Jinfang Ma

Expression of ABCG2 (BCRP) Is Regulated by Nrf2 in Cancer Cells That Confers Side Population and Chemoresistance Phenotype

ATP-binding cassette, subfamily G, member 2 (ABCG2) is expressed in both normal and cancer cells and plays a crucial role in side population (SP) formation and efflux of xenobiotics and drugs. Nrf2, a redox-sensing transcription factor, on constitutive activation in non–small-cell lung cancer cells upregulates a wide spectrum of genes involved in redox balance, glutathione metabolism, and drug detoxification, which contribute to chemoresistance and tumorigenicity. This study examined the mechanism underlying Nrf2-dependent expression of ABCG2 and its role in the multidrug resistance phenotype. In silico analysis of the 5′-promoter flanking region of ABCG2 identified an antioxidant response element (ARE) at −431 to −420 bp. A detailed promoter analysis using luciferase reporter assays showed that ARE at −431 to −420 bp is critical for the Nrf2-mediated expression in lung cancer cells. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays revealed that Nrf2 interacts with the ABCG2 ARE element at −431 to −420 bp in vitro and in vivo. Disruption of Nrf2 expression in lung and prostate cancer cells, by short hairpin RNA, attenuated the expression of ABCG2 transcript and protein, and dramatically reduced the SP fraction in Nrf2-depleted cancer cells. Moreover, depleted levels of ABCG2 in these Nrf2 knockdown cells sensitized them to mitoxantrone and topotecan, two chemotherapy drugs detoxified mainly by ABCG2. As expected, overexpression of Nrf2 cDNA in lung epithelial cells led to an increase in ABCG2 expression and a 2-fold higher SP fraction. Thus, Nrf2-mediated regulation of ABCG2 expression maintains the SP fraction and confers chemoresistance. Mol Cancer Ther; 9(8); 2365–76. ©2010 AACR.

Exposure to Electronic Cigarettes Impairs Pulmonary Anti-Bacterial and Anti-Viral Defenses in a Mouse Model

Electronic cigarettes (E-cigs) have experienced sharp increases in popularity over the past five years due to many factors, including aggressive marketing, increased restrictions on conventional cigarettes, and a perception that E-cigs are healthy alternatives to cigarettes. Despite this perception, studies on health effects in humans are extremely limited and in vivo animal models have not been generated. Presently, we determined that E-cig vapor contains 7x1011 free radicals per puff. To determine whether E-cig exposure impacts pulmonary responses in mice, we developed an inhalation chamber for E-cig exposure. Mice that were exposed to E-cig vapor contained serum cotinine concentrations that are comparable to human E-cig users. E-cig exposure for 2 weeks produced a significant increase in oxidative stress and moderate macrophage-mediated inflammation. Since, COPD patients are susceptible to bacterial and viral infections, we tested effects of E-cigs on immune response. Mice that were exposed to E-cig vapor showed significantly impaired pulmonary bacterial clearance, compared to air-exposed mice, following an intranasal infection with Streptococcus pneumonia. This defective bacterial clearance was partially due to reduced phagocytosis by alveolar macrophages from E-cig exposed mice. In response to Influenza A virus infection, E-cig exposed mice displayed increased lung viral titers and enhanced virus-induced illness and mortality. In summary, this study reports a murine model of E-cig exposure and demonstrates that E-cig exposure elicits impaired pulmonary anti-microbial defenses. Hence, E-cig exposure as an alternative to cigarette smoking must be rigorously tested in users for their effects on immune response and susceptibility to bacterial and viral infections.

Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients

5 5 2 1 jci.org Volume 124 Number 12 December 2014 Retraction Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients Deepti Malhotra, Rajesh K. Thimmulappa, Nicolas Mercado, Kazuhiro Ito, Ponvijay Kombairaju, Sarvesh Kumar, Jinfang Ma, David Feller-Kopman, Robert Wise, Peter Barnes, and Shyam Biswal Original citation: J Clin Invest. 2011;121(11):4289–4302. doi:10.1172/JCI45144. Citation for this retraction: J Clin Invest. 2014;124(12):5521. doi:10.1172/JCI79606. The JCI, with the agreement of the corresponding authors and coauthors, is retracting the article “Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients.” The IP: streptavidin/IB: anti-HDAC2 immunoblot panel in Figure 3B was recently identified as an inverted image of the right four lanes of the anti-H4 acetyl CHIP gel presented in Figure 2A. Additionally, the IP: anti-streptavidin/IB: anti-DDK immunoblot presented in Supplemental Figure 3A was identified as an inverted image of the left four lanes of the anti-H4 acetyl CHIP gel presented in Figure 2A. Further, in Figure 9B, the incorrect anti-HDAC2 immuno blot was presented. The authors sincerely apologize for any misinterpretation of the data as a result of these errors.

Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients (Retracted article. See vol. 124, pg. 5521, 2014)

Chronic obstructive pulmonary disease (COPD), which is caused primarily by cigarette smoking, is a major health problem worldwide. The progressive decline in lung function that occurs in COPD is a result of persistent inflammation of the airways and destruction of the lung parenchyma. Despite the key role of inflammation in the pathogenesis of COPD, treatment with corticosteroids - normally highly effective antiinflammatory drugs - has little therapeutic benefit. This corticosteroid resistance is largely caused by inactivation of histone deacetylase 2 (HDAC2), which is critical for the transrepressive activity of the glucocorticoid receptor (GR) that mediates the antiinflammatory effect of corticosteroids. Here, we show that in alveolar macrophages from patients with COPD, S-nitrosylation of HDAC2 is increased and that this abolishes its GR-transrepression activity and promotes corticosteroid insensitivity. Cys-262 and Cys-274 of HDAC2 were found to be the targets of S-nitrosylation, and exogenous glutathione treatment of macrophages from individuals with COPD restored HDAC2 activity. Treatment with sulforaphane, a small-molecule activator of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), was also able to denitrosylate HDAC2, restoring dexamethasone sensitivity in alveolar macrophages from patients with COPD. These effects of sulforaphane were glutathione dependent. We conclude that NRF2 is a novel drug target for reversing corticosteroid resistance in COPD and other corticosteroid-resistant inflammatory diseases.

Plasma miRNA as Biomarkers for Assessment of Total-Body Radiation Exposure Dosimetry

The risk of radiation exposure, due to accidental or malicious release of ionizing radiation, is a major public health concern. Biomarkers that can rapidly identify severely-irradiated individuals requiring prompt medical treatment in mass-casualty incidents are urgently needed. Stable blood or plasma-based biomarkers are attractive because of the ease for sample collection. We tested the hypothesis that plasma miRNA expression profiles can accurately reflect prior radiation exposure. We demonstrated using a murine model that plasma miRNA expression signatures could distinguish mice that received total body irradiation doses of 0.5 Gy, 2 Gy, and 10 Gy (at 6 h or 24 h post radiation) with accuracy, sensitivity, and specificity of above 90%. Taken together, these data demonstrate that plasma miRNA profiles can be highly predictive of different levels of radiation exposure. Thus, plasma-based biomarkers can be used to assess radiation exposure after mass-casualty incidents, and it may provide a valuable tool in developing and implementing effective countermeasures.

The Twist box domain is required for Twist1-induced prostate cancer metastasis

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial–mesenchymal transition (EMT) that promotes cancer metastasis. Structure–function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Implications: Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential. Mol Cancer Res; 11(11); 1387–400. ©2013 AACR.

Prolonged sulforaphane treatment does not enhance tumorigenesis in oncogenic K-ras and xenograft mouse models of lung cancer

