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Prolactin-inducible protein (PIP) is a multifunctional glycoprotein that is highly expressed and found in the secretions of apocrine glands such as salivary, lacrimal, and sweat glands including the mammary glands. PIP has been implicated in various diseases, including breast cancer, gross cystic disease of the breast, keratoconus of the eye, and the autoimmune Sjögren’s syndrome. Here we have generated a Pip knockout (KO) mouse using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRSPR-associated (Cas)9 system. The Cas9 protein and two single guide RNAs targeting specific regions for both exons 1 and 2 of the Pip gene were microinjected into mouse embryos. The deletions and insertions promoted by CRISPR/Cas9 system on the Pip gene successfully disrupted Pip protein coding, as confirmed by PCR genotyping, sequencing, and ultimately Western blot analysis. This mouse model was generated in the inbred C57Bl/6J mouse, which exhibits lower genetic variation. This novel CRISPR Pip KO mouse model will not only be useful for future studies to interrogate the multifunctional role of PIP in physiological processes but will facilitate a broader understanding of the function of PIP in vivo while providing unprecedented insight into its role in a spectrum of diseases attributed to the deregulation of the PIP gene.

The emerging significance of the bitter taste receptors (T2Rs) role in the extraoral tissues alludes to their potential role in many pathophysiological conditions. The dysregulation of T2R expression and function in disease conditions has now been demonstrated in airways diseases, neurological disorders, and in some cancers. However, the role of T2Rs in the pathophysiology of breast cancer is unexplored thus far. Previously, we demonstrated differential expression of the 25 T2Rs in breast cancer (BC) cells. Based on our previous findings we selected two T2Rs, T2R4 and T2R14 for this work. The objective of the current study is to investigate the expression of T2R4 and T2R14 in BC clinical samples and to examine their physiological role using highly metastatic BC and non-cancerous cell lines. Using approaches, which involve receptor knockdown, pharmacological activation and biochemical assays we report that (i) T2R4 and T2R14 expression patterns are dissimilar, with decreased levels of T2R4 and increased levels of T2R14 in BC clinical samples compared to non-cancerous controls. (ii) Activation of T2Rs with their respective agonist elicited physiological responses in metastatic breast cancer cells, and no responses were seen in non-tumorigenic breast epithelial cells. (iii) Agonist activation of T2Rs (irrespective of T2R subtype) induced anti-proliferative, pro-apoptotic, and anti-migratory responses in highly metastatic breast cancer cells. Taken together, our findings demonstrate that the chemosensory T2R signaling network is involved in evoking physiological responses in the metastatic breast cancer cell line.

Deemed a putative tumor suppressor in breast cancer, the tight junction protein claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype. Moreover, recent in vitro studies show that claudin 1 can regulate breast cancer cell motility and proliferation. Herein, we investigated whether microRNA (miRNA) dysregulation is associated with alterations in the level of claudin 1. Using next-generation sequencing (NGS), we identified seven miRNAs (miR-9-5p, miR-9-3p, let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) that were deregulated as a consequence of claudin 1 overexpression in the MDA-MB231 human breast cancer (HBC) cell line. Most of these miRNAs have been associated with tumor suppression in a variety of cancers, including breast cancer. Moreover, through gene expression profiling analysis, we identified epithelial-mesenchymal transition-related genes, including platelet-derived growth factor receptor-beta (PDGFRB) and cadherin 1 (CDH1, E cadherin), whose downregulation correlated with claudin 1 overexpression. Collectively, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Our data suggest that the dysregulated expression of these miRNAs, in conjunction with the high claudin 1 levels, could serve as a useful biomarker that identifies a subset of tumors within the poorly characterized basal-like subtype of breast cancer. Further studies are warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer.

Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.

