Anne Blanchard

Claudins 1, 3, and 4 protein expression in ER negative breast cancer correlates with markers of the basal phenotype

In the present study we investigated the protein expression of claudins 1, 3, and 4 and their relationship to clinical variables and outcome in a cohort of ER−ve and ER+ve human invasive breast cancers. Immunohistochemical analysis was performed on tissue microarrays representing a total of 412 tumors and interpretable data was derived from 314, 299, and 306 tumors for claudins 1, 3, and 4, respectively. In the ER+ve subset, 5%, 89%, and 52%, and in the ER−ve subset, 39%, 79%, and 79% of tumors stained positively for claudins 1, 3, and 4, respectively (p < 0.0001, p = 0.026, p < 0.0001). Thus, in the two subsets, a significantly higher number of tumors were positive for claudins 3 and 4, compared to claudin 1. In addition, protein expressions of claudins 1 and 4 were significantly higher in those tumors that displayed characteristics of the basal-like subtype of breast cancers (ER−ve, Her-2−ve, EGFR+ve, CK5/6+ve). This study shows a unique pattern of expression for the different claudins in ER−ve and ER+ve tumors. Our data also suggests that increased expression of claudins 1 and 4 was associated with the basal-like subtype of breast cancers, a subtype generally linked to poor outcome.

Claudin 1 in Breast Tumorigenesis: Revelation of a Possible Novel “Claudin High” Subset of Breast Cancers

Claudins are the major component of the tight junctions in epithelial cells and as such play a key role in the polarized location of ion channels, receptors, and enzymes to the different membrane domains. In that regard, claudins are necessary for the harmonious development of a functional epithelium. Moreover, defective tight junctions have been associated with the development of neoplastic phenotype in epithelial cells. Breakdown of cell-cell interactions and deregulation of the expression of junctional proteins are therefore believed to be key steps in invasion and metastasis. Several studies suggest that the claudins are major participants in breast tumorigenesis. In this paper, we discuss recent advances in our understanding of the potential role of claudin 1 in breast cancer. We also discuss the significance of a subset of estrogen receptor negative breast cancers which express “high” levels of the claudin 1 protein. We propose that claudin 1 functions both as a tumor suppressor as well as a tumor enhancer/facilitator in breast cancer.

Expression of both Estrogen Receptor-beta 1 (ER-β1) 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-β1) and its co-regulator Steroid Receptor RNA Activator Protein (SRAP) in breast cancer remain unclear. Previously, ER-β1 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-β1 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-β1 and SRAP were not prognostic. However, high ER-β1 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-β1[hazard ratio = 0.07; 95% confidence interval (CI) 0.01-0.41; P = 0.003] versus those with low SRAP or ER-β1 (interaction test, P = 0.02). The interaction test was not significant in ER-α-positive cohorts. CONCLUSIONS This study provides evidence that both ER-β1 and SRAP could be predictive biomarkers of tamoxifen benefit in ER-α-negative premenopausal EBC.

Steroid Receptor RNA Activator Protein (SRAP): a potential new prognostic marker for estrogen receptor-positive/node-negative/younger breast cancer patients

IntroductionThe steroid receptor RNA activator is a functional RNA suspected to participate in the mechanisms underlying breast tumor progression. This RNA is also able to encode for a protein, Steroid Receptor RNA Activator Protein (SRAP), whose exact function remains to be determined. Our aim was to assess, in a large breast cancer cohort, whether levels of this protein could be associated with outcome or established clinical parameters.MethodsFollowing antibody validation, SRAP expression was assessed by tissue-microarray (TMA) analysis of 372 breast tumors. Clinical follow-up and parameters such as steroid receptor and node status were available for all the corresponding cases. Immunohistochemical scores were independently determined by three investigators and averaged. Statistical analyses were performed using standard univariate and multivariate tests.ResultsSRAP levels were significantly (Mann-Whitney rank sum test, P < 0.05) higher in estrogen receptor-alpha positive (ER+, n = 271), in progesterone receptor positive (PR+, n = 257) and in older patients (age > 64 years, n = 182). When considering ER+ tumors, PR+ tumors, or younger patients (≤ 64 years), cases with high SRAP expression had a significantly (Mantel-Cox test, P < 0.05) worse breast cancer specific survival (BCSS) than those with low SRAP levels. SRAP also appeared as a very powerful indicator of poor prognostic for BCSS in the subset of ER+, node negative and young breast cancer patients (Cox regression analysis, n = 60, BCSS Hazard Ratio = 8.61, P < 0.006).ConclusionsOur data suggest that SRAP levels might provide additional information on potential risk of recurrence and negative outcome in a specific set of patients with otherwise good prognosis when considering only estrogen receptor and nodal status.

