UBC Faculty Research and Publications

Colocalization of somatostatin receptors and epidermal growth factor receptors in breast cancer cells Watt, Heather L; Kumar, Ujendra Mar 6, 2006

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
52383-12935_2005_Article_141.pdf [ 2.47MB ]
Metadata
JSON: 52383-1.0132621.json
JSON-LD: 52383-1.0132621-ld.json
RDF/XML (Pretty): 52383-1.0132621-rdf.xml
RDF/JSON: 52383-1.0132621-rdf.json
Turtle: 52383-1.0132621-turtle.txt
N-Triples: 52383-1.0132621-rdf-ntriples.txt
Original Record: 52383-1.0132621-source.json
Full Text
52383-1.0132621-fulltext.txt
Citation
52383-1.0132621.ris

Full Text

ralCANCER CELLssBioMed CentTIONALINTERNACancer Cell InternationalOpen AccePrimary researchColocalization of somatostatin receptors and epidermal growth factor receptors in breast cancer cellsHeather L Watt1 and Ujendra Kumar*2Address: 1Fraser Laboratories For Diabetes Research, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Quebec, H3A 1A1, Canada  and 2Faculty of Pharmaceutical Sciences, Division of Pharmacology and Toxicology, The University of British Columbia, Vancouver, BC, CanadaEmail: Heather L Watt - heather.watt@mail.mcgill.ca; Ujendra Kumar* - ujkumar@interchange.ubc.ca* Corresponding author    AbstractBackground: Somatostatin receptor (SSTR) expression is positively correlated with tumor sizeand inversely correlated with epidermal growth factor receptor (ErbB) levels and tumordifferentiation. In the present study, we compared SSTR1-5 and ErbB1-4 mRNA and proteinexpression in two breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ERα-).Results: All five SSTRs and four ErbBs were variably expressed as both cell surface and cytoplasmicproteins. In both cell lines, SSTR4 and SSTR1 were highly expressed, followed by SSTR2 and SSTR5with SSTR3 being the least expressed subtype, at the protein level. ErbBs were variably expressedwith ErbB1 as the predominant subtype in both cell lines. ErbB1 is followed by ErbB3, ErbB2 andErbB4 in MCF-7 at both the protein and mRNA levels. In MDA-MB-231 cells, ErbB1 is followed byErbB2, ErbB4 and ErbB3. Our results indicate significant correlations at the level of mRNA andprotein expression in a cell and receptor-specific manner. Using indirect immunofluorescence, wefound that, in MCF-7 cells, SSTR5 was the most prominent subtype coexpressed with ErbBsfollowed by SSTR3, SSTR4, SSTR1 and SSTR2, respectively. In MDA-MB-231 cells, SSTR1colocalized strongly with ErbBs followed by SSTR5, SSTR4, SSTR3 and SSTR2. ErbBs displayedhigher levels of colocalization amongst themselves in MCF-7 cells than in MDA-MB-231 cells.Conclusion: These findings may explain the poor response to endocrine therapy in ER-cancer.Differential distribution of SSTR subtypes with ErbBs in breast cancer cells in a receptor-specificmanner may be considered as a novel diagnosis for breast tumors.BackgroundSomatostatin (SST) is an endogenously produced peptidein neuroendocrine and immune cells. It exists as two bio-logically active forms, SST-14 and SST-28, which are pro-duced by tissue-specific proteolytic processing of acommon precursor [1]. SST is a potent inhibitor of hor-family of G protein-coupled receptors (GPCR) with fiveknown subtypes (SSTR1-5). SST exerts antiproliferativeeffects on normal dividing cells, such as intestinalmucosal cells, activated lymphocytes and inflammatorycells as well as on solid tumors and cultured cells derivedfrom both endocrine and epithelial tumors. These effectsPublished: 06 March 2006Cancer Cell International 2006, 6:5 doi:10.1186/1475-2867-6-5Received: 06 October 2005Accepted: 06 March 2006This article is available from: http://www.cancerci.com/content/6/1/5© 2006 Watt and Kumar; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 19(page number not for citation purposes)mone and growth factor secretion as well as a modulatorof cell proliferation [2,3]. These actions are mediated by ainclude cytostatic (growth arrest) and cytotoxic (apop-totic) actions and are mediated (i) directly by SSTRsCancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5present on tumor cells, and (ii) indirectly via SSTRspresent on non-tumor cell targets. SST inhibits the secre-tion of hormones and growth factors that promote tumorgrowth, inhibits growth factor-induced DNA synthesis,inhibits angiogenesis, promotes vasoconstriction andmodulates immune cell function [1]. Moreover, immuno-reactive SST has been identified, by immunohistochemis-try, in 30% of breast cancer samples and in several breastcancer cell lines [4,5]. Whether SST is synthesized andsecreted from these cells and acts as a paracrine/autocrinegrowth inhibitor remains to be established.All five SSTRs have been implicated in antiproliferativesignaling in a subtype selective manner. When studied asapoptosis [3,6]. Previous studies have demonstrated thepresence of SSTRs in a large variety of tumors and cancercell lines [7-9]. In addition, 15–66% of primary humanbreast tumors are SSTR-positive by binding analysis [10-14]. Consistent with previous studies, we have recentlyshown that SSTRs are expressed in breast cancers in varia-ble amounts and are correlated with various histologicalmarkers in a receptor-specific manner [15]. We have alsoshown the effects of estradiol and tamoxifen on SSTR1and SSTR2 expression in breast cancer cells [16].Epidermal growth factor receptors, members of the type Ireceptor tyrosine kinase (RTK) family commonly knownas ErbBs, are also variably distributed in breast tumors andbreast cancer cell lines as are SSTRs [17,18]. ErbBs can bedetected in all tumors with variable degrees of expression.There are currently four known ErbB receptors with ErbB1(also known as EGFR) and ErbB2 (also known as Neu orHER2) being the most likely to be overexpressed in can-cers, and, therefore, the most studied [19-22]. ErbB3 andErbB4 (also known as HER3 and HER4, respectively) havebeen investigated the least. ErbBs exist as monomers and,upon ligand activation or when overexpressed, formhomo- and heterodimers [23,24].Previous studies showed that ErbB1 is expressed in 40–50% of breast cancer cases and is inversely related withestrogen receptor (ER) levels and survival [25-27]. This isassociated with more aggressive proliferation and unre-sponsiveness to hormone treatment [12,14,27]. Similarly,ErbB2 is present in 10–40% of breast cancer cases and isassociated with poor survival [19,21,25,26]. ErbB3 is alsoexpressed in breast cancer [28,29]. Associations withErbB1 and ER have been shown in some studies but notin others [20]. This discrepancy may be due to the tech-niques employed, antibodies used, sample size or tumortype. In contrast with ErbB1-3, ErbB4 is generally reportedto be associated with favorable prognostic factors[20,21,25,30,31].While ErbBs are involved in tumor growth and cell