Expression of both estrogen receptor-beta 1 (ER-ß1) and its co-regulator steroid receptor RNA activator protein (SRAP) are predictive for benefit from tamoxifen therapy in patients with estrogen receptor-alpha (ER-α)-negative early breast cancer (EBC).

BACKGROUND: Roles of Estrogen Receptor-beta 1 (ER-ß1) and its co-regulator Steroid Receptor RNA Activator Protein (SRAP) in breast cancer remain unclear. Previously, ER-ß1 and SRAP expression were found positively correlated in breast cancer and, therefore, expression of these two molecules could characterize cancers with a distinct clinical outcome. PATIENTS AND METHODS: ER-ß1 and SRAP expression was determined by immunohistochemistry (IHC) in tissue microarrays from a randomized, placebo-controlled trial (NCIC-CTG-MA12), designed to determine the benefit of tamoxifen following chemotherapy in premenopausal early breast cancer (EBC). Expression was dichotomized into low and high using median IHC scores. Relationships with survival used Cox modeling. RESULTS: In the whole cohort, ER-ß1 and SRAP were not prognostic. However, high ER-ß1 and SRAP significantly predicted tamoxifen responsiveness [overall survival, interaction test, P = 0.03; relapse-free survival (RFS), interaction test, P = 0.01]. Stratification by ER-α-status found predictive benefit only in ER-α-negative cases. The difference in RFS between tamoxifen and placebo was greater in patients whose tumors expressed both high SRAP and ER-ß1[hazard ratio = 0.07; 95% confidence interval (CI) 0.01-0.41; P = 0.003] versus those with low SRAP or ER-ß1 (interaction test, P = 0.02). The interaction test was not significant in ER-α-positive cohorts. CONCLUSIONS: This study provides evidence that both ER-ß1 and SRAP could be predictive biomarkers of tamoxifen benefit in ER-α-negative premenopausal EBC.

Influence of mouse prolactin-inducible protein in saliva on the aggregation of oral bacteria.

INTRODUCTION: Mouse prolactin-inducible protein (mPIP) is secreted in mouse saliva and has been found to bind oral bacteria, showing the highest affinity for streptococci. Comparisons between the oral flora of mPIP knockout mice and their wild-type controls showed differences in the genera colonizing the two groups of mice. These findings suggested a role for mPIP in the colonization of the mouse oral cavity, possibly modulating the oral flora. In this in vitro study, we focused on the contribution of this protein to aggregation of oral bacteria, a process thought to promote the clearance of bacteria from the oral cavity, and one that could influence the composition of the oral bacterial community. METHODS: The aggregation of selected human and mouse oral streptococci was measured spectrophotometrically. The aggregation of oral bacteria by saliva from mPIP knockout mice, which lack mPIP, was compared with that of saliva from wild-type mice. RESULTS: Both wild-type saliva and mPIP knockout mouse saliva induced aggregation of human strain Streptococcus gordonii SK120 and mouse streptococci strains M105/6 and M106/2. Bacterial aggregation induced by the saliva of wild-type mice was significantly higher than the aggregation induced by saliva from mPIP knockout mice for all the bacterial strains. CONCLUSION: In this study it was confirmed that mPIP plays a role in the aggregation of oral bacteria. The salivary components promoting aggregation of oral bacteria are considered to be part of the oral defense mechanisms so these findings provide insight into a possible function of mPIP in host defense by promoting aggregation of oral bacteria.

Generation and initial characterization of the prolactin-inducible protein (PIP) null mouse: accompanying global changes in gene expression in the submandibular gland.

The human prolactin-inducible protein/gross cystic disease fluid protein-15 (hPIP/GCDFP-15) is a secretory glycoprotein found primarily in apocrine tissues including the breast and salivary glands. With largely unknown functions, PIP has been implicated in breast cancer and metastasis, host defense processes and T lymphocyte apoptosis. To begin to address PIP function in vivo, we generated the PIP null mouse (Pip-/-). Additionally, to determine the effect of the loss of PIP on gene expression and to gain insight into some of the molecular mechanisms underlying PIP function, microarray analysis of the submandibular gland was also undertaken. Pip-/- mice developed normally with no overt differences in behaviour or gross morphology and were fertile. However, histological examination of 3-month-old Pip-/- mice sometimes showed enlarged submandibular lymph nodes, lymphocytic aggregations within the prostate lobes, and enlarged medulla in the thymus. Functional analysis of gene expression revealed sets of multiple differentially expressed genes associated with cell death and survival, lipid metabolism, inflammation, immune disease, and cancer, as a consequence of mPIP abrogation. Taken together, these studies lend support to an immunomodulatory role for PIP in vivo and provide further insights into potentially novel signaling pathways and regulatory networks for PIP.

