Farnesyltransferase Inhibitor Manumycin TargetsIL1β-Ras-HIF-1α Axis in Tumor Cells of Diverse Origin

Vivek Sharma,1 Sk. Sudipta Shaheen,1 Deobrat Dixit,1 and Ellora Sen1,2

Abstract—We have recently reported that Ras acts as an intermediate coactivator in IL-1β-mediatedhypoxia-inducible factor-1α (HIF-1α) activation in glioblastoma multiforme (GBM). Since HIF-1αplays a crucial role in linking inflammatory and oncogenic pathways, we investigated whether thisIL1β-Ras-HIF-1α signaling axis observed in GBM also exists in other tumors of diverse originunder normoxia. Treatment with IL-1β induced Ras in non-GBM cell lines A549 (lung), HeLa(cervical), and HepG2 (liver), and inhibition of Ras activity attenuated HIF-1α activity. Our findingssuggest that Ras links IL-1β and HIF-1α in tumors of diverse origin. As we have previouslyreported that the farnesyltransferase inhibitor manumycin decreases Ras activity in glioma cells, weinvestigated whether manumycin could regulate IL-1β-mediated HIF-1α activation. Manumycinabrogated IL-1β-induced HIF-1α activation in both glioma and non-glioma tumor cells. In addition,manumycin also decreased IL-1β induced pro-inflammatory responses in tumor cells.

KEY WORDS: Glioblastoma; HIF-1α; Ras; IL-1β.

INTRODUCTION

Hypoxia-inducible factor-1 (HIF-1) is a hetero-dimeric transcription factor composed of HIF-1α andHIF-1β subunits [1]. In solid tumors, HIF-1α isfrequently over-expressed and the elevated HIF-1αcorrelates with progression, aggressiveness, and poorprognosis in many cancer types [1, 2]. Although HIF-1α is a key regulator of cellular response to hypoxia[3], it is also activated under normoxia in response toIL-1β, a pro-inflammatory cytokine [4, 5]. ElevatedIL-1β levels have been reported in non-small-celllung carcinoma, colorectal adenocarcinoma, melanoma[6], and glioblastoma multiforme (GBM) tumorsamples [7]. IL-1β is associated with angiogenesis,metastasis, and tumor growth [8].

Ras is an intermediate component in IL-1β inducedHIF-1α activation in glioma cells, under normoxia [7,9]. We therefore investigated whether the involvement ofRas in IL-1β induced HIF-1α activation is specific to

glioma or exists in other solid tumors, where many pro-tumorigenic signals are driven by HIF-1α. Given thecentral role of HIF-1α in cancer biology, there isconsiderable interest in identifying compounds thatinhibit HIF-1α activity and testing their ability to inhibittumor growth [10]. We have reported that farnesyltrans-ferase inhibitor manumycin, a natural product ofStreptomyces, decreases Ras activity in glioma cells bymodulating redox homeostasis [11]. Importantly, oxida-tive stress and HIF are linked [12]. Ras inhibitiondownregulates HIF-1α activity in GBM [13]. Manumy-cin is known to induce death in human breast, colon, andthyroid cancer cells through induction of oxidative stress[14]. We therefore determined the ability of manumycinto regulate IL-1β-mediated HIF-1α activation andinflammation in glioma and other tumors of diverseorigin.

MATERIALS AND METHODS

Cell Culture and Treatment

Glioblastoma cell line U87MG, A549 (lung), HeLa(cervical), and HepG2 (liver) were cultured in DMEM

1National Brain Research Centre, Manesar, Haryana 122 050, India2 To whom correspondence should be addressed at National BrainResearch Centre, Manesar, Haryana 122 050, India. E-mail: ellora@nbrc.ac.in

0360-3997/12/0200-0516/0 # 2011 Springer Science+Business Media, LLC

Inflammation, Vol. 35, No. 2, April 2012 (# 2011)DOI: 10.1007/s10753-011-9340-6

516

supplemented with 10% FBS. On attaining semi-con-fluence, cells were switched to serum free media (SFM),and after 6 h, cells were treated with 10 ng/ml IL-1β(R&D) in SFM in the presence and absence of 10 microMManumycin for 24 h. All reagents were purchased fromSigma unless otherwise stated. The HIF-1α luciferasereporter was gifted by Chinmay Mukhopadhyay (JNU,India) [15]. DN-RasN17 was purchased from Clontech.

