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Dario C. Altieri, M.D.Academic Role: Professor
Faculty Appointment(s) In:
Other Affiliation(s):
Interface between cell survival and cell proliferation in cancer.
In particular, our laboratory studies a gene family called Inhibitors of Apoptosis (IAP). These IAP molecules have been implicated in two distinct cellular functions: preservation of mitotic transition as demonstrated in lower organisms and direct inhibition of caspases -the effector molecules of apoptosis- in mammalian cells. At least one of the mammalian members of the IAP gene family, survivin, retains the ability to participate in both functions, and has been shown by our laboratory and unanimously independently confirmed in the literature to be over-expressed in virtually every human cancer. First cloned in our laboratory, survivin is the only IAP gene that is cell cycle-regulated and prominently expressed at mitosis in a transcriptionally-controlled pathway. Upon expression in dividing cells, survivin is rapidly recruited to various aspects of the mitotic apparatus, including centrosomes, also called microtubule organizing centers, microtubules of the metaphase and anaphase spindles and midbodies at telophase. A separate nuclear pool of survivin has also been shown to localize to kinetochores of metaphase chromosomes and potentially involved in modulating chromosome dynamics and proper segregation of sister chromatids at cell division (Figure 1). Our laboratory has extensively used cell biological approaches to probe the function of survivin at cell division. For instance, interference with survivin function using molecular antagonists, including antisense, dominant negative mutants and more recently gene silencing by RNA interference unraveled multiple roles of survivin at cell division, ranging from preservation of centrosome numbers, to spindle microtubule assembly to faithful segregation of sister chromatids (Figure 2). As an example, microinjection of an antibody to survivin and analysis by time-lapse videomicroscopy, revealed that antibody interference with survivin resulted in a prolonged metaphase arrest of cervical carcinoma HeLa cells, frequently associated with onset of apoptosis. By immunofluorescence, cells injected with the antibody to survivin exhibited shortened and flattened mitotic spindles severely depleted of microtubules whereas control injected cells had bipolar spindles with a normal complement of microtubules. These data suggest that at least one of the subcellular pools of survivin is required for the assembly of a competent bipolar mitotic spindle and the proper stabilization of spindle microtubules (Figure 3). In addition to a critical role in cell division, which is also highlighted by the dramatic early embryonic lethality of survivin knockout mice, it is also clear that survivin has a function in protecting cells from apoptosis and that this pathway is exploited in nearly every human tumor. Our laboratory has used both biochemical and genetic approaches to study the role of survivin in apoptosis inhibition. Transgenic animals in which expression of survivin is directed to the skin in a tissue-specific manner exhibit strong resistance to apoptosis induced by ultraviolet B irradiation and these animals are more prone to develop aggressive skin cancers in a mouse model of chemical-induced skin carcinogenesis. Conversely, interference with survivin expression or function in tumor cells is sufficient to trigger apoptosis, to enhance the efficacy of conventional anti-tumor treatment and to exert potent anti-tumor activity in vivo. This has been shown by our laboratory and independently by several other groups using various models of human cancer in immunoincompetent mice following treatment with antisense or dominant negative survivin mutants (Figure 4). A third line of investigation in our laboratory focuses on the potential role of survivin as an interface between cell cycle progression and protection from apoptosis. Using biochemical and cell biological approaches, we have identified a critical event in survivin function that links the two properties of the molecule in cell cycle progression and regulation of apoptosis. We found that a unique Thr34 in survivin becomes phosphorylated at mitosis by the main mitotic kinase complex p34cdc2-cyclin B, also known as Cdk1. This phosphorylation event is required to enhance survivin stability at mitosis, whereas inhibition of survivin phosphorylation on Thr34 resulted in initiation of mitochondrial-dependent apoptosis, activation of the caspase cascade and strong anti-cancer activity, in various models of cancer, in vivo. Our ongoing research programs focus on the further elucidation of the checkpoint function of survivin in human cancer. Our working hypothesis is that a detailed mapping of the survivin pathway and its dual role in cell division and apoptosis control may provide a more general paradigm by which mechanisms controlling cell proliferation and cell survival come together in a single interface, and are pathways potentially exploited in human cancer. Our laboratory uses an array of experimental approaches to tackle this problem. These encompass biochemistry of protein-protein interaction of survivin/IAP-associated molecules, cell biology of microtubule dynamics and pathways of spindle formation, and genetic mimicry of survivin function in transgenic or knockout mouse models, in vivo. Our laboratory is divided in working groups addressing the various aspects of the interface between cell proliferation and cell survival and how new knowledge generated in these studies can be translated in pathophysiologically relevant approaches for cancer treatment and therapy.
