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.

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.

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.

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.

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.

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.

Progesterone Receptor Isoforms A and B in Pancreatic Neuroendocrine Tumor.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) have been reported to express progesterone receptor (PR), and its expression has been demonstrated to be a favorable prognostic factor in these patients. We examined the status of the PR isoforms PRA and PRB in the human PNET cell line and their association with cell proliferation of the tumor cells, which is closely related to the clinical outcome of PNET patients. METHODS: Quantitative RT-PCR and cell proliferation assays were performed following treatment with progesterone and RU-486 as a PR antagonist in nontransfected and PRA-transfected cells of the NET cell line QGP-1, which expresses PRB in its native state. PRA, PRB and cyclin D1 (CCND1) were immunolocalized in 87 PNET cases, and the results were compared with clinicopathological parameters. RESULTS: CCND1, c-Fos and c-Jun mRNA levels were all significantly increased by treatment with progesterone in QGP-1 cells with PRB expression compared with PRA-transfected cells (p = 0.02, p = 0.007 and p = 0.001, respectively). The proliferative activity of QGP-1 cells with PRB expression was also significantly stimulated by the administration of progesterone (p = 0.008). PRA immunoreactivity was significantly decreased in higher-grade PNETs (p = 0.04), whereas CCND1 was significantly elevated in higher-grade PNETs (p = 0.035). CONCLUSION: The results of the present study demonstrate that PRA could play an inhibitory role in the cell proliferation of PNETs, possibly by inhibiting PRB-mediated signals in the presence of progesterone, which could result in decreased CCND1 expression. In addition, the status of PRA in tumor cells could be a prognostic factor in PNETs.

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.

Aromatase inhibitor treatment of breast cancer cells increases the expression of let-7f, a microRNA targeting CYP19A1.

Aromatase inhibitors (AIs) are considered the gold standard of endocrine therapy for oestrogen receptor-positive postmenopausal breast cancer patients. AI treatment was reported to result in marked alterations of genetic profiles in cancer tissues but its detailed molecular mechanisms have not been elucidated. Therefore, we profiled miRNA expression before and after treatment with letrozole in MCF-7 co-cultured with primary breast cancer stromal cells. Letrozole significantly altered the expression profiles of cancer miRNAs in vitro. Among the elevated miRNAs following letrozole treatment, computational analysis identified let-7f, a tumour-suppressor miRNA which targeted the aromatase gene (CYP19A1) expression. Quantitative real-time PCR assay using MCF-7 and SK-BR-3 cells as well as clinical specimens of a neoadjuvant study demonstrated a significant inverse correlation between aromatase mRNA and let-7f expression. In addition, high let-7f expression was significantly correlated with low aromatase protein levels evaluated by both immunohistochemistry and the western blotting method in breast cancer cases. Results of 3'UTR luciferase assay also demonstrated the actual let-7f binding sites in CYP19A1, indicating that let-7f directly targets the aromatase gene. Subsequent WST-8 and migration assays performed in let-7f-transfected MCF-7 and SK-BR-3 cells revealed a significant decrement of their proliferation and migration. These findings all demonstrated that let-7f, a tumour suppressor miRNA in breast cancer, directly targeted the aromatase gene and was restored by AI treatment. Therefore, AIs may exert tumour-suppressing effects upon breast cancer cells by suppressing aromatase gene expression via restoration of let-7f.

Runt-related transcription factor 2 in human colon carcinoma: a potent prognostic factor associated with estrogen receptor.