prolif-eration and are often associated with poor response toendocrine therapy and reduced survival, SSTRs play amajor role in the control of tumor growth and tumor cellproliferation [32-34]. SSTR expression is positively corre-lated with tumor size and inversely correlated with ErbBlevels and tumor differentiation [12,14]. Several recentreports have shown GPCRs to directly interact with RTKsvia scaffolding proteins when both receptors are presenttogether in the large signaling complexes [35-37]. Alterna-tively, GPCRs can indirectly transactivate RTKs via G pro-teins which ultimately lead to increased intracellularcalcium levels and activation of PKC [38]. Indirect RTKSemi-quantitative analysis of SSTR1-5 and ErbB1-4 mRNA and protein expression n MCF-7 and MDA-MB-231 breast cance  c llsFigure 1Semi-quantitative analysis of SSTR1-5 and ErbB1-4 mRNA and protein expression in MCF-7 and MDA-MB-231 breast cancer cells. A. Upper panel shows western blot analysis of SSTR1-5 in MCF-7 (left) and MDA-MB-231 (right) cells. Membrane protein (25 µg) was fractionated by SDS-PAGE and probed with affinity-purified SSTR antibodies. Major pro-tein bands of 53 (SSTR1), 57 (SSTR2), 60 (SSTR3), 44 (SSTR4) and 58 kDa (SSTR5) were obtained. Lower panel shows RT-PCR anlaysis of SSTR1-5 mRNA expression in both cell lines. 5 µg of DNA-free RNA was reverse tran-scribed and coamplified with primers specific for SSTR1-5 and β-actin. 8 µL of PCR products were fractionated on aga-rose gels stained with ethidium bromide, visualized under UV lighting and photographed. B. Western blot (upper panel) and RT-PCR (lower panel) analysis of ErbB1-4 expression in MCF-7 (left) and MDA-MB-231 (right) breast tumor cells. Major protein bands of 170 (ErbB1), 185 (ErbB2), 200 (ErbB3) and 175 kDa (ErbB4) were obtained. Experimental conditions were the same as described for panel A except for the specific antibodies and primers.SSTR1SSTR2SSTR3SSTR4SSTR5SSTR1SSTR2SSTR3SSTR4SSTR5MCF-7 MDA-MB-231ErbB1ErbB2ErbB3ErbB4ErbB1ErbB2ErbB3ErbB4Western BlotRT-PCRWestern BlotRT-PCRActinActinSSTR1-5ErbB1-4Page 2 of 19(page number not for citation purposes)individual isotypes, four of the receptors (SSTR1, 2, 4, 5)induce cell cycle arrest whereas SSTR3 uniquely triggerstransactivation has also been reported to occur via mem-brane-bound metalloproteinases (MMPs) or metallopro-Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5teinase-disintegrin proteins (ADAMs) which process ErbBtransmembrane ligands [35,39,40]. In general, RTK trans-activation by GPCRs results in altered mitogen activatedprotein kinase (MAPK) signaling and, subsequently, inaltered cell growth and proliferation [39,41,42]. It is notknown if SSTRs (GPCR) and ErbBs (RTK) are coexpressedwithin the same cells. Hence, before defining the mecha-nisms for functional interactions between ErbBs andSSTRs, it is essential to determine if this occurs. We havetherefore determined, in the current study, SSTR1-5 andErbB1-4 expression at the protein and mRNA levels. Inaddition, since ER has been shown to be associated withErbB levels, we investigated their colocalization in ER-positive (ER+) and negative (ER-) breast cancer cells. Ourdata showed that SSTRs and ErbBs are well expressed inboth cell lines and, significantly, exhibited variable colo-calization.ResultsExpression of SSTRs mRNA and protein in MCF-7 and MDA-MB-231 cellsUsing semi-quantitative RT-PCR, we determined SSTR1-5mRNA expression in MCF-7 (ER+) and MDA-MB-231(ERα-) human breast cancer cells (Fig. 1A). We found sig-nificant differences in overall receptor expression levelsbetween ER+ and ERα – cells. Although SSTR mRNA levelswere greater in MDA-MB-231 than in MCF-7 cells, bothcells lines showed similar patterns of expression. SSTR3was highly expressed, followed by SSTR4, SSTR2 andSSTR5 while SSTR1 was the least expressed subtype, at thelevel of the mRNA.We further determined SSTR1-5 protein expression usingwestern blot and indirect immunofluorescence analyses.Consistent with mRNA results and as detected by westernblot, all SSTR subtypes were expressed at their representa-tive molecular sizes at the protein level (53, 57, 60, 44 and58 kDa for SSTR1-5, respectively) (Table 1 and Fig. 1A).Indirect immunofluorescence analysis of SSTR subtypesrevealed a significant but variable cellular expression ofmultiple SSTRs with all five receptor subtypes expressed asboth membrane and cytoplasmic proteins (Figs. 2, 3, 4, 5,expressed in MCF-7 cells than in MDA-MB-231 cells whileSSTR3 was poorly expressed in both cell lines.Expression of ErbBs mRNA and protein in MCF-7 and MDA-MB-231 cellsAll ErbB subtypes are well expressed at the mRNA level ina significant proportion of breast tumor tissues; however,expression in breast cancer cells is variable [22,43,44].MCF-7 cells expressed all four ErbBs at the level of themRNA with ErbB1 and ErbB3 being the dominant sub-types (Fig. 1B). MDA-MB-231 cells expressed all fourErbBs in a comparable manner, also displaying higherexpression at the mRNA level for ErbB1 and ErbB3. Inter-estingly, in MCF-7 cells, ErbB3 mRNA expression was thestrongest while, in MDA-MB-231 cells, ErbB1 mRNA wasthe most abundant. These results are in agreement with areport by Bièche et al. [43] where MCF-7 cells displayedlower ErbB1 mRNA levels, higher ErbB2 and ErbB3 levelsand equivalent ErbB4 mRNA expression in comparison toMDA-MB-231 cells.Using western blot analysis, ErbB subtypes in MCF-7 andMDA-MB-231 cells displayed variable expression at theprotein level whereby all ErbBs were expressed at theirrepresentative molecular sizes (170, 185, 200 and 175kDa for ErbB1-4, respectively). ErbB1 and ErbB3 were thepredominant subtypes followed by ErbB2 and ErbB4 inMCF-7 cells as determined by western blot analysis (Table2 and Fig. 1B). In contrast, in MDA-MB-231 cells, ErbB1was predominantly expressed followed by ErbB2, ErbB4and ErbB3. Consistent with previous reports, ErbB3 pro-tein expression was strongest in ER+ cells while ErbB1 wasmore abundant in ER-cells [22]. However, our results con-tradict another report with regards to relative ErbB3expression levels [28]. Protein expression was further con-firmed by immunocytochemistry revealing that all ErbBsubtypes were well expressed as membrane and cytoplas-mic proteins in MCF-7 and MDA-MB-231 cells (Figs. 2, 3,4, 5, 6, 7, 8, 9, 10, 11).Colocalization of SSTRs and ErbBs in MCF-7 cellsColocalization between SSTRs and ErbBs revealed signifi-Table 1: Semiquantitative analysis of relative protein expression levels of SSTR1-5 in MCF-7 and MDA-MB-231 cells as determined by western blot analysis.MCF-7 MDA-MB-231SSTR1 ++ +++SSTR2 +++ ++SSTR3 + +SSTR4 ++++ ++++SSTR5 + +++++ strong +++ moderate ++ mild + weakPage 3 of 19(page number not for citation purposes)6, 7, 8, 9). Notably, SSTR1 and 4 were more highly cant variations in a