Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
Glioblastoma multiforme (GBM) is the most common and most deadly primary brain tumor affecting adults. Despite advancements made in surgical, radiological, and chemo-therapies for this grade IV astrocytoma, prognoses have remained very poor: median survival time from diagnosis remains at 9-15 months with less than 10% of patients surviving beyond 5 years.1, 2 Caveolin-1 (Cav-1) is the principle structural protein responsible for the formation of caveolae, or invaginating microdomains in the cell membrane. The capacity for Cav-1 to associate with a wide variety of proteins has implicated it in a number of processes ranging from vesicular transport and cholesterol homeostasis to nitric oxide production and cell migration, among others.3-7 Its ability to regulate cell cycle progression and intracellular signal transduction have resulted in the substantial characterization of Cav-1 in many cancers, where it has been shown to act as both a tumor suppressor and tumor promoter depending on the tissue type.8-11 In gliomas, expression of Cav-1 appears to increase proportionally to tumor grade, with most GBM lesions exhibiting more intense Cav-1 immunoreactivity than their grade II and III counterparts. 12-14 However, little is currently known as to the role of Cav-1 as it relates to GBM in vivo. Recent in vitro studies conducted using the GBM-derived cell line U-87MG have demonstrated that Cav-1 acts as a putative tumor suppressor in GBM by downregulating α5β1 integrin expression and subsequent TGFβ/SMAD pathway activity.15 16
This invention provides a method for treating a subject afflicted with glioblastoma multiforme comprising administering a therapeutically effective regimen of temozolomide to the glioblastoma multiforme-afflicted subject, wherein the subject's glioblastoma multiforme cells are known to be caveolin-1-positive.
This invention also provides a method for determining whether a subject afflicted with glioblastoma multiforme is likely to progress therapeutically in response to a therapeutically effective regimen of temozolomide comprising determining whether the subject's glioblastoma multiforme cells are caveolin-1-positive, whereby if the subject's glioblastoma multiforme cells are caveolin-1-positive, the subject is likely to progress therapeutically in response to a therapeutically effective regimen of temozolomide.
This invention provides a method for treating a subject afflicted with glioblastoma multiforme comprising administering a therapeutically effective regimen of temozolomide to the glioblastoma multiforme-afflicted subject, wherein the subject's glioblastoma multiforme cells are known to be caveolin-1-positive.
Preferably, the subject is human, and the glioblastoma multiforme is newly diagnosed in the subject.
This invention also provides a method for determining whether a subject afflicted with glioblastoma multiforme is likely to progress therapeutically in response to a therapeutically effective regimen of temozolomide comprising determining whether the subject's glioblastoma multiforme cells are caveolin-1-positive, whereby if the subject's glioblastoma multiforme cells are caveolin-1-positive, the subject is likely to progress therapeutically in response to a therapeutically effective regimen of temozolomide.
Preferably, the subject is human, and the glioblastoma multiforme is newly diagnosed in the subject.
The subject can be any animal capable of being afflicted with glioblastoma multiforme.
In this invention, cells that are caveolin-1-positive are determined to be such according to methods known in the art. Such methods include the antibody-based methods described herein.
In one embodiment, the subject's glioblastoma multiforme cells known to be caveolin-1-positive express an amount of caveolin-1 that is a percentage of the amount of caveolin-1 expressed by the lentiviral transduced caveolin-1-overexpressing U-87MG cells described herein, wherein the percentage is selected from below 10%, 10%-20%, 20%-30%, 30%-40%, 40%0-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, 100%-120%, 120%-140%, 140%-160%, 160%-180%, 180%-200%, and above 200%.
Temozolomide (available from Schering-Plough as Temodar® (“Temodar”)) is a known therapeutic for treating glioblastoma multiforme. Typically, it is administered in conjunction with radiotherapy and/or surgery. Although numerous regimens of temozolomide are possible, the regimens set forth in in the Temodar label are preferred. Certain portions of that label are reproduced below, although the label's contents at http://www.accessdata.fda.gov/drugsatfda_docs/label/2006/021029s0121bI.pdf are incorporated herein in their entirety.
