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. 2014 Mar 7;9(3):e89899.
doi: 10.1371/journal.pone.0089899. eCollection 2014.

Attenuation of hedgehog acyltransferase-catalyzed sonic Hedgehog palmitoylation causes reduced signaling, proliferation and invasiveness of human carcinoma cells

Antonios D Konitsiotis  1 , Shu-Chun Chang  1 , Biljana Jovanović  1 , Paulina Ciepla  2 , Naoko Masumoto  2 , Christopher P Palmer  3 , Edward W Tate  2 , John R Couchman  4 , Anthony I Magee  5
Affiliations

Attenuation of hedgehog acyltransferase-catalyzed sonic Hedgehog palmitoylation causes reduced signaling, proliferation and invasiveness of human carcinoma cells

Antonios D Konitsiotis et al. PLoS One. .

Abstract

Overexpression of Hedgehog family proteins contributes to the aetiology of many cancers. To be highly active, Hedgehog proteins must be palmitoylated at their N-terminus by the MBOAT family multispanning membrane enzyme Hedgehog acyltransferase (Hhat). In a pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and transfected HEK293a cells Hhat localized to the endoplasmic reticulum. siRNA knockdown showed that Hhat is required for Sonic hedgehog (Shh) palmitoylation, for its assembly into high molecular weight extracellular complexes and for functional activity. Hhat knockdown inhibited Hh autocrine and juxtacrine signaling, and inhibited PDAC cell growth and invasiveness in vitro. In addition, Hhat knockdown in a HEK293a cell line constitutively expressing Shh and A549 human non-small cell lung cancer cells inhibited their ability to signal in a juxtacrine/paracrine fashion to the reporter cell lines C3H10T1/2 and Shh-Light2. Our data identify Hhat as a key player in Hh-dependent signaling and tumour cell transformed behaviour.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Localization of Hhat in PANC1 cells.
A. Localization of Hhat in PANC1 cells was assessed using Hhat-EGFP transfection and confocal microscopy combined with immunofluorescence localization of ER (PDI) and Golgi (GM130). B. HEK293a Hhat-V5 stable cells were co-stained for the V5 epitope with ER (Calnexin) or Golgi (GM130) and nuclei (DAPI). The data show that both Hhat-EGFP and Hhat-V5 localize primarily in ER with little if any in Golgi apparatus. Scale bar = 10 µm.
Figure 2
Figure 2. Hhat RNAi KD in PANC1 and HEK293a-Hhat-V5 cells.
A. Quantitative RT-PCR was performed using Hhat-specific primers to confirm target gene knockdown in PANC1 cells following Hhat-#1 and Hhat-#2 siRNA transfection. Hhat expression is normalized with GAPDH and compared to Non-targeting siRNA control. Error bar represents the standard error of at least three independent experiments performed in duplicate (**, P<0.01; ***, P<0.001). B. To confirm Hhat KD at the protein level, HEK293a cells stably expressing a pcDNA-DEST40-Hhat-V5 construct at moderate levels were transfected with the Hhat-#1, Hhat-#2 and Non-targeting siRNAs. 72 h post transfection cells were lysed and Hhat expression examined by SDS-PAGE followed by western blotting with an anti-V5 antibody. Blots were probed for tubulin as a loading control. C. Densitometry was performed on the blots in panel B; values were then normalized to the tubulin loading control and compared to the Non-targeting siRNA control.
Figure 3
Figure 3. Hhat KD inhibits Shh palmitoylation and multimeric complex formation.
A. 48 h after Hhat-#1 siRNA transfection PANC1 cells were labeled with YnC15, then medium and cell lysates were collected for 5E1 immunoprecipitation, treated by click chemistry and analyzed by Shh Western blot with H-160 Ab. In both medium and cell lysates, YnC15-labeled Shh was reduced in Hhat-#1 KD cells compared to Mutated Hhat-#1 and control cells. In contrast, YnC15-unlabeled Shh was more abundant in the medium of Hhat-#1 KD cells, but less abundant in cell lysates, demonstrating increased release when Hhat is knocked down. B. To examine the role of Hhat in Shh oligomerization, 72 h after siRNA transfection of PANC1 cells the media were subjected to gel filtration chromatography (Superdex200 10/300 GL column). After TCA precipitation, the fractions were probed by dot blot with anti-Shh H-160 antibody. Untreated and control Mutated Hhat-#1-treated cells showed an abundance of large complexes migrating near the void volume (Vo), whereas Hhat-#1 siRNA-treated KD cells had a much higher proportion of monomer (Vt represents the total volume of the column). Experiments were repeated three times with similar results.
