RegulatorofG-ProteinSignaling5ReducesHeyA8Ovarian...

Hindawi Publishing CorporationBiochemistry Research InternationalVolume 2012, Article ID 518437, 9 pagesdoi:10.1155/2012/518437

Research Article

Regulator of G-Protein Signaling 5 Reduces HeyA8 OvarianCancer Cell Proliferation and Extends Survival in a MurineTumor Model

Molly K. Altman,1 Duy T. Nguyen,1 Santosh B. Patel,1 Jada M. Fambrough,1

Aaron M. Beedle,1 William J. Hardman,2 and Mandi M. Murph1

1 Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, Pharmacy South Building,The University of Georgia, Athens, GA 30602, USA

2 The University of Georgia Medical Partnership, 279 Williams Street, Athens, GA 30602, USA

Correspondence should be addressed to Mandi M. Murph, [email protected]

Received 6 April 2012; Accepted 19 April 2012

Academic Editor: Rolf J. Craven

Copyright © 2012 Molly K. Altman et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The regulator of G-protein signaling 5 (RGS5) belongs to a family of GTPase activators that terminate signaling cascades initiatedby extracellular mediators and G-protein-coupled receptors. RGS5 has an interesting dual biological role. One functional RGS5role is as a pericyte biomarker influencing the switch to angiogenesis during malignant progression. Its other functional role isto promote apoptosis in hypoxic environments. We set out to clarify the extent to which RGS5 expression regulates tumorprogression—whether it plays a pathogenic or protective role in ovarian tumor biology. We thus constructed an inducible geneexpression system to achieve RGS5 expression in HeyA8-MDR ovarian cancer cells. Through this we observed that inducibleRGS5 expression significantly reduces in vitro BrdU-positive HeyA8-MDR cells, although this did not correlate with a reductionin tumor volume observed using an in vivo mouse model of ovarian cancer. Interestingly, mice bearing RGS5-expressing tumorsdemonstrated an increase in survival compared with controls, which might be attributed to the vast regions of necrosis observedby pathological examination. Additionally, mice bearing RGS5-expressing tumors were less likely to have ulcerated tumors. Takentogether, this data supports the idea that temporal expression and stabilization of RGS5 could be a valuable tactic within the contextof a multicomponent approach for modulating tumor progression.

1. Introduction

The regulator of G-protein signaling 5 (RGS5) belongs to afamily of GTPase activators and signal transduction mol-ecules that negatively regulate the function of G-proteins.In other words, RGS proteins terminate cellular signalingcascades initiated by extracellular mediators that bind toand activate G-protein-coupled receptors. More specifically,RGS5 binds to G alpha (i), (q), and (o) subunits within het-erotrimeric G-proteins to terminate signaling and is locatedalong the plasma membrane and within the cytosol [1].RGS5 was isolated in 1997 from mouse pituitary, althoughits preferential expression is in the heart (particularly aorta),skeletal muscle, lung, small intestine, and thyroid [2, 3]. Rgs5is located at 1q23.1, a region on chromosome 1 of interest

for lipid metabolism [4], hypertension [5, 6], blood pressureregulation [7], severity of schizophrenia symptoms [8], andassociation with SNPs that have specific effects dependentupon genetic background [9].

Using a platelet-derived growth factor knockout mousemodel and comparing it to the gene expression of wildtypemice, Bondjers et al. were the first to identify RGS5 as abiomarker of pericytes [10], cells that wrap around the wallsof capillaries. Pericytes are thus involved in the regulationof blood flow and the transformation of new blood vessels.Berger et al. verified that RGS5 is an angiogenic pericytemarker and a component involved in the switch to angiogen-esis during malignant progression [11], with context-specificexpression (i.e., during wound healing). Mitchell et al. con-firmed that the expression of RGS5 is temporally upregulated

2 Biochemistry Research International

during pathological angiogenesis, at approximately 5-6 daysafter corneal scraping, a period when the nascent vesselsprouts acquire their pericyte covering [12].

Looking more broadly at the gene expression of RGS5 inmalignant tumors produces mixed results. Nearly an equiva-lent number of microarray expression experiments archivedin the European Bioinformatics Institute Atlas report that thegene is overexpressed or underexpressed. The interpretationof these mixed results portrays a complex association withintratumor heterogeneity, where gene expression is highlydependent on location [13] and in the specific example ofRGS5, also on several other factors, including hypoxia andvascular remodeling.

However, other reports offer more clearly defined expla-nations. For example, a study by Silini et al. showed that a lowlevel (<1%) of RGS5 fluorescence covered the normal ovary,whereas RGS5 increased to 7.3% coverage in ovarian car-cinoma specimens from patient biopsies. Furthermore, thestaining pattern of RGS5 coincided with vessel-like struc-tures, which is suggestive of a biomarker for cancer vascu-lature and consistent with RGS5 expression predominantlyresulting from the vascular endothelium of carcinoma [14].Therefore, RGS5 levels could be expected to vary accordingto the extent and stage of tumor vascularization, perhapsexplaining the gene expression variability for RGS5 amongsingle-biopsy specimens.