Temodar Capsules for oral administration contain temozolomide, an imidazotetrazine derivative. The chemical name of temozolomide is 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-αs-tetrazine-8-carboxamide.
The material is a white to light tan/light pink powder with a molecular formula of C6H6N6O2 and a molecular weight of 194.15. The molecule is stable at acidic pH (<5), and labile at pH>7, hence Temodar can be administered orally. The prodrug, temozolomide, is rapidly hydrolyzed to the active 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) at neutral and alkaline pH values, with hydrolysis taking place even faster at alkaline pH.
Each capsule contains either 5 mg, 20 mg, 100 mg, 140 mg, 180 mg, or 250 mg of temozolomide. The inactive ingredients for Temodar Capsules are lactose anhydrous, colloidal silicon dioxide, sodium starch glycolate, tartaric acid, and stearic acid. The 5 mg, 20 mg, 100 mg, and 250 mg gelatin capsule shells contain titanium dioxide. The capsules are white and imprinted with pharmaceutical ink. The body of the capsules for the 140 mg and 180 mg strengths is made of gelatin, and is opaque white. The cap is also made of gelatin, and the colors vary based on the dosage strength.
Temodar 5 mg: green imprint contains pharmaceutical grade shellac, anhydrous ethyl alcohol, isopropyl alcohol, n-butyl alcohol, propylene glycol, ammonium hydroxide, titanium dioxide, yellow iron oxide, and FD&C Blue #2 aluminum lake.
Temodar 20 mg: brown imprint contains pharmaceutical grade shellac, anhydrous ethyl alcohol, isopropyl alcohol, n-butyl alcohol, propylene glycol, purified water, ammonium hydroxide, potassium hydroxide, titanium dioxide, black iron oxide, yellow iron oxide, brown iron oxide, and red iron oxide.
Temodar 100 mg: blue imprint contains pharmaceutical glaze (modified) in an ethanol/shellac mixture, isopropyl alcohol, n-butyl alcohol, propylene glycol, titanium dioxide, and FD&C Blue #2 aluminum lake.
Temodar 140 mg: The blue cap contains gelatin, sodium lauryl sulfate, FD&C Blue #2, and titanium dioxide. The capsule body and cap are imprinted with pharmaceutical branding ink, which contains shellac, dehydrated alcohol, isopropyl alcohol, butyl, alcohol, propylene glycol, purified water, strong ammonia solution, potassium hydroxide, and ferric oxide.
Temodar 180 mg: The red cap contains gelatin, sodium lauryl sulfate, titanium dioxide, iron oxide red and iron oxide yellow. The capsule body and cap are imprinted with pharmaceutical branding ink, which contains shellac, dehydrated alcohol, isopropyl alcohol, butyl alcohol, propylene glycol, purified water, strong ammonia solution, potassium hydroxide, and ferric oxide.
Temodar 250 mg: black imprint contains pharmaceutical grade shellac, anhydrous ethyl alcohol, isopropyl alcohol, n-butyl alcohol, propylene glycol, purified water, ammonium hydroxide, potassium hydroxide, and black iron oxide.
Newly Diagnosed Glioblastoma Multiforme. Five hundred and seventy three patients were randomized to receive either Temodar (TMZ)+Radiotherapy (RT) (n=287) or RT alone (n=286). Patients in the Temodar+RT arm received concomitant Temodar (75 mg/m2 ) once daily, starting the first day of RT until the last day of RT, for 42 days (with a maximum of 49 days). This was followed by 6 cycles of Temodar alone (150 or 200 mg/m2) on Day 1-5 of every 28-day cycle, starting 4 weeks after the end of RT. Patients in the control arm received RT only. In both arms, focal radiation therapy was delivered as 60 Gy/30 fractions. Focal RT includes the tumor bed or resection site with a 2-3 cm margin. Pneumocystis carinii pneumonia (PCP) prophylaxis was required during the TMZ+radiotherapy treatment, regardless of lymphocyte count, and was to continue until recovery of lymphocyte count to less than or equal to Grade 1.
