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Regular Articles |
Departments of Pathology*
and Biochemistry and
Molecular Biology,
University of Manitoba,
Faculty of Medicine, Winnipeg, Manitoba, Canada
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently identified psoriasin (S100A7) as a gene that is frequently overexpressed in preinvasive ductal carcinoma in situ (DCIS) relative to adjacent invasive carcinoma, suggesting a role in breast tumor progression.1 Other members of the S100 gene family of calcium-binding proteins have been implicated in a range of biological processes, including tumor metastasis.2 In particular, S100A2 has been shown to be down-regulated in breast tumor cells relative to their normal epithelial cell counterparts,3 whereas up-regulation of S100A4 has been strongly implicated in breast tumor metastasis.4-6 In the latter case this may reflect the ability of S100A4 to influence cell motility,7 the cytoskeleton6,8,9 or cell adhesion molecules.10 Psoriasin was initially identified as a highly abundant protein belonging to the S100 gene family,11 expressed by abnormally proliferating keratinocytes in psoriatic epidermis.12,13 It has subsequently been shown to be a secreted protein that can exert an effect as a chemotactic factor for inflammatory cells.14,15 However, the function of psoriasin in breast cancer remains to be determined.16 In this study we have developed a psoriasin-specific antibody and evaluated the persistence of psoriasin expression in invasive breast tumors with different invasive and metastatic potential as well as host inflammatory response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All breast tumor cases used for this study were selected from the NCIC–Manitoba Breast Tumor Bank (Winnipeg, Manitoba, Canada). As has previously been described,17 tissues accrue to the Bank from cases at multiple centers within Manitoba and are rapidly collected and processed to create matched formalin-fixed embedded and frozen tissue blocks for each case, with mirror-image surfaces oriented by colored inks. The histology of every sample in the Bank is uniformly interpreted in hematoxylin/eosin (H&E)-stained sections from the face of the paraffin tissue block by a pathologist. This information is available in a computerized database along with relevant pathological and clinical information and was used as a guide for the selection of specific paraffin and frozen blocks from cases for this study. For each case interpretations included an estimate of the cellular composition (including the percentage of invasive epithelial tumor cells, collagenous stroma, and fatty stroma), tumor type, and tumor grade for ductal tumors (Nottingham score).18,19 The inflammatory host response was scored semiquantitatively on a scale of 1 (low) to 5 (high). Steroid receptor status was determined for all cases by ligand binding assay performed on an adjacent portion of tumor tissue. Tumors with estrogen and progesterone receptor levels above 20 fmol/mg and 15 fmol/mg of total protein, respectively, were considered ER- or PR-positive.
Two cohorts of tumors were selected. The first cohort comprised 35 invasive ductal carcinomas selected to include six subgroups differing with respect to estrogen receptor status (ER-positive and ER-negative) and tumor grade (low, intermediate, high). Additional selection criteria also included high tissue quality, presence of invasive tumor within >35% of the cross section of the frozen block for invasive ductal cases, and minimal (<5%) normal or in situ epithelial components. The second cohort comprised 23 invasive tumors selected to include four subgroups of different tumor types18 that vary in differentiation and metastatic potential, including invasive lobular (six), medullary (five), tubular (six), and colloid (six). Similar secondary criteria were also used for this cohort.
For analysis of antibody specificity and for positive controls for tumor assays, MCF7 human breast cancer cells obtained from the American Type Culture Collection (Manassas, VA) were used. MCF7 cells were grown as previously described under normal conditions in the presence of 5% fetal bovine serum, to provide a negative control.20 Alternatively MCF7 cells were subjected to estrogen-deprived conditions in the presence of charcoal-stripped serum before stimulation by estradiol (10-8 mol/L) for 48 hours before harvesting to induce psoriasin expression and provide a positive control. As an additional positive control MDA-MB-231 human breast cancer cells were transfected with a plasmid containing the cytomegalovirus (CMV) promotor adjacent to the psoriasin cDNA (Hiller-Hitchcock T, Leygue E, Cummins-Leygue C, Murphy LC, Watson PH, manuscript in preparation), and stable transfectants (CL7FD3 cell clone) expressing psoriasin mRNA were also used.