Not surprisingly given its association with the vascula-ture, RGS5 is also significantly affected by hypoxia, whichis a response initiated by cells in low-oxygen environmentsthat would otherwise succumb to toxic anoxia and cell death.Cancer cells in solid tumors are notorious for adapting tohypoxic environments by downregulating mitochondrialfunction [15] and shifting to aerobic glycolysis [16]. Inter-estingly, Jin et al. showed that endothelial cells exposed to ahypoxic environment (<1% oxygen) display an increase inboth mRNA and protein expression of RGS5 beginning at30 min after exposure and tapering off around 24 hours. Fur-thermore, they identified RGS5 as a hypoxia-inducible genethat stimulates apoptosis and is regulated by the alpha sub-unit hypoxia-inducible factor-1 (HIF-1α) [17]. The HIF-1heterodimeric transcription factor is an important regulatorof angiogenesis because it causes the expression of numeroustarget genes (VEGF, PDGF, TGF-α, PDK1, COX4I2, etc.)which are involved in neovascularization, erythropoiesis,glucose transport, and energy metabolism [18]. Overexpres-sion of VEGF and HIF-1α has been associated with a poorprognosis in ovarian cancer and breast cancer patients [19].

Further studies using an RGS5-deficient RIP1-Tag5transgenic mouse model demonstrated an increased rate oftumorigenesis and reduction in the overall survival of micethrough the development of a normalized network of bloodvessels, decreased hypoxia and reduced vessel permeability[20–22]. Interestingly, Hamzah et al. also reported that RGS5is “a master gene responsible for the abnormal tumor vascu-lar morphology in mice” [20]. We thus questioned whetherthe inducible expression of RGS5 in a tumor model of ovar-ian cancer might counter the effects observed in knockoutmice and support a longer period of survival in vivo. Sincethe role of angiogenesis inhibitors is somewhat controversial

(see Section 4 for more details), such a study may alsoclarify the extent to which RGS5 expression regulates tumorprogression, perhaps via altered vascularization. Herein, weobserved an increase in the survival time in mice bearingtumors with RGS5 expression coupled with increased areasof necrosis and a reduction in tumor ulceration. The controlanimals with tumors expressing the vector alone displayedmore malignant cells within tumors and more had ulceratedtumors. Although all animals eventually succumb to disease,these studies are suggestive that RGS5 expression reducesmalignancy in tumors, thus increasing survival time, andthis is independent of its role in vascular normalization andremodeling.

2. Methods

2.1. Materials. HeyA8-MDR taxane-resistant line of cellswere previously described [23] and are maintained in RPMI1640 medium (Mediatech, Inc., Manassas, VA, USA) supple-mented with 300 ng/mL paclitaxel (Sigma-Aldrich, St. Louis,MO, USA) with 15% fetal bovine serum (PAA Laboratories,Inc., Etobicoke, ON, Canada). Approved fetal bovine serum(Clontech Inc., Mountain View, CA, USA) was used in themedium for the pTet Dual RGS5-modified HeyA8-MDRcells. Doxycycline Hyclate was used to suppress gene expres-sion in the Tet-Off system (Sigma-Aldrich, St. Louis, MO,USA). The compounds nocodazole, BrdU, etoposide, andaphidicolin Assay Kit were purchased from Millipore (Biller-ica, MA, USA).

2.2. Construction of an Inducible RGS5 Cell Line. The Tet-Offadvanced inducible gene expression system (Clontech) wasused to create the RGS5-inducible HeyA8-MDR cell line. ThepTRE-Tight dual RGS5-expressing DNA plasmid was con-structed by standard restriction enzyme cloning to insert anHA-tagged RGS5 coding sequence cassette (Missouri S&TcDNA Resource Center, Rolla, MO, USA) into the pTRE-Tight Dual vector using the restriction enzymes XbaI andNheI. The constructs were verified by DNA sequencing usingspecific primers that were designed to recognize the N′-terminus of our Advanced Vector promoter. In order to cre-ate the doxycycline inducible cell line, 2.5×105 HeyA8-MDRcells were plated in a 6-well dish and transfected at a 2 : 1ratio (plasmid DNA: Lipofectamine 2000, Life Technologies,Grand Island, NY, USA) with pTet Advanced inducible vectorplasmid DNA. Positive clones were selected using G418(Geneticin, Life Technologies). These stable cells were thencotransfected with pTRE-Tight, Dual HA-RGS5 containingplasmid DNA, and the linear hygromycin marker to enableselection with hygromycin. Positive clones were maintainedin paclitaxel, G418, doxycycline, and hygromycin.

