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. 2012 Oct 17;104(20):1576-90.
doi: 10.1093/jnci/djs396. Epub 2012 Oct 5.

Selective inhibition of HER2-positive breast cancer cells by the HIV protease inhibitor nelfinavir

Joong Sup Shim  1 , Rajini Rao, Kristin Beebe, Len Neckers, Inkyu Han, Rita Nahta, Jun O Liu
Affiliations

Selective inhibition of HER2-positive breast cancer cells by the HIV protease inhibitor nelfinavir

Joong Sup Shim et al. J Natl Cancer Inst. .

Abstract

Background: Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is highly aggressive and has higher risk of recurrence than HER2-negative cancer. With few treatment options available, new drug targets specific for HER2-positive breast cancer are needed.

Methods: We conducted a pharmacological profiling of seven genotypically distinct breast cancer cell lines using a subset of inhibitors of breast cancer cells from a screen of the Johns Hopkins Drug Library. To identify molecular targets of nelfinavir, identified in the screen as a selective inhibitor of HER2-positive cells, we conducted a genome-wide screen of a haploinsufficiency yeast mutant collection. We evaluated antitumor activity of nelfinavir with xenografts in athymic nude mouse models (n = 4-6 per group) of human breast cancer and repeated mixed-effects regression analysis. All statistical tests were two-sided.

Results: Pharmacological profiling showed that nelfinavir, an anti-HIV drug, selectively inhibited the growth of HER2-positive breast cancer cells in vitro. A genome-wide screening of haploinsufficiency yeast mutants revealed that nelfinavir inhibited heat shock protein 90 (HSP90) function. Further characterization using proteolytic footprinting experiments indicated that nelfinavir inhibited HSP90 in breast cancer cells through a novel mechanism. In vivo, nelfinavir selectively inhibited the growth of HER2-positive breast cancer cells (tumor volume index of HCC1954 cells on day 29, vehicle vs nelfinavir, mean = 14.42 vs 5.16, difference = 9.25, 95% confidence interval [CI] = 5.93 to 12.56, P < .001; tumor volume index of BT474 cells on day 26, vehicle vs nelfinavir, mean = 2.21 vs 0.90, difference = 1.31, 95% CI = 0.83 to 1.78, P < .001). Moreover, nelfinavir inhibited the growth of trastuzumab- and/or lapatinib-resistant, HER2-positive breast cancer cells in vitro at clinically achievable concentrations.

Conclusion: Nelfinavir was found to be a new class of HSP90 inhibitor and can be brought to HER2-breast cancer treatment trials with the same dosage regimen as that used among HIV patients.

