Role of the Aggresome Pathway in Cancer: Targeting Histone Deacetylase 6–Dependent Protein Degradation

Review

Agustin Rodriguez-Gonzalez,1 Tara Lin,1 Alan K. Ikeda,1 Tiffany Simms-Waldrip,1 Cecilia Fu,1 and Kathleen M. Sakamoto1,2,3

1Division of Hematology-Oncology, Mattel Children’s Hospital and 2Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California; and 3Division of Biology, California Institute of Technology, Pasadena, California

Abstract synthesized in the endoplasmic reticulum (ER) are properly folded Misfolded or aggregated proteins have two fates: they are with the help of ER chaperones. Misfolded proteins are disposed either refolded with the help of chaperones or degraded by the of by ER-associated protein degradation (ERAD). When the level proteasome. Cells also have an alternative pathway that of misfolded proteins exceeds the folding capacity of the ER, cells involves intracellular ‘‘storage bins’’ for misfolded intracellu- activate a feedback mechanism known as the ER stress response lar proteins known as aggresomes. Aggresomes recruit motor (4). Expression of ER chaperones and ERAD-associated proteins is proteins that transport misfolded or aggregated proteins to induced to decrease protein synthesis and, hence, the burden on chaperones and proteasomes for subsequent destruction. the ER. There are four classes of agents that induce ER stress; they There is emerging evidence that inhibiting the aggresome are inhibitors of glycosylation, calcium metabolism, reducing agents, and hypoxia. Finally, the ER stress response can result in pathway leads to accumulation of misfolded proteins and apoptosis in tumor cells through autophagy. We discuss the activation of apoptosis (5). role of aggresomes in cancer and the potential to target Three transmembrane proteins regulate the mammalian ER stress response. PERK is a transmembrane kinase, ATF6 is a this pathway for therapy. [Cancer Res 2008;68(8):2557–60] transmembrane transcription factor, and IRE1 is a transmembrane Protein (Mis)Folding and Aggregation RNase. These three proteins maintain integration of the stress response and are critical for cell survival. ER stress-induced Newly synthesized proteins must overcome several obstacles on apoptotic pathways act through proapoptotic and antiapoptotic their way to becoming functional molecules. Small proteins fold proteins, such as bcl-2, p53, and c-abl. Stress-activated protein through a sequence of folding intermediates. During folding, kinase and c-Jun NH2-terminal kinase (JNK) are also activated (5). partially folded proteins expose hydrophobic domains that lead to Recently, the role of UPR in tumorigenesis has been investigated inappropriate associations and protein aggregation. Aggregation is (6). As tumors increase in size, cells are exposed to several toxic to cells and, due to high concentrations of macromolecules, environmental stressors, including hypoxia, limited nutrients, and causes a significant increase in the association constants of acidosis. Exposure to chemotherapy also activates UPR, resulting in unfolded polypeptides over those in dilute solution. Effects of sensitivity to DNA cross-linking agents (e.g., cisplatin); however, protein aggregation are amplified by the fact that stable folding there is also evidence that activation of the stress response could of a domain cannot occur until the entire protein is synthesized. confer resistance to drugs [e.g., topoisomerase II inhibitors (6)]. This is particularly important during synthesis of identical nascent Clearly, the role of the ERAD and ER stress response is a complex polypeptides on polysomes, where numerous polypeptides expose issue due to the heterogeneity of tumor response. the same aggregation-prone domains leading to increased risk of aggregation (1). Chaperones and Ubiquitin-Proteasome System The ultimate fate of a protein is either correct folding or aggregation (1). Whether a polypeptide chain folds correctly or Molecular chaperones have evolved to assist with folding of whether it aggregates is dependent on particular mutations, newly synthesized proteins and refolding of proteins damaged by modification, mistakes during translation, or unequal synthesis of stress and cellular injury. Chaperones bind to and stabilized subunits (1). Misfolding can also be promoted by pH, temperature, exposed hydrophobic residues through ATP-dependent interac- ionic strength, and redox environment. Because a certain level of tions, allowing the protein to achieve proper folding (7). protein misfolding is inevitable, cells have adapted various quality Chaperones do not seem to catalyze folding, but rather, they control mechanisms to minimize misfolding and to eliminate prevent intermolecular and intramolecular interactions between misfolded proteins before aggregation (2, 3). partially folded or misfolded polypeptides. This is an evolutionarily conserved mechanism from bacteria to eukaryotes to maintain Unfolded Protein Response Pathway proper folding after protein synthesis (1). Second, proteins that are unable to fold properly are targeted for degradation by the Another important pathway in cells to regulate misfolded ubiquitin-proteasome system. The proteasome is a multisubunit proteins is the unfolded protein response (UPR). Proteins complex found in the cytosol and nucleus that degrades cytosolic, nuclear, secretory, and transmembrane proteins into smaller

