increase was blocked byDMAPT (Fig. 6B). Interestingly, H&E staining of muscles did notreveal morphological difference of muscle fibers andmuscle fibersizes among three groups under basic light microscope examina-tion (Fig. 6C). Thus, cancer progressions in PyMTþ mice isassociated with molecular changes in muscle, which may not bemanifested at morphological levels but likely have consequentialeffects on muscle function.

DMAPT alters circulating cytokines/chemokinesWe used a Bio-Plex 200 bead-based suspension system

(Luminex platform) to determine tumor-induced changes

Figure 4.

The effect of DMAPT on cancer-induced changes in circulating and muscle-enriched microRNAs. A, The effect of DMAPT on circulating miRNAs. miR-486levels were lower in plasma of PyMTþ mice, which remained low in DMAPT-treated mice. DMAPT increased circulating miR-146a levels. B, DMAPTtreatment did not restore tumor-induced loss of muscle-enriched microRNAs in hind limb muscle of PyMTþ mice. Average and standard deviation are shown.� , Significance between experimental and wild-type groups, P < 0.05. n ¼ 8–10 mice/group.

Figure 5.

DMAPT restored tumor-induced lossof Pax7, MyoD, and Pgc-1b in skeletalmuscle. A, Pax7 and MyoD proteinlevels. Left, Pax7 and MyoD protein inskeletal muscles of hind limbs. Image Jwas used to quantitate Westernblotting data (right). B, qRT-PCRanalysis of multiple mRNAs in skeletalmuscles of PyMTþ mice. Eight of 10analyzedmRNAswere downregulatedin muscle, whereas two of 10 mRNAswere not changed in PyMTþ micecompared with wild-type mice.DMAPT treatment significantlyreversed tumor-induced loss of Pax7,Pgc-1b, and Hoxa9 mRNA levels.� , Significance between experimentaland wild-type groups, P < 0.05. #,Significance between vehicle-treatedand DMAPT-treated PyMTþ animals.P < 0.05. n ¼ 8–10 mice/group.






in circulating cytokines/chemokines and to determine the effectof DMAPT treatment on their levels. Expression levels ofseven cytokines (Gm-csf, Tnfa, Tgfb2, Tgfb3, Il-6, Vegf, andG-csf) were elevated and six cytokines (Lix, Il-1a, Kc, Il-9, Mip-2, and Tgfb1) were decreased in PyMTþ mice compared withcontrol mice (Fig. 7, Supplementary Table S3 for raw data).DMAPT reversed cancer-induced changes in the expression

of six of these cytokines (i.e., Lix, Il-1a, Il-9, Mip-2, Tgfb1,and GM-csf). Among the cytokines whose expression wasunaffected by tumors, DMAPT reduced the levels of Il-5and Mip-1b. Collectively, these results demonstrate uniqueeffects of PyMT-derived tumors on circulating cytokines/che-mokines levels and pharmacologic approaches to circumventthese changes.

Figure 6.

Cox IV, ECM, and muscle fiber length changes in skeletal muscle of transgenic mice. A, Cox IV levels, a Pgc-1b target, in muscle. Cox IV levels were measured byimmunofluorescence and quantitated using Image J. B,Deposition of ECM in skeletal muscle of PyMTþmice. Top images show histochemical staining by Texas-red-conjugated-wheat germ agglutinin in skeletal muscle of mice, whereas bottom graph shows quantification of ECM by Image J. C, Analysis of skeletal muscle in hindlimb ofmice. Top, H&E staining of quadriceps; bottom, analysis of average area of cross section of each skeletalmuscle fiber fromquadriceps. � , Significance betweenexperimental and wild-type groups, P < 0.05. #, Significance between vehicle-treated and DMAPT-treated PyMTþ animals. P < 0.05. n ¼ 5–6 mice/group.

Wang et al.





Figure 7.

DMAPT altered circulating levels of select cytokines/chemokines. Cytokine/chemokine levels were measured by multiplex ELISA using plasma fromage-matched animals of three groups (n ¼ 6). A value of zero was given to cases with no detectable cytokine/chemokine content. Only data with atleast three animals per group with detectable expression were included. Data were graphed as three panels based on cytokine/chemokine levels.Asterisks indicate significance between experimental and wild-type groups: � , P < 0.05; �� , P < 0.001. #, Significance between vehicle-treated andDMAPT-treated PyMTþ animals. P < 0.05.






