HHS Public Access Author manuscript Author Manuscript
Cancer Res. Author manuscript; available in PMC 2016 November 01. Published in final edited form as: Cancer Res. 2015 November 1; 75(21): 4466–4473. doi:10.1158/0008-5472.CAN-15-0988.
Glioblastomas require integrin αvβ3/PAK4 signaling to escape senescence Aleksandra Franovic1, Kathryn C. Elliott1, Laetitia Seguin1, M. Fernanda Camargo1, Sara M. Weis1, and David A. Cheresh1 1Department
of Pathology and Moores Cancer Center at the University of California, San Diego in La Jolla, California, 92093, USA
Author Manuscript
Abstract
Author Manuscript
Integrin αvβ3 has been implicated as a driver of aggressive and metastatic disease, and is upregulated during glioblastoma progression. Here we demonstrate that integrin αvβ3 allows glioblastoma cells to counteract senescence through a novel tissue-specific effector mechanism involving recruitment and activation of the cytoskeletal regulatory kinase PAK4. Mechanistically, targeting either αvβ3 or PAK4 led to emergence of a p21-dependent, p53-independent cell senescence phenotype. Notably, glioblastoma cells did not exhibit a similar requirement for either other integrins or additional PAK family members. Moreover, αvβ3/PAK4 dependence was not found to be critical in epithelial cancers. Taken together, our findings established that glioblastomas are selectively addicted to this pathway as a strategy to evade oncogene-induced senescence, with implications that inhibiting the αvβ3/PAK4 signaling axis may offer novel therapeutic opportunities to target this aggressive cancer.
Introduction Glioblastoma multiforme, or GBM, is the most aggressive and malignant form of astrocytoma characterized by highly invasive tumor cells. Although these tumors are treated using a combination of surgery, radiotherapy, and chemotherapy, only 5% of patients survive for longer than 5 years after diagnosis. Large-scale efforts have recently provided new clues into gliomagenesis and alterations that characterize this disease (1). Despite identification of new biomarkers and important molecular pathways (2), targeted therapies have not yet elicited durable clinical responses (3).
Author Manuscript
The expression of integrin αvβ3 (ITGAV/ITGB3) and its ligand vitronectin increase during the transition from low-grade astroglial-derived tumors to advanced glioblastoma (4, 5), and we and others have identified αvβ3 as a driver of an aggressive and metastatic tumor phenotype (6, 7). In GBM biopsy samples, αvβ3 expression is prominent in both tumor microvessels and glial tumor cells, and is the most prevalent in highly proliferating and infiltrating areas (8). Cilengitide, designed to target the ligand binding properties of αvβ3
Corresponding Author: David A. Cheresh, Ph.D., Distinguished Professor, Department of Pathology and Moores Cancer Center, University of California, San Diego, 3855 Health Sciences Drive #0803, La Jolla, CA 92093-0803, Phone: 858-822-2232, Fax: 858-822-2630, [email protected]. Conflict of Interest: No potential conflicts of interest were disclosed.
Franovic et al.
Page 2
Author Manuscript
and other αv integrins (9), was tested in combination with temozolomide chemoradiotherapy in a randomized phase III trial for patients with newly diagnosed glioblastoma with methylated MGMT promoters (ClinicalTrials.gov NCT00689221). While some patients responded, Cilengitide failed to meet its primary endpoint of a significant survival advantage (10).
Author Manuscript
In addition to its ligand-dependent signaling role, recent studies suggest αvβ3 has noncanonical cell biological functions that are ligand-independent (6, 7, 11). Since αvβ3 expression correlates with glioblastoma progression, we silenced β3 in a variety of human glioblastoma cells and assessed their growth in vivo and in vitro to evaluate the net contribution of this integrin’s ligand-dependent and –independent functions to glioblastoma biology. To our surprise, glioblastoma cells demonstrated an addiction to αvβ3 as a means to avoid p21 (CDKN1A)-dependent cellular senescence, whereas β3 knockdown did not trigger this effect in a range of histologically distinct epithelial cancers. Loss of αvβ3 led to a concomitant decrease in PAK4 activation, while PAK4 knockdown increased p21 and senescence. These findings reveals a new cell type-specific function for integrin αvβ3, and highlights a particular vulnerability of glioblastoma cells for components of this pathway.