At the time of disease progression, Temodar was administered as salvage therapy in 161 patients of the 282 (57%) in the RT alone arm, and 62 patients of the 277 (22%) in the Temodar+RT arm. The addition of concomitant and maintenance Temodar to radiotherapy in the treatment of patients with newly diagnosed GBM showed a statistically significant improvement in overall survival compared to radiotherapy alone (
Temodar (temozolomide) Capsules are indicated for the treatment of adult patients with newly diagnosed glioblastoma multiforme concomitantly with radiotherapy and then as maintenance treatment.
Dosage of Temodar Capsules must be adjusted according to nadir neutrophil and platelet counts in the previous cycle and the neutrophil and platelet counts at the time of initiating the next cycle. For Temodar dosage calculations based on body surface area (BSA) see assigned Table. For suggested capsule combinations on a daily dose see assigned Table.
Patients with Newly Diagnosed High Grade Glioma:
Temodar is administered orally at 75 mg/m2 daily for 42 days concomitant with focal radiotherapy (60 Gy administered in 30 fractions) followed by maintenance Temodar for 6 cycles. Focal RT includes the tumor bed or resection site with a 2-3 cm margin. No dose reductions are recommended during the concomitant phase; however, dose interruptions or discontinuation may occur based on toxicity. The Temodar dose should be continued throughout the 42 day concomitant period up to 49 days if all of the following conditions are met: absolute neutrophil count ≧1.5×109/L platelet count ≧100×109/L common toxicity criteria (CTC) non-hematological toxicity ≦Grade 1 (except for alopecia, nausea, and vomiting). During treatment, a complete blood count should be obtained weekly. Temozolomide dosing should be interrupted or discontinued during concomitant phase according to the hematological and non-hematological toxicity criteria. PCP prophylaxis is required during the concomitant administration of Temodar and radiotherapy and should be continued in patients who develop lymphocytopenia until recovery from lymphocytopenia (CTC grade ≦1).
Four weeks after completing the Temodar+RT phase, Temodar is administered for an additional 6 cycles of maintenance treatment. Dosage in Cycle 1 (maintenance) is 150 mg/m2 once daily for 5 days followed by 23 days without treatment.
Cycles 2-6:
At the start of Cycle 2, the dose is escalated to 200 mg/m2, if the CTC non hematologic toxicity for Cycle 1 is Grade ≦2 (except for alopecia, nausea, and vomiting), absolute neutrophil count (ANC) is ≧1.5×109/L, and the platelet count is ≧100×109/L. The dose remains at 200 mg/m2 per day for the first 5 days of each subsequent cycle except, if toxicity occurs. If the dose was not escalated at Cycle 2, escalation should not be done in subsequent cycles.
Dose reduction or discontinuation during maintenance:
During treatment, a complete blood count should be obtained on Day 22 (21 days after the first dose of Temodar) or within 48 hours of that day, and weekly until the ANC is above 1.5×109/L, (1,500/μL) and the platelet count exceeds 100×109/L (100,000/μL). The next cycle of Temodar should not be started until the ANC and platelet count exceed these levels. Dose reductions during the next cycle should be based on the lowest blood counts and worst non-hematologic toxicity during the previous cycle.
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
Caveolin-1 (Cav-1), glioblastoma multiforme (GBM), mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), ribosomal protein S6 (RPS6), mammalian target of rapamycin (mTOR), temozolomide (TMZ), transforming growth factor beta (TGFβ), transforming growth motor beta receptor I (TGFβRI), tumor protein 53 (TP53), phosphate and tensin homolog (PTEN), permeability glycoprotein (p-gp)
Caveolin-1 (Cav-1) is a critical regulator of tumor progression in a variety of cancers where it has been shown to act as either a tumor suppressor or tumor promoter. In glioblastoma multiforme, it has been previously demonstrated to function as a putative tumor suppressor. Our studies here, using the human glioblastoma-derived cell line U-87MG, further support the role of Cav-1 as a negative regulator of tumor growth. Using a lentiviral transduction approach, we were able to stably overexpress Cav-1 in U-87MG cells. Gene expression microarray analyses demonstrated significant enrichment in gene signatures corresponding to downregulation of MAPK, PI3K/AKT, and mTOR signaling, as well as activation of apoptotic pathways in Cav-1 overexpressing U-87MG cells. These same gene signatures were later confirmed at the protein level in vitro. To explore the ability of Cav-1 to regulate tumor growth in vivo, we further show that Cav-1 overexpressing U-87MG cells display reduced tumorigenicity in an ectopic xenograft mouse model, with marked hypoactivation of MAPK and PI3K/mTOR pathways. Finally, we demonstrate that Cav-1 overexpression confers sensitivity to the most commonly used chemotherapy for glioblastoma, temozolomide. In conclusion, Cav-1 negatively regulates key cell growth and survival pathways and may be an effective biomarker for predicting response to chemotherapy in glioblastoma.
