Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33

Abstract

Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential.

Figure 1.

HSP90 associates with and stabilizes STK33. (a) Anti-Flag IPs were performed with KRAS WT BT-20 and KRAS mutant MDA-MB-231 breast cancer cell lines stably transduced with empty vector (EV), N-terminally Flag-tagged STK33 (Flag-STK33), or C-terminally Flag-tagged STK33 (STK33-Flag), and the resulting protein complexes were separated by PAGE and stained with Coomassie. Peptides isolated from each lane were analyzed by mass spectrometry. The STK33 band is indicated by an asterisk. SM, size marker. (b) Number of peptides representing proteins highly enriched in STK33-containing IPs. (c) Anti-HA IPs were performed with MDA-MB-231 cells stably transduced with empty vector (EV), N-terminally HA-tagged STK33 (HA-STK33), or C-terminally HA-tagged STK33 (STK33-HA), and immunoblots were probed with antibodies against HSP90A/B, CDC37, and HA. One of three independent experiments is shown. (d) Protein expression of HSP90A, HSP90B, STK33, and cleaved PARP in lung (NCI-H520, A549) and colon (Caco-2, HCT-116) carcinoma cells transduced with a nontargeting control shRNA or a shRNA-targeting HSP90A or HSP90B. One of two independent experiments is shown.

Figure 2.

Pharmacologic inhibition of HSP90 depletes STK33 and preferentially induces apoptosis in mutant KRAS-dependent cancer cells. (a) Immunoblots of lung cancer (NCI-H520, A549), colon cancer (Caco-2, HCT-116), breast cancer (BT-20, MDA-MB-231), and AML (HL-60, SKM-1) cell lines incubated with increasing concentrations of PU-H71 or 17-AAG for 24 h. (b) Caspase 9 activity in KRAS mutant (mut) and KRAS WT cancer cell lines incubated with 0.5 µM PU-H71 for 24 h. Experiments were done in triplicate. Error bars represent mean ± SEM. One of two independent experiments is shown. (c) Viability and proliferation of mutant KRAS-dependent (mut) and KRAS WT cancer cell lines incubated with low PU-H71 concentrations for 48 h. Experiments were done in triplicate. Error bars represent mean ± SEM. One of two independent experiments is shown.

Figure 3.

Causal relationship between degradation of STK33 and killing of mutant KRAS-dependent cancer cells by HSP90 inhibitors. (a) Immunoblots of mutant KRAS-dependent breast cancer (MDA-MB-231) and lung cancer (A549) cell lines incubated with 1 µM PU-H71 for the indicated times. (b) Immunoblot of MDA-MB-231, A549, and Calu-1 cells stably transduced with empty vector (EV) or STK33 and incubated with different concentrations of PU-H71 for 24 h. Cleaved PARP bands were quantified by densitometric analysis using the ImageJ program. Results of two to three independent experiments are shown. RU, relative unit. (c) Caspase 9 activity in KRAS mutant breast cancer (MDA-MB-231) and lung cancer (Calu-1) cell lines transduced with empty vector (EV) or STK33 and incubated with 0.5 µM PU-H71 for 24 h. Experiments were done in triplicate. Error bars represent mean ± SEM. (d) Immunoblot of KRAS WT MDA-MB-453 breast cancer (ERBB2-amplified) and MOLM-14 AML (FLT3-mutated) cells stably transduced with empty vector (EV) or STK33 and incubated with different concentrations of PU-H71 for 24 h. (e) Immunoblot of MDA-MB-231 cells that evaded apoptosis induced by shRNA-mediated STK33 knockdown after incubation with increasing concentrations of PU-H71 for 24 h. All results were confirmed in at least one additional independent experiment.

Figure 4.

Relationship between degradation of AKT1 or RAF1 and killing of mutant KRAS-dependent cancer cells by HSP90 inhibitors. (a) Immunoblots of MDA-MB-231 cells incubated with 1 µM PU-H71 for the indicated times showing depletion of AKT1 and RAF1. (b and c) PARP cleavage in mutant KRAS-dependent lung cancer (A549), breast cancer (MDA-MB-231), and colon cancer (HCT-116) cell lines transduced with shRNAs targeting AKT1 (c) and RAF1 (b), respectively, or a nontargeting control shRNA. (d) Immunoblots of A549, MDA-MB-231, and HCT-116 cells stably transduced with empty vector (EV), AKT1, or RAF1 and incubated with 1 µM PU-H71 for 24 h. All results were confirmed in at least one additional independent experiment.

Figure 5.

