Archive for the ‘Molecular Osteosarcoma Studies’ Category

The Novel Curcumin Analog FLLL32 Decreases STAT3 DNA Binding Activity and Expression, and Induces Apoptosis in Osteosarcoma Cell Lines

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Posted 29 Mar 2011 — by James Street
Category Alternative Therapies, antiangiogenesis, Complementary Therapy, experimental treatments, Follow up Treatment, Local Recurrence, Lung Metastases, Metastases, Molecular Osteosarcoma Studies, Natural Therapies, Osteosarcoma

Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin.

FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3.

Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells.

Methods: Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT(R)), apoptosis (SensoLyte(R) Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2(MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting.

Results: Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA.

FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway.

Conclusions: These data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression.

Future work with FLLL32 will define the therapeutic potential of this compound in vivo.

Author: Stacey FosseyMisty BearJiayuh LinChenglong LiEric SchwartzPui-Kai LiJames FuchsJoelle FengerWilliam KisseberthCheryl London
Credits/Source: BMC Cancer 2011, 11:112

RNAi-mediated knockdown of cyclooxygenase2 inhibits the growth, invasion and migration of SaOS2 human osteosarcoma cells: a case control study

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Posted 06 Mar 2011 — by James Street
Category antiinflammatory, Human osteosarcoma research, Inflamation, Local Recurrence, Lung Metastases, Metastases, metastases, Molecular Osteosarcoma Studies, Osteosarcoma, Understanding Cancer

Cyclooxygenase2 (COX-2), one isoform of cyclooxygenase proinflammatory enzymes, is responsible for tumor development, invasion and metastasis. Due to its role and frequent overexpression in a variety of human malignancies, including osteosarcoma, COX-2 has received considerable attention.

However, the function of COX-2 in the pathogenesis of cancer is not well understood. We examined the role of COX-2 in osteosarcoma.

Methods: We employed lentivirus mediated-RNA interference technology to knockdown endogenous gene COX-2 expression in human osteosarcoma cells (SaOS2) and analyzed the phenotypical changes.

The effect of COX-2 treatment on the proliferation, cell cycle, invasion and migration of the SaOS2 cells were assessed using the MTT, flow cytometry, invasion and migration assays, respectively. COX-2, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) mRNA and protein expression were detected by RT-PCR and western blotting.

Results: Our results indicate that a decrease of COX-2 expression in human osteosarcoma cells significantly inhibited the growth, decreased the invasion and migration ability of SaOS2 cells.

In addition, it also reduced VEGF, EGF and bFGF mRNA and protein expression.

Conclusions: The COX-2 signaling pathway may provide a novel therapeutic target for the treatment of human osteosarcoma.

Author: Qinghua ZhaoChuan WangJiaxue ZhuLei WangShuanghai DongGaoqiao ZhangJiwei Tian
Credits/Source: Journal of Experimental &Clinical Cancer Research 2011, 30:26

Zoledronic acid suppresses lung metastases and prolongs overall survival of osteosarcoma-bearing mice

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Posted 03 Mar 2011 — by James Street
Category Lung Metastases, Metastases, Molecular Osteosarcoma Studies
  1. Benjamin Ory M.D.1,
  2. Marie-Françoise Heymann M.D.2,
  3. Akira Kamijo M.D., Ph.D.3,
  4. François Gouin M.D., Ph.D.1,
  5. Dominique Heymann Ph.D.1,*,†,
  6. Françoise Redini Ph.D.1

Article first published online: 3 NOV 2005

DOI: 10.1002/cncr.21530

Issue

Cancer

Keywords:

  • zoledronic acid;
  • lung metastases;
  • osteosarcoma

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

Although there is no doubt that bisphosphonates (BPs), specific inhibitors of osteoclasts, are beneficial for the treatment of bone metastases, their effects on visceral metastases are unclear. The effect of zoledronic acid (ZOL) was examined in vivo on lung metastasis progression and animal survival, and in vitro on the cellular mechanisms involved.

METHODS

An animal model of lung metastasis was developed in C3H/He mice inoculated intravenously with a spontaneous murine osteosarcoma POS-1 cell line. Lung metastasis was determined at the time of autopsy. ZOL was assessed in vitro on POS-1 cell proliferation, cell cycle progression, and caspase-1 and -3 activities.

RESULTS

The overall survival in five independent experiments (two series treated with ZOL 0.1 mg/kg twice a week, and three series with 0.1 mg/kg five times a week) showed a significant increase of the actuarial survival: 0.422 ± 0.07 in ZOL-treated animals versus 0.167 ± 0.07 in controls (P = 0.036). Lung metastases were absent in all ZOL-treated mice that survived more than 21 days postinjection as revealed by macroscopic and histologic analysis. In vitro, a 48-hour incubation with 10 μM ZOL inhibited POS-1 cell line proliferation associated with cell cycle arrest in S-phase. In addition, ZOL induced a weak increase of caspase-3 activity, but not caspase-1.

CONCLUSION

We demonstrate that ZOL exerts a direct antitumor effect on POS-1 cells in vitro, significantly diminishes osteosarcoma-induced lung metastasis in vivo, thereby prolonging survival of POS-1-inoculated animals. Cancer 2005. © 2005 American Cancer Society.

Bisphosphonates (BPs) are currently the most important class of inhibitors of osteoclast-mediated bone resorption. They are widely and successfully used for the treatment of skeletal diseases, such as Paget disease, postmenopausal osteoporosis, and tumor-induced osteolysis.1 BPs have a high affinity for hydroxyapatite mineral in bone and are taken up selectively and adsorbed to mineral surfaces at sites of increased bone turnover, where they inhibit osteoclast activity.2 In addition to their potent antiosteoclast effects, recent preclinical studies have shown that BPs induce apoptosis of cancer cells from several origins, including human myeloma, breast, and prostate carcinoma cell lines.3 It has also been demonstrated that BPs inhibit cancer cell invasion and angiogenesis.3 In spite of the widely recognized beneficial effects of BPs on bone metastases, the effects of BPs on visceral organs are unclear.4 In preclinical studies, the positive effects of zoledronic acid (ZOL) on nonskeletal metastases have been demonstrated in breast carcinoma models.5, 6 Therefore, it is necessary to extend these studies to nonosseous metastases secondary to cancers from other origins. Osteosarcoma (OS) is the most frequent primary bone tumor that develops mainly in the young, the median age of diagnosis being 18 years. A preference for pulmonary metastases compared with other metastatic sites is a distinct feature of OS and 5-year survival rates after the detection of lung metastasis are less than 30%.7 Despite recent improvements in chemotherapy and surgery, the problem of nonresponse to chemotherapy remains and current strategies for the treatment of high-grade osteosarcoma fail to improve its prognosis.8 Therefore, development of new therapies is needed.

In the present study, we investigated the effect of ZOL, an N-BP of the third generation, on the outcome of lung metastases induced by intravenous (i.v.) inoculation of POS-1 osteosarcoma cells. The POS-1 cell line is derived from an osteosarcoma which developed spontaneously in C3H mice. The tumor can be successfully transplanted in C3H mice or POS-1 cells inoculated into the hind footpad of mice and shows spontaneous metastasis to lung.9 Tumors were first recognized macroscopically at 2 weeks after inoculation and developed in more than 90% of inoculated mice at 5 weeks, lung metastasis being observed in all mice that developed tumors. Using the POS-1 cells, we developed a model of pulmonary metastases without primary bone tumor by inoculating the cells i.v. into the tail vein or by a retro-orbital approach. Therefore, this model was used to test the efficacy of ZOL on the progression of pulmonary metastases.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Animal Model

Four-week-old male C3H/He mice (Elevages Janvier, Le Genest St Isle, France) were housed under pathogen-free conditions at the Experimental Therapy Unit (Medicine Faculty of Nantes, France), in accordance with the institutional guidelines of the French Ethical Committee. The spontaneous murine osteosarcoma cell line POS-1 was kindly provided by the Kanagawa Cancer Centre (Kanagawa, Japan). The cells were cultured in RPMI 1640 medium (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS, Dominique Dutscher, Brumath, France) at 37 °C in a humidified atmosphere (5% CO2/95% air). The mice were anesthetized by inhalation of a mixture of isoflurane/air (1.5%, 1 L/min) combined with an intramuscular injection of Imalgene (100 mg/kg, Merial Laboratories, Lyon, France) prior to i.v. injection of 50 μL of POS-1 cell suspension containing 1.5 × 105 cells. Under these conditions, pulmonary metastases developed rapidly, leading to the death of the animals in 3 weeks after POS-1 cell injection.

