PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. Assassinating Cancer Cells, or Death by Design by Beverly Barton, Ph.D. I am an assassin. Daily I plan the death and destruction of millions of living entities. It’s either them or me, or you, for that matter. Them are cancer cells. So I design molecules specific for killing cancer cells, targeted to gain entry in cancer cells. To do this, I take advantage of what distinguishes malignant cells from benign cells, and I constantly search the literature for the latest information on cell targeting strategies. What makes a malignant cell malignant? Many things affect the phenotype of cells. To narrow my field of inquiry, I examine particular signaling pathways that are aberrantly activated or dysregulated in malignant cells compared to benign cells. For example, the dysregulation of phosphoinositol 3-kinase (PI 3-kinase) results in aberrant activation of the anti-apoptotic protein Akt. I am particularly interested in the persistent activation of STAT3. In benign cells, STAT3 is transiently phosphorylated (activated) by upstream kinases, but in many types of malignant cells, it is persistently or constitutively phosphorylated. Being a transcription factor, STAT3 regulates gene expression when activated. Among the genes it regulates are the anti-apoptotic members of the bcl-2 family, survivin (an anti-apoptotic gene believed by many to regulate bcl-2 family expression as well), several cell cycle progression genes, the angiogenic factor VEGF, the surface protein CD46 (overexpressed by many types of tumor cells, CD46 helps tumor cells escape complement-mediated cytolysis), and various matrix metalloproteases (implicated in metastasis formation). From this limited list, it’s clear that genes under the control of STAT3 add traits to cells that would be consistent with a malignant phenotype (Bowman, Garcia et al. 2000; Aoki, Feldman et al. 2003; Lewis, Winter et al. 2008). What makes cells hyperactivate STAT3 is not fully understood at present (although some model systems and hypotheses abound; for example over-production of a cytokine such as IL-6, which activates STAT3 as part of its signal transduction pathway, is implicated in multiple myeloma (Klein, Zhang et al. 1990)). What is known is that when cells express persistently-activated STAT3, the cells then become addicted to the activated STAT3, requiring it for survival. If activation is inhibited, or protein expression diminished, or STAT3:DNA binding blocked, the cells begin an apoptotic cascade. Thus, activated STAT3 is a proto-oncogene (Bromberg, Wrzeszczynska et al. 1999). Exploiting this addiction to STAT3 by cancer cells is the major anti-cancer strategy I’m using to develop new therapeutic agents for prostate and pancreatic cancer. Strategies for inhibiting STAT3 So we have choices for what we might do to inhibit STAT3. I’ve tried all three strategies, and have been concentrating on the last one, designing molecules that block STAT3:DNA binding. A double-stranded oligonucleotide having the same sequence as a known STAT3 binding site will bind to STAT3 molecules. This type of inhibitor is known as a decoy (Jing and Tweardy 2005; Gao, Xiao et al. 2006). Decoys can be effective; there is literature indicating that decoys cause tumor regression when used to treat head and neck squamous cell carcinoma in a mouse model (Leong, Andrews et al. 2003). I’ve found that even single-stranded oligonucleotides bearing the STAT3 binding sequence can induce apoptosis in prostate and pancreatic cancer cell lines (Barton, Murphy et al. 2004; Lewis, Winter et al. 2008). A major drawback to using oligonucleotides as therapeutic entities, however, is their relatively poor bioavailability. Although I can transfect oligonucleotides at high efficiency in the laboratory, I can’t use a reagent in a clinical setting to help push an oligonucleotide across a cell membrane. Therefore, I’ve become interested in modifying conventional oligonucleotides in two ways: 1) making synthetic analogues lacking the phosphodiester backbone yet still specific for blocking STAT3:DNA binding; 2) adding cell-specific targeting peptides to the synthetic oligonucleotides, to eliminate the need for transfection reagent. To generate oligonucleotides that block STAT3:DNA binding, I searched the literature for published sequences of STAT3 binding sites. The first sequences I used were hybrid sequences, in that the oligonucleotides were used for competition in electrophoretic mobility shift assays across various species (human, mouse, and rat). Although transfecting these sequences into prostate cancer cell lines resulted in marked apoptosis (>50% at 48 hours; (Barton, Murphy et al. 2004)) I wanted to improve the sequences and the therapeutic efficacy. So I decided to synthesize oligonucleotides having authentic human STAT3 sequences. I found four sequences in the literature. Two sequences had little to no activity, relative to scrambled-sequence control oligonucleotides. As for the remaining