receptor and cell-specific manner.Four different cell populations were detected in MCF-7Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 4 of 19(page number not for citation purposes)AFigure 2A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB1 and SSTR1-5 in MCF-7 cells. Localization of ErbB1 (red staining) was visualized using monoclonal antibodies with Cy3-conjugated goat anti-mouse IgG (a-e). The same cells were incubated with polyclonal SSTR1-5 antibodies and visualized (green staining) using FITC-conjugated goat anti-rabbit IgG (f-j). Colocalization of ErbB1 and SSTR1-5 was determined by merging individual red and green images to give orange-labelled cells (k-o). All receptors are expressed as membrane and cytoplasmic protein. Arrows indicate colocalization at the cell surface. Scale bar = 25 µm. B. Quantitative analysis of MCF-7 cells showing colocalization of ErbB1 with SSTR1-5. Cells expressing two receptors together were counted from at least 8 randomly selected vertical and horizontal fields from each coverslip. Data are from three different experiments performed in duplicate and are presented as mean ± SEM for each receptor combination. C. Quantitative analysis of cells showing ErbB1 and SSTR1-5 in distinct locations within the same cell. Data were analyzed as described in B.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 5 of 19(page number not for citation purposes)AFigure 3A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB2 (red staining) and SSTR1-5 (green staining) in MCF-7 cells (for details see legend to Figure 2). Scale bar = 25 µm. B. Quantitative analysis of MCF-7 cells showing colocalization of ErbB2 with SSTR1-5 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB2 and SSTR1-5 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 6 of 19(page number not for citation purposes)AFigure 4A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB3 (red staining) and SSTR1-5 (green staining) in MCF-7 cells (for details see legend to Figure 2). Scale bar = 25 µm. B. Quantitative analysis of MCF-7 cells showing colocalization of ErbB3 with SSTR1-5 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB3 and SSTR1-5 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 7 of 19(page number not for citation purposes)AFigure 5A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB4 (red staining) and SSTR1-5 (green staining) in MCF-7 cells (for details see legend to Figure 2). Scale bar = 25 µm. B. Quantitative analysis of MCF-7 cells showing colocalization of ErbB4 with SSTR1-5 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB4 and SSTR1-5 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 8 of 19(page number not for citation purposes)AFigure 6A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB1 (red staining) and SSTR1-5 (green staining) in MDA-MB-231 cells (for details see legend to Figure 2). Scale bar = 25 µm. B. Quantitative analysis of MDA-MB-231 cells showing colocalization of ErbB1 with SSTR1-5 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB1 and SSTR1-5 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 9 of 19(page number not for citation purposes)AFigure 7A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB2 (red staining) and SSTR1-5 (green staining) in MDA-MB-231 cells (for details see legend to Figure 2). Scale bar = 25 µm. B. Quantitative analysis of MDA-MB-231 cells showing colocalization of ErbB2 with SSTR1-5 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB2 and SSTR1-5 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 10 of 19(page number not for citation purposes)AFigure 8A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB3 (red staining) and SSTR1-5 (green staining) in MDA-MB-231 cells (for details see legend to Figure 2). Scale bar = 25 µm. B. Quantitative analysis of MDA-MB-231 cells showing colocalization of ErbB3 with SSTR1-5 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB3 and SSTR1-5 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 11 of 19(page number not for citation purposes)AFigure 9A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB4 (red staining) and SSTR1-5 (green staining) in MDA-MB-231 cells (for details see legend to Figure 2). Scale bar = 25 µm. B. Quantitative analysis of MDA-MB-231 cells showing colocalization of ErbB4 with SSTR1-5 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB4 and SSTR1-5 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5cells: one expressing SSTRs alone (≤ 7%), a second popu-lation expressing only ErbBs (≤ 15%), a third populationexpressing both receptors in distinct locations within thesame cell (27–70%) and a fourth population of cells dis-playing colocalization (18–62%).In MCF-7 cells, SSTR1 colocalized with ErbB1 (22% ofcells) at the cell membrane and intracellularly (Table 3and Fig. 2). SSTR2 and SSTR4 exhibited similar patterns ofcolocalization with ErbB1 with only 21% of cells coex-pressing both receptors. SSTR3 and SSTR5 colocalizedwith ErbB1 in a greater proportion (30%) of cells. AllSSTR subtypes colocalized with ErbB2 in a comparablemanner at the cell surface as well as intracellularly (Table3 and Fig. 3). ErbB2 and SSTRs colocalized in 40–51% ofcells with SSTR5 displaying the strongest colocalizationwith ErbB2. ErbB3 was coexpressed with SSTR1-5 in acomparable manner to ErbB1 (Table 3 and Fig. 4). SSTR1and SSTR2 colocalized with ErbB3 in 22% of cells whereasSSTR3 and SSTR4 were coexpressed in 28 and 18% ofErbB3-positive cells, respectively. In contrast, SSTR5 colo-calized with ErbB3 in about 41% of cells. In MCF-7 cells,ErbB4 colocalized with all SSTR subtypes (Table 3 and Fig.5). ErbB4 was coexpressed with SSTR2, SSTR3, and SSTR4in a comparable manner (36–39% of cells). On the otherhand, SSTR1 and SSTR5 colocalized with ErbB4 in 47 and63% of cells, respectively. Further colocalization studiesrevealed that SSTR5 was the most prominent SSTR sub-type to colocalize with ErbB1-4 in MCF-7 cells (Table 3and Figs. 2, 3, 4, 5).Colocalization of SSTRs and ErbBs in MDA-MB-231 cellsIn comparison with MCF-7 (ER+) cells, MDA-MB-231(ERα-) cells exhibited significantly variable colocalizationof SSTR1-5 with ErbB1-4. Furthermore, a lower percentageof cells coexpressed both SSTRs and ErbBs in MDA-MB-231 than in MCF-7 cells. Interestingly, using immunocy-tochemistry, 100% of MDA-MB-231 cells expressedErbB1-3. Subsequently, there were no cells that onlyexpressed SSTRs when the cells were double-labeled forSSTRs and ErbB1-3. In contrast, up to 3% of cells showedstaining for SSTRs alone while up to 20% of cells onlyexpressed ErbB4 in cells double-labeled for SSTR1-5 andErbB4. Furthermore, there was a small cell population (≤1%) lacking both receptors.As illustrated in Table 3 and Figure 6, 19% of MDA-MB-231 cells displayed strong colocalization between SSTR1and ErbB1. On the other hand, SSTR2-5 colocalizationwith ErbB1 occurred in only 10–16% of cells (Table 3 andFig. 