- Research article
- Open Access
The role of c-Src in integrin (α6β4) dependent translational control
© Soung et al.; licensee BioMed Central Ltd. 2013
- Received: 10 June 2013
- Accepted: 25 October 2013
- Published: 1 November 2013
Integrin α6β4 contributes to cancer progression by stimulating transcription as well as translation of cancer related genes. Our previous study demonstrated that α6β4 stimulates translation initiation of survival factors such as VEGF by activating mTOR pathway. However, the immediate early signaling events that link α6β4 to mTOR activation needs to be defined.
In the current studies, we demonstrated that c-Src is an immediate early signaling molecule that acts upstream of α6β4 dependent mTOR activation and subsequent translation of VEGF in MDA-MB-435/β4 and MDA-MB-231 cancer cells. m7GTP-Sepharose–binding assay revealed that Src activity is required to form eIF4F complex which is necessary for Cap-dependent translation in α6β4 expressing human cancer cells.
Overall, our studies suggest that integrin β4 and c-Src activation is important early signaling events to lead mTOR activation and cap-dependent translation of VEGF.
Cancer cells must acquire survival advantages including growth signaling autonomy, apoptosis resistance, sustaining of angiogenesis under stress conditions such as nutrient and oxygen deprivation to successfully survive in tumor microenvironment . Although these complicated processes involves regulation of survival related gene expression both at the transcription and translational level, recent evidence suggest that translation initiation is a primary check point that regulates cancer related mRNAs . One of the major mechanisms that cancer cells maintain higher efficiency of translation initiation involves stimulation of translation initiation factor, eIF4E [3, 4].
eIF4E is the rate limiting factor responsible for delivering cellular mRNAs to eIF4F complex (eIF4E, a scaffold protein eIF4G and a RNA helicase eIF4A) through interaction with the 5’-terminal (m7GpppN) Cap structure of mRNAs . Most of the cancer related mRNAs have the highly complex and lengthy 5’ untranslated region, which leads to the low translation initiation efficiency . Therefore, either level or activity of eIF4E needs to be up regulated to maintain active translation of these weak mRNAs. One way to enhance eIF4E activity is through PI3-K/Akt dependent signaling cascade that activates mTOR kinase . Activated mTOR phosphorylates and inactivates eIF4E-binding protein 4E-BP . Upon phosphorylation of 4E-BP, eIF4E is released from 4E-BP and bind to eIF4G to form eIF4F complex which mediates translation initiation [7, 8]. Aggressive cancer cells often take advantage of mitogenic signaling pathways to activate mTOR and free up eIF4E to maintain their survival and growth [9–11].
Our previous studies demonstrated that α6β4 integrin stimulates eIF4E activity to promote translation of survival factor, VEGF via Akt/mTOR pathway in breast carcinoma cells under serum deprivation condition [12, 13]. While α6β4-dependent translation control via ATK/mTOR pathway has been established, the early signaling event to link between α6β4 and mTOR is not well characterized. One of the prime candidates that mediate α6β4 dependent mTOR activation is Src as it is a key immediate early downstream effector of α6β4 and its activity is required for α6β4 signaling competency [14, 15]. Src is an intracellular non-receptor tyrosine kinase which has been implicated in proliferation, metastasis and invasion of various human cancers [16, 17]. For example, oestrogen induced c-Src activation leads to 4E-BP phoshorylation through PI3K/mTOR pathway and consequently promotes translation of HIF-1 α in breast cancer cells . Another study showed that active c-Src up-regulates translation of β-catenin by activation of eIF4E via Ras/ERK pathway and the phosphorylation of 4E-BP via the PI3K/mTOR pathways  Based on these evidences that c-Src stimulate translational initiation via mTOR signaling, we hypothesized that c-Src mediates α6β4 dependent mTOR activation and subsequent assembly of eIF4E machinery to enhance cap-dependent translation of weak mRNAs.
