RORα Binds to E2F1 to Inhibit Cell Proliferation and Regulate Mammary Gland Branching Morphogenesis

Retinoic acid receptor-related orphan nuclear receptor alpha (RORα) is a potent tumor suppressor that reduces cell proliferation and inhibits tumor growth. However, the molecular mechanism by which it inhibits cell proliferation remains unknown. We demonstrate a noncanonical nuclear receptor pathway in which RORα binds to E2F1 to inhibit cell cycle progression. We showed that RORα bound to the heptad repeat and marked box region of E2F1 and suppressed E2F1-regulated transcription in epithelial cells. Binding of RORα inhibited E2F1 acetylation and its DNA-binding activity by recruiting histone deacetylase 1 (HDAC1) to the protein complexes. Knockdown of HDAC1 or inhibition of HDAC activity at least partially rescued transcription factor activity of E2F1 that was repressed by RORα. Importantly, RORα levels were increased in mammary ducts compared to terminal end buds and inversely correlated with expression of E2F1 target genes and cell proliferation. Silencing RORα in mammary epithelial cells significantly enhanced cell proliferation in the ductal epithelial cells and promoted side branching of the mammary ducts. These results reveal a novel link between RORα and E2F1 in regulating cell cycle progression and mammary tissue morphogenesis

Prolyl-4-Hydroxylase α Subunit 2 Promotes Breast Cancer Progression and Metastasis by Regulating Collagen Deposition

BACKGROUND: Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase α subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated. METHODS: Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis. RESULTS: Using gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models. CONCLUSION: These results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression

Image-Adaptive YOLO for Object Detection in Adverse Weather Conditions

Though deep learning-based object detection methods have achieved promising results on the conventional datasets, it is still challenging to locate objects from the low-quality images captured in adverse weather conditions. The existing methods either have difficulties in balancing the tasks of image enhancement and object detection, or often ignore the latent information beneficial for detection. To alleviate this problem, we propose a novel Image-Adaptive YOLO (IA-YOLO) framework, where each image can be adaptively enhanced for better detection performance. Specifically, a differentiable image processing (DIP) module is presented to take into account the adverse weather conditions for YOLO detector, whose parameters are predicted by a small convolutional neural net-work (CNN-PP). We learn CNN-PP and YOLOv3 jointly in an end-to-end fashion, which ensures that CNN-PP can learn an appropriate DIP to enhance the image for detection in a weakly supervised manner. Our proposed IA-YOLO approach can adaptively process images in both normal and adverse weather conditions. The experimental results are very encouraging, demonstrating the effectiveness of our proposed IA-YOLO method in both foggy and low-light scenarios.Comment: AAAI 2022, Preprint version with Appendi

Chaperone Hsp47 Drives Malignant Growth and Invasion by Modulating an ECM Gene Network

The extracellular matrix (ECM) is a determining factor in the tumor microenvironment that restrains or promotes malignant growth. In this report, we show how the molecular chaperone protein Hsp47 functions as a nodal hub in regulating an ECM gene transcription network. A transcription network analysis showed that Hsp47 expression was activated during breast cancer development and progression. Hsp47 silencing reprogrammed human breast cancer cells to form growth-arrested and/or noninvasive structures in 3D cultures, and to limit tumor growth in xenograft assays by reducing deposition of collagen and fibronectin. Coexpression network analysis also showed that levels of microRNA(miR)-29b and -29c were inversely correlated with expression of Hsp47 and ECM network genes in human breast cancer tissues. We found that miR-29 repressed expression of Hsp47 along with multiple ECM network genes. Ectopic expression of miR-29b suppressed malignant phenotypes of breast cancer cells in 3D culture. Clinically, increased expression of Hsp47 and reduced levels of miR-29b and -29c were associated with poor survival outcomes in breast cancer patients. Our results show that Hsp47 is regulated by miR-29 during breast cancer development and progression, and that increased Hsp47 expression promotes cancer progression in part by enhancing deposition of ECM proteins

Increased ROS Production in Non-Polarized Mammary Epithelial Cells Induces Monocyte Infiltration in 3D Culture

Loss of epithelial cell polarity promotes cell invasion and cancer dissemination. Therefore, identification of factors that disrupt polarized acinar formation is crucial. Reactive oxygen species (ROS) drive cancer progression and promote inflammation. Here, we show that the non-polarized breast cancer cell line T4-2 generates significantly higher ROS levels than polarized S1 and T4R cells in three-dimensional (3D) culture, accompanied by induction of the nuclear factor κB (NF-κB) pathway and cytokine expression. Minimizing ROS in T4-2 cells with antioxidants reestablished basal polarity and inhibited cell proliferation. Introducing constitutively activated RAC1 disrupted cell polarity and increased ROS levels, indicating that RAC1 is a crucial regulator that links cell polarity and ROS generation. We also linked monocyte infiltration with disruption of polarized acinar structure using a 3D co-culture system. Gain- and loss-of-function experiments demonstrated that increased ROS in non-polarized cells is necessary and sufficient to enhance monocyte recruitment. ROS also induced cytokine expression and NF-κB activity. These results suggest that increased ROS production in mammary epithelial cell leads to disruption of cell polarity and promotes monocyte infiltration

RORα Suppresses Cancer-Associated Inflammation by Repressing Respiratory Complex I-Dependent ROS Generation