Gene expression was verified in HeyA8-MDR pTet dualHA-tagged RGS5-inducible cells by seeding the cells in amultiple 6-well plates in medium with tetracycline-free fetalbovine serum without doxycycline for 24, 48, and 72 hours.Cells were harvested at the indicated time points, the RNAwas isolated and processed for qRT-PCR using primers todetect RGS5 expression (amplicon size: 153 bp) resultingfrom the Tet-Off Advanced inducible system. The following

Biochemistry Research International 3

primers were selected from PrimerBank (http://pga.mgh.harvard.edu/primerbank/), to confirm gene expressionusing qRT-PCR (RGS5 Fwd: 5′-ATTCAAACGGAGGCT-CCTAAAG-3′ and RGS5 Rev: 5′-CACAAAGCGAGGCAG-AGAATC-3′).

2.3. BrdU Proliferation Analysis. HeyA8-MDR cells wereplated in a 96-well plate at a density of 3,000 cells per well.Half of the plate was grown in medium containing regularfetal bovine serum with doxycycline and the other halfdoxycycline-free with medium containing tetracycline-freefetal bovine serum. Cells were then incubated at 37◦C for72 hours to allow for RGS5-inducible gene expression. Priorto fixation, HeyA8-MDR cells were pulse-treated for 1 hourwith BrdU and then treated for 4 hours with the indicated cellcycle arrest compounds. Cells were then fixed and stained forproliferation and nuclear morphology according to standardprocedures from the manufacturer’s protocol (BrdU AssayKit, Millipore). Representative images were taken using theCellomics ArrayScan VTI HCS Reader (Thermo FisherScientific, Waltham, MA, USA) as previously described [24]and are shown here. High-content scanning analysis softwarewas used to determine the average number of cells per field.The data was retrieved from the manufacturer’s software andresults were plotted with GraphPad Prism (La Jolla, CA,USA).

2.4. Animal Model of Ovarian Cancer with Gene Modulation.Six-week-old female athymic nude mice acclimated to theanimal facility for 1 week prior to the commencement of thestudy. Animals were injected intraperitoneally with Extracelcontaining approximately 5 million cells of either HeyA8-MDR pTet Advanced Vector (N = 10) or HeyA8-MDR pTetDual RGS5 expressing cells (N = 10) per 0.2 μL injection.Injected mice were monitored for tumor formation, weight,and stomach circumference. After one week, 100% of micedisplayed tumor formation. The animals were monitoredover a course of 2 months and euthanized according to theanimal use protocol approved by the University of GeorgiaIACUC committee. The tumor volume (mm3) was calculatedusing the equation tumor volume= (width)2× length/2, andthen graphed using Prism. The time of survival for eachgroup and overall significance was plotted on a Kaplan-Meiersurvival curve also using GraphPad Prism.

2.5. Measurement of Vascular Endothelial Growth Factor. Atnecropsy, blood from all animals was collected in BD micro-tainer tubes with serum separator (Becton Dickinson Co.,Franklin Lakes, NJ, USA). The serum-containing fractionwas isolated using centrifugation, placed into glass vials, andfrozen immediately. After thawing on ice, the mouse serumwas measured for the presence and concentration of vascularendothelial growth factor a mouse VEGF ELISA kit followingthe manufacturer’s protocol (RayBiotech, Inc., Norcross, GA,USA).

2.6. Tissue Collection, Histology, and Immunofluorescence.Mice were euthanized according to standard protocols.

Visible tumors were dissected from the abdomen, measuredfor size, and flash-frozen with cryomatrix (Thermo FisherScientific) in 2-methylbutane (Sigma) cooled to −140◦C.Cryopreserved tumors were cut in 10 μm sections using aThermo Fisher Scientific cryostat and mounted on micro-scope slides. Hematoxylin and eosin staining was performedaccording to standard protocols and imaged using a Leicamicroscope for pathological evaluation.

For immunofluorescence, tissues were blocked for 30minutes in 5% normal donkey serum (Jackson Immun-oResearch Laboratories, Inc.,West Grove, PA, USA) inphosphate-buffered saline (PBS) for 30 minutes at roomtemperature. The tumor sections were incubated in theprimary antibody CD31 (1 : 500, BD Pharmingen, San Diego,CA, USA) at 4◦C overnight in a humidity chamber, washedwith PBS, and detected with the secondary antibody Rho-damine (TRITC)-conjugated AffiniPure F(ab′) 2-FragmentGoat Anti-Rat IgG (H + L) from Jackson ImmunoResearchLaboratories, Inc. (West Grove, PA, USA). DAPI nuclear stain(final 1 : 10,000) was included in the secondary antibodyincubation. After thorough washing with PBS, slides werecoverslipped with Permount (ThermoFisher Scientific).Immunofluorescence was imaged using an X71 invertedmicroscope (Olympus, Center Valley, PA) at 20x magnifica-tion. Images were resized and adjusted identically using Pho-toshop (Adobe, San Jose, CA, USA). Overlapping pictureswere aligned in Microsoft PowerPoint to generate an imageof an entire tumor cryosection. Each compiled tumor sectionwas imported into Image-Pro Express (Media Cybernetics,Bethesda, MD, USA) for analysis. Tumor vascularization wasdetermined by counting the number of complete and partialvessels visible in the tumor section (stained by antibodyCD31). Tumor area was calculated using the polygon toolin Image-Pro Express and normalized vascularization wasplotted (CD31 vessel counts/tumor area in μm2 × 1× 106).

