Jia Kang

Mitochondria: Redox Metabolism and Dysfunction

Mitochondria are the main intracellular location for fuel generation; however, they are not just power plants but involved in a range of other intracellular functions including regulation of redox homeostasis and cell fate. Dysfunction of mitochondria will result in oxidative stress which is one of the underlying causal factors for a variety of diseases including neurodegenerative diseases, diabetes, cardiovascular diseases, and cancer. In this paper, generation of reactive oxygen/nitrogen species (ROS/RNS) in the mitochondria, redox regulatory roles of certain mitochondrial proteins, and the impact on cell fate will be discussed. The current state of our understanding in mitochondrial dysfunction in pathological states and how we could target them for therapeutic purpose will also be briefly reviewed.

Bcl-2: A prime regulator of mitochondrial redox metabolism in cancer cells

SIGNIFICANCE Mitochondria play a critical role as death amplifiers during drug-induced apoptosis in cancer cells by providing pro-apoptotic factors that are released from the mitochondrial inter-membranous space upon the induction of mitochondrial outer membrane permeabilization. This intrinsic death signaling pathway is the preferred mechanism employed by most anticancer compounds, and as such, resistance to drug-induced apoptosis is invariably associated with inhibition of mitochondrial death signaling network. The latter is a function of a balance between the pro- and the anti-apoptotic members of the Bcl-2 family. Bcl-2 is the prototype anti-apoptotic protein that localizes to the mitochondria and blocks the recruitment and activation of pro-apoptotic proteins, such as Bax, to the mitochondria. RECENT ADVANCES AND CRITICAL ISSUES Recent evidence has highlighted a novel mechanism of anti-apoptotic activity of Bcl-2 in addition to its canonical activity in regulating mitochondrial outer membrane permeabilization. This novel activity is a function of cellular redox regulation, in particular, mitochondrial metabolism in cancer cells. FUTURE DIRECTIONS Here we review the current state of our understanding of the death inhibitory activity of Bcl-2 and provide insight into the novel functional biology of this remarkable protein, which could have implications for designing innovative strategies to overcome the problem of drug resistance in the clinical settings.

The small GTPase Rac1 is a novel binding partner of Bcl-2 and stabilizes its antiapoptotic activity

The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis in leukemia cells by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We report here that silencing and functional inhibition of Rac1 block Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. Using confocal, electron microscopy and coimmunoprecipitation, as well as glutathione S-transferase-fusion proteins, we provide evidence for a colocalization and physical interaction between the 2 proteins. This interaction is blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3 peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia cells and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. These data provide a novel facet in the biology of Bcl-2 with potential implications for targeted anticancer drug design.

Crosstalk between Bcl-2 family and Ras family small GTPases: potential cell fate regulation?

Cell fate regulation is a function of diverse cell signaling pathways that promote cell survival and or inhibit cell death execution. In this regard, the role of the Bcl-2 family in maintaining a tight balance between cell death and cell proliferation has been extensively studied. The conventional dogma links cell fate regulation by the Bcl-2 family to its effect on mitochondrial permeabilization and apoptosis amplification. However, recent evidence provide a novel mechanism for death regulation by the Bcl-2 family via modulating cellular redox metabolism. For example overexpression of Bcl-2 has been shown to contribute to a pro-oxidant intracellular milieu and down-regulation of cellular superoxide levels enhanced death sensitivity of Bcl-2 overexpressing cells. Interestingly, gene knockdown of the small GTPase Rac1 or pharmacological inhibition of its activity also reverted death phenotype in Bcl-2 expressing cells. This appears to be a function of an interaction between Bcl-2 and Rac1. Similar functional associations have been described between the Bcl-2 family and other members of the Ras superfamily. These interactions at the mitochondria provide novel opportunities for strategic therapeutic targeting of drug-resistant cancers.

