Triple Negative Breast Cancer Depends on Sphingosine Kinase 1 (SphK1)/Sphingosine-1-Phosphate (S1P)/Sphingosine 1-Phosphate Receptor 3 (S1PR3)/Notch Signaling for Metastasis.

BACKGROUND Triple negative breast cancer (TNBC) has a more aggressive recurrence. Previous reports have demonstrated that sphingosine kinase 1 (SphK1) is a crucial regulator of breast cancer progression. However, the correlation of SphK1 with clinical prognosis has been poorly investigated. Thus, we aimed to elaborate the role of SphK1 in TNBC metastasis. MATERIAL AND METHODS We first determined the level of SphK1 in breast cancer tissue samples and breast cancer cells. Furthermore, the expression of HER2 and phosphor-SphK1 (pSphK1) in human breast cancer tissue samples was determined by immunohistochemical analysis. Associations between SphK1 and clinical parameters of tumors were analyzed. The activity of SphK1 was measured by fluorescence analysis. Extracellular sphingosine-1-phosphate (S1P) was detected using an ELISA kit. Associations between SphK1 and metastasis potential were analyzed by Transwell assay. RESULTS Levels of SphK1 in TNBC patients were significantly higher than levels in other patients with other breast tumors. The expression of SphK1 was positively correlated with poor overall survival (OS) and progression-free survival (PFS), as well as poor response to 5-FU and doxorubicin. The depression of SphK1 thus could repress the Notch signaling pathway, reduce migration, and invasion of TNBC cells in vivo and in vitro. Furthermore, silencing of SphK1 by Ad-SPHK1-siRNA or SphK1 inhibitor PF543 sensitized TNBCs to 5-FU and doxorubicin. Our results also indicated that SphK1 inhibition could effectively counteracts tumors metastasis via Notch signaling pathways, indicating a potentially anti-tumor strategy in TNBC. CONCLUSIONS We found that elevated levels of pSphK1 were positive correlation with high expression of S1P, which in turn promoted metastasis of TNBC through S1P/S1PR3/Notch signaling pathway.

Splicing factor hnRNPA2B1 contributes to tumorigenic potential of breast cancer cells through STAT3 and ERK1/2 signaling pathway.

Increasing evidence has indicated that the splicing factor hnRNPA2B1 plays a direct role in cancer development, progression, gene expression, and signal transduction. Previous studies have shown that knocking down hnRNPA2B1 in breast cancer cells induces apoptosis, but the mechanism and other functions of hnRNPA2B1 in breast cancer are unknown. The goal of this study was to investigate the biological function, clinical significance, and mechanism of hnRNPA2B1 in breast cancer. The expression of hnRNPA2B1 in 92 breast cancer and adjacent normal tissue pairs was analyzed by immunohistochemical staining. Stable clones exhibiting knockdown of hnRNPA2B1 via small hairpin RNA expression were generated using RNA interference technology in breast cancer cell lines. The effects of hnRNPA2B1 on cell proliferation were examined by MTT and EdU assay, and cellular apoptosis and the cell cycle were examined by flow cytometry. A nude mouse xenograft model was established to elucidate the function of hnRNPA2B1 in tumorigenesis in vivo. The role of hnRNPA2B1 in signaling pathways was investigated in vitro. Our data revealed that hnRNPA2B1 was overexpressed in breast cancer tissue specimens and cell lines. Knockdown of hnRNPA2B1 reduced breast cancer cell proliferation, induced apoptosis, and prolonged the S phase of the cell cycle in vitro. In addition, hnRNPA2B1 knockdown suppressed subcutaneous tumorigenicity in vivo. On a molecular level, hnRNPA2B1 knockdown decreased signal transducer and activator of transcription 3 and extracellular-signal-regulated kinase 1/2 phosphorylation. We concluded that hnRNPA2B1 promotes the tumorigenic potential of breast cancer cells, MCF-7 and MDA-MB-231, through the extracellular-signal-regulated kinase 1/2 or signal transducer and activator of transcription 3 pathway, which may serve as a target for future therapies.

DNA Methylation in Genomes of Several Annual Herbaceous and Woody Perennial Plants of Varying Ploidy as Detected by MSAP.

