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Inhibition of tumor promoting signals by activation of SSTR2 and opioid receptors in human breast cancer… Kharmate, Geetanjali; Rajput, Padmesh S; Lin, Yu-Chen; Kumar, Ujendra Sep 23, 2013

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PRIMARY RESEARCH Open AccessInhibition of tumor promoting signals byactivation of SSTR2 and opioid receptors inhuman breast cancer cellsGeetanjali Kharmate, Padmesh S Rajput, Yu-Chen Lin and Ujendra Kumar*AbstractBackground: Somatostatin receptors (SSTRs) and opioid receptors (ORs) belong to the superfamily of G-proteincoupled receptors and function as negative regulators of cell proliferation in breast cancer. In the present study, wedetermined the changes in SSTR subtype 2 (SSTR2) and μ, δ and κ-ORs expression, signaling cascades andapoptosis in three different breast cancer cells namely MCF-7, MDA-MB231 and T47D.Methods: Immunocytochemistry and western blot analysis were employed to study the colocalization and changesin MAPKs (ERK1/2 and p38), cell survival pathway (PI3K/AKT) and tumor suppressor proteins (PTEN and p53) inbreast cancer cell lines. The nature of cell death upon activation of SSTR2 or OR was analysed using flow cytometryanalysis.Results: The activation of SSTR2 and ORs modulate MAPKs (ERK1/2 and p38) in cell dependent and possiblyestrogen receptor (ER) dependent manner. The activation of tumor suppressor proteins phosphatase and tensinhomolog (PTEN) and p53 antagonized the PI3K/AKT cell survival pathway. Flow cytometry analyses reveal increasednecrosis as opposed to apoptosis in MCF-7 and T47D cells when compared to ER negative MDA-MB231 cells.Furthermore, receptor and agonist dependent expression of ORs in SSTR2 immunoprecipitate suggest that SSTR2and ORs might interact as heterodimers and inhibit epidermal growth factor receptor phosphorylation.Conclusion: Taken together, findings indicate a new role for SSTR2/ORs in modulation of signaling pathwaysinvolved in cancer progression and provide novel therapeutic approaches in breast cancer treatment.Keywords: Breast cancer, Mitogen activated protein kinases, Opioid receptors and somatostatin receptor-2BackgroundSomatostatin (SST) is a multifunctional growth hormoneinhibitory neuropeptide, regulating different arrays offunctions in the brain, endocrine and exocrine tissues.One of the prominent functions of SST is the negativeregulation of cell proliferation in normal as well aspathological conditions including pituitary, pancreaticand breast tumors [1-5]. The anti-proliferative effects ofSST occur indirectly through the inhibition of growthfactors such as insulin growth factor-1 and epidermalgrowth factor and angiogenesis [6-9]. The direct anti-proliferative effect of SST is by binding to seven trans-membrane G-protein coupled receptors (GPCRs) namelysomatostatin receptors 1–5 (SSTR1-5). This direct effectof SST is either cytostatic (cell cycle arrest) or cytotoxic(apoptosis) [2,10-12]. SSTR1-5 subtypes are variablyexpressed in various human tumors including breastcancer tissues and cells [4,12,13]. We have previouslyshown a significant correlation between mRNA and pro-tein expression of SSTRs with histological tumormarkers as well as with expression levels of estrogen re-ceptors (ER) and progesterone receptors [4]. These re-sults indicate that the presence of hormone receptorsmight play crucial role on SSTR effectiveness in breastcancer.SST and SSTRs are highly expressed in breast cancercells as well as autopsied breast tissue. However, SSTR2is the prominent receptor subtype expressed ubiqui-tously and abundantly in breast tumor tissues and* Correspondence: ujkumar@mail.ubc.caFaculty of Pharmaceutical Sciences, The University of British Columbia,Vancouver, BC V6T1Z3, Canada© 2013 Kharmate et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Kharmate et al. Cancer Cell International 2013, 13:93http://www.cancerci.com/content/13/1/93cancer cells. Vikic-Topic et al., described that SSTR2transcript is predominantly expressed in all breast tissuesamples and followed by SSTR1, SSTR3 and SSTR4 [14].Additionally, Pfeiffer et al., reported that SSTR2 andSSTR5 as the predominant subtypes expressed in pri-mary breast tumors [15]. Moreover, MCF-7 cells withover-expression of SSTR2 display diminished rate of cellproliferation [16]. SSTR2 exerts its anti-proliferative ef-fect by either activating or suppressing various signaltransduction pathways including mitogen activated pro-tein kinases (MAPK), phosphatidylinositol-3-protein kin-ase (PI3K)/AKT, phosphotyrosine phosphatases such asPTP1 and PTP2 [17-21]. The activation of multiple sig-naling pathways consequently leads to the induction ofcell cycle arrest via activation of cyclin dependent kinaseinhibitor (p27Kip1) as well as apoptosis. SSTRs positivetumors are less malignant with higher survival ratewhereas the lack of SSTRs expression has also been asso-ciated with poorly differentiated and invasive tumor [22].This could partly be attributed to the over-expression ofepidermal growth factor receptors (EGFRs) in breast can-cer that are associated with poor prognosis and patientsurvival rate [13,23,24]. Importantly, in MCF-7 cells, over-expression of SSTR2 resulted in suppression of EGFRexpression [16]. Further in support, our recent studieshave also described attenuation of EGFR phosphorylationand suppression of tumor