Effect of dual inhibition of apoptosis and autophagy in prostate cancer.

PURPOSE: Targeting multiple anti-apoptotic proteins is now possible with the small molecule BH3 domain mimetics such as ABT-737. Given recent studies demonstrating that autophagy is a resistance mechanism to multiple therapeutic agents in the setting of apoptotic inhibition, we hypothesized that hydroxychloroquine (HCQ), an anti-malarial drug that inhibits autophagy, will increase cytotoxicity of ABT-737. EXPERIMENTAL DESIGN: Cytotoxicity of ABT-737 and HCQ was assessed in vitro in PC-3 and LNCaP cells, and in vivo in a xenograft mouse model. The role of autophagy as a resistance mechanism was assessed by siRNA knockdown of the essential autophagy gene beclin1. ROS was measured by flow cytometry, and mitophagy assessed by the mCherry-Parkin reporter. RESULTS: Induction of autophagy by ABT-737 was a mechanism of resistance in prostate cancer cell lines. Therapeutic inhibition of autophagy with HCQ increased cytotoxicity of ABT-737 both in vitro and in vivo. ABT-737 induced LC-3 and decreased p62 expression by immunoblot in cell lines and by immunohistochemistry in tumors in vivo. Assessment of ROS and mitochondria demonstrated that ROS production by ABT-737 and HCQ was a mechanism of cytotoxicity. CONCLUSIONS: We demonstrated that autophagy inhibition with HCQ enhances ABT-737 cytotoxicity in vitro and in vivo, that LC-3 and p62 represent assessable markers in human tissue for future clinical trials, and that ROS induction is a mechanism of cytotoxicity. These results support a new paradigm of dual targeting of apoptosis and autophagy in future clinical studies.

Deficiency of the dual ubiquitin/SUMO ligase Topors results in genetic instability and an increased rate of malignancy in mice.

BACKGROUND: Topors is a nuclear protein that co-localizes with promyelocytic leukemia bodies and has both ubiquitin and SUMO E3 ligase activity. Expression studies implicated Topors as a tumor suppressor in various malignancies. To gain insight into the function of Topors, we generated a Topors-deficient mouse strain. RESULTS: Mice homozygous for a mutant Topors allele exhibited a high rate of perinatal mortality and decreased lifespan. In addition, heterozygotes were found to have an increased incidence of malignancy, involving a variety of tissues. Consistent with this finding, primary embryonic fibroblasts lacking Topors exhibited an increased rate of malignant transformation, associated with aneuploidy and defective chromosomal segregation. While loss of Topors did not alter sensitivity to DNA-damaging or microtubule-targeting agents, cells lacking Topors exhibited altered pericentric heterochromatin, manifested by mislocalization of HP1alpha and an increase in transcription from pericentric major satellite DNA. Topors-deficient cells exhibited a transcriptional profile similar to that of cells treated with histone deacetylase inhibitors, and were resistant to the anti-proliferative effects of the histone deacetylase inhibitor trichostatin A. CONCLUSION: These results indicate a unique role for Topors in the maintenance of genomic stability and pericentric heterochromatin, as well as in cellular sensitivity to histone deacetylase inhibitors.

Correction: The topoisomerase I- and p53-binding protein topors is differentially expressed in normal and malignant human tissues and may function as a tumor suppressor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of phosphorylation sites of TOPORS and a role for serine 98 in the regulation of ubiquitin but not SUMO E3 ligase activity.

TOPORS is the first example of a protein that possesses both ubiquitin and SUMO E3 ligase activity. The ubiquitination activity maps to a conserved RING domain in the N-terminal region of the protein, which is not required for sumoylation activity. Similar to other E3 ligases, it is likely that the ubiquitin and sumoylation activities of TOPORS are regulated by post-translational modifications. Therefore, we employed mass spectrometry to identify post-translational modifications of TOPORS. Several putative phosphorylated regions were identified in conserved regions of the protein. We investigated the role of phosphorylation of serine 98, which is adjacent to the RING domain, in both cells and in vitro. Mutation of serine 98 to aspartic acid resulted in an increase in the ubiquitin ligase activity of TOPORS both in cells and in vitro. In addition, this mutation increased the binding of TOPORS to the E2 enzyme UbcH5a both in vitro and in cells. Conversely, a phospho-deficient mutant (S98A) exhibited little change in ubiquitin ligase activity compared to wild-type TOPORS, both in cells and in vitro. Neither of the mutants affected the localization of TOPORS to punctate nuclear regions. In addition, neither mutant affected the SUMO ligase activity of TOPORS in cells or in vitro. Molecular modeling studies support a role for serine 98 in regulating TOPORS-E2 interactions. Our findings indicate that phosphorylation of serine 98 regulates the ubiquitin but not the SUMO ligase activity of TOPORS, consistent with a potential binary switch function for TOPORS in protein ubiquitination versus sumoylation.

