ECM-transmitted shear stress induces apoptotic cell extrusion in early breast gland development.

Epithelial cells of human breast glands are exposed to various mechanical ECM stresses that regulate tissue development and homeostasis. Mechanoadaptation of breast gland tissue to ECM-transmitted shear stress remained poorly investigated due to the lack of valid experimental approaches. Therefore, we created a magnetic shear strain device that enabled, for the first time, to analyze the instant shear strain response of human breast gland cells. MCF10A-derived breast acini with basement membranes (BM) of defined maturation state and basoapical polarization were used to resemble breast gland morphogenesis in vitro. The novel biophysical tool was used to apply cyclic shear strain with defined amplitudes (≤15%, 0.2 Hz) over 22 h on living spheroids embedded in an ultrasoft matrix (<60 Pa). We demonstrated that breast spheroids gain resistance to shear strain, which increased with BM maturation and basoapical polarization. Most intriguingly, poorly developed spheroids were prone to cyclic strain-induced extrusion of apoptotic cells from the spheroid body. In contrast, matured spheroids were insensitive to this mechanoresponse-indicating changing mechanosensing or mechanotransduction mechanisms during breast tissue morphogenesis. Together, we introduced a versatile tool to study cyclic shear stress responses of 3D cell culture models. It can be used to strain, in principle, all kinds of cell clusters, even those that grow only in ultrasoft hydrogels. We believe that this approach opens new doors to gain new insights into dynamic shear strain-induced mechanobiological regulation circuits between cells and their ECM.

A Simple Evaluation Tool (ET-CET) Indicates Increase of Diagnostic Skills From Small Bowel Capsule Endoscopy Training Courses: A Prospective Observational European Multicenter Study.

Small bowel capsule endoscopy (SBCE) has become a first line diagnostic tool. Several training courses with a similar format have been established in Europe; however, data on learning curve and training in SBCE remain sparse.Between 2008 and 2011, different basic SBCE training courses were organized internationally in UK (n = 2), Italy (n = 2), Germany (n = 2), Finland (n = 1), and nationally in Germany (n = 10), applying similar 8-hour curricula with 50% lectures and 50% hands-on training. The Given PillCam System was used in 12 courses, the Olympus EndoCapsule system in 5, respectively. A simple evaluation tool for capsule endoscopy training (ET-CET) was developed using 10 short SBCE videos including relevant lesions and normal or irrelevant findings. For each video, delegates were required to record a diagnosis (achievable total score from 0 to 10) and the clinical relevance (achievable total score 0 to 10). ET-CET was performed at baseline before the course and repeated, with videos in altered order, after the course.Two hundred ninety-four delegates (79.3% physicians, 16.3% nurses, 4.4% others) were included for baseline analysis, 268 completed the final evaluation. Forty percent had no previous experience in SBCE, 33% had performed 10 or less procedures. Median scores for correct diagnosis improved from 4.0 (IQR 3) to 7.0 (IQR 3) during the courses (P < 0.001, Wilcoxon), and for correct classification of relevance of the lesions from 5.0 (IQR 3) to 7.0 (IQR 3) (P < 0.001), respectively. Improvement was not dependent on experience, profession, SBCE system, or course setting. Previous experience in SBCE was associated with higher baseline scores for correct diagnosis (P < 0.001; Kruskal-Wallis). Additionally, independent nonparametric partial correlation with experience in gastroscopy (rho 0.33) and colonoscopy (rho 0.27) was observed (P < 0.001).A simple ET-CET demonstrated significant improvement of diagnostic skills on completion of formal basic SBCE courses with hands-on training, regardless of preexisting experience, profession, and course setting. Baseline scores for correct diagnoses show a plateau after interpretation of 25 SBCE before courses, supporting this number as a compromise for credentialing. Experience in flexible endoscopy may be useful before attending an SBCE course.

Nuclear transport receptor karyopherin-α2 promotes malignant breast cancer phenotypes in vitro.

