Search

PDF file 105K, Supplementary methods and figure legends

PDF file - 124K

PDF file - 270K

PDF file 2097K, Knockdown of hTOP3alpha and p53, but not BLM, reduced cellular senescence and enhanced anchorage-independent growth of AGS cells

PDF file - 93.5K

PDF file 2053K, DNA damage-induced expression of p53, p5315P and p21 is greatly reduced in hTOP3α-deficient cells

Purpose: Solid tumors encounter a growth-limiting hypoxic microenvironment as they develop. Hypoxia-inducible factors (HIF) play important roles in hypoxia-associated tumor development and therapeutic resistance. Targeting the HIF pathway (especially HIF-1α) represents a promising cancer treatment strategy. Here, we report a novel class of HIF-1α inhibitors and the possible molecular basis of inhibition.Experimental Design: We analyzed the inhibitory effects of clinically used topoisomerase II (TOP2)–targeting drugs on HIF-1α expression with a primary focus on mitoxantrone. The potential role of TOP2 in mitoxantrone-inhibited HIF-1α expression was studied using pharmacologic inhibition, a knockdown approach, and TOP2 mutant cells. Moreover, involvement of mitoxantrone in proteasome-mediated degradation, transcription, and translation of HIF-1α was examined.Results: The TOP2-targeting mitoxantrone, but neither doxorubicin nor etoposide (VP-16), strongly inhibited HIF-1α expression under hypoxic conditions in a dose- and time-dependent manner. Surprisingly, the mitoxantrone-mediated inhibition of HIF-1α expression was largely independent of two TOP2 isozymes, proteasomal degradation, and transcription. Furthermore, mitoxantrone inhibited HIF-1α expression and function in a similar fashion as cycloheximide, suggesting that mitoxantrone might inhibit HIF-1α via a blockage at its translation step. In vitro translation experiments using HIF-1α mRNA further confirmed inhibition of HIF-1α translation by mitoxantrone. Interestingly, levels of the polysome-bound HIF-1α and VEGF-A mRNA were elevated and decreased after mitoxantrone treatment, respectively.Conclusions: We have identified the TOP2-targeting compound, mitoxantrone, as an HIF-1α inhibitor possibly through a translation inhibition mechanism, suggesting the possibility of an additional anticancer activity for mitoxantrone. Clin Cancer Res; 17(15); 5026–37. ©2011 AACR.

PDF file - 97.5K

PDF file 1548KB, hTOP3alpha regulates TP53 mRNA and p53 protein stability

PDF file 3495K, Reduced expression of hTOP3alpha in renal and stomach tumor samples compared to the matched normal tissues

Purpose: Human DNA topoisomerase III alpha (hTOP3α) is involved in DNA repair surveillance and cell-cycle checkpoints possibly through formatting complex with tumor suppressors. However, its role in cancer development remained unsolved.Experimental Design: Coimmunoprecipitation, sucrose gradient, chromatin immunoprecipitation (ChIP), real time PCR, and immunoblotting analyses were performed to determine interactions of hTOP3α with p53. Paired cell lines with different hTOP3α levels were generated via ectopic expression and short hairpin RNA (shRNA)-mediated knockdown approaches. Cellular tumorigenic properties were analyzed using cell counting, colony formation, senescence, soft agar assays, and mouse xenograft models.Results: The hTOP3α isozyme binds to p53 and cofractionizes with p53 in gradients differing from fractions containing hTOP3α and BLM. Knockdown of hTOP3α expression (sh-hTOP3α) caused a higher anchorage-independent growth of nontumorigenic RHEK-1 cells. Similarly, sh-hTOP3α and ectopic expression of hTOP3α in cancer cell lines caused increased and reduced tumorigenic abilities, respectively. Genetic and mutation experiments revealed that functional hTOP3α, p53, and p21 are required for this tumor-suppressive activity. Mechanism-wise, ChIP data revealed that hTOP3α binds to the p53 and p21 promoters and positively regulates their expression. Two proteins affect promoter recruitments of each other and collaborate in p21 expression. Moreover, sh-hTOP3α and sh-p53 in AGS cells caused a similar reduction in senescence and hTOP3α mRNA levels were lower in gastric and renal tumor samples.Conclusion: We concluded that hTOP3α interacts with p53, regulates p53 and p21 expression, and contributes to the p53-mediated tumor suppression. Clin Cancer Res; 20(6); 1489–501. ©2014 AACR.

