SGLT1 as an adverse prognostic factor in invasive ductal carcinoma of the breast.

PURPOSE: Sodium/glucose cotransporter (SGLT) 1 and 2 expression in carcinoma cells was recently examined, but their association with the clinicopathological factors of the patients and their biological effects on breast carcinoma cells have remained remain virtually unknown. Therefore, in this study, we explored the expression status of SGLT1 and SGLT2 in breast cancer patients and examined the effects of SGLT1 inhibitors on breast carcinoma cells in vitro. METHODS: SGLT1 and SGLT2 were immunolocalized and we first correlated the findings with clinicopathological factors of the patients. We then administered mizagliflozin and KGA-2727, SGLT1 specific inhibitors to MCF-7 and MDA-MB-468 breast carcinoma cell lines, and their growth-inhibitory effects were examined. Protein arrays were then used to further explore their effects on the growth factors. RESULTS: The SGLT1 high group had significantly worse clinical outcome including both overall survival and disease-free survival than low group. SGLT2 status was not significantly correlated with clinical outcome of the patients. Both mizagliflozin and KGA-2727 inhibited the growth of breast cancer cell lines. Of particular interest, mizagliflozin inhibited the proliferation of MCF-7 cells, even under very low glucose conditions. Mizagliflozin downregulated vascular endothelial growth factor receptor 2 phosphorylation. CONCLUSION: High SGLT1 expression turned out as an adverse clinical prognostic factor in breast cancer patient. This is the first study demonstrating that SGLT1 inhibitors suppressed breast carcinoma cell proliferation. These results indicated that SGLT1 inhibitors could be used as therapeutic agents for breast cancer patients with aggressive biological behaviors.

Induction of SGK1 via glucocorticoid-influenced clinical outcome of triple-negative breast cancer patients.

PURPOSE: Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive breast malignancy. Glucocorticoid (GC)-glucocorticoid receptor (GR) pathway plays a pivotal role in the cellular responses to various stresses including chemotherapy. Serum- and glucocorticoid-induced kinase-1 (SGK1) is known as an important downstream effector molecule in the GR signaling pathway, we attempted to explore its clinicopathological and functional significance in TNBC in which GR is expressed. METHODS: We first immunolocalized GR and SGK1 and correlated the results with clinicopathological variables and clinical outcome in 131 TNBC patients. We also evaluated the effects of SGK1 on the cell proliferation and migration in TNBC cell lines with administration of dexamethasone (DEX) to further clarify the significance of SGK1. RESULTS: The status of SGK1 in carcinoma cells was significantly associated with adverse clinical outcome in TNBC patients examined and was significantly associated with lymph node metastasis, pathological stage, and lymphatic invasion of the patients. In particular, SGK1 immunoreactivity was significantly associated with an increased risk of recurrence in GR-positive TNBC patients. Subsequent in vitro studies also demonstrated that DEX promoted TNBC cell migration and the silencing of gene expression did inhibit the cell proliferation and migration of TNBC cells under DEX treatment. CONCLUSIONS: To the best of our knowledge, this is the first study to explore an association between SGK1 and clinicopathological variables and clinical outcome of TNBC patients. SGK1 status was significantly positively correlated with adverse clinical outcome of TNBC patients and promoted carcinoma cell proliferation and migration of carcinoma cells.

Breast Cancer, Diabetes Mellitus and Glucagon-Like Peptide-1 Receptor Toward Exploring Their Possible Associations.

PURPOSE: Diabetes Mellitus (DM) has been one of the well known risk factors of breast cancer (BC) development and also associated with adverse clinical outcomes of BC patients. Glucagon-like peptide-1 (GLP-1) receptor agonists have been used as antidiabetic therapeutic agents and recent epidemiological studies have reported their use to be correlated with increased BC risks. However, biological or pathological details have remained unknown. Therefore, in this study, we examined the status of GLP-1 receptor (GLP-1R) in BC with and without DM and correlated the findings with the clinicopathological factors of the patients to explore the possible involvement of GLP-1 in BC pathology. METHODS: We immunolocalized GLP-1R in cancer and adjacent non-pathological breast tissues in BC patients with DM (125 cases) and without DM (58 cases). We then compared the status of GLP-1R with that of fibroblast growth factor 7 (FGF7) and fibroblast growth factor receptor 2 (FGFR2), Ki-67 labeling index (Ki-67 LI) and disease free survival (DFS) of the patients and also between cancerous and non-pathological breast tissues. RESULTS: GLP-1R immunoreactivity was significantly higher (p = 0.044) in the patients with DM than without in carcinoma tissues. However, this was detected only in invasive carcinoma (p < 0.01) and not in non-invasive carcinoma nor non-pathological mammary glands. FGF7 was significantly correlated with the status of GLP-1R in BC (p = 0.045). In addition, in ER positive BC cases, those with GLP-1R positive status tended to have higher Ki-67 LI of more than 14% (p = 0.070). CONCLUSION: These findings all demonstrated the possible association between GLP-1R status and biological features of BC, especially of invasive BC in DM patients.