DiscussionFunctional limitations and sarcopenia are the validated

systemic effects of breast cancer (5, 38). However, to ourknowledge, none of the currently available transgenic modelshave been used to understand these systemic effects and to testfor therapies that have dual effects on cancer and systemiceffects or that may complement chemotherapy to improvequality of life. Numerous cachexia models have been devel-oped over the years, but most of these involve xenograft modelswith acute onset of cachexia (39, 40). We initiated this studywith three goals: (i) To determine whether transgenic modelsof breast cancer mimic functional limitations observed inpatients. (ii) If so, what are the molecular changes in musclethat accompany mammary tumor progression? (iii) Can thesemodels be used to identify therapies that might overcomesystemic effects with or without antitumor effects? Our resultsclearly show that PyMTþ mice develop functional limitations,which are accompanied with molecular changes in skeletalmuscle, particularly the expression levels of satellite cell-spe-cific transcription factors. Because many cancer patients expe-rience parainflammation of epithelial origin and cytokinesinvolved in this parainflammation can have paracrine effectson muscle (41), drugs that inhibit the function of cytokinescould be effective in reducing cancer-associated systemic effects.The drug DMAPT inhibits NF-kB signaling downstream ofmany cytokines (22, 27, 42) and was effective in reducingsystemic effects of mammary tumor. Although part of thebenefits of DMAPT treatment on muscle function can beattributed to reduced tumor burden, we believe that the drughad additional direct effects on skeletal muscle. Untreated andDMAPT-treated mice showed similar metastatic burden, whichindicates that the drug only limits the primary tumor growthand paracrine factors from metastasis that mediate systemiceffects remain the same in untreated and drug-treated groups.In addition, circulating levels of several cytokines, particularlyG-csf, which promotes breast cancer metastasis through neu-trophil polarization (43), were elevated in PyMTþ mice andwere unaffected by DMAPT treatment. The failure of DMAPT asa single agent to inhibit metastasis is not unexpected and isconsistent with our previous report using parthenolide inxenograft models (44). However, the primary tumor–specificeffect of the drug provides support for the recent observationthat early dissemination seeds metastasis and metastasisevolves independent of primary tumor with distinct signalingnetworks (45).

Genes that undergo changes in expression in cancer-associ-ated skeletal muscle can be classified into two groups; onegroup of DMAPT-responsive genes that is integral to the NF-kBpathway, and the other DMAPT-insensitive group. The DMAPT-sensitive group included genes/proteins in the muscle satellitecell function including Pax7 and Hoxa9. We observed thatthe levels of both of these genes being lower in muscle oftumor-bearing mice compared with wild-type mice. Hoxa9deregulation in muscle has been studied in the context of agingbut not functional limitation, and the current study is the firstreport to show tumor-induced loss of Hoxa9 expression inmuscle (33). Our observation on Pax7 depletion in the muscleof tumor-bearing transgenic mice is different from the observedaccumulation of Pax7 in muscle under acute cachexia due toblock in satellite cell differentiation (24). In this respect, the

duration of the experiment may have contributed to thesedisparate results. The fact that both Pax7 and the other satellitefactor Hoxa9 are reduced in muscle in our model clearlysuggests that cancer advances aging of muscle. In this respect,we also observed increased circulating level of GM-CSF inPyMTþ mice, which has previously been shown to mediateage-associated muscle loss in elderly patients (8). The reductionin MyoD and Pgc-1b in skeletal muscle of tumor-bearing miceis similar to reports in acute models of cachexia (46, 47).