promoting signals in breastcancer cells as well as in human embryonic kidney (HEK)-293 cells transfected with SSTR1 or SSTR5 [13,20,21].Like SSTRs, opioid receptor (ORs), namely μ, δ and κare also the members of GPCR family. SSTRs and ORsshare >40% structural homology and are well expressedin various breast cancer cells as well as in solid breasttumor tissues [25,26]. ORs are well characterized fortheir analgesic role and like SSTRs have recently beenreported as negative regulators of cell growth in varioustumors including, prostrate, lung, kidneys and breastcancer [27-29]. It has been previously shown that >50%of invasive ductal carcinomas are positive for opioidpeptide like immunoreactivity [30]. Furthermore, humanadrenocarcinoma and breast cancer cell lines exhibitedthe binding site for opioids. The opioid agonist [D-Ala2,D-Leu2] enkephalin (DADLE), displayed inhibition of cellproliferation in a concentration dependent manner andwas reversed in the presence of antagonist naloxone[29,31,32].SSTR and OR subtypes constitute functional heteromericcomplexes within same sub-family and other GPCRs andmodulate receptor trafficking and signaling properties[25,33-37]. SSTR5 and dopamine receptor 2 (D2R) hetero-dimerization synergistically control the hyper-secretion ofgrowth hormone and prolactin in pituitary adenomas[38,39]. These observations have led to the application ofnew chimeric molecules of D2R and SSTR5 for thetreatment of pituitary tumor acromegaly [1,38-40]. Further-more, SSTR2 interfere with PI3K signaling via disruption ofthe SSTR2/p85 subunit complex consequently inhibitingthe cell proliferation and tumor growth [17]. HEK-293 cellsco-transfected with SSTR2 and μOR constituted stableheterodimers thereby regulating the receptor phosphoryl-ation, internalization and desensitization [15]. WhetherSSTR2 functionally interacts with ORs in breast cancer cellsexpressing these receptors endogenously and function insimilar manner as described in heterologous system islargely elusive. We hypothesize that the simultaneous acti-vation of SSTR2 and ORs may exert pronounced anti-proliferative effect via changes in signaling pathways inbreast cancer cells. Multiple studies have documented thatestrogen upregulated the expression of SSTR2 mRNA andprotein via ER in T47D and ZR75-1 (ER + ve) breast cancercells. These findings may anticipate the role of SSTR2 inER responsiveness of breast cancer [41-43]. However,SSTR2 and OR subtypes mediated effect on signaling path-ways in part are dependent on the presence of ER in breastcancer cells is still elusive. In the present study, we focus todetermine the expression of SSTR2 and ORs and thechanges in receptor expression and signaling pathwaysupon treatment with receptor specific agonist in humanbreast cancer cell lines; MCF-7 (ER + ve), MDA-MB231(ER-ve), and T47D (ER + ve).Materials and methodsChemicals and reagentsSSTR2 specific non-peptide agonist L-779,976 was pro-vided by Dr. Rohrer from Merck & Co. Specific agonistsfor μOR (DAMGO), δOR ([D-Ala2]- Deltorphin II) andκOR (±)-U-50488 hydrochloride) were purchased fromTocris Biosciences (Ellisville, MO). Polyclonal rabbitanti-SSTR2 antibody was developed in our laboratory[44,45]. The antibodies against ORs (μ, δ and κ), phos-phorylated and total-EGFR were obtained from SantaCruz Biotechnology (Santa Cruz, CA). Rabbit polyclonalantibodies against phosphorylated and total-ERK1/2,p38, PI3K, AKT, PTEN and p53 were purchased fromCell Signaling Technology (Mississauga, ON). Goat anti-rabbit or donkey anti-goat Alexa Fluor-488 and AlexaFluor-594 were purchased from Invitrogen (Burlington,ON). Annexin-APC V was obtained from BD Biosciences(Mississauga, ON). All experiments were performed incompliance with Office of Research and Biosafety Com-mittee guidelines at the University of British Columbia.Cell lines and cultureHuman breast cancer cell lines, MCF-7 and T47D (ER + ve)were maintained in RPMI 1640 medium supplemented with10% (v/v) fetal bovine serum (FBS) and 1% antibiotic (peni-cillin/streptomycin) at 37°C, 5% CO2 as previously de-scribed [4,13]. MDA-MB231 (ER-ve) cells were maintainedKharmate et al. Cancer Cell International 2013, 13:93 Page 2 of 13http://www.cancerci.com/content/13/1/93in Leibovitz’s L-15 medium supplemented with 10% (v/v)FBS and 1% antibiotic (penicillin/streptomycin) at 37°C ina CO2 free atmosphere.Indirect immunofluorescence immunocytochemistryBreast cancer cells were processed for indirect immuno-fluorescence immunocytochemistry as described earlier[13,21]. Briefly, cells were washed and fixed with 4%paraformaldehyde and followed by treatment with TritonX-100 for 10 min at room temperature. The cells wereincubated with anti-goat μ, δ, and κ -OR (1:300) primaryantibodies overnight at 4°C and followed by incubation ingoat-anti-rabbit Alexa 488 (1:700) conjugated secondaryantibodies for 1 h at room temperature for final color de-velopment. The cells were viewed and photographed on aLeica DMLB microscope attached to a Retiga 2000R cam-era. The specificity of immunoreactivity was determinedin absence of primary antibodies or in presence of pre-immune serum as described earlier [45].Western blot analysesMembrane extracts as well as whole cell lysate preparedfrom control and treated cells were fractionated on SDS-PAGE as described previously [13,21]. To determine thereceptor expression in membrane extracts, blots wereincubated