Heterogeneous Nuclear Ribonucleoprotein K Is Involved in the Estrogen-Signaling Pathway in Breast Cancer.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) transcripts are abundant in estrogen receptor (ER)- or progesterone receptor (PR)-positive breast cancer. However, the biological functions of hnRNPK in the ER-mediated signaling pathway have remained largely unknown. Therefore, this study analyzes the functions of hnRNPK expression in the ER-mediated signaling pathway in breast cancer. We initially evaluated hnRNPK expression upon treatment with estradiol (E2) and ICI 182,780 in the ERα-positive breast carcinoma cell line MCF-7. The results revealed that E2 increased hnRNPK; however, hnRNPK expression was decreased with ICI 182,780 treatment, indicating estrogen dependency. We further evaluated the effects of hnRNPK knockdown in the ER-mediated signaling pathway in MCF-7 cells using small interfering RNAs. The results revealed that hnRNPK knockdown decreased ERα expression and ERα target gene pS2 by E2 treatment. As hnRNPK interacts with several other proteins, we explored the interaction between hnRNPK and ERα, which was demonstrated using immunoprecipitation and proximity ligation assay. Subsequently, we immunolocalized hnRNPK in patients with breast cancer, which revealed that hnRNPK immunoreactivity was significantly higher in ERα-positive carcinoma cells and significantly lower in Ki67-positive or proliferative carcinoma cells. These results indicated that hnRNPK directly interacted with ERα and was involved in the ER-mediated signaling pathway in breast carcinoma. Furthermore, hnRNPK expression could be an additional target of endocrine therapy in patients with ERα-positive breast cancer.

Significance of glucocorticoid signaling in triple-negative breast cancer patients: a newly revealed interaction with androgen signaling.

PURPOSE: Chemotherapy is the only current effective systemic treatment for triple-negative breast cancer (TNBC) patients. Therefore, the identification of active biological pathways that could become therapeutic targets is crucial. In this study, considering the well-reported biological roles of glucocorticoid and androgen receptors (GR, AR) in TNBC, we attempted to explore the effects of glucocorticoids (GCs) on cell kinetics as well as the potential interaction between GR and AR in TNBC. METHODS: We first explored the association between the status of GR, AR, and/or GCs-metabolizing enzymes such as 11ß-hydroxysteroid dehydrogenase (11ßHSD) 1 and 2 and the clinicopathological variables of the TNBC patients. Thereafter, we also studied the effects of dexamethasone (DEX) with/without dihydrotestosterone (DHT) on TNBC cell lines by assessing the cell proliferation, migration and GC response genes at the transcriptional level. RESULTS: GR positivity in carcinoma cells was significantly associated with adverse clinical outcome of the patients and AR positivity was significantly associated with lower histological grade and Ki-67 labeling index of the cases examined. In particular, AR positivity was significantly associated with decreased risks of developing recurrence in GR-positive TNBC patients. The subsequent in vitro studies revealed that DEX-promoted cell migration was inhibited by the co-treatment with DHT in GR/AR double-positive HCC38 cells. In addition, DHT inhibited the DEX-increased serum and glucocorticoid-regulated kinase-1 (SGK1) mRNA expression. CONCLUSION: This is the first study to reveal that the interaction of GR and AR did influence the clinical outcome of TNBC patients and GCs induced cell migration in TNBC cells.

The interaction between carcinoembryonic antigen-related cell adhesion molecule 6 and human epidermal growth factor receptor 2 is associated with therapeutic efficacy of trastuzumab in breast cancer.