PDF file - 172K

PDF file - 57.5K

PDF file 1796K, Forced expression of hTOP3alpha induced expression of p5315P, p21 and {gamma}H2AX, but not ATM1981P under DNA damaging-agent stimulation

Purpose: Human DNA topoisomerase III alpha (hTOP3α) is involved in DNA repair surveillance and cell-cycle checkpoints possibly through formatting complex with tumor suppressors. However, its role in cancer development remained unsolved. Experimental Design: Coimmunoprecipitation, sucrose gradient, chromatin immunoprecipitation (ChIP), real time PCR, and immunoblotting analyses were performed to determine interactions of hTOP3α with p53. Paired cell lines with different hTOP3α levels were generated via ectopic expression and short hairpin RNA (shRNA)-mediated knockdown approaches. Cellular tumorigenic properties were analyzed using cell counting, colony formation, senescence, soft agar assays, and mouse xenograft models. Results: The hTOP3α isozyme binds to p53 and cofractionizes with p53 in gradients differing from fractions containing hTOP3α and BLM. Knockdown of hTOP3α expression (sh-hTOP3α) caused a higher anchorage-independent growth of nontumorigenic RHEK-1 cells. Similarly, sh-hTOP3α and ectopic expression of hTOP3α in cancer cell lines caused increased and reduced tumorigenic abilities, respectively. Genetic and mutation experiments revealed that functional hTOP3α, p53, and p21 are required for this tumor-suppressive activity. Mechanism-wise, ChIP data revealed that hTOP3α binds to the p53 and p21 promoters and positively regulates their expression. Two proteins affect promoter recruitments of each other and collaborate in p21 expression. Moreover, sh-hTOP3α and sh-p53 in AGS cells caused a similar reduction in senescence and hTOP3α mRNA levels were lower in gastric and renal tumor samples. Conclusion: We concluded that hTOP3α interacts with p53, regulates p53 and p21 expression, and contributes to the p53-mediated tumor suppression. Clin Cancer Res; 20(6); 1489–501. ©2014 AACR.

Purpose: The IFN-stimulated gene 15 (ISG15)- and ubiquitin-conjugation pathways play roles in mediating hypoxic and inflammatory responses. To identify interaction(s) between these two tumor microenvironments, we investigated the effect of ISG15 on the activity of the master hypoxic transcription factor HIF-1α. Experimental Design: IFN and desferoxamine treatments were used to induce the expression of ISGs and HIF-1α, respectively. Interactions between HIF-1α and the ISG15 and ISGylation system were studied using knockdown of mRNA expression, immunoblotting, coimmunoprecipitation, and pull-down analyses. Effects of the ISG15 and ISGylation system on the HIF-1α–directed processes were examined using reporter, reverse transcription polymerase chain reaction (RT-PCR), and tumorigenic growth assays. Results: We found that the level of the free form of HIF-1α is differentially regulated by IFN treatment, and that the free ISG15 level is lower under hypoxia. Mechanism-directed studies have shown that HIF-1α not only interacts physically with ISG15, but is also ISGylated in multiple domains. ISG15 expression disrupts the functional dimerization of HIF-1α and -1β. Subsequently, expression of the ISG15 and/or ISGylation system attenuates HIF-1α–mediated gene expression and tumorigenic growth. Conclusion: In summary, our results revealed cross-talk between inflammatory and hypoxic pathways through the ISGylation of HIF-1α. On the basis of these results, we propose a novel negative feedback loop for the HIF-1α–mediated pathway involving the regulation of HIF-1α via IFN-induced ISGylation. Clin Cancer Res; 19(21); 5927–39. ©2013 AACR.