with primary antibodies against SSTR2 (1:400)and μ, δ and κ-OR (1:500). The status of signaling mole-cules were examined by incubating the immunoblotswith antibodies against phosphorylated and/or totalextracellular regulated protein kinase 1/2 (ERK1/2), p38,PI3K, AKT, PTEN and p53 (1:1000). Membrane wasincubated with peroxidase conjugated secondary anti-bodies respectively. The bands were detected using che-miluminescence in accordance to the manufacturer’sinstructions (Amersham Biosciences). Images were cap-tured using the Alpha Innotech FluorChem 8800 gel boximager and FluorChem software was used to quantifythe blots. Tubulin was used as the loading control.Co-Immunoprecipitation (Co-IP)Breast cancer cells were treated with specific agonists forSSTR2, L-779,976 (10 nM) and μOR (DAMGO; 1 μM),δOR (Deltorphin-II; 1 μM), or κOR (U50488HCl; 1 μM)alone or in combination for 15 min at 37°C. 200 μg of totalmembrane protein was solubilized in 1 ml of radio-immune precipitation assay (RIPA) buffer (150 mM NaCl,50 mM Tris–HCl, 1% Nonidet P-40, 0.1% SDS, 0.5% so-dium deoxycholate, pH 8.0) for 1 h at 4°C as describedearlier [20,21]. Samples were incubated with anti-SSTR2antibody (1:200) for immunoprecipitation and purifiedwith protein A/G-agarose beads overnight at 4°C. Purifiedproteins were subjected to 7% SDS-PAGE and probed forthe expression of ORS using anti-μ, δ and κ-OR antibodies(1:500) respectively as described previously [20,21].Flow cytometry analysis for apoptosis/necrosisCells were harvested and treated with L-779,976,DAMGO, Deltorphin-II, or U50488HCl alone and/or incombination for 30 min at 37°C. Annexin V-APC stain-ing was performed according to the manufacturer’sprotocol (BD Biosciences). Cells were washed twice withcold PBS and resuspended in 1X binding buffer (0.1 MHepes, pH 7.4, 1.4 M NaCl, 25 mM CaCl2) at a cell dens-ity of 1 × 105cells/ml. Annexin V-APC and propidium iod-ide were added to the cell suspension for 15 min. Prior toflow cytometry, 400 μl of 1X binding buffer was added andcells were analyzed using FACS Calibur flow cytometer(Becton Dickinson, San Jose, CA). A minimum of 10,000events were recorded for each sample. Cells positive forAnnexin V-APC were identified as apoptotic whereas cellspositive for both Annexin V-APC and propidium iodidewere characterized as necrotic.Statistical analysisThe changes in the expression of proteins were quantifiedusing ANOVA and post hoc Dunnett’s or Bonferroni’stests. Statistical analysis was performed using GraphPadPrism 4.0 to determine the significant changes. Significantstatistical differences were taken at *p < 0.05. Results arepresented as mean ± SEM from three independent experi-ments (n = 3).ResultsComparative distribution of SSTR2 and ORs in MCF-7,MDA-MB231 and T47D cellsSSTR2 and ORs expression at the cellular levels and inmembrane fractions was accomplished by immunofluores-cence immunocytochemistry and western blot analysisrespectively. MCF-7 cells, displayed strong membrane ex-pression of SSTR2 and μ, δ, κ-ORs whereas intracellularexpression of SSTR2 was weak than the ORs (Figure 1A).In MDA-MB231 cells, SSTR2 and μ, δ and κ-ORs like im-munoreactivity was observed at the cell surface with adominant expression of δ and κOR, whereas, the receptorsexpression in the cytoplasmic compartment was compar-able (Figure 1A). In contrast, T47D cells displayed strongexpression of SSTR2 and μ, δ, κ-ORs at the cell surface aswell as intracellularly (Figure 1A).To support the cellular distribution by immunocyto-chemistry receptor like immunoreactivity was also con-firmed using Western blot analysis. As shown in Figure 1B,SSTR2 was well expressed at the expected molecular sizeof ~57 kDa with relatively higher expression in MDA-MB231 cells in comparison to MCF-7 and T47D. Theexpression level of SSTR2 was comparatively less in T47Dcells than MCF-7 and MDA-MB231 cells. In contrast, μOR(~50 kDa) and κOR (~46 kDa) were well expressed inMCF-7 cells. Conversely, the expression of κOR in mem-brane extract prepared from MDA-MB231 and T47D cellsKharmate et al. Cancer Cell International 2013, 13:93 Page 3 of 13http://www.cancerci.com/content/13/1/93Figure 1 Differential expression of SSTR2 and μ, δ, κ-ORs in human breast cancer cells. (A) Indirect immunofluorescence staining showingmembrane (non-permeabilized, NP) and intracellular (permeabilized, P) expression of SSTR2 and μ, δ, κ-ORs in breast cancer cells. In therepresentative panels receptor expression at the cell surface is indicated by arrows whereas arrowheads indicate intracellular expression.(B) Cell membrane extracts obtained from MCF-7, MDA-MB231 and T47D breast cancer cells were subjected to Western blot analysis and probedwith specific primary antibodies to detect the receptor expression. The immunoblots show differential expression of SSTR2 (~57 kDa) and μ(~50 kDa), δ (~48 kDa), κ (~46 kDa)-ORs in a cell specific manner. Histogram represents densitometric analysis of the receptor expressionnormalized by using tubulin as loading control. Results are expressed as mean ± SEM (n = 3). **p < 0.01, MCF-7 vs. MDA-MB231 or T47D cells;##p < 0.01; MDA-MB231 vs. T47D. Scale bar 10 μm.Kharmate et al. Cancer Cell International 2013, 13:93 Page 4 of 13http://www.cancerci.com/content/13/1/93was relatively weak (Figure 1B). The expression of δOR(~48 kDa) was comparable in all three cell lines. Theseobservations indicate