Human epidermal growth factor receptor 2 (HER2) is a target of the HER2 inhibitor trastuzumab, which has been administered to HER2-positive breast cancer patients. However, the therapeutic effects of HER2 inhibitor monotherapy are not always clinically effective as compared with cotreatment with chemotherapy. Therefore, it has become pivotal to predict the therapeutic efficacy of trastuzumab monotherapy prior to administration. Recently, carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) has been reported to be a HER2-related factor. The aim of the present study was to explore the therapeutic mechanism of trastuzumab, including the relevance of CEACAM6 expression. CEACAM6/HER2-double-positive human breast carcinoma cell lines BT-474, HCC-1419 and MDA-MB-361 were used in this study. CEACAM6 knockdown decreased the inhibitory effects of trastuzumab in the trastuzumab-sensitive BT-474 and HCC-1419 cells, but not in the trastuzumab-resistant MDA-MB-361 cells. We examined the interaction between CEACAM6 and HER2 by using a proximity ligation assay (PLA). The interaction was detected in BT-474 and HCC-1419 cells, but not in MDA-MB-361 cells, and was significantly associated with in vitro trastuzumab therapeutic sensitivity. We further analysed the status of CEACAM6 and HER2 and their interaction in archival pathology specimens, also using PLA. The interaction was detected only in CEACAM6/HER2-double-positive breast cancer cases, and their PLA score was significantly associated with the efficacy of trastuzumab treatment. Therefore, evaluation of the CEACAM6-HER2 interaction could serve as a marker to predict the efficacy of trastuzumab monotherapy in breast cancer patients. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The significance of lipid accumulation in breast carcinoma cells through perilipin 2 and its clinicopathological significance.

Both systemic and intratumoral lipid metabolism have been recently reported to play pivotal roles in both tumor development and progression in various human malignancies including breast cancer. However, its details have remained largely unknown in breast cancer patients. Therefore, in this study, we focused on perilipin 2, which is involved in constituting the intracellular lipid composition. Perilipin 2 was first immunolocalized in 105 cases of breast cancer. The status of perilipin 2 immunoreactivity was significantly positively associated with histological grade, Ki-67 labeling index and HER2 status and negatively with estrogen receptor status of these patients. Subsequent in vitro study also revealed that its mRNA expression in triple negative breast carcinoma cells was higher than cells of other subtypes. We then examined the correlation between perilipin 2 immunoreactivity and intracellular lipid droplet evaluated by Oil-red O stating in 13 cases of breast carcinoma tissues. A significantly positive correlation was detected between the status of perilipin 2 and Oil-red O staining. These findings above did indicate that perilipin 2 could represent the status of intracellular lipid droplets in surgical pathology specimens of breast cancer and perilipin 2 was also associated with its more aggressive biological phenotypes.

Exploring Protein⁻Protein Interaction in the Study of Hormone-Dependent Cancers.

Estrogen receptors promote target gene transcription when they form a dimer, in which two identical (homodimer) or different (heterodimer) proteins are bound to each other. In hormone-dependent cancers, hormone receptor dimerization plays pivotal roles, not only in the pathogenesis or development of the tumors, but also in the development of therapeutic resistance. Protein⁻protein interactions (PPIs), including dimerization and complex formation, have been also well-known to be required for proteins to exert their functions. The methods which could detect PPIs are genetic engineering (i.e., resonance energy transfer) and/or antibody technology (i.e., co-immunoprecipitation) using cultured cells. In addition, visualization of the target proteins in tissues can be performed using antigen⁻antibody reactions, as in immunohistochemistry. Furthermore, development of microscopic techniques (i.e., electron microscopy and confocal laser microscopy) has made it possible to visualize intracellular and/or intranuclear organelles. We have recently reported the visualization of estrogen receptor dimers in breast cancer tissues by using the in situ proximity ligation assay (PLA). PLA was developed along the lines of antibody technology development, and this assay has made it possible to visualize PPIs in archival tissue specimens. Localization of PPI in organelles has also become possible using super-resolution microscopes exceeding the resolution limit of conventional microscopes. Therefore, in this review, we summarize the methodologies used for studying PPIs in both cells and tissues, and review the recently reported studies on PPIs of hormones.

OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy.