Overexpression of Bcl-2 induces STAT-3 activation via an increase in mitochondrial superoxide

We recently reported a novel interaction between Bcl-2 and Rac1 and linked that to the ability of Bcl-2 to induce a pro-oxidant state in cancer cells. To gain further insight into the functional relevance of this interaction, we utilized computer simulation based on the protein pathway dynamic network created by Cellworks Group Inc. STAT3 was identified among targets that positively correlated with Rac1 and/or Bcl-2 expression levels. Validating this, the activation level of STAT3, as marked by p-Tyr705, particularly in the mitochondria, was significantly higher in Bcl-2-overexpressing cancer cells. Bcl-2-induced STAT3 activation was a function of GTP-loaded Rac1 and NADPH oxidase (Nox)-dependent increase in intracellular superoxide (O2•−). Furthermore, ABT199, a BH-3 specific inhibitor of Bcl-2, as well as silencing of Bcl-2 blocked STAT3 phosphorylation. Interestingly, while inhibiting intracellular O2•− blocked STAT3 phosphorylation, transient overexpression of wild type STAT3 resulted in a significant increase in mitochondrial O2•− production, which was rescued by the functional mutants of STAT3 (Y705F). Notably, a strong correlation between the expression and/or phosphorylation of STAT3 and Bcl-2 was observed in primary tissues derived from patients with different sub-sets of B cell lymphoma. These data demonstrate the presence of a functional crosstalk between Bcl-2, Rac1 and activated STAT3 in promoting a permissive redox milieu for cell survival. Results also highlight the potential utility of a signature involving Bcl-2 overexpression, Rac1 activation and STAT3 phosphorylation for stratifying clinical lymphomas based on disease severity and chemoresistance.

Abstract 3097: Structural and biophysical characterization of anti-apoptotic protein Bcl-2 and GTPase Rac1 interaction

We have recently reported a novel mechanism linking the pro-oxidant activity of the anti-apoptotic protein Bcl-2 to its interaction with the small GTPase Rac1. In this current study, we investigate the nature of this interaction using activated (GTP loaded) Rac1 and Bcl-2 peptides (spanning the BH3 domain and its adjacent loop region) using coimmunoprecipitation, isothermal titration calorimetry (ITC), X-ray crystallography, docking/modeling, fluorescence spectroscopy, molecular dynamics and mutational studies. Our results will develop to translational relevance in the treatment of human cancers that are refractory to chemotherapy due to overexpression of Bcl-2. Citation Format: Shani Ajumal, Jia Kang, Thomas Joseph, Shazib Pervaiz, Kunchithapadam Swaminathan. Structural and biophysical characterization of anti-apoptotic protein Bcl-2 and GTPase Rac1 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3097.

Abstract 13: Biophysical evidence for the existence of a functional interaction between the small GTPase Rac-1 and the anti-apoptotic protein Bcl-2

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We recently reported a novel mechanism of anti-apoptotic activity of Bcl-2 by demonstrating a link between an altered mitochondrial metabolism and apoptosis resistance in cells overexpressing Bcl-21. This increase in mitochondrial metabolism was linked to induction of complex IV activity (Cytochrome C oxidase) as well as oxygen consumption2. The pro-oxidant activity of Bcl-2 was a function of an interaction with the small GTPase Rac1. This interaction was validated in a small number of clinical tissues derived from patients with aggressive B cell lymphomas. Interestingly, interrupting the interaction between Bcl-2 and Rac1 restored apoptosis sensitivity of Bcl-2 overexpressing cell lines3. In the present study we set out to identify the domains within the two proteins responsible for the interaction. We show that the pro-oxidant activity of Bcl-2 and its interaction with Rac1 are dependent on the phosphorylation of Bcl-2 at serine 70 within the non-structured loop region adjacent to the BH3 domain. Also, the activation status of Rac1 appears important in this interaction as GDP loading almost completely inhibits, whereas GTP loading facilitates interaction. To further substantiate those findings, isothermal titration calorimetry (ITC) was carried out using Rac1-GTPγS and Bcl-2 BH3 peptides to gain insight into the energetics of binding of BH3 peptide to Rac1-GTPγS. Indeed, the thermodynamic parameters ΔH and ΔS in the ITC experiments of Rac1- GTPγS and BH3 peptide were -2.55 × 104 Joules/mol and +35.6 joules/mol/deg, respectively, and the ΔG values were negative, indicating that the binding of Rac1-GTPγS with BH3 peptide was spontaneous and exothermic and that hydrophobic interactions played a major role; however, hydrogen bonds could not be discounted due to the positive ΔS value and the presence of polar amino acids in the peptide. Similar results were obtained with the S70 phosphorylation mimetic peptide of Bcl-2 (S70E) but not with the S70A phosphorylation dead peptide. Furthermore, the fluorescence intensity of Rac1 decreased steadily with increasing Bcl-2 peptide concentration, indicating an interaction between Rac1 and Bcl-2 peptides. These data provide strong evidence for the existence of a functional interaction between Bcl-2 and Rac1 and for the translational relevance of this interaction in human cancers that are refractory to chemotherapy due to the overexpression of Bcl-2. 1. Chen, Z, X. and Pervaiz, S. Cell Death Diff. 14(9): 1617-27, 2007. 2. Chen ZX, and Pervaiz S. Cell Death Differ. 2010 Mar;17(3):408-20. 3. Velaithan R, et al. Blood. 2011 Jun 9;117(23):6214-26. Citation Format: Jia Kang, Shani Ajumal, Kunchithapadam Swaminathan, Shazib Pervaiz. Biophysical evidence for the existence of a functional interaction between the small GTPase Rac-1 and the anti-apoptotic protein Bcl-2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 13. doi:10.1158/1538-7445.AM2015-13

