3 Results
4.2.5 Conclusions and outlook
In their function as regulators of gene expression, miRNAs have emerged as promising tools as novel biomarkers and for therapeutic application. In this thesis, we focused especially on the role of miRNAs in two processes that are of major interest to breast cancer research: the ErbB2/ErbB3/Akt signaling pathway contributing to therapy resistance, and cell motility being of central importance for cancer metastasis.
With regard to the ErbB2/ErbB3/Akt signaling pathway, we revealed that the selected subset of screen hit miRNAs may exert their regulatory function by coordinated suppression of mul-tiple proteins within the pathway, including the key molecule ErbB3. Loss of ErbB3 was very likely the major cause for the observed decrease in pathway activity and the reduced HRG-dependent proliferation. In the future, further experiments will be necessary to validate direct targeting or to reveal the mechanism by which downregulation of the proteins is achieved.
Furthermore, the specific inhibition of the endogenous miRNA by LNA-modified antisense oligonucleotides should revert the phenotype, confirming the physiological importance of the-se miRNAs in HRG signaling. Moreover, to validate the tumor-suppressive function of our miRNA subset, clinical datasets should be analyzed to prove clinical significance and finally experiments should be performed in the appropriate cancer specific context. Regarding miR-149 function, we have already been able to address some of these aspects in the second part of this thesis. Based on clinical datasets revealing reduced miR-149 levels in basal-like breast cancer, we investigated miR-149 function in different basal-like breast cancer cell lines. We were able to show very clearly that miR-149 possesses anti-metastatic function in vitro and in vivo. We also addressed the underlying mechanisms, providing evidence that miR-149 interfered with signaling downstream of integrin receptors and RTKs at multiple lev-els by impairing Rac activation. Moreover, we shed light on the fact that Rac activity was re-duced by the downregulation of several Rac regulatory proteins, including Rap1A and Rap1B, and Vav2. Taken together, our present study, in line with other publications, sug-gests that miR-149 is a potential candidate for prognostic and therapeutic application. How-ever, one obstacle is still the appropriate application of miRNA into the human body. Not only are more and more miRNAs now being integrated into clinical trials as biomarkers for prog-nosis and clinical response, they are also promising as anti-cancer therapeutics. For exam-ple, MRX34, an intravenously injected liposome-formulated miR-34 mimic, has recently en-tered clinical trials for patients with advanced or metastatic liver cancer (Bader, 2012). The increased understanding of miRNA biology and function together with improved targeted de-livery systems will hopefully also promote the development of miRNA-based agents for the treatment of basal breast cancer.
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List of Figures
Figure 1: Cancer development ...18 Figure 2: ErbB receptor family. ...19 Figure 3: Signaling downstream of the ErbB2-ErbB3 dimer ...21 Figure 4: PI3K/Akt signaling pathway ...23 Figure 5: Biogenesis of miRNAs ...28 Figure 6: miRNA-mRNA interaction ...50 Figure 8: miR-149 is predicted to bind ErbB3 ...51 Figure 9: Expression of miR-149-5p affects Akt activation by directly targeting ErbB3...53 Figure 11: Workflow of the screening procedure ...54 Figure 12: Correlation analysis. ...55 Figure 13: MicroRNA screen data ...56 Figure 14: Bioinformatical analysis of the screen data ...56 Figure 15: Analysis of the screen data ...57 Figure 16: miRNAs-target gene interaction network ...60 Figure 18: miRNA-protein interaction network for negative regulators of HRG signaling ...61 Figure 19: miR-148b, miR-149, miR-326 and miR-520a-3p target key molecules of the pathway ...62 Figure 20: miR-148b, miR-326 and miR-520a-3p affect the ErbB2-ErbB3 signaling pathway