Measurement of Ras Activity

The Ras activity was performed using a commer-cially available Ras activation assay kit purchased fromUpstate Biotechnology, as described previously [7].

Cytokine Bead Array

The human inflammation cytometric bead array kit(BD Biosciences) was used to quantitatively measurecytokines in the conditioned medium collected fromcells treated with or without IL-1β in the presence andabsence of manumycin, as described previously [16].The results are expressed as picograms of cytokines permilligrams of protein.

Luciferase Assay

Cells at ~60–70% confluence in 24-well plates weretransiently transfected with 0.3 μg of HIF-1α reporterplasmid and 10 ng of the Renilla luciferase expressionvector pRL-TK as a transfection control using Lipofect-amine 2000 (Life Technologies/Invitrogen) as describedpreviously [7]. For determining HIF-1α transcriptionalactivity in the presence of dominant negative Rasconstruct, cells were co-transfected with 0.3 μg ofRasN17 along with the HIF-1α reporter construct. Co-transfection experiments with DN-construct were com-pared with control transfection using the appropriateempty vectors for each construct.

RESULTS

IL-1β Increases Ras Activity in Tumor Cellsof Diverse Origin

We have previously reported IL-1β induced Rasactivity in glioma cells [7]. In this study, we investigatedwhether this ability of IL-1β to trigger activation of Rasis conserved across solid tumor cell lines of diverseorigin. Treatment with IL-1β induced Ras activity in

non-GBM tumor cell lines A549 (lung), HeLa (cervical),and HepG2 (liver) (Fig. 1a).

IL-1β Induced Ras Activity Regulates HIF-1αActivity in Tumor Cells of Diverse Origin

Based on our reports demonstrating the existence ofIL-1β induced Ras-NFκB-HIF-1α pathway in gliomacells [7], we investigated the importance of IL-1β-mediated increase in Ras on HIF-1α transcriptionalactivity in non-glioma cells. We determined HIF-1αtranscriptional activity in cells transfected with DN-RasN17 in the presence of IL-1β. Transfection with DN-RasN17 prevented IL-1β-mediated HIF-1α activation(Fig. 1b). Inhibition of Ras activity attenuated HIF-1αactivity in all the tumor cell lines tested, indicating IL-

Fig. 1. IL-1β-induced Ras activation regulates HIF-1α activity in tu-mor cell lines of diverse origin. a Treatment of A549 (lung), HeLa(cervical), and HepG2 (liver) with IL-1β increases Ras activity. Rasactivity in IL-1β-treated cancer cells was determined by the ability ofRas-GTP to bind to a specific protein domain of Raf in the form of aGST-fusion protein. An increase in Ras activity was observed in cellstreated with IL-1β for 30 min. The figure is representative from threeindependent experiments with similar results. b DN-RasN17 inhibitsthe ability of IL-1β to induce HIF-1α activity in A549, HeLa, andHepG2 cells. Cells co-transfected with RasN17 and HIF-1α luciferasereporter constructs were treated with IL-1β, and luciferase reporter a-ssay was performed after 24 h to determine HIF-1α activity. The graphrepresents fold change in reporter activity over control vector. Valuesrepresent the means±SEM from three independent experiments. Aste-risk significant increase from untreated control, number sign significantdecrease from IL-1β-treated cells (P<0.05).

517Manumycin Downregulates Interleukin-1β-Mediated HIF-1α Activation

1β-Ras-HIF-1α pathway is conserved across tumors ofdiverse origin (Fig. 1b).