BACKGROUND: Glioblastoma is the most malignant human brain tumor and has a dismal prognosis; however, some patients show long-term survival. The interaction between the costimulatory molecule OX40 and its ligand OX40L generates key signals for T-cell activation. The augmentation of this interaction enhances antitumor immunity. In this present study, we explored whether OX40 signaling is responsible for antitumor adaptive immunity against glioblastoma and also established therapeutic antiglioma vaccination therapy. METHODS: Tumor specimens were obtained from patients with primary glioblastoma (n = 110) and grade III glioma (n = 34). Quantitative polymerase chain reaction (PCR), flow cytometry, and immunohistochemistry were used to analyze OX40L expression in human glioblastoma specimens. Functional consequences of OX40 signaling were studied using glioblastoma cell lines, mouse models of glioma, and T cells isolated from human subjects and mice. Cytokine production assay with mouse regulatory T cells was conducted under hypoxic conditions (1.5% O2). RESULTS: OX40L mRNA was expressed in glioblastoma specimens and higher levels were associated with prolonged progression-free survival of patients with glioblastoma, who had undergone gross total resection. In this regard, OX40L protein was expressed in A172 human glioblastoma cells and its expression was induced under hypoxia, which mimics the microenvironment of glioblastoma. Notably, human CD4 T cells were activated when cocultured in anti-CD3-coated plates with A172 cells expressing OX40L, as judged by the increased production of interferon-γ. To confirm the survival advantage of OX40L expression, we then used mouse glioma models. Mice bearing glioma cells forced to express OX40L did not die during the observed period after intracranial transplantation, whereas all mice bearing glioma cells lacking OX40L died. Such a survival benefit of OX40L was not detected in nude mice with an impaired immune system. Moreover, compared with systemic intraperitoneal injection, the subcutaneous injection of the OX40 agonist antibody together with glioma cell lysates elicited stronger antitumor immunity and prolonged the survival of mice bearing glioma or glioma-initiating cell-like cells. Finally, OX40 triggering activated regulatory T cells cultured under hypoxia led to the induction of the immunosuppressive cytokine IL10. CONCLUSION: Glioblastoma directs immunostimulation or immunosuppression through OX40 signaling, depending on its microenvironment.

An activation of LC3A-mediated autophagy contributes to de novo and acquired resistance to EGFR tyrosine kinase inhibitors in lung adenocarcinoma.

The development of therapeutic resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs, ie erlotinib or gefitinib) has been the major clinical problem when treating lung adenocarcinoma patients with these agents. However, its mechanisms have not necessarily been well studied to this date. Autophagy has been recently considered to play pivotal roles in escaping from the effects of anti-neoplastic agents. Therefore, in this study, we examined its roles in the development of resistance to EGFR-TKIs in lung adenocarcinoma. We first established erlotinib-resistant cell lines (PC9/ER) from parental PC9 cells by exposing the cells to erlotinib. In PC9/ER, autophagy-related LC3A expression came to be up-regulated and constitutive activation of LC3A-mediated autophagy became more pronounced through the process of acquiring therapeutic resistance. In addition, inhibition of LC3A or autophagy restores sensitivity to EGFR-TKIs in PC9/ER. LC3A was also activated at the transcriptional level in de novo resistant cells via demethylation of the MAP1LC3A gene. We then evaluated the status of LC3A in 169 lung adenocarcinoma patients using immunohistochemistry. LC3A immunoreactivity was only detected in carcinoma cells (89/169 patients), not in non-tumoural cells. In addition, LC3A immunoreactivity was significantly correlated with progression-free survival (p = 0.0039) and overall survival (p = 0.0040) of 35 patients treated with EGFR-TKIs. The results of our present study demonstrated that LC3A-mediated autophagy in carcinoma cells was involved in the development of resistance to EGFR-TKIs, and that LC3A could serve as a promising therapeutic target for overcoming resistance to EGFR-TKIs and a novel predictor of response to EGFR-TKIs in lung adenocarcinoma patients.

Progesterone receptor-Grb2 interaction is associated with better outcomes in breast cancer.