on multiple levels ...63 Figure 21: miR-148b, miR326 and miR-520a-3p affect HRG-dependent cell growth ...64 Figure 22: Reduced expression of miR-149 in basal breast cancer. ...65 Figure 23: Reduced expression of miR-149 in basal breast cancer cells ...66 Figure 24: Expression of miR-149 reduces cell migration and invasion in MDA-MB-231. ...66 Figure 25: Expression of miR-149 impairs the directionality of cell movement ...67 Figure 26: miR-149 interferes with cell spreading on collagen ...68 Figure 27: miR-149 affects polarization of actin cytoskeleton during cell spreading on collagen. ...69 Figure 28: miR-149 interferes with cell spreading on collagen ...69 Figure 29: miR-149 targets molecules downstream of the integrin pathway ...70 Figure 30: mir-149 targets molecules downstream of the integrin pathway ...71 Figure 31: miR-149 impairs Rac activation MDA-MB-231 ...72 Figure 32: Rac inhibition blocks cell migration and active Rac restores spreading of miR-149 expressing cells ...73 Figure 33: miR-149 impairs with integrin-dependent signaling and cell motility in the prostate cancer cell line PC3 ...74 Figure 34: miR-149 blocks lung colonization in vivo. ...74
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List of Tables
Table 1: List of commonly used target prediction algorithms. ...30 Table 2: List of equipment used in this thesis ...35 Table 3: List of chemicals used in this thesis ...36 Table 4: List of consumables used in this thesis ...37 Table 5: List of buffers and solutions used in this thesis ...37 Table 6: List of cell lines used in this thesis ...38 Table 7: List of cell culture reagents used in this thesis ...38 Table 8: List of miRNAs used in this thesis. ...39 Table 9: List of siRNAs used in this thesis ...39 Table 10: List of SMARTpools used in this thesis ...39 Table 11: List of primers used in this thesis ...40 Table 12: List of qPCR primers used in this thesis ...40 Table 13: List of qPCR primers used in this thesis ...40 Table 14: List of plasmid vectors used in this thesis ...40 Table 15: List of fluorescence labeled primary antibodies used in this thesis ...41 Table 16: List of primary antibodies used in this thesis ...41 Table 17: List of secondary antibodies used in this thesis ...42 Table 18: List of kits and enzymes used in this thesis...42 Table 19: Reagents for PCR ...44 Table 20: program for the thermal cycler ...44 Table 21: Program used for reverse transcription and qPCR. ...47 Table 22: Program used for RT-qPCR. ...47 Table 23: List of predicted miR-149 target genes according to their KEGG pathway annotation. ...70 Table 24: Detailed list of the Dharmacon miRIDIAN® microRNA Library. ... 107 Table 25: Screen data results. ... 112 Table 26: miRNA target list predicted by microRNA.org. ... 124 Table 27: miRNA target list predicted by MicroCosm. ... 127 Table 28: miRNA target list predicted by miRDB. ... 129
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5 List of References
Alemayehu, M., Dragan, M., Pape, C., Siddiqui, I., and Sacks, D.B., et al. (2013). β-Arrestin2 regulates lysophosphatidic acid-induced human breast tumor cell migration and invasion via Rap1 and IQGAP1. PLoS ONE 8, e56174.
Ambros, V., Bartel, B., Bartel, D.P., Burge, C.B., and Carrington, J.C., et al. (2003). A uni-form system for microRNA annotation. RNA 9, 277-279.
Arcaroli, J.J., Quackenbush, K.S., Powell, R.W., Pitts, T.M., and Spreafico, A., et al. (2012).
Common PIK3CA mutants and a novel 3' UTR mutation are associated with increased sensitivity to saracatinib. Clin. Cancer Res. 18, 2704-2714.
Arthur, W.T., Quilliam, L.A., and Cooper, J.A. (2004). Rap1 promotes cell spreading by local-izing Rac guanine nucleotide exchange factors. J Cell Biol 167, 111-122.
Atlas, E., Cardillo, M., Mehmi, I., Zahedkargaran, H., and Tang, C., et al. (2003). Heregulin is sufficient for the promotion of tumorigenicity and metastasis of breast cancer cells in vivo.
Mol. Cancer Res. 1, 165-175.
Bader, A.G. (2012). miR-34 - a microRNA replacement therapy is headed to the clinic. Front Genet 3, 120.