Treatment of Mice with Zoledronic Acid

To determine the effect of zoledronic acid (ZOL, kindly provided as the disodium salt by Pharma Novartis, Basel, Switzerland) on lung metastasis development and mouse survival in the POS-1 osteosarcoma model, 24 mice were injected with POS-1 osteosarcoma cells as described above. At Day 2 after tumor cell inoculation, 6 mice were treated with vehicle alone (phosphate buffered saline, PBS), and 18 with ZOL in PBS at 3 different concentrations and sequences: 1) ZOL 100 μg/kg, twice a week; 2) ZOL 100 μg/kg 5 times a week; and 3) ZOL 1 mg/kg, twice a week. Treatment continued until each animal showed signs of morbidity, which included cachexia or respiratory distress, at which point they were sacrificed by cervical dislocation. Lung tumor dissemination was assessed by analyzing the number of tumor foci. Five independent experiments were performed.

Histologic Analysis

Lungs were fixed in 10% buffered formaldehyde, then embedded in paraffin. Sections (5 μM thick) were mounted on glass slides and stained with hematoxylin-eosin-safran (HES) and picrosirius red for collagen.

In Vitro Analyses

Cell proliferation

Replicate subconfluent cell cultures of POS-1 cells in 96-well plates were treated for 1–3 days with increasing concentrations of ZOL (10−7 to 10−4 M, diluted in RPMI). Cell viability was determined by the sodium 3′[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) cell proliferation reagent assay kit (Roche Molecular Biomedicals, Mannheim, Germany).

Caspase activity

POS-1 cells (2 × 104) grown in 24-well plates were treated with 1 or 10 μM ZOL for the indicated times, washed once with PBS, and lysed with 50 μL of RIPA buffer for 30 minutes. The cells were then scraped off and the protein amount was quantified using the BCA (bicinchominic acid + Copper II sulfate) test (Pierce Chemical, Rockford, IL). Caspase-1 and -3 activity was assessed on 10 μL of cell lysate with the CaspACE assay kit (Promega, Madison, WI) following the manufacturer’s recommendations. Cells treated with UV light for 30 seconds 24 hours before harvesting were used as a positive control for caspase activity.

Cell cycle analysis

Confluent POS-1 cells (treated with increasing concentrations of ZOL for 24 and 48 hr) were removed from culture dishes by trypsinization, washed twice in PBS, and incubated in PBS containing 0.12% Triton X-100, 0.12 mM EDTA, and 100 μg/mL DNase-free ribonuclease A (Sigma Chemical, St. Louis, MO). Then, 50 μg/mL propidium iodide (Sigma) were added for each sample for 20 minutes at 4 °C in the dark. The stained nuclei were analyzed by flow cytometry (FACScan, BD Biosciences, Franklin Lake, NJ) using CellQuest software. Cell cycle distribution was based on 2N and 4N DNA content.

Statistical Analysis

Cell proliferation data are expressed as mean ± SE. Comparison between groups was performed by the Mann-Whitney U-test. The effect of ZOL on disease-free survival was determined using the log rank test.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

ZOL Increases Mice Survival by Inhibiting Pulmonary Metastases Development

Five independent experiments were performed in which mice received 1.5 × 105 POS-1 cells, leading to the development of lung metastasis in 80–90% of mice, with death occurring between Days 17 and 24 after cell inoculation. First, a dose–response of ZOL was performed to determine the optimal sequence and concentration able to affect animal survival. ZOL was well tolerated, without any overt clinical signs of adverse effects. The results presented in Figure 1A reveal that the three sequences improved survival: ZOL 0.1 and 1 mg/kg twice a week induced a survival rate of 40% 45 days after POS-1 cell inoculation. ZOL 0.1 mg/kg 5 times per week maintained survival at 83% up to day 31, and then 66% at 45 days after POS-1 cells injection. A representation of the overall survival rate of five independent experiments (two series treated with 0.1 mg/kg twice a week, and three series with 0.1 mg/kg five times a week) shows a significant increase of the actuarial survival: 0.422 ± 0.07 in ZOL-treated animals versus 0.167 ± 0.07 in controls (P = 0.036, Fig. 1B).

Figure 1. Zoledronic acid (ZOL) prolongs survival of POS-1 cell-inoculated mice. (A) A typical dose–response experiment for ZOL is shown. Four-week-old male C3H/He mice received 1.5 × 105 POS-1 cells intravenously. Two days after inoculation the mice were separated into 4 groups (n = 6) treated subcutaneously as follows: control mice received vehicle (PBS) alone (full line), ZOL 1 mg/kg, twice a week (solid squares, dotted line), 0.1 mg/kg, five times a week (solid triangles, dotted line), 0.1 mg/kg twice a week (solid circles, dotted line). **P < 0.05, ***P < 0.005. (B) Overall survival in five independent series of mice treated with ZOL (solid squares, dotted line: 1 mg/kg, twice or five times a week) as compared with controls (full line). **P < 0.05.

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A high incidence of pulmonary metastases was observed in POS-1 cells-inoculated mice: 87% of mice were positive, with more than 50 tumor foci in each case.

The pulmonary tissue was invaded by tumor foci characterized by high-grade proliferating tumor cells and numerous venous emboli (Fig. 2A,B). Lung metastases were absent in all ZOL-treated mice that survived more than 21 days postinjection, as revealed by macroscopic observation. No metastases were observed at the histologic level 45 days after injection (Fig. 2C,D). The treated tissue is characterized by heterogeneous lung alveolar parenchyma and small fibrotic areas corresponding to regenerative healing tissue (Fig. 2C,D). The presence of collagen in this regenerative tissue was confirmed by picrosirius red staining (data not shown).

Figure 2. Zoledronic acid inhibits the development of lung metastasis in the mouse model of POS-1 cell-induced osteosarcoma. (A,B) Intravenously inoculated POS-1 cells induced numerous lung metastases. The pulmonary tissue is then invaded by high-grade proliferating cell foci (*) and venous emboli. (C,D) No lung metastatic foci were observed after zoledronic acid treatment and small areas of healing tissue can be observed (solid triangle, D). Animals were sacrificed at 45 days postinjection. Original magnification ×40 (A,C); ×200 (B,D).

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ZOL Inhibits POS-1 Cell Proliferation In Vitro and Induces Caspase-3 Activation

To determine whether the in vivo antitumor activity of ZOL could be mediated by a direct effect on POS-1 cell proliferation, ZOL effects were assessed in vitro. The XTT viability test showed that ZOL inhibited POS-1 cell proliferation, with an IC50 value of 44.28 μM (Fig. 3A). Hoechst 33258 staining and caspase activation were investigated to see whether the ZOL-induced inhibition of POS-1 cell proliferation was caused by apoptosis. No modification of nuclear morphology that is characteristic of apoptosis was observed in ZOL-treated POS-1 cells after Hoechst staining (not shown). The results showed that ZOL did not induce any activation of caspase-1 in POS-1 cells (not shown), but it did increase caspase-3 activity at a concentration of 10 μM (72 hr), as compared to positive (UV-treated cells) and negative controls (Fig. 3B).

Figure 3. In vitro effects of zoledronic acid (ZOL) on POS-1 cell proliferation and caspase activation. (A) Proliferation of POS-1 cells was determined after exposure to ZOL (0–10−4M) for 3 days with 10% fetal bovine serum, using the XTT quick proliferation kit as described in Materials and Methods. Changes in absorbance at 490 nm were measured and the results were plotted as percentage of untreated cell proliferation. ***P < 0.005. (B) POS-1 cells were treated with 1 or 10 μM ZOL for the indicated times. Caspase-3 activity was determined in cell lysates using a caspase-3-specific fluorogenic substrate as described in Materials and Methods. CT+ represents a positive control of cells treated with UV light for 30 seconds. CT– represents the POS-1 cells alone as a negative control. Results of three independent experiments are presented and the caspase activity is expressed as a change in absorbance at 460 nm per μg of protein.

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ZOL Induces S-phase Arrest in POS-1 Cells

Flow cytometry analysis of DNA content was performed with osteosarcoma POS-1 cells to identify cell cycle perturbations after ZOL treatment for 48 hours. The results presented in Figure 4 show a 1.8-fold increase in the number of cells arrested in S-phase after ZOL treatment (the number of cells in S-phase increase from 14% in control cells to 19% and 25% in the presence of 1 and 10 μM ZOL, respectively). This observation was concomitant with a reduction of cells in the G0/G1 phase: 55% and 53% for 1 and 10 μM ZOL, respectively (48 hr incubation) versus 63% in the control untreated POS-1 cells (Fig. 4).

