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.

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.

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.

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.

Heart rate variability in the course of chemotherapy and haematopoietic stem cell transplantation for peadiatric patients with haematological malignancies.

BACKGROUND: High-dose chemotherapy and haematopoietic stem cell transplantation are essential for patients with paediatric haematologic diseases, although cardiotoxicity remains a concern. Heart rate variability analysis can evaluate autonomic nervous function interactions with cardiac function. OBJECTIVE: This study aimed to characterise heart rate variability differences between patients undergoing chemotherapy and controls, and the effects of haematopoietic stem cell transplantation on the autonomic nervous system in patients with haematological malignancies. METHODS: Nineteen patients (11 male, median age: 11.6 years) who received conventional chemotherapy followed by transplantation and 19 non-transplant patients (10 male, median age: 11.5 years) receiving chemotherapy only between 2006 and 2018 for haematological malignancies were retrospectively enrolled. Data from 24-hour Holter monitoring were recorded after chemotherapy and before and after transplantation. Heart rate variability was analysed in patients and 32 matched normal controls. RESULTS: There were significant differences between patients and normal controls in all heart rate variability analysis parameters apart from coefficient of variation of RR interval and standard deviation of the average normal RR interval for all 5-minute segments during sleeping. There was a significant difference in the cumulative anthracycline dose and heart rate variability during sleep between the non-transplant and pre-transplant groups. We observed no remarkable differences in time-domain analysis parameters between before and after transplantation, although the low-frequency component of power-spectrum analysis during awake hours was significantly decreased after transplantation. CONCLUSION: Conventional chemotherapy for paediatric haematologic diseases may be a risk factor for autonomic dysfunction. Further declines in heart rate variability after transplantation appear minor.

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.

S100P and Ezrin promote trans-endothelial migration of triple negative breast cancer cells.

PURPOSE: Triple negative breast cancer (TNBC) patients generally have an adverse clinical outcome because their tumors often recur and metastasize to distant sites in the first 3 years after surgery. Therefore, it has become pivotal to identify potential factors associated with metastasis. Here, we focused on the effects of S100P and Ezrin on the trans-endothelial migration (TEM) of TNBC cells, as they have both been suggested to play a role in this process in other malignancies. METHODS: The expression of S100P and Ezrin was examined by immunohistochemistry in 58 primary TNBC samples. The mRNA and protein levels of S100P and Ezrin were assessed in breast cancer-derived cell lines using qRT-PCR and Western blotting, respectively. Proliferation and migration assays were performed using TNBC-derived MFM-223 and SUM-185-PE cells transfected with S100P and Ezrin siRNAs. Two different timeframes were employed for TEM assays using TNBC-derived cells and human umbilical vein endothelial-derived cells, respectively. Correlations between the status of EzrinThr-567 expression and various clinicopathological features were analyzed by immunohistochemistry. RESULTS: We found that S100P and Ezrin double negative TNBC cases were significantly associated with a better disease-free survival. We also found that single and double siRNA-mediated knockdown of S100P and Ezrin in TNBC-derived cells significantly inhibited their TEM and destabilized the intercellular junctions of endothelial cells. In addition, we found that EzrinThr-567 immunoreactivity significantly correlated with vascular invasion in TNBC patients. CONCLUSIONS: From our data we conclude that S100P, Ezrin and EzrinThr-567 are involved in the trans-endothelial migration of TNBC cells and that they may serve as potential targets in TNBC patients.

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.