Requests for reprints: Kathleen M. Sakamoto, Division of Hematology-Oncology, peptides (8, 9). Misfolded secretory and transmembrane proteins Mattel Children’s Hospital University of California at Los Angeles, David Geffen School are retained in the lumen or membrane of the ER and then of Medicine, Los Angeles, CA 90095-1752. Phone: 310-794-7007; Fax: 310-206-8089; retrotranslocated back to the cytosol and delivered to the E-mail: [email protected]. I2008 American Association for Cancer Research. proteasome (1). When correct folding is difficult or impossible doi:10.1158/0008-5472.CAN-07-5989 and degradation is not performed rapidly, proteins interact with www.aacrjournals.org 2557 Cancer Res 2008; 68: (8). April 15, 2008

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Research other unfolded or partially folded proteins, leading to the aggresomes showed that these particles consist of multiple loosely formation of aggregates (3). Cells then destroy protein aggregates associated particles. through the aggresome pathway. The Aggresome Pathway Recruitment of Aggresomes to the Degradation Recent studies have shown a proteasome-independent pathway Machinery that eliminates misfolded polyubiquitinated proteins, known as Aggresomes are not static compartments for misfolded proteins the aggresome (Fig. 1; ref. 1). The initial aggregation process seems (1). They also recruit cytosolic components, including chaperones, to occur cotranslationally, as nascent chains are coming off the ubiquitination enzymes, and proteasome subunits, to facilitate polysome. If nascent peptides do not fold correctly, they will clearance of aggregated proteins. Several chaperones have been coaggregate to form a single aggresomal particle. In cells, these identified [e.g., HSC70, heat shock protein (HSP) 40, and HSP70], particles are uniform in size, supporting the idea that a fixed and some have been targeted for cancer therapy. Chaperones seem number of proteins aggregate to form a single particle (1). These to reduce aggregation; misfolded proteins associate with ER particles are produced throughout the cytosol. After their chaperones first followed by association with cytosolic chaperones formation, the aggresomal particles are transported toward the (1). Recently, heat shock factor 1 (HSF1), the master regulator microtubule organizing center (MTOC), where they are sequestered of the heat shock response in eukaryotes and highly conserved, into a single large cellular garbage bin-like structure known as the was reported to play a major role in carcinogenesis. Elimination of aggresome (1). Movement of the aggresome particle is an active HSF1 protects mice from tumors induced by mutations of the process and requires intact microtubules and association with RAS oncogene or in the tumor suppressor p53 (10). motor dynein. In cells treated with the microtubule-depolarizing Similar to chaperones, proteasomes interact with aggresomes. agent nocodazole, the aggresome remains distributed throughout Proteasomes associate with aggresomes relatively late after small the cytosol. Furthermore, electron microscopy examination of aggresomal particles are delivered to the MTOC. The pathways