The set of downregulated genes in skeletal muscle of PyMTþ

mice compared with wild-type mice and that are insensitive toDMAPT include Acvr2b, Dmpk, Prkag1, andmicroRNAsmiR-486and miR-206. Activin type II receptor (Acvr2b) regulates skeletalmuscle growth by binding with its ligands such as myostatin oractivin (48). Myotonic dystrophy protein kinase (Dmpk) is a Ser/Thr protein kinase, mainly expressed in smooth, skeletal, andcardiac muscles. Decreased Dmpk protein levels may contributeto the pathology of Myotonic Dystrophy, as revealed by genetargeting studies (49). Protein kinase AMP-activated noncatalyticsubunit gamma 1 (Prkag1) is the regulatory subunit of AMP-activated protein kinase (AMPK), which is an important energy-sensing enzyme that monitors cellular energy status (50). Failureof DMAPT to restore the expression of these genes/microRNAscould be the reason for partial recovery of functional limitationsand additional agents that restore the expression and/or functionof these genes may provide better protection against cancer-induced systemic effects.

Our studies show that DMAPT had an effect outside themuscletissue, which may have helped in reversing systemic effectsof cancer and reversing NF-kB–driven inflammation. Weobserved elevated levels of miR-146a in the circulation butnot in muscle of PyMTþ mice treated with DMAPT. miR-146aattenuates the inflammatory response by targeting cytokine recep-tors that promote NF-kB–driven inflammation (51) and its upre-gulation in circulation of DMAPT-treated mice through anunknown mechanism and from an unknown source indicatestreatment-induced attenuation of inflammation (28). ElevatedmiR-146a levels may also be responsible for restored musclefunction in DMAPT-treated mice despite the failure of the drugto reduce the levels of pro-inflammatory cytokines such as Tnfa,Il-6, Tgfb2, Tgfb3, and G-csf, many of which have been linked tocancer-induced changes in skeletal muscle function in at least invitro studies (39). These results also highlight the difficulty intranslating results from in vitro studies that show the effect of thesecytokines on skeletal muscle differentiation into the in vivocondition.

In summation, this report documents skeletal muscle changesin a chronic mammary tumor model that represents a luminal B–intrinsic subtype of breast cancer that are partially different fromchanges observed in acute tumor/cachexia models, and the fea-sibility of reversing these changes by utilizing drugs with NF-kB–inhibitory activity. CirculatingmiR-146a andGm-csf levels aswellas expression levels of Pax7, Hoxa9, MyoD, Pgc-1b, and Cox IV inmuscle could be used as biomarkers to document cancer-inducedfunctional limitations and the specificity of drugs that overcomefunctional limitations.

Disclosure of Potential Conflicts of InterestP.A. Crooks has ownership interest (including patents) in Leuchemix. No

potential conflicts of interest were disclosed by the other authors.

Wang et al.





Authors' ContributionsConception and design: R. Wang, H. NakshatriDevelopment of methodology: R. Wang, M.B. Padua, G. Sandusky,N. Penthala, H. NakshatriAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Wang, M.S. Prasad, M. Anjanappa, M. Jacobson,P.A. Crooks, T. Zimmers, H. NakshatriAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Wang, M.S. Prasad, C. Finnearty, V. Sefcsik,K. McElyea, R. Redmond, G. Sandusky, H. NakshatriWriting, review, and/or revision of the manuscript: R. Wang, P.A. Crooks,H. NakshatriAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Bhat-Nakshatri, M. Jacobson, H. NakshatriStudy supervision: H. NakshatriOther (synthesized the compound DMAPT Fumarate): N. Penthala

AcknowledgmentsWe thank Immunohistochemistry Core for tissue staining and the Multiplex

Analysis Core at the Indiana University Melvin and Bren Simon Cancer Centerfor providing support for analyzing plasma samples.

This work was supported by the Department of Veterans Affairs merit awardBX002764 (to H. Nakshatri). M.B. Padua was supported by a fellowship fromthe Vera Bradley Foundation for Breast Cancer Research.

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 July 28, 2017; revised September 12, 2017; accepted September 21,2017; published OnlineFirst October 4, 2017.

References1. Shachar SS, Deal AM, Weinberg M, Nyrop KA, Williams GR, Nishijima TF,

et al. Skeletalmusclemeasures as predictors of toxicity, hospitalization, andsurvival in patients with metastatic breast cancer receiving taxane-basedchemotherapy. Clin Cancer Res 2017;23:658–65.

2. Kroenke CH, Rosner B, Chen WY, Kawachi I, Colditz GA, Holmes MD.Functional impact of breast cancer by age at diagnosis. J Clin Oncol2004;22:1849–56.