In addition to mediating nuclear transcription, PR mediates extranuclear functions mainly through the PR polyproline domain (PPD) interaction with the SH3 domain of cytoplasmic signaling molecules. PR-PPD-SH3 interaction inhibits EGF-mediated signaling and decreases lung cancer cell proliferation. Grb2 is an essential adaptor molecule with an SH2 domain flanked by two SH3 domains. In this study, we examined whether PR, through interaction between PR-PPD and Grb2-SH3, can interact with Grb2 in cells and breast cancer tissues. Our previous study shows that interaction between PR-PPD and Grb2 could interfere with cytoplasmic signaling and lead to inhibition of EGF-mediated signaling. GST-pulldown analysis shows that PR-PPD specifically interacts with the SH3 domains of Grb2. Immunofluorescence staining shows colocalization of PR and Grb2 in both the nucleus and cytoplasm in BT-474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we show that PR and Grb2 interact in breast cancer cells through the Grb2-SH3 domain. Proximity Ligation Assay (PLA) analysis of 43 breast cancer specimens shows that PR-Grb2 interaction is associated with low histological stage and negatively correlates with lymph node invasion and metastasis in breast cancer. These results, together with our previous findings, suggest that PR-PPD interaction with Grb2 plays an essential role in PR-mediated growth factor signaling inhibition and could contribute significantly to better prognosis in PR- and Grb2-positive breast cancer. Our finding provides a basis for additional studies to explore a novel therapeutic strategy for cancer treatment.

In Situ Proximity Ligation Assay to Visualize Protein-Protein Interactions in Tumor Specimens.

Protein-protein interactions (PPI) are the basis of various biological phenomena, such as intracellular signal transduction, gene transcription, and metabolism. PPI are also considered to be involved in the pathogenesis and development of various diseases, including cancer. PPI phenomenon and their functions have been elucidated by gene transfection and molecular detection technologies. On the other hand, in histopathological analysis, although immunohistochemical analyses provide information pertaining to protein expression and their localization in pathophysiological tissues, it has been difficult to visualize the PPI of these proteins. An in situ proximity ligation assay (PLA) was developed as a microscopic visualization technique for PPI in formalin-fixed, paraffin-embedded (FFPE) tissues as well as in cultured cells and frozen tissues. PLA using histopathological specimens enables cohort studies of PPI, which can clarify the significance of PPI in pathology. We have previously shown the dimerization pattern of estrogen receptors and significance of HER2-binding proteins using breast cancer FFPE tissues. In this chapter, we describe a methodology for the visualization of PPI using PLA in pathological specimens.

The Pro-Tumorigenic Role of Chemotherapy-Induced Extracellular HSP70 from Breast Cancer Cells via Intratumoral Macrophages.

Tumor-associated macrophages (TAMs) contribute to tumor progression and chemoresistance; it is therefore important to clarify the altered functions of macrophages following chemotherapy. While extracellular heat shock protein (HSP) 70 is associated with therapeutic resistance, the effects of HSP70 on TAMs remain largely unknown. Here, we conducted in vitro experiments and immunohistochemistry in 116 breast carcinoma specimens to determine whether the secretion of HSP70 from breast cancer cells following chemotherapy affects macrophage function. It was revealed that the interaction of epirubicin (EPI)-exposed breast cancer cells with macrophages enhanced tumor progression, and EPI promoted the secretion of extracellular HSP70 from breast cancer cells. The expression of pro-tumorigenic macrophage marker CD163 was decreased in macrophages treated with a conditioned medium (CM) from HSP70-silenced breast cancer cells. Breast cancer cells treated with CM from HSP70-silenced breast cancer cells showed decreased expression of transforming growth factor (TGF)-ß, and the pro-tumorigenic effects of macrophages were impaired when TGF-ß signaling was inhibited. Immunohistochemistry demonstrated that HSP70 served as a poor prognostic factor in conjunction with macrophage infiltration. It was therefore concluded that extracellular HSP70 levels increased following chemotherapy and enhanced the pro-tumorigenic effects of TAMs, either directly or indirectly, by regulating TGF-ß expression in breast cancer cells.

Intratumoral cortisol associated with aromatase in the endometrial cancer microenvironment.

Glucocorticoids bind to glucocorticoid receptors (GR). In the peripheral tissues, active cortisol is produced from inactive cortisone by 11ß-hydroxysteroid dehydrogenase (HSD)1. 11ß-HSD2 is responsible for this reverse catalysis. Although GR and 11ß-HSDs have been reported to be involved in the malignant behavior of various cancer types, the concentration of glucocorticoids in cancer tissues has not been investigated. In this study, we measured glucocorticoids in serum and cancer tissues using liquid chromatography-tandem mass spectrometry and clarified, for the first time, the intratumoral "intracrine" production of cortisol by 11ß-HSD1/2 in endometrial cancer. Intratumoral cortisol levels were high in the high-malignancy type and the cancer proliferation marker Ki-67-high group, suggesting that cortisol greatly contributes to the malignant behavior of endometrial cancer. A low expression level of the metabolizing enzyme 11ß-HSD2 is more important than a high expression level of the synthase 11ß-HSD1 for intratumoral cortisol action. Intratumoral cortisol was positively related to the expression/activity of estrogen synthase aromatase, which involved GR expressed in fibroblastic stromal cells but not in cancer cells. Blockade of GR signaling by hormone therapy is expected to benefit patients with endometrial cancer.