Polyploidization is known to accompany altered DNA methylation in higher plants, which plays an important role in gene expression regulation and maintaining genome stability. While the characteristics of DNA methylation in different polyploid plants are still to be elucidated; here, status of genomic DNA methylation in a series of diploid, triploid, and tetraploid annual herbaceous plants (watermelon and Salvia) and woody perennials (pear, Poplar, and loquat) were explored by methylation-specific amplified polymorphism analysis. The results indicated that levels of DNA methylation in triploid watermelon and Salvia were lower than their diploid parents. In triploid Poplar and pear, higher levels of DNA methylation were detected, and no significant difference was observed between triploid and tetraploid in all tested materials. Further data analysis suggested that about half of the total detected sites underwent changes of DNA methylation patterns in triploid watermelons and Salvia, as well as an obvious trend towards demethylation. However, the changes of DNA methylation patterns in three triploid woody perennials were only 17.54-33.40%. This implied that the characteristics of DNA methylation are significantly different during the polyploidization of different plant species. Furthermore, the results suggested that the level of DNA methylation was nonlinearly related to the ploidy level, and triploid plants displayed more interesting DNA methylation status. The characteristics and possible functions of DNA methylation in different ploidy series are further discussed.

Differential diagnosis of plasma cell mastitis and invasive ductal carcinoma using multiparametric MRI.

BACKGROUND: Evaluate the potential of multiparametric magnetic resonance imaging (MRI) for the differential diagnosis of plasma cell mastitis (PCM) and invasive ductal carcinoma (IDC). METHODS: A total of 465 female patients, including 197 PCM (42.4%) and 268 IDC (57.6%), were examined using breast MRI scanning using routine sequences, dynamic contrast-enhanced MRI (DCE-MRI), diffusion-weighted imaging (DWI) and MR spectroscopy (MRS). The MRI features of PCM and IDC lesions were analyzed and compared to the histological results. RESULTS: Compared to IDC, the PCM lesions were more frequent in the subareolar regions, hyperintense on T2WI (P<0.01) and showed an initial signal increase ≤90%, a persistent and plateau pattern of time-intensity curves, non-mass enhancement, multiple rim enhancements, central hyperintensity on DWI, a higher ADC value, and total choline (tCho) peak negative and tCho peak integral <29.95 AU (P<0.01). The following breast-associated findings were also observed frequently in PCM: Ipsilateral breast enlargement, nipple retraction, skin thickening, peripheral edema and axillary lymphadenopathy. However, no significant difference was observed between the two groups for the shape, border and adjacent vessel signs of the lesion. CONCLUSIONS: Some of the MRI features of PCM and IDC lesions were different. An integrated analysis of these multiparametric MRI features can thus assist in the differential diagnosis of PCM and IDC lesions.

Huaier polysaccharide inhibits the stem-like characteristics of ERα-36high triple negative breast cancer cells via inactivation of the ERα-36 signaling pathway.

Triple negative breast cancer (TNBC) is a highly aggressive cancer and lack of targeting therapies. It is believed that the breast cancer stem cells (BCSCs) are responsible for the aggressive characteristics of TNBC. Hence, developing BCSC-targeting agents may provide new therapeutic strategies for the patients. Huaier polysaccharide (HP), an active ingredient extracted from the mushroom Trametes robiniophila Murr, has been widely used in clinical anti-cancer treatments in China. Here we demonstrated that HP could target BCSCs in TNBC cells, resulting in decreased mammosphere formation, downregulated expression of stem-related genes and reduced proportion of aldehyde dehydrogenase positive cells in vitro, and inhibited xenograft tumor formation in vivo. Mechanically, HP markedly reduced the expression of estrogen receptor α-36 (ERα-36), a recently identified subtype of estrogen receptor α, and attenuated ERα-36-mediated activation of AKT/ß-catenin signaling in ERα-36high TNBC cells. This study provides a new insight into the mechanism of HP on BCSC-targeting therapy and new ideas for comprehensive treatment strategies for TNBC.

SPHK1 promotes metastasis of thyroid carcinoma through activation of the S1P/S1PR3/Notch signaling pathway.

