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Background: According to the WHO, breast cancer is the most common cancer among women, affecting over 2.1 million people annually. The prolactin inducible protein (PIP) was identified by our group as an abundantly secreted protein in some human breast cancer cell lines. Studies show that over 90% of breast cancers express PIP to varying degrees and PIP expression is associated with better prognosis and patient response to chemotherapy. However, a definitive role for PIP in breast cancer pathogenesis is not known. We previously found that in addition to its role in innate immunity, PIP plays a role in adaptive immune response because its deficiency was associated with defective type 1 T helper cell (Th1) activity, a critical immune component necessary for anti-tumor immunity. Here, we directly assessed the role of PIP in the pathogenesis of breast cancer by developing transplantable mouse models of breast cancer using PIP over-expressing 4T1 mouse breast cancer cell line. Methods: Lentiviral transduction was used to generate PIP overexpressing 4T1 and its corresponding empty vector control. Fluorescent microscopy, flow cytometry and Western blot were used to confirm successful transduction and PIP expression. In vitro functional assays were performed to characterise this cell line, including XTT assay and cell counting to assess cell viability and proliferation; and scratch and Transwell migration assays to assess cell migration. The effect of PIP expression on sensitivity to doxorubicin, cisplatin, etoposide and tamoxifen was evaluated in vitro. To assess the impact of PIP expression on breast tumorigenesis in vivo, the cells were injected into syngeneic immunocompetent mice and tumor latency, size and progression were monitored. The immune phenotype and cytokine response were evaluated in the tumors, spleens and lymph nodes by flow cytometry. Results: Western blot analysis using cell lysate and culture media confirmed PIP expression and secretion respectively. In vitro functional assays showed comparable rates of proliferation, migration and response to chemotherapeutic compounds in PIP overexpressing 4T1 compared to control. In vivo studies showed that overexpression of PIP leads to delayed tumor onset, smaller tumor size in the PIP group compared to the control group. PIP group showed increased frequency of NK cells and dendritic cells and reduced frequency of CD4+IL4+ T-cells in the tumor. Conclusion: Collectively, these studies show that PIP overexpression had no significant effect on cancer cell proliferation, migration, and response to chemotherapeutic agents in vitro but affects the onset and progression of tumor in mice, suggesting that PIP does not act directly on breast cancer cells but may do so indirectly by altering the nature of immune response. Citation Format: Chidalu A. Edechi, Anne Blanchard, Sam Kung, Jude Uzonna, Yvonne Myal. Overexpression of prolactin inducible protein (PIP) in 4T1 cells leads to delayed tumor onset and reduced tumor size in experimental model of mouse breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-315.

MiRNAs have been shown to regulate numerous cellular functions including metastasis of cancer cells, and are considered promising biomarkers of disease. The major tight junction protein of epithelial cells, claudin 1, is down regulated or absent in human invasive breast cancer and is therefore considered a putative tumor suppressor. However, claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype and in vitro studies revealed that claudin 1 can regulate breast cancer cell motility and proliferation. To gain further understanding of how claudin 1 mediates its activities in breast cancer, we examined changes in global micro RNA (miRNA) gene expression when claudin 1 was over-expressed in the phenotypically basal-like human breast cancer (HBC) cell line MDA-MB231. Using next generation sequencing, followed by qPCR validation, we identified seven miRNAs (miR-9-5p, miR-9-3p, miR- let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) whose expression were altered as a consequence of claudin 1 over expression. Most of these miRNAs were down regulated and associated with tumor suppression in a variety of cancers including breast. Additionally, using gene expression profiling analysis of the claudin 1 over expressing clones, we identified a number of down regulated EMT related genes, including PDGFRB and CDH1 (E- cadherin). Expression of these genes is frequently lost during breast tumor progression. Interestingly, SPP1 and SERPINE 1, genes often associated with tumor cell migration and motility, were up regulated in the claudin 1 over expressing clones. Employing the miRNA target prediction program miRWalk, a number of validated miRNA target sites were identified on some of these deregulated genes. A target site for miR-9-5p was identified on the CDH1 sequence, whereas a target site of miR-99-5p was identified on SERPINE 1. Altogether, over 500 target genes have been validated as targets for the deregulated miRNAs. Overall, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Moreover, our data provides further evidence to suggest that claudin 1 has the potential to be a useful biomarker that identifies a subset of tumors, within the aggressive but currently poorly characterized basal-like subtype. Our study also suggests that miRNAs may be used in conjunction with claudin 1 to better characterize these cancers. Further studies are now warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer. Citation Format: Anne AA Blanchard, Anna Majer, Sarah Medina, Stephanie A. Booth, Yvonne Myal. Claudin 1 expression levels affect miRNA dynamics in human basal-like breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1923.