Antibody Reagents
A psoriasin-specific chicken IgY polyclonal antibody was generated by immunization of chickens with a 14-amino acid peptide corresponding to the carboxy terminus of psoriasin (KQSHGAAPCSGGSQ; Bionostics, Toronto, and Aves Labs). A >90% pure IgY fraction from chicken egg yolk was obtained in phosphate-buffered saline (PBS) and then further purified by passing it over a psoriasin peptide affinity column made by binding the synthetic peptide to N-hydroxy-succinimide-activated Sepharose 4B (Pharmacia Biotech), according to the manufacturer’s instructions. The bound IgY was then eluted with 5.0 mol/L sodium thiocyanate, followed by dialysis against PBS. Additional antibodies used included a commercial anti-S100 antibody (Sigma, St. Louis, MO) as well as a rabbit polyclonal antibody, raised against the recombinant protein (kindly provided by Prof J. Celis, University of Aarhus, Aarhus, Denmark).
Western Blot Analysis
For tumors, multiple sections (10–20 x 20 µm) were cut from the face of frozen tissue blocks immediately adjacent to the face of the matching paraffin block.17 For cell lines, trypsinized cell pellets were obtained from breast cancer cell lines (grown to ~80% confluence). Total protein lysates were extracted from both the cell line pellets and frozen tissue sections, using Tri-reagent (Sigma), as described by the manufacturer. The recovered protein was dissolved in SDS isolation buffer (50 mmol/L Tris, pH 6.8, 20 mmol/L EDTA, 5% sodium dodecyl sulfate (SDS), 5 mmol/L ß-glycerophosphate) and a cocktail of protease inhibitors (Boehringer Mannheim, Laval, PQ). Protein concentrations were determined using the Micro-BCA protein assay kit (Pierce, Rockford, IL). Sixty micrograms of total protein lysates were run on a 16.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) mini gel, using Tricine SDS-PAGE to separate the proteins,21 and then transferred to 0.2-µm Nitrocellulose (Bio-Rad, Mississauga, ON). After blocking in 10% skimmed milk powder in Tris-buffered saline-0.05% Tween (TBST buffer), blots were incubated with chicken IgY anti-psoriasin antibody (~15 µg/ml in TBST), followed by incubation with secondary antibody, rabbit IgG anti-chicken IgY conjugated to horseradish peroxidase (1:5000 dilution in TBST; Jackson ImmunoResearch Laboratories), and visualization by incubation with Supersignal (Pierce), per the manufacturer’s instructions. Exposed x-ray films were photographed, and the band intensities were determined by video image analysis, using MCID M4 software (Imaging Research, ST. Catherine’s, ON). All signals were adjusted with reference to the psoriasin-transfected MDA-MB-231 cell control (CL7FD3), run on each blot.
Immunohistochemistry
Immunohistochemistry was performed on 5-µm paraffin-embedded breast tumor tissue sections from tissue blocks fixed in 10% neutral buffered formalin for 18–24 hours. After deparaffinizing, clearing, and hydrating to PBS buffer (pH 7.4) containing 0.05% Tween 20 (Mallinckrodt), the sections were pretreated with hydrogen peroxide (3%) for 10 minutes to remove endogenous peroxidases, and nonspecific binding was blocked with normal rabbit serum (1:50; Sigma). Primary chicken IgY anti-psoriasin antibody (1:500 dilution in PBS) was applied for 1 hour at 37°C followed by washing and incubation with the secondary antibody, peroxidase-conjugated affinity purified rabbit anti-chicken (1:200 dilution), for 1 hour at room temperature. Detection was performed with 3,3'-diaminobenzidine tetrahydrochloride peroxidase substrate (Sigma) and counterstaining with methyl green (2%), followed by dehydration, clearing, and mounting. A positive tissue control and a negative reagent control (normal rabbit serum only/no primary antibody) were run in parallel in all experiments. Immunostaining was scored semiquantitatively by assessing the average signal intensity (on a scale of 0 to 3) and the proportion of tumor cells showing a positive nuclear signal (0, none; 0.1, less than one-tenth; 0.5, less than one-half; 1.0 greater than one-half). The intensity and proportion scores were then multiplied to give an overall score, and tumors with a score equal to or higher than 1.0 were deemed positive.