Figure 1. Localization of survivin to the mitotic apparatus. HeLa cells were stained with monoclonal antibodies (mAb) recognizing immunochemically distinct subcellular pools survivin localized to spindle microtubules (A, mAb 8E2) or kinetochores of metaphase chromosomes (B, mAb 32.1). In A, image merging is shown of dual labeling for survivin (green) and tubulin (red).
Figure 2. Cell division defects induced by molecular antagonists of survivin. HeLa cells were transfected with a survivin antisense oligonucleotide or a survivin Cys84→Ala dominant negative mutant and analyzed by fluorescence microscopy for integrity of the mitotic apparatus (A, B, microtubules), ploidy (C, D, nuclear morphology) and centrosome number (E, F, centrosomes).
Figure 3. Alternative dimeric structures of mouse survivin. The three dimer arrangements resolved by X-Ray crystallography of mouse survivin are shown.
Figure 4. Adenoviral targeting of survivin inhibits tumor growth, in vivo. Breast cancer xenografts grown in immunocompromised mice were injected with replication-defective adenovirus constructs encoding GFP (pAd-GFP) or a survivin Thr34→Ala dominant negative mutant and analyzed for kinetics of tumor growth (A), or proliferation in situ (B) by Ki-67 staining and immunohistochemistry. Representative publicationsAmbrosini G, Adida C and Altieri DC. (1997) A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nature Med. 3:917-921. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC and Altieri DC. (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396:580-584. Li F, Ackermann EJ, Bennett CF, Rothermel AL, Plescia J, Tognin S, Villa A, Marchisio PC and Altieri DC. (1999) Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nature Cell Biology 1:461-466. Muchmore SW, Chen J, Jakob C, Zakula D, Matayoshi, ED, Wu W, Zhang H, Li F, Ng S-C,and Altieri DC. (2000) Crystal structure and mutagenic analysis of the Inhibitor-of-Apoptosis protein survivin. Mol. Cell 6:173-182. O'Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio PC and Altieri DC. (2000) Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc. Natl. Acad. Sci. U.S.A. 97:13103-13107. Smith SD, Wheeler MA, Plescia J, Colberg JW, Weiss RM and Altieri DC. (2001) Urine detection of survivin and diagnosis of bladder cancer. JAMA 285:324-328. Mesri M, Wall NR, Li J, Kim RW and Altieri DC. (2001) Cancer gene therapy using a survivin mutant adenovirus. J. Clin. Invest. 108:981-990. Grossman D, Kim PJ, Blanc-Brude OP, Brash DE, Tognin S, Marchisio PC and Altieri DC. (2001) Transgenic expression of survivin in keratinocytes counteracts UVB-induced apoptosis and cooperates with loss of p53. J. Clin. Invest. 108:991-999. Blanc-Brude OP, Yu J, Simosa H, Conte MS, Sessa WC and Altieri DC. (2002) Inhibitor of apoptosis protein survivin regulates vascular injury. Nature Med. 8:987-994 O'Connor DS, Wall NR, Porter ACG and Altieri DC. (2002) A p34cdc2 survival checkpoint in cancer. Cancer Cell 2:43-54. Kim PJ, Plescia J, Clevers H, Fearon ER and Altieri DC. (2003) Survivin and molecular pathogenesis of colorectal cancer. Lancet 362:205-209. Fortugno P, Beltrami E, Plescia J, Fontana J, Pradhan D, Marchisio PC, Sessa WC and Altieri DC. (2003) Regulation of survivin function by Hsp90.Proc. Natl. Acad. Sci. U.S.A. 100:13791-13796. Dohi T, Okada K, Xia F, Wilford CE, Samuel T, Welsh K, Marusawa H, Zou H, Armstrong R, Matsuzawa SI, Salvesen GS, Reed JC and Altieri DC. (2004) An IAP-IAP complex inhibits apoptosis. J. Biol. Chem. 279:34087-34090. Dohi T, Beltrami E, Wall NR, Plescia J and Altieri DC. (2004) Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J. Clin. Invest. 