6). In MDA-MB-231 cells, ErbB2 weakly colocalizedwith all SSTR subtypes at the cell surface in only 11–18%of cells (Table 3 and Fig. 7). SSTR1 was coexpressed withErbB3 in 24% of cells (Table 3 and Fig. 8). SSTR2, SSTR3and SSTR4 colocalized with ErbB3 at the cell surface andintracellularly in approximately 12, 20 and 14%, respec-tively, of the cell population (Table 3 and Fig. 8). Mean-while, SSTR5 displayed colocalization (17% of cells) withErbB3 mainly at the cell surface. In MDA-MB-231 cells,SSTR1-4 colocalized with ErbB4 at the cell surface in 8–12% of cells (Table 3 and Fig. 9). In contrast, SSTR5 andErbB4 colocalization was seen in 31% of cells. Notably,colocalization of SSTRs with ErbB4 occurred mainly in the"apical" endings of the cells.Colocalization of ErbBs in MCF-7 cells and MDA-MB-231 cellsTo better understand whether there is any preferential andselective colocalization between ErbB subtypes in ER+ andER-cells, we determined the colocalization of ErbBs inTable 2: Semiquantitative analysis of relative protein expression levels of ErbB1-4 in MCF-7 and MDA-MB-231 cells as determined by western blot analysis.MCF-7 MDA-MB-231ErbB1 ++++ ++++ErbB2 ++ +++ErbB3 +++ +ErbB4 ++ ++++++ strong +++ moderate ++ mild + weakTable 3: Colocalization of SSTR1-5 with ErbB1-4 in MCF-7 and MDA-MB-231 cells.MCF-7 MDA-MB-231ErbB1 ErbB2 ErbB3 ErbB4 ErbB1 ErbB2 ErbB3 ErbB4SSTR1 + ++ + +++ ++ + +++ +SSTR2 + ++ + ++ + + + +SSTR3 +++ ++ ++ ++ ++ + ++ +SSTR4 + +++ + ++ ++ ++ + +Page 12 of 19(page number not for citation purposes)SSTR5 +++ +++ +++ ++++ + + ++ +++Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 13 of 19(page number not for citation purposes)AFigure 10A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB1-3 and ErbB2-4 in MCF-7 cells. Localization of ErbB1-3 (red staining) was visualized using monoclonal antibodies with Cy3-conjugated goat anti-mouse IgG (a-f). The same cells were incubated with polyclonal ErbB2-4 antibodies and visualized (green staining) using FITC-conju-gated goat anti-rabbit (g-l). Colocalization of ErbB1-3 and ErbB2-4 was determined by merging individual red and green images to give orange-labelled cells (m-r). All receptors are expressed as membrane and cytoplasmic protein. Arrows indicate colocal-ization at the cell surface. Scale bar = 25 µm. B. Quantitative analysis of MCF-7 cells showing colocalization of ErbB1-3 with ErbB2-4 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB1-3 and ErbB2-4 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5Page 14 of 19(page number not for citation purposes)AFigure 11A. Representative photomicrographs illustrating double immunofluorescence localization of ErbB1-3 and ErbB2-4 in MDA-MB-231 cells (for details see legend to Figure 10). Scale bar = 25 µm. B. Quantitative analysis of MDA-MB-231 cells showing colo-calization of ErbB1-3 with ErbB2-4 (for details see legend to Figure 2). C. Quantitative analysis of cells showing ErbB1-3 and ErbB2-4 in distinct locations within the same cell. Data were analyzed as described in Figure 2.Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5MCF-7 and MDA-MB-231 cells. As shown in MCF-7 cells(Fig. 10), ErbB2, ErbB3 and ErbB4 colocalized with ErbB1in 23%, 31% and 26% of cells, respectively. Furthermore,ErbB3 and ErbB4 were coexpressed with ErbB2 in 26%and 39%, respectively, while ErbB3 and ErbB4 colocalizedin 22% of MCF-7 cells. In contrast, MDA-MB-231 cellsdemonstrated lesser degrees of colocalization than MCF-7cells with the exception of ErbB1 and ErbB3 (Fig. 11).ErbB1 colocalized with ErbB2, ErbB3 and ErbB4 in 11%,39% and 19% of cells, respectively. Meanwhile, ErbB3and ErbB4 colocalized with ErbB2 in 15% and 20% ofcells, respectively, and ErbB3 and ErbB4 were coexpressedin 14% of the cell population.DiscussionThe present study represents the first comprehensivedescription showing SSTR1-5 and ErbB1-4 colocalizationin ER+ and ER-breast cancer cells. All five SSTRs weredetected in MCF-7 and MDA-MB-231 with a rich expres-sion of subtypes 1 and 4, moderate expression of SSTR2and relatively weak expression of subtypes 3 and 5. Ourdata also demonstrate a potential correlation betweenSSTR and ErbB expression and estrogen dependency. Wefound higher levels of expression of ErbB1 and lower lev-els of SSTR1, SSTR4 and ErbB3 in ERα – (MDA-MB-231)cells when compared to ER+ (MCF-7) breast cancer cells.In addition, we showed that there was more colocaliza-tion of SSTRs with ErbBs in MCF-7 cells than in MDA-MB-231 cells. We also detected preferential colocalizationamong ErbBs in both MCF-7 and MDA-MB-231 cells.Overall expression levels of SSTR subtypes in culturedbreast cancer cell lines were comparatively less than insolid tumors. Significantly, SSTR3, which is well expressedin breast tumor tissues, was relatively poorly expressed inthese cell lines [15]. These results indicate that the variousbreast cancer cell lines, although useful for studying SSTRbiology, do not necessarily reflect endogenous tumorSSTR expression or function. Possible explanations for thedifference are the probable induction of SSTR expressionin solid tumors by circulating hormones, or, locally, bygrowth factors, cytokines, and other mediators producedfrom peritumoral structures such as the stroma, blood ves-sels and immune cells [45]. Increasing evidence points tothe occurrence of multiple SSTR subtypes in many differ-ent types of tumor cells as well as normal cells [46,47]. Allfive SSTR isoforms bind the natural ligands SST-14 andSST-28 with nanomolar affinity and share common sign-aling pathways, such as the inhibition of adenylyl cyclase,making the functional significance of expressing morethan one SSTR subtype in the same cell unclear [2].Whether the different SSTRs subserve different biologicalroles in the same cell or cooperate through dimerizationand SSTR5 heterodimerization, in stably transfected HEKand CHO-K1 cells, results in a new receptor withenhanced signaling properties [48,49]. We further antici-pate such a possibility of heterodimerization betweenSSTR1 and SSTR5 and, additionally, between SSTRs andErbBs in breast cancer cells.Whereas SSTRs have been associated with antiprolifera-tive signaling, several previous studies, using a variety oftumors including MCF-7 and MDA-MB-231 cells, havecorrelated ErbBs with tumor progression and poor prog-nosis [19,22,50,51]. However, the data have been incon-sistent and controversial [52-54]. These inconsistenciesmay have arisen due to the techniques employed, the var-iation between cell stocks studied in different laboratoriesand, most significantly, the different passages at which thecells were used [45]. In this regard, we have seen signifi-cant variation in receptor expression/levels