Radiosensitization of HSF-1 Knockdown Lung Cancer Cells by Low Concentrations of Hsp90 Inhibitor NVP-AUY922.

The inhibition of heat shock protein 90 (Hsp90) a molecular chaperone for multiple oncogenic client proteins is considered as a promising approach to overcome radioresistance. Since most Hsp90 inhibitors activate HSF-1 that induces the transcription of cytoprotective and tumor-promoting stress proteins such as Hsp70 and Hsp27, a combined approach consisting of HSF-1 knockdown (k.d.) and Hsp90 inhibition was investigated. A specific HSF-1 k.d. was achieved in H1339 lung cancer cells using RNAi-Ready pSIRENRetroQ vectors with puromycin resistance. The Hsp90 inhibitor NVP-AUY922 was evaluated at low concentrations-ranging from 1-10 nM-in control and HSF-1 k.d. cells. Protein expression (i.e., Hsp27/Hsp70, HSF-1, pHSF-1, Akt, ß-actin) and transcriptional activity was assessed by western blot analysis and luciferase assays and radiosensitivity was measured by proliferation, apoptosis (Annexin V, active caspase 3), clonogenic cell survival, alkaline comet, γH2AX, 53BP1, and Rad51 foci assays. The k.d. of HSF-1 resulted in a significant reduction of basal and NVP-AUY922-induced Hsp70/Hsp27 expression levels. A combined approach consisting of HSF-1 k.d. and low concentrations of the Hsp90 inhibitor NVP-AUY922 reduces the Hsp90 client protein Akt and potentiates radiosensitization, which involves an impaired homologous recombination mediated by Rad51. Our findings are key for clinical applications of Hsp90 inhibitors with respect to adverse hepatotoxic effects.

Role of membrane Hsp70 in radiation sensitivity of tumor cells.

BACKGROUND: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in the cytosol and integrated in the plasma membrane of tumor cells via lipid anchorage. Following stress such as non-lethal irradiation Hsp70 synthesis is up-regulated. Intracellular located Hsp70 is known to exert cytoprotective properties, however, less is known about membrane (m)Hsp70. Herein, we investigate the role of mHsp70 in the sensitivity towards irradiation in tumor sublines that differ in their cytosolic and/or mHsp70 levels. METHODS: The isogenic human colon carcinoma sublines CX(+) with stable high and CX(-) with stable low expression of mHsp70 were generated by fluorescence activated cell sorting, the mouse mammary carcinoma sublines 4 T1 (4 T1 ctrl) and Hsp70 knock-down (4 T1 Hsp70 KD) were produced using the CRISPR/Cas9 system, and the Hsp70 down-regulation in human lung carcinoma sublines H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD was achieved by small interfering (si)RNA against Heat shock factor 1 (HSF-1). Cytosolic and mHsp70 was quantified by Western blot analysis/ELISA and flow cytometry; double strand breaks (DSBs) and apoptosis were measured by flow cytometry using antibodies against γH2AX and real-time PCR (RT-PCR) using primers and antibodies directed against apoptosis related genes; and radiation sensitivity was determined using clonogenic cell surviving assays. RESULTS: CX(+)/CX(-) tumor cells exhibited similar cytosolic but differed significantly in their mHsp70 levels, 4 T1 ctrl/4 T1 Hsp70 KD cells showed significant differences in their cytosolic and mHsp70 levels and H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD lung carcinoma cell sublines had similar mHsp70 but significantly different cytosolic Hsp70 levels. γH2AX was significantly up-regulated in irradiated CX(-) and 4 T1 Hsp70 KD with low basal mHsp70 levels, but not in their mHsp70 high expressing counterparts, irrespectively of their cytosolic Hsp70 content. After irradiation γH2AX, Caspase 3/7 and Annexin V were up-regulated in the lung carcinoma sublines, but no significant differences were observed in H1339 ctrl/H1339 HSF-1 KD, and EPLC-272H ctrl/EPLC-272H HSF-1 KD that exhibit identical mHsp70 but different cytosolic Hsp70 levels. Clonogenic cell survival was significantly lower in CX(-) and 4 T1 Hsp70 KD cells with low mHsp70 expression, than in CX+ and 4 T1 ctrl cells, whereas no difference in clonogenic cell survival was observed in H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/ EPLC-272H HSF-1 KD sublines with identical mHsp70 but different cytosolic Hsp70 levels. CONCLUSION: In summary, our results indicate that mHsp70 has an impact on radiation resistance.

NZ28-induced inhibition of HSF1, SP1 and NF-κB triggers the loss of the natural killer cell-activating ligands MICA/B on human tumor cells.

The activity of natural killer (NK) cells is regulated by activating and inhibiting receptors, whereby the C-type lectin natural killer group 2D (NKG2D) receptor serves as the major activating receptor on NK cells which recognizes major histocompatibility class I chain-related proteins A and B (MICA/B). The MICA/B expression has been described to be regulated by the transcription factor heat shock factor 1 (HSF1). Inhibition of heat shock protein 90 (Hsp90) is known to induce the heat shock response via activation of HSF1 which is associated with tumor development, metastasis and therapy resistance and also with an increased susceptibility to NK cell-mediated lysis. Therefore, we compared the effects of Hsp90 inhibitor NVP-AUY922, HSF1 inhibitor NZ28 and HSF1 knockdown on the sensitivity of lung (H1339) and breast (MDA-MB-231, T47D) cancer cells to NK cell-mediated cytotoxicity and the expression of the NKG2D ligands MICA/B. Although NVP-AUY922 activates HSF1, neither the MICA/B surface density on tumor cells nor their susceptibility to NK cell-mediated lysis was affected. A single knockdown of HSF1 by shRNA decreased the surface expression of MICB but not that of MICA, and thereby, the NK cell-mediated lysis was only partially blocked. In contrast, NZ28 completely blocked the MICA/B membrane expression on tumor cells and thereby strongly inhibited the NK cell-mediated cytotoxicity. This effect might be explained by a simultaneous inhibition of the transcription factors HSF1, Sp1 and NF-κB by NZ28. These findings suggest that new anticancer therapeutics should be investigated with respect to their effects on the innate immune system.

Sensitizing tumor cells to radiation by targeting the heat shock response.

Elevated levels of heat shock proteins (HSPs) contribute to tumor cell survival and mediate protection against radiation-induced cell death. Hsp90 inhibitors are promising radiosensitizers but also activate heat shock factor 1 (HSF1) and thereby induce the synthesis of cytoprotective Hsp70. In this study the heat shock response inhibitor NZ28 either alone or in combination with the Hsp90 inhibitor NVP-AUY922 was investigated for radiosensitizing effects, alterations in cell cycle distribution and effects on migratory/invasive capacity of radioresistant tumor cells. NZ28 reduced the constitutive and NVP-AUY922-induced Hsp70 expression by inhibition of the HSF1 activity and inhibited migration and invasion in human lung and breast tumor cells. Treatment of tumor cells with NZ28 significantly increased their radiation response. One possible mechanism might be a decrease of the radioresistant S-phase. When combined with the Hsp90 inhibitor NVP-AUY922 the concentration of NZ28 could be significantly reduced (1/10th-1/20th) to achieve the same radiosensitization. Our results demonstrate that a dual targeting of Hsp70 and Hsp90 with NZ28 and NVP-AUY922 potentiates the radiation response of tumor cells that are otherwise resistant to ionizing radiation.