Heat Shock Protein 70 (Hsp70) Suppresses RIP1-Dependent Apoptotic and Necroptotic Cascades

Abstract

Hsp70 is a molecular chaperone that binds to "client" proteins and protects them from protein degradation. Hsp70 is essential for the survival of many cancer cells, but it is not yet clear which of its clients are involved. Using structurally distinct chemical inhibitors, we found that many of the well-known clients of the related chaperone, Hsp90, are not strikingly responsive to Hsp70 inhibition. Rather, Hsp70 appeared to be important for the stability of the RIP1 (RIPK1) regulators: cIAP1/2 (BIRC1 and BIRC3), XIAP, and cFLIP_S/L (CFLAR). These results suggest that Hsp70 limits apoptosis and necroptosis pathways downstream of RIP1. Consistent with this model, MDA-MB-231 breast cancer cells treated with Hsp70 inhibitors underwent apoptosis, while cotreatment with z-VAD.fmk switched the cell death pathway to necroptosis. In addition, cell death in response to Hsp70 inhibitors was strongly suppressed by RIP1 knockdown or inhibitors. Thus, these data indicate that Hsp70 plays a previously unrecognized and important role in suppressing RIP1 activity.Implications: These findings clarify the role of Hsp70 in prosurvival signaling and suggest IAPs as potential new biomarkers for Hsp70 inhibition. Mol Cancer Res; 16(1); 58-68. ©2017 AACR.

Figure 1. Inhibition of Hsp70 Causes Rapid Anti-Proliferative Activity

(A) Chemical structures of Hsp70 inhibitors (JG-98, JG-84, MAL3-101 and VER-155008), controls (JG-258) and inhibitors of Hsp90 (17-DMAG) and the proteasome (bortezomib). (B) JG-98 (5 μM) suppress growth of MDA-MB-231 cancer cells with relatively rapid kinetics compared to 17-DMAG (5 μM) or bortezomib (40 nM), as monitored by MTT assays. Results are the average of two independent experiments performed in quintuplicates. The error bars represent the standard error of the mean (SEM). (C) Hsp70 inhibitors induce rapid cell growth effects, as measured by Cell Titer Glo. Results are the average of experiments performed in triplicate. Errors are SEM. (D) Chaperone clients are degraded relatively late after treatment with JG-98 (10 μM), after onset of effects on cell growth. Results are representative of experiments performed in duplicate.

Figure 2. Hsp70 Inhibitor Activity is Independent of Bcl Status

(A) Overexpression of Bcl-xL does not block JG-98 cytotoxicity. MDA-MB-231 cells were treated for 24 hrs with JG-98 (10 μM), 17-DMAG (10 μM), bortezomib (40 nM) or etoposide (20 μM). MTT results are the mean average of triplicates and error bars represent SEM. *p value < 0.05. (B) Jurkat cells over-expressing Bcl-xL were treated with indicated compounds for 24 hours. Viability was determined by trypan blue staining. Results are the average of two experiments performed in triplicate. Error is SEM. **p value < 0.01. (C) Bcl-xL overexpression provides partial resistance to compounds, except JG-98. MDA-MB-231 cells were treated for 72 hours and cell growth determined by MTT assays. Results are the average of two independent experiments performed in triplicate. Error is SEM.

Figure 3. Inhibition of Caspases with z-VAD-fmk Does not Block JG-98’s Activity

(A) Inhibition of caspases by z-VAD.fmk does not suppress the activity of JG-98. Results are the average of three independent experiments performed in quintuplicate. Error bars represent SEM. Cells were pretreated with z-VAD.fmk (40 μM) for 1 hour prior to addition of compounds (same concentrations as in panel A) and MTT assays performed after 24 hrs. **p value < 0.01. ns = not significant. Error is SEM. (B) Cells were treated as in panel A and visualized using an Olympus IX83 Inverted Microscope. Black arrows indicate apoptotic cells; white arrows indicate necrotic cells. (C) Hoechst 33258 staining of MDA-MB-231 cells pretreated with z-VAD.fmk (40 μM) for 1 hour prior to addition of JG-98 (10 μM) for 24 hours. Cells pre-treated with z-VAD.fmk show no nuclear fragmentation (white arrows), even after co-administration of JG-98. Scale bar is 10 μm.

