Targeting circadian transcriptional programs in triple negative breast cancer through a cis-regulatory mechanism.

Circadian clock genes are emerging targets in many types of cancer, but their mechanistic contributions to tumor progression are still largely unknown. This makes it challenging to stratify patient populations and develop corresponding treatments. In this work, we show that in breast cancer, the disrupted expression of circadian genes has the potential to serve as biomarkers. We also show that the master circadian transcription factors (TFs) BMAL1 and CLOCK are required for the proliferation of metastatic mesenchymal stem-like (mMSL) triple-negative breast cancer (TNBC) cells. Using currently available small molecule modulators, we found that a stabilizer of cryptochrome 2 (CRY2), the direct repressor of BMAL1 and CLOCK transcriptional activity, synergizes with inhibitors of proteasome, which is required for BMAL1 and CLOCK function, to repress a transcriptional program comprising circadian cycling genes in mMSL TNBC cells. Omics analyses on drug-treated cells implied that this repression of transcription is mediated by the transcription factor binding sites (TFBSs) features in the cis-regulatory elements (CRE) of clock-controlled genes. Through a massive parallel reporter assay, we defined a set of CRE features that are potentially repressed by the specific drug combination. The identification of cis -element enrichment may serve as a new way of defining and targeting tumor types through the modulation of cis -regulatory programs, and ultimately provide a new paradigm of therapy design for cancer types with unclear drivers like TNBC.

The interplay between the circadian clock and abiotic stress responses mediated by ABF3 and CCA1/LHY.

Climate change is a global concern for all life on our planet, including humans and plants. Plants' growth and development are significantly affected by abiotic stresses, including adverse temperature, inadequate or excess water availability, nutrient deficiency, and salinity. The circadian clock is a master regulator of numerous developmental and metabolic processes in plants. In an effort to identify new clock-related genes and outputs through bioinformatic analysis, we have revealed that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) play a crucial role in regulating a wide range of abiotic stress responses and target ABSCISIC ACID RESPONSIVE ELEMENTS-BINDING FACTOR3 (ABF3), a key transcription factor in the plant hormone Abscisic acid (ABA)-signaling pathway. Specifically, we found that CCA1 and LHY regulate the expression of ABF3 under diel conditions, as well as seed germination under salinity. Conversely, ABF3 controls the expression of core clock genes and orchestrates the circadian period in a stress-responsive manner. ABF3 delivers the stress signal to the central oscillator by binding to the promoter of CCA1 and LHY. Overall, our study uncovers the reciprocal regulation between ABF3 and CCA1/LHY and molecular mechanisms underlying the interaction between the circadian clock and abiotic stress. This finding may aid in developing molecular and genetic solutions for plants to survive and thrive in the face of climate change.

E-box binding transcription factors in cancer.

E-boxes are important regulatory elements in the eukaryotic genome. Transcription factors can bind to E-boxes through their basic helix-loop-helix or zinc finger domain to regulate gene transcription. E-box-binding transcription factors (EBTFs) are important regulators of development and essential for physiological activities of the cell. The fundamental role of EBTFs in cancer has been highlighted by studies on the canonical oncogene MYC, yet many EBTFs exhibit common features, implying the existence of shared molecular principles of how they are involved in tumorigenesis. A comprehensive analysis of TFs that share the basic function of binding to E-boxes has been lacking. Here, we review the structure of EBTFs, their common features in regulating transcription, their physiological functions, and their mutual regulation. We also discuss their converging functions in cancer biology, their potential to be targeted as a regulatory network, and recent progress in drug development targeting these factors in cancer therapy.

Harnessing matrix stiffness to engineer a bone marrow niche for hematopoietic stem cell rejuvenation.

Hematopoietic stem cell (HSC) self-renewal and aging are tightly regulated by paracrine factors from the bone marrow niche. However, whether HSC rejuvenation could be achieved by engineering a bone marrow niche ex vivo remains unknown. Here, we show that matrix stiffness fine-tunes HSC niche factor expression by bone marrow stromal cells (BMSCs). Increased stiffness activates Yap/Taz signaling to promote BMSC expansion upon 2D culture, which is largely reversed by 3D culture in soft gelatin methacrylate hydrogels. Notably, 3D co-culture with BMSCs promotes HSC maintenance and lymphopoiesis, reverses aging hallmarks of HSCs, and restores their long-term multilineage reconstitution capacity. In situ atomic force microscopy analysis reveals that mouse bone marrow stiffens with age, which correlates with a compromised HSC niche. Taken together, this study highlights the biomechanical regulation of the HSC niche by BMSCs, which could be harnessed to engineer a soft bone marrow niche for HSC rejuvenation.