3. Results

3.1. Expression of RGS5 Reduces In Vitro Proliferation ofHeyA8-MDR Ovarian Cancer Cells. Although RGS5 is a bio-marker for tumor vasculature, we sought to understandwhether RGS5 itself plays a pathogenic or protective role inovarian tumor biology. To address this question, we con-structed RGS5 in an inducible gene expression system toinduce high levels of RGS5 protein when cells were culturedin the absence of the antibiotic doxycycline. The Tet-Offinducible system was necessary because RGS proteins reg-ulate G-protein-coupled receptor signaling cascades, whichare required for cancer cells survival and are often critical tocells with oncogenic addictions to survival pathways. Whencells were grown in the absence of doxycycline and mediumcontaining FBS free of tetracycline, the expression of RGS5protein reached ∼7 fold after 48 hours and ∼4.5 fold after72 hours (Figure 1(a)).

When we compared the vector control cells (+doxy-cycline) versus RGS5-expressing HeyA8-MDR cells (−dox-ycycline), we observed a significant reduction in the numberof proliferating cells among the latter group with inducedexpression of RGS5 (Figure 1(b)). Representative images are


Rel

ativ

e ra

tio

(nor

mal

ized

toβ

-mic

rogl

obu

lin)

0

5

10

RGS5

24 hrs

48 hrs

72 hrs

(a)

(b)(c)

Brd

UE

topo

side

Noc

odaz

ole

BrdU Etoposide Nocodazole

400

300

200

100

0

Valid object count

∗∗∗∗∗∗

∗∗∗

+Doxycycline

−Doxycycline

+Doxycycline −DoxycyclineA

vera

ge n

um

ber

of c

ells

per

fiel

d

Figure 1: RGS5 inducible expression in HeyA8-MDR cells reduces proliferation. (a) HeyA8-MDR pTet dual RGS5 inducible cells were seededin a multiple 6-well and cultured in medium with tetracycline-free fetal bovine serum without doxycycline for 24, 48, and 72 hours. Cells wereharvested; the RNA was isolated and processed for qRT-PCR using primers to detect RGS5 expression resulting from the Tet-Off Advancedinducible system. Without the presence of doxycycline or other members of the tetracycline antibiotics, RGS5 expression was observed. (b)HeyA8-MDR cells were plated in a 96-well plate at a density of 3,000 cells per well. Half of the plate was grown in medium containing regularFBS with doxycycline and the other half containing doxycycline-free media. Cells were then incubated at 37◦C for 72 hours to allow forRGS5-inducible gene expression. Prior to fixation, HeyA8 MDR cells were pulse-treated for 1 hour with BrdU and then treated for 4 hourswith the indicated cell cycle arrest compounds. Cells were then fixed and stained for proliferation and nuclear morphology. Representativeimages taken using the Cellomics ArrayScan VTI HCS Reader and are shown here. (c) High-content scanning analysis software was used todetermine the average number of cells per field.

shown from high-throughput scanning (see Section 2.3).We next analyzed cancer cell proliferation using automatedquantification of cells detected per field after a pulse withbromodeoxyuridine (BrdU), a nucleoside analogue andmarker for proliferating cells. Cells engineered to induciblyexpress RGS5 showed a significant reduction in the numberof BrdU-positive proliferating cells (∗∗∗P < 0.001, compar-ing +dox with –dox). Treating the cells with either antineo-plastic reagents etoposide, a topoisomerase II inhibitor, ornocodazole, an inhibitor of microtubule polymerization, fur-ther reduced the average number of cells measured per field

(Figures 1(b) and 1(c); ∗∗∗P < 0.001), although the ratiowas similar between the nontreated and treated conditions.As a control, we measured no net change in the mean differ-ence of “target” BrdU fluorescence intensity among the spe-cific cell cycle compounds, suggesting no net bias effect of thefluorescence. Taken together, these data suggest that RGS5expression reduces the proliferative capacity of HeyA8-MDRovarian cancer cells.

3.2. Expression of RGS5 in an Ovarian Tumor Model IncreasesSurvival. Previous studies have examined the absence of


3000

2000

1000

0

4 9 14 18 23 28 32 36 40 45 55

Tum

or v

olu

me

(mm

3)

Vector

51

pTet dual RGS5

Time (days)

(a)

0 10 20 30 40 50 60

0

20

40

60

80

100

VectorRGS5

Days

P = 0.0143

Kaplan-Meier survival curve

Surv

ival

(%

)

(b)

400

300

200

100

0

VE

GF

(pg/

mL

)

ns

Vector RGS5

(c)

Figure 2: Expression of RGS5 in ovarian tumors did not significantly reduce the rate of growth, but did increase the overall survival timeof mice bearing such tumors. (a) Female athymic nude mice were given intraperitoneal injections of Extracel containing approximately 5million cells of either HeyA8 MDR pTet Advanced Vector (N = 10) or HeyA8-MDR pTet Dual RGS5 expressing cells (N = 10) per 0.2 μLinjection. All mice were monitored for tumor formation and measurements of weight and stomach circumference were routinely taken. Thegraph plots tumor volume (mm3). (b) Mice bearing HeyA8-MDR pTet Dual RGS5-expressing tumors had a significant increase in survivaltime (in days, ∗P = 0.0143, compared to their pTet Advanced Vector alone counterparts). (c) Blood was collected from mice at necropsy.The serum-containing fraction was isolated and measured for vascular endothelial growth factor. Results are nonsignificant (ns) betweenthe groups.