In this study, we assessed the role of c-Src in α6β4 dependent translational control. Pharmacologic inhibition of c-Src as well as knockdown of its expression by shRNA showed that c-Src plays an essential role in mediating α6β4 dependent mTOR activation in MDA-MB-435/β4 and MDA-MB-231 cancer cells. Src is also required to form eIF4F complex and enhance cap-dependent translation of VEGF mRNA. These results suggest that c-Src is an important immediate early signaling molecule to connect α6β4 signaling to mTOR, which eventually contribute to translation of survival factors such as VEGF.
Src activity is required for α6β4 dependent mTOR phosphorylation
c-Src contributes to α6β4 dependent TORC1 and TORC2 activation
Inhibition of c-Src blocks α6β4 dependent translation of VEGF mRNA
Inhibition of Src prevents assembly of eIF4F complexes
A number of studies demonstrated the role of integrins in translation of survival and growth factors through enhancing eIF4E function , but the exact mechanism by which integrins control translation initiation of cancer related mRNAs remains to be determined. In the previous study, we showed that α6β4 integrin promotes the translation of VEGF mRNA through the AKT/mTOR/eIF4E signaling axis . In the current studies, we investigated the role of c-Src as an immediate early signaling effector that mediates α6β4 dependent mTOR activation. We provided evidence that c-Src inhibition by PP2 or shRNA blocks mTOR pathway and the subsequent assembly of eIF4F complexes. This is first report to define the early signaling event that link between α6β4 and mTOR pathway.
Our studies indicated that c-Src is one of early α6β4 signaling effectors that mediate mTOR activation. As c-Src represents one isoform of Src Family Kinases (SFKs), it is possible that other isoform of SFKs could play a role in α6β4 dependent mTOR activation. This is more likely due to the previous report that Fyn becomes activated to mediate α6β4 dependent pro-invasive migration of breast carcinoma cells . α6β4 dependent Fyn activation requires the recruitment of SHP2 to the phosphorylated cytoplasmic domain of integrin β4 . It remains to be seen whether α6β4 dependent c-Src activation also requires the involvement of SHP2. Another possibility is the involvement of Focal Adhesion Kinase (FAK) in c-Src activation. FAK was shown to be activated by α6β4  and FAK mediates Src activation in integrin signaling such as α5β1 or α4β1 . If we establish the mechanism by which a6b4 activates multiple isoforms of SFKs including Fyn and c-Src, we may need to perform sequential knockdown of each SFK isoform expression by shRNAs to test the role of other SFKs in mTOR activation. The assays will test whether multiple SFK isoform synergistically contribute to α6β4 dependent mTOR activation, or the loss of one SFK isoform could simply be compensated by others.
While our current studies mostly focused on translation initiation aspects of mTOR signaling (mostly through TORC1 pathway), TORC2 pathway is likely activated by α6β4/c-Src signaling axis (Figure 2B). Enhancement of eIF-4E function by α6β4 is known to be mediated by TORC1 pathway as we previously showed that TORC1 specific inhibitor, rapamycin blocked α6β4 dependent eIF-4E activation . It remains to be determined how TORC2 signaling pathway contributes to α6β4 dependent phenotypes of breast carcinoma cells such proliferation, survival, cell motility and invasion. Knockdown of TORC2 specific components such as Rictor or Sin1 [26, 27] will address this issue.
It is currently unknown how activated c-Src by α6β4 mediates downstream signaling events leading to mTOR activation. Both Akt and MAPK seem to be prime candidates in mediating c-Src dependent mTOR activation as both involves 4E-BP1 phosphorylation, which is a key event for mTOR activation [19, 28]. Activated Src was shown to mediate both Akt  and MAPK . Alternatively, c-Src could enhance the functional crosstalk between α6β4 and growth factor receptors such as EGFR and c-Met  and this interaction was shown to enhance both Akt  and MAPK signaling . All these evidences suggest that c-Src could be an important therapeutic target that could affect growth factor receptor signaling as well as downstream events such as mTOR signaling. Considering that the role of α6β4 in breast carcinoma progression is well established, but no therapeutic agent against α6β4 is available yet, targeting Src activity will merit consideration against tumors that express high levels of α6β4.
