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.

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.

Forkhead Box I1 in Breast Carcinoma as a Potent Prognostic Factor.

Forkhead box (FOX) proteins are family of transcriptional factors and regulate cell growth and differentiation as well as embryogenesis and longevity. Previous studies have demonstrated that several FOX members regulate growth or metastasis of breast carcinoma, but clinical significance of total FOX members remains unclear. We first examined associations between expression of 40 FOX genes and TNM status of 19 breast carcinoma using microarray data. Subsequently, we immunolocalized FOXI1 in 140 breast carcinomas and evaluated its clinicopathological significance. In the microarray analysis, we newly identified that gene expression of FOXI1 was most pronouncedly linked to metastasis of the breast carcinoma among the FOX members examined. However, clinicopathological significance of FOXI1 has not been examined in the breast carcinoma. FOXI1 immunoreactivity was positive in 44 out of 140 (31%) of breast carcinomas, and it was significantly associated with stage, lymph node metastasis and distant metastasis. The FOXI1 status was significantly associated with worse prognosis of the breast cancer patients, and it turned out to be an independent prognostic factor for both distant disease-free survival and breast cancer-specific survival. These findings suggest that FOXI1 plays important roles in the metastasis of breast carcinoma and immunohistochemical FOXI1 status is a potent prognostic factor.

Androgens enhance the ability of intratumoral macrophages to promote breast cancer progression.

Androgens are produced locally in breast carcinoma tissues by androgen­producing enzymes such as 5α­reductase type 1 (5αRed1) and affect not only breast cancer cells but the tumor microenvironment as well. Tumor­associated macrophages (TAMs) are primary components of the tumor microenvironment and contribute to tumor progression. Although previous studies suggest that androgen/androgen receptor (AR) signaling in macrophages has important roles in human diseases, androgen action on TAMs has remained largely unknown. We immunolocalized macrophage marker CD163 as well as AR and 5αRed1 in 116 breast carcinomas and correlated them with clinicopathological parameters and clinical outcomes. Moreover, we examined the roles of androgens on macrophages in breast cancer progression using cell lines 4T1 (mouse breast cancer) and RAW264.7 (macrophage) in a tumor­bearing female BALB/c mouse model. Double immunohistochemistry revealed that AR was sporadically expressed in the macrophages in breast carcinoma tissues. Macrophage infiltration was significantly correlated with an aggressive phenotype of breast carcinomas and worse prognosis, especially in the 5αRed1­positive group. In a sphere­forming assay using 4T1 and RAW­AR cells, which stably express AR, the sphere size was significantly increased due to androgens when 4T1 cells were cocultured with RAW­AR cells. Furthermore, in vivo experiments revealed that tumor growth and Ki67, a cell proliferation marker, were increased when androgens were stably produced in breast cancer cells and AR was expressed in macrophages. In conclusion, AR is expressed in intratumoral macrophages and is associated with an aggressive phenotype of breast carcinomas, especially when breast cancer cells actively produce androgens. Thus, androgens may enhance the ability of macrophages to promote breast cancer progression.

Stromal CCL5 Promotes Breast Cancer Progression by Interacting with CCR3 in Tumor Cells.

Chemokines secreted from stromal cells have important roles for interactions with carcinoma cells and regulating tumor progression. C-C motif chemokine ligand (CCL) 5 is expressed in various types of stromal cells and associated with tumor progression, interacting with C-C chemokine receptor (CCR) 1, 3 and 5 expressed in tumor cells. However, the expression on CCL5 and its receptors have so far not been well-examined in human breast carcinoma tissues. We therefore immunolocalized CCL5, as well as CCR1, 3 and 5, in 111 human breast carcinoma tissues and correlated them with clinicopathological characteristics. Stromal CCL5 immunoreactivity was significantly correlated with the aggressive phenotype of breast carcinomas. Importantly, this tendency was observed especially in the CCR3-positive group. Furthermore, the risk of recurrence was significantly higher in the patients with breast carcinomas positive for CCL5 and CCR3 but negative for CCR1 and CCR5, as compared with other patients. In summary, the CCL5-CCR3 axis might contribute to a worse prognosis in breast cancer patients, and these findings will contribute to a better understanding of the significance of the CCL5/CCRs axis in breast carcinoma microenvironment.

p16 in highly malignant esophageal carcinomas: the correlation with clinicopathological factors and human papillomavirus infection.