Materials and Methods Cell lines
Author Manuscript
All cells were purchased from American Type Culture Collection (ATCC) within the past 5 years: glioblastoma (U87MG, LN229, LN18, U373, U118, U251), medulloblastoma (DAOY), renal (7860), colorectal (SW620), pancreatic (PANC1), breast (MDA-MB-231, BT20), and lung (A549, H23). Cell line authentication was performed by the ATCC using short tandem repeat DNA profiles. Upon receipt, each cell line was expanded, cryopreserved as low-passage stocks, and tested routinely for mycoplasma. RNA interference and expression constructs For transient knockdown, cells were transfected using the HiPerFect (Qiagen) with AllStars siRNA (Qiagen) for negative control (1027280), ITGB3 (SI00004585), ITGB5 (SI02780617), PAK4 (SI00082341), CDKN1A (SI00008547), or TP53 (SI00011655). For stable knockdown, cells were infected with shRNA targeting ITGB3 (Open Biosystems; TRCN0000003234) using a lentiviral system. Immunoblotting
Author Manuscript
Lysates made in 4% SDS were quantified using the Pierce BCA kit (ThermoScientific) and 25–50 μg protein loaded onto a denaturing SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membranes, blotted with HRP-conjugated secondary antibodies (Bio-Rad), and bands detected by enhanced chemiluminescence (Advansta). Antibodies include β3 (Abcam); β-Actin (Sigma); FAK-pY861 (Invitrogen); FAK and p130Cas (BD Trandsduction Laboratories); β5, p21 Waf1/Cip1, p27 Kip1 (D69C12), p53-pS392), p53 (7F5), Rb-pS795, Rb (D20), PAK4, p130Cas-pY410, PAK4-pS474/PAK5-pS602/PAK6pS560, PAK1, PAK2, and PAK1-pS144/PAK2-pS141 (Cell Signaling).
Cancer Res. Author manuscript; available in PMC 2016 November 01.
Franovic et al.
Page 3
Proliferation and cell cycle
Author Manuscript
The colorimetric BrdU Cell Proliferation Assay kit (Millipore) was used with absorbance at 450/550 nm relative to control. Cells were stained using propidium iodide and subjected to flow cytometry analysis for cell cycle. Animals Animal protocols were approved by the UCSD Institutional Animal Care and Use Committee. 6–8 week old female athymic nu/nu mice were purchased from the UCSD Animal Care Program. Flank tumor xenografts
Author Manuscript
Mice were injected subcutaneously with 106 tumor cells in 200 μl PBS. Tumor size was measured weekly with calipers. Orthotopic brain tumor xenografts Mice were anesthetized by intramuscular injection of ketamine, dexmedetomidine, and buprenorphine. Using a stereotaxic frame (Stoelting Co.), a small burr hole was made in the skull 2 mm anterior and 2 mm lateral to the bregma. A 31-gauge Hamilton needle/syringe was inserted 3 mm, and 0.25 μl/minute was dispensed (105 tumor cells in 2 μl media). Animals were monitored daily and those exhibiting signs of morbidity were euthanized. Tumor spheroids
Author Manuscript
Multicellular spheroids were prepared by seeding 105 cells per 24-well pre-coated with heated 1% Seaplaque agarose (Lonza) in serum-free medium. After 7–10 days, spheroids were collected, fixed in 4% paraformaldehyde, and embedded in paraffin. SA-β-galactosidase staining Cell senescence was measured by the Senescence β-Galactosidase Staining Kit (Cell Signaling). Immunofluorescence microscopy Cells on glass coverslips were fixed with 4% paraformaldehyde and processed for immunofluorescence as previously described (6) for imaging on a Nikon Eclipse C1 confocal microscope with 1.4 NA 60× oil-immersion lens. Antibodies include integrin αvβ3 (LM609), p21, phospho-PAK4, pan-methyl-histone H3 (Lys9) (D54) (Cell Signaling).