cells-specific expression of SSTR2and ORs.SSTR2 and ORs modulate MAPKs in a cell-specific mannerWe next determined whether receptor activation regu-late MAPKs (ERK1/2 and p38) in breast cancer cells. InMCF-7 cells, L-779,976, DAMGO and Deltorphin-IIalone inhibit the phosphorylation of ERK1/2 (p-ERK1/2)(Figure 2A). Furthermore, L-779,976 in the presence ofDAMGO or Deltorphin-II displayed p-ERK1/2 com-parable to control. In contrast, U50488HCL, alone orin combination with L-779,976 significantly elevated p-ERK1/2 in MCF-7 cells (Figure 2A). In MDA-MB231cells, L-779,976 alone had no significant effect on p-ERK1/2. DAMGO alone induced p-ERK1/2 whereas incombined treatment with L-779,976 decreased p-ERK1/2.In contrast, Deltorphin-II alone had no significant effecton p-ERK1/2 whereas in combination with L-779,976enhanced the levels of p-ERK1/2 in MDA-MB231 cells(Figure 2A). Furthermore, in MDA-MB231 cells the acti-vation of κOR enhanced p-ERK1/2 which was significantlydecreased to the control level upon combined treatmentwith SSTR2 agonist L-779,976. In T47D cells, L-779,976maintained p-ERK1/2 comparable to control. The acti-vation of μOR displayed comparable p-ERK1/2 but signi-ficantly increased in combination with L-779,976. Thestatus of p-ERK1/2 was not changed upon activation ofδOR alone whereas expression level was diminishedsignificantly in presence of L-779,976 and Deltorphin-II.Activation of κOR alone had no effect on p-ERK1/2 how-ever simultaneous activation of SSTR2/κOR inhibited p-ERK1/2 when compared to control (Figure 2B).In addition to ERK1/2, in tumor cells, p38 is a crucialmediator of apoptosis, cell-cycle arrest, cell differenti-ation and tumor suppression [46,47]. The pro- and/oranti-apoptotic role of p38 is attributed to the cell-typeand stimuli. Of note, p38 phosphorylation remainedcomparable to control in MCF-7 cells upon treatmentswith SSTR2 and ORs agonists alone or in combination(Figure 2B). Unlike MCF-7 cells, the p-p38 was notdetected in MDA-MB231 and T47D cells across all indi-cated treatments. Collectively, these data suggest thatbasal expression of p-p38 is relatively higher in MCF-7cell in comparison to MDA-MB231 or T47D cells with-out any discernible changes upon SSTR2 and ORsactivation.Figure 2 MAPKs (ERK1/2 and p38) are modulated in a receptor and cell-specific manner. Whole cell lysates obtained from MCF-7,MDA-MB231 and T47D cells following treatment with SSTR2 and ORs agonists alone and/or in combination were subjected to western blotanalysis and probed for phospho-and total ERK1/2 and p38 (1:1000). (A) Immunoblots illustrating agonist mediated changes in phosphorylatedERK1/2 in cell-specific manner. (B) Immunoblots displaying changes in the phosphorylation of p38 upon specific agonist treatments in breastcancer cells. SSTR2 and ORs activation inhibited p38 phosphorylation upon indicated treatment. Histograms depict changes in the expressionlevels of ERK1/2 using densitometric analysis. The data presented here is a representation mean ± SEM of three independent experiments.Significant difference was considered at *p < 0.05 vs. control.Kharmate et al. Cancer Cell International 2013, 13:93 Page 5 of 13http://www.cancerci.com/content/13/1/93SSTR2 and ORs maintained basal activation of PI3K/AKTThe aberrant activation and/or mutations in PI3K areassociated with tumor growth and failure of hormonaltherapy [48-50]. Accordingly, we next determined thestatus of phosphorylated PI3K/AKT in control and cellstreated with SSTR2 and ORs specific agonists. No sig-nificant changes in the status of PI3K phosphorylationwas observed in all cell lines upon indicated treatments,albeit lower levels of p-PI3K when compared to control(Figure 3A). Furthermore, irrespective to treatment andcell lines the status of AKT phosphorylation was withoutany significant changes across all treatments in all threebreast cancer cells as indicated (Figure 3A).SSTR2 and ORs modulate tumor suppressor proteinsPTEN and p53 in cell dependent mannerTumor suppressor proteins, PTEN and p53 serve asnegative regulators of cell proliferation in breast cancerand mutations in PTEN and p53 are often associatedwith the activation of AKT cell survival pathway [49]. InMCF-7 cells, L-779,976 alone significantly enhanced thephosphorylation of PTEN whereas DAMGO alone or incombination with L-779,976 had no effect on p-PTEN(Figure 4A). Upon treatment with Deltorphin-II aloneMCF-7 cells displayed elevated p-PTEN however co-activation of SSTR2 and δOR resulted in significant in-hibition of p-PTEN comparable to control. Furthermore,in presence of U50488HCl alone or in combination withL-779,976 the status of p-PTEN was without any signifi-cant effect in MCF-7 cells (Figure 4A). Conversely, inMDA-MB231 cells there was no discernible change in p-PTEN across all the treatments as indicated. In T47Dcells, L-779,976 alone and in combination with DAMGOsignificantly increased p-PTEN whereas DAMGO aloneenhanced PTEN phosphorylation insignificantly differentthan the control. Furthermore, Deltorphin-II alone andin combination with L-779,976 displayed significant in-crease in the p-PTEN. In T47D cells, κOR agonistresulted in significant activation of PTEN whereas sucheffect was not significantly different from control uponsynergistic activation of SSTR2 and κOR (Figure 4A).As shown in Figure 4A, MCF-7 displayed no discerniblechanges in the expression of p53 across all