Bader AG, Lammers P (2011). The therapeutic potential of microRNAs. Innovations in Phar-maceutical Technology., 52-55.
Bartel, D.P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233.
Baselga, J., and Swain, S.M. (2009). Novel anticancer targets: revisiting ERBB2 and discov-ering ERBB3. Nat. Rev. Cancer 9, 463-475.
Basu, A., and Sivaprasad, U. (2007). Protein kinase Cepsilon makes the life and death deci-sion. Cell. Signal. 19, 1633-1642.
Bellis, S.L., Perrotta, J.A., Curtis, M.S., and Turner, C.E. (1997). Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin. Biochem. J. 325 ( Pt 2), 375-381.
Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series B 57, 289-300.
Berezikov, E. (2011). Evolution of microRNA diversity and regulation in animals. Nat. Rev.
Genet. 12, 846-860.
Berger, M.B., Medrola, J.M., and Lemmon, M.A. (2004). ErbB3/HER3 does not homodimerize upon neuregulin binding at the cell surface. FEBS Letters 569, 332-336.
Bernstein, E., Kim, S.Y., Carmell, M.A., Murchison, E.P., and Alcorn, H., et al. (2003). Dicer is essential for mouse development. Nat. Genet. 35, 215-217.
Bertucci, M.C., and Mitchell, C.A. (2013). Phosphoinositide 3-kinase and INPP4B in human breast cancer. Ann. N. Y. Acad. Sci. 1280, 1-5.
94
Betel, D., Wilson, M., Gabow, A., Marks, D.S., and Sander, C. (2008). The microRNA.org resource: targets and expression. Nucleic Acids Res. 36, D149-53.
Bill, A., Schmitz, A., Albertoni, B., Song, J.-N., and Heukamp, L.C., et al. (2010). Cytohesins are cytoplasmic ErbB receptor activators. Cell 143, 201-211.
Blenkiron, C., Goldstein, L.D., Thorne, N.P., Spiteri, I., and Chin, S.-F., et al. (2007).
MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol. 8, R214.
Bos, P.D., Nguyen, D.X., and Massagué, J. (2010). Modeling metastasis in the mouse. Curr Opin Pharmacol 10, 571-577.
Breuleux, M. (2007). Role of heregulin in human cancer. Cell. Mol. Life Sci. 64, 2358-2377.
Burgess, A.W., Cho, H.-S., Eigenbrot, C., Ferguson, K.M., and Garrett, Thomas P J, et al.
(2003). An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors.
Mol. Cell 12, 541-552.
Bustelo, X.R., Sauzeau, V., and Berenjeno, I.M. (2007). GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo. Bioessays 29, 356-370.
Cai, J., Fang, L., Huang, Y., Li, R., and Yuan, J., et al. (2013). miR-205 targets PTEN and PHLPP2 to augment AKT signaling and drive malignant phenotypes in non-small cell lung cancer. Cancer Res. 73, 5402-5415.
Calin, G.A., Dumitru, C.D., Shimizu, M., Bichi, R., and Zupo, S., et al. (2002). Frequent dele-tions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. U.S.A. 99, 15524-15529.
Campbell, M.R., Amin, D., and Moasser, M.M. (2010). HER3 comes of age: new insights into its functions and role in signaling, tumor biology, and cancer therapy. Clin. Cancer Res. 16, 1373-1383.
Cantley, L.C. (2002). The phosphoinositide 3-kinase pathway. Science 296, 1655-1657.
Cantley, L.C., and Neel, B.G. (1999). New insights into tumor suppression: PTEN suppress-es tumor formation by rsuppress-estraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl.
Acad. Sci. U.S.A. 96, 4240-4245.
Cargnello, M., and Roux, P.P. (2011). Activation and function of the MAPKs and their sub-strates, the MAPK-activated protein kinases. Microbiol. Mol. Biol. Rev. 75, 50-83.
Carpten, J.D., Faber, A.L., Horn, C., Donoho, G.P., and Briggs, S.L., et al. (2007). A trans-forming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 448, 439-444.
Chan, S.-H., Huang, W.-C., Chang, J.-W., Chang, K.-J., and Kuo, W.-H., et al. (2014).