Figure 1. The aggresome pathway. Unfolded or misfolded proteins can originate from translating polysomes, from proteins retrotranslocated from the ER to the cytosol, or from proteins damaged by stress. If these unfolded/misfolded proteins fail to fold correctly and are not degraded by the proteasome, they can form aggregates throughout the cells. These aggregates are transported in a microtubule-dependent manner to the MTOCthat requires the dynein/dynactin motor compl ex. HDAC6 acetylates a-tubulin and associates with dynein to facilitate transport of aggregated bodies through the cytosol to lysosomes for degradation. HDAC6 coordinates the cell response to protein aggregate formation. Balance between HDAC6 and its partner p97/VCP determines the fate of polyubiquitinated misfolded proteins. The recruitment of HDAC6 to ubiquitinated proteins leads to the induction of a HSP90-dependent pathway that triggers protection against cell stress (1, 18).

Cancer Res 2008; 68: (8). April 15, 2008 2558 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Aggresomes in Cancer regulating interaction between aggresomes and chaperones or HDAC6 controls major cell response pathways to cytotoxic proteasomes have yet to be determined. Finally, clearance of accumulation of protein aggregates (18). Studies by Boyault et al. aggregated proteins seems to activate autophagy. Autophagy is (18) showed that HDAC6 senses ubiquitinated cellular aggregates one of the primary pathways by which large structures, such as and induces the expression of major cellular chaperones by mitochondria and peroxisomes, are degraded in cells (1). Aggre- triggering the dissociation of a repressive HDAC6/HSF1/HSP90 somes are thought to be engulfed by autophagosomes, which then complex and subsequent HSF1 activation. The other known target fuse to lysosomes, resulting in degradation of protein matter by of HDAC6 is HSP90 chaperone activity that adds to the multi- lysosomal hydrolases. functionality of the protein and provides further rationale to target Autophagy is activated by cellular stress, including those that HDAC6 for cancer therapy (18). Therefore, HDAC6 seems to be a trigger the aggresome pathway leading to degradation by lyso- master regulator of the cell protective response to cytotoxic protein somes. There is recent evidence that the aggresome and autophagy aggregate formation. pathways are linked. Studies on parkin-mediated K63-linked The cellular concentration of HDAC6 and its partner, p97/VCP, polyubiquitination seems to couple misfolded proteins to the determines the outcome of polyubiquitinated misfolded proteins dynein motor complex through interaction with histone deacetylase (Fig. 1). An excess of HDAC6 favors the accumulation of ubiqui- 6 (HDAC6), resulting in the formation of aggresomes and clearance tinated, misfolded proteins, resulting in the formation of aggre- by autophagy (11). Furthermore, studies on viruses have shown that somes. The abundance of p97/VCP results in the release of HDAC6 viral replication and assembly often occur in inclusions that form at and delivery of ubiquitinated proteins to the proteasome for pericentriolar sites close to the MTOC or in specialized nuclear degradation. The accumulation of ubiquitinated proteins results in domains called ND10/PML bodies, similar to aggresomes (12). HDAC6-mediated transport along microtubules into aggresomes Protein degradation by basal constitutive autophagy is important to and final degradation by the lysosomes. In this manner, HDAC6 avoid accumulation of polyubiquitinated protein aggregates and regulates the recruitment of the autophagic machinery to destroy development of diseases, such as Huntington’s, Parkinson’s, or the aggregates (1, 18). Alzheimer’s. The polyubiquitin-binding protein p62/SQSTM1 is degraded by lysosomes. Recent articles suggest that p62 is required Role of HDAC6 in Cancer for both the formation and the degradation of polyubiquitin- containing bodies by autophagy (13). It is likely, therefore, that There is increasing evidence that HDAC6 plays a role in cancer HDAC6 autophagy and aggresome formation in cancer cells are linked and cells and may be a target for drug