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Targeting HSF1 sensitizes cancer cells to HSP90 inhibition.

Chen Y, Chen J, Loo A, Jaeger S, Bagdasarian L, Yu J, Chung F, Korn J, Ruddy D, Guo R, McLaughlin ME, Feng F, Zhu P, Stegmeier F, Pagliarini R, Porter D, Zhou W - Oncotarget (2013)

Bottom Line: A striking combinational effect was observed when HSF1 knockdown plus with HSP90 inhibitors treatment in various cancer cell lines and tumor mouse models.Interestingly, HSF1 is highly expressed in hepatocellular carcinoma (HCC) patient samples and HCC is sensitive to combinational treatment, indicating a potential indication for the combinational treatment.To understand the mechanism of the combinational effect, we identified that a HSF1-target gene DEDD2 is involved in attenuating the effect of HSP90 inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.

ABSTRACT
The molecular chaperone heat shock protein 90 (HSP90) facilitates the appropriate folding of various oncogenic proteins and is necessary for the survival of some cancer cells. HSP90 is therefore an attractive drug target, but the efficacy of HSP90 inhibitor may be limited by HSP90 inhibition induced feedback mechanisms. Through pooled RNA interference screens, we identified that heat shock factor 1(HSF1) is a sensitizer of HSP90 inhibitor. A striking combinational effect was observed when HSF1 knockdown plus with HSP90 inhibitors treatment in various cancer cell lines and tumor mouse models. Interestingly, HSF1 is highly expressed in hepatocellular carcinoma (HCC) patient samples and HCC is sensitive to combinational treatment, indicating a potential indication for the combinational treatment. To understand the mechanism of the combinational effect, we identified that a HSF1-target gene DEDD2 is involved in attenuating the effect of HSP90 inhibitors. Thus, the transcriptional activities of HSF1 induced by HSP90 inhibitors provide a feedback mechanism of limiting the HSP90 inhibitor's activity, and targeting HSF1 may provide a new avenue to enhance HSP90 inhibitors activity in human cancers.

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Combination of HSF1 knockdown and HSP90 inhibitor leads to a decreased level of p-ERK and an increased cell apoptosis rateA. Western blotting analysis of HCT116 cells expressing the inducible shHSF1 treated with different doses of NVP-HSP990. shNTC or shHSF1 transduced HCT116 cells were treated with or without Doxycycline for 3 days and were further treated with different doses of NVP-HSP990 for 48h. B. Western blotting analysis of A375 cells expressing the inducible shRNA treated with different doses of NVP-HSP990. shNTC or shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. C. Cell cycle analysis of A375 cells treated with NVP-HSP990 with or without HSF1 knockdown. shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. The percentage of S+G2M cells were determined by PI staining. D. Cell apoptosis analysis of A375 cells with HSF1 knockdown and NVP-HSP990 treatment. shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. The apoptotic cells represented by 7AAD+AnnexinV+ were determined by FACS.
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Figure 4: Combination of HSF1 knockdown and HSP90 inhibitor leads to a decreased level of p-ERK and an increased cell apoptosis rateA. Western blotting analysis of HCT116 cells expressing the inducible shHSF1 treated with different doses of NVP-HSP990. shNTC or shHSF1 transduced HCT116 cells were treated with or without Doxycycline for 3 days and were further treated with different doses of NVP-HSP990 for 48h. B. Western blotting analysis of A375 cells expressing the inducible shRNA treated with different doses of NVP-HSP990. shNTC or shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. C. Cell cycle analysis of A375 cells treated with NVP-HSP990 with or without HSF1 knockdown. shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. The percentage of S+G2M cells were determined by PI staining. D. Cell apoptosis analysis of A375 cells with HSF1 knockdown and NVP-HSP990 treatment. shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. The apoptotic cells represented by 7AAD+AnnexinV+ were determined by FACS.