3. Prado CM, Baracos VE, McCargar LJ, Reiman T, Mourtzakis M, Tonkin K,et al. Sarcopenia as a determinant of chemotherapy toxicity and time totumor progression in metastatic breast cancer patients receiving capecita-bine treatment. Clin Cancer Res 2009;15:2920–6.

4. Fox KM, Brooks JM, Gandra SR, Markus R, Chiou CF. Estimation ofcachexia among cancer patients based on four definitions. J Oncol2009;2009:693458.

5. Braithwaite D, Satariano WA, Sternfeld B, Hiatt RA, Ganz PA, KerlikowskeK, et al. Long-term prognostic role of functional limitations amongwomenwith breast cancer. J Natl Cancer Inst 2010;102:1468–77.

6. Patel HJ, Patel BM. TNF-alpha and cancer cachexia: molecular insights andclinical implications. Life Sci 2017;170:56–63.

7. Remels AH,GoskerHR, Schrauwen P,Hommelberg PP, Sliwinski P, PolkeyM, et al. TNF-alpha impairs regulation of muscle oxidative phenotype:implications for cachexia? FASEB J 2010;24:5052–62.

8. Calvani R, Marini F, Cesari M, Buford TW, Manini TM, Pahor M, et al.Systemic inflammation, body composition, and physical performance inold community-dwellers. J Cachexia Sarcopenia Muscle 2017;8:69–77.

9. Perkins ND. The diverse and complex roles of NF-kappaB subunits incancer. Nat Rev Cancer 2012;12:121–32.

10. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ Jr, Sledge GW Jr.Constitutive activation of NF-kappaB during progression of breast cancerto hormone-independent growth. Mol Cell Biol 1997;17:3629–39.

11. Bakkar N, Guttridge DC. NF-kappaB signaling: a tale of two pathways inskeletal myogenesis. Physiol Rev 2010;90:495–511.

12. Cai D, Frantz JD, Tawa NE Jr, Melendez PA, Oh BC, Lidov HG, et al.IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell2004;119:285–98.

13. Chacon-Cabrera A, Fermoselle C, Urtreger AJ, Mateu-Jimenez M, DiamentMJ, de Kier Joffe ED, et al. Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy, structure, andweakness. J Cell Physiol 2014;229:1660–72.

14. ChenD,GoswamiCP, Burnett RM,AnjanappaM,Bhat-Nakshatri P,MullerW, et al. Cancer affects microRNA expression, release, and function incardiac and skeletal muscle. Cancer Res 2014;74:4270–81.

15. Horak M, Novak J, Bienertova-Vasku J. Muscle-specific microRNAs inskeletal muscle development. Dev Biol 2016;410:1–13.

16. Dey BK, Gagan J, Dutta A. miR-206 and -486 induce myoblast differen-tiation by downregulating Pax7. Mol Cell Biol 2011;31:203–14.

17. Buckingham M, Rigby PW. Gene regulatory networks and transcriptionalmechanisms that control myogenesis. Dev Cell 2014;28:225–38.

18. Small EM, O'Rourke JR, Moresi V, Sutherland LB, McAnally J, Gerard RD,et al. Regulation of PI3-kinase/Akt signaling by muscle-enriched micro-RNA-486. Proc Natl Acad Sci U S A 2010;107:4218–23.

19. Alexander MS, Casar JC, Motohashi N, Vieira NM, Eisenberg I, Marshall JL,et al. MicroRNA-486-dependent modulation of DOCK3/PTEN/AKT sig-naling pathways improves muscular dystrophy-associated symptoms.J Clin Invest 2014;124:2651–67.

20. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, et al. Progression tomalignancy in the polyoma middle T oncoprotein mouse breast cancermodel provides a reliable model for human diseases. Am J Pathol2003;163:2113–26.

21. Pfefferle AD, Herschkowitz JI, Usary J, Harrell JC, Spike BT, Adams JR, et al.Transcriptomic classification of genetically engineered mouse models ofbreast cancer identifies human subtype counterparts. Genome Biol2013;14:R125.

22. NakshatriH, AppaiahHN,AnjanappaM,GilleyD, TanakaH, Badve S, et al.NF-kappaB-dependent and -independent epigenetic modulation using thenovel anti-cancer agent DMAPT. Cell Death Dis 2015;6:e1608.