development. is an targeting both with inhibitors could be potentially synergistic. estrogen-regulated gene that has prognostic significance in estrogen Molecular studies to understand the aggresome pathway have receptor (ER)-positive breast cancer cells (19). Overexpression of implicated specific signaling molecules. One question is: how do HDAC6 in MCF-7 breast cancer cells increased cell motility, cells recognize aggregated proteins and result in the formation of suggesting a role for HDAC6 in metastases (19). One study showed aggresomes? Published reports suggest that the sequestration of that elevated HDAC6 protein expression by immunohistochemical aggregated proteins into single aggresome is induced by cellular staining in 139 consecutively archived human breast cancer tissues signaling mechanisms. Activation of the mitogen-activated protein was associated with improved survival in patients who were ER kinase kinase (MEKK1) increases formation of aggresomes (14). positive and received tamoxifen (19). The combination of farnesyl MEKK1 seems to act at a relatively late stage of aggresome transferase inhibitor lonafarnib and paclitaxel seems to enhance formation and affects sequestration of particles into pericentriolar HDAC6-dependent tubulin deacetylation in both breast and small aggresomes. The kinase activity of MEKK1 is required, suggesting cell lung carcinoma cells (20). HDAC6 has additional functions in that phosphorylation of downstream substrates is critical for integrating signaling and cytoskeleton remodeling. Zhang et al. (21) aggresome formation. Previous studies showed that MEKK1- showed that cortactin, which localizes to regions of cells undergoing regulated pathways involve JNK, extracellular signal-regulated active membrane remodeling, is a genuine substrate of HDAC6. kinase, and p38. Interestingly, the effect of MEKK1 on aggresome Furthermore, cortactin has been found to be overexpressed in formation does not seem to involve any of these kinases, suggesting several carcinomas (22). Therefore, HDAC6 could be a viable target that there are novel yet unidentified signaling pathways activated for cancer therapy. by cellular stress resulting from aggresomes. Tubacin Is an Inhibitor of HDAC6 HDAC6 and the Aggresome Pathway Recently, Stuart Schreiber’s laboratory at Massachusetts Institute HDAC6 is a member of the class II HDAC family and is known to of Technology/Broad Institute isolated a small-molecule inhibitor of deacetylate a-tubulin and increase cell motility. HDAC6 is mainly HDAC6 identified through a multidimensional chemical genetic localized in the cytoplasm and has two catalytic domains with screen of 7,392 small molecules and cell-based assay (23, 24). This deacetylase activity. The COOH-terminal domain is able to inhibitor, known as tubacin, inhibits a-tubulin deacetylation in deacetylate a-tubulin both in vitro and in vivo, and this activity mammalian cells. TSA is a broad HDAC inhibitor, whereas tubacin is reversibly inhibited by trichostatin A (TSA; ref. 15). HDAC6 plays is specific for the tubulin deacetylase activity of HDAC6. These an essential role in aggresomal protein degradation because it can compounds inhibit the HDAC6 deacetylase activity by chelating a bind to both polyubiquitinated proteins and dynein proteins, Zn2+ cation and may also alter the formation of complexes of thereby recruiting protein cargo to dynein motors to transport HDAC6 with other intracellular proteins, such as HSP90, Dia2, and misfolded proteins to aggresomes (16). Previous work suggests protein phosphatase-1 (15). Tubacin does not seem to affect global that targeting both the proteasome-dependent pathways with histone deacetylation, gene expression profiling, or cell cycle bortezomib and the aggresome pathway in tumor cells could progression (23, 24). This drug binds to one of the catalytic domains induce greater accumulation of polyubiquitinated proteins and of HDAC6 that contains the tubulin deacetylase activity. Recent significant cell stress followed by activation of apoptosis (17). data suggest that tubacin treatment does not affect microtubule www.aacrjournals.org 2559 Cancer Res 2008; 68: (8). April 15, 2008