Figure 4. Flow cytometry of zoledronic acid (ZOL)-treated POS-1 cells. POS-1 cells were incubated for 24–48 hours in the absence (control) or presence of 1 or 10 μM ZOL. At each point cells were harvested, fixed, and stained with propidium iodide. The positions on the histograms of the hypodiploid sub-G0/G1 (APO), G0/G1, S and G2/M peaks, and the percentage of cells in each of the cycle phases are indicated from a representative experiment.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Using a rat model of osteosarcoma, we previously demonstrated that ZOL was able to reduce primary tumor growth and prolong rat survival by decreasing lung metastases dissemination associated with a primary bone tumor.10 However, a direct effect on pulmonary metastases alone could not be identified in the rat model. Using a murine model of lung metastases induced by i.v. injection of osteosarcoma cells, we here demonstrate that ZOL significantly reduced lung metastasis progression, thus extending animal survival. The overall available data on the effects of BPs on visceral metastases in clinical and preclinical studies remain controversial. In a human study, Diel et al.11 initially described that clodronate had adjuvant inhibitory effects on metastases in visceral organs in breast cancer. Later, however, they found no significant effects of clodronate on visceral metastases in the same populations of patients in the extended follow-up.12 Moreover, McCloskey et al.13 did not observe adjuvant effects of clodronate. In contrast, Saarto et al.14 reported that adjuvant treatment with clodronate increased the development of nonskeletal metastases in breast carcinoma patients. Thus, the evidence from the completed clinical trials remain conflicting, as also revealed by preclinical studies in experimental animal models. Indeed, some data suggest that BPs may increase tumor burden and metastases in soft tissues.15, 16 In contrast, the experimental bisphosphonate YH529 reduced nonosseous metastases in the MDA-MB-231 model of breast carcinoma in nude mice.17 More recently, Michigami et al.5 reported that ibandronate reproducibly reduced bone metastases in two animal models of breast carcinoma, but in one of these models (the 4T1 mouse model), neither the preventive nor therapeutic administration of ibandronate caused any effects on lung metastases. In the MDA-MB-231 model of breast cancer, therapeutic administration of ibandronate showed no effects on adrenal metastases.5 More recently, using the 4T1 mouse model, Hiraga et al.6 demonstrated that zoledronic acid significantly suppressed lung and liver metastases and prolonged overall survival of tumor-bearing mice. Using the same model, Nobuyuki et al.18 showed that i.v. ZOL decreased tumor burden not only in bone but also in the liver and lungs of treated mice. In another model of mammary carcinoma cells injected into the medullar space of the proximal tibia of Fisher rats, alendronate reduced lung nodule counts by 95%.19 Here, we confirm the antitumor effect of ZOL on lung metastases progression using an experimental model different from breast carcinoma-derived lung metastases. In the present study, no metastases could be observed macroscopically 21 days after injection, and histologically 45 days postinjection. It can be suggested that micrometastases were present at 21 days, inducing the death of some animals between Days 21 and 45 (Fig. 1), and that at Day 45 the alive animals do not exhibit any further lung metastases. At that time, necrosis could not be observed in ZOL-treated animals. To explain why all of these treated animals do not survive, it can be suggested that in vivo some tumor cells escape to ZOL-induced inhibition of proliferation, probably by developing resistance to ZOL treatment. This phenomenon has been reported in the case of myeloma cells treated with alendronate, another N-BP.20 In this study, although N-BP induced apoptosis of myeloma cells in vitro, most in vivo studies fail to demonstrate a corresponding antitumor effect. This discrepancy might reflect the development of a metabolic resistance to the antitumor effect of N-BP in myeloma cells when they are exposed to N-BP for a prolonged time. In our laboratory, we developed a rat model of osteosarcoma in vivo and used the corresponding cell lines for in vitro experiments.10 When these cells were maintained for a long time in culture, part of these cells became resistant to ZOL treatment, so we can suggest that the same phenomenon happened with POS-1 cells.

Thus, ZOL, which seems to be one of the most effective antiresorptive BPs in vitro, may also exert potent antitumor activity in vivo against the progression of visceral metastases. Inhibition of several cellular mechanisms such as vascularization, adhesion, invasion, and migration have been proposed to explain this phenomenon.3 These mechanisms could be studied with the POS-1 cells used to induce pulmonary metastases in the present model. Indeed, the availability of the corresponding cells will allow testing the effects of ZOL on migration and invasion of POS-1 cells in vitro, and also integrin expression and cell adhesion to endothelial cells.

The ZOL doses used in the present study are justified, as 0.1 mg/kg is clearly equivalent to the clinical dose (4 mg i.v. every 3–4 weeks is equivalent to approximately 100 μg/kg of the research grade disodium salt). However, even if a dosing frequency of twice or five times a week is much greater, it could be justified by the very aggressive nature of lung metastases in the murine model and the short survival times. The doses and schedules of ZOL used in this preclinical study are in agreement with the subchronic and chronic toxicity data given by the Novartis Pharma Laboratories (pers. commun.). Therefore, ZOL doses used in the present study are related to the achievable, safe levels in humans. The observation that lung metastases were reduced suggests a direct effect of ZOL on tumor cells that was not dependent on its effect on the bone microenvironment. However, given the low transient levels of ZOL in blood and soft tissues, we cannot discriminate between a direct effect on tumor cell dissemination from the primary injection site or growth inhibition of the secondary lung metastases. Among the mechanisms hypothesized for the BP antitumor activity, data from the literature report that caspase-dependent apoptosis appears to be the major mechanism responsible for BP-induced tumor cell apoptosis, and caspase-3 is certainly the major player in this response.21 In our experimental model, ZOL caused a direct inhibition of POS-1 cell proliferation and an accumulation of cells in the S-phase of the cycle. Similar results were observed by Evdokiou et al.22 Indeed, using a panel of human osteogenic sarcoma cell lines, those authors demonstrated that ZOL reduced cell numbers in a dose- and time-dependent manner, due either to cell cycle arrest in the S-phase or to the induction of apoptosis.22 A comparable mechanism was also described for N-containing BPs such as ZOL in other cell types—for example, melanoma cells.23 In our study, cell cycle arrest in the S-phase was accompanied by a weak activation of caspase-3 activity, a well-characterized effect of N-BPs.21 However, it is difficult to conclude which is the exact mechanism involved in this model, as Hoescht staining did not reveal any modification of nuclear morphology in ZOL-treated cells (not shown). Moreover, in the cell cycle analysis (Fig. 4), no cells in the sub-G0/G1 phase were observed that could represent apoptotic cells. Therefore, the weak caspase-3 activation observed in our study may be nonspecific, associated with a general cytotoxic effect of the high concentration of ZOL. TUNEL staining, performed on rat osteosarcoma tumor samples, were negative for ZOL-treated animals. Another mechanism could be envisaged, such as anoikis, previously reported in human osteogenic sarcoma cells treated with ZOL by Evdokiou et al.22

In conclusion, using a model of lung metastases different in its origin from the well-studied breast carcinoma, we confirm the direct antitumor effect of ZOL both in vitro (antiproliferative and apoptotic effect) and in vivo on lung metastasis progression, leading to a significant prolongation of disease-free survival in animals. This result reveals that this compound could benefit patients with nonskeletal metastases, including osteosarcoma patients who remain at high risk of eventual relapse, with overt metastatic disease, with tumors that recur after treatment, or that show a low degree of necrosis after administration of chemotherapy and continue to have an unsatisfactory outcome.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Dr. Jonathan Green for helpful discussions and C. Bailly, A. Hivonnait, and C. Le Corre from the Experimental Therapy Unit of the IFR26 (Nantes, France) for technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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    Sato M, Grasser W, Endo N, et al. Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest. 1991; 88: 2095–2105.

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    Michigami T, Hiraga T, Williams PJ, et al. The effect of the bisphosphonate ibandronate on breast cancer metastasis to visceral organs. Breast Cancer Res Treat. 2002; 75: 249–258.

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    Hiraga T, Williams PJ, Ueda A, Tamura D, Yoneda T. Zoledronic acid inhibits visceral metastases in the 4T1/luc mouse breast cancer model. Clin Cancer Res. 2004; 10: 4559–4567.

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    Tsuchiya H, Kanazawa Y, Abdel-Wanis ME, et al. Effect of timing of pulmonary metastases identification on prognosis of patients with osteosarcoma: the Japanese Musculoskeletal Oncology Group study. J Clin Oncol. 2002; 20: 3470–3477.