In Situ Hybridization
In situ hybridization was performed on paraffin sections (5 µm) according to a previously described protocol.1 Linearized psoriasin plasmid cDNA (1.0 µg/µl) was used to generate UTPS35-labeled sense and antisense RNA probes with the Riboprobe System (Promega, Madison, WI) according to the manufacturer’s instructions. Sense and antisense probes were equalized by diluting 1 x 106 cpm/µl in hybridization solution. These were then applied to paraffin sections (approximately 30 µl of probe per section) that had undergone postfixation with 4% paraformaldehyde (pH 7.4) in PBS and further pretreatments with triethanolamine/acetic anhydride and proteinase K before hybridization. Sections were then coverslipped, sealed, and incubated overnight in a humid chamber at 42°C. After coverslip removal, sections underwent incubation in posthybridization solution and buffered RNase A (20 µg/ul), followed by several washes in descending dilutions of standard saline citrate buffer to remove weakly bound nonspecific label. After dehydration in ethanol containing 300 mmol/L ammonium acetate, the sections were coated in NTB-2 Kodak emulsion, subsequently developed after various time intervals from 2 to 5 weeks, and counterstained with Lee’s methylene blue and basic fuchsin. Psoriasin expression was assessed by bright-field microscopic examination at low power (10x objective) magnification with reference to the negative sense and positive control tumor sections run with each batch. Levels were scored semiquantitatively as previously described22 by assessing the average signal intensity (on a scale of 0 to 3) and the proportion of tumor cells showing a positive signal (0, none; 0.1, less than one-tenth; 0.5, less than one-half; 1.0 greater than one-half). The intensity and proportion scores were then multiplied to give an overall score, and tumors with a score equal to or higher than 1.0 were deemed positive.
Reverse Transcriptase-Polymerase Chain Reaction Analysis
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed based on extracted RNA (600 ng) that was reverse transcribed in a total volume of 20 µl as described previously.1 Briefly, reverse transcription was completed with the following reaction mixture: for each sample, 200 ng (2 µl of 0.1 µg/µl) of total RNA was added to 16 µl of RT mix (4 µl of 5x RT buffer; 1 µl of each of dATP, dCTP, dGTP, and dTTP, all at 2.5 mmol/L; 2 µl of 0.1% bovine serum albumin; 2 µl of 0.1 mol/L dithiothreitol; 1 µl of 0.25 mol/L random hexamer primer; 2 µl of dimethyl sulfoxide (DMSO), and 1 µl of 200 units/µl of Moloney murine leukemia virus reverse transcriptase) and incubated at 37°C for 1.5 hours. Each PCR was performed in 50-µl volume, using 1 µl of the completed RT reaction (cDNA); 30.8 µl of sterile water; 5 µl of 10x PCR buffer; 5 µl of 25 mmol/L MgCl2; 200 mmol/L each of dATP, dCTP, dGTP, and dTTP; 1 µl of DMSO; 1 unit of Taq DNA polymerase; and 0.5 µl of 50 mmol/L PCR primers. The psoriasin primers were sense (5'-AAG AAA GAT GAG CAA CAC-3') and antisense (5'-CCA GCA AGG ACA GAA ACT-3') corresponding to the cDNA sequence,13 or alternatively, PCR was performed with GAPDH primers, sense (5'-ACC CAC TCC TCC ACC TTT G-3') and antisense (5'-CTC TTG TGC TCT TGC TGG G-3').23 For PCR amplification the reaction comprised an initial step of 5 minutes at 94°C, and then 45 cycles (30 seconds at 94°C, 30 seconds at 56°C, 30 seconds at 72°C) for psoriasin or 35 cycles (45 seconds at 93°C, 45 seconds at 58°C, 30 seconds at 72°C) for GAPDH. PCR products of the two genes amplified from the same RT reaction were loaded into the same wells onto a 1.5% agarose gel before electrophoresis and ethidium bromide staining to visualize psoriasin (246 bp) and GAPDH (198 bp) cDNAs under UV illumination.