Elevated expression of ISG15 in tumor cells interferes with the ubiquitin/26S proteasome pathway.

IFN-stimulatory gene factor 15 (ISG15) is a ubiquitin-like protein, which is conjugated to many cellular proteins. However, its role in protein degradation is unclear. Here, we show that ISG15 is highly elevated and extensively conjugated to cellular proteins in many tumors and tumor cell lines. The increased levels of ISG15 in tumor cells were found to be associated with decreased levels of polyubiquitinated proteins. Specific knockdown of ISG15 expression using ISG15-specific small interfering RNA (siRNA) was shown to increase the levels of polyubiquitinated proteins, suggesting an antagonistic role of ISG15 in regulating ubiquitin-mediated protein turnover. Moreover, siRNA-mediated down-regulation of the major E2 for ISG15 (UbcH8), which blocked the formation of ISG15 protein conjugates, also increased the levels of polyubiquitinated proteins. Together, our results suggest that the ISG15 pathway, which is deregulated during tumorigenesis, negatively regulates the ubiquitin/proteasome pathway by interfering with protein polyubiquitination/degradation.

Differential effects of the breast cancer resistance protein on the cellular accumulation and cytotoxicity of 9-aminocamptothecin and 9-nitrocamptothecin.

Breast cancer resistance protein (BCRP)/MXR/ABCG2 is a new member of the family of ATP-dependent drug efflux proteins. Whereas overexpression of another member of this family, P-glycoprotein, minimally affects the cytotoxicity of camptothecins (CPTs), overexpression of wild-type as well as certain mutant BCRPs confers resistance to CPT analogues that are used clinically, including topotecan and irinotecan. Relatively little is known regarding the effects of BCRP on other CPT analogues. We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T). The results indicate that overexpression of either P-glycoprotein, multidrug resistance protein type 1, or multidrug resistance protein type 2 has little effect on the cytotoxicity of 9-NC or 9-AC. By contrast, overexpression of either wild-type or R482T BCRP confers resistance to 9-AC, but not to 9-NC. Furthermore, overexpression of wild-type or mutant BCRP is associated with reduced intracellular accumulation of 9-AC, but not 9-NC. In addition, immunoblotting studies indicate that whereas increased BCRP expression is evident in cells selected for resistance to irinotecan, BCRP expression is not detectable in two different cell lines selected for resistance to 9-NC. Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC. Furthermore, the results suggest that polar groups at the 9 or 10 position of the CPT A ring facilitate interaction with BCRP and have implications for the clinical development of new CPT analogues.

The topoisomerase I- and p53-binding protein topors is differentially expressed in normal and malignant human tissues and may function as a tumor suppressor.

Topors was identified recently as a human protein that binds to topoisomerase I and p53. Topors contains a highly conserved RING domain and localizes in promyelocytic leukemia nuclear bodies. Relatively little is known regarding topors expression patterns or function. We now demonstrate that topors mRNA and protein are widely expressed in normal human tissues. By contrast, topors mRNA and protein levels are decreased or undetectable in colon adenocarcinomas relative to normal colon tissue, and expression of the topors protein is not detectable in several colon cancer cell lines. The human TOPORS gene is located on chromosome 9p21, with loss of heterozygosity in this region frequently observed in several different malignancies. While we were unable to detect loss of heterozygosity of the TOPORS gene in 16 sporadic colon cancer cases, increased methylation of a CpG island in the TOPORS promoter was evident in colon adenocarcinoma specimens relative to matched normal tissues. Additional studies indicate that forced expression of topors inhibits cellular proliferation and is associated with an accumulation of cells in the G(0)/G(1) phase of the cell cycle. This effect is independent of the topors RING domain and maps to a C-terminal region of the protein. These results suggest that topors functions as a negative regulator of cell growth, and possibly as a tumor suppressor.