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    Scotlandi K, Serra M, Nicoletti G, et al. Multidrug resistance and malignancy in human osteosarcoma. Cancer Res. 1996; 56: 2434–2439.

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    Nitto H, Koshino T, Mitsugi N, Hiruma T. Growth of a murine osteosarcoma-derived cell sarcoma increases serum immunosuppressive acidic protein levels. Cancer J. 2000; 60: 5625–5629.
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    Heymann D, Ory B, Blanchard F, et al. Enhanced tumor regression and tissue repair when zoledronic acid is combined with ifosfamide in rat osteosarcoma. Bone. 2005; 37: 74–86.

  • 11
    Diel IJ, Solomayer E-F, Costa SD, et al. Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Engl J Med. 1998; 339: 357–363.

  • 12
    Diel IJ, Solomayer E-F, Gollan C, Shutz F, Bastert G. Bisphosphonates in the reduction of metastases in breast cancer — results of the extended follow-up of the first study population. Proc ASCO 2001 (Abstr. 314).
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    McCloskey EV, Powles T, Paterson AHG, Ashley S, Kanis JA. Clodronate reduces incidence of skeletal metastases in women with primary breast cancer. Bone. 1998; 23: S189.
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    Saarto T, Blomqvist C, Virkkunen P, Eloma II. Adjuvant clodronate treatment does not reduce the frequency of skeletal metastases in node-positive breast cancer patients: 5-year results of a randomised controlled trial. J Clin Oncol. 2001; 19: 10–17.

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    Sasaki A, Boyce BF, Story B, et al. Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice. Cancer Res. 1995; 55: 3551–3557.

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    Stearns ME, Wang M. Effects of alendronate and taxol on PC-3ML cell bone metastases in SCID mice. Invasion Metastasis. 1996; 16: 116–131.

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    Sasaki A, Kitamura K, Alcade RE et al. Effect of a newly developed bisphosphonate, YH529, on osteolytic bone metastases in nude mice. Int J Cancer. 1998; 77: 279–285.

  • 18
    Nobuyuki H, Hiraga T, Williams PJ, et al. The bisphosphonate zoledronic acid inhibits metastases to bone and liver with suppression of osteopontin production in mouse mammary tumor. J Bone Miner Res. 2001; 16: S191.

  • 19
    Alvarez E, Galbreath EJ, Westmore M, et al. Properties of bisphosphonates in the 13762 syngenic rat mammary carcinoma model of tumor induced bone resorption. Proc Am Assoc Cancer Res. 2002; 43: 316.
  • 20
    Salomo M, Jurlander J, Nielsen LB, Gimsing P. How myeloma cells escape bisphosphonate-mediated killing: development of specific resistance with preserved sensitivity to conventional chemotherapeutics. Br J Haematol. 2003; 122: 202–210.

  • 21
    Benford HL, McGowan NW, Helfrich MH, Nuttall ME, Rogers MJ. Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro. Bone. 2001; 28: 465–473.

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    Evdokiou A, Labrinidis A, Bouralexis S, Hay S, Findlay DM. Induction of cell death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis. Bone. 2003; 33: 216–228.

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    Forsea AM, Muller C, Riebeling C, Orfanos CE, Geilen CC. Nitrogen-containing bisphosphonates inhibit cell cycle progression in human melanoma cells. Br J Cancer. 2004; 91: 803–810.

Epeius Biotechnologies’ REXIN-G, A Tumor-Targeted Genetic Medicine For Metastatic Cancer, Gains Phase 3 Product Designation From The U.S. FDA

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Posted 03 Mar 2011 — by James Street
Category genetic, genetic research, genetic research, Local Recurrence, Lung Metastases, Metastases, Molecular Osteosarcoma Studies, Osteosarcoma clinical trials, Targeted Cancer Therapy

01 Mar 2011

Epeius Biotechnologies announced that the U.S. FDA has granted Phase 3 status for the Company’s lead anti-cancer agent, Rexin-G, the first, and so far only, targeted gene delivery system developed to seek out and destroy metastatic cancer. According to Dr. Maria Gordon, Chief Medical Officer of Epeius, “What this means, in terms of clinical development, is that the Rexin-G product, with its advanced GMP manufacturing, bio-processing, and final formulation, meets rigorous FDA standards for obtaining a marketing license in the future; and that Epeius Biotech can now proceed with its strategic, diversified Phase 3 drug development program for pancreatic cancer, osteosarcoma and soft tissue sarcoma.”

In addition to these high-priority programs, Rexin-G has demonstrated significant anti-tumor activity in chemotherapy-resistant breast cancer, hormone-refractory prostate cancer, ovarian cancer, squamous cell carcinoma, and certain hematologic malignancies, such as large B-cell lymphoma.

Rexin-G® was granted accelerated approval for the treatment of all chemotherapy-resistant solid malignancies in the Republic of the Philippines in 2007. In the U.S.A., Rexin-G gained Orphan Drug Designation and market protections from the FDA for pancreatic cancer in 2003, followed by Orphan Drug Status for both osteosarcoma and soft tissue sarcoma in 2008. More recently, Epeius Biotechnologies completed a series of Phase 1 and Phase 2 clinical trials in the U.S., establishing the thresholds for bioactivity and dose-dependent efficacy for Rexin-G against a number of otherwise intractable cancers, as well as the product’s overall safety over extended survival times and a notable lack of either safety issues or dose-limiting toxicities.

With these development-stage accomplishments at hand, Epeius is gearing up to open a series of pivotal studies for both pancreatic cancer and sarcomas in the U.S., while continuing to advance the clinical utility and market development of Rexin-G worldwide. With the completion of the enabling platform development and the clinical validation of its foremost oncology product, Epeius Biotechnologies continues to lead the field of genetic medicine with the development of its product pipeline, which includes Reximmune-C, a tumor-targeted gene delivery system for ‘personalized’ cancer vaccinations, administered to stimulate a long-lasting anti-tumor immunity.

Source: Epeius Biotechnologies Corporation

Article URL: http://www.medicalnewstoday.com/articles/217745.php

Main News Category: Cancer / Oncology

Also Appears In:  Pharma Industry / Biotech Industry,  Clinical Trials / Drug Trials,  Regulatory Affairs / Drug Approvals,

Lombardi research teams hone in on treatments for osteosarcoma and Ewing’s sarcoma

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Posted 21 Jan 2011 — by James Street
Category Antiagiogenesis, Dog Osteosarcoma, Finance and Politics of cancer research and treatment, genetic research, Human osteosarcoma research, Local Recurrence, Lung Metastases, Molecular Osteosarcoma Studies, Osteosarcoma, Osteosarcoma Outcomes, Osteosarcoma Treatment Centers

Washington, DC – Most cancers arise from the epithelium, the tissue that lines the body and the organs, but sarcomas come from connective tissue cells, like the bones. At Georgetown Lombardi Comprehensive Cancer Center, research have engaged in a full-court press to develop new therapies to treat osteosarcoma and Ewing’s sarcoma, the two most common bone tumors in children, adolescents and young adults.

At the AACR 101st Annual Meeting 2010, they offer new molecular insights into the translocation that causes Ewing’s sarcoma, a genetic exchange between chromosomes that results in a fused gene that produces an oncogenic protein. These findings, coupled with use of sophisticated drug design technology, have led to identification of three new targets for potential treatment of this rare cancer. Additionally, researchers have identified two small molecules that have the potential to prevent or treat spread of osteosarcoma, a very aggressive cancer. (Embargoes listed with each abstract summary that follows).

“Scientific studies in the past decade identified very promising molecular targets that play a major role in tumor progression and invasion. With the help of a multidisciplinary team of scientists at Lombardi, we are now focusing on developing small molecules that can hit these targets,” says Aykut Üren, M.D., assistant professor in the Department of Oncology at Lombardi. “With this truly translational experimental approach, we may be able to optimize our small molecules for clinical trials in the near future.”