RGS5 expression in vivo using knockout mice [22]. In con-trast with these studies, we created an in vivo model oftumorigenesis with inducible expression of RGS5 to measurewhether there was an effect on tumor regression. Athymicnude female mice were injected intraperitoneally withtumors containing either the vector alone or RGS5-express-ing tumors. We routinely measured tumor volume, but wereunable to detect any differences between these groups(Figure 2(a)). In contrast, control animals with empty vectortumors displayed lower body conditioning scores than micebearing RGS5 tumors and therefore had to be monitoredmore frequently. Mice bearing RGS5 tumors displayed moreactive behavior and appeared healthier over a longer periodof time with higher body conditioning scores in comparison(data not shown).

We also measured the difference in survival timesbetween the two groups. The control mice with empty vector

tumors began to die (or were euthanized because theyreached humane endpoints) at 22 days and all succumbed todisease by 47 days (Figure 2(b)). In comparison, the firstmouse from the group bearing RGS5 tumors died in 28 days,and the last two in the group died after 55 days. The resultssuggest a significant increase in survival time (P = 0.0143)with RGS5 expression, although there were no animals thatultimately survived the disease.

Since RGS5 has been shown to be a hypoxia-induciblegene regulated by HIF-1α [17], we measured vascularendothelial growth factor (VEGF) in serum. Solid tumorswill develop hypoxic regions, which would then upregulateRGS5 and possibly interfere with our results. We chose VEGFbecause HIF-1 regulates VEGF expression as well as RGS5.If we detected significantly altered levels of VEGF betweenthe groups, then this could indicate a problem with the data.However, there was no significant difference between


Table 1: Histological examination of tumor sections by a pathologist.

Vector controls Tissue type Tumor (%) Necrosis Mitoses (mm2) Karyorrhexis

1 Mesentery 70 Not identified <1 Yes

2 Skin 95 Ulcer/focal <1 Yes

3 Mesentery >90 Not identified <1 Yes

RGS5-expressing

1 Mesentery >90 Broad area 1 Yes

2 Mesentery >90 Broad area(s) 1 Yes

3 Mesentery 60 Scattered 1 Yes

8

6

4

2

0

ControlRGS5-expressing

(N=

10/g

rou

p)

Ulcerated tumors No ulceration

Nu

mbe

r of

tu

mor

bea

rin

g m

ice

Figure 3: Mice bearing RGS5-expressing ovarian tumors displayedless frequent tumor ulceration. The number of mice with ulceratedtumors observed at necropsy was recorded (N = 10 per group).Tumor sizes were relatively similar between the two groups.

the serum levels of VEGF between the groups (Figure 2(c)),suggesting that endogenous RGS5 regulation wasunchanged.

3.3. Histological Studies Suggest Large Areas of Necrosis inRGS5-Expressing Tumors. We randomly selected tumorsfrom each group for sectioning and histological analysis.Interestingly, the hematoxylin and eosin stained tumor sec-tions showed several important differences between controland RGS5-expressing tumors. In the control group of micewith tumors containing the empty vector, diffuse sheets ofmalignant cells comprised 70–95% of the sampled tissue,demonstrating extensive involvement of the tumor (Table 1).In contrast, mice bearing tumors expressing RGS5 hadregions of necrosis that varied from scattered necrotic areasto broad and large areas of central necrosis. RGS5-expressingtissue was also composed of∼60–90% tumor. In the necroticareas of RGS5-expressing tumors, pyknotic nuclei and darkeosinophil cytoplasm were observed in the malignant cellsalong the peripheral areas of the necrotic regions. Finally, thehistological tumor samples of the control mice showed areasof skin ulceration, whereas the RGS5-expressing sections didnot. This is in agreement with the overall observations where

we observed a reduced presence of tumor ulceration in micebearing RGS5-expressing tumors (Figure 3).

3.4. Tumor Angiogenesis. To clarify the functional contribu-tion of RGS5 expression in tumor angiogenesis, we randomlyselected tumors from each group for sectioning and analysisof positively stained structures of the cluster of differentia-tion 31 (CD31) or platelet/endothelial cell adhesion molecule1 (PECAM-1), which is a glycoprotein biomarker expressedon vascular endothelial cells used to assess the degreeof angiogenesis. We observed CD31-positive structures intumor specimens from both groups of animals (Figure 4(a)).The RGS5-expressing tumors displayed a greater frequencyof CD31-positive vessel-like structures, compared with thevector-expressing tumors (Figure 4(b), ∗∗P < 0.01). This isconsistent with the role of RGS5 as a pericyte biomarker thatis temporally upregulated during the switch to angiogenesisin malignancy. The data is suggestive that introducing RGS5into the solid tumor likely influenced the balance of thisswitch in favor of angiogenesis.