Preliminary experiments were performed with cell line and tumor RNA samples to establish the appropriate RNA input and PCR cycle number conditions to achieve amplification with both psoriasin and GAPDH primers in the linear range in a typical sample. Tumors from each cohort were processed as a batch, from frozen sectioning to RNA extraction, reverse transcription in triplicate, and then duplicate PCRs from each RT reaction. For each batch controls included RT-negative and RNA-negative controls and both psoriasin-positive (estradiol-stimulated MCF7) and psoriasin-negative (untransfected, wild-type MDA-MB-231 cells) RNA controls. All primary tumor PCR signals were assessed in gels and autoradiographs by video image capture and with a MCID-M4 image analysis program. Psoriasin expression was standardized to GAPDH expression assessed from the same RT reaction in separate PCR reactions and run in parallel on the same gel, and the mean of each duplicate PCR was then expressed relative to the levels in the MCF7 cell line standard. The invasive tumor component within each section was also assessed in the adjacent mirror image paraffin section, and the percentage area occupied by tumor was used to correct for differences in epithelial cell content of the tumor sections used for RNA extraction.
Statistical Analysis
For analysis of associations, standardized psoriasin mRNA levels
were used either as a continuous variable or transformed into low- or
high-expression categories, using a level of one relative density unit.
This cutpoint was selected to correspond to the lowest level at which
protein could be detected by Western blot. Correlations with estrogen
(ER) and progesterone (PR) receptor levels and inflammation were tested
using Spearman’s test. Associations with categorical variables were
tested by either Mann-Whitney or analysis of variance tests for
selected dependent variables, or unpaired t-test for
independent variables, or a 
2
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiple S100 proteins are expressed in individual tissues and
cells. To specifically distinguish psoriasin expression within archival
formalin-fixed and paraffin-embedded tissues we raised a polyclonal
antibody in chicken against a synthetic peptide that corresponded to
the COOH terminus of psoriasin. This 14-amino acid region was selected
on the basis of very low homology to other S100 proteins. Western blot
analysis of an MDA-MB-231 breast cell line transfected with a plasmid
incorporating psoriasin cDNA under the control of a CMV promotor (and
known to express psoriasin mRNA by Northern blot; unpublished data) and
breast tumors showed a single band corresponding to a protein of approx
11.7 kd with the chicken IgY antibody (Figure 1A)
. This signal could be completely
inhibited by preincubation of the primary antibody with psoriasin
synthetic peptide (data not shown) and was absent from the wild-type
and vector-alone transfected MDA-MB-231 control cells. By comparison, a
commercial anti-S100 antibody (Sigma), known to detect several S100
proteins in MDA-MB-231 cells,24
weakly recognized the same
11.7-kd protein in transfected cells as well as several other S100
proteins in most samples (Figure 1B)
. Both antibodies reacted with
additional higher molecular mass bands in tumor samples. However,
specificity of the 11.7-kd psoriasin signal was further confirmed by
Western blot using another anti-psoriasin polyclonal rabbit antibody
previously raised against a recombinant psoriasin protein (data not
shown).
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To assess cellular localization of psoriasin we studied paraffin-embedded tissue blocks from breast, skin, and larynx by immunohistochemistry. The breast tumors studied possessed either high (six cases) or low (seven cases) levels of psoriasin mRNA and total protein expression (determined by Western blot and RT-PCR analysis of protein and RNA extracted from sections cut from the adjacent mirror-image frozen tissue blocks). Skin biopsies from the margins of two psoriatic lesions and a squamous carcinoma of larynx were also studied, as psoriasin was originally identified as a highly expressed protein in psoriatic skin and has also been identified as an expressed sequence tag in a cDNA library from laryngeal squamous carcinoma (http://www.ncbi.nlm.nih.gov/UniGene/Hs.112408). All cases were subjected to both immunohistochemistry and in situ hybridization on adjacent paraffin sections, and both signals were assessed independently, using a semiquantitative scoring system as described in Materials and Methods.
In breast tumors psoriasin protein was detected predominantly within
epithelial tumor cells and was localized within both tumor cell nuclei
as well as cytoplasm. Psoriasin was also present within some stromal
cells and in some cases also on the luminal aspects of endothelial
cells within small vessels (Figure 2)
.
