HUMAN IMMUNODEFICIENCY VIRUS-1 TAT PROTEIN UPREGULATES IL-6 AND IL-8 EXPRESSION IN HUMAN BREAST CANCER CELLS Yong Woo Lee 1, Natasha Kyprianou 1, Avindra Nath 2, and Michal Toborek 1 1 Department of Surgery, University of Kentucky, Lexington, KY, 2 Department of Neurology, Johns Hopkins University, Baltimore, MD ABSTRACT The aim of the present study It has been proposed that the inflammatory tumor microenvironment via overexpression of inflammatory mediators is associated with progression of breast cancer metastasis in human immunodeficiency virus (HIV)-infected women. The cellular and molecular regulatory mechanisms underlying this process, however, are not fully understood. In the present study, we hypothesize that HIV-1 Tat protein may increase the metastatic potential of human breast cancer cells through upregulation of cytokines and chemokines that induce the inflammatory tumor microenvironment. Real-time reverse transcriptase-polymerase chain reaction and the quantitative sandwich enzyme immunoassay analyses showed that exposure of MDA-MB-231 cells to HIV-1 Tat protein resulted in a significant and dose-dependent upregulation of interleukin-6 (IL-6) and interleukin-8 (IL-8) mRNA and protein expression. HIV-1 Tat protein also markedly increased NF-kB DNA-binding activity and transactivation in MDA-MB-231 cells. Additionally, pretreatment with NF-kB inhibitors significantly attenuated the ability of HIV-1 Tat protein to upregulate IL-6 and IL-8 expression. These results suggest that HIV-1 Tat protein upregulates expression of IL-6 and IL-8 in human breast cancer cells by NF-kB- dependent pathway. These data may contribute to exploration of the new molecular mechanisms leading to novel approaches for the therapeutic drug developments specifically targeted against the inflammatory pathways of breast cancer metastasis in AIDS patients. INTRODUCTION It has been proposed that the cancer metastasis can be regulated by the inflammatory tumor microenvironment. For example, significantly higher levels of pro-inflammatory cytokines, such as TNF- , IL-1  and IL-6, were detected in patients with metastatic breast cancer. Additionally, it was demonstrated a critical role for IL-8 in promoting the metastatic potential of breast cancer cells. These basic and clinical studies support the idea that inflammatory tumor microenvironment via overexpression of inflammatory mediators may play a crucial role in the establishment of breast cancer metastasis. Even though the overall health and survival of HIV-infected individuals have improved since the introduction of highly active antiretroviral therapy (HAART), the effect of HAART on the cancer incidence rates in HIV-infected people remains unclear. Recent studies have raised concerns that HAART has shifted the spectrum of HIV-related diseases from opportunistic infections toward longer-term complications of HIV such as malignancies. Indeed, several epidemiological data indicate that the risk of development of breast cancer is increased in HIV-infected patients. Breast cancer appears to be more aggressive in HIV- positive women while HIV infection does not have a direct tumorigenic effect on the pathogenesis of breast cancer. These results suggest that HIV infection may play a potential role in the progression of breast cancer metastasis. Previous in vitro and in vivo studies have demonstrated an association between inflammatory pathways with breast cancer progression and metastasis in HIV-infected individuals. The cellular and molecular mechanisms underlying this process, however, are not fully understood. To investigate the effects of HIV-1 Tat protein on the inflammatory tumor microenvironment, which is critical for the development of breast cancer metastasis. 1. Cells : Human breast cancer cells, MDA-MB HIV-1 Tat protein : Recombinant Tat Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) – Gene specific TaqMan® assay 4. Enzyme-linked immunosorbent assay (ELISA) 5. Electrophoretic mobility shift assay (EMSA) 6. Transient transfection and dual luciferase assay Experimental Procedures Tat upregulates expression of IL-6 and IL-8 in MDA-MB-231 cells (A) (B) Cycle Number Delta Rn Cycle Number Control Tat, 1.0 Tat, 10 Tat, 100 Control Tat, 1.0 Tat, 10 Tat, 100 IL-6 IL-8 (A) (B) HIV-1 Tat protein increases production of interleukin-6 (IL-6) and interleukin-8 (IL-8) protein in human breast cancer cells. MDA-MB-231 cells were treated with the indicated concentrations of HIV-1 Tat protein (1.0, 10 and 100 nM) for 24 h. Aliquots of culture media were collected and the concentrations of IL-6 (A) and IL-8 (B) were measured by ELISA. Data shown are means ± SD of 6 determinations. *Statistically significant compared with the control cultures. HIV-1 Tat protein upregulates the mRNA expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) in human breast cancer cells. MDA-MB-231 