Background: Sulforaphane (SFN), an activator of nuclear factor erythroid-2 related factor 2 (Nrf2), is a promising chemopreventive agent which is undergoing clinical trial for several diseases. Studies have indicated that there is gain of Nrf2 function in lung cancer and other solid tumors because of mutations in the inhibitor Kelch-like ECH-associated protein 1 (Keap1). More recently, several oncogenes have been shown to activate Nrf2 signaling as the main prosurvival pathway mediating ROS detoxification, senescence evasion, and neoplastic transformation. Thus, it is important to determine if there is any risk of enhanced lung tumorigenesis associated with prolonged administration of SFN using mouse models of cancer. Materials and Methods: We evaluated the effect of prolonged SFN treatment on oncogenic K-ras (K-rasLSL-G12D)-driven lung tumorigenesis. One week post mutant-K-ras expression, mice were treated with SFN (0.5 mg, 5 d/wk) for 3 months by means of a nebulizer. Fourteen weeks after mutant K-ras expression (K-rasLSL-G12D), mice were sacrificed, and lung sections were screened for neoplastic foci. Expression of Nrf2-dependent genes was measured using real time RT-PCR. We also determined the effect of prolonged SFN treatment on the growth of preclinical xenograft models using human A549 (with mutant K-ras and Keap1 allele) and H1975 [with mutant epidermal growth factor receptor (EGFR) allele] nonsmall cell lung cancer cells. Results: Systemic SFN administration did not promote the growth of K-rasLSL-G12D-induced lung tumors and had no significant effect on the growth of A549 and H1975 established tumor xenografts in nude mice. Interestingly, localized delivery of SFN significantly attenuated the growth of A549 tumors in nude mice, suggesting an Nrf2-independent antitumorigenic activity of SFN. Conclusions: Our results demonstrate that prolonged SFN treatment does not promote lung tumorigenesis in various mouse models of lung cancer.

Abstract 1493: The Twist box is required for Twist1-induced prostate cancer metastasis.

The Twist1 gene has diverse roles during development and pathologic states such as cancer. Twist1 is best known for its roles in cancer by inducing an epithelial-mesenchymal transition (EMT) transcriptional program implicated in facilitating tumorigenesis, tumor progression and treatment resistance. Twist1 is a bHLH transcription factor that has both repressor and transactivation functions, but the importance of these different activities for Twist1 cancer phenotypes are unknown. We hypothesized Twist1 may mediate these various functions using distinct structural domains and/or motifs. We disrupted the putative transactivation domain in the Twist box of Twist1 by mutating a critical phenylalanine residue (F191) to glycine. We then created stable isogenic prostate cancer cell lines overexpressing wildtype and F191G versions of Twist1. We assessed the role of the Twist box using in vitro and in vivo assays, which mimic the various stages of cancer progression to metastasis. These include loss of homotypic cell-cell contacts, cell migration and invasion, anoikis resistance and soft agar colony formation. We also observed biophysical cell traction forces on a fabricated substratum and finally performed experimental lung metastasis assays. The overexpression of Twist1 in prostate cancer cells lead to an EMT biomarker phenotype and the F191G mutant lacked expression of some of these markers. The F191G mutant was deficient for transcriptional activity using promoter reporter based assays. Using single cell measurements we found that Twist1 expressing Myc-CaP cells exert more force on the substratum than vector control cells. Additional in vitro assays suggest Twist1 can confer cellular properties associated with increased tumor aggressiveness including increased migration/invasion, cell death/anoikis resistance and in vitro tumorigenic potential by soft agar colony formation. The Twist box mutant, F191G, displayed compromised activity compared to wildtype Twist1 in many of the in vitro assays described above revealing that the Twist box is necessary for many of the pro-metastatic functions of Twist1. We compared the gene expression profile of Twist1 and F191G overexpressing prostate cancer cells by microarray and observed that the F191G mutant had an expression profile that was similar to wildtype Twist1 but attenuated. Lastly, Twist1 overexpression compared to vector control prostate cancer cells showed an increased frequency of metastatic lung tumors using the experimental lung metastasis assay. Interestingly, Twist1 overexpression also resulted in the appearance of extra-thoracic metastases. The F191G mutant was less able to confer prostate cancer cells the ability to colonize metastatic lesions in the lung and resulted in no extra-thoracic metastases. Our results show that F191G mutation behaves as loss of function and is necessary for Twist1-induced metastasis of prostate cancer cells. Citation Format: Rajendra P. Gajula, Sivarajan T. Chettiar, Russell D. Williams, Saravanan Thiyagarajan, Yoshinori Kato, Khaled Aziz, Ruoqi Wang, Nishant Gandhi, Aaron T. Wild, Farhad Vesuna, Jinfang Ma, Tarek Salih, Jessica Cades, Elana Fertig, Shyam Biswal, Timothy F. Burns, Christine Chung, Charles M. Rudin, Venu Raman, Joseph M. Herman, Russell K. Hales, Steven An, Phuoc T. Tran. The Twist box is required for Twist1-induced prostate cancer metastasis. [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 1493. doi:10.1158/1538-7445.AM2013-1493