In order to establish durable expression of Cav-1 over time in a cell line model, we chose to use a lentiviral transduction approach over the transient transfection methods used in previous in vitro studies.15,16 After selection with puromycin, U-87MG cells transduced with lentiviral constructs stably expressing full length Cav-1 cDNA (LV105 Cav-1) were shown to effectively upregulate Cav-1 compared to an empty control lentivirus (LV105 Control) as demonstrated by western immunoblot (
Using a microarray consisting of >20,000 transcript probes, we were able to identify 2,001 genes (˜10%) significantly modulated by Cav-1 overexpression (Tables 1, 2, S1 and S2). Gene set enrichment analyses performed on microarray expression data obtained from LV105 Control and LV105 Cav-1 U-87MG cells indicates that Cav-1 expression corresponds to changes in a variety of cancer-associated gene signatures. Specifically, by comparing expression data to biological process gene ontology sets, it was found that Cav-1-overexpressing U-87MG cells demonstrated significant (p<0.001) enrichment among gene sets related to negative regulation of signal transduction, MAP-kinase activity, cell proliferation, and transcription (Tables 2 and S1). Signatures related to caspase activation, apoptosis, and the transforming growth factor β pathway were also highly enriched (Tables 2 and S1). When expression data was compared to a curated canonical pathway database, gene sets related to PI3K/AKT, mTOR, and ERK signaling, as well as cell death and extracellular matrix signaling were found to be significantly enriched (Tables 2 and S1).
To validate the results obtained from our microarray analyses, we next sought to confirm Cav-1 mediated modulation of intracellular signaling pathways at the protein level. Overexpression of Cav-1 in U-87MG cells results in abrogated activity of proliferative pathways as shown by reduced phosphorylation of ERK1/2 and decreased expression of the cell cycle driver cyclin D1 when compared to control as shown by western immunoblot (
To evaluate the ability of Cav-1 to regulate tumorigenicity in vivo, U-87MG cells infected with either Cav-1-expressing or control lentivirus were injected subcutaneously into the flanks of athymic nu/nu male mice. After 4 weeks, mice were sacrificed and tumors were collected, weighed, and measured. Importantly, mice harboring Cav-1 overexpressing tumors demonstrated markedly reduced (˜7 fold) tumor weights and volumes as compared to their control counterparts (p<0.001,
Similar to results obtained in vitro, our immunohistochemical analyses show that explanted xenograft tumors overexpressing Cav-1 demonstrate fewer cells staining positive for Phospho-ERK1/2 and cyclin D1 (
To further examine the effect of Cav-1 on chemotherapeutic-induced apoptosis, U-87MG cells were stained for the cell death marker Annexin V and measured by flow cytometry. Cav-1 overexpressing U-87MG cells cultured for 72 hours in the presence of 500 μM of temozolomide (TMZ), the most commonly used chemotherapeutic for GBM, showed significant reductions in cell viability when compared to TMZ treated LV105 control cells (5.5%, p<0.01,
Although it has been, demonstrated that Cav-1 expression in glioma increases variably in accordance with grade, little is currently known about its biological effects on tumor onset and progression.12-14, 17, 18 Previous in vitro studies using transient transfection techniques have shown that loss of Cav-1 in U-87MG cells resulted in the adoption of a more proliferative and invasive phenotype whereas its forced overexpression conferred the opposite effects.15 In line with previous studies, we here show that Cav-1 functions as a putative tumor suppressor in glioblastoma. Using a novel lentivirus transduction system we created a stable Cav-1 overexpressing cell line based on the U-87MG background.