PU-H71 induces ubiquitination and proteasomal degradation of STK33. (a) Immunoblot of A549 cells treated with 20 mM NH₄Cl for 2 h, followed by incubation with 0.5 µM PU-H71 for 16 h. One of two independent experiments is shown. (b) Immunoblot of A549 cells treated with 10 µM MG-132 for 2 h, followed by incubation with 0.5 µM PU-H71 for 16 h. (c) IP of HA-tagged STK33 from 293T cells treated with or without 1 µM PU-H71 for 7 h and 10 µM MG-132 for the final 3 h. Ubiquitinated STK33 appears as a smear with high molecular weight. One of three independent experiments is shown. (d) Anti-Flag IPs were performed with KRAS WT BT-20 and mutant KRAS-dependent MDA-MB-231 cells stably transduced with empty vector (EV), N-terminally Flag-tagged STK33 (Flag-STK33), or C-terminally Flag-tagged STK33 (STK33-Flag), and the resulting protein complexes were analyzed by mass spectrometry. The enrichment of peptides representing proteins involved in the ubiquitin/proteasome system is shown. (e) Immunoblot of MDA-MB-231 and A549 cells transduced with empty vector (EV) or BAG2 and treated with 0.5 µM PU-H71 for 24 h. (f) Immunoblot of HCT-116 and A549 cells transduced with a nontargeting control shRNA or shRNAs targeting BAG2. (g) Anti-Flag IPs were performed with MDA-MB-231 cells stably transduced with empty vector (EV) or Flag-tagged STK33, and immunoblots were probed with antibodies against BAG2 and Flag. One of two independent experiments is shown. (h) Immunoblot of 293T cells transfected with HA-tagged STK33 or Xpress-tagged USP15 and USP5, respectively, and treated with or without 0.5 µM PU-H71 for 24 h. One of two independent experiments is shown. (i) MDA-MB-231 cells were transfected with a nontargeting control shRNA or a shRNA targeting HERC2. After 3 d, cells were incubated with 1 µM PU-H71 for 18 h, as indicated. Protein levels were analyzed by Western blotting using the indicated antibodies. One of two independent experiments is shown.

Figure 6.

HSP90 inhibition impairs the growth of mutant KRAS-dependent colon tumors on chicken CAM. (a) Tumor formation on chicken CAM of KRAS WT (Caco-2) and mutant KRAS-dependent (HCT-116) colon cancer cell lines treated with 5 µM 17-AAG or 1 µM PU-H71 or vehicle for 48 h. Representative photographs of tumors (cells were deposited within a 5-mm silicon ring to allow drug administration) and tumor areas (error bars represent mean ± SEM of four to six tumors) are shown. Bar, 1.5 mm. (b and c) IHC analysis of CAM tumors shown in a. STK33 protein expression (b) and TUNEL staining (c) of Caco-2 and HCT-116 tumors. Representative photographs of tissue sections and the proportion of TUNEL-positive cells (error bars represent mean ± SEM of four microscopic fields with 600 cells) are shown. NS, not significant. Insets in c show details of the corresponding photographs. Bar: 125 µm; inset, 25 µm.

Figure 7.

STK33 overexpression restores the growth of mutant KRAS-dependent breast tumors treated with HSP90 inhibitors. (a) Tumor formation on chicken CAM of mutant KRAS-dependent MDA-MB-231 breast cancer cells stably transduced with Flag-tagged STK33 or empty vector (EV) and treated with 5 µM 17-AAG or 1 µM PU-H71 for 48 h. Representative photographs of tumors and tumor areas (error bars represent mean ± SEM of four to six tumors) are shown. NS, not significant. Bar, 1.5 mm. (b) IHC analysis of CAM tumors shown in a. Representative photographs of tissue sections and the proportion of TUNEL-positive cells (error bars represent mean ± SEM of four microscopic fields with 600 cells) are shown. EV, empty vector. Insets show details of the corresponding photographs. Bar: 125 µm; inset, 25 µm.

Figure 8.

HSP90 inhibition impairs the growth of mutant KRAS-dependent tumors in nude mice. (a) Immunoblot of HCT-116 tumors. After tumor formation, mice were injected once with 75 mg per kg body weight PU-H71 or PBS, and tumors were harvested after the indicated times. One mouse per condition and time point was analyzed. (b) Effects of PU-H71 on tumor formation of mutant KRAS-dependent (SW-480, HCT-116) and KRAS WT (Caco-2, Colo-320HSR) colon cancer cell lines in nude mice. Photographs of representative tumors and tumor volumes (error bars represent mean ± SEM of 7–14 tumors from 4–7 mice) are shown. (c) IHC analysis of tumors shown in b. Representative photographs of tissue sections and the proportion of TUNEL-positive cells (error bars represent mean ± SEM of four microscopic fields with 600–800 cells) are shown. NS, not significant. Bar, 125 µm. (d) Immunoblot of tumors shown in b demonstrating STK33 expression in treated mice. Tumors were harvested 24 h after the last injection.

Figure 9.

HSP90 inhibition impairs the survival of primary KRAS mutant colon tumor-initiating cells. (a) Characteristics of primary human colon cancer samples used for the generation of sphere cultures. (b) Photographs of colon cancer sphere cultures treated with 0.5 µM PU-H71 or PBS for 48 h. Bar, 200 µm. (c) Viability and proliferation of colon cancer sphere cultures incubated with 0.5 µM PU-H71 for 48 h. 6–12 independent experiments were done. Error bars represent mean ± SD. (d) Cumulative analysis of the response to PU-H71 of the different patient-derived sphere cultures according to KRAS status. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.