Figure 4
Figure 4. Hhat KD reduces Shh signaling in PANC1 cells.
To study the role of Hhat in Shh signaling, expression levels of Shh targets Ptch and Gli1 were examined by both RT-PCR and Western blot after Hhat-#1 KD (A, C). Quantification of results is provided in B and D. Interestingly, cell-associated Shh protein is reduced by Hhat-#1 KD (C, bottom panel) but Shh mRNA level is unchanged (A), suggesting that Hhat-#1 KD reduces Shh retention by cells. In combination, this suggests that un-palmitoylated Shh has less ability to associate to cell membranes, but instead is released to the medium.
Figure 5
Figure 5. Hhat KD inhibits PANC1 proliferation and matrix invasion.
A. PANC1 cells were labeled with CFSE at the start of the experiment and treated with Shh neutralizing antibody 5E1 on Day 1 (5E1 D1) to test whether PANC1 proliferation is Shh dependent. 5E1 treatment on Day 4 (5E1 D4) was used to mimic the kinetics of siRNA KD. Cells were allowed to grow for 8 days during which CFSE dilution gave a measure of cell division. 8 days after transfection with Hhat-#1 siRNA the cells were analyzed by flow cytometry. The Y-axis shows the CFSE mean fluorescence intensity (MFI) observed from 30,000 cells in each condition. The experiments were repeated three times in triplicate, and statistical significance was measured by using the two-tailed t test (***, P<0.001). B, C. 72 h after transfection with Hhat-1 siRNA or control Mutated Hhat siRNA PANC1 cells were plated onto Matrigel invasion chambers for 24 h. Cells that had migrated from the upper to the lower side of the filter were photographed (B) and counted with a light microscope (40 fields/filter, C). Non-treated (NT) and 5E1-treated cells are shown for comparison in B. The experiments were repeated four times, and statistical significance was measured by using the two-tailed t test (***, P<0.001).
Figure 6
Figure 6. Hhat KD inhibits Hh-mediated juxtacrine/paracrine signaling.
A. Hhat-#2 siRNA KD in A549 cells was assessed by qPCR 24–96 h after siRNA transfection showing 20–60% decrease in Hhat mRNA expression. B. A549 cells co-cultured with C3H10T1/2 cells (1∶2 ratio) were treated with cyclopamine or 5E1 anti-Shh blocking antibody for 2 days and ALP induction in C3H10T1/2 cells was then determined using p-nitrophenyl phosphate in an ALP assay and measuring absorbance at 405 nm. C. A549 cells treated with Hhat-#2 siRNA for 2 days were co-cultured with C3H10T1/2 cells (1∶2 ratio) and ALP induction was assessed after additional 2 days as in B. The results show a significant reduction (*, P<0.05) in the ability of A549 cells treated with Hhat-#2 compared to cells treated with non-targeting (NT) siRNA pool to induce ALP production in C3H10T1/2 cells. The experiments were repeated four times and statistical significance was measured using the paired t test. D. PANC1 and HEK293a-Shh cells induce luciferase activity in the Shh-Light2 cells when co-cultured for 72 h, while the HEK29a cell line, which does not express Shh, did not show luciferase activity higher than background levels (Shh-Light2 cells alone). The data shown are representative of five independent experiments and are the means ± standard error of triplicates. Data were normalized to the Shh-Light2 cell monoculture response (*, P<0.05; **, P<0.01). E. PANC1 cells were transfected with Hhat-#1, Hhat-#2 or Non-targeting siRNAs. 48 h post-transfection, Shh-Light2 cells were mixed with the transfected cells (2∶1 ratio) and they were co-cultured for 72 h. The data shown are representative of three independent experiments and are the means ± standard error of triplicates. Data were normalized to the Non-targeting siRNA response (*, P<0.05; **, P<0.01).
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