Runt-related transcription factor 2 (RUNX2) belongs to the RUNX family of heterodimeric transcription factors, and is mainly associated with osteogenesis. Previous in vitro studies demonstrated that RUNX2 increased the cell proliferation of mouse and rat colon carcinoma cells but the status of RUNX2 has remained unknown in human colon carcinoma. Therefore, we examined clinical significance and biological functions of RUNX2 in colon carcinoma. RUNX2 immunoreactivity was examined in 157 colon carcinoma tissues using immunohistochemistry. RUNX2 immunoreactivity was evaluated as percentage of positive carcinoma cells [i.e., labeling index (LI)]. We used SW480 and DLD-1 human colon carcinoma cells, expressing estrogen receptor-ß (ER) in subsequent in vitro studies. RUNX2 immunoreactivity was detected in colon carcinoma cells, and the median value of RUNX2 LI was 67%. RUNX2 LI was significantly associated with Dukes' stage, liver metastasis and ERß status. In addition, RUNX2 LI was significantly associated with adverse clinical outcome of the colon carcinoma patients, and turned out an independent prognostic factor following multivariate analysis. Results of in vitro studies demonstrated that both SW480 and DLD-1 cells transfected with small interfering RNA against RUNX2 significantly decreased their cell proliferation, migration and invasive properties. In addition, RUNX2 mRNA level was significantly decreased by ER antagonist in these two cells. These findings all suggest that RUNX2 is a potent prognostic factor in human colon carcinoma patients through the promotion of cell proliferation and invasion properties, and is at least partly upregulated by estrogen signals through ERß of carcinoma cells.

Nucleobindin 2 in human breast carcinoma as a potent prognostic factor.

It is well-known that estrogens immensely contribute to the progression of human breast carcinoma, but their detailed molecular mechanisms remain largely unclear. In this study, we identified nucleobindin 2 (NUCB2) as a gene associated with recurrence based on microarray data of estrogen receptor (ER)-positive breast carcinoma cases (n = 10), and subsequent in vitro study showed that NUCB2 expression was upregulated by estradiol in ER-positive MCF-7 cells. However, NUCB2 has not yet been examined in breast carcinoma, and its significance remains unknown. Therefore, we further examined the biological functions of NUCB2 in breast carcinoma using immunohistochemistry and in vitro studies. NUCB2 immunoreactivity was detected in carcinoma cells in 77 of 161 (48%) breast cancer cases, and positively associated with lymph node metastasis and ER status of the patients. In addition, NUCB2 status was significantly associated with an increased risk of recurrence and adverse clinical outcome of the patients using both univariate and multivariate analyses. Results of siRNA transfection experiments showed that NUCB2 significantly increased cell proliferation, and migration and invasion properties in both MCF-7 and ER-negative SK-BR-3 cells. These results suggest that NUCB2 is upregulated by estrogens and plays an important role, especially in the process of metastasis, in breast carcinomas. NUCB2 status is considered a potent prognostic factor in human breast cancer.

Estrogen receptor α and ß in esophageal squamous cell carcinoma.

A gender difference has been reported in the morbidity of esophageal squamous cell carcinoma (ESCC). Estrogens have been proposed to play a role in this difference but the details have not yet been clarified. Therefore, in the present study, we examined the status of estrogen receptor (ER)α and ERß in 90 Japanese ESCC patients. ERα and ERß immunoreactivity was detected in the nuclei of ESCC cells (41.1 and 97.8%, respectively). There was a significant positive association between the ERß H score and histological differentiation (P = 0.0403), TNM-pM (LYM) (P = 0.00164) and Ki67/MIB1 LI of carcinoma cells (P = 0.0497, r = 0.207). In addition, the ERß status of carcinoma cells was significantly correlated with unfavorable clinical outcome of the patients. Multivariate analysis further revealed the ERß status in carcinoma cells as an independent unfavorable prognostic factor of these patients. We further examined the effects of estrogen treatment on ESCC cell line (ECGI-10) transfected with ERα or ERß in vitro. The number of ECGI-10 transfected with ERß was increased by estradiol or ERß specific agonist but estradiol did not exert any effect upon the cell number of ECGI-10 transfected with ERα. In summary, the results of the present study clearly demonstrate that the status of ERß in ESCC was closely associated with the unfavorable prognosis, possibly through altering cell proliferation of carcinoma cells.

Nudix-type motif 2 in human breast carcinoma: a potent prognostic factor associated with cell proliferation.