at differentpassages (data not shown). In keeping with ErbBs roles intumor progression and poor prognosis, overexpression ofErbBs in breast carcinomas has been correlated with a lackof ER [44,52]. Furthermore, blocking ER using antisensestrategies resulted in increased ErbB1, no change in ErbB2and a slight decrease in ErbB3 expression in breast cancercells [22]. Consistent with these observations, we foundhigher levels of expression of ErbB1 and decreased levelsof ErbB3 in ERα – (MDA-MB-231) than in ER+ (MCF-7)cells. In accordance with previous studies, our findingsstrongly support the concept that the presence of ER couldbe a determining factor in ErbB expression in both breastcancer cells and tumors.Previous reports state that specific ErbB heterodimers, i.e.,ErbB1/ErbB2 and ErbB2/ErbB3, result in increased tumorgrowth and cell proliferation. We report that, in MCF-7and MDA-MB-231 cells, there is preferential colocaliza-tion of ErbBs with other ErbBs. We found greater colocal-ization between ErbB1 and ErbB3 in both MCF-7 andMDA-MB-231 cells. We also detected a high degree ofcolocalization between ErbB2 and ErbB4 in MCF-7 cells.These data strongly support previous observationswhereby heterodimerization between ErbB1 and ErbB2was correlated with tumor progression [22,51]. Thesealternate heterodimer pairs, i.e., ErbB1/ErbB3 and ErbB2/ErbB4, may account for the less aggressive proliferationrates reported for both cell lines. Furthermore, in agree-ment with previous studies, we detected fewer cells show-ing ErbB colocalization in ERα – cells (MDA-MB-231)than in ER+ (MCF-7) cells with the exception of thosecoexpressing ErbB1 and ErbB3. Altogether, the higherdegree of colocalization of ErbBs in MCF-7 cells than inMDA-MB-231 cells may be partially associated withslower tumor growth and better response to hormonalPage 15 of 19(page number not for citation purposes)to create greater signaling diversity remains to be deter-mined. In this regard, we have recently shown that SSTR1therapy. Our data provide direct evidence that ErbB1 andErbB3 are the prominent subtypes which may interact asCancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5heterodimers, in these cells. Nothing is currently knownregarding the physiological responses and functional con-sequences of these observations suggesting that furtherstudies are required in this direction.In addition to heterodimerization within receptor sub-families, there have been several reports demonstratingthat crosstalk between RTKs and GPCRs modulates down-stream signaling pathways [35-37]. Even so, direct evi-dence for functional interactions between ErbBs andSSTRs have not yet been demonstrated despite the criticalroles they play in tumor progression. We showed here thatthere was increased colocalization of SSTRs with ErbBs inMCF-7 cells (ER+) compared with MDA-MB-231 (ERα-)cells. This may help elucidate why estrogen-sensitivetumors show less aggressive proliferation than estrogen-insensitive tumors. This pattern of colocalization mayalso explain the superior response of ER+ patients to SSTanalog therapy [55]. In MCF-7 cells, the preferentiallygreater colocalization of SSTRs with ErbB2 may serve tocounteract any deleterious effects of ErbB2. Whether thiscolocalization exists in vivo and is lost during tumor pro-gression needs to be determined. Furthermore, colocaliza-tion of SSTR1 and SSTR5 with ErbB4 supports theantiproliferative effects of both SSTRs. SSTR interactionswith ErbB4 may also serve to potentiate ErbB4's previ-ously reported role in differentiation and apoptosis [30].Furthermore, by preventing ErbB4's downregulation,SSTRs may be indirectly circumventing ErbB1-3's growthpromoting effects. However, whether such interactionsexist in vivo in solid tumors needs to be determined.Despite SSTR and ErbB colocalization, low abundance ofSSTRs alongside high expression of ErbBs within the samecell may account for the failure of SST treatment of breasttumor or other ErbB-expressing tumors. Furthermore, it isanticipated but not yet proven that SSTRs would reversethe effects of ErbBs with respect to MAPK activation andsubsequent cell proliferation [56-58]. In addition, somereports suggest that the ER is involved in MAPK activation[59-61]. Previous studies have also demonstrated that ERpresence is required for cbl-induced ubiquitination ofErbB1 and that ubiquitination of ErbB1 results in its deg-radation [62]. This could result in different levels of acti-vation of downstream pathways in ER+ (MCF-7) and ERα– (MDA-MB-231) breast cancer cells. In addition, SST-induced internalization and subsequent downregulationof SSTR2-5 on the membrane may release ErbBs fromcomplexes and result in cell proliferation [63-65]. Alto-gether, this suggests that not only do we need to activateSSTRs to counteract ErbBs effects on cell proliferation butwe also need a mechanism to upregulate, or at least main-tain, SSTRs on the membrane in order to reduce or modifyConclusionIn summary, the present results have important func-tional and therapeutic implications. Predominant SSTR1expression and weak SSTR5 expression in breast cancercells may help explain their poor sensitivity to hormonaltherapy. These data may also explain the differentialeffects of the SST analog octreotide in breast cancer ther-apy. Since there is evidence of crosstalk between GPCRsand RTKs, cells displaying SSTR colocalization with ErbBsuggest that, within these cells, both receptor families mayfunctionally interact through hetero-oligomerization. Ifsuch a process exists, it may account for the diversificationof receptor signaling. Most significantly, developing a newtherapeutic agent that could both activate SSTRs andinhibit ErbB overexpression could potentially be a way toblock tumor progression.Materials and methodsMaterials and reagentsRPMI 1640 and L-15 culture media were purchased fromInvitrogen (Burlington, Ontario). Fetal bovine serum(FBS) and Antibiotic-Antimycotic solution were pur-chased from Wisent (St. Bruno, Quebec). The proteaseinhibitor cocktail used for protein extraction was suppliedby Sigma-Aldrich Canada Ltd (Oakville, Ontario). Nor-mal goat serum (NGS) was purchased from Vector Labo-ratories (Burlington, Ontario). Polyclonal rabbit anti-SSTR antibodies were developed in the lab and their spe-cificity has been previously described [66,67]. Purifiedmouse anti-ErbB1 (sc-101), ErbB2 (sc-08), ErbB3 (sc-7390), rabbit anti-ErbB1 (sc-03), ErbB2 (sc-284), ErbB3(sc-285), ErbB4 (sc-283) and goat anti-ErbB4 (sc-283-G)were purchased from Santa Cruz Biotechnology (SantaCruz, California). The secondary FITC- and Cy3-conju-gated goat anti-mouse or anti-rabbit and Cy3-conjugateddonkey anti-sheep IgG antibodies were obtained fromJackson ImmunoResearch Laboratories (West Grove, Pen-sylvania).Cell cultureMCF-7 cells