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COX-2-dependent stabilization of survivin in non-small cell lung cancer¹

KOSTYANTYN KRYSAN, FARRUKH H. MERCHANT^||,,, LI ZHU, MARIAM DOHADWALA, JIE LUO, YING LIN, NATHALIE HEUZE-VOURC’H, MEHIS PÕLD, DAVID SELIGSON^,¶, DAVID CHIA^,,¶, LEE GOODGLICK^,,¶, HEJING WANG, ROBERT STRIETER^*,, SHERVEN SHARMA and STEVEN DUBINETT^*,,,2

David Geffen School of Medicine at UCLA,
^* Division of Pulmonary and Critical Care;
UCLA Lung Cancer Research Program, Jonsson Comprehensive Cancer Center, Los Angeles;
Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles;
Department of Pathology and Laboratory Medicine;
^|| Department of Human Genetics;
Jonsson Comprehensive Cancer Center;
^¶ Early Detection Research Network and the UCLA Defined Tumor Marker Evaluation Center;
Department of Biomathematics, Los Angeles, California, USA

²Correspondence: David Geffen School of Medicine at UCLA, Division of Pulmonary and Critical Care Medicine, 37-131 CHS, 10833 Le Conte Ave., Los Angeles, CA 90095-1690, USA. E-mail: sdubinett{at}mednet.ucla.edu

SPECIFIC AIMS

Cyclooxygenase 2 (COX-2) is frequently overexpressed in a variety of malignancies. The overall objective of this study was to determine the mechanisms whereby COX-2 overexpression modulates the apoptosis resistance of human non-small cell lung cancer (NSCLC) cells.

PRINCIPAL FINDINGS

1. COX-2 overexpression increases whereas COX-2 inhibition limits apoptosis resistance in NSCLC cells
The effect of COX-2 overexpression on apoptosis resistance was studied in A549 and H157 NSCLC cell lines stably expressing COX-2 cDNA in sense (S) and antisense (AS) orientations as well as on vector-transduced (V) and parental (P) cells. Likewise, pretreatment of the NSCLC cells with exogenous PGE2 (10 µg/mL) for 4 h prior to apoptosis induction significantly decreased apoptosis whereas pretreatment with the specific COX-2 inhibitor sc-58236 (25 µM) rendered cells more sensitive to apoptosis induction.

2. Expression of the anti-apoptotic protein survivin is suppressed in COX-2 antisense cells
We tested the expression of cIAP1, cIAP2, XIAP, and survivin in P, V, AS, and S A549 and H157 NSCLC cells and found that only survivin was differentially expressed in COX-2 gene-modified cells (Fig. 1 A). Treatment of A549P and H157P cells with the COX-2-specific inhibitor sc58236 for 24 h significantly reduced survivin levels (Fig. 1B ) without notable induction of apoptosis. Consistent with these data, after apoptotic stimuli we found increased caspase 9 activation in COX-2AS cells and a profound decrease of caspases 3 and 9 activation in COX-2S cells (Fig. 1C, D ). Similar patterns of COX-2-dependent survivin expression were observed in vivo when COX-2 gene-modified human NSCLC cells were used to induce tumors in SCID mice. COX-2AS-derived tumors also had significantly lowered tumorigenicity compared with sense- and vector-derived tumors.

View larger version (41K): [in this window] [in a new window]   Figure 1. Survivin levels are significantly decreased in A549 and H157 AS cells. A) Western blot with cell lysates from P, V, AS, and S cells. Lysates were separated in SDS-PAGE, transferred to the nitrocellulose membrane and probed with anti-survivin antibody (top panel). The same filter was washed and reprobed with anti-actin antibody to ensure that equal amounts of proteins were loaded on the gel (lower panel). B) Survivin expression is suppressed by the COX-2 specific inhibitor sc58236. Cells were treated with sc58236 and survivin expression was assessed by Western blot (top panel). The same filter was reprobed with anti-actin antibodies (lower panel). COX-2 overexpression suppresses activation of caspase 9 (C) and caspase 3 (D) in NSCLC cells exposed to apoptotic stimuli. Cleaved caspase 9 and 3 levels were not detectable in nonapoptotic cells. The antibodies were specific only to cleaved forms of caspases 3 and 9 (17 and 35 kDa, respectively). The same filters were reprobed with anti-actin antibodies (C, D, lower panels). Data are representative of 3 independent experiments.