Immunolocalization of Cytoplasmic ER in ER-negative Breast Carcinoma as a Potent Favorable Prognostic Predictor.

It is known that estrogen receptor (ER) has extranuclear signaling functions in addition to classical genomic pathway, and estrogenic actions have been reported in ER-negative breast carcinoma cells. However, significance of cytoplasmic-ER immunoreactivity has not been reported in ER-negative breast carcinoma tissues. We immunolocalized cytoplasmic ER in 155 ER-negative breast carcinoma tissues and evaluated its clinicopathological significance including the prognosis. As a comparative cohort set, we also used 142 ER-positive breast carcinomas. Cytoplasmic-ER immunoreactivity was detected in the carcinoma cells, but not in the non-neoplastic mammary epithelium. Cytoplasmic-ER immunoreactivity was positive in the 35 out of 155 (23%) ER-negative breast carcinoma cases, whereas it was detected only in 2 out of 142 (1.4%) ER-positive cases. Cytoplasmic ER status was positively associated with cytoplasmic-PR status, but inversely associated with Ki67 labeling index or distant free-relapse survival rate. Moreover, cytoplasmic-ER status turned out to be an independent good prognostic factor for both distant relapse-free survival and breast cancer specific survival. These findings suggested that cytoplasmic ER plays important roles in the ER-negative breast carcinoma, and cytoplasmic ER is a potent good prognostic factor. Among the ER-negative breast cancer patients, clinical benefit of chemotherapy may be limited in the cytoplasmic-ER positive cases.

Visualization of the protein-protein interactions of hormone receptors in hormone-dependent cancer research.

In hormone-dependent cancers, the activation of hormone receptors promotes the progression of cancer cells. Many proteins exert their functions through protein-protein interactions (PPIs). Moreover, in such cancers, hormone-hormone receptor binding, receptor dimerization, and cofactor mobilization PPIs occur primarily in hormone receptors, including estrogen, progesterone, glucocorticoid, androgen, and mineralocorticoid receptors. The visualization of hormone signaling has been primarily reported by immunohistochemistry using specific antibodies; however, the visualization of PPIs is expected to improve our understanding of hormone signaling and disease pathogenesis. Visualization techniques for PPIs include Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation analysis; however, these techniques require the insertion of probes in the cells for PPI detection. Proximity ligation assay (PLA) is a method that could be used for both formalin-fixed paraffin-embedded (FFPE) tissue as well as immunostaining. It can also visualize hormone receptor localization and post-translational modifications of hormone receptors. This review summarizes the results of recent studies on visualization techniques for PPIs with hormone receptors; these techniques include FRET and PLA. In addition, super-resolution microscopy has been recently reported to be applicable to their visualization in both FFPE tissues and living cells. Super-resolution microscopy in conjunction with PLA and FRET could also contribute to the visualization of PPIs and subsequently provide a better understanding of the pathogenesis of hormone-dependent cancers in the future.

FE65 defines the efficacy of tamoxifen treatment via osteopontin expression in estrogen receptor-positive breast cancer.

BACKGROUND: Metastasis and endocrine therapy resistance are clinical challenges in the treatment of estrogen receptor (ER) -positive breast tumors. Therefore, mechanistic exploration of tamoxifen (TAM) resistance is considered pivotal to improve the prognosis of ER positive breast cancer patients. We previously demonstrated the correlation between FE65 and ER, and subsequently explored the effects of FE65 on TAM and potential interaction between FE65 and Osteopontin (OPN) in ER-positive breast cancer. METHODS: We immunolocalized FE65 and OPN in ER-positive breast cancers and correlated the results with their clinicopathological variables. We then performed proximity ligation and proliferation assays to correlate TAM resistance with FE65 expression. The RT2 Profiler Human PCR Array Human Estrogen Receptor Signaling was also used to profile 96 ER related genes. Hoechst 33342 Staining was used to evaluate apoptosis. RESULTS: FE65 immunoreactivity was significantly associated with higher pathological N factor of the cases examined, and a potential correlation with tamoxifen resistance of the ER-positive patients. FE65 knockdown significantly increased the proportion of apoptotic carcinoma cells. The statistically significant positive correlation between FE65 and OPN was detected in this study. Subsequent immunohistochemical analyses revealed that OPN status was significantly associated with cancer metastasis and overall survival of 142 patients and FE65 status. CONCLUSIONS: We firstly demonstrated the clinicopathological significance of FE65 in ER-positive breast cancer patients and results indicated that the effects of FE65 on ER-positive breast cancer patients were mediated through OPN expression. In addition, results suggested the clinical value of FE65 as potential prognostic factor and surrogate marker of TAM therapy in ER-positive breast cancer patients.

