Integrin αv Expression Is Required for the Acquisition of a Metastatic Stem/Progenitor Cell Phenotype in Human Prostate Cancer
2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.07.011
ISSN1525-2191
AutoresChristel van den Hoogen, Geertje van der Horst, Henry Cheung, Jeroen T. Buijs, Rob C.M. Pelger, Gabri van der Pluijm,
Tópico(s)Prostate Cancer Treatment and Research
ResumoIntegrins participate in multiple cellular processes, including cell adhesion, migration, proliferation, survival, and the activation of growth factor receptors. Recent studies have shown that expression of αv integrins is elevated in the prostate cancer stem/progenitor cell subpopulation compared with more differentiated, committed precursors. Here, we examine the functional role of αv integrin receptor expression in the acquisition of a metastatic stem/progenitor phenotype in human prostate cancer. Stable knockdown of αv integrins expression in PC-3M-Pro4 prostate cancer cells coincided with a significant decrease of prostate cancer stem/progenitor cell characteristics (α2 integrin, CD44, and ALDHhi) and decreased expression of invasion-associated genes Snail, Snail2, and Twist. Consistent with these observations, αv-knockdown strongly inhibited the clonogenic and migratory potentials of human prostate cancer cells in vitro and significantly decreased tumorigenicity and metastatic ability in preclinical models of orthotopic growth and bone metastasis. Our data indicate that integrin αv expression is functionally involved in the maintenance of a highly migratory, mesenchymal cellular phenotype as well as the acquisition of a stem/progenitor phenotype in human prostate cancer cells with metastasis-initiating capacity. Integrins participate in multiple cellular processes, including cell adhesion, migration, proliferation, survival, and the activation of growth factor receptors. Recent studies have shown that expression of αv integrins is elevated in the prostate cancer stem/progenitor cell subpopulation compared with more differentiated, committed precursors. Here, we examine the functional role of αv integrin receptor expression in the acquisition of a metastatic stem/progenitor phenotype in human prostate cancer. Stable knockdown of αv integrins expression in PC-3M-Pro4 prostate cancer cells coincided with a significant decrease of prostate cancer stem/progenitor cell characteristics (α2 integrin, CD44, and ALDHhi) and decreased expression of invasion-associated genes Snail, Snail2, and Twist. Consistent with these observations, αv-knockdown strongly inhibited the clonogenic and migratory potentials of human prostate cancer cells in vitro and significantly decreased tumorigenicity and metastatic ability in preclinical models of orthotopic growth and bone metastasis. Our data indicate that integrin αv expression is functionally involved in the maintenance of a highly migratory, mesenchymal cellular phenotype as well as the acquisition of a stem/progenitor phenotype in human prostate cancer cells with metastasis-initiating capacity. 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Prostate cancer specific integrin alphavbeta3 modulates bone metastatic growth and tissue remodeling.Oncogene. 2007; 26: 6238-6243Crossref PubMed Scopus (164) Google Scholar In this study, we generated human prostate cancer cell lines with a stable knockdown of αv integrins. Data are presented that indicate an essential role for these αv integrins in tumor growth and metastasis via the induction of prostate cancer cells with a stem/progenitor phenotype. The human osteotropic PC-3M-Pro4 prostate cancer cells were generated from PC-3 cells (ATCC, Manassas, VA; no. CRL-1435) by injecting PC-3 cells into athymic mouse prostates and selecting for clones with increasing metastatic potential by several rounds of re-injecting cells from xenograft tumors back into the mouse prostate. PC-3M-Pro4 cells were stably transfected with a cytomegalovirus promoter-driven mammalian expression vector containing firefly-luciferase (pcDNA3.1 CMV-ff-luc). One clone with high luciferase activity was selected with neomycin (800 μg/mL; Life Technologies, Basel, Switzerland) and successfully used for in vivo bioluminescent imaging (BLI).12van den Hoogen C. van der Horst G. Cheung H. Buijs J.T. Lippitt J.M. Guzman-Ramirez N. Hamdy F.C. Eaton C.L. Thalmann G.N. Cecchini M.G. Pelger R.C. van der Pluijm G. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer.Cancer Res. 2010; 70: 5163-5173Crossref PubMed Scopus (291) Google Scholar, 33Buijs J.T. Rentsch C.A. van der Horst G. van Overveld P.G. Wetterwald A. Schwaninger R. Henriquez N.V. Ten Dijke P. Borovecki F. Markwalder R. Thalmann G.N. Papapoulos S.E. Pelger R.C. Vukicevic S. Cecchini M.G. Lowik C.W. van der Pluijm G. BMP7, a putative regulator of epithelial homeostasis in the human prostate, is a potent inhibitor of prostate cancer bone metastasis in vivo.Am J Pathol. 2007; 171: 1047-1057Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar The human prostate cancer cell lines PC-3M-Pro4lucA6 (Pro4luc) and C4-2B were maintained as described previously.12van den Hoogen C. van der Horst G. Cheung H. Buijs J.T. Lippitt J.M. Guzman-Ramirez N. Hamdy F.C. Eaton C.L. Thalmann G.N. Cecchini M.G. Pelger R.C. van der Pluijm G. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer.Cancer Res. 2010; 70: 5163-5173Crossref PubMed Scopus (291) Google Scholar, 33Buijs J.T. Rentsch C.A. van der Horst G. van Overveld P.G. Wetterwald A. Schwaninger R. Henriquez N.V. Ten Dijke P. Borovecki F. Markwalder R. Thalmann G.N. Papapoulos S.E. Pelger R.C. Vukicevic S. Cecchini M.G. Lowik C.W. van der Pluijm G. BMP7, a putative regulator of epithelial homeostasis in the human prostate, is a potent inhibitor of prostate cancer bone metastasis in vivo.Am J Pathol. 2007; 171: 1047-1057Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar Puromycin in a concentration of 1 μg/mL was added for cells with stable short hairpin RNA interference (shRNAi) knockdown (see further below). HEK293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. All cell lines were grown in a humidified incubator at 37°C and 5% CO2. shRNAi constructs (integrin αv clone nos. TRCN0000003239, TRCN0000003240, TRCN0000000768, and TRCN000000769) were derived from the MISSION library of Sigma-Aldrich (St. Louis, MO). HEK293T cells were lentivirally transfected with the short hairpin constructs together with the packaging plasmids REV, GAG, and VSV in a 1:1:1:1 ratio with the use of Fugene HD (Roche, Indianapolis, IN) as transfection reagent. The supernatant fluid of the culture medium containing the lentiviral vector was collected 48 hours after transfection. Cells (Pro4lucA6 and C4-2B) were mixed with 1 mL of shRNA-lentiviral vector, and 8 μg of Polybrene (Sigma-Aldrich) was added. The mixture was incubated for 1 to 2 hours at room temperature. Scrambled shRNA (clone no. TRC1/1.5), which was used as control, lacks identity with any mammalian mRNA sequence. Cells stably expressing the shRNA were selected with puromycin (1 μg/mL; Sigma-Aldrich). The effects of integrin αv knockdown described in this study represent activities of the heterogeneous cell populations transduced with high efficiency by the lentivirus and not single-cell selected clones. The integrin αv knockdown cell line is further referred to as αv-kd-Pro4luc or αv-kd-C42B cells and the nontargeting control cell line as NT-Pro4luc or NT-C42B cells. Expression of integrin αv, and a number of previously described stem and EMT markers, was measured by fluorescence-activated cell sorting (FACS) analysis with the use of the Calibur2 flow cytometer (BD Biosciences, San Jose, CA) and FCS Express 3 software (De Novo Software, Los Angeles, CA). The cells (1 × 105) were incubated for 45 minutes at 4°C in a solution of 90 μL of FACS wash buffer containing PBS + 1% fetal calf serum + 0.1% natriumazide NaN3 and 10 μL of antibody (αv-phosphatidylethanolamine, α2-fluorescein isothiocyanate, CD44-allophycocyanin, CD44v6-allophycocyanin; Miltenyi Biotec Inc., Auburn, CA). To determine E-cadherin/vimentin ratios, cells were harvested and labeled with E-cadherin-fluorescein isothiocyanate (BD Biosciences; 1:10) in FACS buffer for 30 minutes at 4°C in the dark. Then