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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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Related in: MedlinePlus

HSF1 knockdown sensitizes hepatocellular cancer cells to HSP90 inhibitor in vitro and in vivoA. The mRNA level of HSF1 was measured among tumor samples from HCC patients compared to normal control. B. IHC showed the expression of HSF1 in primary human hepatocellular cancer. C. The cell growth curve of Hep3B cell with or without HSF1 knockdown at different time points. shNTC or shHSF1 transduced Hep3B cells were treated with or without Doxycycline for 7 days. Relative cell growth (average of at least 3 independent experiments) was measured by CellTiter-Glo and normalized to cells without Doxycycline treatment. D. Cell colony formation assay of HSF1 knockdown in Hep3B cells. shNTC or shHSF1 transduced Hep3B cells were treated with Doxycycline for 12 days. E. Western blotting analysis of Hep3B cells expressing the indicated shRNA and cDNA contruct. The inducible lenti-virus construct (HSF1R cDNA) was used to transduce Hep3B cells and expression of HSF1R cDNA with or without HSF1 knockdown were tested. HSF1, HSP70 and GAPDH were detected by western blotting. F. Cell colony formation assay of over-expression of HSF1R cDNA in Hep3B cells with or without HSF1 knockdown. G. The comparison of dose response of NVP-HSP990 in Hep3B cells with inducible expression of either HSF1 shRNA alone or both HSF1 shRNA and HSF1R cDNA. HSF1 shRNA alone or both HSF1 shRNA and HSF1R cDNA transduced cancer cells were treated with or without Doxycycline for 3 days, then followed by being treated for 5 days with serial dilutions of NVP-HSP990. H. Western blotting analysis of tumor samples. Tumor samples were collected at the end of studies and western blotting analysis of HSF1, HSP70 and GAPDH were performed. I. The combinational effect of HSF1 knockdown and HSP90 inhibitor in Hep3B xenograft mouse model. Tumor growth rate of Hep3B cells expressing inducible control shRNA or shRNA against HSF1 under Doxycycline and/or NVP-HSP990 were compared at different time points.
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Figure 3: HSF1 knockdown sensitizes hepatocellular cancer cells to HSP90 inhibitor in vitro and in vivoA. The mRNA level of HSF1 was measured among tumor samples from HCC patients compared to normal control. B. IHC showed the expression of HSF1 in primary human hepatocellular cancer. C. The cell growth curve of Hep3B cell with or without HSF1 knockdown at different time points. shNTC or shHSF1 transduced Hep3B cells were treated with or without Doxycycline for 7 days. Relative cell growth (average of at least 3 independent experiments) was measured by CellTiter-Glo and normalized to cells without Doxycycline treatment. D. Cell colony formation assay of HSF1 knockdown in Hep3B cells. shNTC or shHSF1 transduced Hep3B cells were treated with Doxycycline for 12 days. E. Western blotting analysis of Hep3B cells expressing the indicated shRNA and cDNA contruct. The inducible lenti-virus construct (HSF1R cDNA) was used to transduce Hep3B cells and expression of HSF1R cDNA with or without HSF1 knockdown were tested. HSF1, HSP70 and GAPDH were detected by western blotting. F. Cell colony formation assay of over-expression of HSF1R cDNA in Hep3B cells with or without HSF1 knockdown. G. The comparison of dose response of NVP-HSP990 in Hep3B cells with inducible expression of either HSF1 shRNA alone or both HSF1 shRNA and HSF1R cDNA. HSF1 shRNA alone or both HSF1 shRNA and HSF1R cDNA transduced cancer cells were treated with or without Doxycycline for 3 days, then followed by being treated for 5 days with serial dilutions of NVP-HSP990. H. Western blotting analysis of tumor samples. Tumor samples were collected at the end of studies and western blotting analysis of HSF1, HSP70 and GAPDH were performed. I. The combinational effect of HSF1 knockdown and HSP90 inhibitor in Hep3B xenograft mouse model. Tumor growth rate of Hep3B cells expressing inducible control shRNA or shRNA against HSF1 under Doxycycline and/or NVP-HSP990 were compared at different time points.

Mentions: To identify the cancer type which may be useful for the stratification of the combinational treatment, we examined HSF1 expression in TCGA database and found that HSF1 is over-expressed in several tumor types (Supplementary Figure S1). Among them, we are particularly interested in HCC since HSF1 has been reported to be a key modulator of HCC development in mouse model [26]. HSF1 mRNA was significantly elevated in HCC tumor in compared to normal control (Fig. 3A). To examine the protein expression of HSF1 in HCC patients, immunohistochemistry (IHC) study for HSF1 was performed on primary human HCC samples and non-neoplastic liver samples and the HSF1 antibody was validated by using Hep3B cell pellets with inducible HSF1 shRNAs. HSF1 staining was significantly decreased in Hep3B cells treated with Doxycycline in comparison with cells without Doxycycline treatment (Supplementary Fig. S2). HSF1 staining was then scored in 50 human HCC tumor samples. Samples that were not immunoreactive (Ki67 negative) or had insufficient tumor were excluded from the analysis. HSF1 expression was not observed in non-neoplastic human hepatocytes of the 45 HCC tumor samples were evaluable, 35 HCC cases showed positive HSF1 staining (Fig. 3B and Table 2). The intensity of staining varied considerably from tumor to tumor. These results suggest that both mRNA and protein level of HSF1 expression are increased in HCC.