the treatments.In contrast, in MDA-MB231 cells, L-779,976 alone waswithout any effect on p53 expression. DAMGO aloneenhanced the expression of p53 significantly whereasin combination with L-779,976, p53 level was similar tothe control. Deltorphin-II alone and in combination withL-799,796 significantly elevated the levels of p53. Further-more, p53 expression was unchanged upon activation ofFigure 3 Activation of SSTR2 and ORs modulate PI3K/AKT cell survival pathway. Representative western blots illustrating the changes inthe expression of PI3K (A) and AKT (B) in MCF-7, MDA-MB231 and T47D cells. Activation of SSTR2 and ORs individually or simultaneouslyexhibited no discernible changes in phosphorylation of PI3K and AKT, thus maintaining the status of PI3K/AKT at control levels. Bar graphsrepresent the densitometric analysis of PI3K/AKT expression in different tumor cells. The data presented are a representation oftriplicate experiments.Kharmate et al. Cancer Cell International 2013, 13:93 Page 6 of 13http://www.cancerci.com/content/13/1/93κOR alone whereas the combined treatment displayed sig-nificant increase in p53 expression in MDA-MB231 cells(Figure 4B). In T47D cells, L-779,976 alone and in com-bination with either DAMGO or Deltorphin-II exhibitedno change in p53 expression. In contrast, DAMGO andDeltorphin-II alone significantly enhanced the p53 ex-pression in comparison to the control. U50488HCl alonedid not affect p53 expression however, in combinationwith L-779,976, the p53 levels significantly decreased(Figure 4B). Taken together these observations suggestthat SSTR2 and ORs upregulated the PTEN and p53expression in a receptor and cell dependent manner.SSTR2 and ORs induced early apoptosis and predominantcytostatic effect in cell dependent mannerThe anti-proliferative effect of SSTR2 is mediated via twodifferent mechanisms; cytostatic and cytotoxic. Accord-ingly, applying flow cytometry, we investigated the cellularresponse upon receptor specific agonist treatments. Theresults indicated apoptotic (9.05%) and necrotic (19%)cells in control MCF-7 cells whereas upon single treat-ment with L-779,976 number of the cells displaying apop-tosis and necrosis was enhanced (14.5% and 23.4%respectively) (Figure 5A). In presence of DAMGO aloneor with L-779,976 cells displayed higher extent of necrosis(25% and 27.8% respectively) without any significantchanges in apoptosis (11.8% and 12%). Furthermore, thenumber of cells displaying apoptosis and necrosis wasenhanced upon combined treatment with L-779,976 andDeltorphin-II (12% and 26.7%) than Deltorphin-II alone(9 and 23.6%, respectively). In contrast, the activationof κOR with U50488HCl alone caused higher necrosis(29.9%), but was decreased to 23.4% upon combined treat-ment with L-779,976 whereas cells entering apoptosis waslimited to ~10% (Figure 5A).As illustrated in Figure 5B, MDA-MB231 cells upontreatments with SSTR2 or μ, δ and κ-ORs agonists aloneor in combination displayed <4% of apoptotic cell popula-tion. Conversely, cells undergoing necrosis increased(12.4%) upon treatment with L-779,976 alone in compari-son to the control (10.8%). DAMGO alone enhanced thenecrotic cells (13.5%) whereas upon combined treatmentwith L-779,976, the number of cells going through necrosiswere enhanced to 15.3% in MDA-MB231 cells (Figure 5B).Deltorphin-II alone enhanced the necrosis to 16%; how-ever, combined treatment with L-779,976 decreased necro-sis to 12.9%, similar to that seen with L-779,976 alone(12.4%). Similarly, treatment with U50488HCl led to 14.7%necrosis and was decreased to 11.6% upon combined treat-ment with L-779,976 (Figure 5B).In basal condition T47D cells number of the cellsgoing through apoptosis and necrosis cells were 3.65%Figure 4 Tumor suppressor proteins PTEN and p53 are modulated in cell and receptor specific manner in tumor cells. Representativeimmunoblots showing the effect of SSTR2 or ORs activation on the expression of PTEN (A) and p53 (B) in MCF-7, MDA-MB231 and T47D cellsrespectively. (A) The expression of PTEN is enhanced in MCF-7 as well as T47D cells; however the MDA-MB231 cells displayed no discerniblechanges in the levels of PTEN. (B) The expression of p53 was in a receptor and cell-specific manner. Note the comparable changes in PTEN andp53 expression in MCF-7 and T47D cells. Histograms illustrate densitometric analysis of PTEN and p53 levels using tubulin as the loading control.The results presented here are representation of mean ± SEM, n = 3. Significant difference was considered at *p < 0.05 vs. control.Kharmate et al. Cancer Cell International 2013, 13:93 Page 7 of 13http://www.cancerci.com/content/13/1/93Figure 5 (See legend on next page.)Kharmate et al. Cancer Cell International 2013, 13:93 Page 8 of 13http://www.cancerci.com/content/13/1/93and 22% respectively (Figure 5C). The treatment with L-779,976 enhanced the apoptosis (11.5%), however necro-sis was elevated (34%) when compared to the control.Interestingly, upon treatments with DAMGO, alone orin combination with L-779,976, the population of nec-rotic cells (36.9% and 38%) was enhanced significantly ascompared to the early apoptotic cells (8.79% and 7.37%).Deltorphin-II alone or in combination with L-779,976displayed higher necrosis (40.7% and 36.7%) than apop-tosis (5.7% and 7.62%) (Figure 5C). Of note, combinedtreatment of U50488HCl and L-779,976 markedly in-creased the necrosis (52.4%) whereas exhibited only4.2% apoptosis (Figure 5C). These data indicate that ER+ ve cells (MCF-7 and T47D) are more susceptible tonecrosis upon simultaneous activation of SSTR2 andORs than ER-ve cells (MDA-MB231).Expression of ORs in SSTR2 immunoprecipitate is cell andreceptor-specific in tumor cellsPrevious studies using HEK-293 cells have demonstratedthat ORs and SSTR2 constituted functional heterodimerswith enhanced signaling properties [15]. To decipher theunderlying mechanism by which SSTR2/ORs functionallyinteract in tumor cells expressing these receptors endogen-ously, SSTR2 immunoprecipitate was processed for the ex-pression of ORs by CO-IP. In MCF-7 cells, SSTR2/μORexists in a heteromeric complex at the expected molecularsize of ~120 kDa in control as well as upon activation ofboth receptors independently (Figure 6A). However, withcombined treatment of L-779,976 +DAMGO, there wassignificant loss in SSTR2/μOR complex (Figure 6A). InMDA-MB231 cells, complex formation of SSTR2/μORwas weak in basal state. The SSTR2/ μOR complex wasenhanced significantly upon activation of SSTR2 and thereceptor heterodimerization was lost upon treatmentswith DAMGO alone or in combination with L-779,876(Figure 6A). SSTR2 immunoprecipitate prepared fromcontrol T47D cells was devoid of μOR expression. How-ever, upon treatments with L-779,876 or DAMGO alone orin combination, SSTR2 immunoprecipitate displayed μORexpression at the expected size of ~120 kDa (Figure 6A).In comparison to control, the heterodimerizationbetween SSTR2/δOR strengthened upon treatments withspecific agonists in MCF-7 cells (Figure 6B). In contrast,MDA-MB231 cells in basal condition displayed strongSSTR2/δOR complex formation and led to the disso-ciation upon agonists treatment (Figure 6B). In T47Dcells, no interaction between SSTR2/δOR in basal aswell as upon treatment with L-779,976 was seen. How-ever, upon treatments with Deltorphin-II alone or incombination with L-779,976, cells exhibited significantexpression of δOR in SSTR2 immunoprecipitate.SSTR2 immunoprecipitate prepared from control andU50488HCl treated MCF-7 cells were devoid of κOR ex-pression (Figure 6C). Whereas cells treated with specificagonists for SSTR2 alone or in combination with κORinduced the complex formation between SSTR2/κOR(Figure 6C). In MDA-MB231 cells, activation of SSTR2alone displayed increased complex formation betweenSSTR2/κOR however, upon treatments with U50488HClalone or in combination with L-779,976; the expressionof κOR in SSTR2 immunoprecipitate was comparable tocontrol (Figure 6C). Conversely, T47D cells, exhibited com-parable expression of κOR in SSTR2 immunoprecipitate incontrol as well as upon treatment with SSTR2 agonist.However in comparison SSTR2/κOR complex formationincreased upon treatment with κOR agonist alone and incombination with SSTR2 agonist (Figure 6C). These resultsstrengthen the concept of ligand, cell dependency in pos-sible heterodimerization between SSTR2 and ORs respect-ively. The specificity of immunoreactivity was confirmed inabsence of primary antibodies and incubation with second-ary antibodies alone as previously described [20,21].EGFR phosphorylation in breast cancer cells is abolishedin presence of synergistic activation of SSTR2 and ORsThe observations that breast tumor progression associatedwith EGFR over-expression, phosphorylation and homo-and/or heterodimerization suggested a crucial role ofEGFR in breast cancer [13,20,21,51]. In order to identifythe possible link between EGFR and SSTR2/ORs we deter-mined the effect of SSTR2 and ORs activation on EGFRphosphorylation. In MCF-7 cells, SSTR2 agonist L-779,976 alone or in combination with ORs agonists decreasedthe phosphorylation of EGFR although not significantlydifferent than the control (Figure 6D). Interestingly,MDA-MB231 and T47D cells displayed no EGFR phos-phorylation across all treatments (Figure 6D). These dataindicate that SSTR2 and ORs together maintained EGFRphosphorylation comparable to control in MCF-7 cells.The inhibition of EGFR phosphorylation is crucial inbreast cancer, however, whether the changes seen in thedownstream signaling pathways are in-part due to the lackof EGFR phosphorylation needs further investigation.(See figure on previous page.)Figure 5 Representative flow cytometry analysis displaying apoptosis and necrosis after exposure to SSTR2 and OR agonist in breastcancer cells. Cells were harvested and treated with SSTR2 or ORs specific agonists alone and in combination for 30 min at 37°C as described inMaterial and Method section. The percentage of apoptosis (lower right quadrant) and necrosis (upper right quadrant) was evaluated in MCF-7(A), MDA-MB231 (B) and T47D (C) cells upon treatment as indicated. Flow cytometry profile illustrating APC-Annexin V staining on X-axis whereasPI staining on Y-axis. A minimum of 10,000 events were recorded for each sample.Kharmate et al. Cancer Cell International 2013, 13:93 Page 9 of 13http://www.cancerci.com/content/13/1/93DiscussionIn the present study we unfold the effects of activation ofSSTR2 and ORs alone or in combination on signaling cas-cades and cell proliferation in human breast cancer cellsincluding MCF-7, MDA-MB231 and T47D. The activationof SSTR2 and ORs modulate the MAPK pathway and in-hibit the cell survival PI3K/AKT signaling molecules andthereby enhancing the expression of tumor suppressorproteins PTEN/p53 in