Phase I evaluation of sequential topoisomerase targeting with irinotecan/cisplatin followed by etoposide in patients with advanced malignancy.

PURPOSE: To investigate pharmacologically guided addition of etoposide to a weekly irinotecan/cisplatin chemotherapy. PATIENTS AND METHODS: Patients with advanced nonhematologic malignancies were eligible. Treatment consisted of i.v. administration of 50 mg/m(2) irinotecan and 20 mg/m(2) cisplatin on days 1, 8, 15, and 22 of a 42-day cycle or on days 1 and 8 of a 21-day cycle. Etoposide was administered in a dose-escalating fashion 2 days after each dose of irinotecan/cisplatin, either i.v. as a single dose or p.o. as two doses administered 12 h apart. Pharmacologic analyses included measurement of plasma concentrations of irinotecan, SN-38, and SN-38 glucuronide, as well as quantitation of topoisomerase protein levels in peripheral blood mononuclear cells (PBMNCs). RESULTS: A total of 40 patients with a variety of malignancies received 122 cycles of therapy. Dose-limiting toxicities included neutropenia and diarrhea, with the 21-day cycle tolerated better than the 42-day cycle. For the 21-day cycle, the maximum tolerated dose was 75 mg/m(2) for i.v. etoposide and 85 mg/m(2) for oral etoposide. Objective responses were observed in four patients with previously treated mesothelioma, gastric, breast, and ovarian cancer, respectively. PBMNC levels of topoisomerase IIalpha were increased at the time of etoposide administration in two patients, with these patients having the highest SN-38 glucuronide peak-plasma-concentration and area-under-the-curve values among 15 patients with available pharmacokinetic data. One of these patients had a partial response to therapy. CONCLUSIONS: Pharmacologically guided administration of etoposide in combination with irinotecan/cisplatin using a 21-day cycle is associated with acceptable toxicity and significant antitumor activity. The finding that PBMNC topoisomerase IIalpha protein levels increased after irinotecan/cisplatin treatment in two of six patients supports the continued development of sequential topoisomerase targeting in the treatment of malignancy.

Effects of drug efflux proteins and topoisomerase I mutations on the camptothecin analogue gimatecan.

Clinically relevant resistance to the currently approved camptothecins, irinotecan and topotecan, is poorly understood but may involve increased expression of ATP-dependent drug transporters such as ABCG2 (breast cancer resistant protein, BCRP). Gimatecan (ST1481) is a lipophilic 7-substituted camptothecin derivative that exhibits potent anti-tumor activity in a variety of preclinical cancer models and is under investigation in the clinic. Previous studies reported that gimatecan cytotoxicity was not affected by expression of ABCG2. To confirm and extend this finding, we assessed the cytotoxicity of gimatecan in pairs of isogenic cell lines consisting of transfectants expressing either ABCG2 (including wild-type, R482T, or R482G mutants), ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2), or ABCC4 (MRP4). Expression of wild-type or mutant ABCG2 in human cell lines conferred resistance to topotecan but not to gimatecan. Similarly, intracellular accumulation of gimatecan was unaffected by expression of wild-type ABCG2. Furthermore, expression of P-glycoprotein or MRP2 did not alter gimatecan cytotoxicity. Whereas expression of MRP1 had a minor effect on gimatecan cytotoxicity, expression of ABCC4 was found to significantly reduce the anti-proliferative effects of this drug. Cells containing resistance-conferring mutations in topoisomerase I were also resistant to gimatecan. These results suggest that gimatecan may be more effective than irinotecan or topotecan in cancers that express ABCG2, but not in cancers that express high levels of ABCC4 or contain certain topoisomerase I (TOP1) mutations.

Sequential topoisomerase targeting and analysis of mechanisms of resistance to topotecan in patients with acute myelogenous leukemia.

