Yulong Zou

The Calcium-Binding Protein S100A6 Accelerates Human Osteosarcoma Growth by Promoting Cell Proliferation and Inhibiting Osteogenic Differentiation

Background/Aims: Although osteosarcoma (OS) is the most common primary malignancy of bone, its molecular pathogenesis remains to be fully understood. We previously found the calcium-binding protein S100A6 was expressed in ∼80% of the analyzed OS primary and/or metastatic tumor samples. Here, we investigate the role of S100A6 in OS growth and progression. Methods: S100A6 expression was assessed by qPCR and Western blotting. Overexpression or knockdown of S100A6 was carried out to determine S100A6s effect on proliferation, cell cycle, apoptosis, tumor growth, and osteogenic differentiation. Results: S100A6 expression was readily detected in human OS cell lines. Exogenous S100A6 expression promoted cell proliferation in vitro and tumor growth in an orthotopic xenograft model of human OS. S100A6 overexpression reduced the numbers of OS cells in G1 phase and increased viable cells under serum starvation condition. Conversely, silencing S100A6 expression induced the production of cleaved caspase 3, and increased early stage apoptosis. S100A6 knockdown increased osteogenic differentiation activity of mesenchymal stem cells, while S100A6 overexpression inhibited osteogenic differentiation. BMP9-induced bone formation was augmented by S100A6 knockdown. Conclusion: Our findings strongly suggest that S100A6 may promote OS cell proliferation and OS tumor growth at least in part by facilitating cell cycle progression, preventing apoptosis, and inhibiting osteogenic differentiation. Thus, it is conceivable that targeting S100A6 may be exploited as a novel anti-OS therapy.

Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells.

Ovarian cancer is the most lethal gynecologic malignancy with an overall cure rate of merely 30%. Most patients experience recurrence within 12–24 months of cure and die of progressively chemotherapy-resistant disease. Thus, more effective anti-ovarian cancer therapies are needed. Here, we investigate the possibility of repurposing antibiotic monensin as an anti-ovarian cancer agent. We demonstrate that monensin effectively inhibits cell proliferation, migration and cell cycle progression, and induces apoptosis of human ovarian cancer cells. Monensin suppresses multiple cancer-related pathways including Elk1/SRF, AP1, NFκB and STAT, and reduces EGFR expression in ovarian cancer cells. Monensin acts synergistically with EGFR inhibitors and oxaliplatin to inhibit cell proliferation and induce apoptosis of ovarian cancer cells. Xenograft studies confirm that monensin effectively inhibits tumor growth by suppressing cell proliferation through targeting EGFR signaling. Our results suggest monensin may be repurposed as an anti-ovarian cancer agent although further preclinical and clinical studies are needed.

A thermoresponsive polydiolcitrate-gelatin scaffold and delivery system mediates effective bone formation from BMP9-transduced mesenchymal stem cells

Successful bone tissue engineering requires at the minimum sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we investigated the potential use of a biodegradable citrate-based thermosensitive macromolecule, poly(polyethyleneglycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin (PPCNG) as a scaffold for the delivery of BMP9-stimulated MSCs to promote localized bone formation. The addition of gelatin to PPCN effectively enhanced the cell adhesion and survival properties of MSCs entrapped within the gel in 3D culture. Using the BMP9-transduced MSC line immortalized mouse embryonic fibroblasts (iMEFs), we found that PPCNG facilitated BMP9-induced osteogenic differentiation of iMEFs in vivo and promoted the formation of well-ossified and vascularized trabecular bone-like structures in a mouse model of ectopic bone formation. Histologic evaluation revealed that vascularization of the bony masses retrieved from the iMEFs  +  PPCNG group was significantly more pronounced than that of the direct cell injection group. Accordingly, vascular endothelial growth factor (VEGF) expression was shown to be significantly higher in the bony masses recovered from the iMEFs  +  PPCNG group. Taken together, our results suggest that PPCNG may serve as a novel biodegradable and injectable scaffold and carrier for gene and cell-based bone tissue engineering.