A BMP-2-triggered in vivo osteo-organoid for cell therapy.

Cell therapies and regenerative medicine interventions require an adequate source of therapeutic cells. Here, we demonstrate that constructing in vivo osteo-organoids by implanting bone morphogenetic protein-2-loaded scaffolds into the internal muscle pocket near the femur of mice supports the growth and subsequent harvest of therapeutically useful cells including hematopoietic stem/progenitor cells (HSPCs), mesenchymal stem cells (MSCs), lymphocytes, and myeloid cells. Profiling of the in vivo osteo-organoid maturation process delineated three stages-fibroproliferation, osteochondral differentiation, and marrow generation-each of which entailed obvious changes in the organoid structure and cell type distribution. The MSCs harvested from the osteochondral differentiation stage mitigated carbon tetrachloride (CCl4)-induced chronic liver fibrosis in mice, while HSPCs and immune cells harvested during the marrow generation stage rapidly and effectively reconstituted the impaired peripheral and solid immune organs of irradiated mice. These findings demonstrate the therapeutic potentials of in vivo osteo-organoid-derived cells in cell therapies.

Effect of sulfated chitosan hydrogel on vascularization and osteogenesis.

Bone regeneration and vascularization have presented a clinical challenge for decades. Considering the importance of stem cells, such as mesenchymal stem cells (MSCs), in bone regeneration, endothelial progenitor cells (EPCs) are crucial during bone repair. This paper presented sulfated chitosan (SCS)-based hydrogel scaffolds to accelerate bone tissue regeneration, vascularization enhancement, and improve bone repair. Thus, these scaffolds played a crucial role in the regeneration of blood vessels, with the increased presentation of epithelial progenitors and immune cells in this microenvironment. In vivo experiments showed that the biological impact of SCS was critical for angiogenesis and vascularization, in conjunction with bone morphogenetic protein-2 (BMP-2) and MSCs. Therefore, the BMP-2-/hydrogel system established in this study promoted angiogenesis, stimulated MSC proliferation, and enhanced bone tissue formation. In addition, this paper highlighted the angiogenic role of SCS in creating a micro-environment for effective bone repair and provides insight into the future development of new bone regeneration material.

Clocking cancer: the circadian clock as a target in cancer therapy.

Disruption of the cellular pathway modulating endogenous 24-h rhythms, referred to as "the circadian clock", has been recently proven to be associated with cancer risk, development, and progression. This pathway operates through a complex network of transcription-translation feedback loops generated by a set of interplaying proteins. The expression of core circadian clock genes is frequently dysregulated in human tumors; however, the specific effects and underlying mechanisms seem to vary depending on the cancer types and are not fully understood. In addition, specific oncogenes may differentially induce the dysregulation of the circadian clock in tumors. Pharmacological modulation of clock components has been shown to result in specific lethality in certain types of cancer cells, and thus holds great promise as a novel anti-cancer therapeutic approach. Here we present an overview of the rationale and current evidence for targeting the clock in cancer treatment.

Tuning the bioactivity of bone morphogenetic protein-2 with surface immobilization strategies.

Bone morphogenetic protein-2 (BMP-2) involved therapy is of great potential for bone regeneration. However, its clinical application is restricted due to the undesirable bioactivity and relevant complications in vivo. Immobilization of recombinant BMP-2 (rhBMP-2) is an efficient strategy to mimic natural microenvironment and retain its bioactivity. Herein, we present evidences indicating that osteoinductive capacity of rhBMP-2 can be regulated via variant immobilizing approaches. Three representative superficial immobilizing models were employed to fabricate rhBMP-2-immobilized surfaces including physical adsorption (Au/rhBMP-2), covalent grafting (rhBMP-2-SAM-Au) and heparin binding (Hep-SAM-Au/rhBMP-2) (SAM: self-assembled monolayer). Loading capacity, releasing behavior, osteogenic differentiation and signaling pathways involved, as well as the cellular recognition of rhBMP-2 under various immobilization modes were systematically investigated. As a result, disparate immobilizing approaches not only have effects on loading capacity, but also lead to disparity of osteoinduction at the same dosage. Notably, heparin could reinforce the recognition between rhBMP-2 and its receptors (BMPRs) whereas weaken its binding to its antagonist Noggin. Owing to this "selective" binding feature, the favorable osteoinduction and maximum ectopic bone formation can be achieved with the heparin-binding approach. In particular, manipulation of orientation-mediated BMP-2-cell recognition efficiency may be a potential target to design more therapeutic efficient rhBMP-2 delivery system. STATEMENT OF SIGNIFICANCE: Bone morphogenetic protein-2 (BMP-2) is crucial in bone regeneration. However, its clinical application is challenged due to its shorten half-life and supra-physiological dose associated complications. In this study, three representative superficial immobilizing patterns were fabricated through physical adsorption, covalent grafting and electrostatic interaction with heparin respectively. We provided evidences indicating an dose-dependent osteoinductive capacity of immobilized BMP-2. Further, a possible mechanism of rhBMP-2-cell recognition at the interface was presented, highlighting the superior effect of heparin on rhBMP-2 bioactivity. Finally, We proposed a dual mechanism of tuning the bioactivity of immobilized rhBMP-2 through surface immobilization approaches: regulation of the saturated loading capacity and orientation-mediated rhBMP-2-cell recognition. These results provide novel insights into designing criterion of efficient delivery vehicle for rhBMP-2.