Purpose: Solid tumors encounter a growth-limiting hypoxic microenvironment as they develop. Hypoxia-inducible factors (HIF) play important roles in hypoxia-associated tumor development and therapeutic resistance. Targeting the HIF pathway (especially HIF-1α) represents a promising cancer treatment strategy. Here, we report a novel class of HIF-1α inhibitors and the possible molecular basis of inhibition. Experimental Design: We analyzed the inhibitory effects of clinically used topoisomerase II (TOP2)–targeting drugs on HIF-1α expression with a primary focus on mitoxantrone. The potential role of TOP2 in mitoxantrone-inhibited HIF-1α expression was studied using pharmacologic inhibition, a knockdown approach, and TOP2 mutant cells. Moreover, involvement of mitoxantrone in proteasome-mediated degradation, transcription, and translation of HIF-1α was examined. Results: The TOP2-targeting mitoxantrone, but neither doxorubicin nor etoposide (VP-16), strongly inhibited HIF-1α expression under hypoxic conditions in a dose- and time-dependent manner. Surprisingly, the mitoxantrone-mediated inhibition of HIF-1α expression was largely independent of two TOP2 isozymes, proteasomal degradation, and transcription. Furthermore, mitoxantrone inhibited HIF-1α expression and function in a similar fashion as cycloheximide, suggesting that mitoxantrone might inhibit HIF-1α via a blockage at its translation step. In vitro translation experiments using HIF-1α mRNA further confirmed inhibition of HIF-1α translation by mitoxantrone. Interestingly, levels of the polysome-bound HIF-1α and VEGF-A mRNA were elevated and decreased after mitoxantrone treatment, respectively. Conclusions: We have identified the TOP2-targeting compound, mitoxantrone, as an HIF-1α inhibitor possibly through a translation inhibition mechanism, suggesting the possibility of an additional anticancer activity for mitoxantrone. Clin Cancer Res; 17(15); 5026–37. ©2011 AACR.

Activated inflammatory leukocytes generate a variety of reactive nitrogen and species (RNOS) which have been proposed to contribute to genome instability and cancer development. We previously reported that oxidative stress induces topoisomerase II (TOP2)-mediated DNA breaks via TOP2 cleavable complex (TOP2cc) formation. Moreover, drug-induced TOP2αcc and TOP2βcc are suggested to be anti-cancer and carcinogenic, respectively. Here we show that either inflammation-produced or chemical-released nitric oxide (NO), like TOP2-targeting etoposide (VP-16), induce TOP2cc-meidated DNA breakage, mutagenesis and carcinogenesis: (i) NO-releaser S-nitrosoglutathione (GSNO) induces formation of skin melanomas in 9,10-dimethyl-1,2-benzanthracene (DMBA)-initiated mice; (ii) the co-culture experiments revealed that inflamed macrophages causes activation of DNA damage response and chromosome DNA breakage in target cells (iii) NO production and two TOP2 isozymes are responsible for the generation of chromosome DNA breaks; (iv) consistently, treatment of a NO releaser GSNO induces hTOP2αcc and hTOP2βcc formation; (v) GSNO also increases mutation rate and kill cells in TOP2-dependent manner; and (vi) the incidences of GSNO- and VP-16-induced melanomas are found to be lower in skin-specific top2β-knockout mice. Thus, our results provide the first demonstration for the functional role of TOP2 in the NO-caused DNA damage, mutagenesis and carcinogenesis. The TOP2 involvement in pathological settings such as chronic inflammation-associated mutagenesis and subsequent cancer development were discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-394. doi:1538-7445.AM2012-LB-394

Growth factor receptor bound protein-7 (Grb7) regulates diverse cellular functions as a result of its multi-domain functionality in protein-protein interactions. A number of reports have indicated that Grb7 is essential for EGFR-mediated tumorigenesis and cancer progression. However, it remains unclear how Grb7 potentiates tumor progression. To understand the underlying mechanisms by which Grb7 participates in EGFR-mediated tumorigenesis, we utilized microarray analyses and yeast two-hybrid screening to explore Grb7-mediated signaling. Of which, a migration related protein, EphA4, was markedly up-regulated in associated with Grb7 overexpression but down-regulated while knocking down Grb7. Eventually, in response to EGF stimulation, we found that Grb7 was trans-located into the nucleus from the cytoplasm, which in turn promoted STAT3 transcriptional activity on EphA4 gene expression. The significance of this finding is further highlighted by the co-upregulation of Grb7 and EphA4 is concurrent with the highly invasive capability of lung adenocarcinoma. Moreover, we found that EphA4 could also potentiate EGF-induced reorganization of cytoskeleton, ERK1/2 phosphorylation, and cancer cell functions such as proliferation, migration and anchorage-independent growth. Based on these findings, we hypothesize that EphA4 can act as an amplifier involved EGF-mediated cancer progression in an EGF/Grb7/Stat3-mediated transcriptional manner. This study reveals a novel mechanism by which Grb7 regulates tumor progression through the EGF- by triggering EphA4 expression and then prolonging EGFR signaling in regulation of diverse cellular functions involved in cancer progression, thereby highlighting the potential strategy to target Grb7 for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-28. doi:1538-7445.AM2012-LB-28