Resistance to topoisomerase I (TOP1)-targeting drugs such as topotecan often involves upregulation of topoisomerase II (TOP2), with accompanying increased sensitivity to TOP2-targeting drugs such as etoposide. This trial was designed to investigate sequential topoisomerase targeting in the treatment of patients with high-risk acute myelogenous leukemia. An initial cohort of patients received topotecan and cytosine arabinoside daily for 5 days. Serial samples of circulating mononuclear cells were examined to evaluate peak elevations of TOP2-alpha protein expression. In subsequent cohorts, etoposide was administered daily for 3 days, beginning 6 h after initiation of the topotecan infusion. The etoposide dose was escalated to determine a maximum-tolerated dose. Circulating mononuclear cells were analyzed for TOP1 mutations and ABCG2 protein expression. In addition, systemic and intracellular topotecan concentrations were measured. Thirty-one patients were enrolled. On the basis of TOP1-alpha protein levels in three patients with peripheral blast counts greater than 50%, etoposide administration began 6 h after initiation of the topotecan/cytosine arabinoside infusion. Using this schedule of administration, the maximum-tolerated dose of etoposide was 90 mg/m. No TOP1 mutations were identified, but increases in ABCG2 expression during the infusion were observed in mononuclear cells from two of four evaluable patients. Administration of etoposide 6 h after initiation of a topotecan/cytosine arabinoside infusion is feasible and is associated with clinical activity. Analysis of TOP2-alpha protein levels in this small number of patients indicated that peak increases occurred earlier than expected based on earlier publications. Upregulation of ABCG2 was detected in circulating cells and may represent an inducible form of drug resistance that should be investigated further.

TOPORS functions as a SUMO-1 E3 ligase for chromatin-modifying proteins.

TOPORS is the first example of a protein with both ubiquitin and SUMO-1 E3 ligase activity and has been implicated as a tumor suppressor in several different malignancies. To gain insight into the cellular role of TOPORS, a proteomic screen was performed to identify candidate sumoylation substrates. The results indicate that many of the putative substrates are involved in chromatin modification or transcriptional regulation. Transfection studies confirmed mammalian Sin3A as a sumoylation substrate for TOPORS. These findings suggest that TOPORS may function as a tumor suppressor by regulating mSin3A and other proteins involved in chromatin modification.

The topoisomerase I-binding RING protein, topors, is associated with promyelocytic leukemia nuclear bodies.

We previously identified topors as a topoisomerase I-binding protein that localizes in punctate nuclear regions when expressed as a GFP fusion protein. We now demonstrate that both the GFP-topors fusion protein and endogenous topors are associated with promyelocytic leukemia (PML) nuclear bodies in exponentially growing HeLa cells. Studies using isogenic PML+/+ and PML-/- murine embryonic fibroblasts indicate that the punctate nuclear localization of topors is dependent on PML. A basic C-terminal region but not the N-terminal RING domain of topors is required for the punctate nuclear localization of this protein. Additional studies indicate that topors, but not PML, rapidly relocalizes from nuclear bodies to the nucleoplasm in cells exposed to the transcription inhibitor dichloro-1-beta-d-ribofuranolsylbenzimidazole or to the topoisomerase I-targeting drug camptothecin. These results identify topors as a new member of the group of proteins that associate dynamically with PML nuclear bodies and suggest that topors may be involved in the cellular response to camptothecin.

Topors functions as an E3 ubiquitin ligase with specific E2 enzymes and ubiquitinates p53.

The human topoisomerase I- and p53-binding protein topors contains a highly conserved, N-terminal C3HC4-type RING domain that is homologous to the RING domains of known E3 ubiquitin ligases. We demonstrate that topors functions in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UbcH5a, UbcH5c, and UbcH6 but not with UbcH7, CDC34, or UbcH2b. Additional studies indicate that a conserved tryptophan within the topors RING domain is required for ubiquitination activity. Furthermore, both in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Similar to MDM2, overexpression of topors results in a proteasome-dependent decrease in p53 protein expression in a human osteosarcoma cell line. These results are similar to the recent finding that a Drosophila topors orthologue ubiquitinates the Hairy transcriptional repressor and suggest that topors functions as a ubiquitin ligase for multiple transcription factors.