MicroRNA-149 targets GIT1 to suppress integrin signaling and breast cancer metastasis.
Oncogene 0. e10.1038/onc.2014.10
Chen, J.-F., Mandel, E.M., Thomson, J.M., Wu, Q., and Callis, T.E., et al. (2006). The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat.
Genet. 38, 228-233.
95
Chen, W.S., Xu, P.Z., Gottlob, K., Chen, M.L., and Sokol, K., et al. (2001). Growth retarda-tion and increased apoptosis in mice with homozygous disrupretarda-tion of the Akt1 gene. Genes Dev. 15, 2203-2208.
Cho, H., Mu, J., Kim, J.K., Thorvaldsen, J.L., and Chu, Q., et al. (2001). Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta).
Science 292, 1728-1731.
Cimino, D., Pitta, C. de, Orso, F., Zampini, M., and Casara, S., et al. (2013). miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signa-ture by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1. The FASEB Journal 27, 1223-1235.
Cimmino, A., Calin, G.A., Fabbri, M., Iorio, M.V., and Ferracin, M., et al. (2005). miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc. Natl. Acad. Sci. U.S.A. 102, 13944-13949.
Citterio, C., Menacho-Márquez, M., García-Escudero, R., Larive, R.M., and Barreiro, O., et al. (2012). The rho exchange factors vav2 and vav3 control a lung metastasis-specific transcriptional program in breast cancer cells. Sci Signal 5, ra71.
Collado, M., Blasco, M.A., and Serrano, M. (2007). Cellular senescence in cancer and aging.
Cell 130, 223-233.
Cottonham, C.L., Kaneko, S., and Xu, L. (2010). miR-21 and miR-31 converge on TIAM1 to regulate migration and invasion of colon carcinoma cells. J. Biol. Chem. 285, 35293-35302.
Croft, D., O'Kelly, G., Wu, G., Haw, R., and Gillespie, M., et al. (2011). Reactome: a data-base of reactions, pathways and biological processes. Nucleic Acids Res. 39, D691-7.
Dangi-Garimella, S., Yun, J., Eves, E.M., Newman, M., and Erkeland, S.J., et al. (2009). Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7. EMBO J. 28, 347-358.
DiGiovanna, M.P., Chu, P., Davison, T.L., Howe, C.L., and Carter, D., et al. (2002). Active signaling by HER-2/neu in a subpopulation of HER-2/neu-overexpressing ductal carcinoma in situ: clinicopathological correlates. Cancer Res. 62, 6667-6673.
Djebali, S., Davis, C.A., Merkel, A., Dobin, A., and Lassmann, T., et al. (2012). Landscape of transcription in human cells. Nature 489, 101-108.
Downward, J. (2003). Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Can-cer 3, 11-22.
Duffy, M.J., McKiernan, E., O'Donovan, N., and McGowan, P.M. (2009). Role of ADAMs in cancer formation and progression. Clin. Cancer Res. 15, 1140-1144.
Dummler, B., Tschopp, O., Hynx, D., Yang, Z.-Z., and Dirnhofer, S., et al. (2006). Life with a single isoform of Akt: mice lacking Akt2 and Akt3 are viable but display impaired glucose homeostasis and growth deficiencies. Mol. Cell. Biol. 26, 8042-8051.
Duronio, V. (2008). The life of a cell: apoptosis regulation by the PI3K/PKB pathway.
Biochem. J. 415, 333-344.
96
Easton, R.M., Cho, H., Roovers, K., Shineman, D.W., and Mizrahi, M., et al. (2005). Role for Akt3/protein kinase Bgamma in attainment of normal brain size. Mol. Cell. Biol. 25, 1869-1878.
Eleniste, P.P., and Bruzzaniti, A. (2012). Focal adhesion kinases in adhesion structures and disease. J Signal Transduct 2012, 296450.
Elkabets, M., Vora, S., Juric, D., Morse, N., and Mino-Kenudson, M., et al. (2013). mTORC1 inhibition is required for sensitivity to PI3K p110α inhibitors in PIK3CA-mutant breast can-cer. Sci Transl Med 5, 196ra99.