By subjecting transiently transfected U-87MG cells to a panel of reverse transcription-PCR printers, Martin et al. identified genes pertaining to cell invasion, metastasis, and cell adhesion as those being the most differentially regulated by Cav-1 expression. Particularly, they showed the integrin genes ITGA1, ITGA3, ITGA5, ITGAV, ITGB1, and ITGB5 were significantly down regulated in Cav-1 overexpressing cells, with cells treated with Cav-1 specific siRNA demonstrating marked upregulation of these same genes. Matrix metallopeptidase genes MMP1 and MMP2, as well as transforming growth factor beta receptor I (TGFRBI) were also shown to be significantly modulated by Cav-1.15 A follow up study using Cav-1 silenced U-87MG cells further clarified a mechanism in which Cav-1 acts as a negative regulator of integrin signaling by inhibiting the expression of these integrins themselves as well as sequestering downstream TGFβ/TGFβRI/SMAD2 and ERK pathways.16 Here, we implemented a similar, albeit much more expansive microarray-based approach to study gene perturbations as a result of Cav-1 overexpression. Using gene set enrichment analyses, we indeed show similar expression profiles to those found previously, with gene sets related to integrin interactions, as well as regulation of TGFβ receptor/SMAD pathways showing significant enrichment. In our study, however, we detected a multitude of other significantly enriched gene sets which have not been demonstrated previously in Cav-1 expressing GBM cells. For instance, U-87MG cells overexpressing Cav-1 demonstrated significant upregulation of genes responsible for negative regulation of signal transduction, particularly within ERK, PI3K/AKT, and mTOR pathways. Although it has long been known that Cav-1 serves to negatively regulate the activity of p42/p44 (ERK1/2) signaling proteins of the MAPK pathway, our evidence also suggests it has the capability to sequester PI3K and mTOR activity.20-26 This is notable due to the fact that ERK, PI3K and mTOR signaling axes are frequently unregulated in GBM, suggesting that loss of Cav-1 could lead to unchecked activation of these pathways.27-31 Two of the most commonly silenced genes in GBM are the tumor suppressor proteins PTEN and TP53, which serve to antagonize the PI3K/AKT/mTOR pathway and regulate cell cycle, response to DNA damage and cell death, respectively.32 Of note is that these two genes were among the most unregulated in cells overexpressing Cav-1, which would likely explain the gene signatures corresponding to downregulation of signaling pathways and reduced invasiveness.
A major hallmark of GBM is the ability of tumor cells to invariably metastasize to distant sites in the CNS despite aggressive treatment. This is often attributed to the excessive release of matrix metallopeptidases and urokinase plasminogen activator.33 Here we show that the genes MMP1, MMP3, and PLAU (urokinase plasminogen activator) are highly downregulated in our Cav-1 overexpressing U-87MG cells, which is consistent with reports that Cav-1 negatively regulates tumor invasiveness.15, 34-36 These genes have been shown to be regulated by Erk and TP53, therefore, their reduction may be secondary to Cav-1 modulation of these pathways.37-40
Of note, we also found that genes responsible for sequestering cell cycle progression and transcription were overexpressed in LV105 Cav-1 cells (FOXN3, HDAC5, VHL, CDKNIC, among others). Conversely, genes responsible for progression through cell cycle such as CCND1 (cyclin D1) were found to be significantly downregulated in Cav-1 overexpressing cells, consistent with previous reports that Cav-1 transcriptionally represses cyclin D1.8 Perhaps our most notable finding, however, is that a substantial number of genes involved in the activation of apoptotic and cell death pathways are increased as a result of Cav-1 overexpression (TP53, MOAP1, CASP3, CASP9, BCL2L11, BAK1, BID among others). Although the role of Cav-1 in apoptosis is contentious, with reports indicating both pro- and anti-cell death roles, it may be possible that expression of Cav-1 promotes apoptotic activity in U-87MG cells by inhibiting the BIRC5 gene product, survivin, as is suggested here and in previous reports.41-45 In support of these microarray data, we were able to demonstrate, at the protein level, silencing of ERK, AKT, mTOR, RPS6, and cyclin D1 pathways with corresponding activation and cleavage of the key apoptosis initiator caspase 3.