The steroid receptor RNA activator protein (SRAP) is a newly described protein modulating the activity of multiple transcription factors including the estrogen receptor (ER). We have recently reported the immunodetection by Western blot of multiple SRAP peptides in breast tissue. High expression of these peptides, assessed by tissue micro-array (TMA) analysis, was associated with poor prognosis in patients whose primary tumors were ER positive (ER+). In such studies, it is recognized that intensity as well as specificity of the signal detected directly depends upon the antibody used as well as the position of the epitope recognized. To confirm the potential relevance of SRAP as a new prognostic factor, it is critical to establish whether similar results are obtained with independent antibodies.Two commercial anti-SRAP antibodies (742A and 743A), respectively, recognizing the N- and C-terminal extremity of the protein, were first used to analyze by Western blot SRAP expression in protein extracts from frozen breast tumor tissue sections. These antibodies were further used to investigate by immunohistochemistry (IHC) SRAP location in paraffin-embedded breast tumors. Comparative TMA analysis of 170 ER+ tumors was eventually performed in order to establish the potential associations existing between SRAP expression and clinical outcome.Multiple SRAP peptides were differentially detected by Western blot. Both antibodies led to similar nuclear and cytoplasmic staining in breast tissue section. A solid correlation was found (Spearman r = 0.46, P0.001) between 742A and 743A IHC scores. Results from both antibodies independently showed that dividing expression levels into lower 25 percentile, 26-75 percentile, and highest 25 percentile demonstrated a hazard ratio (HR) of 1.82 (P = 0.0042) for 742A antibody and 1.35 (P = 0.14) for 743A antibody. When both scores are combined, double high expressor (by 742A and 743A) was associated with a poor prognosis of breast-cancer-specific survival (Mantel-Cox: P = 0.005, HR = 2.24).Overall, our data suggest the existence in breast tumor tissue of multiple SRAP-like peptides. Assessing their expression in primary breast tumors can predict clinical outcome in ER+ breast cancer patients.

The propensity for breast cancers to elicit immune responses in patients is well established. The accumulation of tumor infiltrating lymphocytes within the primary breast tumor has been linked to better prognosis and better response to therapy. The prolactin-inducible protein (PIP) is a 15 kD protein that is expressed under physiological conditions of the breast and is regarded as a marker of mammary differentiation. While highly expressed under pathological conditions of the mammary gland, including breast cancers, PIP is expressed in very few other cancers. Although the function of PIP is not well elucidated, numerous studies suggest that its primary role may be related to host defense and immune modulation. However, evidence to show a direct link between PIP and the immune response has been lacking. In this review, we discuss our recent work with Pip-deficient mice, linking PIP not only to a role in innate immunity but for the first time, providing evidence for a role in cell-mediated immunity. These functional studies in Pip null mice lend new insight into the role of PIP in immunity and suggest that PIP may play a similar immune-regulatory role in breast cancer.

Background The claudin 1 tight junction protein, solely responsible for the barrier function of epithelial cells, is frequently down regulated in invasive human breast cancer. The underlying mechanism is largely unknown, and no obvious mutations in the claudin 1 gene (CLDN1) have been identified to date in breast cancer. Since many genes have been shown to undergo deregulation through splicing and mis-splicing events in cancer, the current study was undertaken to investigate the occurrence of transcript variants for CLDN1 in human invasive breast cancer. Methods RT-PCR analysis of CLDN1 transcripts was conducted on RNA isolated from 12 human invasive breast tumors. The PCR products from each tumor were resolved by agarose gel electrophoresis, cloned and sequenced. Genomic DNA was also isolated from each of the 12 tumors and amplified using PCR CLDN1 specific primers. Sanger sequencing and single nucleotide polymorphism (SNP) analyses were conducted. Results A number of CLDN1 transcript variants were identified in these breast tumors. All variants were shorter than the classical CLDN1 transcript. Sequence analysis of the PCR products revealed several splice variants, primarily in exon 1 of CLDN1; resulting in truncated proteins. One variant, V1, resulted in a premature stop codon and thus likely led to nonsense mediated decay. Interestingly, another transcript variant, V2, was not detected in normal breast tissue samples. Further, sequence analysis of the tumor genomic DNA revealed SNPs in 3 of the 4 coding exons, including a rare missense SNP (rs140846629) in exon 2 which represents an Ala124Thr substitution. To our knowledge this is the first report of CLDN1 transcript variants in human invasive breast cancer. These studies suggest that alternate splicing may also be a mechanism by which claudin 1 is down regulated at both the mRNA and protein levels in invasive breast cancer and may provide novel insights into how CLDN1 is reduced or silenced in human breast cancer.