Enerly, E., Steinfeld, I., Kleivi, K., Leivonen, S.-K., and Aure, M.R., et al. (2011). miRNA-mRNA integrated analysis reveals roles for miRNAs in primary breast tumors. PLoS ONE 6, e16915.
Engelman, J.A., Luo, J., and Cantley, L.C. (2006). The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat. Rev. Genet. 7, 606-619.
Engelman, J.A., Zejnullahu, K., Mitsudomi, T., Song, Y., and Hyland, C., et al. (2007). MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.
Science 316, 1039-1043.
Esteller, M. (2011). Non-coding RNAs in human disease. Nat. Rev. Genet. 12, 861-874.
Fantozzi, A., and Christofori, G. (2006). Mouse models of breast cancer metastasis. Breast Cancer Res. 8, 212.
Farazi, T.A., Hoell, J.I., Morozov, P., and Tuschl, T. (2013). MicroRNAs in human cancer.
Adv. Exp. Med. Biol. 774, 1-20.
Fedele, C.G., Ooms, L.M., Ho, M., Vieusseux, J., and O'Toole, S.A., et al. (2010). Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers. Proc. Natl. Acad. Sci. U.S.A. 107, 22231-22236.
Fiddes, R.J., Campbell, D.H., Janes, P.W., Sivertsen, S.P., and Sasaki, H., et al. (1998).
Analysis of Grb7 recruitment by heregulin-activated erbB receptors reveals a novel target selectivity for erbB3. J. Biol. Chem. 273, 7717-7724.
Fornari, F., Milazzo, M., Galassi, M., Callegari, E., and Veronese, A., et al. (2014).
p53/mdm2 Feedback Loop Sustains miR-221 Expression and Dictates the Response to Anticancer Treatments in Hepatocellular Carcinoma. Mol. Cancer Res.
Fortier, A.-M., Asselin, E., and Cadrin, M. (2011). Functional specificity of Akt isoforms in cancer progression. BioMolecular Concepts 2, 1-11.
Franceschini, A., Szklarczyk, D., Frankild, S., Kuhn, M., and Simonovic, M., et al. (2013).
STRING v9.1: protein-protein interaction networks, with increased coverage and integra-tion. Nucleic Acids Res. 41, D808-15.
Frische, E.W., and Zwartkruis, F J T (2010). Rap1, a mercenary among the Ras-like GTPases. Dev. Biol. 340, 1-9.
Fry, W.H., Kotelawala, L., Sweeney, C., and Carraway, K.L. (2009). Mechanisms of ErbB receptor negative regulation and relevance in cancer. Experimental Cell Research 315, 697-706.
97
Gao, Y., Dickerson, J.B., Guo, F., Zheng, J., and Zheng, Y. (2004). Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A 101, 7618-7623.
Geiger, B., Spatz, J.P., and Bershadsky, A.D. (2009). Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21-33.
Gijsen, M., King, P., Perera, T., Parker, P.J., and Harris, A.L., et al. (2010). HER2 Phosphor-ylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Her-ceptin in Breast Cancer. PLoS Biol 8, e1000563.
Gong, C., Yao, Y., Wang, Y., Liu, B., and Wu, W., et al. (2011). Up-regulation of miR-21 me-diates resistance to trastuzumab therapy for breast cancer. J. Biol. Chem. 286, 19127-19137.
Gotoh, N. (2008). Regulation of growth factor signaling by FRS2 family docking/scaffold adaptor proteins. Cancer Sci. 99, 1319-1325.
Griffiths-Jones, S., Saini, H.K., van Dongen, S., and Enright, A.J. (2008). miRBase: tools for microRNA genomics. Nucleic Acids Res. 36, D154-8.
Grimson, A., Farh, K.K.-H., Johnston, W.K., Garrett-Engele, P., and Lim, L.P., et al. (2007).
MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91-105.
Guertin, D.A., Stevens, D.M., Thoreen, C.C., Burds, A.A., and Kalaany, N.Y., et al. (2006).
Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev. Cell 11, 859-871.
Harbinski, F., Craig, V.J., Sanghavi, S., Jeffery, D., and Liu, L., et al. (2012). Rescue screens with secreted proteins reveal compensatory potential of receptor tyrosine kinases in driving cancer growth. Cancer Discov 2, 948-959.
Hatley, M.E., Patrick, D.M., Garcia, M.R., Richardson, J.A., and Bassel-Duby, R., et al.
(2010). Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21. Cancer Cell 18, 282-293.
He, L., and Hannon, G.J. (2004). MicroRNAs: small RNAs with a big role in gene regulation.
Nature reviews. Genetics 5, 522-531.
Hennessy, B.T., Smith, D.L., Ram, P.T., Lu, Y., and Mills, G.B. (2005). Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 4, 988-1004.
Hinohara, K., Kobayashi, S., Kanauchi, H., Shimizu, S., and Nishioka, K., et al. (2012). ErbB receptor tyrosine kinase/NF-κB signaling controls mammosphere formation in human breast cancer. Proc. Natl. Acad. Sci. U.S.A. 109, 6584-6589.
Holbro, T., Beerli, R.R., Maurer, F., Koziczak, M., and Barbas, C.F., et al. (2003). The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 100, 8933-8938.
Hornstein, I., Alcover, A., and Katzav, S. (2004). Vav proteins, masters of the world of cyto-skeleton organization. Cell. Signal. 16, 1-11.
98
Huang, Q., Gumireddy, K., Schrier, M., Le Sage, C., and Nagel, R., et al. (2008). The mi-croRNAs miR-373 and miR-520c promote tumour invasion and metastasis. Nat. Cell Biol.
10, 202-210.
Humphries, J.D., Wang, P., Streuli, C., Geiger, B., and Humphries, M.J., et al. (2007).
Vinculin controls focal adhesion formation by direct interactions with talin and actin. J. Cell Biol. 179, 1043-1057.
Hynes, N.E., and Lane, H.A. (2005). ERBB receptors and cancer: the complexity of targeted inhibitors. Nat. Rev. Cancer 5, 341-354.
Ikenoue, T., Kanai, F., Hikiba, Y., Obata, T., and Tanaka, Y., et al. (2005). Functional analy-sis of PIK3CA gene mutations in human colorectal cancer. Cancer Res. 65, 4562-4567.
Iliopoulos, D., Polytarchou, C., Hatziapostolou, M., Kottakis, F., and Maroulakou, I.G., et al.
(2009). MicroRNAs differentially regulated by Akt isoforms control EMT and stem cell re-newal in cancer cells. Sci Signal 2, ra62.
Iorio, M.V., Ferracin, M., Liu, C.-G., Veronese, A., and Spizzo, R., et al. (2005). MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 65, 7065-7070.
Jefferson, A.B., and Majerus, P.W. (1995). Properties of type II inositol polyphosphate 5-phosphatase. J. Biol. Chem. 270, 9370-9377.
Jin, Y., Tymen, S.D., Chen, D., Fang, Z.J., and Zhao, Y., et al. (2013). MicroRNA-99 Family Targets AKT/mTOR Signaling Pathway in Dermal Wound Healing. PLoS ONE 8, e64434.
Ju, X., Katiyar, S., Wang, C., Liu, M., and Jiao, X., et al. (2007). Akt1 governs breast cancer progression in vivo. Proc. Natl. Acad. Sci. U.S.A. 104, 7438-7443.
Kadamur, G., and Ross, E.M. (2013). Mammalian phospholipase C. Annu. Rev. Physiol. 75, 127-154.
Kanehisa, M., Goto, S., Sato, Y., Furumichi, M., and Tanabe, M. (2011). KEGG for integra-tion and interpretaintegra-tion of large-scale molecular data sets. Nucleic Acids Research 40, D109.
Kastan, M.B., and Bartek, J. (2004). Cell-cycle checkpoints and cancer. Nature 432, 316-323.