4. Discussion

In this study, we used Tet-off inducible expression to studythe role of RGS5, in vitro and in vivo, in tumor proliferationand pathology. We found that mice bearing RGS5-expressingtumors survived longer than controls and displayed largeregions of necrosis within their tumors. They were also lesslikely to have ulcerated tumors in comparison to controlmice. Our study is consistent with previous work thatproduced RGS5-deficient mice and suggested that RGS5 lossaccelerated tumor development, enhanced tumor growth (inthe later tumor stages), reduced survival, decreased hypoxia,and decreased vessel permeability [20–22].

Hamzah et al. created an RGS5 knockout mouse modelusing a mix of normal 129 and C57BL/6 mice crossed withthe C3H background, which then allowed the assessment ofimmune function. Although not statistically significant, theRGS5 knockout mouse model resulted in the opening of solidtumors to spontaneous immune effector T-cell infiltrationinto the tumor parenchyma. In addition, this model showedprolonged survival among tumor-bearing mice with thetransfer of activated and specific T cells [20]. Our studyused athymic immunodeficient nude mice, which manifestan inhibition of the immune system and thus will not mountan immune response to xenograft injection. It is interesting


CD31

RG

S5V

ecto

r

100 μm

100 μm

(a)

70

60

50

40

30

20

10

0

No.

CD

31-p

osit

ive

vess

els

per

tota

l

VectorRGS5

tum

or t

issu

e ar

eaμ

m2×

106

CD31-positive vessels

∗

(b)

Figure 4: RGS5-expressing ovarian tumors increase the number of CD31-positive vessel-like structures. Tumor specimens were sectionedand prepared on slides for immunofluorescence. (a) Tumor sections were imaged for CD31 expression (red, 20x magnification). (b) CD31-positive vasculature was quantified from compiled whole tumor images (N = 4 vector, N = 4 RGS5) using CellSens and GraphPad Prismsoftware. ∗∗P < 0.01, comparing vector versus RGS5.

that we observed vast regions of necrosis in the solid tumorswithout the immune system modulating this response inRGS5-expressing tumors. This necrosis is likely due to twofactors: hypoxia resulting from aberrant tumor vasculatureand RGS5 induction of apoptosis [17].

Although we observed an increase in the survival time inmice bearing RGS5-expressing tumors, the overall increase intime was modest. Too many chemotherapies and biologicalagents also achieve mediocre increases in overall survival,leading the industry to focus instead on quality-of-lifeparameters for measuring drug “successes.” The results ofour study dampen enthusiasm for pursuing RGS5 as a singletarget for therapeutics in tumorigenesis. However, our studydoes provide support for including RGS5 as one importantcomponent of a multicomponent approach to help modulatetumor progression. As the treatment of cancer is a multi-faceted discipline, and cytotoxic chemotherapy is combina-torial, momentum for combinatorial biological therapies isalso gaining, even among “magic bullet” therapeutics (e.g.,imatinib and vemurafenib). The shift is being driven bychemoresistance to therapy, which is relevant in this settingbecause RGS proteins are involved in chemoresistance [23]as well as hypoxia—a driver of chemoresistance [25].

Our study also demonstrates that RGS5 effects activecellular proliferation in HeyA8-MDR cells in vitro using theBrdU assay. This result is in contrast with other studiessuggesting that the overexpression of RGS5 reduces the rateof growth without affecting cell proliferation [17]. The dif-ferences between the two studies are likely the model system.Whereas we are using rapidly proliferating ovarian cancercells resistant to paclitaxel, the previous study used human

umbilical vein endothelial cells. Thus, our model system ishighly aggressive, tumorigenic, and drug-resistant with orwithout RGS5 expression.

The addition of chemotherapeutic agents (etoposide ornocodazole) to the culture of RGS5-inducible HeyA8-MDRcells further reduced the average number of cells present andproliferating. Although very exciting, the fact that RGS5 hasa dual role tumor biology (i.e., vascularization versus tumorgrowth) makes it unclear how modulation of RGS expressionwould affect therapy. For example, RGS5 modulation couldsignificantly complicate drug delivery into solid tumorparenchyma. On one hand, RGS5 loss results in normal-ization of the vasculature in vivo, which would allow pen-etration of T cells and chemotherapy, but otherwise RGS5loss enhances tumor growth [20]. On the other hand, RGS5is overexpressed in aberrant tumor vasculature [11], but itsexpression induces endothelial apoptosis [17], reduces cellproliferation, and increases overall survival. Indeed, in ourstudy the mice bearing RGS5-expressing tumors that showedlarge areas of central necrosis were not treated with cytotoxicchemotherapy; however, if we had treated these mice withintravenous cisplatin or carboplatin, it is unclear whetherthese drugs could have reached the tumor parenchymawithout a robust vasculature and what the effect on overallsurvival would have been.