Original article: http://www.eurekalert.org/pub_releases/2010-04/gumc-lrt040710.php

Inhibitory effects of 22-oxa-calcitriol and all-trans retinoic acid on the growth of a canine osteosarcoma derived cell-line in vivo and its pulmonary metastasis in vivo

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Posted 21 Jan 2011 — by James Street
Category Dog Osteosarcoma, Lung Metastases, Molecular Osteosarcoma Studies, Mouse Osteosarcoma Studies, Osteosarcoma, Osteosarcoma Outcomes
Author(s): Barroga EF, Kadosawa T, Okumura M, Fujinaga T
Source: RESEARCH IN VETERINARY SCIENCE    Volume: 68    Issue: 1    Pages: 79-87    Published: FEB 2000
Times Cited: 7     References: 23
Abstract: Pulmonary metastasis is a major cause of death and a major obstacle to the successful treatment of canine osteosarcoma. However, the residual capacity of the neoplasia for differentiation and its susceptibility to undergo apoptosis may be used to suppress its growth and metastatic properties. The highly metastasizing POS (HMPOS) canine osteosarcoma cell line which preferentially metastasize to the lungs was used to test the possible efficacy of 22-oxa-calcitriol (OCT) and all-trans retinoic acid (ATRA) to inhibit growth and pulmonary metastasis of the subcutaneously grown osteosarcoma in nude mice. Treatments in vitro, morphologically elongated and increased alkaline phosphatase activity and staining of cells. Tumour growth in vivo was inhibited significantly and the combination treatment of OCT and ATRA (OCT + ATRA) exerted a synergistic and stronger suppression at concentration of 1.0 mu g kg(-1) body weight when given subcutaneously three times a week for 5 weeks. The subcutaneous rumours of the control mice consisted of osteoblast-like cells and isolated chondroblast-like cells, but formed several areas of osteoid and increased amount of collagen tissue in all treated mice. Pinpoint macrometastatic nodules developed only in all control mice. Micrometastatic nodule developed only in two of six mice treated with ATRA. However, nodule size and number, and lung wet weight were all reduced significantly. Metastasis were not seen in the mice treated with OCT or OCT + ATRA. This study demonstrated that inhibition of growth and pulmonary metastasis was induced by subcutaneous treatment with these drugs and suggest that both its differentiating and apoptotic inducing activities may be responsible for the antitumour effects. These drugs may be useful in the clinic as an adjunct for the treatment of canine osteosarcoma. (C) 2000 Harcourt Publishers Ltd.
Document Type: Article
Language: English
Reprint Address: Barroga, EF (reprint author), Hokkaido Univ, Grad Sch Vet Med, Dept Vet Clin Sci, Lab Vet Surg, Sapporo, Hokkaido 0600818 Japan
Addresses:
1. Hokkaido Univ, Grad Sch Vet Med, Dept Vet Clin Sci, Lab Vet Surg, Sapporo, Hokkaido 0600818 Japan
Publisher: W B SAUNDERS CO LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND
Subject Category: Veterinary Sciences
IDS Number: 287TP
ISSN: 0034-5288

Rapamycin Inhibits Ezrin-Mediated Metastatic Behavior in a Murine Model of Osteosarcoma

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Posted 21 Jan 2011 — by James Street
Category Local Recurrence, Lung Metastases, Metastases, Molecular Osteosarcoma Studies, Mouse Osteosarcoma Studies, Osteosarcoma

1. Xiaolin Wan,
2. Arnulfo Mendoza,
3. Chand Khanna, and
4. Lee J. Helman

+ Author Affiliations

1.
Molecular Oncology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland

1. Requests for reprints:
Lee J. Helman, Molecular Oncology Section, Pediatric Oncology Branch, Building 10, Room 1 West-3750, National Cancer Institute, NIH, Bethesda, MD 20892-1106. Phone: 301-496-4257; Fax: 301-480-4318; E-mail: helmanl@nih.gov.

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Abstract

Osteosarcoma is the most frequent primary malignant tumor of bone with a high propensity for metastasis. We have previously showed that ezrin expression is necessary for metastatic behavior in a murine model of osteosarcoma (K7M2). In this study, we found that a mechanism of ezrin-related metastatic behavior is linked to an Akt-dependent mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (S6K1)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) pathway. Suppression of ezrin expression either by antisense transfection or by small interfering RNAs or disruption of ezrin function by transfection of a dominant-negative ezrin-T567A mutant led to decreased expression and decreased phosphorylation of both S6K1 and 4E-BP1. Proteosomal inhibition by MG132 reversed antisense-mediated decrease of S6K1 and 4E-BP1 protein expression, but failed to affect the effect of ezrin on phosphorylation of S6K1 and 4E-BP1. Blockade of the mTOR pathway with rapamycin or its analog, cell cycle inhibitor-779 led to significant inhibition of experimental lung metastasis in vivo. These results suggest that blocking the mTOR/S6K1/4E-BP1 pathway may be an appropriate target for strategies to reduce tumor cell metastasis.

* ezrin
* S6K1
* 4E-BP1
* metastasis
* rapamycin

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Introduction

Ezrin is a member of the ezrin/radixin/moesin (ERM) family of proteins that link the cell membrane to the actin cytoskeleton and are involved in cytoskeletal organization (1, 2) . Ezrin is believed to be involved in intracellular signal transduction that is related to cell migration and metastasis because ezrin is reported to be a substrate for tyrosine kinases (3, 4) and binds adhesion molecules such as CD43, CD44, intercellular adhesion molecule-1, and intercellular adhesion molecule-2 (5–7) . Of note, high levels of CD44 seem to be dependent on ezrin expression and are associated with invasion and metastatic behavior of tumor cells (8). The discovery that merlin/schwannomin, the neurofibromatosis-2–associated tumor-suppressor protein, is related to the ezrin/radixin/moesin family has provided additional insights into the relationship between ezrin and tumorigenesis (9). Recently, we have found ezrin expression in murine osteosarcoma and rhabdomyosarcoma to be necessary for metastatic behavior (10–12) . Suppression of ezrin protein expression by antisense transfection or stable expression of short hairpin RNA, or disruption of ezrin function by transfection of a dominant-negative ezrin significantly reduced the metastatic behavior in both murine models and was associated with decreased Akt and mitogen-activated protein kinase (MAPK) activity (11, 12) . However, the specific mechanism or mechanisms by which ezrin mediates the metastatic process remains to be elucidated.

Rapamycin and analogues such as cell cycle inhibitor-779 (CCI-779) are currently undergoing clinical and preclinical evaluations as an anticancer agent. The anticancer property of rapamycin is attributed to the inhibition of mammalian target of rapamycin (mTOR) signaling pathway, which controls mRNA translation and cell proliferation. Rapamycin binds to the FK506 binding protein (FKBP-12), and this complex interacts with mTOR. This interaction inhibits mTOR kinase activity and subsequently decreases phosphorylation and activation of p70 ribosomal protein S6 kinase (p70S6K, S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) that play fundamental roles in ribosome biogenesis and cap-dependent translation, respectively (13, 14) . A previous study has found that the ability of rapamycin to inhibit metastatic tumor growth and angiogenesis in in vivo mouse models is linked to reduced translational production of vascular endothelial growth factor vascular endothelial growth factor and to blockage of vascular endothelial growth factor–induced endothelial cell signaling (15). Most recently, investigators have showed that rapamycin can reverse resistance to doxorubicin in a mouse model of an Akt-driven aggressive lymphoma (16). These data suggest that blockade of the mTOR pathway might also have an inhibitory effect on both resistance to cytotoxic therapy as well as tumor metastasis.

In this study, we linked ezrin-related metastatic behavior to activation of S6K1 and 4E-BP1 signaling. We found that antisense-mediated and small interfering RNA (siRNA)–induced reduction of ezrin expression or disruption of ezrin function by transfection of a dominant-negative ezrin-T567A mutant led to decreased expression and decreased phosphorylation of both S6K1 and 4E-BP1. Proteosomal inhibition by MG132 reversed antisense-mediated decrease of S6K1 and 4E-BP1 protein expression, but failed to affect the effect of ezrin on phosphorylation of S6K1 and 4E-BP1. Finally treatment with rapamycin and its analogue, CCI-779 led to a significant inhibition of experimental metastasis in vivo. These results indicate that blockade of the mTOR/S6K1/4E-BP1 pathway by rapamycin could be of potential therapeutic benefit for reducing tumor cell metastasis in osteosarcoma.
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Materials and Methods

Cell Culture. The K12, K7M2 murine OSA cell lines, ezrin-antisense clones 13, 1.46 and 1.52 cells, and dominant-negative ezrin (T567A mutant) clones T567A-GFP-7, and T567A-GFP-8 as well as empty-GFP clones empty-GFP-2.5, empty-GFP-2.7 cells generated from K7M2 have been previously described (13, 14) . These cells were maintained in DMEM containing 10% fetal bovine serum, l-glutamine (2 mmol/L), penicillin (100 units/mL), and streptomycin (100 units/mL, BioSource International Inc, Camarillo, CA) at 37°C in a humidified CO2 incubator.