cell and possibly ER dependentmanner. These changes in modulation of downstream sig-naling were corroborated using flow cytometry describingthe nature of cell death which showed eminent necrosisindicating the anti-proliferative effect of SSTR2 and ORsin breast cancer cells. In addition, our results describe re-ceptor and agonist dependent expression of ORs in SSTR2immunoprecipitate suggesting that SSTR2 and ORs mightinteract as heterodimers. In tumor cells, activation ofSSTR2 and ORs inhibit the phosphorylation of EGFR.Although, there is growing understanding for the synergis-tic effect of many GPCRs in cells transfected with one ormore receptor, this is the first comprehensive descriptionof SSTR2 and ORs in tumor cells expressing both receptorsubtypes endogenously. Our results demonstrate agonistdependent internalization of SSTR2 and ORs in receptorand cell-specific manner. The activation of SSTR2 andORs modulate MAPKs (ERK1/2 and p38) and the expres-sion of tumor suppressor proteins, PTEN and p53 re-sulting in the suppression of PI3K/AKT pathway. Wefurther correlated the effects of SSTR2/ORs mediatedsignaling with the functional consequences by measuringthe apoptosis and necrosis using flow cytometry. The re-sults described here also revealed heterodimerization be-tween SSTR2/OR and inhibition of EGFR phosphorylation.Our findings for the first time highlight the molecularmechanisms for the role of SSTR2 and ORs mediatedantagonism of tumorigenic signaling pathways in humanbreast cancer cells in receptor dependent manner.The co-expression of given receptors is a pre-requisitefor heterodimerization and this was further supportedby expression of ORs in SSTR2 immunoprecipitate.Figure 6 Agonist dependent complex formation between SSTR2/ORs and inhibition of EGFR phosphorylation. Co-immunoprecipitationshowing the expression of μOR (A), δOR (B) and κOR (C) in SSTR2 immunoprecipitate obtained from MCF-7, MDA-MB231 and T47D cellsfollowing indicated treatment. The agonist-induced heterodimerization between SSTR2 and μOR, δOR or κOR is receptor and cell-specific. (D)Western blot showing SSTR2 and ORs mediated inhibition of EGFR phosphorylation in breast cancer cells. MCF-7 cells displayed EGFRphosphorylation comparable to control without any discernible difference upon indicated treatment. Note the lack of EGFR phosphorylation inMDA-MB231 despite basal EGFR expression whereas T47D cells were devoid of EFGR expression and phosphorylation. Tubulin was used as aloading control.Kharmate et al. Cancer Cell International 2013, 13:93 Page 10 of 13http://www.cancerci.com/content/13/1/93Consistent with previous studies in heterologous system,our results showed that SSTR2 exist as pre-formedheterodimers with μ, δ and κ-ORs in breast cancer cells inreceptor and cell-specific manner [15]. Moreover, SSTR2formed heterodimers with δ and κORs in agonist and cellspecific manner. Our results strengthen the concept thatthe activation of one receptor is capable of inducing recep-tor complex formation while second protomer may eitherstabilize or dissociate the heteromeric complex.There is preponderance of evidence suggesting themechanistic significance of GPCRs expression, traffick-ing and heterodimerization in modulation of down-stream signaling pathways [18,20,21,52]. Studies suggestthat the activation of ERKs and p38 in various cancercells lead to aberrant cell proliferation [53]. Consistentwith the existing data, activation of SSTR2 or ORs aloneor in combination inhibit ERK1/2 in MCF-7 and T47Dcells more than the MDA-MB231 cells [28]. Interest-ingly, p38 mediated anti-apoptotic effects typically seenin breast cancer are remarkably diminished in MDA-MB231 and T47D cells upon activation of SSTR2 andORs. Surprisingly, T47D and MCF-7 cells are ER + ve,but the cellular response to SSTR2/OR activation andinhibition of p38 was pronounced in T47D cells. Al-though such discrepancy in inhibition of p38 may beattributed due to the changes in other isoforms of p38in addition to the difference in the origin of these cells;MCF-7 (adenocarcinoma) and T47D (ductal carcinoma).However, the intensity of expression levels of ER in thesecells cannot be avoided from the discussion and futurestudies are warranted in this direction. Moreover resultsdescribed here are consistent with the previous studiesshowing an enhanced pro-apoptotic effect in MCF-7cells over-expressing SSTR2 [16].PI3K/AKT hyper-phosphorylation, uncontrolled breastcancer progression and resistance to hormonal therapyis well established [48,54]. In all three tumor cell lines,the status of phosphorylated PI3K/AKT was not signifi-cantly different from the control. Moreover, this block-ade of PI3K/AKT specifically in MCF-7 and T47D cellswas accompanied by an increased expression of tumorsuppressor PTEN supporting the notion that loss ofPTEN is due to PI3K/AKT activation [48,49]. Notably,the activation of PI3K/AKT induced ERα expression inMCF-7 cells and spared them from tamoxifen mediatedapoptosis [54]. This suggests that enhanced PTEN ex-pression, predominantly in ER + ve MCF-7 and T47Dcells as described here may alleviate TAM resistance.The activation of PI3K/AKT has also been shown todown regulate p53 induced apoptosis in breast cancer[55]. Previous reports suggested that in MCF-7 cells withover-expression of ErbB2 significantly activate PI3K anddecreased the expression of p53, however, this effect