YB-1 bridges neural stem cells and brain tumor-initiating cells via its roles in differentiation and cell growth.

The Y-box binding protein 1 (YB-1) is upregulated in many human malignancies including glioblastoma (GBM). It is also essential for normal brain development, suggesting that YB-1 is part of a neural stem cell (NSC) network. Here, we show that YB-1 was highly expressed in the subventricular zone (SVZ) of mouse fetal brain tissues but not in terminally differentiated primary astrocytes. Conversely, YB-1 knockout mice had reduced Sox-2, nestin, and musashi-1 expression in the SVZ. Although primary murine neurospheres were rich in YB-1, its expression was lost during glial differentiation. Glial tumors often express NSC markers and tend to loose the cellular control that governs differentiation; therefore, we addressed whether YB-1 served a similar role in cancer cells. YB-1, Sox-2, musashi-1, Bmi-1, and nestin are coordinately expressed in SF188 cells and 9/9 GBM patient-derived primary brain tumor-initiating cells (BTIC). Silencing YB-1 with siRNA attenuated the expression of these NSC markers, reduced neurosphere growth, and triggered differentiation via coordinate loss of GSK3-ß. Furthermore, differentiation of BTIC with 1% serum or bone morphogenetic protein-4 suppressed YB-1 protein expression. Likewise, YB-1 expression was lost during differentiation of normal human NSCs. Consistent with these observations, YB-1 expression increased with tumor grade (n = 49 cases). YB-1 was also coexpressed with Bmi-1 (Spearmans 0.80, P > 0.001) and Sox-2 (Spearmans 0.66, P > 0.001) based on the analysis of 282 cases of high-grade gliomas. These proteins were highly expressed in 10/15 (67%) of GBM patients that subsequently relapsed. In conclusion, YB-1 correlatively expresses with NSC markers where it functions to promote cell growth and inhibit differentiation.

Expression of N-myc downstream regulated gene 1 (NDRG1) in central neurocytoma.

N-myc downstream regulated gene 1 (NDRG1), also known as Cap43, Drg-1, and rit42, is expressed in various normal tissues and cancers, in which it is often associated with a favorable prognosis. It also plays a critical role in central nervous system development, with NDRG1 deficiency resulting in neural defects in mice. Central neurocytoma (CN) is a relatively rare tumor of the neurocytes in the brain, which occurs mainly in young adults. In the present study, we found that tissue samples from four patients with CN had both nuclear and cytoplasmic/membranous expression of NDRG1 protein in highly differentiated CN tumor cells. NDRG1 was also expressed in intratumoral microvessels. Immunohistochemical study of serial sections from the same patients revealed a marked association between the expression pattern of NDRG1 and that of neuron-specific enolase, a tumor differentiation marker. The data presented in this study suggest that NDRG1 could be considered a potential differentiation marker for CN.

Impaired ovarian development and reduced fertility in female mice deficient in Skp2.

p27 is a major negative regulator of somatic cellular proliferation, and its down-regulation has been shown to be associated with cancer development. Targeted disruption ofp27 results in complete loss of fertility in female mice, suggesting that it plays a significant role in the development of female gametes and the surrounding environment. We have now investigated the effect of loss of Skp2, an F-box protein that mediates ubiquitin-dependent degradation of p27, on female gamete production. The female Skp2-deficient mice showed accumulation of p27 in the ovary and severely compromised gamete development from the embryonic stage to follicular growth in the adult ovary, eventually leading to a decreased functional gamete reserve. Additional deletion of p27 resulted in relatively normal ovarian folliculogenesis, suggesting that accumulating p27 is primarily responsible for the compromised ovarian development. Embryonic ovaries of Skp2(-/-) mice manifested massive apoptosis as evidenced by cleavage of pro-caspase 3 and poly(ADP-ribose) polymerase-1. This in turn resulted in a significant decrease in the remaining pool of functional gametes in Skp2(-/-) mice shortly after sexual maturity and premature ovarian failure. The increased apoptosis seemed to be attributable to the polyploidy of granulosa cells. These results suggest that proper progression of the cell cycle, regulated by the p27-Skp2 axis, is pivotal for the maintenance of fertility, and that defects in this system may underlie the pathogenesis of abnormal gamete production and premature ovarian failure during the reproductive life of women.

N-myc downstream-regulated gene 1 (NDRG1) a differentiation marker of human breast cancer.

