MicroRNA-409-5p is upregulated in breast cancer and its downregulation inhibits cancer development through downstream target of RSU1.

We investigated the expression and function of miR-409-5p in human breast cancer. Quantitative real-time polymerase chain reaction was conducted to evaluate endogenous miR-409-5p expression in breast cancer tumors and breast cancer cell lines. Lentiviral transduction was performed to stably downregulate miR-409-5p in breast cancer cell lines MDA-MB-231 and MCF-7 and cells. The effects of miR-409-5p downregulation on breast cancer proliferation, migration, and xenograft development were then evaluated. Downstream target gene of miR-409-5p, Ras suppressor protein 1, was examined by dual-luciferase activity assay, quantitative real-time polymerase chain reaction, and western blot in lentiviral-transduced breast cancer cells. Ras suppressor protein 1 was also inhibited in miR-409-5p-downregulated breast cancer cells to examine its functional effect on breast cancer proliferation and migration. MiR-409-5p was aberrantly upregulated in both breast cancer tumors and cell lines. Lentiviral transduction successfully downregulated endogenous miR-409-5p expression as well as suppressed proliferation, migration, and xenograft development in MDA-MB-231 and MCF-7 cells. Ras suppressor protein 1 was confirmed to be directly targeted by miR-409-5p in breast cancer cells. Small interfering RNA-mediated Ras suppressor protein 1 inhibition reversely promoted cancer proliferation and migration in miR-409-5p-downregualted breast cancer cells. MiR-409-5p is downregulated in breast cancer and its inhibition has anti-cancer effect on breast cancer development both in vitro and in vivo. The regulatory effect of miR-409-5p inhibition is likely through the inverse upregulation of Ras suppressor protein 1 in breast cancer.

A prospective, randomized study on hepatotoxicity of anastrozole compared with tamoxifen in women with breast cancer.

Tamoxifen and anastrozole are widely used as adjuvant treatment for early stage breast cancer, but their hepatotoxicity is not fully defined. We aimed to compare hepatotoxicity of anastrozole with tamoxifen in the adjuvant setting in postmenopausal breast cancer patients. Three hundred and fifty-three Chinese postmenopausal women with hormone receptor-positive early breast cancer were randomized to anastrozole or tamoxifen after optimal primary therapy. The primary end-point was fatty liver disease, defined as a liver-spleen ratio <0.9 as determined using a computed tomography scan. The secondary end-points included abnormal liver function and treatment failure during the 3-year follow up. The cumulative incidence of fatty liver disease after 3 years was lower in the anastrozole arm than that of tamoxifen (14.6% vs 41.1%, P < 0.0001; relative risk, 0.30; 95% CI, 0.21-0.45). However, there was no difference in the cumulative incidence of abnormal liver function (24.6% vs 24.7%, P = 0.61). Interestingly, a higher treatment failure rate was observed in the tamoxifen arm compared with anastrozole and median times to treatment failure were 15.1 months and 37.1 months, respectively (P < 0.0001; HR, 0.27; 95% CI, 0.20-0.37). The most commonly reported adverse events were 'reproductive system disorders' in the tamoxifen group (17.1%), and 'musculoskeletal disorders' in the anastrozole group (14.6%). Postmenopausal women with hormone receptor-positive breast cancer receiving adjuvant anastrozole displayed less fatty liver disease, suggesting that this drug had a more favorable hepatic safety profile than tamoxifen and may be preferred for patients with potential hepatic dysfunction.

(99)mTc-3PRGD2 scintimammography in palpable and nonpalpable breast lesions.