In conclusion, we defined that c-Src is an immediate early signaling molecule that connects α6β4 to mTOR signaling axis. c-Src mediates α6β4 dependent mTOR activation and subsequent enhancement of cap-dependent translation of weak mRNAs such as VEGF. Our finding suggests that c-Src could be an important target of therapy for tumors that express high levels of α6β4.
Cell lines and cultures
The MDA-MB-231 human breast carcinoma cells and MDA-MB-435 human cancer cells were obtained from the Lombardi Breast Cancer Depository at Georgetown University. The generation of MDA-MB-435 subclones (MDA-MB-435/mock (vector only) and MDA-MB-435/β4 (β4 over-expression)) was done as previously described . MDA-MB-231 cells were stably infected with lentivirus that expressed shRNA targeted against β4 integrin or Src and MDA-MB-435/β4 cells were infected against Src as previously described . GFP shRNA was used as control and puromycin (2 μg/ml) was used for the selection of infected cells. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/low glucose (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA).
Antibodies and reagents
The integrin β4 (clone H-101) and actin (clone C-11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the p-mTOR (S2448), p-Src (Y416), p-Akt (S473), p-S6 ribosomal protein (S235/236), p-4E-BP1 (S65), 4E-BP1, mTOR, Src (clone 36D10), and Akt antibodies were obtained from Cell Signaling Technology (Beverly, MA). Also, integrin β4 (Y1494, phospho-specific) antibody was obtained from ECM bioscience (Versailles, KY) and PP2 (Src kinase inhibitor) was purchased from EMD chemicals Inc. (San Diego, CA). The antibodies against eIF4G and eIF4E were kindly provided by Dr. Rhoads (LSUHSC, Shreveport). For the pharmacological inhibition, cells were incubated with or without 10–50 μM PP2 for 24 hours before lysis for Western blot analysis.
Western blot analysis
Cells were lysed using 50 mM Tris buffer, pH 7.4, containing 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor (Pierce, Rockford, IL) and scraped, collected, and then protein concentration was determined using BCA protein assay kit (Pierce, Rockford, IL). Total protein was resolved on the 4-20% gradient SDS-PAGE, transferred to polyvinylidene fluoride membranes and incubated with a primary antibody. After three 10 min washes in 50 mM Tris buffer, pH 7.5, containing 0.15 M NaCl and 0.1% Tween-20, protein was detected, in turn, by means of a peroxidase - or alkaline phoaphatase - conjugated secondary antibody and visualized using the Luminol and Oxidizing solutions (Boston Bioproducts, Worcester, MA) or BCIP/NBT Color development substrate (Promega, Madison, WI).
The MDA-MB-231 cells and MDA-MB-435/β4 cells were maintained in low serum (0.5% FBS) medium and then pretreated with 0.1% DMSO (as a solvent for PP2) or 10 μM PP2 for 24 h. The MDA-MB-231 cells and MDA-MB-435/β4 cells were infected with lentivirouses expressing GFP- or Src shRNA. Before cell lysis, cells were treated with 50 μg/ml cycloheximide (VWR) and then incubated for 5–10 min at 37°C. After washing with PBS containing 100 μg/ml cycloheximide, cells were lysed in 0.5 ml buffer containing 50 mM Tris–HCl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 0.5% NP-40, 2 mM DTT, 100 μg/ml cycloheximide, 50 μg/ml heparin, RNasin 0.5 U/μl (Applied Biosystems), and Complete™ EDTA-free protease inhibitor cocktail (Roche), incubated on ice for 10 min and centrifuged for 5 min at 10,000 × g, 4°C. The supernatants were collected and frozen at -80°C. One hundred μg aliquots of total lysates were used for m7GTP-Sepharose binding experiments. An equal volume of lysate was applied to a 15 to 45% (w/v) sucrose gradient containing 100 μg/ml cycloheximide and then centrifuged in a Beckman SW41Ti rotor at 38,000 rpm at 4°C for 3 h. Gradients were fractionated (1 ml) and then monitored for absorbency at 254 nm using an ISCO syringe pump with UV-6 detector.