Mentions: To understand the mechanism of the combination effects of HSF1 knockdown and HSP90 inhibition, we tested: 1) whether HSF1 knockdown may facilitate the degradation of HSP90 client protein by HSP90 inhibition, such as BRAF or HER2 oncogenic proteins; 2) whether HSP90 inhibition may enhance the attenuation of MAPK signaling mediated by HSF1 knockdown as recent finding suggests that HSF1 deficiency attenuates MAPK signaling in mice[27] and 3) HSF1 may regulate other target genes rather than HSP70, which may play a role in attenuating the effect of HSP90 inhibition. Therefore, we examined the status of HSP90 client proteins and the downstream effects in cell treated with either HSF1 shRNA or HSP90 inhibitor or combination of HSF1 shRNA and HSP90 inhibitor. HCT116 cells were treated with different doses of NVP-HSP990 and HSF1 knockdown in combination with NVP-HSP990 (5nM) reduced the HSP70, p-ERK and HER2 levels significantly while NVP-HSP990 or HSF1 knockdown alone did not(Fig. 4A). HSF1 knockdown in combination with NVP-HSP990 (25nM) led increased cleaved PARP (Fig. 4A). The combination also led an enhanced degradation of BRAF in A375 cells (Fig. 4B). A decreased level of HSP70, p-ERK and increased level of cleaved PARP were also observed in melanoma cells (Fig. 4B) and hepatocellular cancer cells (Supplementary Fig. S3). To understand how HSF1 knockdown affects the cell proliferation under HSP90 inhibitor treatment, cell cycle analysis was performed. HSF1 knockdown didn't affect the percentage of cancer cells in cell cycle while HSP90 inhibitor caused more cancer cells into S+G2M phase (data not shown). In contrast, the percentage of cancer cells in the S+G2M phase was significantly decreased in HSF1 knockdown group than in the control group under HSP90 inhibitor treatment(Fig. 4C), indicating that under HSP90 inhibition the knockdown of HSF1 blocks cancer cells to enter the cell cycle, thereby decrease the proliferation of cancer cells. Next, we examined whether HSF1 knockdown may enhance apoptosis of cancer cells under HSP90 inhibitor treatment by staining the cells with 7AAD and Annexin V. Similarly, HSF1 knockdown didn't affect the apoptosis of cancer cells while HSP90 inhibitor induced the apoptosis of cancer cells (data not shown). HSF1 knockdown further enhanced the apoptotic proportion of cancer cells under HSP90 inhibitor treatment (Fig. 4D). Thus, the combination treatment of HSF1 knockdown and HSP90 inhibition facilitates the degradation of HSP90 client proteins, such as BRAF and HER2, inhibits MAPK growth signaling and results in cell cycles arrest and cell apoptosis.


Targeting HSF1 sensitizes cancer cells to HSP90 inhibition.

Chen Y, Chen J, Loo A, Jaeger S, Bagdasarian L, Yu J, Chung F, Korn J, Ruddy D, Guo R, McLaughlin ME, Feng F, Zhu P, Stegmeier F, Pagliarini R, Porter D, Zhou W - Oncotarget (2013)