p16 is generally considered to be a surrogate maker of human papillomavirus (HPV) infection and also a predictive marker of favorable clinical outcome of patients with squamous cell carcinoma of the oropharynx. p16 overexpression is also known to be induced by deregulation of RB1 in neuroendocrine carcinomas. In highly malignant esophageal neoplasms, however, the status of p16 has remained largely unknown. We immunolocalized p16 and Rb1 in 82 surgically resected esophageal high-grade squamous cell carcinomas (46 poorly differentiated and 36 basaloid squamous cell carcinomas) and 15 esophageal small-cell carcinomas in order to clarify the clinical and biological significance of p16. p16 immunoreactivity was detected in 7/82 (9%) high-grade squamous cell carcinomas and 15 (100%) small-cell carcinomas. p16 immunoreactivity was significantly associated with Rb1 protein loss in both groups (P < 0.001). HPV was detected in none of the p16-positive cases examined. Clinical outcome of the p16-positive high-grade squamous cell carcinomas was not different from that of the p16-negative counterparts (P = 0.687) but significantly better than those with the small-cell carcinomas (P = 0.023). p16 was therefore considered to be induced through an inactivation of the RB1 signaling pathway and not through HPV infection in highly malignant esophageal neoplasms. Nevertheless, patients' clinical outcome of these neoplasms significantly differs; therefore, small-cell carcinomas have to be carefully differentiated from other neoplasms. In addition, p16 overexpression is not predictive of favorable clinical outcome in high-grade squamous cell carcinomas of the esophagus.

Enhancer remodeling promotes tumor-initiating activity in NRF2-activated non-small cell lung cancers.

Transcriptional dysregulation, which can be caused by genetic and epigenetic alterations, is a fundamental feature of many cancers. A key cytoprotective transcriptional activator, NRF2, is often aberrantly activated in non-small cell lung cancers (NSCLCs) and supports both aggressive tumorigenesis and therapeutic resistance. Herein, we find that persistently activated NRF2 in NSCLCs generates enhancers at gene loci that are not normally regulated by transiently activated NRF2 under physiological conditions. Elevated accumulation of CEBPB in NRF2-activated NSCLCs is found to be one of the prerequisites for establishment of the unique NRF2-dependent enhancers, among which the NOTCH3 enhancer is shown to be critical for promotion of tumor-initiating activity. Enhancer remodeling mediated by NRF2-CEBPB cooperativity promotes tumor-initiating activity and drives malignancy of NRF2-activated NSCLCs via establishment of the NRF2-NOTCH3 regulatory axis.

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 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.

The crosstalk between aldosterone and calcium metabolism in primary aldosteronism: A possible calcium metabolism-associated aberrant "neoplastic" steroidogenesis in adrenals.

Intracellular calcium (Ca) levels play pivotal roles in aldosterone biosynthesis. Several somatic mutations of ion channels associated with aldosterone over-production were reported to result in over-inflow of Ca ion. Recently, the main regulators of extracellular Ca including VDR, CaSR and PTH1R were also reported to regulate steroidogenesis including aldosterone production. Therefore, not only intracellular but also extracellular Ca levels could regulate aldosterone biosynthesis. In addition, primary aldosteronism (PA) is clinically associated with not only more frequent cardiovascular events but also secondary metabolic disorders including abnormal calcium metabolism, osteoporosis and others. However, the details of Ca metabolic abnormalities associated with, including the potential correlation between those abnormalities and aldosterone overproduction, have remained virtually unknown. Therefore, in this study, we first immunolocalized Ca metabolism-related receptors (CaSR, VDR and PTH1R) in normal adrenal glands (NAs), aldosterone-producing adenomas (APAs) and cortisol-producing adenoma (CPA). We then compared the findings with clinicopathological parameters of these patients and the patterns of KCNJ5 somatic mutation of the tumors among APA patients. In vitro study was also performed to further explore the potential effects of extracellular Ca, PTH, Vitamin D and ionophore on aldosterone production. Ca metabolism-related receptors were predominantly localized in aldosterone-producing cells (ZG and APA) in both immunohistochemistry and qRT-PCR analysis. CYP11B2 mRNA was significantly increased by CaCl2 treatment and further by adding ionophore. All the key enzymes related to aldosterone and cortisol biosynthesis including CYP11B2, CYP17A1 and CYP11B1 were upregulated by PTH treatment in this model and PTH could serve as a co-stimulator of ANG II to increase CYP11B2 expression. VDR mRNA levels were positively correlated with those of CYP11B2, CYP17A1 and CYP11B1 in APA tumor tissues and significantly higher in KCNJ5 mutated APAs than wild type. CYP11B1 levels were also significantly increased by VitD treatment. PTH1R mRNA levels were positively correlated with those of CYP17A1 and CYP11B1, both involved in cortisol production. In addition, the status of VDR was correlated with TRACP-5b levels, and that of PTH1R with serum Ca levels as well as urinary Ca excretion, respectively. Results of our present study did firstly demonstrate that aldosterone-producing cells were more sensitive to the fluctuations of extracellular Ca levels and Ca metabolism could directly influence steroidogenesis, especially "neoplastic" co-secretion of aldosterone and cortisol in APA patients.