Elevated production of proprotein convertases (PCs), proteolytic enzymes that posttranslationally modify the biological activities of diverse groups of cellular proteins, is a common occurrence in human breast carcinomas. A transgenic mouse model was developed to gain insight into the significance of PC production in breast development and neoplasia. Mammary epithelium-specific and early expression of PC1 was targeted by the use of the mouse mammary tumor virus promoter/enhancer. Whole-mount examinations revealed that the mammary glands of 83-day-old virgin PC1 transgenic mice exhibited an accelerated lobuloalveolar development compared with that of age-matched wild-type mice (p < 0.001). This phenotypic change was accompanied by extensive alterations in gene expression assessed by gene expression microarray analyses. Pathway analysis of PC1-induced alterations in gene expression has revealed possible mechanism of action of PC1 in the mammary gland. PC1 expression alone, however, did not promote spontaneous mammary tumorigenesis in the transgenic mice. PC1 transgene expression resulted in a significantly higher incidence (p = 0.008) and accelerated growth (p = 0.023) of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas. The present study therefore shows that PC1 expression can promote normal and neoplastic mammary development and growth and suggests that proprotein convertases may be important etiological factors in human breast neoplasia.

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−/−mouse). 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.

The steroid receptor RNA activator gene (SRA1) produces both a functional RNA (SRA) and a protein (SRAP), whose exact physiological roles remain unknown. To identify cellular processes regulated by SRAP we compared the transcriptome of Hela and MDA-MB-231 cancer cells upon depletion of the SRA/SRAP transcripts or overexpression of the SRAP protein. RNA-seq and Ontology analyses pinpointed cellular movement as potentially regulated by SRAP. Using live cell imaging, we found that SRA/SRAP depletion and SRAP overexpression lead respectively to a decrease and increase in cancer cell motility. Our results highlight for the first time a link existing between SRA1 gene expression and cell motility.

Background Knowledge of the mouse salivary proteome is not well documented and as a result, very limited. Currently, several salivary proteins remain unidentified and for some others, their function yet to be determined. The goal of the present study is to utilize mass spectrometry analysis to widen our knowledge of mouse salivary proteins, and through extensive database searches, provide further insight into the array of proteins that can be found in saliva. A comprehensive mouse salivary proteome will also facilitate the development of mouse models to study specific biomarkers of many human diseases. Results Individual saliva samples were collected from male and female mice, and later pooled according to sex. Two pools of saliva from female mice (2 samples/pool) and 2 pools of saliva from male mice were used for analysis utilizing high performance liquid chromatograph mass spectrometry (nano-RPLC-MS/MS). The resulting datasets identified 345 proteins: 174 proteins were represented in saliva obtained from both sexes, as well as 82 others that were more female specific and 89 that were more male specific. Of these sex linked proteins, twelve were identified as exclusively sex-limited; 10 unique to males and 2 unique to females. Functional analysis of the 345 proteins identified 128 proteins with catalytic activity characteristics; indicative of proteins involved in digestion, and 35 proteins associated with stress response, host defense, and wound healing functions. Submission of the list of 345 proteins to the BioMart data mining tool in the Ensembl database further allowed us to identify a total of 283 orthologous human genes, of which, 131 proteins were recently reported to be present in the human salivary proteome. Conclusions The present study is the most comprehensive list to date of the proteins that constitute the mouse salivary proteome. The data presented can serve as a useful resource for identifying potentially useful biomarkers of human health and disease. Electronic supplementary material The online version of this article (doi:10.1186/s12953-015-0068-3) contains supplementary material, which is available to authorized users.