Manipulation of VEGF-induced angiogenesis by 2-N, 6-O-sulfated chitosan.

Emerging evidence suggests that vascular endothelial growth facto (VEGF) is important in the treatment of various ischemic and cardiovascular diseases. However, it often suffers from high cost and easy deactivation with a short half-life. Here, we describe a synthetic 2-N, 6-O-sulfated chitosan (26SCS) with a high affinity to VEGF promoting the binding of the signaling protein to its VEGF receptor 2 (VEGFR2), activating receptor phosphorylation and pro-angiogenic related genes expression, and further stimulating downstream VEGF-dependent endothelial cell viability, migration, tube formation and rat aortic rings outgrowth. Interestingly, the obvious recruitment of mural cells were occurred to stabilize the sprouted microvessels. In addition, the pro-angiogenic potential of 26SCS composited VEGF was confirmed in vivo using the chick embryo chorioallantoic membrane (CAM) assay with an extensive perfusable vascular network. A longer monitoring was administered subcutaneously to mice in a biocompatible gelatin sponge and showed that VEGF with 26SCS had the capability to efficiently enhance neovascularization. These findings highlight that 26SCS, the semi-synthetic natural polymer, may be a promising coagent with VEGF for vascular therapy. STATEMENT OF SIGNIFICANCE: Vascular endothelial growth factor (VEGF) is crucial for facilitating angiogenesis to supply oxygen and nutrient during wound healing and tissue regeneration. However, appropriate use of VEGF is an ongoing challenge due to its rapidly clearance and severe side effects at higher dosage. In this study, we described a synthetic 2-N, 6-O-sulfated chitosan (26SCS) with a high affinity to VEGF, which could significantly promote its binding capacity to VEGF receptor 2 and further stimulate the angiogenic behavior of endothelial cells. We further confirmed that 26SCS was spatially combined with VEGF in a "lying manner", and this spatial arrangement was more conducive to exposure of the receptor binding domain of VEGF. Additionally, it also promoted in vivo angiogenesis in a chicken chorioallantoic membrane assay and mouse subcutaneous implant model. This strategy may afford a new avenue to enhance pro-angiogenic capacity of VEGF.

Enhancement of BMP-2-mediated angiogenesis and osteogenesis by 2-N,6-O-sulfated chitosan in bone regeneration.

Sulfated polysaccharides are attractive semi-synthesized materials that can be used as a mimic of heparan sulfate to modulate the protein activity and other physiological processes. In this study, we employed sulfated chitosan to enhance the angiogenic capacity of bone morphogenic protein-2 (BMP-2). Bone marrow stromal cells (BMSCs) cultured in a combination of BMP-2 and 2-N,6-O-sulfated chitosan (SCS) group exhibited a higher cell viability and sprouting ability. The cells also secreted more VEGF and NO. The expression patterns of angiogenic and osteogenic genes were analyzed, and VEGFR2 signaling was found to play a role in the enhancing effect of SCS. The chick embryo chorioallantoic membrane (CAM) assay revealed an enhanced angiogenic effect of BMP-2 when SCS was involved. In addition, we investigated angiogenesis and osteogenesis in mouse using a BMP-2-induced ectopic bone model. Histological and immune-staining analysis revealed that both bone and vascular tissue were enhanced when SCS was added. These data prove that SCS can improve the angiogenic potential of BMP-2 and thus lead to better bone regeneration.