114:1117-1127. Ghosh JC, Dohi T, Raskett CM, Kowalik TF and Altieri DC. (2006) Activated checkpoint kinase 2 provides a survival signal for tumor cells. Cancer Res. Dec 15;66(24):11576-9. Ghosh JC, Dohi T, Kang BH and Altieri DC. (2007) Hsp60 regulation of tumor cell apoptosis. J Biol Chem. 283: 5188-5194 Xia F, Lee CW and Altieri DC. (2008) Tumor cell dependence on Ran-GTP-directed mitosis. Cancer Res. 68:1826-33. Xia F, Canovas PM, Guadagno TM and Altieri DC. (2008) A survivin-Ran complex regulates spindle formation in tumor cells. Mol. Cell. Biol. 28: 5299-311. Lee CW, Raskett CM, Prudovsky I and Altieri DC. (2008) Molecular dependence of estrogen receptor-negative breast cancer on a Notch-survivin signaling axis. Cancer Res. 68:5273-5281. Jiang J, Nishimura W, Devor-Henneman D, Kusewitt D, Wang H, Holloway MP, Dohi T, Sabo E, Robinson ML, Altieri DC, Sharma A and Altura RA. (2008). Postnatal expansion of the pancreatic b-cell mass is dependent on survivin. Diabetes 57:2718-2727. Altieri DC. (2008) New wirings in the survivin networks. Oncogene 27:6276-6284. Luo J and Altieri DC. (2008) SIRTing through breast cancer is just a survivin’ game. Mol. Cell 32:159-160. Torti FM, Altieri DC, Broach J, Fan H, Lotze M, Manfredi J, Matrisian L, Yu D, Giaccia A, The Cancer Biology Training Consortium (2008) Ph.D. training in cancer biology. Cancer Res. 68:9122-9124. Lee CW, Simin K, Liu Q, Plescia J, Guha M, Khan A, Hsieh C-C and Altieri DC. (2008) A functional Notch-survivin gene signature in basal breast cancer. Breast Cancer Res. 10:R97. Xia F, Lee CW and Altieri DC. (2009) Ran-GTP control of tumor cell mitosis. Cancer Res. 69:1240-1241 [response to comment]. Kang BY, Plescia J, Song HY, Meli M, Colombo G, Beebe K, Scroggins B, Neckers L, and Altieri DC. (2009) Combinatorial drug design targeting multiple cancer signaling networks controlled by mitochondrial Hsp90. J. Clin. Invest. 119:445-8. Filion TM, Qiao M, Ghule PN, Mandeville M, van Wijnen AJ, Stein JL, Lian JB, Altieri DC and Stein GS. (2009) Survival responses of human embryonic stem cells to DNA damage.J Cell Physiol. [Epub ahead of print] Guha M, Plescia J, Leav I, Li J, Languino LR and Altieri DC. (2009) Endogenous Tumor Suppression Mediated by PTEN Involves Survivin Gene Silencing.Cancer Res . [Epub ahead of print] Altieri DC, Languino LR, Lian JB, Stein JL, Leav I, van Wijnen AJ, Jiang Z and Stein GS. (2009) Prostate cancer regulatory networks.J Cell Biochem. [Epub ahead of print] Additional information about lab rotations within the CPHR program can be found at http://www.umassmed.edu/cphr/program/rotations.aspx
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Our laboratory is interested in how tumor cells evade from a normal process of cellular suicide also called apoptosis or programmed cell death. It is now known that abnormally extended cell viability through inhibition of apoptosis is an invariant molecular hallmark of perhaps all human tumors, and is thought to contribute to the onset and progression of the disease by promoting the accumulation of transforming mutations and facilitating the insurgence of resistance to chemo or radiation therapy. Apoptosis is one of the most intensely investigated fields in cellular and molecular biology, and this reflects its extraordinary evolutionary conservation, its genetic complexity with multiple intersecting signaling pathways, and its critical impact on human diseases, obviously including cancer. The study of apoptosis has also provided new therapeutic opportunities for rational cancer therapy, by identifying means and pathways to lower a cellular survival threshold in cancer cells and restore their sensitivity to conventional anti-cancer drugs.
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