Antibodies and Reagents. Anti-ezrin monoclonal antibody was purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies to phospho-S6K1 (Thr389), S6K1, phospho-4E-BP1 (Thr37), 4E-BP1, phospho-Akt (Ser473), Akt, phospho-4E-BP1 (Ser2448), phospho-p44/42 MAPK, and p44/42 MAPK were purchased from Cell Signaling Technology Inc. (Beverly, MA). Anti-actin antibody was from Amersham Pharmacia Biotech (Piscataway, NJ). MG132 was purchased from Calbiochem (San Diego, CA). U0126 was purchased from Promega Corp. (Madison, WI). LY294002 was purchased from Sigma Chemical Co.. Rapamycin was purchased from LC Laboratories (Woburn, MA). CCI-779 was obtained from Developmental Therapeutics Program, National Cancer Institute (Bethesda, MD) and Wyeth Laboratories (Philadelphia, PA).

Transfection. Myc-tagged, activated Akt, dominant-negative Akt (Akt K179M), and empty vector (pUSE) were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). K7M2 cells were transfected with dominant-negative Akt and empty vector by using electroperation in a gene Pulsar (0.22 kV/cm; capacitance, 960 μF; Bio-Rad, Richmond, CA). After selection in medium containing G418, single clones were isolated and expanded. Ezrin-antisense clones 1.46 and 1.52 cells were transfected with activated Akt and empty vector by using electroperation in a Bio-Rad gene Pulsar (0.22 kV/cm; capacitance, 960 μF). After 72 hours, these cells were harvested and subjected to Western blot analysis.

Western Blot Analysis. Confluent cells were lysed in lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L sodium chloride, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pryophosphate, 1 mmol/L β-glycerolphosphate, 1 mmol/L sodium orthovanadate, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin). Protein lysates (20-50 μg per lane), as determined by Bio-Rad protein assay, were separated in 10% to 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% nonfat dried milk in TBS-T (20 mmol/L Tris-HCl, pH 7.5, 8 g/l of sodium chloride, 0.1% Tween 20) and then incubated with primary antibodies. Horseradish peroxidase conjugated anti-rabbit IgG (Cell Signaling) was used as secondary antibody. Protein was visualized using enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).

S6K1 Assay. S6K1 activity was determined in vitro as described previously (17).

Gene Silencing with siRNAs. We obtained annealed, 21-bp siRNA duplexes from Dharmacon Research Inc. (Lafayette, CO). The target sequence for ezrin was 5′-AAGGAAUCCUUAGCGAUGAGA-3′, corresponding to position 440 to 460 in the human ezrin mRNA. A siRNA targeting a nonspecific sequence was purchased from Dharmacon Research Inc. and served as a negative control. We applied siRNA duplexes at a final concentration of 100 nmol/L using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA).

In vivo Experimental Metastasis Assay. Four- to 5-week-old female beige severe combined immunodeficient (SCID) mice (Charles River Laboratories, Wilmington, MA) were inoculated with 1 × 106 K7M2 cells per mouse via the tail vein and then randomly assigned to treatment groups. Mice were treated i.p. daily × 5 days for 5 to 6 consecutive weeks with 5 mg/kg rapamycin, 5 mg/kg CCI-779, 20 mg/kg CCI-779, or vehicle alone. All mice underwent complete necropsy for confirmation of pulmonary metastases. All animal work was done with the approval of the Animal Care and Use Committee of the National Cancer Institute.

Lung Histopathology. Lung tissue was fixed in 10% formalin and embedded in paraffin. Paraffin sections (5 μm) were stained with H&E.
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Results

Reduction of S6K1 and 4E-BP-1 Phosphorylation by Suppression of Ezrin Expression. Previous experiments using cDNA microarray and Northern blot analysis have showed that the highly metastatic K7M2 cell line has much higher ezrin expression compared with the less metastatic K12 cell line (10). To study the specific role of ezrin and ezrin-mediated signaling pathways, we generated stable K7M2 clones expressing antisense ezrin cDNA (11). The expression levels of ezrin protein in G418-resistant clones were analyzed by Western blotting using a monoclonal ezrin antibody. Both 1.46 and 1.52 clones transfected with antisense vector showed a marked decrease of ezrin protein expression, whereas clone 13 had a minimal reduction in ezrin expression compared with K7M2 cells ( Fig. 1A ).
Figure 1.
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Figure 1.

Suppression of ezrin expression leads to reduced S6K1 and 4E-BP1 phosphorylation and expression. A, ezrin expression was analyzed by Western blotting in K12, K7M2, and ezrin antisense transfectant cells using anti-ezrin antibody. B, S6K1 phosphorylation and expression were determined by Western blot analysis in K12, K7M2, and ezrin-antisense transfected cell lines. C, S6K1 activity was measured in K12, K7M2, and ezrin-antisense transfected cell lines using a peptide substrate (AKRRRLSSLRA) as described. Columns, mean; bars, SE. D, 4E-BP1 phosphorylation and expression were determined by Western blot analysis in K12, K7M2, and ezrin-antisense transfected cell lines. E, K7M2 cells plated in six-well plates were transfected with 100 nmol/L control or ezrin siRNA using LipofectAMINE 2000. After 5 days, cells were lysed in lysis buffer and then subjected to Western blot analysis. Blotting with antibody against actin was used to confirm equal loading of the protein. Similar results were achieved in three independent experiments.

We sought to determine whether S6K1 and 4E-BP-1, two major downstream targets of mTOR that play critical roles in translation regulation, were involved in ezrin-mediated metastatic signaling because our previous data showed ezrin-mediated effects on Akt activity. We first examined the phosphorylation and expression status of S6K1 and 4E-BP-1 in K12 and K7M2 cell lines. Our data revealed that S6K1 and 4E-BP-1 are both more highly phosphorylated and expressed in K7M2 cells compared with K12 cells ( Fig. 1B and D). Furthermore, S6K1 activity, as evaluated by in vitro kinase assays, is significantly elevated in K7M2 cells compared with K12 cells ( Fig. 1C). Down-regulation of ezrin expression decreased phosphorylation and expression of S6K1 and 4E-BP-1 as well as S6K1 activity ( Fig. 1B-D). To further test the effects of ezrin on the regulation of S6K1 and 4E-BP1, we next targeted ezrin by siRNA. Inhibition of ezrin expression by siRNA led to decreased phosphorylation of S6K and 4E-BP1, with minimal reduction of S6K1 and 4E-BP1 expression ( Fig. 1E). These data suggest that S6K1 and 4E-BP1 are associated with ezrin-mediated signaling.

Proteosomal Inhibition Reverses Ezrin-Antisense–Mediated Suppression of S6K1 and 4E-BP1 Expression, but Does Not Affect S6K1 and 4E-BP1 Phosphorylation. We sought to determine whether the observed decrease in S6K1 activity and 4E-BP1 phosphorylation was a direct result of decreased protein expression or an independent activity of ezrin suppression. We therefore treated K7M2 antisense clones with MG132, a proteasome inhibitor, to block protein degradation. As shown in Fig. 2 , exposure of cells to MG132 (50 μmol/L) for 6 hours reversed the decrease in S6K1 and 4E-BP1 protein expression induced by ezrin-antisense transfection but failed to affect S6K1 and 4E-BP1 phosphorylation. Similar results were also observed after 6 hours of treatment with doses as low as 2 μmol/L MG132 (data not shown). Neither 2 nor 50 μmol/L had any significant affect on cell viability as determined by visual inspection and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (data not shown). These data suggest that suppression of S6K1 and 4E-BP1 expression by ezrin-antisense transfection is mediated via enhanced degradation of S6K1 and 4E-BP1 proteins through a proteasome-dependent pathway, and that down-regulation of S6K1 and 4E-BP1 phosphorylation induced by ezrin-antisense transfection is an independent effect of ezrin down-regulation.
Figure 2.
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Figure 2.

Proteosomal inhibition with MG132 reverses ezrin anti-sense–mediated reduction of S6K1 and 4E-BP1 expressions but does not affect S6K1 and 4E-BP1 phosphorylation. 13, 1.46, and 1.52 cells were treated with MG132 (50 mmol/L) for 6 hours and then lysed in lysis buffer for Western blot analysis of S6K1 and 4E-BP1 phosphorylation and expression. Similar results were achieved in three independent experiments.