The aim of this study was to explore the diagnostic performance of 99mTc-3(poly-(ethylene glycol),PEG)4-RGD2 (99mTc-3PRGD2) scintimammography (SMM) in patients with either palpable or nonpalpable breast lesions and compare SMM to mammography to assess the possible incremental value of SMM in breast cancer detection. We also investigated the αvß3 expression in malignant and benign breast lesions. Ninety-four patients with 110 lesions were included in this study. Mammograms were evaluated according to the Breast Imaging Reporting and Data System (BI-RADS) by a specialized imaging radiologist. Prone SMM was performed 1 hour after injection of 99mTc-3PRGD2. Scintigraphic images were interpreted independently by two experienced nuclear medicine physicians using a three-point system, and the kappa value was calculated to determine the interreader agreement. The McNemar test was used to compare SMM and mammography with respect to sensitivity, specificity, and accuracy. Diagnostic values for breast cancer detection were evaluated for each lesion. Immunohistochemistry was performed to evaluate integrin αvß3 expression. Histopathology revealed 46 malignant lesions and 64 benign lesions. The overall sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of SMM were 83%, 73%, 77%, 69%, and 85%, respectively. The kappa value between the two reviewers was 0.63. The diagnostic values of SMM were higher than those of mammography in evaluating overall breast lesions. A sensitivity of 91% was achieved when SMM and mammography results were combined with 60% of all false-negative mammography findings classified as true-positive results by SMM. Integrin αvß3 expression was positively identified using SMM imaging. SMM is a promising tool to avoid unnecessary biopsies when used in addition to mammography and can be used to image αvß3 expression in breast cancer with good image quality.

Neogenin expression is inversely associated with breast cancer grade in ex vivo.

BACKGROUND: Neogenin is closely related to the human tumor suppressor gene deleted in colorectal cancer and plays a role in mammary morphogenesis. This study aimed to assess neogenin expression in breast cancer for any clinically significant association. METHODS: A total of 54 breast cancer patients who underwent modified radical mastectomy were enrolled for immunohistochemical and quantitative real-time PCR analysis of neogenin expression in their cancerous breast tissues in comparison to matching distant non-cancerous tissues. RESULTS: The data showed that the neogenin protein was either strongly or moderately expressed in the cytoplasm of the distant non-cancerous cells. In contrast, neogenin protein was either weakly or not expressed in the cytoplasm of 51/54 (94.4%) breast cancer cells, among which 13 breast cancer cases did not express neogenin protein at all (13/54, 24.1%). Similarly, levels of neogenin mRNA were significantly lower in breast cancer tissues than that of the matched distant non-cancerous tissues (51/54, 94.4%). Neogenin expression was inversely associated with breast cancer grade; that is, grade III breast cancer expressed much less neogenin than grade I-II (P<0.05). CONCLUSIONS: This study indicates that neogenin expression in breast cancer tissues is inversely associated with tumor grade.

Induction of autophagy by the MG­132 proteasome inhibitor is associated with endoplasmic reticulum stress in MCF­7 cells.

The aim of the present study was to investigate whether endoplasmic reticulum (ER) stress is involved in MG­132­induced autophagy, and to determine the effects of the inhibition of autophagy and ER stress on cell viability following MG­132 treatment. The proteasome inhibitor, MG­132, was used to induce autophagy in MCF­7 cells, and 3­methyladenine (3­MA) and salubrinal were used to inhibit autophagy and ER stress, respectively. An MTT assay was used to analyze cell viability. Apoptosis and the cell cycle were analyzed using flow cytometry. The expression levels of apoptosis­ and ER stress­associated genes were investigated using western blot and reverse transcription­quantitative polymerase chain reaction analyses. MG­132 inhibited cell proliferation, and induced apoptosis and cell cycle arrest at the G2 phase of the cell cycle. Notably, MG­132 increased the autophagy­associated conversion of microtubule­associated protein 1 light chain 3 (LC3)­I to LC3­II, which was partially attenuated by the ER stress inhibitor, salubrinal. In addition, MG­132 inhibited the protein expression of the anti­apoptotic protein, B­cell lymphoma (Bcl)­2, whereas the expression levels of Bcl­2­associated X protein and caspase­3 were upregulated. These effects were enhanced by co­treatment with either 3­MA or salubrinal. Furthermore, the mRNA and protein levels of the ER stress­associated genes, glucose­regulated protein 78, growth arrest and DNA damage induced gene­153, and caspase­12, were upregulated by MG132, and these levels were significantly inhibited by co­treatment of the cells with salubrinal. Taken together, the results of the present study indicated that the induction of autophagy by the proteasome inhibitor was associated with ER stress in the MCF­7 cells, and that the inhibition of autophagy or ER stress enhanced MG­132­induced apoptosis. These findings suggest the potential application of inhibitors of ER stress and autophagy, in combination with proteasomal inhibitors, for the development of combinatorial targeted cancer therapy.