International audience; 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. 1 (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.

Products of the steroid receptor RNA activator (SRA1) gene have the unusual property to function both at the RNA and the protein levels. SRA-RNA has long been known to increase the activity of multiple nuclear receptors. It has more recently been proposed than steroid receptor RNA activator protein (SRAP) also modulates steroid receptors activity. Herein, we show for the first time that SRAP physically interacts with multiple transcription factors and is recruited to specific promoter regions. Artificially recruiting SRAP to the promoter of a luciferase reporter gene under the control of the strong transcriptional activator VP16 leads to a decrease in transcription. Altogether we propose that SRAP could be a new transcriptional regulator, able to function as a repressor through direct association with promoters.Structured summaryMINT-7761068: SRAP (uniprotkb:Q9HD15) physically interacts (MI:0915) with HDAC2 (uniprotkb:Q92769) by anti bait coimmunoprecipitation (MI:0006)

The steroid receptor RNA activator gene (SRA1) encodes for a functional RNA (SRA) as well as a protein (SRAP). While several groups reported on SRA-RNA mechanism of action, SRAP exact function remains to be elucidated, mainly due to a lack of studies investigating the function of the protein independently of its RNA counterpart. Using two independent models to examine its specific functions, SRAP was found to enhance estrogen receptor alpha activity in a ligand and response-element dependent manner. Our data therefore suggest that both transcript and protein products of the SRA1 gene co-modulate the transcriptional activity of steroid receptors.

Products of the Steroid Receptor RNA Activator gene (SRA1) have the unusual property to modulate the activity of steroid receptors and other transcription factors both at the RNA (SRA) and the protein (SRAP) level. Balance between these two genetically linked entities is controlled by alternative splicing of intron-1, whose retention alters SRAP reading frame. We have previously found that both fully-spliced SRAP-coding and intron-1-containing non-coding SRA RNAs co-exist in breast cancer cell lines. Herein, we report a significant (Student's t-test, P < 0.003) higher SRA–intron-1 relative expression in breast tumors with higher progesterone receptor contents. Using an antisense oligoribonucleotide, we have successfully reprogrammed endogenous SRA splicing and increased SRA RNA–intron-1 relative level in T5 breast cancer cells. This increase is paralleled by significant changes in the expression of genes such as plasminogen urokinase activator and estrogen receptor beta. Estrogen regulation of other genes, including the anti-metastatic NME1 gene, is also altered. Overall, our results suggest that the balance coding/non-coding SRA transcripts not only characterizes particular tumor phenotypes but might also, through regulating the expression of specific genes, be involved in breast tumorigenesis and tumor progression.

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.