23. Sau A, Lau R, Cabrita MA, Nolan E, Crooks PA, Visvader JE, et al. Persistentactivation of NF-kappaB in BRCA1-deficient mammary progenitors drivesaberrant proliferation and accumulation of DNA damage. Cell Stem Cell2016;19:52–65.

24. HeWA,Berardi E, CardilloVM,Acharyya S, AulinoP, Thomas-Ahner J, et al.NF-kappaB-mediated Pax7 dysregulation in themusclemicroenvironmentpromotes cancer cachexia. J Clin Invest 2013;123:4821–35.

25. Shintaku J, Peterson JM, Talbert EE, Gu JM, Ladner KJ, Williams DR, et al.MyoD regulates skeletal muscle oxidative metabolism cooperatively withalternative NF-kappaB. Cell Rep 2016;17:514–26.

26. Neelakantan S, Nasim S, Guzman ML, Jordan CT, Crooks PA. Ami-noparthenolides as novel anti-leukemic agents: discovery of the NF-kappaB inhibitor, DMAPT (LC-1). Bioorg Med Chem Lett 2009;19:4346–9.

27. Shanmugam R, Kusumanchi P, Appaiah H, Cheng L, Crooks P, Neelakan-tan S, et al. A water soluble parthenolide analog suppresses in vivo tumorgrowth of two tobacco-associated cancers, lung and bladder cancer, bytargeting NF-kappaB and generating reactive oxygen species. Int J Cancer2011;128:2481–94.

28. Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB-dependentinduction of microRNA miR-146, an inhibitor targeted to signaling pro-teins of innate immune responses. Proc Natl Acad Sci U S A 2006;103:12481–6.

29. Georgantas RW, Streicher K, Greenberg SA, Greenlees LM, Zhu W,Brohawn PZ, et al. Inhibition of myogenic microRNAs 1, 133, and206 by inflammatory cytokines links inflammation and muscle degen-eration in adult inflammatory myopathies. Arthritis Rheumatol2014;66:1022–33.

30. Tang X, Tian X, Zhang Y,WuW, Tian J, Rui K, et al. Correlation between thefrequency of Th17 cell and the expression of microRNA-206 in patientswith dermatomyositis. Clin Dev Immunol 2013;2013:345347.

31. Fry CS, Kirby TJ, Kosmac K,McCarthy JJ, PetersonCA.Myogenic progenitorcells control extracellular matrix production by fibroblasts during skeletalmuscle hypertrophy. Cell Stem Cell 2017;20:56–69.

32. Eisele PS, Salatino S, Sobek J, Hottiger MO, Handschin C. The peroxisomeproliferator-activated receptor gamma coactivator 1alpha/beta (PGC-1)






coactivators repress the transcriptional activity of NF-kappaB in skeletalmuscle cells. J Biol Chem 2013;288:2246–60.

33. Schworer S, Becker F, Feller C, Baig AH, Kober U, Henze H, et al. Epigeneticstress responses induce muscle stem-cell ageing by Hoxa9 developmentalsignals. Nature 2016;540:428–32.

34. Trivedi CM, Patel RC, Patel CV. Differential regulation of HOXA9 expres-sion by nuclear factor kappa B (NF-kappaB) and HOXA9. Gene2008;408:187–95.

35. Summermatter S, Bouzan A, Pierrel E, Melly S, Stauffer D, Gutzwiller S,et al. Blockade of metallothioneins 1 and 2 increases skeletal muscle massand strength. Mol Cell Biol 2017;37, e00305-16.

36. Calve S, Odelberg SJ, Simon HG. A transitional extracellular matrixinstructs cell behavior during muscle regeneration. Dev Biol 2010;344:259–71.

37. Hatfield KJ, Reikvam H, Bruserud O. The crosstalk between the matrixmetalloprotease system and the chemokine network in acute myeloidleukemia. Curr Med Chem 2010;17:4448–61.

38. Pardo I, LillemoeHA, Blosser RJ, ChoiM, Sauder CA,DoxeyDK, et al. Next-generation transcriptome sequencing of the premenopausal breast epithe-lium using specimens from a normal human breast tissue bank. BreastCancer Res 2014;16:R26.