Tumorigenesis and tumor progression are associated with dysfunction of the nuclear transport machinery at the level of import and export receptors (karyopherins). Recent studies have shown that the nuclear import factor karyopherin-α2 (KPNA2) is a novel prognostic marker for poor prognosis in human breast cancer. Based on the well-defined hallmarks of cancer progression, we performed a detailed in vitro characterization of the phenotypic effects caused by KPNA2 overexpression and KPNA2 silencing in benign and malignant human breast cells. KPNA2 overexpression clearly increased proliferation of MCF7 tumor cells and further led to a reduction of cell-matrix adhesion in benign MCF10A cells, whereas cell migration was significantly increased (P<0.0001) in both tumor models. Remarkably, these individual effects of KPNA2 overexpression on proliferation, cell-matrix adhesion and migration resulted in an increased colony spreading of benign MCF10A breast cells and malignant MCF7 tumor cells (P<0.001), which is a hallmark of cancer progression. Conversely, RNA interference-mediated KPNA2 silencing caused a complete inhibition of MCF7 tumor cell proliferation and migration (P<0.0001). In addition, in these experiments apoptosis was increased (P<0.05) and formation of tumor cell colonies was reduced (P<0.01). Thus, KPNA2 overexpression provoked increased aggressiveness of malignant MCF7 breast tumor cells and induced a shift in benign MCF10A breast cells toward a malignant breast cancer phenotype. In conclusion, we demonstrate for the first time in experimental tumor models that forced KPNA2 expression drives malignant features relevant for breast cancer progression, while its silencing is required for the remission of those progressive phenotypes. This study gives clear evidence that KPNA2 acts as a novel oncogenic factor in human breast cancer, in vitro.

Intermediate filament dynamics and breast cancer: aberrant promoter methylation of the Synemin gene is associated with early tumor relapse.

Synemin (SYNM) is a type IV intermediate filament that has recently been shown to interact with the LIM domain protein zyxin, thereby possibly modulating cell adhesion and cell motility. Owing to this multiplicity of potential functions relevant to cancer development, we initiated a study to decipher SYNM expression and regulation in benign human breast tissue and breast cancer. Dot blot array analysis showed significant SYNM mRNA downregulation in 86% (n=100, P<0.001) of breast cancers compared with their normal tissue counterparts, a result that was confirmed by real-time PCR analysis (n=36, P<0.0001). Immunohistochemistry analysis showed abundant SYNM protein expression in healthy myoepithelial breast cells, whereas SYNM expression loss was evident in 57% (n=37, P<0.001) of breast cancer specimens. Next, we analyzed methylation of the SYNM promoter to clarify whether the SYNM gene can be silenced by epigenetic means. Indeed, methylation-specific PCR analysis showed tumor-specific SYNM promoter methylation in 27% (n=195) of breast cancers. As expected, SYNM promoter methylation was tightly associated (P<0.0001) with SYNM expression loss. In-depth analysis of the SYNM promoter by pyrosequencing showed extensive CpG methylation of DNA elements supposed to regulate gene transcription. Demethylating treatment of SYNM methylated breast cancer cell lines with 5-aza-2-deoxycytidine clearly reestablished the SYNM expression. Statistical analysis of the patient cohort showed a close association between SYNM promoter methylation and unfavorable recurrence-free survival (hazard ratio=2.941, P=0.0282). Furthermore, SYNM methylation positively correlated with lymph node metastases (P=0.0177) and advanced tumor grade (P=0.0275), suggesting that SYNM methylation is associated with aggressive forms of breast cancer. This is the first study on the epigenetic regulation of the SYNM gene in a cancer entity. We provide first hints that SYNM could represent a novel putative breast tumor suppressor gene that is prone to epigenetic silencing. SYNM promoter methylation may become a useful predictive biomarker to stratify breast cancer patients' risk for tumor relapse.

[Promoter methylation of ID4. A marker for recurrence-free survival in human breast cancer]. / Promotormethylierung von ID4. Ein Marker für die Rezidivbildung des humanen Mammakarzinoms.

The aim of this study was to unravel the role of the transcription factor inhibitor of DNA binding 4 (ID4) in human breast carcinogenesis in more detail, especially the impact of ID4 promoter methylation on disease progression. Demethylating treatment of breast cancer cell lines was associated with ID4 reexpression. ID4 promoter methylation was frequently observed in primary breast cancer samples (68.9%, 117/170). We found a very tight correlation (p<0.001) between ID4 promoter methylation and loss of ID4 mRNA expression in these specimens. For breast tissue as the first tumour entity analyzed in detail, we could show a direct correlation between ID4 promoter methylation and loss of ID4 expression on both the mRNA and protein level. Interestingly, ID4 promoter methylation was a factor for unfavourable recurrence-free survival (p=0.036) and increased the patient's risk for lymph node metastases (p=0.030). Our data suggest that ID4 is a potential tumour suppressor gene in human breast tissues that undergoes epigenetic silencing during carcinogenesis, leading to an increased risk for tumour relapse. Thus, ID4 methylation status could serve as a prognostic biomarker in human breast cancer.