Disruption of Ezrin Function by Transfection of Dominant-Negative Ezrin (T567A) Inhibited Akt, S6K1, and 4E-BP1 Phosphorylation. Our previous data showed that disruption of ezrin function by stable transfection of dominant-negative ezrin (T567A) completely inhibited experimental metastases in mice (11). To further determine whether the role of functional ezrin in K7M2 cell metastasis is also linked to Akt/mTOR signaling pathway, we analyzed both phosphorylation and expression of Akt, S6K1 and 4E-BP1 in cell lines transfected with dominant-negative ezrin (T567A) or empty vector. As shown in Fig. 3A , Akt, S6K1 and 4E-BP1 phosphorylation were inhibited in dominant-negative ezrin (T567A) clones compared with empty vector clones. In addition, stable transfection of dominant-negative ezrin (T567A) led to reduction of S6K1 and 4E-BP1 protein expression but failed to influence the levels of Akt protein. These data are consistent with our findings in ezrin-antisense transfection cells ( Fig. 1). We have previously shown phosphorylation and activity of p44/42 MAPK are reduced when ezrin protein expression is suppressed by stable transfection of ezrin antisense (11). However, p44/42 MAPK phosphorylation is increased, not decreased, in cells expressing ezrin-T567A ( Fig. 3), whereas the total expression of p44/42 MAPK is not significantly changed in these clone cells ( Fig. 3A). Thus, phosphorylation of ezrin-T567 residue seems to be a crucial site for ezrin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway, but not for ezrin-mediated activation of the p44/42 MAPK signaling pathway.
Figure 3.
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Figure 3.

Disruption of ezrin function resulted in decreased phosphorylation of Akt, S6K1, and 4E-BP1 (A) and ezrin-associated phosphorylation of S6K1 and 4E-BP1 is rapamycin sensitive (B). A, phosphorylation and expression of Akt, S6K1, 4E-BP1, and p44/42 MAPK were analyzed by Western blotting in empty vector-GFP and dominant-negative ezrin (T567A) clones. B, confluent cells were treated with rapamycin (100 nmol/L), LY294002 (10 μmol/L), or U0126 (10 μmol/L) for 1 hour, lysed, and then subjected to Western blot analysis of Akt, S6K1, and 4E-BP1 phosphorylation and expression. Similar results were achieved in two separate experiments.

Ezrin-Linked S6K1 and 4E-BP1 Phosphorylation Is Rapamycin Sensitive. To further determine whether the ezrin-linked S6K1 and 4E-BP1 phosphorylation is mTOR dependent, K7M2 cells were treated with an mTOR inhibitor rapamycin, as well as a PI3K inhibitor LY294002, and a MAPK inhibitor U0126, for 1 hour. As shown in Fig. 3B, rapamycin inhibited S6K1 and 4E-BP1 phosphorylation but did not affect Akt phosphorylation. LY294002 completely inhibited not only Akt phosphorylation but also S6K1 and 4E-BP1 phosphorylation. U0126 affected neither Akt phosphorylation nor S6K1 and 4E-BP1 phosphorylation. These inhibitors failed to alter the expression of Akt, S6K1, and 4E-BP1. Taken together, these data suggest that the ezrin-related S6K1 and 4E-BP1 phosphorylation in K7M2 cells is rapamycin sensitive and downstream of PI3K.

Suppression of Experimental Metastasis in the K7M2 Murine Osteosarcoma Model by Rapamycin and Its Analogue CCI-779. To determine the potential effect of mTOR inhibition on experimental metastases in the K7M2 murine osteosarcoma model, we evaluated the effect of rapamycin and its analogue CCI-779. Mice were treated i.p. daily × 5 days every week for 5 to 6 weeks with 5 mg/kg rapamycin, 5 mg/kg CCI-779, 20 mg/kg CCI-779, or vehicle alone. Treatment with rapamycin and CCI-779 significantly prolonged the survival (morbidity associated with pulmonary metastasis) of SCID beige mice ( Fig. 4A ). In the control group, only 25% (2 of 8) mice survived beyond 38 days, whereas 100% (9 of 9) in both 5 mg/kg rapamycin- and 20 mg/kg CCI-779–treated groups were alive at 38 days. Two of 9 mice treated with 5 mg/kg CCI-779 died, but no gross pulmonary metastasis were detected in the two dead mice. At necropsy, no evidence of tumor was observed. Therefore, the cause of death in these mice is uncertain. All eight mice in the control group after 38 days of injection developed multiple lung metastases, whereas 1 of 7 mice in the 5 mg/kg CCI-779–treated group and 2 of 9 mice in the 5 mg/kg rapamycin–treated group developed single lung metastases. We failed to detect gross lung metastasis in any mouse treated with 20 mg/kg CCI-779 ( Fig. 4B). Histopathologic examination of H&E-stained sections of lungs was done in all animals. In contrast to multiple, large pulmonary metastases seen in all control-treated mice ( Fig. 4C, left), we found single small micrometastases in 6 of 9 mice treated with 5 mg/kg rapamycin (not shown), 6 of 9 mice treated with 5 mg/kg CCI-779 ( Fig. 4C, middle, micrometastasis indicated by arrow), and in only 2 of 9 mice treated at 20 mg/kg CCI-779 ( Fig. 4C, right, indicate normal lung only).
Figure 4.
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Figure 4.

Impact of rapamycin and its analogue CCI-779 on survival (A) and experimental lung metastases (B) in tumor-inoculated SCID beige mice. K7M2 cells (1 × 106 per mouse) were injected into the tail vein of SCID beige mice. These mice were treated i.p. daily × 5 in each of 5 to 6 consecutive weeks with 5 mg/kg rapamycin, 5 mg/kg CCI-779, 20 mg/kg CCI-779, or vehicle alone. All mice underwent complete necropsy and confirmation of metastases. C, representative H&E-stained lung sections (40× power). Left, the entire field is composed of tumor nodules. Middle, note only small microscopic tumor nodule (arrow). Right, no tumor nodules are noted, only normal lung. Bar, 100 μm.
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Discussion

Ezrin is known to be involved in a variety of cellular functions, such as cell cytoskeletal organization, cell motility, and morphogenesis. The high levels of ezrin expression in cell lines of endometrial (18), colorectal (19), and pancreatic carcinoma with high metastatic potential (20) have suggested that its expression has been associated with events that may promote tumor progression and metastasis. Consistent with that, our recent studies found that high expression of ezrin in K7M2 murine osteosarcoma cells is associated with highly metastatic behavior (10). Suppression of ezrin protein by antisense transfection and disruption of ezrin function significantly reduced lung metastases in two distinct mouse tumor models (11, 12) , providing an excellent experimental model to investigate the mechanisms of ezrin-mediated metastasis. In this report, we show that both blockade of ezrin expression either by antisense transfection or by siRNA and disruption of ezrin function by stable transfection of dominant-negative ezrin (T567A) led to inhibition of S6K1 and 4E-BP1 phosphorylation ( Figs. 1B, D, and E and 3A), which both lie downstream of mTOR and play fundamental roles in ribosome biogenesis and cap-dependent translation, respectively (21, 22) . These results indicate that ezrin signaling is involved in regulating mRNA translation and provide, for the first time, a linkage between ezrin and mTOR signaling.

Recent studies reported that S6K1 and 4E-BP1 also are regulated through the PI3K/Akt-signaling pathway (23). These studies raise the possibility of a direct signaling pathway from PI3K/Akt to mTOR. Our previous studies show that the inhibition of ezrin expression resulted in markedly reduced Akt phosphorylation and activity (11). Both S6K1 and 4E-BP1 phosphorylation were completely inhibited by the PI3K inhibitor LY294002 in K7M2 cells ( Fig. 3B). Furthermore, stable transfection of the dominant-negative Akt (K179M mutant) into K7M2 cells led to reduction of Akt phosphorylation as well as S6K1 and 4E-BP1 phosphorylation (data not shown). On the other hand, transient transfection of activated Akt into ezrin-antisense clones 1.46 and 1.52 cells led to up-regulation of S6K1 and 4E-BP1 phosphorylation (data not shown). These data are consistent with a PI3K/Akt/mTOR pathway in these cells. Phosphorylation of ezrin at T567 has been identified to play an important role in its conformational activation. Inactive, cytosolic ezrin, in a closed conformation through head-to-tail interaction between the amino- and carboxyl-terminal domains, requires phosphorylation at residue T567 and interaction with phosphatidylinositol 4,5-bisphosphate to cause unfolding, translocation to the plasma membrane, and cross-linking between integral membrane proteins and cytoskeleton (24–27) . Disruption of ezrin function by transfection of ezrin-T567A mutant significantly reduced lung metastases in two distinct mouse tumor models (11, 12) . In this study, we found that transfection of ezrin-T567A mutant into K7M2 cells not only inhibited Akt phosphorylation but also inhibited S6K1 and 4E-BP1 phosphorylation ( Fig. 3), which is consistent with our findings in ezrin-antisense transfected cells ( Fig. 1). As noted, ezrin has been found to directly bind PI3K (28). Thus, ezrin-mediated regulation of mTOR targets S6K1 and 4E-BP1 seems to be indirect through a direct interaction of ezrin with PI3K or phosphatidylinositol 4,5-bisphosphate leading to sequential activation of PI3K/Akt/mTOR signaling cascades.