International audience; TABLE OF CONTENTS 1. Abstract 2. Introduction 2. Material and Methods 2.1. Cell Culture 2.2. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) 2.3. Rapid amplification of the 5'-cDNA end (RACE) 2.4. Identification of transcription factors binding sites within the 87-bp enhancer region (ENH) 2.5. Plasmids 2.6. Transient DNA transfections and luciferase assays 3. Results 3.1. Identification of the transcription initiation sites 3.2. Endogenous SBEM promoter activity in mammary and non-mammary cancer cells 3.3. Analysis of SBEM promoter activity in mammary and non-mammary cancer cells 3.4. The ENH region (-357/-270) drives a strong breast-specific promoter activity 3.5. Importance of octamer-binding transcription factors motif in the SBEM promoter activity 3.6. Oct1 and Oct2 enhance both exogenous and endogenous SBEM promoter activities 4. Discussion 5. Acknowledgement 6. References 1. ABSTRACT The human small breast epithelial mucin (SBEM) gene has been identified as being preferentially expressed in mammary epithelial cells and over-expressed in breast tumors. In this report, we have characterized the promoter of SBEM gene in order to identify sequences responsible for this strong mammary expression. A series of SBEM promoter/luciferase constructs were transiently transfected into both breast (MCF-7, BT-20) and non-breast (HeLa and HepG2) cell lines. In addition to the minimal promoter and to a repressor region, we have identified an 87-bp sequence (-357/-270) driving a strong breast-specific expression. Site-directed mutagenesis of a putative octamer-binding transcription factor binding site located within this latter region led to a strong decrease of the transcriptional activity of the SBEM promoter. Furthermore, transient over-expression of Oct1 and Oct2 not only increased SBEM promoter reporter activity, but also enhanced endogenous SBEM mRNA level. Overall, the data suggest that octamer-binding transcription factors participate in the strong expression of SBEM gene in breast tissues. Clarifying the SBEM gene regulation will help to dissect mechanisms underlying transcription of normal breast and breast cancer-associated genes.

The Steroid Receptor RNA Activator 1 (SRA1) has originally been described as a noncoding RNA specifically activating steroid receptor transcriptional activity. We have, however, identified, in human breast tissue, exon- 1 extended SRA1 isoforms containing two initiating AUG codons and encoding a protein we called SRAP. We recently reported a decreased estrogen receptor activity in breast cancer cells overexpressing SRAP, suggesting antagonist roles played by SRA1 RNA and SRAP. SRA1 appears to be the first example of a molecule active both at the RNA and at the protein level. No data are currently available regarding the mechanisms possibly involved in the generation of coding and noncoding functional SRA1 RNAs. Using 5'-Rapid Amplification of cDNA Extremities (5'-RACE), we have herein identified several putative transcription initiation sites surrounding the second methionine codon and used to generate coding SRA1 transcripts. In the process, we also identified an alternatively spliced noncoding SRA1 transcript still containing an intron-1 sequence. Using targeted RT-PCR approaches, we confirmed the presence in breast cancer cell lines of SRA1 RNAs containing a full as well as a partial intron-1 sequence and established that the relative proportion of these RNAs varied within breast cancer cell lines. Using a "minigene" strategy, we also showed that artificial RNAs containing the SRA1 intron-1 sequence are alternatively spliced in breast cancer cell lines. Interestingly, the splicing pattern of the minigene products parallels the one of the endogenous SRA1 transcripts. Altogether, our data suggest that the primary genomic sequence in and around intron-1 is sufficient to lead to a differential splicing of this intron. We propose that alternative splicing of intron-1 is one mechanism used by breast cancer cells to regulate the balance between coding and functional noncoding SRA1 RNAs.

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.

Background The human psoriasin (S100A7) gene has been implicated in inflammation and tumor progression. Implementation of a mouse model would facilitate further investigation of its function, however little is known of the murine psoriasin gene. In this study we have cloned the cDNA and characterized the expression of the potential murine ortholog of human S100A7/psoriasin in skin inflammation and mammary tumorigenesis. Methods On the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities of the patterns of mouse S100A7/psoriasin gene expression (measured by RT-PCR and in-situ hybridization) with those of human S100A7/psoriasin, we propose that mouse S100A7/psoriasin is the murine ortholog of human psoriasin/S100A7. Results Although mouse S100A7/psoriasin is poorly conserved relative to other S100 family members, its pattern of expression parallels that of the human psoriasin gene. In murine skin S100A7/psoriasin was significantly upregulated in relation to inflammation. In murine mammary gland expression is also upregulated in mammary tumors, where it is localized to areas of squamous differentiation. This mirrors the context of expression in human tumor types where both squamous and glandular differentiation occur, including cervical and lung carcinomas. Additionally, mouse S100A7/psoriasin possesses a putative Jab1 binding motif that mediates many downstream functions of the human S100A7 gene. Conclusion These observations and results support the hypothesis that the mouse S100A7 gene is structurally and functionally similar to human S100A7 and may offer a relevant model system for studying its normal biological function and putative role in tumor progression.