Thyroid carcinoma is characterized by an aggressive behavior, lack of effective targeted therapies and a high rate of relapse. Sphingosine kinase 1 (SPHK1) has been reported to be a critical regulatory factor in the progression of thyroid carcinoma, but the correlation between SPHK1 and clinical prognosis of patients with thyroid carcinoma has remained to be fully elucidated. The present study aimed to systematically assess the roles of SPHK1 in thyroid carcinoma metastasis and further investigate the possible underlying mechanisms. First, the expression of SPHK1 was detected in tissue samples from 53 thyroid carcinoma patients and in thyroid carcinoma cell lines by reverse transcription-quantitative polymerase chain reaction analysis. Furthermore, the level of phospho-(p)-SPHK1 was immunohistochemically detected in human thyroid carcinoma tissue samples. The activity of SPHK1 was measured with a commercial SPHK1 Activity Assay kit. A sphingosine-1-phosphate (S1P) competitive ELISA kit was used to determine the extracellular S1P levels. The metastatic potential was assessed by a Transwell assay. In addition, the association between SPHK1 and clinicopathological features of the patients was analyzed. The results indicated that the expression of SPHK1 in thyroid carcinoma samples was significantly higher than in paired adjacent normal thyroid tissues. High levels of SPHK1 were positively correlated with poor overall survival and progression-free survival. Downregulation of SPHK1 by lentiviral vector expressing SPHK1 small interfering (si)RNA evidently repressed Notch signaling and reduced the migration and invasion of thyroid carcinoma cells in vitro and in a NOD/SCID mouse model. Furthermore, inhibition of SPHK1 by siRNA or treatment with SPHK1 inhibitor 5C sensitized thyroid carcinoma to cisplatin and doxorubicin. In addition, it was demonstrated that silencing of SPHK1 effectively inhibits processes associated with thyroid carcinoma metastasis through the Notch signaling pathway, and SPHK1 may therefore represent a potential therapeutic target in thyroid carcinoma. In conclusion, the present study indicated that high levels of p-SPHK1 were positively correlated with high levels of S1P which in turn promoted thyroid carcinoma metastasis via the S1P/S1P receptor 3/Notch signaling pathway, suggesting possible prognostic markers and therapeutic targets.

Tamoxifen enhances stemness and promotes metastasis of ERα36+ breast cancer by upregulating ALDH1A1 in cancer cells.

The 66 kDa estrogen receptor alpha (ERα66) is the main molecular target for endocrine therapy such as tamoxifen treatment. However, many patients develop resistance with unclear mechanisms. In a large cohort study of breast cancer patients who underwent surgery followed by tamoxifen treatment, we demonstrate that ERα36, a variant of ERα66, correlates with poor prognosis. Mechanistically, tamoxifen directly binds and activates ERα36 to enhance the stemness and metastasis of breast cancer cells via transcriptional stimulation of aldehyde dehydrogenase 1A1 (ALDH1A1). Consistently, the tamoxifen-induced stemness and metastasis can be attenuated by either ALDH1 inhibitors or a specific ERα36 antibody. Thus, tamoxifen acts as an agonist on ERα36 in breast cancer cells, which accounts for hormone therapy resistance and metastasis of breast cancer. Our study not only reveals ERα36 as a stratifying marker for endocrine therapy but also provides a promising therapeutic avenue for tamoxifen-resistant breast cancer.

Identification of MsHsp20 Gene Family in Malus sieversii and Functional Characterization of MsHsp16.9 in Heat Tolerance.

Heat shock proteins (Hsps) are common molecular chaperones present in all plants that accumulate in response to abiotic stress. Small heat shock proteins (sHsps) play important roles in alleviating diverse abiotic stresses, especially heat stress. However, very little is known about the MsHsp20 gene family in the wild apple Malus sieversii, a precious germplasm resource with excellent resistance characteristics. In this study, 12 putative M. sieversii Hsp20 genes were identified from RNA-Seq data and analyzed in terms of gene structure and phylogenetic relationships. A new Hsp20 gene, MsHsp16.9, was cloned and its function studied in response to stress. MsHsp16.9 expression was strongly induced by heat, and transgenic Arabidopsis plants overexpressing MsHsp16.9 displayed improved heat resistance, enhanced antioxidant enzyme activity, and decreased peroxide content. Overexpression of MsHsp16.9 did not alter the growth or development under normal conditions, or the hypersensitivity to exogenous ABA. Gene expression analysis indicated that MsHsp16.9 mainly modulates the expression of proteins involved in antioxidant enzyme synthesis, as well as ABA-independent stress signaling in 35S:MsHsp16.9-L11. However, MsHsp16.9 could activate ABA-dependent signaling pathways in all transgenic plants. Additionally, MsHsp16.9 may function alongside AtHsp70 to maintain protein homeostasis and protect against cell damage. Our results suggest that MsHsp16.9 is a protein chaperone that positively regulates antioxidant enzyme activity and ABA-dependent and independent signaling pathway to attenuate plant responses to severe stress. Transgenic plants exhibited luxuriant growth in high temperature environments.

High plasma fibrinogen is correlated with poor response to trastuzumab treatment in HER2 positive breast cancer.