39. Miyamoto Y, Hanna DL, Zhang W, Baba H, Lenz HJ. Molecular pathways:cachexia signaling-A targeted approach to cancer treatment. Clin CancerRes 2016;22:3999–4004.

40. Ballaro R, Costelli P, Penna F. Animal models for cancer cachexia. CurrOpin Support Palliat Care 2016;10:281–7.

41. AranD, Lasry A, ZingerA, BitonM, Pikarsky E,HellmanA, et al.Widespreadparainflammation in human cancer. Genome Biol 2016;17:145.

42. GuzmanML,RossiRM,NeelakantanS,LiX,CorbettCA,HassaneDC,et al.Anorally bioavailable parthenolide analog selectively eradicates acute myelog-enous leukemia stem and progenitor cells. Blood 2007;110:4427–35.

43. Coffelt SB, KerstenK,DoornebalCW,Weiden J, VrijlandK,HauCS, et al. IL-17-producing gammadelta T cells and neutrophils conspire to promotebreast cancer metastasis. Nature 2015;522:345–8.

44. Sweeney CJ, Mehrotra S, SadariaMR, Kumar S, Shortle NH, Roman Y, et al.The sesquiterpene lactone parthenolide in combination with docetaxelreduces metastasis and improves survival in a xenograft model of breastcancer. Mol Cancer Ther 2005;4:1004–12.

45. Hosseini H, Obradovic MM, Hoffmann M, Harper KL, Sosa MS, Werner-KleinM, et al. Early dissemination seedsmetastasis in breast cancer. Nature2016 Dec 14. [Epub ahead of print].

46. GuttridgeDC,MayoMW,Madrid LV,WangCY, Baldwin AS Jr. NF-kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay andcachexia. Science 2000;289:2363–6.

47. Sacheck JM, Hyatt JP, Raffaello A, Jagoe RT, Roy RR, Edgerton VR, et al.Rapid disuse and denervation atrophy involve transcriptional changessimilar to those of muscle wasting during systemic diseases. FASEB J2007;21:140–55.

48. Zhou X, Wang JL, Lu J, Song Y, Kwak KS, Jiao Q, et al. Reversal of cancercachexia and muscle wasting by ActRIIB antagonism leads to prolongedsurvival. Cell 2010;142:531–43.

49. Kaliman P, Llagostera E. Myotonic dystrophy protein kinase (DMPK) andits role in the pathogenesis of myotonic dystrophy 1. Cell Signal2008;20:1935–41.

50. Schonke M, Myers MG Jr, Zierath JR, Bjornholm M. Skeletal muscle AMP-activated protein kinase gamma1(H151R) overexpression enhances wholebody energy homeostasis and insulin sensitivity. Am J Physiol EndocrinolMetab 2015;309:E679–90.

51. Li K, Ching D, Luk FS, Raffai RL. Apolipoprotein E enhances micro-RNA-146a in monocytes and macrophages to suppress nuclear factor-kappaB-driven inflammation and atherosclerosis. Circ Res 2015;117:e1–e11.


Wang et al.




2017;16:2747-2758. Published OnlineFirst October 4, 2017.Mol Cancer Ther Ruizhong Wang, Poornima Bhat-Nakshatri, Maria B. Padua, et al. Functional Limitations in a Transgenic Model of Breast CancerPharmacological Dual Inhibition of Tumor and Tumor-Induced

Updated version

10.1158/1535-7163.MCT-17-0717doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2017/10/04/1535-7163.MCT-17-0717.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/16/12/2747.full#ref-list-1

This article cites 49 articles, 14 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/16/12/2747To 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-17-0717

http://mct.aacrjournals.org/content/suppl/2017/10/04/1535-7163.MCT-17-0717.DC1

http://mct.aacrjournals.org/content/16/12/2747.full#ref-list-1

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

mailto:[email protected]

http://mct.aacrjournals.org/content/16/12/2747


Documents

Pharmacological Dual Inhibition of Tumor and Tumor-Induced ......Small Molecule Therapeutics Pharmacological Dual Inhibition of Tumor and Tumor-Induced Functional Limitations in a