two oligonucleotides, one had nearly as much activity as the original and the other was superior to the original (Lewis, Winter et al. 2008). I conclude that my strategy for creating transcription factor inhibitors is a viable one. Although I myself work only on STAT3, I expect that other groups working on other transcription factors will make prototype inhibitors and publish their findings in the future. Synthetic oligonucleotide analogues These molecules-peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids-although different in the specifics, share several important features (Egholm, Buchardt et al. 1992; Aartsma-Rus, Kaman et al. 2004). They all lack the phosphodiester backbone found in DNA; thus there no longer is a charge barrier to overcome in order for these molecules to enter cells. Another consequence of their chemistry is that they form triple helices with double-stranded DNA. In fact, their affinity for double-stranded DNA is greater than the affinity of protein for DNA. Thus in theory they can kick off bound STAT3 from the genome, situating themselves instead on STAT3 binding sites, and remain seated at least until apoptosis is complete. By adding cell targeting peptides or ligand molecules such as dihydrotesosterone, these anti-STAT3 nucleic acid analogues become in essence “magic bullets,” accumulating preferentially in cancer cells and not in other cells (Boffa, Scafi et al. 2000; Bendifallah, Rasmussen et al. 2006). A cell-targeting strategy not only would in theory reduce adverse side-effects, it would help bring costs down as less of an expensive therapeutic agent would be needed to effect a cure. Prototype molecules are being examined for use in the treatment of HIV (Tripathi, Chaubey et al. 2007), for imaging (Tian, Aruva et al. 2005; Tian, Chakrabarti et al. 2005), and soon hopefully for cancer (Chen, Koeneman et al. 2003). My laboratory will be switching to PNAs from oligonucleotides after we finish up studies designed to help us choose the best rationally-designed cell-penetrating peptide for targeting to the cancers of interest. It’ll be fun to report on those findings when they become available. References Cited Aartsma-Rus, A., W. E. Kaman, et al. (2004). "Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells." Gene Ther 11(18): 1391-8. Aoki, Y., G. M. Feldman, et al. (2003). "Inhibition of STAT3 signaling induces apoptosis and decreases survivin expression in primary effusion lymphoma." Blood 101(4): 1535-1542. Barton, B. E., T. F. Murphy, et al. (2004). "Novel Single-Stranded Oligonucleotides that Inhibit STAT3 Induce Apoptosis In Vitro and In Vivo in Prostate Cancer Cell Lines." Mol Cancer Ther 3(10): 1183-1191. Bendifallah, N., F. W. Rasmussen, et al. (2006). "Evaluation of Cell-Penetrating Peptides (CPPs) as Vehicles for Intracellular Delivery of Antisense Peptide Nucleic Acid (PNA)." Bioconjugate Chem. 17(3): 750-758. Boffa, L. C., S. Scafi, et al. (2000). "Dihydrotestosterone as selective cellular /nuclear localization vector for anti-peptide nucleic acid in prostatic carcinoma cells." Cancer Res. 60: 2258-2262. Bowman, T., R. Garcia, et al. (2000). "STATs in oncogenesis." Oncogene 19: 2474-2488. Bromberg, J. F., M. H. Wrzeszczynska, et al. (1999). "Stat3 as an oncogene." Cell 98(3): 295-33. Chen, Z., K. S. Koeneman, et al. (2003). "Consequences of telomerase inhibition and combination treatments for the proliferation of cancer cells." Cancer Res 63(18): 5917-25. Egholm, M., O. Buchardt, et al. (1992). "Peptide nucleic acids (PNA). Oligonucleotide analogues with achiral peptide backbone." J. Am. Chem. Soc. 114: 1895-1897. Gao, H., J. Xiao, et al. (2006). "A single decoy oligodeoxynucleotides targeting multiple oncoproteins produces strong anticancer effects." Mol Pharmacol 70(5): 1621-9. Jing, N. and D. J. Tweardy (2005). "Targeting Stat3 in cancer therapy." Anticancer Drugs 16(6): 601-7. Klein, B., X. G. Zhang, et al. (1990). "Interleukin-6 is the central tumor growth factor in vitro and in vivo in multiple myeloma." Eur. Cytokine Net. 1: 193-. Leong, P. L., G. A. Andrews, et al. (2003). "Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth." Proc. Natl. Acad. Sci. USA 100(7): 4138-4143. Lewis, H. D., A. Winter, et al. (2008). "STAT3 Inhibition in Prostate and Pancreatic Cancer Lines by STAT3 Binding Sequence Oligonucleotides: Differential Activity Between 5’ and 3’ Ends." Mol Cancer Therapeutics In Press. Tian, X., M. R. Aruva, et al. (2005). "Tumor-targeting peptide-PNA-peptide chimeras for imaging overexpressed oncogene mRNAs." Nucleosides Nucleotides Nucleic Acids 24(5-7): 1085-91. Tian, X., A. Chakrabarti, et al. (2005). "External Imaging of CCND1, MYC, and KRAS Oncogene mRNAs with Tumor-Targeted Radionuclide-PNA-Peptide Chimeras." Ann N Y Acad Sci 1059: 106-44. Tripathi, S., B. Chaubey, et al. (2007). "Anti HIV-1 virucidal activity of polyamide nucleic acid-membrane transducing peptide conjugates targeted to primer binding site of HIV-1 genome." 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