In addition, whether or not angiogenesis is an optimaltarget for therapy against solid tumors is another growingcontroversy. Recent studies in glioblastoma hypothesize thatvascular normalization improves survival through tumorperfusion [25]. Furthermore, another study of bevacizumab-treated rats bearing human glioblastoma multiforme tumors


demonstrated, “a strong and highly significant increase inthe number of tumor cells invading the normal brain” [26],suggesting a negative effect on tumor biology in that setting.

In 2011, the Food and Drug Administration revoked itsapproval of bevacizumab for therapy in metastatic breastcancer because it lacked efficacy and increased the riskfor lethal side effects. Some speculated that this review bythe FDA was also necessary due to bevacizumab’s extraor-dinarily high yearly cost without the possibility of achiev-ing monotherapeutic cure. For example, bevacizumab isindicated for use as combination therapy in metastatic col-orectal cancer, nonsquamous nonsmall cell lung cancer andmetastatic renal cell carcinoma and as a single agent forglioblastoma patients based on objective response rate, notsurvival [27]. Thus, combination regimens with angiogenesisinhibitors have substantially increased the expense of cancertreatment through drug costs and the costs associated withhospitalization for adverse drug responses [28]. A betterunderstanding of the tumor vasculature and its impact onthe biology of solid tumors and therapy is needed to addressthis controversial approach.

Future studies might explore the roles of combinations ofRGS5 with other RGS proteins in ovarian cancer. For exam-ple, on chromosome 1q23.3–1q31 there are 5 genes encodingRGS family members (RGS2, RGS4, RGS5, RGS8, andRGS16), and these have overlapping cellular functions [8]. Itis unclear whether combinations of these RGS proteinswould further modulate the aggressiveness of ovarian cancercells or tumors in mice. Since RGS proteins turn off signalingcascades from growth factors, future studies could also assessthe modulation of these with other proteins affecting G-protein-coupled receptors and receptor inhibitors. Much isleft to learn about RGS proteins and their role in tumorigen-esis and therapy.

Acknowledgments

The authors do not have any conflict of interests with thecontent of the paper. This work was supported in part bygrants from the Georgia Cancer Coalition and the NationalInstitutes of Health (1R15CA151006-01, American Recoveryand Reinvestment Act).

References

[1] J. Zhou, K. Moroi, M. Nishiyama et al., “Characterization ofRGS5 in regulation of G protein-coupled receptor signaling,”Life Sciences, vol. 68, no. 13, pp. 1457–1469, 2001.

[2] C. Chen, B. Zheng, J. Han, and S. C. Lin, “Characterizationof a novel mammalian RGS protein that binds to Gα proteinsand inhibits pheromone signaling in yeast,” The Journal ofBiological Chemistry, vol. 272, no. 13, pp. 8679–8685, 1997.

[3] N. Seki, S. Sugano, Y. Suzuki et al., “Isolation, tissue expres-sion, and chromosomal assignment of human RGS5, a novelG-protein signaling regulator gene,” Journal of Human Genet-ics, vol. 43, no. 3, pp. 202–205, 1998.

[4] B. Xiao, Y. Zhang, W. Q. Niu, P. J. Gao, and D. L. Zhu,“Haplotype-based association of regulator of G-protein sig-naling 5 gene polymorphisms with essential hypertension

and metabolic parameters in Chinese,” Clinical Chemistry andLaboratory Medicine, vol. 47, no. 12, pp. 1483–1488, 2009.

[5] Y. P. C. Chang, X. Liu, J. D. O. Kim et al., “Multiple genes foressential-hypertension susceptibility on chromosome 1q,”American Journal of Human Genetics, vol. 80, no. 2, pp. 253–264, 2007.

[6] M. U. Faruque, G. Chen, A. Doumatey et al., “Association ofATP1B1, RGS5 and SELE polymorphisms with hypertensionand blood pressure in African-Americans,” Journal of Hyper-tension, vol. 29, no. 10, pp. 1906–1912, 2011.

[7] H. Cho, C. Park, I. Y. Hwang et al., “Rgs5 targeting leadsto chronic low blood pressure and a lean body habitus,”Molecular and Cellular Biology, vol. 28, no. 8, pp. 2590–2597,2008.

[8] D. B. Campbell, L. A. Lange, T. Skelly, J. Lieberman, P. Levitt,and P. F. Sullivan, “Association of RGS2 and RGS5 vari-ants with schizophrenia symptom severity,” SchizophreniaResearch, vol. 101, no. 1–3, pp. 67–75, 2008.

[9] E. N. Smith, C. S. Bloss, J. A. Badner et al., “Genome-wideassociation study of bipolar disorder in European Americanand African American individuals,” Molecular Psychiatry, vol.14, no. 8, pp. 755–763, 2009.