were maintained in RPMI 1640 medium sup-plemented with 0.35 µM insulin, 10% (v/v) FBS and 1%(v/v) Antibiotic-Antimycotic solution at 37°C in anatmosphere of 5% CO2/95% air. MDA-MB-231 cells weremaintained in L-15 medium supplemented with 10% FBSand 1% Antibiotic-Antimycotic solution at 37°C in flaskswith phenolic caps.Expression of SSTR1-5 mRNA in MCF-7 and MDA-MB-231 breast cancer cellsSSTR1-5 and ErbB1-4 mRNA levels were measured bysemi-quantitative RT-PCR in MCF-7 (ER+) and MDA-MB-231 (ERα-) breast cancer cells as previously described withPage 16 of 19(page number not for citation purposes)ErbB signaling. some modifications [15,68]. Briefly, 5 µg of DNA-freeCancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5RNA was reverse transcribed and the resulting cDNA sam-ples were amplified by PCR using the following primers:hSSTR1 forward 5'-TGGTGGGCTTCGTGTTGT-3'reverse 5'-GATGACCGACAGCTGACTCA-3'hSSTR2 forward 5'-ATCTGGGGCTTGGTACACAG-3'reverse 5'-GAAGACAGCCACCACGAT-3'hSSTR3 forward 5'-TCATCTGCCTCTGCTACCTG-3'reverse 5'-TTGAAGCGGTAGGAGAGGAA-3'hSSTR4 forward 5'-CGCTCGGAGAAGAAAATCAC-3'reverse 5'-CCCACCTTTGCTCTTGAGAG-3'hSSTR5 forward 5'-CTCTCTCTGGACCTTGTGCC-3'reverse 5'-ACGAGCAAACAGGTACGCTT-3'hErbB1 forward 5'-AGTCGCCCAAAGTTCCGTGAGT-3'reverse 5'-TGGGAGGAAGGTGTCGTCTATG-3'hErbB2 forward 5'-AACTCACCTACCTGCCCACCAA-3'reverse 5'-GTGGTATTGTTCAGCGGGTCTC-3'hErbB3 forward 5'-CAGGTCTACGATGGGAAGTTTG-3'reverse 5'-CTCACGATGTCCCTCCAGTCAA-3'hErbB4 forward 5'-ACCCTTCAGCACCCAGACTACC-3'reverse 5'-GACCACCAGAGAAAGAGAGGGG-3'β-actin forward 5'-ATCATGAAGTGTGACGTGGAC-3'reverse 5'-AACCGACTGCTGTCACCTTCA-3'The PCR products were separated by electrophoresis on1.5% agarose gels stained with ethidium bromide, visual-ized under UV illumination and photographed using anAlpha Innotech FluorChem 8800 (Alpha Innotech Co.,San Leandro, CA).Western blot analysisCrude membrane extracts from MCF-7 and MDA-MB-231cells were prepared using a glass homogenizer in 20 mMTris-HCl, pH 7.5 (1:300 protease inhibitor cocktail) asmM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01%bromophenol blue and 5% β-mercaptoethanol. Sampleswere placed in boiling water for 5 min and fractionated byelectrophoresis on a 10% SDS-polyacrylamide gel asdescribed by Laemmli [70]. The fractionated proteinswere transferred by electrophoresis to a 0.2 µm nitrocellu-lose membrane (Trans-Blot Transfer Medium, Bio-Rad) intransfer buffer consisting of 0.025 M Tris, 0.19 M glycineand 15% methanol. Western Blot analysis was performedas previously described with slight modifications [71].Briefly, membranes were blotted with anti-SSTRs polyclo-nal (dilution 1:400) and anti-ErbB polyclonal (dilution1:600–1500) antibodies. Blocking of membranes, incuba-tion with primary and secondary antibodies and detectionby chemiluminescence were performed with the Western-Breeze® kit according to manufacturer's instructions.Molecular weights were estimated using the MagicMarkXP Western Protein Standard (Invitrogen). Images werecaptured using an Alpha Innotech FluorChem 8800 gelbox imager.ImmunocytochemistryMCF-7 and MDA-MB-231 cells were plated on glass cover-slips in 24-well plates and processed for indirect immun-ofluorescence for colocalization as previously describedwith slight modifications [16]. Cells were washed once inPBS and fixed with 4% paraformaldehyde on ice for 20minutes. After two subsequent washes in PBS, cells wereincubated with 5% NGS (diluted in PBS) for 1.5 hours fol-lowed by incubation with SSTR (1:500) and ErbB (1:150)antibodies in 1% NGS (in PBS) for 48 h at 4°C. Cells werethen washed twice in PBS followed by incubation withCy3-conjugated goat anti-mouse (1:500) or Cy3-conju-gated donkey anti-sheep (1:500) and FITC-conjugatedgoat anti-rabbit (1:100) secondary antibodies for 3 hours.After two subsequent washes in PBS, cells were mountedand viewed under a Leica DMLB microscope attached to aCoolSnap CCD camera. Adobe Photoshop was used, in aconsistent manner, to create the overlays and to adjust thecontrast and brightness of all images.Quantitative analysisCounting of SSTR-, ErbB- and SSTR+ErbB-positive cellswas performed directly at high magnification (40×) undera Leica DMLB microscope. At least 8 horizontal and 8 ver-tical fields per coverslip were randomly selected for eachreceptor combination. Total number of cells positive foreither one or both receptors was considered as 100% andpercent colocalization was calculated accordingly. Totalnumber of cells counted per coverlip ranged from 205 to877.AbbreviationsPage 17 of 19(page number not for citation purposes)previously described [69]. Membrane protein (25 µg) wassolubilized in Laemmli sample buffer containing 62.5SSTR, somatostatin receptor; ErbB, epidermal growth fac-tor receptor; ER, estrogen receptor; SST, somatostatin;Cancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5GPCR, G protein-coupled receptor; RTK, receptor tyrosinekinase; MAPK, mitogen activated protein kinase; FBS, fetalbovine serum; NGS, normal goat serumAuthors' contributionsHLW carried out all experiments, participated in thedesign of the study, performed the statistical analysis andhelped to draft the manuscript. UK conceived the study,participated in its design and helped to draft the manu-script.AcknowledgementsThis work was funded Canadian Institute of Health Research (CIHR) grants MOP-10411, MOP-74465 and MOP-6196 (UK). We thank Archana Venugopalan for technical support and Maria Correia for secretarial help.References1. Patel YC: Somatostatin and its receptor family.  Frontiers in Neu-roendocrinology 1999, 20:157-198.2. Patel YC, Srikant CB: Somatostatin receptors.  Trends EndocrinolMetab 1997, 8:398-405.3. Patel YC: Basic aspects of somatostatin receptors.  In Advancesin Molecular and Cellular Endocrinology Edited by: LeRoith D. Green-wich, CT , JAI Press; 1998. 4. Ciocca DR, Puy LA, Fasoli LC, Tello O, Aznar JC, Gago FE, Papa SI,Sonego R: Corticotropin releasing hormone, leutinizing hor-mone releasing hormone, growth hormone releasing hor-mone, and somatostatin-like immunoreactivities in biopsiesfrom breast cancer patients.  Breast Cancer Res  Treat 1990,15:175-184.5. Nelson J, Cremin M, Murphy RF: Synthesis of somatostatin bybreast cancer cells and their inhibition by exogenous soma-tostatin and sandostatin.  Br J Cancer 1989, 59:739-742.6. Sharma K, Srikant CB: Induction of wild-type p53 Bax and acidicendonuclease during somatostatin signaled apoptosis inMCF-7 human breast cancer cells.  Int J Cancer 1998, 76:259-266.7. Papotti M, Kumar U, Volante M, Pecchioni C, Patel YC: Immunohis-tochemical detection of somatostatin receptor types 1-5 inmedullary carcinoma of the thyroid.  Clin Endocrinol (Oxf) 2001,54:641-649.8. Panetta R, Patel YC: Expression of mRNA for all five humansomatostatin receptors (hSSTR1-5) in pituitary tumors.  