Notch Signaling Augments BMP9-Induced Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells (MSCs)

Background/Aims: Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into several lineages including bone. Successful bone formation requires osteogenesis and angiogenesis coupling of MSCs. Here, we investigate if simultaneous activation of BMP9 and Notch signaling yields effective osteogenesis-angiogenesis coupling in MSCs. Methods: Recently-characterized immortalized mouse adipose-derived progenitors (iMADs) were used as MSC source. Transgenes BMP9, NICD and dnNotch1 were expressed by adenoviral vectors. Gene expression was determined by qPCR and immunohistochem¡stry. Osteogenic activity was assessed by in vitro assays and in vivo ectopic bone formation model. Results: BMP9 upregulated expression of Notch receptors and ligands in iMADs. Constitutively-active form of Notch1 NICD1 enhanced BMP9-induced osteogenic differentiation both in vitro and in vivo, which was effectively inhibited by dominant-negative form of Notch1 dnNotch1. BMP9- and NICD1-transduced MSCs implanted with a biocompatible scaffold yielded highly mature bone with extensive vascularization. NICD1 enhanced BMP9-induced expression of key angiogenic regulators in iMADs and Vegfa in ectopic bone, which was blunted by dnNotch1. Conclusion: Notch signaling may play an important role in BMP9-induced osteogenesis and angiogenesis. It’s conceivable that simultaneous activation of the BMP9 and Notch pathways should efficiently couple osteogenesis and angiogenesis of MSCs for successful bone tissue engineering.

A Blockade of IGF Signaling Sensitizes Human Ovarian Cancer Cells to the Anthelmintic Niclosamide-Induced Anti- Proliferative and Anticancer Activities

Background/Aims: Ovarian cancer is the most lethal gynecologic malignancy, and there is an unmet clinical need to develop new therapies. Although showing promising anticancer activity, Niclosamide may not be used as a monotherapy. We seek to investigate whether inhibiting IGF signaling potentiates Niclosamides anticancer efficacy in human ovarian cancer cells. Methods: Cell proliferation and migration are assessed. Cell cycle progression and apoptosis are analyzed by flow cytometry. Inhibition of IGF signaling is accomplished by adenovirus-mediated expression of siRNAs targeting IGF-1R. Cancer-associated pathways are assessed using pathway-specific reporters. Subcutaneous xenograft model is used to determine anticancer activity. Results: We find that Niclosamide is highly effective on inhibiting cell proliferation, cell migration, and cell cycle progression, and inducing apoptosis in human ovarian cancer cells, possibly by targeting multiple signaling pathways involved in ELK1/SRF, AP-1, MYC/MAX and NFkB. Silencing IGF-1R exert a similar but weaker effect than that of Niclosamides. However, silencing IGF-1R significantly sensitizes ovarian cancer cells to Niclosamide-induced anti-proliferative and anticancer activities both in vitro and in vivo. Conclusion: Niclosamide as a repurposed anticancer agent may be more efficacious when combined with agents that target other signaling pathways such as IGF signaling in the treatment of human cancers including ovarian cancer.

Noncanonical Wnt signaling plays an important role in modulating canonical Wnt-regulated stemness, proliferation and terminal differentiation of hepatic progenitors

The liver provides vital metabolic, exocrine and endocrine functions in the body as such pathological conditions of the liver lead to high morbidity and mortality. The liver is highly regenerative and contains facultative stem cells that become activated during injury to replicate to fully recover mass and function. Canonical Wnt/β-catenin signaling plays an important role in regulating the proliferation and differentiation of liver progenitor cells during liver regeneration. However, possible roles of noncanonical Wnts in liver development and regeneration remain undefined. We previously established a reversibly-immortalized hepatic progenitor cell line (iHPx), which retains hepatic differentiation potential. Here, we analyze the expression pattern of the essential components of both canonical and noncanonical Wnt signaling pathways at different postnatal stages of mouse liver tissues and iHPx cells. We find that noncanonical Wnt4, Wnt5a, Wnt9b, Wnt10a and Wnt10b, are highly expressed concordantly with the high levels of canonical Wnts in late stages of liver tissues. Wnt5a, Wnt9b, Wnt10a and Wnt10b are able to antagonize Wnt3a-induced β-catenin/TCF activity, reduce the stemness of iHPx cells, and promote hepatic differentiation of liver progenitors. Stem cell implantation assay demonstrates that Wnt5a, Wnt9b, Wnt10a and Wnt10b can inhibit cell proliferation and promote hepatic differentiation of the iHPx progenitor cells. Our results strongly suggest that noncanonical Wnts may play an important role in fine-tuning Wnt/β-catenin functions during liver development and liver regeneration. Thus, understanding regulatory mechanisms governing proliferation and differentiation of liver progenitor cells may hold great promise to facilitate liver regeneration and/or progenitor cell-based therapies for liver diseases.

NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells

Background: BMP9 induces both osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). Nell1 is a secretory glycoprotein with osteoinductive and anti-adipogenic activities. We investigated the role of Nell1 in BMP9-induced osteogenesis and adipogenesis in MSCs. Methods: Previously characterized MSCs iMEFs were used. Overexpression of BMP9 and NELL1 or silencing of mouse Nell1 was mediated by adenoviral vectors. Early and late osteogenic and adipogenic markers were assessed by staining techniques and qPCR analysis. In vivo activity was assessed in an ectopic bone formation model of athymic mice. Results: We demonstrate that Nell1 expression was up-regulated by BMP9. Exogenous Nell1 potentiated BMP9-induced late stage osteogenic differentiation while inhibiting the early osteogenic marker. Forced Nell1 expression enhanced BMP9-induced osteogenic regulators/markers and inhibited BMP9-upregulated expression of adipogenic regulators/markers in MSCs. In vivo ectopic bone formation assay showed that exogenous Nell1 expression enhanced mineralization and maturity of BMP9-induced bone formation, while inhibiting BMP9-induced adipogenesis. Conversely, silencing Nell1 expression in BMP9-stimulated MSCs led to forming immature chondroid-like matrix. Conclusion: Our findings indicate that Nell1 can be up-regulated by BMP9, which in turn accelerates and augments BMP9-induced osteogenesis. Exogenous Nell1 may be exploited to enhance BMP9-induced bone formation while overcoming BMP9-induced adipogenesis in regenerative medicine.

Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses

Background/Aims: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production. Methods: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity. Results: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection. Conclusion: The RAPA cells are highly efficient for adenovirus production and amplification.

Anthelmintic mebendazole enhances cisplatin’s effect on suppressing cell proliferation and promotes differentiation of head and neck squamous cell carcinoma (HNSCC)

Head and neck squamous cell carcinoma (HNSCC) is one of the most common and aggressive types of human cancers worldwide. Nearly a half of HNSCC patients experience recurrence within five years of treatment and develop resistance to chemotherapy. Thus, there is an urgent clinical need to develop safe and novel anticancer therapies for HNSCC. Here, we investigate the possibility of repurposing the anthelmintic drug mebendazole (MBZ) as an anti-HNSCC agent. Using the two commonly-used human HNSCC lines CAL27 and SCC15, we demonstrate MBZ exerts more potent anti-proliferation activity than cisplatin in human HNSCC cells. MBZ effectively inhibits cell proliferation, cell cycle progression and cell migration, and induces apoptosis of HNSCC cells. Mechanistically, MBZ can modulate the cancer-associated pathways including ELK1/SRF, AP1, STAT1/2, MYC/MAX, although the regulatory outcomes are context-dependent. MBZ also synergizes with cisplatin in suppressing cell proliferation and inducing apoptosis of human HNSCC cells. Furthermore, MBZ is shown to promote the terminal differentiation of CAL27 cells and keratinization of CAL27-derived xenograft tumors. Our results are the first to demonstrate that MBZ may exert its anticancer activity by inhibiting proliferation while promoting differentiation of certain HNSCC cancer cells. Its conceivable the anthelmintic drug MBZ can be repurposed as a safe and effective agent used in combination with other frontline chemotherapy drugs such as cisplatin in HNSCC treatment.

The Prodomain-Containing BMP9 Produced from a Stable Line Effectively Regulates the Differentiation of Mesenchymal Stem Cells

Background: BMPs play important roles in regulating stem cell proliferation and differentiation. Using adenovirus-mediated expression of the 14 types of BMPs we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs), which was undetected in the early studies using recombinant BMP9 proteins. Endogenous BMPs are expressed as a precursor protein that contains an N-terminal signal peptide, a prodomain and a C-terminal mature peptide. Most commercially available recombinant BMP9 proteins are purified from the cells expressing the mature peptide. It is unclear how effectively these recombinant BMP9 proteins functionally recapitulate endogenous BMP9. Methods: A stable cell line expressing the full coding region of mouse BMP9 was established in HEK-293 cells by using the piggyBac transposon system. The biological activities and stability of the conditioned medium generated from the stable line were analyzed. Results: The stable HEK-293 line expresses a high level of mouse BMP9. BMP9 conditioned medium (BMP9-cm) was shown to effectively induce osteogenic differentiation of MSCs, to activate BMP-R specific Smad signaling, and to up-regulate downstream target genes in MSCs. The biological activity of BMP9-cm is at least comparable with that induced by AdBMP9 in vitro. Furthermore, BMP9-cm exhibits an excellent stability profile as its biological activity is not affected by long-term storage at -80ºC, repeated thawing cycles, and extended storage at 4ºC. Conclusions: We have established a producer line that stably expresses a high level of active BMP9 protein. Such producer line should be a valuable resource for generating biologically active BMP9 protein for studying BMP9 signaling mechanism and functions.