RNA preparation and quantitative real time PCR
Before RNA isolation, four hundred aliquots from each fraction after ribosome fractionation were spiked with 100 pg of GFP mRNA (internal control). Then, the RNA was purified from using an E.Z.N.A. Total RNA Kit (Omega bio-tek) according to manufacturer’s instructions. Reverse transcription was performed with random primers and reverse transcriptase from the TaqMan® Reverse Transcription Reagents kit (Applied Biosystems) following the manufacturer’s protocol. Quantitative real time PCR was used to measure the GFP and VEGF mRNAs level in each fraction. Amplification and detection were performed using the iCycler IQ Real-time PCR detection system with IQ™ SYBRgreen Supermix (Bio-Rad). The VEGF mRNA levels were normalized with the GFP internal control. Then, relative amount of VEGF in each fraction was expressed as a percentage of the sum of this mRNA in all fractions. To assist statistical significance of the changes in the VEGF mRNA redistribution along the sucrose density gradients, the percentage of VEGF mRNA co-sedimented with untranslated complexes (U, fractions #1-3), light polyribosomes, containing weakly translated mRNA (fractions #4-8) or heavy polyribosomes, containing efficiently translated mRNAs (H, fractions #9-12), was calculated as a sum of VEGF mRNA in the corresponding fractions from the original data.
Protein binding assays on m7GTP-sepharose
One hundred μg of lysates were prepared as described in the “Ribosome Fractionation” section and then diluted in equal volume of buffer containing 50 mM Tris–HCl (pH 7.5) and 2 mM DTT. The samples were mixed with 50 μl m7GTP-Sepharose (GE Healthcare), 50% slurry in buffer containing 20 mM Tris–HCl (pH 7.5), 100 mM KCl, 1 mM DTT, and 10% (v/v) glycerol. After 2 h incubation at 4°C with rotation, the resin was washed three times with 200-μl aliquots of buffer B. Proteins were eluted in 20 μl SDS-electrophoresis buffer and analyzed by Western blotting. To assist statistical significance of the changes in the eIF4E and 4EBP1 binding, the bands of corresponding proteins were scanned and analyzed with ImageQuant TL software.
This study is supported by American Cancer Society (RSG-09-091-01-CSM: JC) and NIH-NCI (R01CA163657-01A1: JC). We thank Dr. Rhoads (LSUHSC-Shreveport) for providing antibodies against eIF4G and eIF4E.
- Hanahan D, Weinberg RA: The hallmarks of cancer. Cell. 2000, 100: 57-70. 10.1016/S0092-8674(00)81683-9.View ArticlePubMedGoogle Scholar
- Graff JR, Zimmer SG: Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs. Clin Exp Metastasis. 2003, 20: 265-273. 10.1023/A:1022943419011.View ArticlePubMedGoogle Scholar
- Sonenberg N, Gingras AC: The mRNA 5’ cap-binding protein eIF4E and control of cell growth. Curr Opin Cell Biol. 1998, 10: 268-275. 10.1016/S0955-0674(98)80150-6.View ArticlePubMedGoogle Scholar
- Goodfellow IG, Roberts LO: Eukaryotic initiation factor 4E. Int J Biochem Cell Biol. 2008, 40: 2675-2680. 10.1016/j.biocel.2007.10.023.PubMed CentralView ArticlePubMedGoogle Scholar
- Raught B, Gingras AC: eIF4E activity is regulated at multiple levels. Int J Biochem Cell Biol. 1999, 31: 43-57. 10.1016/S1357-2725(98)00131-9.View ArticlePubMedGoogle Scholar