[10] C. Bondjers, M. Kalen, M. Hellstrom et al., “Transcriptionprofiling of platelet-derived growth factor-B-deficient mouseembryos identifies RGS5 as a novel marker for pericytes andvascular smooth muscle cells,” American Journal of Pathology,vol. 162, no. 3, pp. 721–729, 2003.

[11] M. Berger, G. Bergers, B. Arnold, G. J. Hammerling, and R.Ganss, “Regulator of G-protein signaling-5 induction inpericytes coincides with active vessel remodeling during neo-vascularization,” Blood, vol. 105, no. 3, pp. 1094–1101, 2005.

[12] T. S. Mitchell, J. Bradley, G. S. Robinson, D. T. Shima, and Y.S. Ng, “RGS5 expression is a quantitative measure of pericytecoverage of blood vessels,” Angiogenesis, vol. 11, no. 2, pp. 141–151, 2008.

[13] M. Gerlinger, A. J. Rowan, S. Horswell et al., “Intratumorheterogeneity and branched evolution revealed by multiregionsequencing,” The New England Journal of Medicine, vol. 366,pp. 883–892, 2012.

[14] A. Silini, C. Ghilardi, S. Figini et al., “Regulator of G-proteinsignaling 5 (RGS5) protein: a novel marker of cancer vas-culature elicited and sustained by the tumor’s proangiogenicmicroenvironment,” Cellular and Molecular Life Sciences, vol.69, no. 7, pp. 1167–1178, 2012.

[15] I. Papandreou, R. A. Cairns, L. Fontana, A. L. Lim, and N.C. Denko, “HIF-1 mediates adaptation to hypoxia by activelydownregulating mitochondrial oxygen consumption,” CellMetabolism, vol. 3, no. 3, pp. 187–197, 2006.

[16] O. Warburg, “On respiratory impairment in cancer cells,”Science, vol. 124, no. 3215, pp. 269–270, 1956.

[17] Y. Jin, X. An, Z. Ye, B. Cully, J. Wu, and J. Li, “RGS5, a hypoxia-inducible apoptotic stimulator in endothelial cells,” TheJournal of Biological Chemistry, vol. 284, no. 35, pp. 23436–23443, 2009.

[18] R. A. Weinberg, Ed., The Biology of Cancer, Garland Science,Taylor & Francis Group, New York, NY, USA, 2007.

[19] S. M. Weis and D. A. Cheresh, “Pathophysiological conse-quences of VEGF-induced vascular permeability,” Nature, vol.437, no. 7058, pp. 497–504, 2005.

[20] J. Hamzah, M. Jugold, F. Kiessling et al., “Vascular nor-malization in Rgs5-deficient tumours promotes immunedestruction,” Nature, vol. 453, no. 7193, pp. 410–414, 2008.


[21] M. Manzur, J. Hamzah, and R. Ganss, “Modulation of the“blood-tumor” barrier improves immunotherapy,” Cell Cycle,vol. 7, no. 16, pp. 2452–2455, 2008.

[22] M. Manzur, J. Hamzah, and R. Ganss, “Modulation of Gprotein signaling normalizes tumor vessels,” Cancer Research,vol. 69, no. 2, pp. 396–399, 2009.

[23] S. B. Hooks, P. Callihan, M. K. Altman, J. H. Hurst, M. W. Ali,and M. M. Murph, “Regulators of G-Protein signaling RGS10and RGS17 regulate chemoresistance in ovarian cancer cells,”Molecular Cancer, vol. 9, article 289, 2010.

[24] W. Jia, J. O. Eneh, S. Ratnaparkhe, M. K. Altman, and M. M.Murph, “MicroRNA-30c-2∗ expressed in ovarian cancercells suppresses growth factor-induced cellular proliferationand downregulates the oncogene BCL9,” Molecular CancerResearch, vol. 9, pp. 1732–1745, 2011.

[25] G. Sorensen, K. Emblem, P. Polaskova et al., “Increased sur-vival of glioblastoma patients who respond to antiangiogenictherapy with elevated blood perfusion,” Cancer Research, vol.72, no. 2, pp. 402–407, 2012.

[26] O. Keunen, M. Johansson, A. Oudin et al., “Anti-VEGF treat-ment reduces blood supply and increases tumor cell invasionin glioblastoma,” Proceedings of the National Academy ofSciences of the United States of America, vol. 108, no. 9, pp.3749–3754, 2011.

[27] I. Genentech, “U.S. BL 125085 Supplement: Bevacizumab,” inSouth San Francisco, I. Genentech, Ed., pp. 1–23, 2011.

[28] D. B. Jackson and A. K. Sood, “Personalized cancer medi-cine—advances and socio-economic challenges,” NatureReviews Clinical Oncology, vol. 8, pp. 735–741, 2011.

Submit your manuscripts athttp://www.hindawi.com

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation http://www.hindawi.com

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttp://www.hindawi.com

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research


International Journal of

Microbiology

RegulatorofG-ProteinSignaling5ReducesHeyA8Ovarian...

Documents

Transcript of RegulatorofG-ProteinSignaling5ReducesHeyA8Ovarian...