Co-expression of nuclear heterogeneous nuclear ribonucleic protein K and estrogen receptor α in endometrial cancer.

Heterogeneous nuclear ribonucleic protein K (hnRNPK) regulates the expression of various genes, but has contradictory roles as a tumor promoter and a tumor suppressor. We recently reported that the expression of hnRNPK is negatively associated with malignant behavior of breast cancer where it was induced by estrogen, and bound to estrogen receptor α (ERα) in the nucleus of breast cancer cells. However, the significance of hnRNPK in endometrial cancer, also an estrogen-dependent cancer, remains unclear. In this study, we first examined the localization of hnRNPK and ERα in normal endometrium and endometrial cancer. hnRNPK and ERα immunoreactivity was detected in the nuclei of endometrial glandular and carcinoma cells. In normal endometria, hnRNPK labeling index/immuno-intensity was significantly higher in the proliferative phase than in the secretory phase. In endometrial cancer tissues, hnRNPK labeling index/immuno-intensity was significantly higher in the adjacent non-malignant glandular cells compared to that in carcinoma cells. Immunohistochemistry results for ERα were identical to that of hnRNPK both in normal endometrium and endometrial cancer. In normal and cancerous tissues, the median value of the hnRNPK labeling index was significantly higher in the ERα-high group. Intratumoral estrogen, but not androgen, measured using liquid chromatography-tandem mass spectrometry, was significantly positively correlated with the hnRNPK labeling index in endometrial cancer tissues. Database analysis revealed that the hnRNPK high expression group had a significantly better prognosis for both overall and disease-free survival. These results suggest that hnRNPK interacts with ERα to regulate endometrial changes during the menstrual cycle and suppress the malignant behavior of endometrial cancer.

FE65 in breast cancer and its clinicopathological significance.

BACKGROUND: Transcription coregulator adapter protein FE65 is well known to play pivotal roles in pathogenesis of Alzheimer's disease by regulating amyloid precursor protein (APP) expression and processing. APP was recently reported to be also involved in development of human malignancies. Therefore, in this study, we studied FE65 status in different subtypes of human breast cancer and correlated the results with cell proliferation and migration of carcinoma cells and clinicopathological features of breast cancer patients to explore its biological and clinical significance in breast cancer. METHODS: We first immunolocalized FE65 and APP in 138 breast cancer patients and correlated the results with their tumor grade. Then, we did further exploration by proximity ligation assay, WST-8, and wound-healing assay. RESULTS: FE65 immunoreactivity in carcinoma cells was significantly associated with lymph-node metastasis, ERα, and high pathological N factor. APP immunoreactivity was significantly positively correlated with high pathological N factor. FE65, APP, and p-APP were all significantly correlated with shorter disease-free survival of breast cancer patients. In addition, the status of FE65 was significantly associated with overall survival. Results of in vitro analysis revealed that FE65 promoted the migration and proliferation of T-47D and ZR-75-1 breast carcinoma cells. In situ proximity ligation assay revealed that FE65 could bind to APP in the cytoplasm. FE65 was also associated with APP and ERα in carcinoma cells, suggesting their cooperativity in promoting carcinoma cell proliferation and migration. APP was also significantly associated with adverse clinical outcome of the patients. CONCLUSIONS: This is the first study to explore the clinical significance of FE65 in human breast cancer. The significant positive correlation of FE65 with poor clinical outcome, direct binding to APP, and promotion of carcinoma cell proliferation and migration indicated that FE65-APP pathway could serve as the potential candidate of therapeutic intervention in breast cancer patients.