However, in situ hybridization demonstrated that mRNA
expression was limited to epithelial tumor cells in all cases (Figure 2)
. The nuclear immunohistochemical staining was completely abolished
by competition with the immunizing peptide and was not present in
tumors that were negative for psoriasin but showed additional
immunoreactive bands on Western blot (eg, see case 13402, Figure 1
, and
case 8840, Figure 4
). Immunohistochemistry and Western blot were
concordant in 12/13 cases. In one case Western blot analysis was
negative and weak focal staining was seen by immunohistochemistry.
Specificity of the nuclear signal was further confirmed by the fact
that the presence of immunohistochemically detected protein expression,
assessed on the basis of nuclear staining, was highly concordant (92%)
with expression detected by in situ hybridization mRNA.
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. The adjoining normal skin was negative.
Occasional positive immunohistochemical staining, but no mRNA signal,
was also observed in stromal cells in the dermis underlying the
psoriatic lesion. As seen in breast tumor cells, psoriasin protein was
localized both to the nucleus and cytoplasm within keratinocytes
(Figure 2)
. The same nuclear and cytoplasmic localization was also
detected in a squamous laryngeal carcinoma (data not shown). However,
the polyclonal rabbit anti-psoriasin antibody previously shown to
provide immunofluorescent staining in frozen skin
sections13,25
did not detect any signal on paraffin
sections from skin or breast. Additional experiments were performed
with the chicken IgY anti-psoriasin antibody on skin and breast tumor
sections in which immunohistochemical conditions (microwave
versus protease antigen retrieval) and tissue
treatment/fixation conditions (formalin versus alcohol
versus paraformaldehyde versus frozen) were
varied, and nuclear localization persisted under all conditions (data
not shown). Expression of Psoriasin mRNA in Invasive Breast Tumors
The changes in psoriasin expression previously observed in
association with the transition from in situ to invasive
carcinoma suggested a functional role in the early stages of
progression. However, alteration of psoriasin expression in normal skin
has also been associated with abnormal keratinocyte differentiation. To
examine further the relationship of psoriasin with differentiation and
invasiveness, we used RT-PCR and Western blot to examine psoriasin mRNA
and protein levels in a cohort of invasive tumors. These tumors
included several different tumor types and a range of differentiation,
as determined by tumor grade and estrogen receptor status (Table 1)
.
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. Within the invasive ductal subgroup
there was no significant difference in psoriasin expression with tumor
grade. There was also no significant difference between tumor size or
type, although there was a trend toward lower levels of expression in
both well-differentiated tumor types, tubular and mucinous carcinomas,
whereas lobular and medullary carcinomas showed a trend toward higher
expression than invasive ductal tumors. However, higher levels of
psoriasin mRNA expression showed a significant inverse correlation with
both ER and PR levels (r = -0.66,
P = 0.0001; r = -0.47,
P = 0.0003, Spearman) and with ER and PR negative
status (ER-ve vs. ER+ve; n = 28
vs. 29, mean (SD) 1.032 (0.7) vs. 0.32 (0.36),
P < 0.0001 Mann-Whitney; PR-ve vs. PR+ve,
n = 25 vs. 32, 1.05 (0.72) vs.
0.37 (0.40), P < 0.0001) in all tumors and within the
invasive ductal subgroup. Psoriasin expression was also higher in
axillary node-positive cases in all tumors (mean (SD) = 0.86
(0.73) vs. 0.59 (0.66), and the difference was statistically
significant for the invasive ductal subgroup (mean (SD) = 0.88
(0.79) vs. 0.38 (0.28), P = 0.035,
t-test). These relationships with ER, PR, and nodal status
(Table 2)
were also evident and remained
statistically significant after correction of psoriasin levels for the
relative tumor cell content, assessed as a percentage within the
paraffin sections adjacent to the frozen tissue sections studied.
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and Figure 4
). These tumors (six
ductal, two lobular, two medullary) corresponded to those with the
highest mRNA levels observed by RT-PCR (above 1.0 arbitrary expression
units). Also consistent with RT-PCR analysis, Western blot-positive
invasive ductal tumors were also significantly associated with
ER-negative (P < 0.0001) and PR-negative
(P < 0.0012) and node-positive
(P = 0.0143) status (Table 2)
.