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Research stability but rather affects cell motility in lymphocytes (24). Conclusions Recently, more selective inhibitors of HDAC6 have been identified Recent studies on the aggresome pathway and inhibitors of (25). Several compounds were shown to act synergistically with HDAC6 suggest an emerging field in cancer therapy. Tubacin has a paclitaxel in ER -positive breast cancer cells. been shown to act synergistically with other agents in multiple The effects of tubacin has been studied in normal and neoplastic myeloma cells to increase cellular stress and induce apoptosis. We cells (17). Overexpression of HDAC6 in primary lymphocytes and have found tubacin to be effective to inhibit growth of acute T-cell lines increases cell migration mediated by cytokines. lymphoblastic leukemia cell lines as a single agent (data not Knockdown of HDAC6 in T cells decreases chemotactic mobility shown). Further investigation is warranted to elucidate the independent of its enzymatic activity (15). Treatment of multiple signaling pathways that regulate HDAC6-induced apoptosis, myeloma cells with tubacin resulted in decreased cell growth at mechanisms of tubacin action, and role of the aggresome pathway A an IC50 of 2 to 5 mol/L (17). No toxicity was observed in normal in other tumors. peripheral blood mononuclear cells. Tubacin treatment in combi- a nation with bortezomib resulted in increased -tubulin acetylation Acknowledgments and accumulation of polyubiquitinated proteins in multiple myeloma cells (17). These cells undergo apoptosis through a Received 10/26/2007; revised 12/27/2007; accepted 1/2/2008. Grant support: Department of Defense grant W81XWH-06-1-0192 and MEC/ caspase-8–dependent pathway. Tubacin was also found to inhibit Fulbright fellowship grant EX2005-0517 (A. Rodriguez-Gonzalez); NIH postdoctoral interaction of HDAC6 with dynein and augmented activation of fellowship grants T32HL086345 (T. Lin) and T32CA9056 (A.K. Ikeda); American Academy of Pediatrics (T. Simms-Waldrip); and NIH grants HL75826 and HL83077, JNK, caspase-3, caspase-8, and caspase-9. Furthermore, treatment American Cancer Society grant RSG-99-081-01-LIB, Department of Defense grant with the proteasome inhibitor bortezomib and tubacin together CM050077, and Leukemia and Lymphoma Society Translational Research Grant 6019-07 induced synergistic antitumor activity in multiple myeloma cells (K.M. Sakamoto). K.M. Sakamoto is a Scholar of the Leukemia and Lymphoma Society. The costs of publication of this article were defrayed in part by the payment of page (17). Published data therefore provide rationale for combined charges. This article must therefore be hereby marked advertisement in accordance therapy in clinical trials for patients with multiple myeloma. with 18 U.S.C. Section 1734 solely to indicate this fact.

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Cancer Res 2008; 68: (8). April 15, 2008 2560 www.aacrjournals.org

Correction: Review Article on Aggresomes in Cancer

In the Review Article on aggresomes in cancer in the April 15, 2008 issue of Cancer Research (1), several sentences from an article by Garcia-Mata and colleagues (2) were cited but should have been quoted as well. Rodriguez-Gonzales and colleagues apologize for this oversight.

1. Rodriguez-Gonzales A, Lin T, Ikeda AK, Simms-Waldrip T, Fu C, Sakamoto KM. Role of the aggresome pathway in cancer: targeting histone deacetylase 6–dependent protein degradation. Cancer Res 2008;68:2557–60. 2. Garcia-Mata R, Gao YS, Sztul E. Hassles with taking out the garbage: aggravating aggresomes. Traffic 2002;3:388–96.

I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-69-9-COR1

Cancer Res 2009; 69: (9). May 1, 2009 4092 www.aacrjournals.org Role of the Aggresome Pathway in Cancer: Targeting Histone Deacetylase 6−Dependent Protein Degradation

Agustin Rodriguez-Gonzalez, Tara Lin, Alan K. Ikeda, et al.

Cancer Res 2008;68:2557-2560.

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