Kawano, S., Ikeda, W., Kishimoto, M., Ogita, H., and Takai, Y. (2009). Silencing of ErbB3/ErbB2 signaling by immunoglobulin-like Necl-2. J. Biol. Chem. 284, 23793-23805.
Ke, Y., Zhao, W., Xiong, J., and Cao, R. (2013). miR-149 Inhibits Non-Small-Cell Lung Can-cer Cells EMT by Targeting FOXM1. Biochem Res Int 2013, 506731.
Kefas, B., Godlewski, J., Comeau, L., Li, Y., and Abounader, R., et al. (2008). microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. Cancer Res. 68, 3566-3572.
Keklikoglou, I., Koerner, C., Schmidt, C., Zhang, J.D., and Heckmann, D., et al. (2012).
MicroRNA-520/373 family functions as a tumor suppressor in estrogen receptor negative breast cancer by targeting NF-κB and TGF-β signaling pathways. Oncogene 31, 4150-4163.
99
Kim, J., Krichevsky, A., Grad, Y., Hayes, G.D., and Kosik, K.S., et al. (2004). Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proc. Natl.
Acad. Sci. U.S.A. 101, 360-365.
Klein, U., Lia, M., Crespo, M., Siegel, R., and Shen, Q., et al. (2010). The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell 17, 28-40.
Koressaar, T., and Remm, M. (2007). Enhancements and modifications of primer design program Primer3. Bioinformatics 23, 1289-1291.
Kramer, F., Bayerlova, M., Klemm, F., Bleckmann, A., and Beissbarth, T. (2013).
rBiopaxParser--an R package to parse, modify and visualize BioPAX data. Bioinformatics 29, 520-522.
Laketa, V., Zarbakhsh, S., Traynor-Kaplan, A., Macnamara, A., and Subramanian, D., et al.
(2014). PIP₃ induces the recycling of receptor tyrosine kinases. Sci Signal 7, ra5.
Le Clainche, C., and Carlier, M.-F. (2008). Regulation of actin assembly associated with pro-trusion and adhesion in cell migration. Physiol. Rev. 88, 489-513.
Lee, R.C., Feinbaum, R.L., and Ambros, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843-854.
Lee, S.-L., Chou, C.-C., Chuang, H.-C., Hsu, E.-C., and Chiu, P.-C., et al. (2013). Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines. PLoS ONE 8, e67149.
Leivonen, S.-K., Sahlberg, K.K., Mäkelä, R., Due, E.U., and Kallioniemi, O., et al. (2014).
High-throughput screens identify microRNAs essential for HER2 positive breast cancer cell growth. Mol Oncol 8, 93-104.
Liang, J., Zubovitz, J., Petrocelli, T., Kotchetkov, R., and Connor, M.K., et al. (2002). PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest.
Nat. Med. 8, 1153-1160.
Liang, Z., Li, Y., Huang, K., Wagar, N., and Shim, H. (2011). Regulation of miR-19 to Breast Cancer Chemoresistance Through Targeting PTEN. Pharm Res 28, 3091-3100.
Liang, Z., Wu, H., Xia, J., Li, Y., and Zhang, Y., et al. (2010). Involvement of miR-326 in chemotherapy resistance of breast cancer through modulating expression of multidrug re-sistance-associated protein 1. Biochem. Pharmacol. 79, 817-824.
Liao, W.-T., Li, T.-T., Wang, Z.-G., Wang, S.-Y., and He, M.-R., et al. (2013). microRNA-224 promotes cell proliferation and tumor growth in human colorectal cancer by repressing PHLPP1 and PHLPP2. Clin. Cancer Res. 19, 4662-4672.
Ling, H., Fabbri, M., and Calin, G.A. (2013). MicroRNAs and other non-coding RNAs as tar-gets for anticancer drug development. Nat Rev Drug Discov 12, 847-865.
Lopez, S.M., Hodgson, M.C., Packianathan, C., Bingol-Ozakpinar, O., and Uras, F., et al.
(2013). Determinants of the tumor suppressor INPP4B protein and lipid phosphatase activ-ities. Biochem. Biophys. Res. Commun. 440, 277-282.