The insulin-like growth factor I receptor (IGF-IR) is expressed in many cell types and is critical for normal growth and development. In the healthy mammary gland, the role of IGF-IR is not fully elucidated. However, IGF-IR, which is primarily expressed in the mammary epithelial cells, is known to play an obligatory role in cellular transformation, facilitating the progression to breast cancer. We have utilized the tetracycline regulatory (tet-on) system to generate an in vitro model system to allow us to further investigate IGF-I/IGF-IR function in mammary epithelial cells. A plasmid construct containing a mutant IGF-I receptor (IGF-IR-DN) fused to the tetracycline operator (tetOPh(CMV)-IGF-IR-DN) was stably transfected into MCF-7 human breast cancer cells. The conditional regulation of the IGF-IR-DN gene expression was studied in four independent clonal lines. The translated IGF-IR-DN protein was detected only in the stably transfected doxycycline- induced cells, and its expression was up-regulated (three- to sixfold) following induction. IGF-I stimulated cell proliferation diminished (twofold) in doxycycline- induced cells compared to uninduced cells, demonstrating that the transgene construct was functional and ruling out any pleiotropic effect that may be attributed to doxycycline. Interestingly, autophosphorylation of the IGF-IR and phosphorylation of the downstream substrate, insulin receptor substrate-1 (IRS-1), was not inhibited in doxycycline/IGF-I treated cells, suggesting the possibility that activation of downstream substrates other than the IRS-1 may be critical for optimal cell proliferation. This novel in vitro model should allow us to more directly examine the role of IGF-I/IGF-IR signaling and function in mammary epithelial cells.

The function of the mouse submaxillary gland/prolactin inducible protein (mSMGP/mPIP), the homologue of the human gross cystic disease fluid protein 15 (GCDFP-15)/prolactin inducible protein (hPIP) remains unknown. The human gene, normally expressed in apocrine glands of healthy individuals, is aberrantly expressed in human breast cancers where it is regulated by hormones including androgens, and in prostate cancers. We have previously reported that in the adult mouse and rat, gene expression is tissue-specific for the salivary and lacrimal glands, and is hormonally regulated. In this study, we examine the endogenous pattern of mouse SMGP/PIP (mSMGP/mPIP) gene expression in mid- and late-embryonic, and in early postnatal development. Gene expression was analyzed by RT-PCR followed by Southern blot analysis, and by in situ hybridization. Gene expression was detected in the submandibular gland as early as embryonic day 14 (E14), a period that coincides with the initiation of submandibular gland development in the embryo, suggesting that mSMGP/mPIP may have a functional role in the developing gland. Nearing the end of gestation, E18, mSMGP/mPIP transcripts were localized in the proacinar cells of the gland, and gene expression continued to be maintained following birth. In addition, during early postnatal development, mSMGP/mPIP gene expression was detected in the other two major salivary glands, the sublingual and parotid, as well as in the lacrimal gland and in reproductive tissues. In the prostate, gene expression was turned off by 10 weeks of age. The spatial and temporal pattern of the mSMGP/mPIP gene expression, in addition to our recent demonstration that mSMGP/mPIP is found in mouse saliva and can bind bacteria, suggest that this protein may have a protective role in the mouse.