Manumycin Decreases IL-1β-Mediated HIF-1αActivation. Ras inhibitors downregulate HIF-1α in glio-mas [13]. Since we have previously demonstrated thatmanumycin downregulates activated Ras in glioma cells[11], we investigated whether downregulation of Ras uponmanumycin treatment effects IL-1β induced HIF-1αactivation. We determined HIF-1α activity in cells treatedwith IL-1β in the absence or presence of 10 μMmanumycin. This dose was chosen as it effectively blockedRas activation in glioma cell [11].While exposure to IL-1βresulted in a significant increase in HIF-1α activity,treatment with manumycin reversed IL-1β mediatedincrease in HIF-1α activity in all cancer cell lines tested(Fig. 2). Treatment with manumycin alone had no effect onHIF-1α activity (Fig. 2).

Manumycin Attenuates IL-1β-Mediated Releaseof Pro-inflammatory Mediators

We have demonstrated the IL-1β induces an IL-1β-HIF-1α autocrine loop that sustains a pro-inflammatorymilieu in glioma cells [7]. Since inflammation plays animportant role in tumor progression, we performedcytokine bead array (CBA) to investigate the status ofpro-inflammatory cytokines in IL-1β treated tumor celllines in the presence and absence of manumycin. A

general trend toward increase in the release of pro-inflammatory cytokine IL-6 and IL-8 was observed inIL-1β-treated tumor cells (Fig. 3). Treatment of cellswith IL-1β in the presence of manumycin abrogated theability of IL-1β to induce pro-inflammatory cytokinerelease (Fig. 3a, b).

Fig. 2. Manumycin inhibits IL-1β-induced HIF-1α activity. Followingtransient transfection of U87MG, A549, HeLa, and HepG2 cells withHIF-1α luciferase reporter construct, cells were treated with either IL-1β or manumycin or both. After 24 h luciferase reporter assay wasperformed to determine HIF-1α activity. The graph represents fold c-hange in activity over control vector. Values represent the means±SEMfrom three independent experiments. Asterisk significant increase fromuntreated control, number sign significant decrease from IL-1β-treatedcells (P<0.05).

Fig. 3. Manumycin alters the release of pro-inflammatory cytokinesreleased from tumor cells. Expression of IL-6 and IL-8 in U87MG,A549, HeLa, and HepG2 cells treated with either IL-1β or manumycinor both for 24 h, as observed by CBA. Increase in pro-inflammatorycytokines observed upon IL-1β treatment was significantly suppressedin the presence of manumycin. Values represent mean±SEM from th-ree individual experiments. Asterisk significant increase from control,number sign significant decrease from IL-1β-treated cells (P<0.05).

518 Sharma, Shaheen, Dixit, and Sen

DISCUSSION

Our result indicates that IL-1β induces HIF-1αactivation in lung, cervical, and hepatocellular carci-noma cell lines. This induction in HIF-1α activation isdependent on IL-1β-mediated Ras activation, as IL-1β-induced HIF-1α activation is abrogated in cells trans-fected with RasN17. We have recently documented thedependence of IL-1β-induced HIF-1α activity on Ras[7, 9]. This study establishes Ras as an importantcomponent in IL-1β-induced HIF-1α activation undernormoxia not only in glioblastoma but also across awide range of tumors. The seminal finding of our studyis that Ras activation triggered by IL-1β regulates HIF-1α activation in different cancer cells of different origin.Also, Ras inhibitor manumycin effectively abrogated IL-1β-induced HIF-1α activation and pro-inflammatorycytokine response in all tumor cell lines tested. AsHIF-1α is considered to be one of the most importantanti-cancer targets [10], the potential ability of manu-mycin to regulate HIF-1α activation and inflammatoryresponse in cancers harboring activated Ras warrantsfurther investigation.

REFERENCES

1. Rankin, E.B., and A.J. Giaccia. 2008. The role of hypoxia-inducible factors in tumorigenesis. Cell Death and Differentiation15(4): 678–685.

2. Semenza, G.L. 2010. Defining the role of hypoxia-induciblefactor 1 in cancer biology and therapeutics. Oncogene 29(5):625–634.

