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. 2015 Jan 8;34(2):263-8.
doi: 10.1038/onc.2013.575. Epub 2014 Jan 27.

Hedgehog acyltransferase as a target in pancreatic ductal adenocarcinoma

E Petrova  1 , A Matevossian  2 , M D Resh  3
Affiliations

Hedgehog acyltransferase as a target in pancreatic ductal adenocarcinoma

E Petrova et al. Oncogene. .

Abstract

Sonic Hedgehog (Shh) is abnormally expressed in pancreatic cancer and is associated with disease onset and progression. Inhibition of Shh signaling is thus an attractive clinical target for therapeutic intervention. Most efforts to block Shh signaling have focused on inhibitors of Smoothened, which target the canonical Shh signaling pathway. These approaches have met with limited success, in part due to development of resistance-conferring mutations and contributions from non-canonical signaling pathways. Here, we show that Hedgehog acyltransferase (Hhat), the enzyme responsible for the attachment of palmitate onto Shh, is a novel target for inhibition of Shh signaling in pancreatic cancer cells. Depletion of Hhat with lentivirally delivered small hairpin RNA decreased both anchorage-dependent and independent proliferation of human pancreatic cancer cells. In vivo, Hhat knockdown led to reduction of tumor growth in a mouse xenograft model of pancreatic cancer. RU-SKI 43, a small molecule inhibitor of Hhat recently developed by our group, reduced pancreatic cancer cell proliferation and Gli-1 activation through Smoothened-independent non-canonical signaling. In addition, RU-SKI 43 treatment inhibited two key proliferative pathways regulated by Akt and mTOR. This work demonstrates that Hhat has a critical role in pancreatic cancer and that a small molecule inhibitor of Hhat can successfully block pancreatic cancer cell proliferation. It also highlights the importance of developing optimized Hhat inhibitors to be used as therapeutics in pancreatic cancer, as well as in other malignancies characterized by Shh overexpression.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Hhat inhibition blocks Shh signaling in pancreatic cancer cells. Expression of Hhat (a), Shh (b) and Gli-1 (c) mRNAs in different pancreatic cancer cell lines, and a control cervical cancer (HeLa) cell line, was measured by quantitative reverse transcriptase–PCR. Bars represent mean±s.d. (n = 2). (d and e) qRT–PCR analysis of Hhat (d) and Gli-1 (e) expression after Hhat was depleted in AsPC-1 cells using lentivirally delivered shRNA (TRCN0000035601, OpenBiosystems, Huntsville, AL, USA). A shRNA construct, carrying a scrambled sequence, was used as a control. shRNA-expressing lentiviruses were produced by co-transfecting confluent 293T cells in 15 cm plates with the shRNA plasmid, the HIV packaging vector pHRD8.2, and pcDNA3.1 VSV-G. Virus was collected 48 and 72 h later. The medium was cleared from debris by centrifugation at 500 × g for 5 min; the supernatant was filtered through a 0.45 mm filter and centrifuged at 38720 × g for 2 h at 4 °C. The pelleted virus was resuspended in medium and added to cells, and cells were selected in puromycin. Bars represent mean±s.d. (n = 2). The experiment was performed twice. (f and g) qRT–PCR analysis of Shh (f) and Gli-1 (g) expression after Shh was depleted in AsPC-1 cells using lentivirally delivered shRNA (TRCN0000033304, OpenBiosystems) as in (d) and (e). Bars represent mean±s.d. (n = 2). The experiment was performed twice. (h) AsPC-1 cells were treated with DMSO or 10 μM RU-SKI 43 for 72 h. Gli-1 mRNA levels were measured by qRT–PCR. Data were normalized to Gli-1 levels in DMSO-treated samples. Each bar represents mean±s.d. (n = 3). The experiment was performed 2–5 times. (i) Stable lines of Panc-1 cells expressing scrambled, Shh or Hhat shRNA were generated. The cells were co-cultured with C3H10T1/2 cells (1:4 ratio) for 4 days. The SensoLyte FDP Alkaline Phosphatase Assay Kit (AnaSpec, Fremont, CA, USA) was used to measure alkaline phosphatase levels in the cell lysates by monitoring fluorescence for 30 min at 5-min intervals on a Tecan Infinite F500 plate reader (Männdorf, Switzerland). Each point represents mean±s.d. (n = 3). The experiment was performed three times. (j) Panc-1 and C3H10T1/2 cells were co-cultured and incubated with medium containing DMSO or RU-SKI 43 for 72 h (DMSO or drug was replenished 48 h after the initial addition). Alkaline phosphatase activity in cell lysates was measured as in (i). Each point represents mean±s.d. (n = 3). The experiment was performed 2–5 times.
