Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However, an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study, we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1 (ALDH1), side-population (SP), and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore, knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised NOD/SCID mice, supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion, but not for CSC maintenance. Taken together, our study provides evidence that KLF4 plays a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus, targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer.

Melatonin, an endogenously produced neurohormone secreted by the pineal gland, has a variety of physiological functions and neuroprotective effects. It can modulate the functions of neural stem cells (NSCs) including proliferation and differentiation in embryonic brain tissue but its effect and mechanism on the stem cells in hypoxia remains to be explored. Here, we show that melatonin stimulates proliferation of NSCs during hypoxia. Additionally, it also promoted the differentiation of NSCs into neurons. However, it did not appear to exert an obvious effect on the differentiation of astrocytes. The present results have further shown that the promotional effect of NSCs proliferation by melatonin involved the MT1 receptor and increased phosphorylation of ERK1/2. The effect of melatonin on differentiation of NSCs is linked to altered expression of differentiation-related genes. In the light of these findings, it is suggested that melatonin may be beneficial as a supplement for treatment of neonatal hypoxic-ischemic brain injury for promoting the proliferation and differentiation of NSCs.

Purpose The improved soft tissue contrast of magnetic resonance imaging (MRI) compared to computed tomography (CT) makes it a useful imaging modality for radiotherapy treatment planning. Even when MR images are acquired for treatment planning, the standard clinical practice currently also requires a CT for dose calculation and x‐ray–based patient positioning. This increases workloads, introduces uncertainty due to the required inter‐modality image registrations, and involves unnecessary irradiation. While it would be beneficial to use exclusively MR images, a method needs to be employed to estimate a synthetic CT (sCT) for generating electron density maps and patient positioning reference images. We investigated 2D and 3D convolutional neural networks (CNNs) to generate a male pelvic sCT using a T1‐weighted MR image and compare their performance. Methods A retrospective study was performed using CTs and T1‐weighted MR images of 20 prostate cancer patients. CTs were deformably registered to MR images to create CT‐MR pairs for training networks. The proposed 2D CNN, which contained 27 convolutional layers, was modified from the state‐of‐the‐art 2D CNN to save computational memory and prepare for building the 3D CNN. The proposed 2D and 3D models were trained from scratch to map intensities of T1‐weighted MR images to CT Hounsfield Unit (HU) values. Each sCT was generated in a fivefold cross‐validation framework and compared with the corresponding deformed CT (dCT) using voxel‐wise mean absolute error (MAE). The sCT geometric accuracy was evaluated by comparing bone regions, defined by thresholding at 150 HU in the dCTs and the sCTs, using dice similarity coefficient (DSC), recall, and precision. To evaluate sCT patient positioning accuracy, bone regions in dCTs and sCTs were rigidly registered to the corresponding cone‐beam CTs. The resulting paired Euler transformation vectors were compared by calculating translation vector distances and absolute differences of Euler angles. Statistical tests were performed to evaluate the differences among the proposed models and Han’s model. Results Generating a pelvic sCT required approximately 5.5 s using the proposed models. The average MAEs within the body contour were 40.5 ± 5.4 HU (mean ± SD) and 37.6 ± 5.1 HU for the 2D and 3D CNNs, respectively. The average DSC, recall, and precision for the bone region (thresholding the CT at 150 HU) were 0.81 ± 0.04, 0.85 ± 0.04, and 0.77 ± 0.09 for the 2D CNN, and 0.82 ± 0.04, 0.84 ± 0.04, and 0.80 ± 0.08 for the 3D CNN, respectively. For both models, mean translation vector distances are less than 0.6 mm with mean absolute differences of Euler angles less than 0.5°. Conclusions The 2D and 3D CNNs generated accurate pelvic sCTs for the 20 patients using T1‐weighted MR images. Statistical tests indicated that the proposed 3D model was able to generate sCTs with smaller MAE and higher bone region precision compared to 2D models. Results of patient alignment tests suggested that sCTs generated by the proposed CNNs can provide accurate pa...

Aims/hypothesis: Maternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs. Materials and methods: Cell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2′-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT-PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically. Results: High glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells. Conclusions/interpretation: The changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the basis for the defective neural tube patterning observed in embryos of diabetic pregnancies.

Overexpression of HOXB7 in breast cancer cells induces an epithelial–mesenchymal transition and promotes tumor progression and lung metastasis. However, the underlying mechanisms for HOXB7-induced aggressive phenotypes in breast cancer remain largely unknown. Here, we report that phosphorylation of SMAD3 was detected in a higher percentage in primary mammary tumor tissues from double-transgenic MMTV-Hoxb7/Her2 mice than tumors from single-transgenic Her2/neu mice, suggesting activation of TGFβ/SMAD3 signaling by HOXB7 in breast tumor tissues. As predicted, TGFβ2 was high in four MMTV-Hoxb7/Her2 transgenic mouse tumor cell lines and two breast cancer cell lines transfected with HOXB7, whereas TGFβ2 was low in HOXB7-depleted cells. HOXB7 directly bound to and activated the TGFβ2 promoter in luciferase and chromatin immunoprecipitation assays. Increased migration and invasion as a result of HOXB7 overexpression in breast cancer cells were reversed by knockdown of TGFβ2 or pharmacologic inhibition of TGFβ signaling. Furthermore, knockdown of TGFβ2 in HOXB7-overexpressing MDA-MB-231 breast cancer cells dramatically inhibited metastasis to the lung. Interestingly, HOXB7 overexpression also induced tumor-associated macrophage (TAM) recruitment and acquisition of an M2 tumor-promoting phenotype. TGFβ2 mediated HOXB7-induced activation of macrophages, suggesting that TAMs may contribute to HOXB7-promoted tumor metastasis. Providing clinical relevance to these findings, by real-time PCR analysis, there was a strong correlation between HOXB7 and TGFβ2 expression in primary breast carcinomas. Taken together, our results suggest that HOXB7 promotes tumor progression in a cell-autonomous and non–cell-autonomous manner through activation of the TGFβ signaling pathway.