- Svitkin YV, Herdy B, Costa-Mattioli M, Gingras A-C, Raught B, Sonenberg N: Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation. Mol Cell Biol. 2005, 25: 10556-10565. 10.1128/MCB.25.23.10556-10565.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang X, Proud CG: The mTOR pathway in the control of protein synthesis. Physiol Bethesda Md. 2006, 21: 362-369. 10.1152/physiol.00024.2006.View ArticleGoogle Scholar
- Averous J, Proud CG: When translation meets transformation: the mTOR story. Oncogene. 2006, 25: 6423-6435. 10.1038/sj.onc.1209887.View ArticlePubMedGoogle Scholar
- De Benedetti A, Graff JR: eIF-4E expression and its role in malignancies and metastases. Oncogene. 2004, 23: 3189-3199. 10.1038/sj.onc.1207545.View ArticlePubMedGoogle Scholar
- Graff JR, Konicek BW, Lynch RL, Dumstorf CA, Dowless MS, McNulty AM, Parsons SH, Brail LH, Colligan BM, Koop JW, Hurst BM, Deddens JA, Neubauer BL, Stancato LF, Carter HW, Douglass LE, Carter JH: eIF4E activation is commonly elevated in advanced human prostate cancers and significantly related to reduced patient survival. Cancer Res. 2009, 69: 3866-3873. 10.1158/0008-5472.CAN-08-3472.View ArticlePubMedGoogle Scholar
- Bianchini A, Loiarro M, Bielli P, Busà R, Paronetto MP, Loreni F, Geremia R, Sette C: Phosphorylation of eIF4E by MNKs supports protein synthesis, cell cycle progression and proliferation in prostate cancer cells. Carcinogenesis. 2008, 29: 2279-2288. 10.1093/carcin/bgn221.View ArticlePubMedGoogle Scholar
- Chung J, Kim TH: Integrin-dependent translational control: Implication in cancer progression. Microsc Res Tech. 2008, 71: 380-386. 10.1002/jemt.20566.View ArticlePubMedGoogle Scholar
- Chung J, Bachelder RE, Lipscomb EA, Shaw LM, Mercurio AM: Integrin (alpha 6 beta 4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells. J Cell Biol. 2002, 158: 165-174. 10.1083/jcb.200112015.PubMed CentralView ArticlePubMedGoogle Scholar
- Dutta U, Shaw LM: A Key tyrosine (Y1494) in the β4 integrin regulates multiple signaling pathways important for tumor development and progression. Cancer Res. 2008, 68: 8779-8787. 10.1158/0008-5472.CAN-08-2125.PubMed CentralView ArticlePubMedGoogle Scholar
- Bertotti A, Comoglio PM, Trusolino L: β4 integrin activates a Shp2–Src signaling pathway that sustains HGF-induced anchorage-independent growth. J Cell Biol. 2006, 175: 993-1003. 10.1083/jcb.200605114.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim LC, Song L, Haura EB: Src kinases as therapeutic targets for cancer. Nat Rev Clin Oncol. 2009, 6: 587-595. 10.1038/nrclinonc.2009.129.View ArticlePubMedGoogle Scholar
- Irby RB, Yeatman TJ: Role of Src expression and activation in human cancer. Oncogene. 2000, 19: 5636-5642. 10.1038/sj.onc.1203912.View ArticlePubMedGoogle Scholar
- Sudhagar S, Sathya S, Lakshmi BS: Rapid non-genomic signalling by 17β-oestradiol through c-Src involves mTOR-dependent expression of HIF-1α in breast cancer cells. Br J Cancer. 2011, 105: 953-960. 10.1038/bjc.2011.349.PubMed CentralView ArticlePubMedGoogle Scholar
- Karni R, Gus Y, Dor Y, Meyuhas O, Levitzki A: Active Src elevates the expression of beta-catenin by enhancement of cap-dependent translation. Mol Cell Biol. 2005, 25: 5031-5039. 10.1128/MCB.25.12.5031-5039.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim TH, Kim HI, Soung YH, Shaw LA, Chung J: Integrin (alpha6beta4) signals through Src to increase expression of S100A4, a metastasis-promoting factor: implications for cancer cell invasion. Mol Cancer Res MCR. 2009, 7: 1605-1612. 10.1158/1541-7786.MCR-09-0102.View ArticlePubMedGoogle Scholar