Expression of small breast epithelial mucin (SBEM) protein in tissue microarrays (TMAs) of primary invasive breast cancers.

AIMS: Small breast epithelial mucin (SBEM) is a recently described gene product that shows promise as a new breast biomarker. The aim was to investigate for the first time SBEM protein expression in a large cohort (n = 300) of invasive breast cancers, its relationship to established clinical variables and its association with clinical outcome. METHODS AND RESULTS: Immunohistochemical analysis was performed on tissue microarrays consisting of 149 oestrogen receptor (ER) alpha- and 151 ERalpha+ breast cancers. Overall, 18% of tumours were SBEM+ (n = 53/300). However, SBEM protein was more frequently observed in ER- (22%) than in ER+ cancers (13%; P = 0.049). A significant association with psoriasin/S100A7 expression (P < or = 0.0001) was observed in the entire cohort. SBEM was also positively associated with HER-2 (P = 0.046) in ER- cancers, and increased levels of SBEM were strongly associated with higher tumour grade (P = 0.0015). Furthermore, SBEM expression showed a trend towards an association with reduced overall survival and relapse-free survival in the ER+ cohort (P = 0.063 and P = 0.072, respectively). CONCLUSIONS: Our results suggest that SBEM may identify a unique subset of breast cancers with poor prognosis and may have future implications for therapeutic management of this disease.

Human small breast epithelial mucin: the promise of a new breast tumor biomarker.

Breast cancer remains one of the most frequently diagnosed cancers today. In developed countries, one in eight women is expected to present with breast cancer within her lifetime and an estimated 1,000,000 cases are detected each year worldwide (Canadian Cancer Statistics, http://www.cancer.ca/vgn/images/ portal/cit_86751114/14/33/1959864 11niw_stats2004_en.pdf). For women with recurrent disease, the median time of survival is about 2 years. Despite optimal surgery, adjuvant irradiation, hormonal treatment, and chemotherapy, approximately 30% of patients with localized breast cancer finally develop distant metastases. Early detection, which enables intervention at a localized and potentially curable stage, remains a central goal in breast cancer treatment. Indeed, the 5-year survival rate for women with breast cancer has been shown to increase dramatically when the disease is diagnosed at an early stage: from less than 25% in women with disseminated cancer to about 75% in patients with regional disease and over 95% in women with a localized tumor (Breast Cancer Facts and Figures, 2001-2002, http://www.cancer.org/downloads/STT/BrCaFF 2001.pdf). Unfortunately, only 60% of all breast cancers are diagnosed at a local stage. Any improvement in early detection through identification of tumor biomarkers would have a significant impact on reducing overall breast cancer mortality.

Identification of mouse submaxillary gland protein in mouse saliva and its binding to mouse oral bacteria.

The mouse submaxillary gland protein (mSMGP) is highly expressed in the submandibular gland of the adult mouse and rat. It shares 51% identity at the amino-acid level with a human protein, the prolactin-inducible protein (PIP)/gross cystic disease fluid protein 15 (GCDFP-15), which has been found in saliva, tears, sweat, seminal plasma, submucosal glands of the lung and amniotic fluid. More recently, the human PIP has been reported to bind to bacterial strains normally found in the mouth, ear canal and human skin. Sequence analysis of mSMGP/PIP earlier identified the presence of a signal peptide, suggesting that it is a secreted protein. Here, by Western blotting, mSMGP/PIP has been identified in mouse saliva. To investigate further the role of this secreted protein, its ability to bind specifically to oral bacteria was examined; the hypothesis was that mSMGP/PIP is involved in non-immune host defence by binding to bacteria. Several bacterial strains, found to belong to the genera Streptococcus, Aerococcus, Pseudomonas, Staphylococcus, Sphingomonas, Vibrio and Aeromonas, were isolated from the mouse oral cavity. Following incubation of these bacteria with (35)S-labeled, in vitro-translated mSMGP/PIP, the protein was found to bind specifically and selectively to several but not all strains tested, showing the highest affinity for the streptococci. The protein also bound specifically to an Aerococcus sp., and a low binding interaction with the Pseudomonas and Staphylococcus spp. was observed. The conservation of SMGP sequences among several animal species suggests that this protein may play an important part in the biology of the submandibular gland. As the function of the mSMGP/PIP is still undetermined, these findings provide insight into a possible involvement of this protein in host defence.