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Figures

Figure 1.
Figure 1.
Effect of nelfinavir (NFV) on human epidermal growth factor receptor 2 (HER2) signaling pathway. A) Effect of increasing concentrations of NFV on HER2 signaling pathway and caspase cascades in HER2-positive and -negative breast cancer cells. Cells were treated with various concentrations of NFV for 24 hours, and each protein level was analyzed by immunoblots. B) A time-course experiment of the NFV effect on HER2 signaling pathway and caspase cascades in HER2-positive breast cancer, HCC1954 cells. C) Effect of proteasome inhibitors and caspase inhibitors on NFV effects on HER2 signaling pathway and caspase cascades in HCC1954 cells. For the cotreatment experiments, cells were pretreated with either caspase inhibitors or proteasome inhibitors for 30 minutes and then incubated with NFV for 24 hours. z-DEVD (30 µM z-DEVD-fmk, caspase-3 inhibitor), z-IETD (30 µM z-IETD-fmk, caspase-8 inhibitor), z-VAD (30 µM z-VAD-fmk, pan caspase inhibitor), MG132 (0.4 µM, proteasome inhibitor), and BTZ (20nM bortezomib, proteasome inhibitor) were used for this study. Act-cas-3 = active form of caspase 3; Act-cas-8 = active form of caspase 8; Act-cas-9 = active form of caspase 9; NS = nonspecific band; pERK1 and -2; phospho ERK1 and -2; pHER2 = phospho-HER2; Pro-cas-8 = proform of caspase 8; Pro-cas-9 = proform of caspase 9.
Figure 2.
Figure 2.
Identification of molecular target of nelfinavir. A) Nelfinavir (NFV) sensitive, heterozygote yeast strains identified from the screen. The yeast strains colored in gray are sensitive to NFV by at least two-fold compared with the wild-type parental yeast strain. The yeast strains colored in white, including heat shock protein 82 (HSP82), are genetically or physically linked strains with the NFV-sensitive strains. Solid lines indicate a physical interaction between two genes, and dotted lines represent a genetic interaction. B) Effect of nelfinavir on interaction between heat shock protein 90 (HSP90) and co-chaperones in rabbit reticulocyte lysates. Rabbit reticulocyte lysates were preincubated with drugs (NFV, 5 µM and 20 µM; MG132, 5 µM; geldanamycin [GA], 5 µM; novobiocin [NB], 0.2mM) for 2 hours on ice and were diluted in a buffer for the immunoprecipitation assay with HSP90 antibody. DMSO = dimethyl sulfoxide; HOP = HSP70–HSP90 organizing protein; HSP70 = heat shock protein 70; IgG = immunoglobulin G; IP = immunoprecipitation; WB = immunoblot.
Figure 3.
Figure 3.
Proteolytic footprinting to assess heat shock protein 90 (HSP90) conformational changes under the nelfinavir (NFV) treatment. A) A schematic illustration of HSP90 domain structures and trypsin cleavage sites. Major peptide fragments of HSP90 generated by trypsin digestion were labeled based on the observed molecular size of each peptide. N+M and M+C domains of HSP90 were cloned and purified. B and C) Effect of drugs on trypsin digestion profile of the full-length HSP90. Purified full-length (FL) HSP90 (150ng) was incubated with NFV (100 µM), geldanamycin (GA) (50 µM), or novobiocin (NB) (10mM) for 2 hours at room temperature prior to trypsin digestion. The cleavage fragments were then detected using a C-terminal (B) or an N-terminal (C) HSP90 antibody. D) FL HSP90 (600ng) was incubated with NFV (100 µM) or NB (10mM) for 30 minutes at room temperature prior to trypsin digestion. The cleavage fragments were then analyzed using an N-terminal HSP90 antibody. E and F) Effect of drugs on trypsin digestion profile of HSP90 truncation domains. Two micrograms of HSP90 N+M domain (E) or M+C domain (F) were incubated with GA (50 µM), NFV (100 µM), and NB (10mM) for 1 hour at room temperature prior to trypsin digestion. The cleavage products were then analyzed by Coomasie brilliant blue staining after separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis. CL = charged linker domain; DMSO = dimethyl sulfoxide; EEVD = glutamic acid, glutamic acid, valine, aspartic acid.
Figure 4.
Figure 4.
Effect of nelfinavir (NFV) on conformational change of heat shock protein 90 (HSP90) domains. A) A time-course experiment of the trypsin digestion of HSP90 M+C domain. The M+C domain (2 µg) was incubated with dimethyl sulfoxide (DMSO) or NFV for 1 hour at room temperature and digested with trypsin on ice for indicated time points. The cleavage products were then analyzed by Coomasie brilliant blue staining after separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis. Major cleavage fragments, 50 kD and 35 kD, are indicated by arrows. A representative gel image from three independent experiments is shown. B) Quantitation of the amount of M+C domain and its cleavage products shown in (A). The ratio was calculated by dividing the amount of uncleaved M+C domain with the amount of total sum of M+C domain, 50 kD fragment and 35 kD fragment. The number on each bar is the relative ratio (average from three independent