cells were either untreated or treated with the indicated concentrations of HIV-1 Tat protein (1.0, 10 and 100 nM) for 4 h. The mRNA levels of IL-6 (A) and IL- 8 (B) were determined by quantitative real-time reverse transcriptase- polymerase chain reaction (RT-PCR). Upper panels: Amplification plots of real-time PCR analysis for the detection of IL-6 and IL-8 mRNA. The fluorescence emission (Delta Rn) is plotted against the cycle number. Lower panels: Relative quantification of IL-6 (A) and IL-8 (B) mRNA expression. Using the 2 –ΔΔCT method, the data are presented as the fold change in gene expression normalized to a housekeeping gene,  -actin, and relative to the untreated control. Data shown are means ± SE of 4 determinations. *Statistically significant compared with the control cultures. Tat increases NF-  B DNA-binding and transactivation in MDA-MB-231 cells Probe Tat (nM) Competitor (A) (B) (C) A: Representative electrophoretic mobility shift assay (EMSA) analysis of the effects of HIV-1 Tat protein on NF-  B DNA-binding activity in human breast cancer cells. MDA-MB-231 cells were either untreated or treated with the indicated concentrations of HIV-1 Tat protein (1.0, 10 and 100 nM) for 2 h. Nuclear extracts were prepared and analyzed by EMSA. Competition study was performed by the addition of excess unlabeled NF-  B DNA-binding consensus sequence using nuclear extracts from cells treated with 100 nM Tat. B: Densitometric quantification of EMSA analysis. Experiments were repeated four times, and the intensities of the NF-  B-specific bands were measured and statistically analyzed. The results are expressed as -fold increase over control values. Data shown are the means ± SE. *Statistically significant as compared with control cultures. C: HIV-1 Tat protein increases NF-  B transactivation in human breast cancer cells as measured by transient transfection and dual luciferase assay. MDA-MB-231 cells were transfected with the p[NF-  B]Luc plasmid and treated with increasing concentrations of HIV-1 Tat protein (1.0, 10 and 100 nM) for 24 h. The cells also were co-transfected with the internal Renilla luciferase control vector (pRL-SV40) to normalize transfection rates. Values represent means ± SE of 4 determinations. *Statistically significant as compared with control cultures. NF-  B inhibitors attenuate Tat-mediated upregulation of IL-6 and IL-8 expression in MDA-MB-231 cells (A) (B) NF-  B inhibitors attenuate mRNA expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) in human breast cancer cells stimulated with HIV-1 Tat protein. MDA-MB-231 cells were pretreated with the indicated concentrations of NF-  B inhibitors, such as TPCK (A) or TLCK (B), for 1 h and exposed to 100 nM HIV-1 Tat protein for 4 h. The mRNA levels of IL-6 (upper panels) and IL-8 (lower panels) were determined by real-time reverse transcriptase- polymerase chain reaction (RT-PCR). Data are means ± SE of 4 determinations. *Statistically significant as compared with control cultures. #Values in the groups treated with Tat plus TPCK or TLCK are significantly different from the Tat-treated group. (A) (B) NF-  B inhibitors attenuate protein expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) in human breast cancer cells stimulated with HIV-1 Tat protein. MDA-MB-231 cells were pretreated with the indicated concentrations of NF-  B inhibitors, such as TPCK (A) or TLCK (B), for 1 h and exposed to 100 nM HIV-1 Tat protein for 24 h. Aliquots of culture media were collected and the concentrations of IL-6 (upper panels) and IL-8 (lower panels) were measured by ELISA. Data are means ± SD of 6 determinations. *Statistically significant as compared with control cultures. #Values in the groups treated with Tat plus TPCK or TLCK are significantly different from the Tat-treated group. The present study provides the first evidence for the role of HIV-1 Tat protein to upregulate IL-6 and IL-8 expression in human breast cancer cells by triggering NF-  B-mediated molecular signaling pathways. These results may open up new areas of investigation in AIDS- related cancer research and lead to the development of an effective clinical strategy toward the prevention and/or treatment of disease progression specifically targeted against the inflammatory pathways of breast cancer metastasis in AIDS patients. CONCLUSION 1.HIV-1 Tat protein significantly upregulates mRNA and protein expression of interleukin-6 and interleukin-8 in a dose-dependent manner in human breast cancer cells. 2. HIV-1 Tat protein markedly enhances NF-  B DNA-binding activity and facilitates NF-  B-mediated transcription in human breast cancer cells. 3. NF-  B inhibitors, TPCK or TLCK, significantly inhibits Tat- induced overexpression of IL-6 and IL-8 in human breast cancer cells. SUMMARY This work was supported by NIH COBRE P20 RR 15592, NS39254, MH63022, and AA ACKNOWLEDGEMENTS