Importantly, we here show for the first time that forced expression of Cav-1 in vivo results in a dramatic reduction of tumor burden in U-87MG xenografts. Although Cosset et al. have demonstrated that explanted human glioma tissue lacking Cav-1 expression results in increased expression of α5β1 integrin subunits, we were able to demonstrate a direct inverse relationship with Cav-1 expression and cell proliferation in an animal model.16 In line with our in vitro data, these xenograft tumors displayed reduced activity of ERK, RPS6, and mTOR pathways. As these pathways have been previously shown to play major roles in glioma progression, it is likely that Cav-1 could act as a critical regulator of tumor growth and protein synthesis in a clinical setting. As examples of this, studies have shown that exogenous administration of cavtratin, or a soluble peptide consisting of the Cav-1 scaffolding domain fused to an internalization domain, results in reduced MAPK activity in oligodendroglial cells in vivo, as well as reduced tumor volumes in a xenograft model of Lewis Lung Carcinoma.23,46 A separate study demonstrated that in vitro administration of full length Cav-1 prevented invasion of three different GBM-derived cell lines using a Boyden-chamber assay.47 In this regard, it may be suggested that Cav-1 be explored as a therapeutic agent in GBM.
Lastly, we have also demonstrated that expression of Cav-1 confers sensitivity to the most commonly used chemotherapeutic in GBM, temozolomide. This could be due in part to the action of the permeability glycoprotein (P-gp) transporter, a multidrug exporter which normally prevents the influx of drugs across the blood brain barrier. However, cancerous cells can also express this protein, rendering treatment of GBM with conventional chemotherapeutics less effective.48 Cav-1 has been shown to associate with P-gp and negatively regulate its activity, therefore overexpression of Cav-1 most likely results in improved access of TMZ to the intracellular compartment of U-87MG cells in our model.49, 50 Interestingly, a separate study showed that treatment with TMZ resulted in upregulation of Cav-1 expression in vivo using orthotopic GBM xenograft models.47 In light of our data, this could suggest a positive feedback loop exists in which treatment with TMZ serves to auto-sensitize GBM cells through a Cav-1 dependent mechanism. This finding implicates Cav-1 as a potential biomarker predicting response to chemotherapies for GBM as it has been shown for other cancers such as breast, lung, and oral squamous cell carcinomas.51-53
Taken together, these studies confirm and expand upon previous work identifying Cav-1 as a putative tumor suppressor in GBM. We here show that stable overexpression of Cav-1 in a widely used model of GBM results in silencing of key proliferative and cell survival pathways in vitro as well as in vivo. Additionally, we have demonstrated its ability to modulate sensitivity to commonplace chemotherapeutics for GBM. These findings highlight the potential of Cav-1 to serve as a novel biomarker indicating potential response to therapy and also a candidate therapy for treatment of GBM.
The human glioblastoma-derived cell line U-87MG was obtained from American Type Culture Collection (ATCC) and cultured in Eagle's Modified Essential Medium (EMEM, ATCC) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies). Cells were cultured in the presence of 5% CO2 at 37° C. Temozolomide (TMZ) was obtained from Sigma-Aldrich and dissolved in DMSO to a concentration of 100 mM. The following antibodies were used: mouse anti-Caveolin-1 (2297, BD Bioscience), rabbit anti-Caveolin-1 (N-20), mouse anti-cyclin D1 (DCS-6, Santa Cruz Biotechnology), rabbit anti-phospho mTOR (Se2448, D9C2, Cell Signaling), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling), rabbit anti-phospho-AKT (Ser473, D9E, Cell Signaling), rabbit anti-phospho-ribosomal S6 (Ser235/236, 91B2, Cell Signaling) rabbit anti-ERK1/2 (Cell Signaling), rabbit anti-AKT (Cell Signaling), rabbit anti-ribosomal S6 (5G10, Cell Signaling), rabbit anti-cleaved caspase 3 (Asp175, Cell Signaling), rabbit anti-caspase 3 (Cell Signaling), and mouse anti-GAPDH (6C5, Fitzgerald Industries).