Analysis of tissue- and hormone-specific regulation of the human prolactin-inducible protein/gross cystic disease fluid protein-15 gene in transgenic mice.

The human prolactin-inducible protein/gross cystic disease fluid protein-15 (PIP/GCDFP-15) gene is expressed in more than 90% of human breast cancer biopsies but not in the normal mammary gland. However, it is expressed in several normal human apocrine glands such as the lacrimal and salivary glands. In human breast cancer cell lines, the gene is regulated by a number of hormones including androgen and prolactin. It is not known whether gene expression in normal tissues is under similar hormonal control. To understand the mechanisms by which hormone- and tissue-specific expression of the human PIP/GCDFP-15 gene are regulated in vivo, we generated transgenic mice using a 13.7 kb genomic DNA fragment containing the entire 7 kb human gene, together with 2.9 kilobases of 5' and 3.8 kilobases of 3' flanking sequences. The human PIP/GCDFP-15 transgene was found to be expressed in both the lacrimal and salivary glands but was not expressed in the mammary glands of transgenic mice. This tissue-specific pattern of the transgene expression in the mouse was very similar to that of the endogenous human PIP/GCDFP-15 gene, and to the endogenous mouse,gene. In the mouse salivary glands, the transgene expression was highest in the parotid, considerably less in the submaxillary (submandibular) and absent in the sublingual glands. In the mouse lacrimal gland, as in the human breast cancer cell lines, the human PIP/GCDFP-15 transgene was also up-regulated by androgen. These studies demonstrate that the human gene with its 6.3 kb flanking sequences is able to confer gene expression in vivo in a tissue-specific and hormone-responsive manner.

A new member of the hormonally regulated rodent submaxillary gland glycoprotein gene family: cDNA cloning and tissue specific expression.

Polymerase chain reaction was used to amplify and identify two related rat submaxillary gland glycoprotein (rSMGGP and rSMGGP1) cDNAs. They were 489 bp and 594 bp long respectively. The shorter cDNA (rSMGGP) was identical to the previously published rat spot-I protein. The longer cDNA (rSMGGP1) had an additional (117 bp) unique nucleotide sequence in the 3' coding region, and the overall homology between the two cDNAs was 78%. rSMGGP also had a 68% homology to the mouse submaxillary gland glycoprotein (mSMGGP) cDNA. The predicted translated product of rSMGGP1 was 130 amino acids long, 39 amino acids longer than the rSMGGP. The region of greatest diversity between the putative peptides of the two rat cDNAs and the mouse cDNA was in the carboxy terminus. Northern blot analysis, using both rat cDNAs as probes, showed hybridization to an mRNA transcript (650 bases) in the submaxillary and lacrimal gland of the normal adult male and female rat. A larger transcript (approximately 700 bases) was induced under conditions of altered hormonal profiles: hypophysectomy, pregnancy/lactation, and castration. Dihydrotestosterone administration inhibited expression of the two transcripts in both the lacrimal and submaxillary glands of male and female rats. The labelled 117 bp DNA fragment unique to the rSMGGP1 cDNA hybridized only to the 700 base transcript in the rat lacrimal and submaxillary gland suggesting that differential exon usage produces the two variant mRNAs. The regulation of the SMGGP gene expression may provide yet another useful model for studying the mechanism of down-regulation of genes by androgen and the identification of tissue specific factors in the lacrimal and submaxillary gland.

Age-related changes in peptide-23/pancreatitis-associated protein and pancreatic stone protein/reg gene expression in the rat and regulation by growth hormone-releasing hormone.