N-myc downstream-regulated gene 1 (NDRG1), also called differentiation-related gene-1 (Drg1) and Cap43, is expressed in various normal tissues and suppressed in several malignancies. In this study, whether NDRG1 expression was correlated with differentiation of human breast cancer cells has been investigated. Endogenous expression level of NDRG1 was closely correlated with differentiation status of breast cancer cell lines. Furthermore, sodium butyrate (NaB), an inducer of cellular differentiation, increased the expression of ß-casein, a milk-related differentiation marker, together with up-regulation of NDRG1 in breast cancer cells. In contrast, inhibition of NDRG1 by its siRNA resulted in reduced accumulation of ß-casein. Immunohistochemical analysis showed co-expression of NDRG1 and ß-casein or milk fat protein (MFP), another differentiation marker of breast tissue, in the mouse xenograft model of breast cancer. Furthermore, overexpression of NDRG1 expanded the differentiated area in the xenograft model of breast cancer. In human breast cancer, using samples from 45 patients, we also showed close relationship between NDRG1 and ß-casein or MFP expression. Altogether, in vitro and in vivo data demonstrated a possible role of NDRG1 in differentiation of breast cancer. We concluded that NDRG1 could be used as a biomarker for differentiation of breast cancer for both diagnostic and therapeutic purposes.

Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas.

Rhabdomyosarcoma, consisting of alveolar (aRMS) and embryonal (eRMS) subtypes, is the most common type of sarcoma in children. Currently, there are no targeted drug therapies available for rhabdomyosarcoma. In searching for new molecular therapeutic targets, we carried out genome-wide small interfering RNA (siRNA) library screens targeting human phosphatases (n = 206) and kinases (n = 691) initially against an aRMS cell line, RH30. Sixteen phosphatases and 50 kinases were identified based on growth inhibition after 72 hours. Inhibiting polo-like kinase 1 (PLK1) had the most remarkable impact on growth inhibition (approximately 80%) and apoptosis on all three rhabdomyosarcoma cell lines tested, namely, RH30, CW9019 (aRMS), and RD (eRMS), whereas there was no effect on normal muscle cells. The loss of PLK1 expression and subsequent growth inhibition correlated with decreased p-CDC25C and Cyclin B1. Increased expression of WEE 1 was also noted. The induction of apoptosis after PLK1 silencing was confirmed by increased p-H2AX, propidium iodide uptake, and chromatin condensation, as well as caspase-3 and poly(ADP-ribose) polymerase cleavage. Pediatric Ewing's sarcoma (TC-32), neuroblastoma (IMR32 and KCNR), and glioblastoma (SF188) models were also highly sensitive to PLK1 inhibition. Finally, based on cDNA microarray analyses, PLK1 mRNA was overexpressed (>1.5 fold) in 10 of 10 rhabdomyosarcoma cell lines and in 47% and 51% of primary aRMS (17 of 36 samples) and eRMS (21 of 41 samples) tumors, respectively, compared with normal muscles. Similarly, pediatric Ewing's sarcoma, neuroblastoma, and osteosarcoma tumors expressed high PLK1. We conclude that PLK1 could be a promising therapeutic target for the treatment of a wide range of pediatric solid tumors including rhabdomyosarcoma.

Effect of combined treatment with alendronate and calcitriol on femoral neck strength in osteopenic rats.

BACKGROUND: Hip fracture is associated with pronounced morbidity and excess mortality in elderly women with postmenopausal osteoporosis. Many drugs have been developed to treat osteoporosis and to reduce the risk of osteoporotic fractures. We investigated the effects of combined alendronate and vitamin D3 treatment on bone mass and fracture load at the femoral neck in ovariectomized (OVX) rats, and evaluated the relationship between bone mass parameters and femoral neck strength. METHODS: Thirty 12-week-old female rats underwent either a sham-operation (n = 6) or OVX (n = 24). Twenty weeks later, OVX rats were further divided into four groups and received daily doses of either saline alone, 0.1 mg/kg alendronate, 0.1 microg/kg calcitriol, or a combination of both two drugs by continuous infusion via Alzet mini-osmotic pumps. The sham-control group received saline alone. After 12 weeks of treatment, femoral necks were examined using peripheral quantitative computed tomography (pQCT) densitometry and mechanical testing. RESULTS: Saline-treated OVX rats showed significant decreases in total bone mineral content (BMC) (by 28.1%), total bone mineral density (BMD) (by 9.5%), cortical BMC (by 26.3%), cancellous BMC (by 66.3%), cancellous BMD (by 29.0%) and total cross-sectional bone area (by 30.4%) compared with the sham-control group. The combined alendronate and calcitriol treatments improved bone loss owing to estrogen deficiency. On mechanical testing, although OVX significantly reduced bone strength of the femoral neck (by 29.3%) compared with the sham-control group, only the combined treatment significantly improved the fracture load at the femoral neck in OVX rats to the level of the sham-controls. The correlation of total BMC to fracture load was significant, but that of total BMD was not. CONCLUSION: Our results showed that the combined treatment with alendronate and calcitriol significantly improved bone fragility of the femoral neck in OVX osteopenic rats.

Tyrosine-kinase inhibition results in EGFR clustering at focal adhesions and consequent exocytosis in uPAR down-regulated cells of head and neck cancers.