High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor.

We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer.

miR-506 regulates breast cancer cell metastasis by targeting IQGAP1.

MicroRNA (miRNA or miR)-506 is a novel miRNA related to the survival of breast cancer patients. However, the mechanism underlying miRNA-506 involvement in breast carcinogenesis remains unclear. In the present study, we found that miR-506 was downregulated in human breast malignant tissues and breast cancer cell lines by RT-qPCR analysis, and the expression level of miR-506 was decreased with the increasing of tumor stage. Subsequently, gain-of-function and loss-of-function experiments were performed in vitro, and the results from MTT assay, Transwell-Matrigel invasion assay and cell adhesion assay revealed that miR-506 suppresses cell proliferation, invasion and adhesion of breast cancer cells. Luciferase reporter assay revealed that IQ motif containing GTPase activating protein 1 (IQGAP1) is a direct target of miR-506. miR-506 represses the expression of IQGAP1 and its downstream extracellular signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathways, as demonstrated by the RT-qPCR and western blot analysis. Furthermore, we found that IQGAP1 rescues the effect of miR-506 on cell proliferation, invasion, adhesion, and the activation of ERK MAPK signaling. In conclusion, the present study is the first to provide evidence that miR-506 acts as a tumor suppressor, at least partially, by directly downregulating IQGAP1 in breast cancer cells. The miR-506/IQGAP1/ERK pathway may be a novel therapeutic target in breast cancer.

Tumor suppressive microRNA-193b promotes breast cancer progression via targeting DNAJC13 and RAB22A.

Breast cancer is still a leading cause of cancer deaths in women. Despite improvements in therapeutic approaches in local control, metastatic relapse is almost always incurable, underlining the importance to better understand the biological bases that contribute to disease progression. In this study, we demonstrated that miR-193b was significantly down-regulated in two primary human breast cancer cell lines (MDA-MB-231 and MCF-7). Reconstitution of miR-193b expression resulted in decreasing cell proliferation, clonogenicity, migration and invasion. By using in silico prediction algorithms approach for target identification, we identified DNAJC13 (HPS40) and RAB22A to be direct targets of miR-193b. Concordantly, Re-expression of miR-193b decreased DNAJC13 (HPS40) and RAB22A expression. Luciferase reporter assays confirmed the direct interaction of miR-193b with both DNAJC13 (HPS40) and RAB22A. Our findings have demonstrated that miR-193b as a novel tumor suppressor plays an important role in breast cancer progression, understanding the mechanisms could account for the aggressive behaviour of breast cancer.

miRNA profiling reveals a potential role of milk stasis in breast carcinogenesis.

The tumor microenvironment plays an important role in breast carcinogenesis. Milk acts as an important microenvironment of breast cancer, but its role in breast carcinogenesis is largely unknown. Milk stasis may exist in the breast for a number of years after breastfeeding. In the present study, we reported the first microRNA (miRNA) profiling of milk from patients with milk stasis. We identified 266 known miRNAs and 271 novel miRNAs in 10 milk stasis only samples, 271 known miRNAs and 140 novel miRNAs in 10 milk stasis plus breast neoplasm samples by deep sequencing. miRNA profiles were different between the two groups. Furthermore, nine tumor suppressor miRNAs such as miR-29a, miR-146 and miR-223 were significantly downregulated, while seven oncogenic miRNAs such as miR-451, miR-486, miR-107, miR-92 and miR-10 were significantly upregulated in the milk of milk stasis plus neoplasm patients. Three of the identified miRNAs (miR-140, miR-21 and let-7a) were selected using real-time PCR, confirming that these miRNAs were highly expressed. The results also showed that the three miRNAs detected were more abundant in the milk than in the blood. In summary, the data suggested that miRNAs in milk from milk stasis patients may contribute to breast carcinogenesis and that they are more sensitive biomarkers for breast cancer than miRNAs in the blood.