Plasmids Ex-Neg-LV105 (empty control vector) and Ex-D0159-LV105 (Cav-1 c-DNA vector) were obtained from Genecopoeia and transfected into the packaging cell line Genecopoeia 293Ta using the Lenti-Pac HIV Expression Packaging Kit as per manufacturer's instructions, 48 hours post-transfection, lentivirus containing supernatants were collected and centrifuged at 500×g for 10 minutes to clear cellular debris. U-87MG cells were cultured in viral supernatants supplemented with 5 μg/ml polybrene (Santa Cruz) for 24 hours prior to changing back into complete medium containing 2.5 μg/ml puromycin hydrochloride (Santa Cruz) to select for lentiviral-transduced cells. After one week of selection, cells were allowed to grow in complete medium without puromycin.
Cells at 70% confluence were collected, pelleted at 300×g, washed twice with Dulbecco's PBS (DPBS) and resuspended in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, pH 7.5) including Complete Protease Inhibitor Cocktail (Roche Diagnostics) and Halt Phosphatase Inhibitor Cocktail (Thermo-Scientific). Lysates were sonicated and centrifuged at 10,000×g for 10 min to clear cellular debris prior to protein quantification by BCA assay (Thermo-Scientific) as per manufacturer's instructions. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 8-12% acrylamide), transferred to nitrocellulose membranes (Whatman), and blocked for 1 hour in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 8.0) with 5% bovine serum albumin (BSA). Membranes were incubated with primary antibodies diluted in TBST+1% BSA overnight at 4° C. followed by incubation in either horseradish peroxidase (HRP) conjugated anti-mouse (Thermo-Scientific) or anti-rabbit (BD Biosciences) antibodies. Detection of bound antibodies was accomplished with the use of Supersignal chemiluminescent substrates (Thermo-Scientific).
U-87MG cells grown on glass coverslips in 6 -well plates were fixed in ice-cold methanol for 20 min, washed with PBS, and incubated with anti-Cav-1 primary antibody (BD Bioscience) in immunofluorescence (IF) buffer (PBS+5% BSA, 0.5% NP40) for 30 min at 37° C. before incubation with secondary fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (Jackson Labs) in IF buffer. Cells were counterstained with Hoechst nuclear dye (Life Technologies) prior to coverslipping and visualization with a Zeiss LSM 510 confocal microscope (Carl Zeiss Microscopy).
DNA microarray analysis was performed using the Human Whole Genome OneArray v2 (Phalanx Biotech, Belmont, Calif.). RNA quality and integrity were determined utilizing an Agilent 2100 Bioanalyzer (Agilent Technologies) and absorbance at A260/A280. Only high quality RNA, having a RIN of >7.0, and an A260/280 absorbance ratio of >1.8, was utilized for further experimentation, RNA was synthesized to double-stranded cDNA and amplified using in vitro transcription that included amino-allyl UTP, and the aRNA product was subsequently conjugated with with Cy5 NHS ester (GE Healthcare Lifesciences). Fragmented aRNA was hybridized at 42° C. overnight using the HybBag mixing system with 1X OneArray Hybridization Buffer (Phalanx Biotech), 0.01 mg/ml sheared salmon sperm DNA (Promega), at a concentration of 0.025 mg/ml labeled target. After hybridization, the arrays were washed according to the OneArray protocol. Raw intensity signals for each microarray were captured using a Molecular Dynamics™ Axon 4100A scanner, measured using GenePixPro™ Software. Significantly up- or down-regulated genes in LV105 Cav-1 cells were identified as having normalized intensities above background (>50), a fold change of ±1.5 compared to control, and p<0.05.