Abstract 5577: STAT3 phosphorylation and Bcl-2 expression as a predictive signature for stratifying clinical lymphomas.

Overexpression of Bcl-2 presents a major therapeutic challenge in the management of hematopoietic and other malignancies. By conventional dogma Bcl-2 blocks drug-induced apoptosis by inhibiting mitochondrial permeabilization and downstream death amplification. Recently we demonstrated a novel mechanism of death inhibition by Bcl-2 via inducing an increase in mitochondrial oxygen consumption and superoxide production. Interestingly, transfection of cells with a dominant negative mutant of Rac1 or its knockdown sensitized Bcl-2 expressing cells to apoptosis. Furthermore, using a human cancer cell lines and tissue biopsies from patients with a variety of lymphomas revealed that Rac1 was localized at the mitochondria and interacted with Bcl-2. Intrigued by these data from clinical lymphomas we set out to investigate the downstream effector pathways involved in Bcl-2-Rac-1 interaction-mediated survival signaling. We took two independent approaches to address this. Firstly, we employed a computer simulation driven predictive experimental strategy based on the protein pathway dynamic network of cancer physiology created by Cellworks Group Inc. An HCT116 human colorectal cancer cell line with KRAS over-activation, PI3K overexpression, CDKN2A deletion, β-catenin overexpression and Bcl-2 overexpression was created as a baseline. Phenotypic indices of cell proliferation, viability, metastasis, and angiogenesis were amplified in a variant cell line when Rac1 was overexpressed based on the above computer modeled HCT116. Of note, the expression level of the cell proliferation inducer/transcription factor STAT3 and β-catenin were significantly upregulated in the variant cell line; however knockdown of either Bcl-2 or Rac1 in both the base and the variant cell lines resulted in a significant increase in protein involved in apoptosis signaling. Based on these pharmacodynamic computer simulation driven predictions, we set out to verify the correlation between STAT3 signaling (its phosphorylation at Y705 and S727) and Bcl-2-Rac1 interaction. Indeed, we obtain evidence for a cause-en effect relationship between Rac1 activation, Bcl-2 phosphorylation at S70 and the activation of STAT3. More importantly, using primary cells from patients with a variety of lymphomas, we analyzed the activation of STAT3 and Bcl-2 expression and asked if a correlation existed in malignant lymphomas. Interestingly, based on a small number of clinical samples, there appears to be tight correlation between the phosphorylation status of STAT3 and the expression of Bcl-2 in a specific sub-group of B cell lymphomas. These data provide strong translational relevance for a novel predictive signature for stratifying lymphomas with potential clinical implications and validate the use of computer modeling to predict clinical outcomes. Citation Format: Jia Kang, Vignette Z. Q. Ooi, Shireen Vali, Shweta Kapoor, Ansu Kumar, Taher Abbasi, Shazib Pervaiz. STAT3 phosphorylation and Bcl-2 expression as a predictive signature for stratifying clinical lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5577. doi:10.1158/1538-7445.AM2013-5577 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2003: STAT3 is a critical mediator of the pro-survival effect of Bcl-2-Rac1 interaction in human cancer cells