- Ma XM, Blenis J: Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol. 2009, 10: 307-318. 10.1038/nrm2672.View ArticlePubMedGoogle Scholar
- Dancey J: mTOR signaling and drug development in cancer. Nat Rev Clin Oncol. 2010, 7: 209-219. 10.1038/nrclinonc.2010.21.View ArticlePubMedGoogle Scholar
- Yang X, Dutta U, Shaw LM: SHP2 mediates the localized activation of Fyn downstream of the α6β4 integrin to promote carcinoma invasion. Mol Cell Biol. 2010, 30: 5306-5317. 10.1128/MCB.00326-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Abdel-Ghany M, Cheng H-C, Elble RC, Pauli BU: Focal adhesion kinase activated by beta(4) integrin ligation to mCLCA1 mediates early metastatic growth. J Biol Chem. 2002, 277: 34391-34400. 10.1074/jbc.M205307200.View ArticlePubMedGoogle Scholar
- Wu L, Bernard-Trifilo JA, Lim Y, Lim S-T, Mitra SK, Uryu S, Chen M, Pallen CJ, Cheung N-K, Mikolon D, Mielgo A, Stupack DG, Schlaepfer DD: Distinct FAK-Src activation events promote alpha5beta1 and alpha4beta1 integrin-stimulated neuroblastoma cell motility. Oncogene. 2008, 27: 1439-1448. 10.1038/sj.onc.1210770.PubMed CentralView ArticlePubMedGoogle Scholar
- Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, Huang Q, Qin J, Su B: SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell. 2006, 127: 125-137. 10.1016/j.cell.2006.08.033.View ArticlePubMedGoogle Scholar
- Liu L, Parent CA: Review series: TOR kinase complexes and cell migration. J Cell Biol. 2011, 194: 815-824. 10.1083/jcb.201102090.PubMed CentralView ArticlePubMedGoogle Scholar
- Vojtechová M, Turecková J, Kucerová D, Sloncová E, Vachtenheim J, Tuhácková Z: Regulation of mTORC1 signaling by Src kinase activity is Akt1-independent in RSV-transformed cells. Neoplasia New York N. 2008, 10: 99-107. 10.1593/neo.07905.View ArticleGoogle Scholar
- Haynes MP, Li L, Sinha D, Russell KS, Hisamoto K, Baron R, Collinge M, Sessa WC, Bender JR: Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen. J Biol Chem. 2003, 278: 2118-2123. 10.1074/jbc.M210828200.View ArticlePubMedGoogle Scholar
- Li JD, Feng W, Gallup M, Kim JH, Gum J, Kim Y, Basbaum C: Activation of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells. Proc Natl Acad Sci U S A. 1998, 95: 5718-5723. 10.1073/pnas.95.10.5718.PubMed CentralView ArticlePubMedGoogle Scholar
- Lipscomb EA, Mercurio AM: Mobilization and activation of a signaling competent alpha6beta4integrin underlies its contribution to carcinoma progression. Cancer Metastasis Rev. 2005, 24: 413-423. 10.1007/s10555-005-5133-4.View ArticlePubMedGoogle Scholar
- Shaw LM, Rabinovitz I, Wang HH-F, Toker A, Mercurio AM: Activation of phosphoinositide 3-OH kinase by the α6β4 integrin promotes carcinoma invasion. Cell. 1997, 91: 949-960. 10.1016/S0092-8674(00)80486-9.View ArticlePubMedGoogle Scholar
- Trusolino L, Bertotti A, Comoglio PM: A signaling adapter function for alpha6beta4 integrin in the control of HGF-dependent invasive growth. Cell. 2001, 107: 643-654. 10.1016/S0092-8674(01)00567-0.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.