Figure 2
Figure 2
Hhat inhibition blocks pancreatic cancer cell growth. (a) AsPC-1 cells stably expressing scrambled, Shh, or Hhat shRNAs were seeded at 2 × 104 cells/well in six-well plates and grown for 6 days; then cell numbers were quantified. Bars represent mean±s.d. (n = 2). The experiment was performed three times. (b and c) AsPC-1 cells (b) or Hs766t cells (c) were seeded at 5 × 104 cells/well in six-well plates, and 24 h later DMSO, 10 μM RU-SKI 43, or 10 μM C-2 was added to the medium. Cells were grown in the presence of drugs for 6 days (drugs were replenished every 48 h) and cell numbers were quantified. Bars represent mean±s.d. (n = 2). The experiment was performed 2–5 times. (d and e) AsPC-1 cells were seeded at 5 × 104 cells/well in six-well plates and 24 h later were treated with varying concentrations of RU-SKI 43. Cells were grown in the presence of the drug for 6 days (drugs were replenished every 48 h), at which time cell number was counted (d) and Gli-1 mRNA levels were measured by qRT–PCR (e). Each bar represents mean±s.d. (n = 3). The experiment was performed twice. (f) Panc-1 cells were transduced with lentivirus encoding Hhat-HA or LacZ, and selected in Blasticidin S. Expression of Hhat-HA was verified by western blot (inset). A total of 5 × 104 cells/well were seeded in six-well plates and 24 h later medium with DMSO or 10 μM RU-SKI 43 was added to the cells. After 6 days of treatment (drugs were replenished every 48 h) cell number was determined. Cell number was normalized to that of the DMSO-treated samples. Each bar represents mean±s.d. (n = 3). (g) AsPC-1 cells were seeded at 5 × 105 cells/well in six-well plates and transfected on days 0 and 3 with control siRNA or Shh siRNA. Cells were treated with buffer or 1 μM Shh (C24II) once a day, starting on day 1, and cell numbers were quantified on day 6. Bars represent mean±s.d. (n = 2). The experiment was performed twice. Relative Shh expression in control siRNA and Shh siRNA-transfected cells was quantified by qRT-PCR (inset).
Figure 3
Figure 3
Hhat inhibition blocks anchorage-independent pancreatic cancer cell growth. (a and b) AsPC-1 (a) and Panc-1 (b) cells stably expressing scrambled, Shh or Hhat shRNAs were seeded at 2 × 104 cells/well in 24-well low-adherence plates, and cell numbers were quantified 14 days later. Bars represent mean±s.d. (n = 2). Each experiment was performed twice. (c and d) AsPC-1 (c) and Panc-1 (d) cells were seeded in 24-well low-adherence plates (2 × 104 cells/well), and after 24 h DMSO or 10 μM RU-SKI 43 was added to the medium. Drugs were replenished every 48 h. Cell numbers were quantified 14 days later. Bars represent mean±s.d. (n = 2). Each experiment was performed twice.
Figure 4
Figure 4
Hhat inhibition affects Akt and mTOR pathways in vitro and reduces tumor growth in vivo. (a and b) Panc-1 cells were seeded in 60 mm plates (0.5 × 106 cells/plate), and 24 h later cells were treated with DMSO or 10 μM RU-SKI 43 for 48 h. Cell lysates were analyzed for protein phosphorylation or protein cleavage using the PathScan Intracellular Signaling Array kit (Cell Signaling, Danvers, MA, USA). Each signal was quantified and normalized to DMSO-treated samples. Graphs depict changes in the phosphorylation of proteins from two pathways: Akt signaling (a) and mTOR signaling (b). The experiment was performed in duplicate, and each sample was tested twice. Bars represent mean±s.d. (n = 2). The experiment was performed twice. (c) 5 × 104 Panc-1 cells/well were seeded in six-well plates and 24 h later were treated with DMSO, 10 μM RU-SKI 43, 50 nM Rapamycin (Selleckchem, Houston, TX, USA) or RU-SKI +Rapamycin. Cell number was determined after 6 days of treatment (drugs were replenished every 48 h). Cell number was normalized to that of the DMSO-treated samples. Each bar represents mean±s.d. (n = 3). The experiment was repeated twice. (d) Panc-1 cells were stably transfected with vectors carrying Shh, Hhat or a scrambled shRNA sequence. A total of 15 × 106 cells were injected into each flank of athymic female mice (2 mice per shRNA construct). Growth of the tumor xenografts was measured for 72 days (2 measurements per week). Each point represents mean±s.d. (n = 4). All experiments were performed in accordance with MSKCC IACUC-approved protocols.
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