experiments) between DMSO and NFV. DMSO vs NFV (in each time point), *P < .05, calculated by two-sided Student’s t test. Error bars = 95% confidence intervals. C) Effect of cotreatment of NFV and NB (novobiocin) on the trypsin digestion profile of full-length (FL) HSP90. The HSP90 was pretreated with NFV (100 µM) or NB (10mM) for 30 minutes at room temperature and then treated with NB or NFV for 1 hour at room temperature. The order of treatment is indicated by arrows. The cleavage profile was analyzed by the N-terminal HSP90 antibody. D) Effects of drugs on trypsin digestion profiles of HSP90 are summarized. The trypsin cleavage rate on each site was illustrated with different size of arrows. The bigger arrows are the faster cleavage occurs. GA = geldanamycin.
Figure 5.
Figure 5.
A systematic comparison of nelfinavir (NFV) effect on human epidermal growth factor receptor 2 (HER2)–positive breast cancer cells with that of known heat shock protein 90 (HSP90) inhibitors and a proteasome inhibitor. A and B) Effect of drugs on the protein levels of HSP90 client proteins, co-chaperones, an estrogen receptor (ER) stress marker, an apoptosis marker, and ubiquitinated proteins in HCC1954 cells. The cells were treated with drugs for 24 hours, and immunoblot analyses were conducted. C) Immunofluorescence of HER2 and ubiquitinated proteins in HCC1954 cells, bar = 30 μm. D and E) Effect of NFV and novobiocin (NB) on interaction between HSP90 and HER2. HCC1954 cells were treated with NFV or NB for 5 hours, and HSP90 was immunoprecipitated to analyze the binding status of HER2 (D). The cells were treated with NFV for 3 hours, and HER2 was immunoprecipitated to analyze the binding status of HSP90 (E). Whole cell lysates (WCLs) were used to analyze the input. AHA1 = activator of heat shock protein ATPase homolog 1; CDK4 = cyclin dependent kinase 4; CDK6 = cyclin dependent kinase 6; DAPI = 4’,6-diamidino-2-phenylindole; eIF2α = eukaryotic initiation factor 2α; HSP70 = heat shock protein 70; IP = immunoprecipitation; GA = geldanamycin; MG = MG132; PARP = poly ADP ribose polymerase; Ub = ubiquitin; WB = immunoblot.
Figure 6.
Figure 6.
Effect of nelfinavir (NFV) on the growth of human epidermal growth factor receptor 2 (HER2)–positive or HER2-negative breast cancer xenografts in mice. A and B) Analysis of tumor volume index. Twelve female athymic nude mice (BALB/c, nu/nu-NCr) (n = 6 mice per group) bearing HCC1954 HER2-positive human breast cancer cells were injected intraperitoneally with vehicle or NFV (25mg/kg) every day. Mice bearing HCC1937 HER2-negative breast cancer cells (n = 4 mice per group) were also injected intraperitoneally with vehicle or NFV(25mg/kg) every day. Mean tumor volume indices in each group are shown. The tumor volume index was calculated as a ratio of the tumor volume on a given day divided by the tumor volume of day 0. Error bars = 95% confidence intervals. C and D) Analysis of tumor volume index. Ten female athymic nude mice (BALB/c, nu/nu-NCr) (n = 5 mice per group) bearing BT474 HER2-positive human breast cancer cells or MDA-MB-231 (HER2-negative ones) were orally given vehicle or NFV (40mg/kg) every day. Mean tumor volume indices in each group are shown. The tumor volume index was calculated as a ratio of the tumor volume on a given day divided by the tumor volume of day 0. Error bars = 95% confidence intervals. E and F) The protein levels of HER2, phospho-HER2 (p-HER2), AKT, and phospho-AKT (p-AKT) in the BT474 (E) or MDA-MB-231 (F) tumor extracts were analyzed by immunoblots and quantitated using Image J software. Vehicle vs test groups, *P < .05, calculated by two-sided Student’s t test. Error bars = 95% confidence intervals.
Figure 7.
Figure 7.
Effect of nelfinavir on the proliferation of trastuzumab-/lapatinib-resistant, human epidermal growth factor receptor 2 (HER2)–positive breast cancer cells. Cell proliferation was assessed using [3H]-thymidine incorporation assay. A) Effect of trastuzumab on the proliferation of drug-sensitive and -resistant breast cancer cells. The dose-response curves of trastuzumab on BT474 (drug-sensitive, Her2-positive breast cancer cell line), BT474-TrastR (acquired trastuzumab-resistant BT474 cells), MDA-MB-453 (intrinsic trastuzumab-resistant, Her2-positive breast cancer cell line), and JIMT1 (intrinsic trastuzumab-resistant, Her2-positive breast cancer cell line) are shown. The dashed line represents a half maximal inhibitory concentration (IC50) of the drug. Error bars = 95% confidence intervals. B) Effect of lapatinib on the proliferation of drug-sensitive and -resistant breast cancer cells. The dashed line represents an IC50 of the drug. Error bars = 95% confidence intervals. C) Effect of nelfinavir on the proliferation of drug-sensitive and -resistant breast cancer cells. The dashed line represents an IC50 of the drug. Error bars = 95% confidence intervals.
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