3. Iyer, N.V., L.E. Kotch, F. Agani, S.W. Leung, E. Laughner, R.H.Wenger, M. Gassmann, J.D. Gearhart, A.M. Lawler, A.Y. Yu, et al.1998. Cellular and developmental control of O2 homeostasis byhypoxia-inducible factor 1 alpha. Genes & Development 12(2):149–162.

4. Hellwig-Burgel, T., K. Rutkowski, E. Metzen, J. Fandrey, and W.Jelkmann. 1999. Interleukin-1beta and tumor necrosis factor-alpha

stimulate DNA binding of hypoxia-inducible factor-1. Blood 94(5):1561–1567.

5. Jung, Y.J., J.S. Isaacs, S. Lee, J. Trepel, and L. Neckers. 2003. IL-1beta-mediated up-regulation of HIF-1alpha via an NFkappaB/COX-2 pathway identifies HIF-1 as a critical link between inflammation andoncogenesis. The FASEB Journal 17(14): 2115–2117.

6. Elaraj, D.M., D.M. Weinreich, S. Varghese, M. Puhlmann, S.M.Hewitt, N.M. Carroll, E.D. Feldman, E.M. Turner, and H.R.Alexander. 2006. The role of interleukin 1 in growth andmetastasis of human cancer xenografts. Clinical Cancer Research12(4): 1088–1096.

7. Sharma, V., D. Dixit, N. Koul, V.S. Mehta, and E. Sen. 2011. Rasregulates interleukin-1beta-induced HIF-1alpha transcriptionalactivity in glioblastoma. Journal of Molecular Medicine (Berlin,Germany) 89(2): 123–36.

8. Voronov, E., D.S. Shouval, Y. Krelin, E. Cagnano, D. Benharroch,Y. Iwakura, C.A. Dinarello, and R.N. Apte. 2003. IL-1 is requiredfor tumor invasiveness and angiogenesis. Proceedings of theNational Academy of Sciences of the United States of America100(5): 2645–2650.

9. Kaluz, S., and E.G. Van Meir. 2011. At the crossroads of cancerand inflammation: Ras rewires an HIF-driven IL-1 autocrine loop.Journal of molecular medicine (Berlin, Germany) 89(2): 91–94.

10. Semenza, G.L. 2003. Targeting HIF-1 for cancer therapy. NatureReviews Cancer 3(10): 721–732.

11. Dixit, D., V. Sharma, S. Ghosh, N. Koul, P.K. Mishra, and E. Sen.2009. Manumycin inhibits STAT3, telomerase activity and growthof glioma cells by elevating intracellular reactive oxygen speciesgeneration. Free Radical Biology & Medicine 47(4): 364–374.

12. Tormos, K.V., and N.S. Chandel. 2010. Inter-connection betweenmitochondria and HIFs. Journal of Cellular and MolecularMedicine 14(4): 795–804.

13. Blum, R., J. Jacob-Hirsch, N. Amariglio, G. Rechavi, and Y.Kloog. 2005. Ras inhibition in glioblastoma down-regulateshypoxia-inducible factor-1alpha, causing glycolysis shutdown andcell death. Cancer Research 65(3): 999–1006.

14. Pan, J., M. She, Z.X. Xu, L. Sun, and S.C. Yeung. 2005.Farnesyltransferase inhibitors induce DNA damage via reactiveoxygen species in human cancer cells. Cancer Research 65(9):3671–3681.

15. Biswas, S., M.K. Gupta, D. Chattopadhyay, and C.K. Mukhopad-hyay. 2007. Insulin-induced activation of hypoxia-inducible factor-1 requires generation of reactive oxygen species by NADPHoxidase. American Journal of Physiology. Heart and CirculatoryPhysiology 292(2): H758–H766.

16. Sharma, V., C. Joseph, S. Ghosh, A. Agarwal, M.K. Mishra, and E.Sen. 2007. Kaempferol induces apoptosis in glioblastoma cellsthrough oxidative stress. Molecular Cancer Therapeutics 6(9):2544–2553.

519Manumycin Downregulates Interleukin-1β-Mediated HIF-1α Activation