Re-examining HSPC1 inhibitors

Abstract

HSPC1 is a critical protein in cancer development and progression, including colorectal cancer (CRC). However, clinical trial data reporting the effectiveness of HSPC1 inhibitors on several cancer types has not been as successful as predicted. Furthermore, some N-terminal inhibitors appear to be much more successful than others despite similar underlying mechanisms. This study involved the application of three N-terminal HSPC1 inhibitors, 17-DMAG, NVP-AUY922 and NVP-HSP990 on CRC cells. The effects on client protein levels over time were examined. HSPC1 inhibitors were also applied in combination with chemotherapeutic agents commonly used in CRC treatment (5-fluorouracil, oxaliplatin and irinotecan). As HSPA1A and HSPB1 have anti-apoptotic activity, gene-silencing techniques were employed to investigate the significance of these proteins in HSPC1 inhibitor and chemotherapeutic agent resistance. When comparing the action of the three HSPC1 inhibitors, there are distinct differences in the time course of important client protein degradation events. The differences between HSPC1 inhibitors were also reflected in combination treatment-17-DMAG was more effective compared with NVP-AUY922 in potentiating the cytotoxic effects of 5-fluorouracil, oxaliplatin and irinotecan. This study concludes that there are distinct differences between N-terminal HSPC1 inhibitors, despite their common mode of action. Although treatment with each of the inhibitors results in significant induction of the anti-apoptotic proteins HSPA1A and HSPB1, sensitivity to HSPC1 inhibitors is not improved by gene silencing of HSPA1A or HSPB1. HSPC1 inhibitors potentiate the cytotoxic effects of chemotherapeutic agents in CRC, and this approach is readily available to enter clinical trials. From a translational point of view, there may be great variability in sensitivity to the inhibitors between individual patients.

Keywords: Chemoresistance; Chemotherapy; Colorectal cancer; Heat shock proteins; Novel target; Tumour evolvability.

Fig. 1

a Caspase-3. b Propidium iodide. c MTS assays on HT29 cells treated with HSPC1 inhibitors (0–2 μM) for 48 h. d Bright field microscopy at ×40 magnification on HT29 cell treated with HSPC1 inhibitors (1 μM) for 48 h. Data in a, b and c are presented as mean ± SEM. Statistical analysis was performed using the one-way ANOVA with Dunnett’s post hoc test

Fig. 2

Time course analysis of a HER-2, b pNF-kB, c HSPA1A, d HSPB1 and e Annexin V, in HT29 cells treated with HSPC1 inhibitors (1 μM) for 48 h. Data are presented as mean ± SEM. Statistical analysis was performed using the one-way ANOVA with Dunnett’s post hoc test. The inset denotes the Western blots for HSPB1 and HSPA1A following 48 h of HSPC1 inhibitor treatment (1 μM)

Fig. 3

a HSPA1A and b HSPB1 analyses in HT29 cells treated with CHX (2.5 μg/ml) for 1 h before addition of 17-DMAG (1 μM) for 24 h. CHX remained on the cells throughout the 17-DMAG treatment period. Data are presented as absolute mean fluorescence intensity (MFI) values. Statistical analysis was performed using the unpaired t test

Fig. 4

HT29 cellular metabolism (a, c, e) and caspase-3 analysis (b, d, f) following treatment with 12.5 μM 5-FU (a, b), OX (c, d) or IRN (e, f) for 24 h followed by different HSPC1 inhibitors (300 nM) for a further 48 h. Data are presented as percentage of cell metabolism and percentage increase in caspase-3 compared to the control (untreated) population. Statistical analysis was performed using the unpaired t test

Fig. 5

a HSPA1A, b HSPB1 and c caspase-3 analyses in HT29 cells following siRNA-mediated gene silencing prior to HSPC1 inhibitor treatment. Cells were treated with siRNA for 24 h before addition of HSPC1 inhibitor (300 nM) to the culture medium for a further 48 h. Lipofectamine only (Lipo), non-targeting siRNA (NT) and RISC-free siRNA (RF) were used as controls to ensure specific knockdown of HSPA1A or HSPB1. HSPA1A and HSPB1 data are presented as percentage change from levels expressed by untreated cells. Caspase-3 data is presented as percentage of cells that are positive for caspase-3 after 48 h. Statistical analysis was performed using the unpaired t test to compare inhibitor-only treatment with the corresponding inhibitor plus siRNA

Fig. 6

Cell survival analysis analysed by caspase-3 or MTS assay in HT29 treated with HSPB1 or HSPA1A siRNA for 24 h followed by 12.5 μM of a 5-FU, b OX or c IRN for a further 48 h. Statistical analysis was performed using the unpaired t test to compare chemotherapeutic drug alone with the corresponding drug + siRNA-treated samples

Fig. 7

HER-2 levels in HT29 cells treated for 24 h with 1 μM NVP-AUY922 or for comparison, 17-DMAG. NVP-AUY922 was re-applied at varying time intervals during the 24-h period. Twenty-four-hour treatment with NVP-AUY922, no re-application (1). NVP-AUY922 was re-applied at t = 6 and left for the remaining 18 h (2). NVP-AUY922 was re-applied at t = 6 and t = 12 and left for the remaining 12 h (3). NVP-AUY922 was re-applied at t = 6, t = 12 and t = 18 and left for the remaining 6 h (4). Statistical analysis was performed using unpaired t test to compare percentage changes in HER-2 after various treatment to control (untreated) population

Fig. 8

MTS assay on HT29 cells following combined treatment with OX (25 μM) and NVP-AUY922 (300 nM) and for comparison, 17-DMAG (300 nM). OX was applied in isolation for the first 24 h, and NVP-AUY922/17-DMAG was added to the culture media for a further 48 h. NVP-AUY922 was re-applied at specific time intervals over the 48-h period. Forty-eight-hour treatment with NVP-AUY922, no re-application (0). NVP-AUY922 was re-applied at t = 6 and left for the remaining 42 h (1). NVP-AUY922 was re-applied at t = 6 and t = 12 and left for the remaining 36 h (2). NVP-AUY922 was re-applied at t = 6, t = 12 and t = 24 and left for the remaining 24 h (3). NVP-AUY922 was re-applied at t = 6, t = 12, t = 24 and t = 32 and left for the remaining 16 h (4). NVP-AUY922 was re-applied at t = 6, t = 12, t = 24, t = 32 and t = 40 and left for the remaining 8 h (5). Statistical analysis was performed using the one-way ANOVA with Dunnett’s post hoc test, and significant differences from the “OX only” are indicated