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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Chad R Schultz, William A Golembieski, Daniel A King, Stephen L Brown, Chaya Brodie, Sandra A Rempel
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-20) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CRS performed radiation and clonogenic assays, AKT Inhibitor IV assays, Western blot analyses, and participated in experimental design. WAG performed siRNA inhibition experiments, clonogenic assays, and Western blot analyses. DK performed clonogenic assays. SLB participated in experimental design and execution of radiation assays, and interpreted radiation data. CB critically reviewed data and manuscript. SAR conceived of the overall study and design, performed Image J analyses, analyzed and interpreted all data, wrote and submitted the manuscript. All authors read and approved the final manuscript.

Abstract

Background

The current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ), followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival.

Results

Our studies found the following. 1) SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2) Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3) Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4) Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5) However, inhibiting pAKT suppresses tumor cell survival. 6) Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7) There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8) This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1) SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2) Despite this enhanced signaling, SPARC protects cells against TMZ. 3) This protection can be reduced by inhibiting pAKT. 4) Combined inhibition of HSP27 and pAKT is more effective than TMZ treatment alone.

Conclusions

We conclude that inhibition of HSP27 alone, or in combination with pAKT inhibitor IV, may be an effective therapeutic approach to inhibit SPARC-induced glioma cell invasion and survival in SPARC-positive/PTEN-wildtype and SPARC-positive/PTEN-null tumors, respectively.
Zusatzmaterial
Additional file 1: Figure S1. Timing study of TMZ-induced death in U87 cells. C1.1 GFP-expressing cells (1 × 104) were plated overnight in 6-well plates. Cells were treated with 0 (0.1% DMSO) or 100 μM TMZ for 2 days, and the media were changed every 2 days. Lysates were harvested on days 6 and day 8. Western blots were probed as indicated. (TIFF 563 KB)
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Additional file 2: Figure S2. Proposed mechanisms. For all panels, solid lines represent SPARC-induced pro-survival and pro-death signaling, dashed lines represent siRNA or AKT inhibitor IV inhibition of signaling, and changes in protein levels due to siRNA or AKT inhibitor IV inhibition of signaling are indicated by changes in color intensity. Panel A. Proposed mechanisms of forced SPARC-induced signaling in U87 glioma cells alone or treated with temozolomide (TMZ). In the absence of TMZ, SPARC promotes migration via upregulation of the p38MAPK-MAPKAPK2-HSP27 signaling pathway [ 28]. SPARC promotes pro-survival signaling (pHSP27, pAKT) and pro-apoptotic signaling (pro- and cleaved caspase 8 and cleaved caspase 3). Note: pAKT indirectly inhibits cleavage of caspase-3 through inhibition of caspase-9 activation. The dashed blue lines illustrate caspase activation inhibited by HSP27. We propose that the pro-survival and pro-apoptotic signaling cascades balance one another, and cells survive. However, TMZ treatment of SPARC-expressing cells induces caspase 7 and PARP activation as indicated by the green line. Consistent with the literature [ 23], we propose that integrin beta 1 recruits procaspase 8 and AKT. SPARC binds to procaspase 8 to induce chemosensitivity to TMZ. Panel B. Inhibition of HSP27 shifts the balance towards SPARC-induced pro-apoptotic signaling. Dashed blue lines indicate that the loss of HSP27 expression decreases migration and suppresses pro-survival signaling (AKT1, AKT2). The dashed red lines indicate the resultant increase in pro-apoptotic signaling (cleaved caspase 3, 7 and PARP), due to loss of HSP27, resulting in increased apoptosis. Decreased AKT1/2 expression is accompanied by the loss of SPARC-induced sensitivity to TMZ, indicated by the dashed green line. Panel C. Proposed mechanism of endogenous SPARC-regulated signaling in LN443 glioma cells treated with HSP27 siRNA alone or with temozolomide (TMZ). SPARC signaling is proposed to be the same as in U87 cells (Panel A). However, in the absence of forced SPARC expression, HSP27 inhibition suppresses SPARC and pAKT, thereby suppressing SPARC-induced pro-survival and pro-apoptotic signaling, as indicated by the dashed red and blue lines. The loss of HSP27 inhibition permits basal apoptotic signaling through caspase 3, 7 and PARP cleavage. The loss of HSP27 is proposed to decrease migration. In TMZ, the HSP27 inhibition suppresses SPARC-induced sensitivity through caspase 7 and PARP activation, as indicated by the dashed green line. Panel D. Proposed signaling in LN443 glioma treated with SPARC siRNA alone or with TMZ. As expected, loss of SPARC eliminates enhanced pro-survival signaling through HSP27 and pAKT and eliminates pro-apoptotic signaling through caspases 8 and 3. Survival is maintained through basal HSP27 and AKT signaling to inhibit apoptosis, as indicated by the red lines. In TMZ, loss of SPARC eliminates caspase 7and PARP cleavage, indicated by the dashed green line. Panel E. Proposed signaling in LN443 glioma cells treated with AKT1/2/3 siRNA and TMZ. AKT1/2 or AKT3 inhibition results in decreased total AKT1/2 or AKT3, but not pAKT. We propose that SPARC-induced procaspase 8 expression and cleavage are not affected, permitting the cleavage of caspase 3. However, SPARC-induced upregulation of HSP27 inhibits further cleavage of caspases 3, 8 and PARP, as indicated by the red lines. In TMZ, suppression of either AKT1/2 or AKT3 reduced SPARC-induced cleavage of caspase 7 and PARP, as indicated by the dashed green line. Panel F. Proposed signaling in LN443 glioma cells treated with AKT inhibitor IV and TMZ. AKT inhibitor IV suppresses total AKT2, pAKT, SPARC. Loss of pAKT and SPARC permits basal cleavage of caspase 3, but basal levels of HSP27 inhibits further caspase 3 and 8 and PARP cleavage. Unexpectedly, the inhibitor increased cleaved caspase 7 by in the absence or presence of TMZ, but this did not enhance apoptosis. Symbols are courtesy of SABiosciences. (TIFF 4 MB)
Additional file 3: Figure S3. SPARC expression does not protect LN443 cells against temozolomide (TMZ). Average surviving fraction ± SD of LN443 cells in 0, 1, 50, and 100 μM TMZ plating 375 cells/60-mm dish. (TIFF 266 KB)
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Additional file 4: Table S1. Summary of cell Lines. (DOC 38 KB)
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Additional file 5: Figure S4. MGMT expression profile. Westem blot analysis of MGMT protein in glioma lysates. X- empty lane. T98G is a positive control for MGMT. (TIFF 117 KB)
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Authors’ original file for figure 1
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