Pre-processed expression data was subjected to Gene Set Enrichment Analysis using C5.BP.V3.0 (gene ontology: biological processes) and C2.CP.V3.0 (canonical pathways) MSigDB gene sets.54,55 Genes expression data were ordered based on a signal-2-noise metric and compared statistically to existing gene sets at a resolution of 1,000 permutations. Statistical significance of gene set enrichment was assumed at nominal p<0.05, with a false discovery rate (FDR) q<0.25.
All animal studies were conducted in accordance with the guidelines set forth by the National Institutes of Health and the Thomas Jefferson University Institutional Animal Care and Use Committee (IACUC). Briefly, U-87MG cells were washed with DPBS, trypsinized, counted, and resuspended in a volume of complete medium yielding 1×106 cells/50 μl, which was subsequently injected subcutaneously into the flanks of 6-8 week old male athymic nu/nu mice (NCI). After 4 weeks, mice were sacrificed and tumors were excised, weighed and measured prior to further processing.
Explanted xenograft tumors were fixed in 10% phosphate buffered formalin solution for 24 hours prior to dehydration in 70% ethanol, paraffin embedding, and sectioning onto slides. Following xylene deparaffinizaiton and rehydration, slides were subjected to 10 minutes of heat antigen retrieval in 10 mM sodium citrate buffer pH 6.0 and endogenous peroxide quenching in 3% hydrogen peroxide for 20 min. Tissues were blocked in 10% normal goat serum (NGS, Vector Labs, Burlingame, Calif.) for 1 hour at room temperature and incubated overnight with primary antibody in 10% NGS at 4° C. Slides were then washed in PBS and blocked with Biotin-Blocking System (Dako) before incubating with the appropriate secondary antibody in PBS and developing with 3,3-diaminobenzidine (DAB) substrate (Dako). Slides were counterstained with hematoxylin (Sigma), dehydrated, and coverslipped prior to imaging with an Olympus BX51 light microscope equipped with a Micropublisher 5.0 CCD camera (QImaging).
U-87MG cells (50,000/well) were plated in 12-well tissue culture dishes and allowed to attach overnight prior to changing their medium into complete EMEM containing either 500 μM TMZ or DMSO control and culturing them for an additional 72 hours. Cells were then trypsinized, centrifuged at 300×g for 5 min and resuspended in binding buffer with APC-conjugated anti-Annexin V antibody (BP Biosciences) and 0.33 μg/ml Propidium Iodide (PI, KPL, Gaithersburg, Md.). All samples were run on a BD FACSCalibur flow cytometer (BD Biosciences). Cells were quantified according to staining as follows: viable (Annexin V negative, PI negative), early apoptotic (Annexin V positive, PI negative), late apoptotic (Annexin V positive, PI positive), and dead (Annexin V negative, PI positive).
All data were expressed as mean±SEM Differences between groups were evaluated by either unpaired Student's t-test or one-way ANOVA followed by Tukey's multiple-group comparisons test, where appropriate. Statistical significance was assumed at p<0.05.
Top 100 microarray hits demonstrating the most significantly up- and downregulated genes in Cav-1 overexpressing U-87MG cells. For a complete list see Table S2. (n=3 samples from each group).
Enrichment score (ES), normalized enrichment score (NES), nominal p-value, and false discovery rate (FDR) q-value for selected gene sets enriched in LV105 Cav-1 cells vs control using (A) gene ontology: biological process and (B) canonical pathway molecular signature databases (n=3 samples from each group). For a detailed list of genes see Table S1.
Expanded Gene Set Enrichment Analysis for (A) gene ontology: biological process and (B) canonical pathway molecular signature databases (n=3 samples from each group).
Top microarray hits demonstrating, the most significantly up- and downregulated genes in Cav-1 overexpressing U-87MG cells (fold change >±1.5 vs LV105 control, p<0.05, n=3 samples from each group).
This application claims priority of U.S. Provisional Application No. 61/813,724, filed Apr. 19, 2013, the contents of which are incorporated herein by reference in their entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/US2014/034639 | 4/18/2014 | WO | 00 |
Number | Date | Country | |
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61813724 | Apr 2013 | US |