Natural products reveal cancer cell dependence on oxysterol-binding proteins

Abstract

Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)--proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.

Figure 1. Chemical structures

Natural products cephalostatin 1 (1), OSW-1 (2), ritterazine B (3), schweinfurthin A (4), schweinfurthin B (5) and stellettin E. OSW-1 analogs 6–10 and the OSW-1 affinity reagent (OAR). PMbz, para-methoxy benzoyl.

Figure 2. OSBP and ORP4L are high-affinity receptors of ORPphilins

(a) Cephalostatin 1 (1), OSW-1 (2), ritterazine B (3), schweinfurthin A (4), stellettin E and OSW-1 analogs 6–10 are selectively growth-inhibitory toward HCT-116 cells lacking p21. GI₅₀ values of the compounds were determined in HCT-116 p21^+/+ and p21^−/− cells after 48 h using a resazurin redox dye to measure cell viability. Values are mean ± s.d. of three individual experiments. Asterisk denotes value from ref. 5. (b) Affinity chromatography experiment with HeLa S-100 lysate using OSW-1 affinity reagent (OAR) in the absence (left lane) and presence (right lane) of excess OSW-1 (10 μM). The intense band indicated with a red arrow was excised from the gel and identified as oxysterol binding protein (OSBP). (c) Scatter plot depicting 121 proteins identified and quantified by itRAQ-based quantitative proteomics. Competition is plotted on the y axis as log₁₀ fold change for 10 μM OSW-1 over DMSO control, and the P value representing statistical significance is plotted on the x axis. P values are arbitrarily set to 1 for nonsignificant single peptide quantitations. (d,e) Representative competition-binding experiments of [³H]25-OHC (20 nM) with ORPphilins in S100 lysate made from HEK-293t cells overexpressing either OSBP-Myc-His (d) or ORP4L-Myc-His (e). K_i values are mean ± s.d. from at least three independent experiments. An accurate K_i value for ritterazine B with ORP4L could not be determined because of insufficient quantities of compound. 25-OHC, 25-hydroxycholesterol; BFA, brefeldin A; 5-FU, 5-fluorouracil.

Figure 3. shRNA knockdown, protein expression and SAR experiments support OSBP and ORP4L as targets of ORPphilins

(a) Quantitation of OSBP concentrations in nontargeting shRNA (shNT) and OSBP-targeting shRna (shOSBP) cells. Mean ± s.d. of seven individual experiments. ****P ± 0.0001 (n = 7) compared to shNT cells (two-tailed Student's t-test). (b) Knockdown and dose-shift experiment in HCT-116 p21^−/− cells stably transfected with either shOSBP or shNT. Cells were treated with compounds for 72 h, and GI₅₀ values were determined as described in Figure 2. Values are mean ± s.d. of three or four individual experiments. *P = 0.0002 (n = 3), **P = 0.0006 (n = 4), ***P = 0.0003 (n = 3) compared to shNT cells (two-tailed Student's t-test). (c) Western blot of HeLa lysates expressing lacZ-, OSBP- or ORP4L-Myc-His. (d) Effect of overexpression of lacZ, OSBP or ORP4L on the growth-inhibitory activity (48 h) of cephalostatin 1, OSW-1, schweinfurthin A and taxol in HeLa cells. Relative GI₃₀ values are averages from at least three individual experiments. (e,f) Correlation between binding affinity (K_i) to OSBP-Myc-His (e) or ORP4L-Myc-His (f) and growth inhibition (GI₅₀) in HCT-116 cells. Data points represent mean ± s.d. from three individual experiments. Pearson correlation coefficients (r) were calculated using GraphPad Prism 5 (P < 0.001). In f, schweinfurthin A (red) was omitted from the calculation. Uncut, full-gel images of panels a and c are shown in Supplementary Figure 4.

Figure 4. Suppression of antiproliferative activity by 25-OHC administration and effects of lipoprotein-deficient media on proliferation

(a) Effect of 25-OHC coadministration on growth-inhibitory activity (48 h) of cephalostatin 1 or OSW-1 in HCT-116 cells. (b) Removal of lipoproteins from the growth medium sensitizes CHO-7 cells to 25-OHC but not cephalostatin 1, OSW-1 or schweinfurthin A. Graphs represent mean ± s.d. of two individual experiments. CHO-7 is a CHO-K1 subclone that tolerates growth media depleted of lipoproteins.

Figure 5. ORPphilins induce OSBP translocation in cells

(a–e) OSBP (green) and trans-Golgi protein P230 (red) were visualized in HCT-116 cells using indirect immunofluorescence microscopy. Cells were treated with vehicle (a), 5 μM 25-OHC (b), 5 μM schweinfurthin A (c), 10 nM OSW-1 (d) or 20 nM cephalostatin 1 (e) for 4 h and then stained using specific primary antibodies against OSBP (green) and trans-Golgi protein P230 (red), followed by secondary staining with fluorescent antibodies. Nuclei (blue) were stained with Hoescht 33258. Scale bars are 5 μm. In a–c, arrows show colocalization of OSBP and p230. In e, the arrow shows OSBP localized at the plasma membrane.

Figure 6. Cephalostatin 1 and OSW-1 cause proteasome-dependent reduction of cellular OSBP concentrations

(a) Cephalostatin 1 (50 nM) and OSW-1 (10 nM) induce a time-dependent reduction in OSBP protein levels in HCT-116 cells as judged by western blotting. (b) Schweinfurthin A and 25-OHC do not induce reduction in OSBP concentrations after 24 h of treatment but can block the reduction induced by OSW-1. (c) The reduction in OSBP protein concentrations (at 24-h time point) is blocked by addition of the proteasome inhibitors MG-132 or lactacystin. *P < 0.05 (n = 3), **P < 0.01 (n = 3), ***P< 0.001 (n = 3), ****P < 0.0001 (n = 3) relative to vehicle-treated cells; ^#P = 0.0006 (n = 3), ^##P = 0.0196 (n = 3), ^###P = 0.0025 (n = 3) (two-tailed Student's t-test). Uncut, full-gel image of a is shown in Supplementary Figure 4.

Figure 7. ORPphilins block sphingomyelin biosynthesis

Pulse-labeling of sphingomyelin with [³H]serine in CHO-K1 cells. Cells were treated with vehicle or compound for 6 h, and in the last 2 h the cells were pulsed with 10 μCi [³H]serine. Label incorporation into sphingomyelin was measured by scintillation counting after lipid isolation by thin layer chromatography. All data are mean ± s.d. from three individual experiments. *P < 0.05 (n = 3), **P < 0.01 (n = 3), ***P < 0.001 (n = 3), ****P < 0.0001 (n = 3) relative to vehicle-treated cells (two-tailed Student's t-test).