wasreversed upon blocking PI3K pathway [56]. Taken intoconsideration, enhanced expression of p53 and compar-able activation of PI3K/AKT to control also highlightnovel function of SSTR2/ORs in breast cancer cells.Given the receptor dependent role, SSTR2 and ORsregulating the MAPK and PI3K/AKT pathways in breastcancer cells is an indication of inhibition of EGFR func-tions. This speculation is supported by aggressive tumorproliferation in breast cancer displaying over-expressionand hyper-phosphorylation of EGFR [24,51,57]. An in-verse relationship between EGFR and ER has beenestablished. Moreover, the absence of ER expression inhuman breast cancer cell lines including MDA-MB231is associated with higher levels of functional EGFR pro-tein and mRNA [58]. In parallel to previous studies, ourdata revealed complete blockade of EGFR phosphoryl-ation upon activation of SSTR2 and ORs in MDA-MB231 and T47D cells [59,60]. Conversely, in MCF-7cells, EGFR phosphorylation was lower than the controlsuggesting that endogenous EGFR phosphorylation isrelatively higher in comparison to other cancer cells. Weanticipate that the SSTR2/ORs mediated inhibition ofEGFR phosphorylation possibly arrest the downstreamMAPK and PI3K/AKT signaling thereby inducing theexpression of tumor suppressor PTEN and p53. Thissupposition is further supported by our recent studiesshowing the inhibition of EGFR phosphorylation by acti-vation of SSTR1 or SSTR5 leading to the pronounced in-hibition of cell proliferating signals in HEK-293 cells[20,21]. Moreover, the physiological response of tumorcells upon activation of SSTR2/ORs parallel the changesseen in downstream signaling. FACs analysis revealedhigher necrotic cell population (>25%) in MCF-7 andT47D cells than MDA-MB231 cells. The early apoptosisincreased upon specific treatment, however, was limitedto <5%. Consistent with previous studies, our observa-tions suggest a much pronounced cytotoxic role ofSSTR2 and ORs in breast cancer cells as described previ-ously [16]. These observations warrant concentrationand time dependent effect of SSTR2/OR agonist onapoptosis and necrosis and further studies are in pro-gress in this direction.Targeting EGFR hyperactivity and PI3K/AKT pathwayshave been the therapeutic approach for the treatment ofbreast cancer. SSTR2 agonists have been clinically usedfor the treatment of acromegaly and pancreatic tumors,whereas ORs are emerging new members of GPCR fam-ily for their anti-proliferative role in various tumors.Therefore, targeting SSTR2 and specific ORs could pos-sibly be a better therapeutic approach in breast cancertreatment. In addition to the anti-proliferative role, theanalgesic role of ORs in the treatment of breast cancercannot be neglected since patients undergoing chemo-therapy are known to experience pain. Previously, SSTanalogs have been proven to be effective in pain relief inKharmate et al. Cancer Cell International 2013, 13:93 Page 11 of 13http://www.cancerci.com/content/13/1/93cases where opioids therapy failed [61]. It would beworth investigating the dual role of ORs as a tumor sup-pressor and analgesic agent in the treatment of breastcancer in synergism with SSTR subtypes. Our observa-tions uncovered the new role of SSTR2 and ORs in com-bination in regulating the key tumor promoting signalsin breast cancer cells. To determine whether such effectis partly due to direct functional interaction betweenthese receptors further studies are in progress in this dir-ection. Taken together, results presented in this study inpart may be due the presence and expression intensityof ER. Furthermore, additional studies are essential tosupport whether knocking down ER in MCF-7 andT47D and transient expression of ER in MDA-MB231cells display comparable changes. In conclusion, this isthe first comprehensive study unveiling the molecularmechanisms of SSTR2/ORs mediated anti-proliferativesignaling with novel therapeutic implications in breastcancer treatment.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsGK and UK conceived, designed and wrote the manuscript. GK performedexperiments and analyzed the data. PSR performed microscopy and YLperformed western blot. All authors read and approved the manuscript.AcknowledgementsThis work was supported by Canadian Institute of Health Research Grant(MOP 10268 and MOP 74465), grant from Canadian Breast CancerFoundation BC/Yukon and NSERC to UK. UK is a Senior Scholar of MichaelSmith Foundation for Health Research.Received: 13 June 2013 Accepted: 23 August 2013Published: 23 September 2013References1. Ben-Shlomo A, Melmed S: Somatostatin agonists for treatment ofacromegaly. Mol Cell Endocrinol 2008, 286(1–2):192–198.2. Buscail L, Esteve J, Saint-Laurent N, Bertrand V, Reisine T, O’Carroll A, Bell GI,Schally AV, Vaysse N, Susini C: Inhibition of cell proliferation by thesomatostatin analogue RC-160 is mediated by somatostatin receptorsubtypes SSTR2 and SSTR5 through different mechanisms. Proc Nat AcadSci 1995, 92(5):1580–1584.3. Buscail L, Vernejoul F, Faure P, Torrisani J, Susini C: Regulation of cellproliferation by somatostatin. 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Cancer Cell International 2013 13:93.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitKharmate et al. Cancer Cell International 2013, 13:93 Page 13 of 13http://www.cancerci.com/content/13/1/93


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