Co-expression of carcinoembryonic antigen-related cell adhesion molecule 6 and 8 inhibits proliferation and invasiveness of breast carcinoma cells.

The carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 and CEACAM8 form heterodimers and exert their effects. Therefore, we examined the effects of CEACAM6 and CEACAM8 co-expression in breast cancer. We first studied CEACAM6/8 expression using immunohistochemistry in 109 patients with breast cancer. We then established MCF-7 cells that were stably transfected with CEACAM8 and studied cell proliferation, invasion and adhesion. The number of CEACAM6 and CEACAM8 double-positive breast carcinoma cells significantly increased in patients with low histopathological grade and stage. Proximity ligation assay (PLA) confirmed high CEACAM6/8 expression in MCF-7 cells. CEACAM6/8 expression promoted the adhesion of MCF-7 cells to endothelial cell monolayers but inhibited their invasion and proliferation. Furthermore, CEACAM6 status in carcinoma cells was significantly higher in bone than in lung metastases. CEACAM6/8 expression is associated with the inhibition of vascular invasion and cell proliferation. CEACAM6 expression was also considered to be involved in bone metastases of breast cancer. This is the first study to demonstrate the possible role of CEACAM6/8 heterodimer and CEACAM6 expression in breast cancer patients.

OLFM4, LY6D and S100A7 as potent markers for distant metastasis in estrogen receptor-positive breast carcinoma.

Metastatic breast cancer is a highly lethal disease, and it is very important to evaluate the biomarkers associated with distant metastasis. However, molecular features of distant metastasis remain largely unknown in breast cancer. Estrogens play an important role in the progression of breast cancer and the majority of stage IV breast carcinomas express estrogen receptor (ER). Therefore, in this study, we examined molecular markers associated with distant metastasis in ER-positive breast carcinoma by microarray and immunohistochemistry. When we examined the gene expression profile of ER-positive stage IV breast carcinoma tissues (n = 7) comparing ER-positive stage I-III cases (n = 11) by microarray analysis, we newly identified OLFM4, LY6D and S100A7, which were closely associated with the distant metastasis. Subsequently, we performed immunohistochemistry for OLFM4, LY6D and S100A7 in 168 ER-positive breast carcinomas. OLFM4, LY6D and S100A7 immunoreactivities were significantly associated with stage, pathological T factor, distant metastasis and Ki67 status in the ER-positive breast carcinomas. Moreover, these immunoreactivities were significantly associated with a worse prognostic factor for distant metastasis-free and breast cancer-specific survival in ER-positive stage I-III breast cancer patients. However, when we performed immunohistochemistry for OLFM4, LY6D and S100A7 in 40 ER-negative breast carcinomas, these immunoreactivities were not generally associated with the clinicopathological factors examined, including distant metastasis and prognosis of patients, in this study. These results suggest that OLFM4, LY6D and S100A7 immunoreactivity are associated with an aggressive phenotype of ER-positive breast carcinoma, and these are potent markers for distant metastasis of ER-positive breast cancer patients.

Myosin 5a regulates tumor migration and epithelial-mesenchymal transition in esophageal squamous cell carcinoma: utility as a prognostic factor.

Esophageal squamous cell carcinoma (ESCC) is highly malignant. Recently, the expression of myosin 5a, a member of the myosin superfamily, was reported to be associated with increased invasiveness and metastasis in many tumor types. Moreover, myosin 5a is upregulated by Snail and activated by Akt2, both of which are epithelial-mesenchymal transition (EMT) markers. In this study, we confirmed the expression of myosin 5a in ESCC surgical specimens and cell lines, revealing its correlation with tumor invasion, migration, patient prognosis, and expression of EMT-related proteins. The expression of myosin 5a, vimentin, and E-cadherin was immunohistochemically evaluated in 118 patients with ESCC who underwent esophagectomy without chemotherapy or irradiation therapy prior to surgery. We also investigated ESCC cell migration under myosin 5a silencing by siRNA induction. The high expression of myosin 5a was correlated with tumor depth, lymph node metastasis, pathological stage, high vimentin expression, and low E-cadherin expression. Patients with high expression of myosin 5a, including those with pT1 cancer, exhibited significantly worse survival. Moreover, the expression level of vimentin mRNA and the number of migrated ESCC cells decreased significantly following myosin 5a silencing. Our findings demonstrate that high expression of myosin 5a may be an independent prognostic factor in patients with ESCC, even in early invasive carcinoma, and indicate myosin 5a has a role in both cell migration and EMT.