We recently showed that Bcl-2 overexpression induced a pro-oxidant intracellular milieu resulting in an inhibition of apoptotic execution and that reversing the pro-oxidant milieu by inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We provide evidence that the small GTPase Rac1 is functionally involved since gene silencing and functional inhibition of Rac1 blocked Bcl-2-mediated increase in intracellular superoxide production in tumor cells. A co-localization and physical interaction between the two proteins were observed, which could be blocked by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides both in vitro and in vivo. The Bcl-2 BH3 peptides as well as silencing and functional inhibition of Rac1 reversed intracellular superoxide levels and overcame Bcl-2-mediated drug resistance in human leukemia and cervical cancer cells, demonstrating that this interaction is functionally relevant. Notably, the interaction was observed in primary cells from patients diagnosed with B-cell lymphoma with Bcl-2 overexpression but not in noncancerous tissue. Furthermore, computer simulation driven virtual predictive experiments based on the protein pathway dynamic network created by Cellworks Group Inc. were carried out to study the functional implication of this interaction. An HCT116 human colorectal cancer cell line with KRAS over-activation, PI3K overexpression, CDKN2A deletion, β-catenin overexpression and Bcl-2 overexpression was created as a base line. Phenotypic indices of angiogenesis, proliferation, viability, metastasis as well as tumor volume were all found to be amplified in a variant cell line when Rac1 was overexpressed based on the above computer modeled HCT116. Strikingly, the expression levels of both STAT3 and β-catenin were significantly upregulated in the variant cell line; however upon Rac1 or Bcl-2 knockdown in both the base and the variant cell lines, apoptotic markers such as Bax, caspase 3 and cleaved PARP1 were amplified while STAT3 and β-catenin were significantly down-regulated. Intrigued by these predictions, we set out to verify the correlation between STAT3 signaling and Bcl-2-Rac1 interaction. Interestingly, there is indeed a strong correlation between the activation status of STAT3 (pY705) and Bcl-2 expression as well as Rac1 activation status. Furthermore, STAT3 was found to be localized to mitochondria and our preliminary results suggest an association with Bcl-2. 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 2003. doi:1538-7445.AM2012-2003

Abstract C149: STAT3 is a critical mediator of the prosurvival effect of Bcl-2-Rac1 interaction in human cancer cells.

The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. Gene silencing and functional inhibition of Rac1 blocked Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. We provide evidence for a co-localization and physical interaction between the 2 proteins, which could be blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. In an attempt to study the functional implication of this interaction, we carried out a computer simulation driven virtual predictive experiments based on the protein pathway dynamic network created by Cellworks Group Inc. The base line used for the study was a KRAS over-activated, PI3K overexpressed, CDKN2A deleted, β-catenin overexpressed and Bcl-2 overexpressed system aligned to HCT116 human colorectal cancer cell line. When a variant of the above base line was created with Rac1 overexpression, phenotypic indices of angiogenesis, proliferation, viability, metastasis as well as tumor volume were all upregulated. Strikingly, the expression levels of STAT3 and β-catenin were significantly amplified in the computer modeled variant cell line; however upon Rac1 or Bcl-2 knockdown in both the base and the variant cell lines, apoptotic markers such as Bax, caspase 3 and cleaved PARP1 were increased while STAT3, β-catenin, NF-κB and AGER were significantly down-regulated. Intrigued by these predictions, we set out to verify the link between STAT3 signaling and Bcl-2-Rac1 interaction. Interestingly, the activation status of STAT3 (pY705) strongly correlated with Bcl-2 expression as well as Rac1 activation status. Furthermore, STAT3 was found localized to mitochondria and preliminary results suggest an interaction with Bcl-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C149.