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

Claudin 1 is a tight junction protein which contributes to the transepithelial barrier that controls the transport of ions and small molecules in epithelial cells. Claudin 1 is also important for the organization of epithelial cell polarity and thus is crucial in maintaining the differentiated state of epithelial cells. An increasing number of studies have shown that the deregulated expression of the junctional proteins is directly or indirectly involved in cancer progression, including breast cancer. However, although claudin 1 forms the backbone of the tight junction, the role of claudin 1 in breast cancer is not known. Studies from our laboratory and others suggest that it may be that of a tumor suppressor, as it is often down regulated or absent in human invasive breast cancer. In the present study we explored mechanisms that may be involved in the down regulation of claudin 1 in human breast cancer. Using claudin 1 primers, RT-PCR analysis of several human breast cancer tissue samples resulted in the identification of a number of cDNAs, ranging in size from 217bp-615bp. Sequencing of these cDNAs revealed that they were products of variant claudin 1 transcripts, exhibiting various deletions primarily involving exon 1. Further inspection of these sequences showed that the RNA from which they were derived could not be translated into full length claudin 1 protein because of existing frame shifts and premature stop codons which may partially explain the loss of the protein in these tumors. Using genomic DNA derived from the tumors, we sequenced the claudin 1 exon coding regions in order to ascertain whether deletions or mutations could explain the aberrant transcripts. We identified several single nucleotide polymorphisms (SNPs), however, the SNPs did not appear to be directly linked to these alternate splice variants. Additionally, methylation specific PCR assay of exon 1 of the claudin 1 gene suggests distinct methylation patterns within the breast tumors. This data, in conjunction with supporting evidence obtained from the Cancer Genome Atlas study, suggests that methylation is involved in the regulation of claudin 1 expression in human breast cancer. In this study, we have identified for the first time variant claudin 1 transcripts in human breast cancer. These studies provide novel insights and reveal two possible mechanisms that may explain the down regulation of claudin 1during breast cancer progression. Since the breakdown of cell-cell interactions and the deregulated expression of the junctional proteins, are believed to be key steps in invasion and metastasis, understanding the molecular and genetic changes within tight junction molecules such as claudin 1 during breast tumorigenesis will be critical for facilitating more effective patient management. Citation Format: Anne AA Blanchard, Xiuli Ma, Teresa Zelinski, Jiuyong Xie, Steven Cooper, Yvonne Myal. Identification of variant claudin 1 transcripts in human breast tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 751. doi:10.1158/1538-7445.AM2013-751

The claudins are a family of tight junction (TJ) proteins that are crucial for the organization of epithelial cell polarity, contribute to the transepithelial barrier that controls the transport of ions and small molecules and the overall maintenance of the differentiated state of epithelial cells. An absence of, or defects in tight junctions have been associated with development of the neoplastic phenotype. Claudin 1, a member of this family forms the backbone of the TJ strand and is primarily responsible for the regulation of the paracellular pathway function in epithelial cells. However, the role of claudin 1 in breast cancer is not known, but studies from our laboratory and others suggest that it may be that of a tumor suppressor. Paradoxically, our laboratory has also shown that claudin 1 protein expression is significantly higher in a particular subset of breast cancers, the basal-like subtype, an aggressive form of breast cancer associated with poor prognosis. To begin to delineate a direct functional role for claudin 1 in basal-like breast cancer (BLBC), we generated in vitro, a stably transfected shRNA claudin 1 knockdown model, using the basal-like BT20 human breast cancer (HBC) cell line which over-expresses endogenous claudin 1. As well, we over-expressed claudin 1 in another basal-like HBC cell line, MDA-MB231, which expresses low levels of endogenous claudin 1. Claudin 1 gene knockdown/over-expression was confirmed by western blot analysis. Cell cultures were then evaluated for changes in proliferation and migration. The differential expression of genes associated with epithelial-mesenchymal transition in the claudin 1 gene knockdown/over-expression clones was also investigated by qPCR using PCR arrays. Several significantly differentially expressed genes, such as BMP7, PAI1and SPP1 were identified. Additionally, to examine the clinical relevance of high claudin 1 in the basal-like human invasive breast cancers, we generated a BLBC tissue microarray and interrogated the association of high claudin 1 expression and patient survival and disease recurrence. In our cohort we found no correlation with claudin 1 expression with either of these parameters, but a significant association with age (p=0.0022). High claudin 1 expression was significantly associated with BLBC from postmenopausal women > 55 years of age. The heterogeneous nature of the disease presentation of breast cancer and the limitations in identifying clinically relevant subsets of patients are major challenges for current breast cancer diagnostic and therapeutic strategies. These studies will provide information which would further define this subset of breast cancer that is particularly difficult to treat. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 350. doi:1538-7445.AM2012-350

Mouse prolactin-inducible protein (mPIP) is the homologue of the human PIP which is a known marker of both malignant and benign human breast cancer. PIP is shown to be highly expressed and secreted by submandibular, lacrimal and salivary glands. Secreted PIP in saliva has been shown to bind to bacteria suggesting that PIP may play a role in innate host defence. Furthermore, human PIP was shown to be a ligand of the CD4 molecule of T lymphocytes, acting as a potential inhibitor of T cell apoptosis suggesting a role in immune host defence. To address the role of mPIP in innate and adaptive host defence, we generated a PIP knockout mouse (PIP−/−) by targeted gene disruption. Histological as well as physiological and behavioural analyses were used to study the phenotype of these mice. Microarray was also conducted to assess the global effect of mPIP abrogation on gene expression in submandibular glands. PIP−/− mice develop normally with no obvious phenotypic differences that distinguish them from wild-type siblings. However, some adult mutant mice exhibit increased proliferative activity in lymphoid tissues, evident with enlarged submandibular lymph nodes. Light microscopy of these submandibular lymph nodes revealed activated germinal centres with an increased number of immune cells displaying high mitotic and apoptotic rates. Additional analyses showed enlarged medullary regions in the thymus of some PIP−/− mice populated with mature lymphocytes and lymphocytic aggregations within the prostate lobes. As well, analysis of blood cell profiles revealed significant increased in the number of monocytes (p In summary our data suggests a role for mPIP in modulation of adaptive and innate immune response in mice. Furthermore, our microarray analysis has revealed novel potential genes and pathways involved in mPIP signalling. (Supported by: Natural Sciences and Engineering Research Council of Canada) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3817.