Peptide-23 is a 16-kilodalton protein secreted by rat pituitary cells that was first identified because it was regulated by GRF and somatostatin in a similar fashion to GH. Cloning of peptide-23 complementary DNA revealed that it is identical to pancreatitis-associated protein (PAP) and a member of the c-lectin gene family. We examined the expression of peptide-23/PAP and a structurally related protein, pancreatic stone protein (PSP/reg), in the rat gastrointestinal tract. Here we report age-related changes in the expression and GRF regulation of peptide-23. Both peptide-23/PAP messenger RNA (mRNA) and PSP/reg mRNA were virtually undetectable in the small intestine of newborn and 1- and 2-week-old rats. A dramatic increase in the expression of both genes was seen at the time of weaning in the third week postpartum. The abundance of both of these mRNA decreases after 3 and 6 months of age. Peptide-23/PAP mRNA is most abundant in the ileum, whereas PSP/reg is maximally expressed in the pancreas and duodenum. Human GRF analog pellets were implanted sc into adult male rats for 2 weeks to study the chronic effects of GRF on the expression of these genes. Both peptide-23/PAP and PSP/reg mRNA levels in duodenum and jejunum were increased in these rats compared with levels in control rats. However, no increase in peptide-23/PAP mRNA in response to GRF treatment was seen in the ileum, where the level of expression of this gene is very high, and GRF had no effect on peptide-23/PSP expression in the heart, pituitary, or hypothalamus, where expression is normally undetectable. In situ hybridization was used to localize peptide-23/PSP in the small intestine and pancreas of GRF-treated rats. An increase in peptide-23/PAP mRNA was restricted to acinar cells close to islets, whereas little expression was seen in acinar cells distant from islets, suggesting that either peptide-23/PAP may have some paracrine action on the islets, or alternatively, an islet-derived factor may function as a paracrine modulator of peptide-23/PAP expression. These data demonstrate that GRF modulates peptide-23/PAP expression in the gastrointestinal tract in a similar fashion to that previously reported for pituitary cells in primary culture.

Molecular cloning and expression of peptide 23, a growth hormone-releasing hormone-inducible pituitary protein.

Peptide 23 is a newly identified protein secreted by rat pituitary cells in primary culture. Although the secretion of this protein is stimulated by GH-releasing hormone and inhibited by somatostatin, the N-terminal amino acid sequence of peptide 23 shows no homology to rat GH. Using the polymerase chain reaction technique, we cloned and sequenced the peptide 23 complementary DNA (cDNA). By means of the mixed oligonucleotide-primed amplification of cDNA technique, primers corresponding to the NH2-amino acid sequence of peptide 23 were used to amplify, clone, and sequence a 74-basepair cDNA of peptide 23. This polymerase chain reaction product was then used as a primer to amplify the complete peptide 23 cDNA by means of the rapid amplification of cDNA ends procedure. The cDNA of peptide 23 obtained by the rapid amplification of cDNA ends procedure contained 777 nucleotides and encoded a 175-amino acid protein with a 26-amino acid putative signal peptide. The calculated mol wt of the mature protein (16,613 daltons) was in good agreement with that estimated by polyacrylamide gel electrophoresis (16 kilodaltons). Northern blot analysis revealed a major messenger RNA species of about 0.9 kilobase and a minor species of about 1.7 kilobases in cultured rat anterior pituitary cells. In rats, peptide 23 was most abundant in the pancreas and gastrointestinal tract. A GenBank sequence search revealed complete sequence identity between peptide 23 cDNA and pancreatitis-associated protein cDNA, an approximately 73% homology with human hepatocellular carcinoma cDNA from human hepatocellular carcinoma, 64% homology with bovine pancreatic thread protein cDNA, and 55% homology with rat and human reg cDNAs, which have been reported to be expressed in regenerating pancreatic islets. Therefore, peptide 23 is identical to pancreatitis-associated protein and a member of the C-type lectin supergene family.

Tissue-specific androgen-inhibited gene expression of a submaxillary gland protein, a rodent homolog of the human prolactin-inducible protein/GCDFP-15 gene.