Some of HER-2 positive breast cancer patients failed to trastuzumab treatment. Recent reports have indicated the correlation between plasma coagulation parameters and clinical characteristics in breast cancer. The aim of this study was to analyze the role of coagulation parameters in trastuzumab treated patients. Coagulation parameters from trastuzumab treated breast cancer patients were retrospectively studied from 2006 to 2010. The correlation between routine coagulation levels and clinical characteristics were analyzed, including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fib) and D-dimer (DD). The Kaplan-Meier analysis and Cox regression hazard model were applied to assess their effect on prognosis. Totally 102 hospitalized breast cancer patients who received trastuzumab were collected and followed-up. All patients were HER-2 positive advanced breast cancer, with a median age of 45 years old. Extended PT, APTT, and TT were found in trastuzumab treatment non-effective group, as well as increased Fib and DD. But significant increase was only found in Fib. High Fib status (Fib > 2.88 g/L) was correlated with clinical characteristics, such as pathological grade, and reversely correlated with PTEN expression. More importantly, poor disease-free survival (DFS) and overall survival (OS) to trastuzumab treatment were found in high Fib breast cancer patients. This retrospective study suggests high Fib status was correlated with poor treatment response to trastuzumab. Our findings indicated that Fib > 2.88 should alert physicians to consider a pretreatment for reducing Fib levels before trastuzumab treatment in HER-2 positive breast cancer patients.

SRC-1 and Twist1 expression positively correlates with a poor prognosis in human breast cancer.

To evaluate the possible prognostic value of Steroid Receptor Coactivator-1 (SRC-1) and Twist1 expression in human breast cancer, we examined SRC-1 and Twist1 expression using immunohistochemistry on tissue microarray sections containing 137 breast cancer specimens. All patients were followed up for a median of 5 years following surgery. Survival curves were generated using the Kaplan-Meier method. Multivariate analysis was performed using the Cox proportional hazard regression model to assess the prognostic values. The results showed a positive correlation between SRC-1 and Twist1 expression at protein levels (P < 0.001). Also, SRC-1 expression positively correlated with HER2 expression (P = 0.024). The protein expression of Twist1 positively associated with lymph node metastasis (P < 0.001), but inversely correlated with PR status (P = 0.041). Patients with SRC-1 or Twist1-positive expression exhibited poorer overall survival (OS) and disease-free survival (DFS) than did those with SRC-1 or Twist1-negative expression (P < 0.05 for all). In addition, SRC-1-negativeive/Twist1-negative patients had the best OS and DFS (P < 0.01 for both). In multivariate survival analysis, SRC-1 expression, tumor stage, and PR were found to be independent prognostic factors related to OS (P = 0.019, < 0.001 and 0.02, respectively) and Twist1 expression, lymph node status and PR were independent predictors of DFS (P = 0.006, 0.001 and 0.029, respectively). These results suggest that a combined SRC-1/Twist1 expression status could improve the prognostic judgment for breast cancer patients.

TGF-beta1 immunohistochemistry and promoter methylation in chronic renal failure rats treated with Uremic Clearance Granules.

The aim of the study was the explain the mechanism related to therapeutic effects of Uremic Clearance Granules (Niaoduqing Keli in Chinese) on adenine-induced Chronic Renal Failure in rats. Thirty 8-week-old male Wistar rats were selected and randomly divided in to 3 groups: Normal Control Group (NCG)consisted of 10 rats, Chronic Renal Failure Pathological Control Group (PCG) 10 rats, and Uremic Clearance Granules Treatment Group (UCG) 10 rats. Each rat in PCG and UCG was fed with adenine-enriched diets, containing 10 g adenine per kg food for 6 weeks. After fed with adenine, each rat in UCG was administered orally with 2 ml solution of Uremic Clearance Granules for 6 weeks. The concentration of Uremic Clearance Granules solution was 0.42 g/ml which was 10 times of human. On days 42 and 84, the serum levels of creatinine, Blood Urea Nitrogen and homocysteine were determined. The methylation of TGFbeta1 promoter was tested by methylation-specific PCR. TGF-beta1 mRNA and protein expression in rat renal cortex were analyzed by real-time RT-PCR and Immunohistochemistry. (1) Experimented on model of Chronic Renal Failure in rats, the preparation was proved to be able to reduce serum creatinine, Blood Urea Nitrogen, and homocysteine (p<0.05), improve renal function. (2) The expression of TGF-beta1 in mRNA and protein level were down-regulated. (3) TGF-beta1 promoter was demethylated at some loci in PCG, and was recovered in UCG. After treatment with Uremic Clearance Granules, the Chronic Renal Failure Wistar rat's kidney function was recovered. The recovery may be result of the remethylation of TGF-beta1 promoter and then lead to TGF-beta1 be transcripted and translated normally. The experimental study explain the molecular mechanism by which Uremic Clearance Granules treat Chronic Renal Failure.