Author Manuscript
Statistics One way ANOVA or t-tests with P
Cancer Res. Author manuscript; available in PMC 2016 November 01. Published in final edited form as: Cancer Res. 2015 November 1; 75(21): 4466–4473. doi:10.1158/0008-5472.CAN-15-0988.
Glioblastomas require integrin αvβ3/PAK4 signaling to escape senescence Aleksandra Franovic1, Kathryn C. Elliott1, Laetitia Seguin1, M. Fernanda Camargo1, Sara M. Weis1, and David A. Cheresh1 1Department
of Pathology and Moores Cancer Center at the University of California, San Diego in La Jolla, California, 92093, USA
Author Manuscript
Abstract
Author Manuscript
Integrin αvβ3 has been implicated as a driver of aggressive and metastatic disease, and is upregulated during glioblastoma progression. Here we demonstrate that integrin αvβ3 allows glioblastoma cells to counteract senescence through a novel tissue-specific effector mechanism involving recruitment and activation of the cytoskeletal regulatory kinase PAK4. Mechanistically, targeting either αvβ3 or PAK4 led to emergence of a p21-dependent, p53-independent cell senescence phenotype. Notably, glioblastoma cells did not exhibit a similar requirement for either other integrins or additional PAK family members. Moreover, αvβ3/PAK4 dependence was not found to be critical in epithelial cancers. Taken together, our findings established that glioblastomas are selectively addicted to this pathway as a strategy to evade oncogene-induced senescence, with implications that inhibiting the αvβ3/PAK4 signaling axis may offer novel therapeutic opportunities to target this aggressive cancer.
Introduction Glioblastoma multiforme, or GBM, is the most aggressive and malignant form of astrocytoma characterized by highly invasive tumor cells. Although these tumors are treated using a combination of surgery, radiotherapy, and chemotherapy, only 5% of patients survive for longer than 5 years after diagnosis. Large-scale efforts have recently provided new clues into gliomagenesis and alterations that characterize this disease (1). Despite identification of new biomarkers and important molecular pathways (2), targeted therapies have not yet elicited durable clinical responses (3).
Author Manuscript
The expression of integrin αvβ3 (ITGAV/ITGB3) and its ligand vitronectin increase during the transition from low-grade astroglial-derived tumors to advanced glioblastoma (4, 5), and we and others have identified αvβ3 as a driver of an aggressive and metastatic tumor phenotype (6, 7). In GBM biopsy samples, αvβ3 expression is prominent in both tumor microvessels and glial tumor cells, and is the most prevalent in highly proliferating and infiltrating areas (8). Cilengitide, designed to target the ligand binding properties of αvβ3
Corresponding Author: David A. Cheresh, Ph.D., Distinguished Professor, Department of Pathology and Moores Cancer Center, University of California, San Diego, 3855 Health Sciences Drive #0803, La Jolla, CA 92093-0803, Phone: 858-822-2232, Fax: 858-822-2630, [email protected]. Conflict of Interest: No potential conflicts of interest were disclosed.
Franovic et al.
Page 2
Author Manuscript
and other αv integrins (9), was tested in combination with temozolomide chemoradiotherapy in a randomized phase III trial for patients with newly diagnosed glioblastoma with methylated MGMT promoters (ClinicalTrials.gov NCT00689221). While some patients responded, Cilengitide failed to meet its primary endpoint of a significant survival advantage (10).
Author Manuscript
In addition to its ligand-dependent signaling role, recent studies suggest αvβ3 has noncanonical cell biological functions that are ligand-independent (6, 7, 11). Since αvβ3 expression correlates with glioblastoma progression, we silenced β3 in a variety of human glioblastoma cells and assessed their growth in vivo and in vitro to evaluate the net contribution of this integrin’s ligand-dependent and –independent functions to glioblastoma biology. To our surprise, glioblastoma cells demonstrated an addiction to αvβ3 as a means to avoid p21 (CDKN1A)-dependent cellular senescence, whereas β3 knockdown did not trigger this effect in a range of histologically distinct epithelial cancers. Loss of αvβ3 led to a concomitant decrease in PAK4 activation, while PAK4 knockdown increased p21 and senescence. These findings reveals a new cell type-specific function for integrin αvβ3, and highlights a particular vulnerability of glioblastoma cells for components of this pathway.