The human PRL-inducible protein (PIP)/gross cystic disease fluid protein-15 is expressed in pathological conditions of the mammary gland and in several exocrine tissues, such as the lacrimal, salivary, and sweat glands. In human breast cancer cells, the expression of PIP/gross cystic disease fluid protein-15 is stimulated by androgen and PRL, and inhibited by estrogen. However, it is not known whether the expression of PIP in other tissues is under similar hormonal regulation. In the present study we employed reverse transcriptase-polymerase chain reaction followed by rapid amplification of complementary DNA (cDNA) ends to amplify the PIP cDNA homolog, the submaxillary gland protein (SMGP) in the mouse. The mouse PIP/SMGP cDNA encodes a putative secreted peptide of 144 amino acids with a 51% identity with human PIP. Using the mouse PIP/SMGP cDNA as a probe, we examined the tissue- and cell-specific expression of PIP/SMGP messenger RNA by in situ hybridization and Northern blot analysis of mouse and rat tissues. Hormonal regulation was also studied in the rat. PIP/SMGP messenger RNA expression was only detected in the lacrimal and submaxillary glands of the rodents. In the rat submaxillary gland, PIP/SMGP gene expression was confined to the acinar cells. In the male rat lacrimal gland, castration resulted in an increase in expression, and in both male and female rats, androgen replacement abolished PIP/SMGP gene expression. This pattern of regulation was not observed in the submaxillary gland and was actually reversed in human breast cancer cells. PRL had no effect on the regulation of PIP/SMGP in either salivary or lacrimal glands. Our study indicates that tissue-specific factors are important in determining the hormone responsiveness of the PIP/SMGP gene. Regulation of the PIP/SMGP gene in vivo may provide a useful model system to study the mechanism of down-regulation of expression by androgen in a tissue-specific manner.

Identification and cloning of a new category of DNA fragments which are poorly represented in human genomic libraries.

We have developed an alternative strategy for the preparation of genomic libraries that ensures better representation of genomic sequences commonly underrepresented in genomic libraries constructed using standard protocols. To overcome the apparent bias against genomic sequences containing clusters of restriction sites we have used nonoptimized restriction digestions to generate a mixture of DNA fragments which have been cloned into the EMBL3 vector. To validate this protocol we have screened the EMBL3 library to identify a full length genomic clone of the prolactin-inducible gene (PIP). Screening 4 other, commercially available, genomic libraries prepared using standard protocols for restriction digestion of the genomic DNA failed to identify any full length clones. We show that this increase in the representation of the full length PIP gene in the EMBL3 genomic library is attributable to the method of insert preparation used and suggests that an additional subset of sequences that may be poorly represented in, or absent from, established libraries may be cloned using this modified protocol.

PCR detection of the rat brain basic fibroblast growth factor (bFGF) mRNA containing a unique 3' untranslated region.

We utilized the reverse transcription-polymerase chain reaction (RT-PCR) to detect the presence of basic fibroblast growth factor (bFGF) messenger RNA in rat brain, ovary and kidney. The nucleotide sequence of the RT-PCR product revealed a novel 3' untranslated (UT) sequence in the rat basic FGF mRNA. In this sequence, as in the 3' UT regions of many growth regulatory genes, there is a high degree of conservation of A+T rich motifs which have been shown to play a major role in mRNA stability.

The prolactin-inducible protein (PIP/GCDFP-15) gene: cloning, structure and regulation.

The androgen and prolactin responsive prolactin-inducible protein (PIP)/gross cystic disease fluid protein (GCDFP-15) is expressed in benign and malignant human breast tumors and in such normal exocrine organs as sweat, salivary and lacrimal glands. In this paper we report the cloning and structure of the human gene, and describe potential mechanisms involved in its regulation by hormones. The entire PIP gene, 7 kb long, was found in a single recombinant phage clone. The gene has 4 exons ranging from 106 bp to 223 bp in length. Nuclear run-on experiments utilizing PIP genomic fragments to detect nascent PIP transcripts revealed that both androgen and prolactin increased transcription of the PIP gene. Neither hormone had any effect on the stability of PIP precursor RNA or mature mRNA. Therefore the PIP gene is an excellent model by which to study the molecular events associated with the actions of prolactin and androgen in the regulation of gene expression in mammalian cells.

Genetic linkage between the Kell blood group system and prolactin-inducible protein loci: provisional assignment of KEL to chromosome 7.

The Kell blood group locus (KEL) is tightly linked to the prolactin-inducible protein locus (PIP) with zeta = 9.12 at theta = 0.00 for combined paternal and maternal meioses. In view of the regional localization of PIP to 7q32-q36 (Myal et al. 1989a), a similar assignment for KEL is favoured.

The gene for prolactin-inducible protein (PIP), uniquely expressed in exocrine organs, maps to chromosome 7.

The hormonally responsive prolactin-inducible protein (PIP) gene is expressed in benign and malignant breast tumor tissues and in such normal exocrine organs as sweat, salivary, and lacrimal glands. In this communication we report the regional chromosome localization of the PIP gene locus to chromosome 7 by Southern hybridization to DNA from human-hamster somatic cell hybrids, and to 7q32-36 by in situ hybridization.