cells were washed with 1 mL of FACS buffer and fixed with freshly prepared 2% formaldehyde for 15 minutes. Cells were washed with ice-cold PBS and subsequently incubated for 30 minutes at 4°C in the dark with vimentin rabbit polyclonal antibody (1:200 in FACS buffer; Abcam Inc., Cambridge, MA). Cells were washed twice with 1 mL of FACS buffer and incubated for 30 minutes at 4°C with goat anti-rabbit IgG-allophycocyanin antibody (Invitrogen, Carlsbad, CA). After the last incubation step, the cells were washed and centrifuged for 5 minutes, followed by adding 250 μL of FACS wash buffer. ALDH activity was measured as described earlier.12van den Hoogen C. van der Horst G. Cheung H. Buijs J.T. Lippitt J.M. Guzman-Ramirez N. Hamdy F.C. Eaton C.L. Thalmann G.N. Cecchini M.G. Pelger R.C. van der Pluijm G. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer.Cancer Res. 2010; 70: 5163-5173Crossref PubMed Scopus (291) Google Scholar RNA was extracted with the use of Trizol (Invitrogen), according to the manufacturer's instructions. Real-time quantitative PCR (qPCR) was run and analyzed with a Bio-Rad IQ5 cycler (Bio-Rad, Hercules, CA). For primer sequences, see Table 1. Gene expression was measured relative to GAPDH expression with the use of the following formula: relative transcript abundance = 10,000/2(Ctgene−CtGAPDH).Table 1q-PCR Primer SequencesPrimerSequenceAlpha-v forward5′-GCTGGACTGTGGAGAAGAC-3′Alpha-v reverse5′-AAGTGAGGTTCAGGGCATTC-3′Alpha-2 forward5′-TTTGGTAGTGTGCTGTGTTC-3′Alpha-2 reverse5′-GACTCTTCCTTCCTCTTTCTTTAG-3′N-Cadherin forward5′-CAGACCGACCCAAACAGCAAC-3′N-Cadherin reverse5′-GCAGCAACAGTAAGGACAAACATC-3′CD44 forward5′-TGGCACCCGCTATGTCCAG-3′CD44 reverse5′-GTGACAGGGATTCTGTCTG-3′Osteopontin forward5′-CAAAGTCAGCCGTGAATTCCA-3′Osteopontin reverse5′-AACCCAATAAACTGAGAAAGAAGC-3′Snail forward5′-TGCAGGACTCTAATCCAAGTTTACCC-3′Snail reverse5′-GTGGGATGGCTGCCAGC-3′Snail2 forward5′-TGTGTGGACTACCGCTGC-3′Snail2 reverse5′-TCCGGAAAGAGGAGAGAGG-3′Twist forward5′-TGTCCGCGTCCCACTAGC-3′Twist reverse5′-TGTCCATTTTCTCCTTCTCTGGA-3′ALDH7A1 shRNAi clone TRCN00000032395′-CCGGGACTGAGCTAATCTTGAGAATCTCGAGATTCTCAAGATTAGCTCAGTCTTTTT-3′ALDH7A1 shRNAi clone TRCN00000032405′-CCGGCTCTGTTGTATATCCTTCATTCTCGAGAATGAAGGATATACAACAGAGTTTTT-3′ALDH7A1 shRNAi clone TRCN00000007685′-CCGGGTGAGGTCGAAACAGGATAAACTCGAGTTTATCCTGTTTCGACCTCACTTTTT-3′ALDH7A1 shRNAi clone TRCN00000007695′-CCGGCGACAGGCTCACATTCTACTTCTCGAGAAGTAGAATGTGAGCCTGTCGTTTTT-3′Nontarget control TRC1/1.55′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT-3′ Open table in a new tab Cell suspensions were generated12van den Hoogen C. van der Horst G. Cheung H. Buijs J.T. Lippitt J.M. Guzman-Ramirez N. Hamdy F.C. Eaton C.L. Thalmann G.N. Cecchini M.G. Pelger R.C. van der Pluijm G. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer.Cancer Res. 2010; 70: 5163-5173Crossref PubMed Scopus (291) Google Scholar and overlaid onto a 60-mm dish containing a solidified bottom layer of 0.6% Noble agarose (Becton Dickinson, Franklin Lakes, NJ) in medium. Medium (1 mL) was placed on top of the solidified cell layer. Plates were incubated for 1 to 3 weeks until colonies were visible. The colonies on the soft agar plates were counted with light microscopy (Zeiss Axiovert 200M, Sliedrecht, The Netherlands). Three individual and representative fields of each well were counted. The mean number of colonies/field was calculated. Cells were seeded into a 96-well plate containing an average of 1 cell per well. Plates were monitored twice a week and maintained in Dulbecco's modified Eagle's medium/10% FCII medium. After 1 to 3 weeks, colonies were clearly visible, and the mean number of positive wells/plate was counted by microscopy (Zeiss Axiovert 200M). Tumor cell migration was performed in Transwell migration chambers (Costar, Cambridge, MA).12van den Hoogen C. van der Horst G. Cheung H. Buijs J.T. Lippitt J.M. Guzman-Ramirez N. Hamdy F.C. Eaton C.L. Thalmann G.N. Cecchini M.G. Pelger R.C. van der Pluijm G. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer.Cancer Res. 2010; 70: 5163-5173Crossref PubMed Scopus (291) Google Scholar Three random fields were counted for each well, and mean numbers of migrated cells/field were calculated. For apoptotic analysis, harvested cells were stained with Annexin V/propidium iodide (Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit; Invitrogen), incubated for 15 minutes according to the manufacturer's protocol. Samples were analyzed with FACSCalibur2 (BD Biosciences) and FCS Express 3 software (DeNovo Software). Cells were seeded at a density of 2500/cm2 and allowed to grow for 24, 48, and 72 hours, respectively After the cell incubation, 20 μL of MTS was added to the medium, and mitochondrial activity was measured at 490 nm after 2 hours of incubation at 37°C (CellTiter96 Aqueous Non-radioactive Cell proliferation assay; Promega, Madison, WI). Male nude (BALB/c nu/nu) mice were housed in individual ventilated cages under sterile condition according to the local guidelines for the care and use of laboratory animals (DEC07026 and 09052). Mice were anesthetized before surgical and analytical procedures were performed. A 10-μL single-cell suspension of 1 × 105 αv-kd-Pro4luc cells or NT-Pro4luc cells in PBS was combined with 10 μL of growth factor-reduced Matrigel (BD Biosciences) and injected subcutaneously in anesthetized 6-week-old male nude mice. The progression of cancer cell growth was monitored weekly by BLI.12van den Hoogen C. van der Horst G. Cheung H. Buijs J.T. Lippitt J.M. Guzman-Ramirez N. Hamdy F.C. Eaton C.L. Thalmann G.N. Cecchini M.G. Pelger R.C. van der Pluijm G. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer.Cancer Res. 2010; 70: 5163-5173Crossref PubMed Scopus (291) Google Scholar A single-cell suspension of 1 × 105 αv-kd-Pro4luc cells or NT-Pro4luc cells/10 μL of PBS was combined with 10 μL of growth factor-reduced Matrigel (BD Biosciences) and surgically inoculated into the prostate of anesthetized 6-week-old male nude mice.12van den Hoogen C. van der Horst G. Cheung H. Buijs J.T. Lippitt J.M. Guzman-Ramirez N. Hamdy F.C. Eaton C.L. Thalmann G.N. Cecchini M.G. Pelger R.C. van der Pluijm G. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer.Cancer Res. 2010; 70: 5163-5173Crossref PubMed Scopus (291) Google Scholar, 34Buijs J.T. Henriquez N.V. van Overveld P.G. van der Horst G. Que I. Schwaninger R. Rentsch C. Ten D.P. Cleton-Jansen A.M. Driouch K. Lidereau R. Bachelier R. Vukicevic S. Clezardin P. Papapoulos S.E. Cecchini M.G. Lowik C.W. van der Pluijm G. Bone morphogenetic protein 7 in the development and treatment of bone metastases from breast cancer.Cancer Res. 2007; 67: 8742-8751Crossref PubMed Scopus (168) Google Scholar The cutaneous wound was sutured. The progression of cancer cell growth was monitored weekly by BLI. A single-cell suspension of 1 × 105 αv-kd-Pro4luc cells or NT-Pro4luc cells per 100 μL of PBS was injected into the left cardiac ventricle of anesthetized 5-week-old male nude mice, and cancer cell growth was monitored weekly by BLI.14Kong D. Banerjee S. Ahmad A. Li Y. Wang Z. Sethi S. Sarkar F.H. Epithelial to mesenchymal transition is mechanistically linked with stem cell signatures in prostate cancer cells.PLoS One. 2010; 5: e12445Crossref PubMed Scopus (338) Google Scholar Luciferin (Perbio Science Nederland B.V., Etten-Leur, The Netherlands) was injected intraperitoneally. BLI of tumors induced by the luciferase-expressing human prostate cancer cell lines was performed with the Xenogen IVIS100. Analyses for each metastatic site were performed after definition of the region of interest and quantified with Living Image 4.2 (Caliper Life Sciences, Teralfene, Belgium). Values are expressed as photons per second. Statistical analysis was performed with GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, CA) with the use of either t test (for comparison between two groups) or analysis of variance (for comparison between more than two groups). Unless otherwise stated, data are presented as the mean ± SEM. P values ≤ 0.05 were regarded as being statistically significant (*P < 0.05, **P < 0.01, and ***P < 0.001). Strong αv integrin expression is associated with the basal layer of
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