99mTc-3P4-RGD2 scintimammography in the assessment of breast lesions: comparative study with 99mTc-MIBI.

PURPOSE: To compare the potential application of (99m)Tc-3P-Arg-Gly-Asp ((99m)Tc-3P4-RGD2) scintimammography (SMM) and (99m)Tc-methoxyisobutylisonitrile ((99m)Tc-MIBI) SMM for the differentiation of malignant from benign breast lesions. METHOD: Thirty-six patients with breast masses on physical examination and/or suspicious mammography results that required fine needle aspiration cytology biopsy (FNAB) were included in the study. (99m)Tc-3P4-RGD2 and (99m)Tc-MIBI SMM were performed with single photon emission computed tomography (SPECT) at 60 min and 20 min respectively after intravenous injection of 738 ± 86 MBq radiotracers on a separate day. Images were evaluated by the tumor to non-tumor localization ratios (T/NT). Receiver operating characteristic (ROC) curve analysis was performed on each radiotracer to calculate the cut-off values of quantitative indices and to compare the diagnostic performance for the ability to differentiate malignant from benign diseases. RESULTS: The mean T/NT ratio of (99m)Tc-3P4-RGD2 in malignant lesions was significantly higher than that in benign lesions (3.54 ± 1.51 vs. 1.83 ± 0.98, p<0.001). The sensitivity, specificity, and accuracy of (99m)Tc-3P4-RGD2 SMM were 89.3%, 90.9% and 89.7%, respectively, with a T/NT cut-off value of 2.40. The mean T/NT ratio of (99m)Tc-MIBI in malignant lesions was also significantly higher than that in benign lesions (2.86 ± 0.99 vs. 1.51 ± 0.61, p<0.001). The sensitivity, specificity and accuracy of (99m)Tc-MIBI SMM were 87.5%, 72.7% and 82.1%, respectively, with a T/NT cut-off value of 1.45. According to the ROC analysis, the area under the curve for (99m)Tc-3P4-RGD2 SMM (area = 0.851) was higher than that for (99m)Tc-MIBI SMM (area = 0.781), but the statistical difference was not significant. CONCLUSION: (99m)Tc-3P4-RGD2 SMM does not provide any significant advantage over the established (99m)Tc-MIBI SMM for the detection of primary breast cancer. The T/NT ratio of (99m)Tc-3P4-RGD2 SMM was significantly higher than that of (99m)Tc-MIBI SMM. Both tracers could offer an alternative method for elucidating non-diagnostic mammograms.

Evaluation of factors associated with pain experienced during mammary ductoscopy.

BACKGROUND: The aim of this study was to evaluate patient characteristics and findings after mammary ductoscopy in an effort to reduce the pain experienced during the procedure. PATIENTS AND METHODS: We evaluated outpatients in whom mammary ductoscopy was performed under local or intraductal anesthesia without preference, and their clinical characteristics and findings were recorded. Average and maximum pain scores were determined after the examination for statistical analysis. RESULTS: The overall average pain score was 3.74 ± 1.353, and the maximum pain score was 6.40 ± 1.681. The type of anesthesia, total operation time, nipple retraction, and discharge status significantly correlated with the pain score. Intraductal anesthesia lowered the average pain score by 0.60, whereas a total procedure time greater than 12 min increased the average pain score by 0.956. The pain score decreased if patients had nipple retraction, and intraductal anesthesia proved suitable for patients with normal nipples. CONCLUSION: Intraductal anesthesia is suitable for most patients, and ductoscopy should not exceed 12 min to minimize the pain. Nipple retraction does not significantly increase pain, but local anesthesia should be used in patients with retracted nipples. Patient age, breastfeeding history, menstrual stage, and presence of intraductal tumors were not associated with the level of pain experienced.

The Mammotome biopsy system is an effective treatment strategy for breast abscess.