Materials and Methods Cell lines
Author Manuscript
All cells were purchased from American Type Culture Collection (ATCC) within the past 5 years: glioblastoma (U87MG, LN229, LN18, U373, U118, U251), medulloblastoma (DAOY), renal (7860), colorectal (SW620), pancreatic (PANC1), breast (MDA-MB-231, BT20), and lung (A549, H23). Cell line authentication was performed by the ATCC using short tandem repeat DNA profiles. Upon receipt, each cell line was expanded, cryopreserved as low-passage stocks, and tested routinely for mycoplasma. RNA interference and expression constructs For transient knockdown, cells were transfected using the HiPerFect (Qiagen) with AllStars siRNA (Qiagen) for negative control (1027280), ITGB3 (SI00004585), ITGB5 (SI02780617), PAK4 (SI00082341), CDKN1A (SI00008547), or TP53 (SI00011655). For stable knockdown, cells were infected with shRNA targeting ITGB3 (Open Biosystems; TRCN0000003234) using a lentiviral system. Immunoblotting
Author Manuscript
Lysates made in 4% SDS were quantified using the Pierce BCA kit (ThermoScientific) and 25–50 μg protein loaded onto a denaturing SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membranes, blotted with HRP-conjugated secondary antibodies (Bio-Rad), and bands detected by enhanced chemiluminescence (Advansta). Antibodies include β3 (Abcam); β-Actin (Sigma); FAK-pY861 (Invitrogen); FAK and p130Cas (BD Trandsduction Laboratories); β5, p21 Waf1/Cip1, p27 Kip1 (D69C12), p53-pS392), p53 (7F5), Rb-pS795, Rb (D20), PAK4, p130Cas-pY410, PAK4-pS474/PAK5-pS602/PAK6pS560, PAK1, PAK2, and PAK1-pS144/PAK2-pS141 (Cell Signaling).
Cancer Res. Author manuscript; available in PMC 2016 November 01.
Franovic et al.
Page 3
Proliferation and cell cycle
Author Manuscript
The colorimetric BrdU Cell Proliferation Assay kit (Millipore) was used with absorbance at 450/550 nm relative to control. Cells were stained using propidium iodide and subjected to flow cytometry analysis for cell cycle. Animals Animal protocols were approved by the UCSD Institutional Animal Care and Use Committee. 6–8 week old female athymic nu/nu mice were purchased from the UCSD Animal Care Program. Flank tumor xenografts
Author Manuscript
Mice were injected subcutaneously with 106 tumor cells in 200 μl PBS. Tumor size was measured weekly with calipers. Orthotopic brain tumor xenografts Mice were anesthetized by intramuscular injection of ketamine, dexmedetomidine, and buprenorphine. Using a stereotaxic frame (Stoelting Co.), a small burr hole was made in the skull 2 mm anterior and 2 mm lateral to the bregma. A 31-gauge Hamilton needle/syringe was inserted 3 mm, and 0.25 μl/minute was dispensed (105 tumor cells in 2 μl media). Animals were monitored daily and those exhibiting signs of morbidity were euthanized. Tumor spheroids
Author Manuscript
Multicellular spheroids were prepared by seeding 105 cells per 24-well pre-coated with heated 1% Seaplaque agarose (Lonza) in serum-free medium. After 7–10 days, spheroids were collected, fixed in 4% paraformaldehyde, and embedded in paraffin. SA-β-galactosidase staining Cell senescence was measured by the Senescence β-Galactosidase Staining Kit (Cell Signaling). Immunofluorescence microscopy Cells on glass coverslips were fixed with 4% paraformaldehyde and processed for immunofluorescence as previously described (6) for imaging on a Nikon Eclipse C1 confocal microscope with 1.4 NA 60× oil-immersion lens. Antibodies include integrin αvβ3 (LM609), p21, phospho-PAK4, pan-methyl-histone H3 (Lys9) (D54) (Cell Signaling).
Author Manuscript
Statistics One way ANOVA or t-tests with P