Figure 4. Proteolytic Signature of Cells Treated with JG-98 Supports Activation of Both an Apoptotic and Alternative Cell Death Pathway

(A) Schematic overview of the proteolytic profiling study (aka “N-terminal degradomics”). (B) Overview of the peptide fragments produced in response to compound treatments. More than 45% of the substrates identified after JG-98 treatment feature a P1 = D, suggesting the activation of caspases. In general, there was minimal proteolysis in untreated cells or cells treated with JG-98 and z-VAD. Aminobutyric acid (Abu). (C) Logo plots show that JG-98 activates apoptosis in Jurkat cells and NSA is unable to block this activity. However, z-VAD reversed the proteolytic profile to one that approximated untreated cells. (D) The caspase activation profile (P1=D peptides) was similar between cells treated with JG-98 or JG-98 plus NSA, while the pattern of peptides (P1 = all) was similar between JG98 + zVAD and untreated cells. Experiments repeated in biological duplicate and techincal triplicate.

Figure 5. JG-98 Activity Occurs through a RIP1-Dependent Process

(A) RIP1 KO Jurkat cells are resistant to JG-98. Viability was determined by trypan blue exclusion. Cells were treated for 24 hrs with JG-98 (10 μM), 17-DMAG (10 μM), bortezomib (40 nM), etoposide (20 μM). Results are the average of three independent experiments performed in triplicate. ns = not significant; * p < 0.05. (B) JG-98 activity requires RIP1 kinase. MDA-MB-231 cells were pretreated with 20 μM necrostatin-1 for 1 hour prior to addition of compounds. Viability was determined by three independent MTT assays performed in quintuplicate. Error is SEM. ns = not significant; ***p < 0.0001. (C) JG-98 induces degradation of RIP1 modulators. MDA-MB-231 cells were treated for 24 hours. Results represent experiments performed in triplicate. (D) RIP1 regulators are rapidly degraded in response to JG-98. MDA-MB-231 cells were treated with JG-98 (10 μM). Results are representative of duplicates.

Figure 6. JG-98 Activity Requires Both Apoptosis and Necroptosis

(A) Inhibition of necroptosis alone does not restore growth of cells treated with JG-98. Cells were pretreated with necrosulfonamide (NSA, 20 μM) for 1 hour prior to addition of compounds. Error is SEM. (B) Inhibition of both apoptosis and necroptosis is necessary to fully suppress JG-98 activity. Cells were pretreated with both 40 μM z-VAD.fmk and 20 μM Necrosulfonamide for 1 hour prior to addition of compounds. Cell growth was determined by three independent MTT assays performed in quintuplicate. *p value < 0.05, **p value < 0.01, ***p value < 0.001, ns = not significant. (C) Jurkat cells were pretreated with z-VAD.fmk (40 μM), NSA, both, or necrostatin-1 for 1 hour prior to addition of JG-98 (10 μM). Inactivation of both apoptosis and necroptosis was necessary to prevent activity. Cell viability was determined by trypan blue exclusion. Results are the average of three independent experiments performed in triplicate.

Figure 7. Hsp70 Limits RIP1/3 Oligomerization

(A) JG-98 toxicity does not depend on TNF signaling. MDA-MB-231 cells were pre-treated with Enbrel (5 μg/mL) for 1 hour prior to addition of compounds. Viability was determined by MTT assay. Results are the average of three independent experiments performed in quintuplicate. Error is SEM. **p value < 0.01 (B) JG-98 does not induce formation of a RIP1-caspase-8 complex. MDA-MB-231 cells were treated for 24 hours with indicated compounds. SM-164 was used at 100 nM. (C) Treatment with JG-98 (10 μM) favored formation of a high molecular mass oligomer of RIP1 and a reduction of RIP3. Co-treatment with z-VAD-fmk exacerbated this effect. Necrostatin could block the effects of JG-98. Results are representative of experiments performed in triplicate. (D) Treatment with 17-DMAG or bortezomib (BTZ) did not change RIP1 oligomerization.