Nudix-type motif 2 (NUDT2) hydrolyzes diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) associated with various cellular functions. Previous studies demonstrated its regulation through estrogens, suggesting possible importance of NUDT2 in breast carcinoma. NUDT2, however, has not been examined in malignant tissues. Therefore, we examined its expression and functions in breast carcinoma. Immunohistochemistry for NUDT2 was examined by invasive ductal carcinoma (IDC: n = 145) and pure ductal carcinoma in situ (DCIS: n = 82), and NUDT2 mRNA was examined by real-time PCR in 9 DCIS, 19 IDC and 6 non-neoplastic breast tissues. We also used T47D breast carcinoma cells in in vitro studies. NUDT2 immunoreactivity was detected in 78% of DCIS and 63% of IDC, and NUDT2 mRNA level was significantly higher in DCIS or IDC than non-neoplastic breast. NUDT2 status was significantly correlated with Van Nuys classification, HER2 or Ki-67 in DCIS, and with stage, lymph node metastasis, histological grade or HER2 in IDC. NUDT2 status was significantly associated with adverse clinical outcome of IDC patients and proved an independent prognostic factor. Results of transfection experiments demonstrated that proliferation activity of T47D cells was significantly associated with NUDT2 expression level according to the treatment of estradiol and/or tamoxifen. NUDT2 expression was significantly decreased by estradiol, and it was also significantly decreased in T47D cells transfected with HER2 siRNA. These findings suggest that NUDT2 is an estrogen-repressed gene and is also induced by HER2 pathways in breast carcinoma cells. NUDT2 promotes proliferation of breast carcinoma cells and is a potent prognostic factor in human breast carcinomas.

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.

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.

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.

Immunolocalization of estrogen-producing and metabolizing enzymes in benign breast disease: comparison with normal breast and breast carcinoma.

It is well known that estrogens play important roles in the cell proliferation of breast carcinoma. Benign breast disease (BBD) contains a wide spectrum of diseases, and some are considered an important risk factor for subsequent breast carcinoma development. However, the significance of estrogens in BBD has remained largely unknown. Therefore, in this study, we examined tissue concentrations of estrogens and immunolocalization of estrogen-producing/metabolizing enzymes in BBD, and compared these findings with those in the normal breast and ductal carcinoma in situ (DCIS). Tissue concentration of estradiol in BBD (n = 9) was significantly (3.4-fold) higher than normal breast (n = 9) and nearly the same (0.7-fold) as in DCIS (n = 9). Immunoreactivity of estrogen sulfotransferase in BBD was significantly lower (n = 82) than normal breast (n = 28) but was not significantly different from DCIS (n = 28). Aromatase and steroid sulfatase immunoreactivities tended to be higher (P = 0.07) in BBD than in normal breast, and 17ß-hydroxysteroid dehydrogenase type 1 immunoreactivity was significantly higher in BBD than normal breast in the postmenopausal tissues. Immunoreactivity of estrogen and progesterone receptors was also significantly higher in BBD than normal breast. These results suggest that tissue concentration of estradiol is increased in BBD at a level similar to DCIS, which is considered mainly due to loss of estrogen sulfotransferase expression. Increased local estradiol concentration in BBD due to aberrant expression of estrogen-producing/metabolizing enzymes may play important roles in the accumulation of estradiol-mediated growth and/or subsequent development of breast carcinoma.

Runx2 in human breast carcinoma: its potential roles in cancer progression.

Runx2 has been proposed as one of the pivotal factors in the process of osteogenesis and metastasis in human malignancies including breast cancer, but its details have not been evaluated. Therefore, in this study, we evaluated its expression in human breast cancer using immunohistochemistry. One hundred and thirty-seven formalin-fixed and paraffin-embedded breast cancer specimens were used in this analysis of immunohistochemical study. Immunoreactivity was evaluated using the labeling index (LI). Runx2 immunoreactivity was detected in both carcinoma and stromal cells, as well as non-pathological ductal cells. The nuclear LI of Runx2 in carcinoma cells was associated with the clinical stage, histological grade and HER2 status of the patients examined. In addition, among the patients not associated with distant metastasis, those with high Runx2 LI demonstrated a significantly worse clinical outcome than those with a low LI. This was more pronounced in the group of estrogen receptor (ER)-negative cases. In addition, both univariate and multivariate analyses demonstrated that the Runx2 LI in breast carcinoma cells turned out an independent prognostic factor. Results of our present study demonstrated that Runx2 plays very important roles in the progression of breast cancer, especially in those of ER-negative cases.