LifeSci 1995, 56:333-342.9. Reubi JC, Schaer JC, Waser B, Mengod G: Expression and locali-zation of somatostatin receptor SSTR1, SSTR2 and SSTR3mRNAs in primary human tumors using in situ hybridiza-tion.  Cancer Res 1994, 54:3455-3459.10. Prevost G, Hosford D, Thomas F: Receptors for somatostatinand somatostatin analogues in human breast tumors.  Ann NY Acad Sci   1994, 733:147-154.11. Prevost G, Lanson M, Thomas F, Veber N, Gonzalez W, Beaupain R,Starzec A, Bogden A: Molecular heterogeneity of somatostatinanalogue BIM-23014C receptors in human breast carcinomacells using the chemical cross-linking assay.  Cancer Res 1992,52:843-850.12. Foekens JA, van Putten WL, Portengen H, Rodenburg CJ, Reubi JC,Berns PM, Henzen-Logmans SC, van der Burg ME, Alexieva-Figusch J,Klijn JG: Prognostic value of pS2 protein and receptors forepidermal growth factor (EGF-R), insulin-like growth factor-1 (IGF-1-R) and somatostatin (SS-R) in patients with breastand ovarian cancer.  J Steroid Biochem Mol Biol 1990, 37:815-821.13. Fekete M, Wittliff A, Schally AV: Characteristics and distributionof receptors for [D-Trp6]-luteinizing-releasing hormone,somatostatin, epidermal growth factor, and sex steroids in500 biopsy samples of human breast cancer.  J Clin Lab Anal1989, 3:137-147.14. Foekens JA, Portengen H, van Putten WLJ, Trapman A, Reubi JC,Alexieva-Figusch J, Klijn JGM: Prognostic value of receptors for15. Kumar U, Grigorakis SI, Watt HL, Sasi R, Snell L, Watson P, Chaud-hari S: Somatostatin receptors in primary human breast can-cer: quantitative analysis of mRNA for subtypes 1-5 andcorrelation with receptor protein expression and tumorpathology.  Breast Cancer Res  Treat 2005, 92:175-186.16. Rivera JA, Alturaihi H, Kumar U: Differential regulation of soma-tostatin receptors 1 and 2 mRNA and protein expression bytamoxifen and estradiol in breast cancer cells.  Journal of Car-cinogenesis 2005, 4(1):10-19.17. Wiseman SM, Makretsov N, Nielsen TO, Gilks B, Yorida E, CheangM, Turbin D, Gelmon K, Huntsman DG: Coexpression of the type1 growth factor receptor family members HER-1, HER-2,HER-3 has a synergistic negative prognostic effect on breastcarcinoma survival.  Cancer 2005, 103:1770-1777.18. Abd El-Rehim DM, Pinder SE, Paish CE, Bell JA, Rampaul RS, BlameyRW, Robertson JFR, Nicholson RI, Ellis IO: Expression and co-expression of the members of the epidermal growth factorreceptor (EGFR) family in invasive breast carcinoma.  Br JCancer 2004, 91:1532-1542.19. Kilinç N, Yaldiz M: P53, c-erbB-2 expression and steroid hor-mone receptors in breast carcinoma: correlations with his-topathological parameters.  Eur J Gynaecol Oncol 2004,25:606-610.20. Esteva FJ, Hortobagyi GN, Sahin AA, Smith TL, Chin DM, Liang SY,Pusztai L, Buzdar AU, Bacus SS: Expression of erbB/Her recep-tors, Heregulin and p38 in primary breast cancer using quan-titative immunohistochemistry.  Pathology Oncology Research2001, 7:171-177.21. Suo Z, Bjaamer A, Ottestad L, Nesland JM: Expression of EGFRfamily and steroid receptor hormone receptors in ductalcarcinoma in situ of the breast.  Ultrastructural Pathology 2001,25:349-356.22. deFazio A, Chiew YE, Sini RL, Janes PW, Sutherland RL: Expressionof c-erbB receptors, heregulin, oestrogen receptor in humanbreast cell lines.  Int J Cancer 2000, 87:487-498.23. Sweeney C, Carraway KL: Ligand discrimination by ErbB recep-tors: differential signaling through differential phosphoryla-tion site usage.  Oncogene 2000, 19:5568-5573.24. Tzahar E, Pinkas-Kramarski R, Moyer JD, Klapper LN, Alroy I, Lev-kowitz G, Shelly M, Henis S, Eisenstein M, Ratzkin BJ, Sela M, AndrewsGC, Yarden Y: Bivalence of EGF-like ligands drives the ErbBsignaling network.  EMBO J 1997, 16(16):4938-4950.25. Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JMS: Expressionof the HER1-4 family of receptor tyrosine kinases in breastcancer.  Journal of Pathology 2003, 200:290-297.26. Spaventi R, Kamenjicki E, Pecina N, Grazio S, Grazio S, Pavelic J, KusicB, Cvrtila D, Danilovic Z, Spaventi S, Pavelic K, Gluckman J, PavelicZP: Immunohistochemical detection of TGF-a, EGF-R, c-erbB-2, c-H-ras, c-myc, estrogen and progesterone in benignand malignant human breast lesions: a concommitantexpression?  In vivo 1994, 8:183-190.27. Reubi JC, Torhorst J: The relationship between somatostatin,epidermal growth factor, and steroid hormone receptors inbreast cancer.  Cancer 1989, 64(6):1254-1260.28. Naidu R, Yadav M, Nair S, Kutty MK: Expression of c-erbB3 pro-tein in primary breast carcinomas.  Br J Cancer 1998,78:1385-1390.29. Travis A, Pinder SE, Robertson HFR, Bell JA, Wencyk P, Gullick WJ,Nicholson RI, Poller DN, Blamey RW, Elston CW, Ellis IO: C-erbB-3 in human breast carcinoma: expression and relation toprognosis and established prognostic indicators.  Br J Cancer1996, 74(2):229-233.30. Srinivasan R, Gillett CE, Barnes DM, Gullick WJ: Nuclear expres-sion of the c-erbB-4/HER-4 growth factor receptor in inva-sive breast cancers.  Cancer Research 2000, 60:1483-1487.31. Tang CK, Concepcion XZ, Milan M, Gong X, Montgomery E, LippmanME: Ribozyme-mediated down-regulation of ErbB-4 in estro-gen receptor-positive breast cancer cells inhibits prolifera-tion both in vitro and in vivo.  Cancer Research 1999,59:5315-5322.32. Srikant CB: Cell cycle dependent induction of apoptosis bysomatostatin analog SMS 201-995 in AtT-20 mouse pituitarycells.  Biochem Biophys Res Commun 1995, 209(2):400-406.33. Pagliacci MC, Tognellini R, Grignani F, Nicoletti I: Inhibition ofPage 18 of 19(page number not for citation purposes)insulin-like growth factor 1, somatostatin, and epidermalgrowth factor in human breast cancer.  Cancer Res 1989,49:7002-7009.human breast cancer cell (MCF-7) growth in vitro by thesomatostatin analog SMS 201-995: effects on cell cycleCancer Cell International 2006, 6:5 http://www.cancerci.com/content/6/1/5parameters and apoptotic cell death.  Endocrinology 1991,129(5):2555-2562.34. Szende B, Srkalovic G, Schally AV, Lapis K, Groot K: Inhibitoryeffects of analogs of luteinizing hormone-releasing hormoneand somatostatin on pancreatic cancers in hamsters. Eventsthat accompany tumor regression.  Cancer 1990,65(10):2279-2290.35. Carpenter G: EGF receptor transactivation mediated by theproteolytic production of EGF-like agonists.  Sci STKE 2000,2000(15):PE1.36. Daub H, Weiss FU, Wallash C, Ullrich A: Role of transactivationof the EGF receptor in signalling by G-protein coupledreceptors.  Nature 1996, 379:557-560.37. Fergusson SSG: Receptor tyrosine kinase transactivation: fine-tuning synaptic transmission.  Trends in Neurosciences 2003,26:119-122.38. Pierce KL, Premont RT, Lefkowitz RJ: Seven-transmembranereceptors.  Nat Rev Mol Cell Biol 2002, 3:639-650.39. Wetzker R, Böhmer FD: Transactivation joins multiple tracksto the ERK/MAPK cascade.  nat rev mol cell biol 2003, 4:651-657.40. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallash C, Ull-rich A: EGF receptor transactivation by G-protein-coupledreceptors requires metalloproteinase cleavage of proHB-EGF.  Nature 1999, 402:884-888.41. Luttrell LM, Daaka Y, Lefkowitz RJ: Regulation of tyrosine kinasecascades by G-protein-coupled receptors.  Current opinion in cellbiology 1999, 11:177-183.42. Schäfer B, Gshwing A, Ullrich A: Multiple G-protein-coupledreceptor signals converge on the epidermal growth factorreceptor to promote migration and invasion.  Oncogene 2004,23:991-999.43. Bièche I, Onody P, Tozlu S, Driouch K, Vidaud M, Lidereau R: Prog-nostic value of erbB family mRNA expression in breast car-cinomas.  Int J Cancer 2003, 106:758-765.44. Walker RA, Dearing SJ: Expression of epidermal growth factorreceptor mRNA and protein in primary breast carcinomas.Breast Cancer Res Treat 1999, 53:167-176.45. Lacroix M, Leclercq G: Relevance of breast cancer cell lines asmodels for breast tumors: an update.  Breast Cancer Res Treat2004, 83:249-289.46. Kumar U, Laird D, Srikant CB, Escher E, Patel YC: Expression ofthe five somatostatin receptor (SSTR1-5) subtypes in ratpituitary somatotrophes: quantitative analysis by double-label immunofluorescence confocal microscopy.  Endocrinology1997, 138(10):4473-4476.47. Patel YC, Greenwood MT, Warszynska A, Panetta R, Srikant CB: Allfive cloned human somatostatin receptors (hSSTR1-5) arefunctionally coupled to adenylyl cyclase.  Biochem Biophys ResCommun 1994, 198(2):605-612.48. Grant M, Patel R, Kumar U: The role of subtype-specific ligandbinding and the C-tail domain in dimer formation of humansomatostatin receptors.  J Biol Chem 2004, 279(37):38636-38643.49. Rocheville M, Lange DC, Kumar U, Sasi R, Patel RC, Patel YC: Sub-types of the somatostatin receptor assemble as functionalhomo- and heterodimers.  J Biol Chem 2000, 275(11):7862-7869.50. Ciocca DR, Elledge R: Molecular markers for predictingresponse to tamoxifen in breast cancer patients.  Endocrine2000, 13(1):1-10.51. Olayioye MA, Neve RM, Lane H, Hynes NE: The ErbB signalingnetwork: receptor heterodimerization in development andcancer.  The EMBO Journal 2000, 19:3159-3167.52. Mueller H, Loop P, Liu R, Wosikowski K, Kueng W, Eppenberger U:Differential signal transduction of epidermal-growth-factorreceptors in hormone-dependent and hormone-independ-ent human breast cancer cells.  Eur J Biochem 1994,221(2):631-637.53. deFazio A, Chiew YE, Donoghue C, Lee CS, Sutherland RL: Effect ofsodium butyrate on estrogen receptor and epidermalgrowth factor receptor gene expression in human breastcancer cell lines.  J Biol Chem 1992, 267(25):18008-18012.54. Godden J, Leake R, Kerr DJ: The response of breast cancer cellsto steroid and peptide growth factors.  Anticancer Research 1992,12(5):1683-1688.55. Weber C, Merriam L, Koschitzky T, Karp F, Benson M, Forde K,vivo by somatostatin analog SMS 201-995: treatment of nudemouse xenografts.  Surgery 1989, 106(2):416-422.56. Buscail L, Vernejoul F, Faure P, Torrisani J, Susini C: Regulation ofcell proliferation by somatostatin.  Ann Endocrinol (Paris) 2002,63(2 Pt 3):2S13-2S18.57. Cattaneo MG, Taylor JE, Culler MD, Nisoli E, Vicentini LM: Selectivestimulation of somatostatin receptor subtypes: differentialeffects on Ras/MAP kinase pathway and cell proliferation inhuman neuroblastoma cells.  FEBS Letters 2000, 481:271-276.58. Florio T, Thellung S, Arena S, Corsaro A, Bajetto A, Schettini G, StorkPJ: Somatostatin receptor 1 (SSTR1)-mediated inhibition ofcell proliferation correlates with the activation of the MAPkinase cascade: role of the phosphotyrosine phosphatatseSHP-2.  J Physiol Paris 2000, 94(3-4):239-250.59. Keshamouni VG, Mattingly RR, Reddy KB: Mechanism of 17-β-estradiol-induced erk1/2 activation in breast cancer cells.  JBiol Chem 2002, 277:22558-22565.60. Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM:Rapid membrane effects of steroids in neuroblastoma cells:effects of estrogen on mitogen activated protein kinase sig-nalling cascade and c-fos immediate early gene transcrip-tion.  Endocrinology 1997, 138(9):4030-4033.61. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P,Nola E, Auricchio F: Tyrosine kinase/p21ras/MAP-kinase path-way activation by estradiol-receptor complex in MCF-7 cells.EMBO J 1996, 15(6):1292-1300.62. Duan L, Miura Y, Dimri M, Majumder B, Dodge IL, Reddi AL, GhoshA, Fernandes N, Zhou P, Mullane-Robinson K, Rao N, Donoghue S,Rogers RA, Bowtell D, Naramura M, Gu H, Band V, Band H: Cbl-mediated ubiquitinylaiton is required for lysosomal sortingof epidermal growth factor receptor but is dispensible forendocytosis.  J Biol Chem 2003, 278:28950-28960.63. Hipkin RW, Friedman J, Clark RB, Eppler CM, Schonbrunn A: Ago-nist-induced desensitization, internalization, and phosphor-ylation of the sst2A somatostatin receptor.  J Biol Chem 1997,272(21):13869-13876.64. Roth A, Kreienkamp HJ, Meyerhof W, Richter D: Phosphorylationof four amino acid residues in the carboxyl terminus of therat somatostatin receptor subtype 3 is crucial for its desen-sitization and internalization.  J Biol Chem 1997,272(38):23769-23774.65. Hukovic N, Panetta R, Kumar U, Patel YC: Agonist-dependentregulation of cloned human somatostatin receptor types 1-5(hSSTR1-5): subtype selective internalization or upregula-tion.  Endocrinology 1996, 137(9):4046-4049.66. Kumar U: Expression of somatostatin receptor subtypes(SSTR1-5) in alzheimer's disease brain: an immunohisto-chemical analysis.  Neuroscience 2005, 134:525-538.67. Kumar U, Sasi R, Suresh S, Patel A, Thangaraju M, Metrakos P, PatelSC, Patel YC: Subtype-selective expression of the five somato-statin receptors (hSSTR1-5) in human pancreatic islet cells.Diabetes 1999, 48:77-85.68. Khare S, Kumar U, Sasi R, Puebla L, Calderon L, Lemstrom K, HayryP, Patel YC: Differential regulation of somatostatin receptortypes 1-5 in rat aorta after angioplasty.  Faseb J 1999,13:387-394.69. Ramirez JL, Watt HL, Rocheville M, Kumar U: Agonist-induced up-regulation of human somatostatin receptor type 1 is regu-lated by β-arrestin-1 and requires an essential serine residuein the receptor C-tail.  Biochimica et Biophysica Acta 2005,1669:182-192.70. Laemmli UK: Cleavage of structural proteins during theassembly of the head of bacteriophage T4.  Nature 1970,227(5259):680-685.71. Ramirez JL, Grant M, Norman M, Wang XP, Moldovan S, de Mayo FJ,Brunicardi C, Kumar U: Deficiency of somatostatin (SST)receptor type 5 (SSTR5) is associated with sexually dimor-phic changes in the expression of SST and SST receptors inbrain and pancreas.  Molecular and Cellular Endocrinology 2004,221:105-119.Page 19 of 19(page number not for citation purposes)Logerfo P: Inhibition of growth of human breast carcinomas in

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.52383.1-0132621/manifest

Comment

Related Items