The human Prolactin-Inducible-Protein (PIP)/Gross Cystic Disease Fluid Protein 15 (GCDFP-15) gene is abnormally expressed in human invasive breast cancer and gross cystic disease of the breast. In healthy individuals PIP is highly expressed in salivary, sweat and lacrimal glands, and the secreted protein abundantly found in saliva as well as other bodily fluids which bathe various ports of entry. PIP has been shown to bind numerous bacterial strains as well as a number of immunoregulatory molecules including the CD4-T cell receptor and IgG and is therefore implicated to play an immune role in host defense. In the present study we interrogated the salivary proteome of the PIP knockout (Pip KO) mouse by mass spectrometry to determine protein changes that occur in saliva in the absence of PIP. Proteomic analysis identified 165 proteins in the mouse saliva which had a minimum of 2 unique peptides at the 99% confidence level. Nineteen proteins, including 2 proposed markers of disease, S100A9 and adenosine deaminase, were significantly differentially expressed in the saliva of the Pip KO mouse, compared to the saliva of the wild type counterparts. Functional and biological determinations revealed that the differentially expressed proteins were associated with processes that include inflammatory responses, immunity and cell death. This study provides evidence that a number of proteins associated with host defense are affected by the loss of PIP, and lends support for an immunomodulatory role for PIP. Citation Format: Anne Blanchard, Peyman Ezzati, Xiuli Ma, John Wilkins, Yvonne Myal. Analysis of the salivary proteome of the prolactin-inducible-protein knockout mouse. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2014-2488

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.

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.

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.

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.

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.

Human tumors were analyzed for the presence of mRNA coding for basic fibroblast growth factor (basic FGF). Basic FGF transcript levels were consistently elevated in schwannoma samples (five acoustic neuromas and two spinal schwannomas) ranging from 9- to 22-fold higher than the average level of expression in four benign meningioma samples. Acidic extracts of acoustic neuromas contained a potent mitogen which bound to heparin-Sepharose, eluted at 2 M NaCl, and cross-reacted with an N-terminal specific anti-basic FGF antiserum. The present findings indicate that basic FGF appears to be the major heparin-binding endothelial cell mitogen in acoustic neuromas. Southern restriction analysis revealed no evidence of amplification or rearrangement of the gene for basic FGF in schwannomas or in the astrocytoma cell line U87-MG. These findings demonstrate a tumor-specific elevation in basic FGF transcript levels in tumors of Schwann cell origin and suggest that increased transcription or stabilization of basic FGF mRNA may play an autocrine role in the development and progression of these tumors.

We have initiated an analysis of the action of prolactin in a human breast cancer cell line (T-47D) by isolating and sequencing cDNA clones for a prolactin-inducible protein (PIP). PIP cDNA (approximately 600 base pairs) was isolated from a lambda gt11 expression library prepared from mRNA extracted from T-47D cells treated with prolactin and hydrocortisone. The identity of PIP cDNA was confirmed by hybrid-selected translation. PIP mRNA is a single mRNA species of approximately 900 bases which responds to lactogenic hormones (human prolactin and growth hormone) in a time- and dose-dependent manner. Treatment of T-47D cells with human growth hormone (hGH) increased PIP mRNA levels by 4.6-fold, while the combination of hGH and hydrocortisone or dihydrotestosterone had a more dramatic, 12.4-fold, effect. The latter steroid was the most potent. Dihydrotestosterone plus hGH increased transcription of the PIP gene by 3.7-fold. Dihydrotestosterone alone was as effective as dihydrotestosterone plus hGH in increasing the transcription rate, while hGH alone had no effect. Thus, lactogen may regulate PIP gene expression at a post-transcriptional step. PIP mRNA was expressed at low levels in other human breast cancer cell lines which were prolactin receptor-positive; MCF-7 and EFM-19 lines were exceptions. PIP cDNA had an open reading frame of 146 amino acids, sufficient to encode the precursor form of the secreted PIP protein (approximately 14.5 kDa). The similarity of the predicted amino acid sequence of PIP to the sequence of a protein encoded by a gene expressed in the mouse submaxillary gland prompted us to look for PIP in human saliva. Western blot analysis confirmed the presence of PIP in human saliva. Therefore, the PIP gene is useful in studying the molecular actions of the prolactin/growth hormone polypeptide hormone family and the interaction with androgen, in mammary and other potential target cells.