The relationship between psoriasin mRNA and protein expression and host
inflammatory response was also examined (Table 2)
. Psoriasin mRNA
showed a significant positive correlation in the entire cohort
(n = 57, r = 0.47,
P = 0.0002), in the entire cohort excluding the
medullary carcinoma subgroup, which includes inflammatory infiltrates
as a diagnostic criterion (n = 52,
r = 0.42, P = 0.0022), and within the
invasive ductal subgroup alone (n = 34,
r = 0.39, P = 0.023). Cases with
Western blot-detectable psoriasin protein also showed increased
inflammatory infiltrates, both in the entire cohort (mean (SD) =
3.6 (1.1) vs. 2.3 (1.2), P = 0.004) and in
the entire cohort excluding the medullary subgroup (mean (SD) =
3.3 (0.89) vs. 2.1 (0.98), P = 0.007).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Indirect support for a role for S100 genes in breast tumor progression is provided by several observations. Disruption of calcium signaling pathways has been implicated as a central mechanism in tumorigenesis and specifically in the process of invasion and metastasis.26 Moreover, the chromosomal location of the S100 gene family lies in a region of chromosome 1 that frequently (>50%) shows loss of heterozygosity in invasive tumors.27 Furthermore, several S100 genes are expressed in breast cell lines and tumors and are known to manifest alteration of their expression in association with tumorigenesis and progression.11,24 In particular, S100A2 and S100A4 have been identified to be differentially expressed between normal and neoplastic cells3,28,29 and up-regulated in metastatic as compared to nonmetastatic cells in both mouse and rat mammary tumor cell lines.5,30 In vivo studies of breast tumors have also shown a correlation between high levels of S100A4 expression, nodal metastasis, and ER-negative status.31 More direct evidence has emerged from modulation of S100A4 expression in transfected cell lines that have shown that overexpression of S100A4 can also induce the metastatic phenotype in mouse, rat, and human cells.4,6,32 Furthermore, there is evidence that S100A4 may exert its effect on cell cytoskeleton8,9 and motility,7 and it has also been demonstrated that up-regulation of S100A4 in mouse tumor cell lines can down-regulate expression of E- cadherin and disturb the intracellular distribution of B-catenin.10
A possible role for psoriasin (S100A7) in breast cancer first emerged when it was also identified as a cDNA down-regulated in a nodal metastasis relative to a primary breast tumor.33 Nevertheless, the significance of the initial observation was unclear because of the fact that expression was only detectable in a small proportion of cells within invasive primary tumors studied by in situ hybridization and overall could be detected in only 18% of primary tumor specimens assessed by Northern analysis. An explanation for this paradox became apparent when psoriasin was also identified by us as a gene that is particularly highly expressed in the ductal epithelial cells of preinvasive ductal carcinoma in situ,1 which can be present as a significant component with invasive tumor specimens. We have now shown that when higher levels of psoriasin expression persist within invasive tumors, this correlates with indicators of increased metastatic potential. It should be noted that the strong relationship with ER status is compatible with studies of S100A431 and the in vitro observation33 (and our unpublished data) that psoriasin is regulated by estradiol in MCF7 cells. Although it is interesting that the nature of this correlation is different between the in vitro and in vivo situations, similar differences have been found with other genes in breast tumors,34 suggesting that additional external factors may influence psoriasin regulation in vivo.
Although the biological effect of alteration of psoriasin in breast tumors is currently unknown, it is interesting to speculate from this pattern of expression that psoriasin may be important in the invasive phenotype.16 This role might be mediated through an indirect influence on the effector cells of the host immune response or perhaps through a more direct influence on the epithelial tumor cell. The first hypothesis is supported by the correlation seen here with the degree of host inflammatory cell response within breast tumors and the previous evidence that implicates psoriasin as a chemotactic factor.14 However, psoriasin protein was only detected in approximately 50% of medullary and ductal tumors with marked inflammatory responses. The second hypothesis is supported by our observation that psoriasin may not only be secreted13,15 but also can be localized in both nuclear and cytoplasmic compartments in normal skin and breast tumors. Although further studies beyond immunohistochemistry are necessary to confirm this observation, the pattern of expression is consistent between cells in two closely related epithelia, epidermis and breast ductal epithelium, and the detection of nuclear and cytoplasmic signal was unrelated to tissue fixation or immunohistochemistry protocol, which may effect staining with some antibodies.35,36 Dual localization and alteration of the subcellular localization with disease has also been observed with another S100 related keratinocyte protein, profilaggrin, expressed in the epidermis.2,37 Similarly, altered cellular distribution of proteins such as BRCA1 and B-catenin are also recognized to be an important aspect of tumor progression.38-40 Furthermore, other S100 proteins have previously been associated with both extracellular and intracellular actions,41 and previous studies have also indicated potential interactions for S100A4 with both cytoskeletal8,9 and nuclear42 proteins. It has also recently been shown that other secreted S100 proteins can be localized to cytoplasm and nucleus,43,44 and specifically S100A2 has been found in breast cell nuclei, whereas S100A6 localizes to the cytoplasm;24 however, the functional significance of these findings remains unknown.