Claudin 1 expression in basal-like breast cancer is related to patient age

BackgroundDefects in tight junctions, gate-keepers of the integrity of the epidermal barrier function, are known to contribute to cancer development. As such, enhancing our understanding of how the expression of proteins involved in these junctions is regulated in cancer, remains a priority. Although the expression of one of these proteins, claudin 1, is down regulated in most invasive human breast cancers (HBC), we have recently shown that high levels of claudin 1, characterized tumors belonging to the very aggressive basal-like breast cancer (BLBC) subtype. In these tumors, the claudin 1 protein, usually localized in the cell membrane, is often mislocalized to the cytoplasm.MethodsTo examine the clinical relevance of this observation, we have generated and analyzed an invasive HBC tissue microarray consisting of 151 breast tumor samples; 79 of which presented a basal-like phenotype (i.e. ER-ve, PR-ve HER2-ve, CK5/6 or EGFR+ve). We also interrogated the outcome of claudin 1 knockdown in a human BLBC cell line, BT-20.ResultsImmunohistochemical analysis of this patient cohort revealed a significant association between high claudin 1 expression and BLBCs in women 55 years of age and older. Interestingly, no significant association was found between claudin 1 and nodal involvement, tumor grade or tumor size. Regression analysis however, showed a significant positive association between claudin 1 and claudin 4, even though claudin 4 did not significantly correlate with patient age. Claudin 1 knockdown in BT-20 cells resulted in decreased cell migration. It also significantly altered the expression of several genes involved in epithelial-mesenchymal-transition (EMT); in particular, SERPINE 1 (PAI1) and SSP1 (osteopontin), known to inhibit EMT and cancer cell migration. Conversely, genes known to maintain EMT through their interaction, SNAIL2, TCF4 and FOXC2 were significantly down regulated.ConclusionsThe association of high claudin 1 protein levels observed in tumors derived from older women with BLBC, suggests that claudin 1 has the potential to serve as a marker which can identify a specific subgroup of patients within the BLBC subtype and thus, further contribute to the characterization of these ill-defined breast cancers. More importantly, our studies strongly suggest that claudin 1 directly participates in promoting breast cancer progression, possibly through the alteration of expression of EMT genes.

Claudin 1 in Breast Cancer: New Insights.

Claudin 1 is a small transmembrane protein responsible for maintaining the barrier function that exists between epithelial cells. A tight junction protein that regulates the paracellular transport of small ions across adjacent cells, claudin 1 maintains cellular polarity and plays a major role in cell-cell communication and epithelial cell homeostasis. Long considered to be a putative tumor suppressor in human breast cancer, new studies suggest a role much more complex. While most invasive breast cancers exhibit a down regulation or absence of claudin 1, some aggressive subtypes that exhibit high claudin 1 levels have now been described. Furthermore, a causal role for claudin 1 in breast cancer progression has recently been demonstrated in some breast cancer cell lines. In this review we highlight new insights into the role of claudin 1 in breast cancer, including its involvement in collective migration and epithelial mesenchymal transition (EMT).

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.

Developmental changes in insulin-like growth factor I receptor gene expression in the mouse mammary gland.

The insulin‐like growth factor I receptor (IGF‐IR), which mediates the mitogenic action of IGF‐I, has been shown to play an essential role in normal growth and development. However, the precise role of IGF‐IR in the growth and differentiation of the mammary gland has not been elucidated. This study examines the profile of the IGF‐IR gene and protein expression during normal postnatal mammary gland development in order to gain further insight into the role of the IGF‐I/IGF‐IR during mammary gland morphogenesis. Gene and protein expression were examined in developing mouse mammary glands (virgin, pregnant, lactating, involuting) by real time PCR analysis and Western blotting. Both IGF‐IR gene and protein expression levels were high during early pregnancy. Interestingly, the level of gene expression was significantly down‐regulated during late pregnancy (5.4 fold) and lactation (9–13 fold) and significantly up‐regulated (3.9 fold) during late involution, to the level observed in the virgin mammary gland. By in situ hybridization, the IGF‐IR transcripts were localized to the proliferating ductal epithelium of the mammary glands of virgin mice and to the differentiating ductal and alveolar epithelium of the mammary glands during pregnancy and lactation. In the involuting gland, the transcripts were localized to the regressing ductal epithelium. These data are direct evidence that IGF‐IR expression is important for alveolar cell proliferation and suggest that the progression of involution may require the down‐regulation of IGF‐IR gene expression. Altogether, these results demonstrate that a developmental IGF‐IR gene expression pattern exists in the mouse mammary gland and that increases in gene expression at specific phases of development may reflect an important role for IGF‐I/IGF‐IR at those phases of development.

Towards further defining the proteome of mouse saliva

BackgroundKnowledge 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.ResultsIndividual 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.ConclusionsThe 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.

Expression of the mouse homologue for the human GCDFP-15/PIP gene during pre- and early post-natal development

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.