BACKGROUND: Antisense (AS) induced down-regulation of uPAR in ACCS adenoid-cyctic carcinoma cells decreased the cellular adhesion and invasion on various extracellular matrices. Additionally, ACCS-AS cells showed an increased EGFR expression and other behavioral similarities to NA-SCC, a typical highly proliferative but less invasive squamous cell carcinoma (SCC) cell line of the head and neck. ACCS, ACCS-AS and NA-SCC cells were used to elucidate the relationships between uPAR down-regulation and EGFR inhibition. RESULTS: Tyrosine kinase inhibitor Gefitinib (IRESSA, ZD 1839) significantly reduced the chemotactic cell migration and adhesion. This was associated with reduced EGFR and ERK activation. In addition, anti-proliferative effect of gefitinib in uPAR down-regulated ACCS-AS was significantly higher than parental ACCS, to levels comparable to gefitinib-sensitive NA-SCC cells. This was evidenced by both reduced dosage and duration of treatment. Furthermore, time-lapse videography showed that treatment with gefitinib was also associated with cell rounding and loss of pseudopodia, mostly in ACCS-AS rather than parental ACCS cells. There were also evidences of formation and exocytosis of vacuole-like structures in ACCS-AS, as well as NA-SCC, but not in parental ACCS cells. Interestingly, immunocytochemistry showed that the exocytotic vacuoles actually contained de-activated EGFR. CONCLUSION: Our results suggested that down-regulation of uPAR affected the fate of EGFR in high EGFR expressing cells. Furthermore, combining the uPAR down-regulation with EGFR inhibition showed a synergistic anti-tumor effect and might provide an alternative method to increase anti-proliferative effect of tyrosine kinase inhibitors with lower doses and duration to reduce their side effects during cancer control.

Effect of preoperative combined treatment with alendronate and calcitriol on fixation of hydroxyapatite-coated implants in ovariectomized rats.

BACKGROUND: Osteoporosis is a potential risk factor affecting implant stability following total joint arthroplasty. Bisphosphonates are potent inhibitors of osteoblastic bone resorption, and vitamin D is an important hormone involved in the regulation of calcium metabolism. We investigated the benefit of preoperative treatment with alendronate, vitamin D (calcitriol), or a combination of these substances on fixation of hydroxyapatite-coated implants in an ovariectomized rat model of osteoporosis. METHODS: Of sixty-four female rats in the study, fifty-six underwent ovariectomy and eight, a sham operation. A hydroxyapatite-coated femoral implant and an uncoated implant were implanted in all rats seven months after the ovariectomy or the sham operation. Of the fifty-six rats that underwent ovariectomy, eight served as the control group. The remaining forty-eight were treated with alendronate, calcitriol, or a combination of these agents either starting eight weeks before implantation of the rod or starting immediately after implantation. The treatment was continued until four weeks after the implantation. Four weeks after the implantation, the total and cancellous bone mineral density at the proximal part of the tibia and the shear strength of the bone-implant interface were measured in all rats. RESULTS: Although total bone mineral density increased in all of the alendronate-treated groups, compared with that in the ovariectomized control group, cancellous bone mineral density increased only in the group pretreated with both alendronate and calcitriol. The implant stability in the ovariectomized control rats was significantly decreased compared with that in the sham-operation rats. While treatment with alendronate only or calcitriol only did not improve the stability of the implants, treatment with a combination of alendronate and calcitriol, starting preoperatively, significantly improved the stability of the hydroxyapatite-coated implants. CONCLUSIONS: Treatment with a combination of alendronate and calcitriol, starting preoperatively, can improve cancellous bone mineral density and the stability of hydroxyapatite-coated implants in an osteoporotic rat.

Expression of the urokinase receptor regulates focal adhesion assembly and cell migration in adenoid cystic carcinoma cells.

Adenoid cystic carcinoma (AdCC) cell lines (ACCS and ACCT) showed higher migration responses and adhesion to the extracellular matrix (ECM), especially types I and IV collagen, than did the oral squamous cell carcinoma (SCC) lines (NA and TF). The response to collagens was largely and exclusively inhibited by anti-alpha(2) integrin antibody. Moreover, AdCC cell lines expressed higher surface levels of urokinase-type plasminogen activator receptor (uPAR) than did SCC cell lines. When AdCC cells were plated on collagen, the surface level of uPAR was increased, and numerous focal adhesions consisting of uPAR, vinculin, and paxillin were assembled; whereas collagen-stimulated SCC cell counterparts or AdCC cells plated on other types of ECM, such as fibronectin, failed to assemble such definite focal adhesions. In order to elucidate the association of uPAR with collagen-induced events, an ACCS-AS cell line transfected with a vector expressing antisense uPAR RNA was established and shown to have reduced uPAR (about 10% that of parental ACCS at both the protein and mRNA levels). ACCS-AS showed a strong reduction of collagen-stimulated migration and focal adhesion assembly of alpha(2) integrin, vinculin, and paxillin. These findings suggest that AdCC has a proclivity for migrating to types I and IV collagens due to the overexpression of uPAR, which plays a key role in focal adhesion assembly and migration.