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)

HSF1 knockdown sensitizes hepatocellular cancer cells to HSP90 inhibitor in vitro and in vivoA. The mRNA level of HSF1 was measured among tumor samples from HCC patients compared to normal control. B. IHC showed the expression of HSF1 in primary human hepatocellular cancer. C. The cell growth curve of Hep3B cell with or without HSF1 knockdown at different time points. shNTC or shHSF1 transduced Hep3B cells were treated with or without Doxycycline for 7 days. Relative cell growth (average of at least 3 independent experiments) was measured by CellTiter-Glo and normalized to cells without Doxycycline treatment. D. Cell colony formation assay of HSF1 knockdown in Hep3B cells. shNTC or shHSF1 transduced Hep3B cells were treated with Doxycycline for 12 days. E. Western blotting analysis of Hep3B cells expressing the indicated shRNA and cDNA contruct. The inducible lenti-virus construct (HSF1R cDNA) was used to transduce Hep3B cells and expression of HSF1R cDNA with or without HSF1 knockdown were tested. HSF1, HSP70 and GAPDH were detected by western blotting. F. Cell colony formation assay of over-expression of HSF1R cDNA in Hep3B cells with or without HSF1 knockdown. G. The comparison of dose response of NVP-HSP990 in Hep3B cells with inducible expression of either HSF1 shRNA alone or both HSF1 shRNA and HSF1R cDNA. HSF1 shRNA alone or both HSF1 shRNA and HSF1R cDNA transduced cancer cells were treated with or without Doxycycline for 3 days, then followed by being treated for 5 days with serial dilutions of NVP-HSP990. H. Western blotting analysis of tumor samples. Tumor samples were collected at the end of studies and western blotting analysis of HSF1, HSP70 and GAPDH were performed. I. The combinational effect of HSF1 knockdown and HSP90 inhibitor in Hep3B xenograft mouse model. Tumor growth rate of Hep3B cells expressing inducible control shRNA or shRNA against HSF1 under Doxycycline and/or NVP-HSP990 were compared at different time points.
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Figure 3: HSF1 knockdown sensitizes hepatocellular cancer cells to HSP90 inhibitor in vitro and in vivoA. The mRNA level of HSF1 was measured among tumor samples from HCC patients compared to normal control. B. IHC showed the expression of HSF1 in primary human hepatocellular cancer. C. The cell growth curve of Hep3B cell with or without HSF1 knockdown at different time points. shNTC or shHSF1 transduced Hep3B cells were treated with or without Doxycycline for 7 days. Relative cell growth (average of at least 3 independent experiments) was measured by CellTiter-Glo and normalized to cells without Doxycycline treatment. D. Cell colony formation assay of HSF1 knockdown in Hep3B cells. shNTC or shHSF1 transduced Hep3B cells were treated with Doxycycline for 12 days. E. Western blotting analysis of Hep3B cells expressing the indicated shRNA and cDNA contruct. The inducible lenti-virus construct (HSF1R cDNA) was used to transduce Hep3B cells and expression of HSF1R cDNA with or without HSF1 knockdown were tested. HSF1, HSP70 and GAPDH were detected by western blotting. F. Cell colony formation assay of over-expression of HSF1R cDNA in Hep3B cells with or without HSF1 knockdown. G. The comparison of dose response of NVP-HSP990 in Hep3B cells with inducible expression of either HSF1 shRNA alone or both HSF1 shRNA and HSF1R cDNA. HSF1 shRNA alone or both HSF1 shRNA and HSF1R cDNA transduced cancer cells were treated with or without Doxycycline for 3 days, then followed by being treated for 5 days with serial dilutions of NVP-HSP990. H. Western blotting analysis of tumor samples. Tumor samples were collected at the end of studies and western blotting analysis of HSF1, HSP70 and GAPDH were performed. I. The combinational effect of HSF1 knockdown and HSP90 inhibitor in Hep3B xenograft mouse model. Tumor growth rate of Hep3B cells expressing inducible control shRNA or shRNA against HSF1 under Doxycycline and/or NVP-HSP990 were compared at different time points.
Mentions: To identify the cancer type which may be useful for the stratification of the combinational treatment, we examined HSF1 expression in TCGA database and found that HSF1 is over-expressed in several tumor types (Supplementary Figure S1). Among them, we are particularly interested in HCC since HSF1 has been reported to be a key modulator of HCC development in mouse model [26]. HSF1 mRNA was significantly elevated in HCC tumor in compared to normal control (Fig. 3A). To examine the protein expression of HSF1 in HCC patients, immunohistochemistry (IHC) study for HSF1 was performed on primary human HCC samples and non-neoplastic liver samples and the HSF1 antibody was validated by using Hep3B cell pellets with inducible HSF1 shRNAs. HSF1 staining was significantly decreased in Hep3B cells treated with Doxycycline in comparison with cells without Doxycycline treatment (Supplementary Fig. S2). HSF1 staining was then scored in 50 human HCC tumor samples. Samples that were not immunoreactive (Ki67 negative) or had insufficient tumor were excluded from the analysis. HSF1 expression was not observed in non-neoplastic human hepatocytes of the 45 HCC tumor samples were evaluable, 35 HCC cases showed positive HSF1 staining (Fig. 3B and Table 2). The intensity of staining varied considerably from tumor to tumor. These results suggest that both mRNA and protein level of HSF1 expression are increased in HCC.

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