BACKGROUND: Although most breast abscesses can be treated with the current first-line treatment of antibiotics by needle aspiration, the therapeutic duration is lengthy and recurrences often occur. Therefore, we aimed to investigate the clinical efficacy of the Mammotome biopsy system (Johnson & Johnson Corp., New Brunswick, NJ) in a cohort of patients with breast abscesses. METHODS: Forty lactating and 30 nonlactating breast abscess patients with unfavorable outcomes with antibiotic treatment and/or needle aspiration failure were recruited and treated with the Mammotome biopsy system. RESULTS: Skin inflammation of all patients disappeared within 6 days with no recurrence. The clinical outcomes in patients with an abscess size ≤ 3.5 cm was significantly better than those with an abscess size >3.5 cm (P = .025). CONCLUSIONS: The Mammotome biopsy system, an effective treatment strategy that is minimally invasive and less damaging, in combination with appropriate antibiotic therapy can be used safely as the first-line approach to breast abscess management.

Interaction between BRCA1/BRCA2 and ATM/ATR associate with breast cancer susceptibility in a Chinese Han population.

The etiology of breast cancer is associated with several factors, the most important being genetic susceptibility. Both BRCA1/2 and ATM/ATR play a role in DNA repair pathways and breast cancer. The aims of our investigation were to confirm the contributions of the known polymorphisms in these genes and investigate the pattern of their interaction. A total of 360 Han Chinese blood samples were collected from 180 patients with breast cancer and 180 healthy controls. In total, five single-nucleotide polymorphisms (SNP) in these genes were genotyped using polymerase chain reaction-based restriction fragment length polymorphism analysis. Combined effects of paired SNP were tested by the Multifactor Dimensionality Reduction (MDR) method and theory of Information Gain (IG). By MDR analysis, the best model was determined to be a five-locus site model. Interaction tree by the hierarchical clustering method showed that the ATR mutant rs13091637 gave a maximum stand-alone IG value, while the ATM mutant rs611646 showed the lowest IG value but had the top interaction effect. These results suggest that combined effects of the polymorphisms in these genes may confer susceptibility to breast cancer in a Chinese Han cohort (n = 360). Each of the variants, though, performs a different role in the pathogenetic mechanism of breast cancer.

Aberrant p63 and WT-1 expression in myoepithelial cells of pregnancy-associated breast cancer: implications for tumor aggressiveness and invasiveness.

Our recent studies revealed that focal alterations in breast myoepithelial cell layers significantly impact the biological presentation of associated epithelial cells. As pregnancy-associated breast cancer (PABC) has a significantly more aggressive clinical course and mortality rate than other forms of breast malignancies, our current study compared tumor suppressor expression in myoepithelial cells of PABC and non-PABC, to determine whether myoepithelial cells of PABC may have aberrant expression of tumor suppressors. Tissue sections from 20 cases of PABC and 20 cases of stage, grade, and age matched non-PABC were subjected to immunohistochemistry, and the expression of tumor suppressor maspin, p63, and Wilms' tumor 1 (WT-1) in calponin positive myoepithelial cells were statistically compared. The expression profiles of maspin, p63, and WT-1 in myoepithelial cells of all ducts encountered were similar between PABC and non-PABC. PABC, however, displayed several unique alterations in terminal duct and lobular units (TDLU), acini, and associated tumor tissues that were not seen in those of non-PABC, which included the absence of p63 and WT-1 expression in a vast majority of the myoepithelial cells, cytoplasmic localization of p63 in the entire epithelial cell population of some lobules, and substantially increasing WT-1 expression in vascular structures of the invasive cancer component. All or nearly all epithelial cells with aberrant p63 and WT-1 expression lacked the expression of estrogen receptor and progesterone receptor, whereas they had a substantially higher proliferation index than their counterparts with p63 and WT-1 expression. Hyperplastic cells with cytoplasmic p63 expression often adjacent to, and share a similar immunohistochemical and cytological profile with, invasive cancer cells. To our best knowledge, our main finings have not been previously reported. Our findings suggest that the functional status of myoepithelial cells may be significantly associated with tumor aggressiveness and invasiveness.