Coexistence of glandular papilloma and sclerosing pneumocytoma in the bronchiole.

Both glandular papilloma (GP) and sclerosing pneumocytoma (SP) are rare tumors in the lung. We herein report an extremely rare case of coexistence of these two uncommon tumors. The patient was a 40-year-old Japanese woman with no chief complaint. A solitary nodule of the lung was detected using chest computed tomography. The transbronchial biopsy revealed that the tumor histologically corresponded to GP. The patient subsequently underwent partial resection of the right upper lobe. Histological examination of the resected specimens further revealed that the mass contained two different and independent elements and displayed typically histological features of GP and SP. Molecular analysis further revealed the presence of BRAF V600E and AKT1 E17K mutations in GP, whereas only AKT1 mutation was detected in SP. To our knowledge, this is the first case of coexistence of GP and SP in the bronchiole harboring common AKT1 mutation and different BRAF V600E mutational status.

ARHGAP15 in Human Breast Carcinoma: A Potent Tumor Suppressor Regulated by Androgens.

Rho GTPase activating protein 15 (ARHGAP15) is a recently identified GTPase activating protein which enhances intrinsic hydrolysis of GTP-bound Ras-related C3 botulinus toxin substrate (Rac1), resulting in inactivation of Rac1. Although a lot of studies have pointed out the pivotal roles of the Rac1 pathway in the progression of breast carcinomas, the clinical significance of ARHGAP15 has remained largely unknown in human breast carcinomas. Therefore, we immunolocalized ARHGAP15 in one hundred breast carcinoma tissues. ARHGAP15 immunoreactivity was frequently detected in the cytoplasm of carcinoma cells, and was positively correlated with that of Rac1 and androgen receptor labeling index. Furthermore, ARHGAP15 immunoreactivity was significantly correlated with decreased risk of recurrence and improved prognosis, and multivariate analyses demonstrated that ARHGAP15 immunoreactivity was an independent prognostic factor for both disease-free and breast-cancer-specific survival of the patients. In addition, exogenous overexpression of ARHGA15 suppressed cell proliferation and migration of MCF-7 cells and SK-BR-3 cells. On the other hand, ARHGAP15 mRNA was significantly induced by dihydrotestosterone. These findings suggest that ARHGAP15 is an androgen-induced gene and has anti-tumorigenic roles associated with the Rac1 pathway. ARHGAP15 immunoreactivity is therefore considered a potent prognostic factor in human breast carcinomas.

Progesterone arrested cell cycle progression through progesterone receptor isoform A in pancreatic neuroendocrine neoplasm.

In pancreatic neuroendocrine neoplasms (Pan-NEN) progesterone signaling has been shown to have both inhibitory and stimulatory effects on cell proliferation. The ability of progesterone to inhibit tumor proliferation is of particular interest and is suggested to be mediated through the less abundantly expressed progesterone receptor (PR) isoform A (PRA). To date the mechanistic processes underlying this inhibition of proliferation remain unclear. To examine the mechanism of PRA actions, the human Pan-NEN cell line QGP-1, that endogenously expresses PR isoform B (PRB) without PRA, was transfected with PRA. PRA transfection suppressed the majority of cell cycle related genes increased by progesterone including cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2). Importantly, following progesterone administration cell cycle distribution was shifted to S and G2/M phases in the naïve cell line but in PRA-transfected cells, this effect was suppressed. To see if these mechanistic insights were confirmed in patient samples PRA, PRB, CCNA2, CCNB, CDK1 and CDK2 immunoreactivities were assessed in Pan-NEN cases. Higher levels of cell cycle markers were associated with higher WHO grade tumors and correlations between the markers suggested formation of cyclin/CDK activated complexes in S and G2/M phases. PRA expression was associated with inverse correlation of all cell cycle markers. Collectively, these results indicate that progesterone signals through PRA negatively regulates cell cycle progression through suppressing S and G2/M phases and downregulation of cell cycle phases specific cyclins/CDKs.

Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma.