Combination of HSF1 knockdown and HSP90 inhibitor leads to a decreased level of p-ERK and an increased cell apoptosis rateA. Western blotting analysis of HCT116 cells expressing the inducible shHSF1 treated with different doses of NVP-HSP990. shNTC or shHSF1 transduced HCT116 cells were treated with or without Doxycycline for 3 days and were further treated with different doses of NVP-HSP990 for 48h. B. Western blotting analysis of A375 cells expressing the inducible shRNA treated with different doses of NVP-HSP990. shNTC or shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. C. Cell cycle analysis of A375 cells treated with NVP-HSP990 with or without HSF1 knockdown. shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. The percentage of S+G2M cells were determined by PI staining. D. Cell apoptosis analysis of A375 cells with HSF1 knockdown and NVP-HSP990 treatment. shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. The apoptotic cells represented by 7AAD+AnnexinV+ were determined by FACS.
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Figure 4: Combination of HSF1 knockdown and HSP90 inhibitor leads to a decreased level of p-ERK and an increased cell apoptosis rateA. Western blotting analysis of HCT116 cells expressing the inducible shHSF1 treated with different doses of NVP-HSP990. shNTC or shHSF1 transduced HCT116 cells were treated with or without Doxycycline for 3 days and were further treated with different doses of NVP-HSP990 for 48h. B. Western blotting analysis of A375 cells expressing the inducible shRNA treated with different doses of NVP-HSP990. shNTC or shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. C. Cell cycle analysis of A375 cells treated with NVP-HSP990 with or without HSF1 knockdown. shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. The percentage of S+G2M cells were determined by PI staining. D. Cell apoptosis analysis of A375 cells with HSF1 knockdown and NVP-HSP990 treatment. shHSF1 transduced A375 cells were treated with or without Doxycycline for 3 days and were further treated with NVP-HSP990 100nM for 48h. The apoptotic cells represented by 7AAD+AnnexinV+ were determined by FACS.
Mentions: To understand the mechanism of the combination effects of HSF1 knockdown and HSP90 inhibition, we tested: 1) whether HSF1 knockdown may facilitate the degradation of HSP90 client protein by HSP90 inhibition, such as BRAF or HER2 oncogenic proteins; 2) whether HSP90 inhibition may enhance the attenuation of MAPK signaling mediated by HSF1 knockdown as recent finding suggests that HSF1 deficiency attenuates MAPK signaling in mice[27] and 3) HSF1 may regulate other target genes rather than HSP70, which may play a role in attenuating the effect of HSP90 inhibition. Therefore, we examined the status of HSP90 client proteins and the downstream effects in cell treated with either HSF1 shRNA or HSP90 inhibitor or combination of HSF1 shRNA and HSP90 inhibitor. HCT116 cells were treated with different doses of NVP-HSP990 and HSF1 knockdown in combination with NVP-HSP990 (5nM) reduced the HSP70, p-ERK and HER2 levels significantly while NVP-HSP990 or HSF1 knockdown alone did not(Fig. 4A). HSF1 knockdown in combination with NVP-HSP990 (25nM) led increased cleaved PARP (Fig. 4A). The combination also led an enhanced degradation of BRAF in A375 cells (Fig. 4B). A decreased level of HSP70, p-ERK and increased level of cleaved PARP were also observed in melanoma cells (Fig. 4B) and hepatocellular cancer cells (Supplementary Fig. S3). To understand how HSF1 knockdown affects the cell proliferation under HSP90 inhibitor treatment, cell cycle analysis was performed. HSF1 knockdown didn't affect the percentage of cancer cells in cell cycle while HSP90 inhibitor caused more cancer cells into S+G2M phase (data not shown). In contrast, the percentage of cancer cells in the S+G2M phase was significantly decreased in HSF1 knockdown group than in the control group under HSP90 inhibitor treatment(Fig. 4C), indicating that under HSP90 inhibition the knockdown of HSF1 blocks cancer cells to enter the cell cycle, thereby decrease the proliferation of cancer cells. Next, we examined whether HSF1 knockdown may enhance apoptosis of cancer cells under HSP90 inhibitor treatment by staining the cells with 7AAD and Annexin V. Similarly, HSF1 knockdown didn't affect the apoptosis of cancer cells while HSP90 inhibitor induced the apoptosis of cancer cells (data not shown). HSF1 knockdown further enhanced the apoptotic proportion of cancer cells under HSP90 inhibitor treatment (Fig. 4D). Thus, the combination treatment of HSF1 knockdown and HSP90 inhibition facilitates the degradation of HSP90 client proteins, such as BRAF and HER2, inhibits MAPK growth signaling and results in cell cycles arrest and cell apoptosis.

Bottom Line: A striking combinational effect was observed when HSF1 knockdown plus with HSP90 inhibitors treatment in various cancer cell lines and tumor mouse models.Interestingly, HSF1 is highly expressed in hepatocellular carcinoma (HCC) patient samples and HCC is sensitive to combinational treatment, indicating a potential indication for the combinational treatment.To understand the mechanism of the combinational effect, we identified that a HSF1-target gene DEDD2 is involved in attenuating the effect of HSP90 inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.

ABSTRACT
The molecular chaperone heat shock protein 90 (HSP90) facilitates the appropriate folding of various oncogenic proteins and is necessary for the survival of some cancer cells. HSP90 is therefore an attractive drug target, but the efficacy of HSP90 inhibitor may be limited by HSP90 inhibition induced feedback mechanisms. Through pooled RNA interference screens, we identified that heat shock factor 1(HSF1) is a sensitizer of HSP90 inhibitor. A striking combinational effect was observed when HSF1 knockdown plus with HSP90 inhibitors treatment in various cancer cell lines and tumor mouse models. Interestingly, HSF1 is highly expressed in hepatocellular carcinoma (HCC) patient samples and HCC is sensitive to combinational treatment, indicating a potential indication for the combinational treatment. To understand the mechanism of the combinational effect, we identified that a HSF1-target gene DEDD2 is involved in attenuating the effect of HSP90 inhibitors. Thus, the transcriptional activities of HSF1 induced by HSP90 inhibitors provide a feedback mechanism of limiting the HSP90 inhibitor's activity, and targeting HSF1 may provide a new avenue to enhance HSP90 inhibitors activity in human cancers.

Show MeSH
Related in: MedlinePlus