The role of Akt in the regulation of mTOR activation is complex. Although Ser2448 in mTOR has been identified to be a direct phosphorylation target of Akt (29), substitution of Ser2448 by alanine failed to alter the ability of mTOR to activate S6K1 (30). We examined the phosphorylation of serine residue 2448 of mTOR in ezrin-antisense transfected cell lines. Down-regulation of ezrin failed to affect mTOR phosphorylation on Ser2448 (data not shown). Recent study has shown that phosphorylation of Ser2448 does not seem to modulate in vitro 4E-BP1 phosphorylation by mTOR (31). Moreover, mutation of Ser2035 in mTOR inhibited the abilities of mTOR to phosphorylate S6K1 and 4E-BP1 in vitro (32). Furthermore, in our study ezrin-associated phosphorylation of S6K1 and 4E-BP1 is rapamycin sensitive, suggesting that these observed ezrin effects occur through a mTOR signaling pathway. However, the specific mechanism remains to be elucidated.

To further determine the functional significance of ezrin-regulated mTOR/S6K1/4E-BP1 pathways, we studied the effect of mTOR inhibition on in vitro and in vivo metastatic pathways. Suppression of S6K1 and 4E-BP1 by rapamycin led to decreased K7M2 cell migration and invasion compared with untreated cells (data not shown). Treatment of tumor-inoculated SCID beige mice with rapamycin and CCI-779 resulted in prolonged survival and inhibition of pulmonary metastasis ( Fig. 4A and B). These results suggest that mTOR/ S6K1/4E-BP1 pathways play an important role in ezrin-mediated metastatic behavior. Recently, rapamycin has been reported to inhibit metastatic tumor growth in other murine models (15, 33) . Thus, inhibition of the mTOR/S6K1/4E-BP1 pathway by rapamycin or other inhibitors may be worthy of clinical evaluation as an antimetastatic intervention. The challenge will be to develop schedules of rapamycin and its analogues that can be chronically administered without causing significant immunosuppression.
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Acknowledgments

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Sung-Hyeok Hong for help in performing the invasion experiments, and Sally Hausman and Ed Sausville for the supply of CCI-779.
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Footnotes

* Received August 30, 2004.
* Revision received December 13, 2004.
* Accepted January 6, 2005.

* ©2005 American Association for Cancer Research.

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S. Korean scientists find protein that helps predict spread of bone cancer

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Posted 19 Jan 2011 — by James Street
Category Lung Metastases, Metastases, Molecular Osteosarcoma Studies, Osteosarcoma Outcomes, Proteomics

SEOUL, April 26 (Yonhap) — South Korean scientists on Thursday said they have found a method to predict the spread of bone cancer to internal organs that could help saves lives.

Kim Min-suk and Jeon Dae-geun at the Korea Institute of Radiological and Medical Sciences said clinical observations on 64 patients over three years have shown a close correlation between the presence of ezrin protein and bone cancer metastasis.

Bone cancer, also called osteosarcoma, is a rare form of cancer that usually strikes teenagers. The cancer is hard to treat because while surgery can remove malignant tumors, there is a chance that the cancer can metastasize and invade distant tissues and organs. The mortality rate for a person who has contracted the illness is 40 percent.

“Of the 33 people who tested positive for the ezrin protein, 21 suffered from cancerous tumors invading distant tissues and organs,” said Kim. In the 31 cases where no ezrin protein was detected, only one patient had to undergo treatment after the cancer metastasized.

The findings, released in the Journal of Clinical Orthopedics and Related Research, represent the first time that scientists have found a direct link between the protein and the spread of bone cancer. In the past, ezrin was used to determine if cancer cells had been completely destroyed after surgery or other forms of treatment.

The scientists also said that the newly discovered link could help prevent relapse among patients who have already been treated for cancer.

yonngong@yna.co.kr

Ariad’s Ridaforolimus Part I

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Posted 18 Jan 2011 — by James Street
Category Clinical Trials, Drug Testing, Educational, Local Recurrence, Lung Metastases, Metastases, Molecular, Molecular Osteosarcoma Studies, Osteosarcoma, Osteosarcoma Outcomes

January 13th, 2010

Ariad’s Ridaforolimus, Cancer Beater?

Alright pharma investors, today I am going to take you through a brief analysis of Ariad’s potential cancer beater Ridaforolimus in soft-tissue and bone Sarcomas. First, I’ll run through how it works and what it is supposed to do, then I’ll go over the Phase II results and what I expect out of the upcoming Phase III SUCCEED trial results.

What is the difference between Soft tissue and Bone Sarcoma?

Put simply, soft-tissue sarcoma is a cancer that starts off in the body’s supporting tissues such as the muscles, fat, fibrous, and blood vessels. While, bone sarcomas are named because of the site from which they arise: bones. Sarcomas are quite rare with less than 1% of all adult cancer diagnoses resulting in a positive sarcoma diagnosis. The most common type of bone sarcoma is Osteosarcoma which accounts for 35% of all bone tumors. In 2008, it was estimated that 1,270 new cases were diagnosed in the U.S. alone and another 5,150 would die from the disease in 2008. There has been a chronic lack of funding in research for the cancer, with only about $37.1 million dollars being invested into the disease in 2007.

How does Ridaforolimus work?

The drug is from the family of mTOR inhibitors (mTOR stands for Mammalian target of rapamaycin). MTOR is a protein kinase that regulates cell growth, cell proliferation, cell survival, and transcription. And in many cancer patients the mTOR protein runs wild and begins signaling other cells in the body to develop out of control, which in turn causes cancer. Ridaforolimus works by inhibiting the mTOR signaling by binding to it and inhibiting it from telling other cells in the body to run wild. This in turn reduces cancer cell growth, division, metabolism and angiogenesis.

So what about the Phase II Clinical Trial?
The Phase II clinical trial revealed some pretty good results, results good enough for Merck to partner up with the company to develop the drug. In Phase II trials the drug performed well, achieving a 17 month Overall Survival rate compared to a 9 month Overall Survival rate of 9 months for the comparator arm in patients who achieved disease stabilization for at least 16 weeks or tumor regression. With an 8 month extension on life, the drug merited additional studies in Phase III trials.

Malignant Cancer-Causing Molecule Identified

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Posted 12 Jan 2011 — by James Street
Category Educational, Molecular, Molecular Osteosarcoma Studies

Posted on: January 10, 2011

A cancerous cell is not a problem if it is benign. However, if it becomes malignant, it can cause major health problems and even be fatal. Scientists have identified a molecule known as PML which directly affects whether or not a cancer cell
becomes malignant. This line of study may prove to be a breakthrough in cancer research if scientists can figure out how malignant tumors can be converted to benign tumors.

The research was conducted at the University of Montreal’s Department of Biochemistry and the University of Montreal Hospital Research Centre. The team was led by Dr. Gerardo Rebeyre of the University of Montreal.

“We discovered that benign cancer cells produce the PML molecule and display abundant PML bodies, keeping them in a dormant, senescent state. Malignant cancer cells either don’t make or fail to organize PML bodies, and thus proliferate uncontrollably,” said Ferbeyre.

A dormant, senescent state is one in which the cell has matured and is no longer able to reproduce. PML is the body’s natural defense against the cancer spreading to other cells and other parts of the body. Previously, the mechanism for how PML worked was a mystery. However, the team’s research has managed to shine a light on it by collecting samples from hospital patients.

“Our findings unravel the unexpected ability of PML to organize a network of tumor suppressor proteins to repress the expression or the amount of other proteins required for cell proliferation,” explained researcher Véronique Bourdeau. Researcher Mathieu Vernier emphasized that “this is an important finding with implications for our understanding on how the normal organism defends itself from the threat of cancer.”

The research can be found in the journal, Genes and Development, and was funded by the Canadian Cancer Society and the Fonds de la recherché en Sante du Quebec. The researcher’s work creates exciting avenues for future research. According to Ferbeyre, “Our discovery opens new possibilities to explore what other molecules are involved in generating senescence: a goal we consider important if we want to design therapies that turn malignant tumors into benign tumors.”

Posted By: David A Gabel

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