Breast cancer development is associated with macrophage infiltration and differentiation in the tumor microenvironment. Our previous study highlights the crucial function of reactive oxygen species (ROS) in enhancing macrophage infiltration during the disruption of mammary tissue polarity. However, the regulation of ROS and ROS-associated macrophage infiltration in breast cancer has not been fully determined. Previous studies identified retinoid orphan nuclear receptor alpha (RORα) as a potential tumor suppressor in human breast cancer. In the present study, we showed that retinoid orphan nuclear receptor alpha (RORα) significantly decreased ROS levels and inhibited ROS-mediated cytokine expression in breast cancer cells. RORα expression in mammary epithelial cells inhibited macrophage infiltration by repressing ROS generation in the co-culture assay. Using gene co-expression and chromatin immunoprecipitation (ChIP) analyses, we identified complex I subunits NDUFS6 and NDUFA11 as RORα targets that mediated its function in suppressing superoxide generation in mitochondria. Notably, the expression of RORα in 4T1 cells significantly inhibited cancer metastasis, reduced macrophage accumulation, and enhanced M1-like macrophage differentiation in tumor tissue. In addition, reduced RORα expression in breast cancer tissue was associated with an increased incidence of cancer metastasis. These results provide additional insights into cancer-associated inflammation, and identify RORα as a potential target to suppress ROS-induced mammary tumor progression

Comprehensive Fractal Description of Porosity of Coal of Different Ranks

We selected, as the objects of our research, lignite from the Beizao Mine, gas coal from the Caiyuan Mine, coking coal from the Xiqu Mine, and anthracite from the Guhanshan Mine. We used the mercury intrusion method and the low-temperature liquid nitrogen adsorption method to analyze the structure and shape of the coal pores and calculated the fractal dimensions of different aperture segments in the coal. The experimental results show that the fractal dimension of the aperture segment of lignite, gas coal, and coking coal with an aperture of greater than or equal to 10 nm, as well as the fractal dimension of the aperture segment of anthracite with an aperture of greater than or equal to 100 nm, can be calculated using the mercury intrusion method; the fractal dimension of the coal pore, with an aperture range between 2.03 nm and 361.14 nm, can be calculated using the liquid nitrogen adsorption method, of which the fractal dimensions bounded by apertures of 10 nm and 100 nm are different. Based on these findings, we defined and calculated the comprehensive fractal dimensions of the coal pores and achieved the unity of fractal dimensions for full apertures of coal pores, thereby facilitating, overall characterization for the heterogeneity of the coal pore structure

NFkB Disrupts Tissue Polarity in 3D by Preventing Integration of Microenvironmental Signals

The microenvironment of cells controls their phenotype, and thereby the architecture of the emerging multicellular structure or tissue. We have reported more than a dozen microenvironmental factors whose signaling must be integrated in order to effect an organized, functional tissue morphology. However, the factors that prevent integration of signaling pathways that merge form and function are still largely unknown. We have identified nuclear factor kappa B (NFkB) as a transcriptional regulator that disrupts important microenvironmental cues necessary for tissue organization. We compared the gene expression of organized and disorganized epithelial cells of the HMT-3522 breast cancer progression series: the non-malignant S1 cells that form polarized spheres (\u27acini\u27), the malignant T4-2 cells that form large tumor-like clusters, and the \u27phenotypically reverted\u27 T4-2 cells that polarize as a result of correction of the microenvironmental signaling. We identified 180 genes that display an increased expression in disorganized compared to polarized structures. Network, GSEA and transcription factor binding site analyses suggested that NFkB is a common activator for the 180 genes. NFkB was found to be activated in disorganized breast cancer cells, and inhibition of microenvironmental signaling via EGFR, beta1 integrin, MMPs, or their downstream signals suppressed its activation. The postulated role of NFkB was experimentally verified: Blocking the NFkB pathway with a specific chemical inhibitor or shRNA induced polarization and inhibited invasion of breast cancer cells in 3D cultures. These results may explain why NFkB holds promise as a target for therapeutic intervention: Its inhibition can reverse the oncogenic signaling involved in breast cancer progression and integrate the essential microenvironmental control of tissue architecture

Hsp47 Promotes Cancer Metastasis by Enhancing Collagen-Dependent Cancer Cell-Platelet Interaction

Increased expression of extracellular matrix (ECM) proteins in circulating tumor cells (CTCs) suggests potential function of cancer cell-produced ECM in initiation of cancer cell colonization. Here, we showed that collagen and heat shock protein 47 (Hsp47), a chaperone facilitating collagen secretion and deposition, were highly expressed during the epithelial-mesenchymal transition (EMT) and in CTCs. Hsp47 expression induced mesenchymal phenotypes in mammary epithelial cells (MECs), enhanced platelet recruitment, and promoted lung retention and colonization of cancer cells. Platelet depletion in vivo abolished Hsp47-induced cancer cell retention in the lung, suggesting that Hsp47 promotes cancer cell colonization by enhancing cancer cell–platelet interaction. Using rescue experiments and functional blocking antibodies, we identified type I collagen as the key mediator of Hsp47-induced cancer cell–platelet interaction. We also found that Hsp47-dependent collagen deposition and platelet recruitment facilitated cancer cell clustering and extravasation in vitro. By analyzing DNA/RNA sequencing data generated from human breast cancer tissues, we showed that gene amplification and increased expression of Hsp47 were associated with cancer metastasis. These results suggest that targeting the Hsp47/collagen axis is a promising strategy to block cancer cell–platelet interaction and cancer colonization in secondary organs