PURPOSE: The tumor microenvironment plays pivotal roles in promotion of many malignancies. Cancer-associated fibroblasts (CAFs) have been well-known to promote proliferation, angiogenesis, and metastasis but mechanistic understanding of tumor-stroma interactions is not yet complete. Recently, estrogen synthetic enzymes were reported to be upregulated by co-culture with stromal cells in ER positive breast carcinoma (BC) but effects of co-culture on androgen metabolism have not been extensively examined. Therefore, we evaluated roles of CAFs on androgen metabolism in ER-negative AR-positive BC through co-culture with CAFs. METHODS: Concentrations of steroid hormone in supernatant of co-culture of MDA-MB-453 and primary CAFs were measured using GC-MS. Cytokines derived from CAFs were determined using Cytokine Array. Expressions of androgen synthetic enzymes were confirmed using RT-PCR and Western blotting. Correlations between CAFs and androgen synthetic enzymes were analyzed using triple-negative BC (TNBC) patient tissues by immunohistochemistry. RESULTS: CAFs were demonstrated to increase expressions and activities of 17ßHSD2, 17ßHSD5, and 5α-Reductase1. IL-6 and HGF that were selected as potential paracrine mediators using cytokine array induced 17ßHSD2, 17ßHSD5, and 5α-Reductase1 expression. Underlying mechanisms of IL-6 paracrine regulation of 17ßHSD2 and 17ßHSD5 could be partially dependent on phosphorylated STAT3, while phosphorylated ERK could be involved in HGF-mediated 5α-Reductase1 induction. α-SMA status was also demonstrated to be significantly correlated with 17ßHSD2 and 17ßHSD5 status in TNBC tissues, especially AR-positive cases. CONCLUSIONS: Results of our present study suggest that both IL-6 and HGF derived from CAFs could contribute to the intratumoral androgen metabolism in ER-negative BC patients.

p62/sequestosome 1 in human colorectal carcinoma as a potent prognostic predictor associated with cell proliferation.

p62/sequestosome 1 (p62) is a multi-domain protein that functions as a receptor for ubiquitinated targets in the selective autophagy and serves as a scaffold in various signaling cascades. p62 have been reported to be up-regulated in several human malignancies, but the biological roles and significance of p62 are still poorly understood in colorectal carcinoma. We immunohistochemically evaluated p62 in 118 colorectal adenocarcinoma and 28 colorectal adenoma cases. We used four colon carcinoma cells (HCT8, HT29, COLO320, and SW480) in the in vitro studies. p62 immunoreactivity was detected in 11% of colorectal adenoma cases and 31% of adenocarcinoma cases, while it was negligible in the normal epithelium. The immunohistochemical p62 status was significantly associated with synchronous liver metastasis, and it turned out to be an independent adverse prognostic factor in colorectal cancer patients. Following in vitro studies revealed that HCT8 and HT29 cells transfected with p62-specific siRNA showed significantly decreased cell proliferation activity, whereas COLO320 and SW480 cells transfected with p62 expression plasmid showed significantly increased cell proliferation activity. The p62-mediated cell proliferation was not associated with the autophagy activity. These findings suggest that p62 promotes the cell proliferation mainly as a scaffold protein, and that the p62 status is a potent prognostic factor in colorectal carcinoma patients.

In Situ Evaluation of Estrogen Receptor Dimers in Breast Carcinoma Cells: Visualization of Protein-Protein Interactions.

The estrogen receptor (ER) functions as a dimer and is involved in several different biological functions. However ER dimeric proteins have not been identified by in situ methodologies. Structured illumination microscopy (SIM) has been recently developed, which enabled the localization of protein and protein interaction. Therefore, in this study, we firstly demonstrated that ERs formed both homodimers and heterodimers in breast carcinoma cell lines using Nikon's SIM (N-SIM). ERα/α homodimers were detected in the nuclei of both ERα-positive MCF-7 and T-47D cells; 23.0% and 13.4% of ERα proteins formed ERα/α homodimers, respectively. ERα/ß heterodimers were also detected in MCF-7 and T-47D. Approximately 6.6% of both ERα and ERß1 proteins formed ERα/ß1 heterodimers in MCF-7. In addition, 18.1% and 22.4% of ERα and ERß proteins formed ERα/ß2 heterodimers and ERα/ß5 heterodimers in MCF-7, respectively. In addition, by using proximity ligation assay (PLA) in MCF-7, estradiol-induced ERα/α homodimers and ERα/ß1 heterodimers were both detected after 15 to 45 min of treatment and at 15 min, respectively. The percentage of total ER proteins could also be determined using N-SIM. By using both methods, it has become possible to evaluate precise localization and ratio of ER dimers among different cell types.