3. COX-2 and PGE2 regulate survivin ubiquitination in NSCLC cells
Because cellular survivin levels are regulated by the ubiquitin/proteasome pathway, we analyzed survivin ubiquitination in NSCLC cells and found it was notably decreased in COX-2S cells. In contrast, significantly higher levels of ubiquitinated survivin were found in COX-2AS cells. Consistent with this, treatment of the cells with exogenous PGE2 (10 µg/mL for 24 h) resulted in decreased survivin ubiquitination compared with the control cells.

4. Regulation of survivin is cell cycle independent in NSCLC cells
Consistent with previously reported data, we observed cell cycle-dependent survivin expression in HeLa cells. In contrast, NSCLC cells expressed abundant levels of survivin at all phases of the cell cycle.

5. COX-2 and survivin are up-regulated in human lung cancers in situ
We found frequent coexpression of COX-2 and survivin as assessed by immunohistochemistry in human lung cancer specimens (Fig. 2 ). Analysis of medical histories of the patients revealed a marked reduction of tumor survivin levels in patients treated with COX-2 inhibitors prior to surgery.

View larger version (91K): [in this window] [in a new window]   Figure 2. COX-2 and survivin are expressed in human NSCLC in situ. Lung sections were double stained with anti-COX-2 and anti-survivin antibodies. COX-2 is seen as brown/black staining and survivin is red/pink. A) Although most epithelial cells were COX-2 negative in normal lung sections, type II (and to a lesser extent, type I) pneumocytes of the alveolar wall were occasionally positive for COX-2. Survivin expression was not detected (400x). Typical overlapping staining patterns of COX-2 and survivin were observed in B) regions of tumor Ad 8 (200x) and C) regions of tumor Sq 1 (200x). In regions of tumor Ad 9 (D), focal staining of COX-2 was observed with little or no survivin staining (200x). E) Percentage of malignant cells stained positively for COX-2 and survivin in different tumor specimens. Samples were scored based on the percentage of tumor cells staining positive at various intensity levels.

CONCLUSIONS AND SIGNIFICANCE

Our findings demonstrate that overexpression of COX-2 as well as treatment with PGE2 significantly increases the survival of NSCLC cells exposed to apoptotic stimuli. This capacity of COX-2 and PGE2 to enhance cell survival may help explain the COX-2-dependent resistance to radiation and chemotherapy in lung cancer. Thus survivin, expressed in a cell cycle-independent manner in NSCLC, is a candidate protein that may be responsible for elevated apoptosis resistance in COX-2-overexpressing NSCLC cells. We describe a new cellular mechanism of protein level regulation via COX-2- and PGE2-dependent modulation of protein ubiquitination. Our data link COX-2- and PGE2-rich tumor microenvironments to elevated tumor apoptosis resistance through ubiquitination-dependent survivin stabilization (Fig. 3 ). Further studies are required to elucidate the mechanisms that link ubiquitination of cellular proteins, such as survivin to COX-2 and PGE2 production.

View larger version (83K): [in this window] [in a new window]   Figure 3. COX-2 overexpression modulates survivin ubiquitination in NSCLC cells. Left panel: in COX-2 overexpressing cells, survivin is stabilized due to the lowered ubiquitination levels, which may account for the elevated apoptosis resistance of these cells. Right panel: COX-2 suppression leads to increased survivin ubiquitination and proteasomal degradation, thus rendering cells more sensitive to apoptosis induction.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0369fje

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