In conclusion, we have shown that expression of psoriasin (S100A7) mRNA and protein correlates with indicators of poor prognosis in invasive breast tumors, including ER, PR, and nodal status, but is not related to differentiation, as manifested by invasive tumor type or grade. The relationship observed between psoriasin and the inflammatory response is also compatible with a role as a chemotactic factor; however, the possibility of additional intracellular functions is raised by the presence of its nuclear localization in both skin and breast tumors. Further studies will be necessary to confirm the latter observation and pursue the biological functions of psoriasin in relation to breast tumor progression.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Medical Research Council of Canada (MRC) and the U.S. Army Medical Research and Materiel Command (USAMRMC). The Manitoba Breast Tumor Bank is supported by funding from the National Cancer Institute of Canada (NCIC). P. H. W. is an MRC Clinician-Scientist; L. C. M. is an MRC Scientist; E. L. is a recipient of a USAMRMC Postdoctoral Fellowship. T. H.-H. is a recipient of an MRC studentship award.
Accepted for publication August 24, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 S. Porre, S. Heinonen, R. Mantyjarvi, M. Rytkonen-Nissinen, O. Perola, J. Rautiainen, and T. Virtanen Psoriasin, a calcium-binding protein with chemotactic properties is present in the third trimester amniotic fluid Mol. Hum. Reprod., February 1, 2005; 11(2): 87 - 92. [Abstract] [Full Text] [PDF]
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 A. Handra-Luca, H. Bilal, J.-C. Bertrand, and P. Fouret Extra-Cellular Signal-Regulated ERK-1/ERK-2 Pathway Activation in Human Salivary Gland Mucoepidermoid Carcinoma: Association to Aggressive Tumor Behavior and Tumor Cell Proliferation Am. J. Pathol., September 1, 2003; 163(3): 957 - 967. [Abstract] [Full Text] [PDF]
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E. D. Emberley, Y. Niu, C. Njue, E. V. Kliewer, L. C. Murphy, and P. H. Watson Psoriasin (S100A7) Expression Is Associated with Poor Outcome in Estrogen Receptor-negative Invasive Breast Cancer Clin. Cancer Res., July 1, 2003; 9(7): 2627 - 2631. [Abstract] [Full Text] [PDF]
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E. D. Emberley, Y. Niu, E. Leygue, L. Tomes, R. D. Gietz, L. C. Murphy, and P. H. Watson Psoriasin Interacts with Jab1 and Influences Breast Cancer Progression Cancer Res., April 15, 2003; 63(8): 1954 - 1961. [Abstract] [Full Text] [PDF]
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A. Adeyinka, Y. Nui, T. Cherlet, L. Snell, P. H. Watson, and L. C. Murphy Activated Mitogen-activated Protein Kinase Expression during Human Breast Tumorigenesis and Breast Cancer Progression Clin. Cancer Res., June 1, 2002; 8(6): 1747 - 1753. [Abstract] [Full Text] [PDF]
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C. Enerback, D. A. Porter, P. Seth, D. Sgroi, J. Gaudet, S. Weremowicz, C. C. Morton, S. Schnitt, R. L. Pitts, J. Stampl, et al. Psoriasin Expression in Mammary Epithelial Cells in Vitro and in Vivo Cancer Res., January 1, 2002; 62(1): 43 - 47. [Abstract] [Full Text] [PDF]
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