The acute transcriptomic and proteomic response of HC-04 hepatoma cells to hepatocyte growth factor and its implications for Plasmodium falciparum sporozoite invasion

Abstract

The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (<0.1%), poor understanding of steady-state HCC biology, and lack of appropriate tools for trace sample analysis. HC-04 is the only HCC that supports complete development of human malaria parasites. We hypothesized that HCCs are in various intermediate stages of the epithelial-mesenchymal transition (EMT) and HC-04s retain epithelial characteristics that permit infection. We developed a facile analytical approach to test this hypothesis viz. the HC-04 response to hepatocyte growth factor (HGF). We used online two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) to quantify protein expression profiles in HC-04 pre-/post-HGF treatment and validated these results by RT-qPCR and microscopy. We successfully increased protein identification efficiency over offline-2D methods by 12-fold, using less sample material, allowing robust protein quantification. We observed expected up-regulation and down-regulation of EMT protein markers in response to HGF, but also unexpected cellular responses. We also observed that HC-04 is generally more susceptible to HGF-mediated signaling than what was observed for HepG2, a widely used, but poor malaria liver stage-HCC model. Our analytical approach to understanding the basic biology of HC-04 helps us understand the factors that may influence its utility as a model for malaria liver-stage development. We observed that HC-04 treatment with HGF prior to the addition of Plasmodium falciparum sporozoites did not facilitate cell invasion, which suggests unlinking the effect of HGF on malaria liver stage development from hepatocyte invasion. Finally, our 2D-LC-MS/MS approach and broadly applicable experimental strategy should prove useful in the analysis of various hepatocyte-pathogen interactions, tumor progression, and early disease events.

Fig. 1.

Overview of the strategy and the proteomics analytical platform for label-free quantification of hepatocyte proteins. A, Overview of the epithelial-mesenchymal transition (EMT) in the context of our questions. The EMT is a process where epithelial cells lose their established tissue identity (cell polarity and cell-cell adhesion) and is important during development, wound healing, carcinogenesis and the development of cell lines. Here, we assess the effect of HGF on an HCC line in relation to EMT and possible effects on invasion by the malaria parasite. B, Protein sample preparation. Following freezing-thaw lysis of samples a membrane enriched fraction was pelleted by centrifugation prior to FASP digestion. The remaining soluble protein fraction was then processed by standard trypsin digestion. Each sample was then desalted, dried and dissolved in loading buffer prior to online-two dimensional (2D) LC-MS/MS analysis. C, Overview of online-2D LC-MS/MS platform. Our platform was constructed by integrating an SCX column into a chip-cube based RPLC system as the first dimensional separation step. All LC-MS/MS data was searched using Mascot prior to Scaffold for curation and label free quantitation analysis.

Fig. 2.

Evaluation of the online-2D LC-MS/MS platform. A, Overlap of identification of three continuous technical runs. B, Normalized spectral counts of total identified proteins from same three runs. R² values are derived from sample-to-sample comparison for each protein. C, Comparison of the performance of our online-2D system versus a commercial OFFGEL peptide fractionation Offline-2D system.

Fig. 3.

Quantitative proteomic analysis of HC-04 following HGF treatment using the online-2D LC-MS/MS platform revealed significant up-regulation of proteins according to cellular compartment. A, Stacked bar graphs of select Gene Ontology (GO) terms associated with our identified proteins. Abundance in functional categories is very similar across each group in this qualitative analysis. B, Volcano plot of quantifiable proteome comparing HC-04 protein levels before and after HGF treatment. C-D, Volcano plots of different quantifiable subsets of proteins based on cell compartment as determined by fractionation into membrane (C) and soluble (D) protein samples.

Fig. 4.

Immunohistochemistry analyses supported our predictions and proteomics results whereas transcriptomics results suggested discordance between transcript and protein expression levels at 24 h post HGF treatment. A, Tight junction protein ZO-1 localized at cell-cell interfaces in naïve cells and decreased in abundance following HGF treatment. B, Less staining with lectin (EBL), which binds sialic acids (produced by SIAS) was found in HC-04 following HGF treatment. C, SUMO1 and D, β-catenin both decreased slightly following HGF treatment with SUMO1 localizing primarily in the nuclei, whereas β -catenin was seen throughout the cytoplasm and cell surface. E–F, Cytoskeletal protein β-tubulin (E) staining intensity changed slightly with HGF treatment, whereas α-tubulin (F) localization remained stable.

Fig. 5.

Proteomic analysis of HepG2 following HGF treatment compared with the HC-04 response to HGF. We applied online 2D methods to profile protein expression in naïve and HGF treated HepG2 (supplemental Table S7). A total of 1759 and 1757 protein groups were identified in HepG2-naive samples and HGF-treated samples, respectively. A, Volcano plot of the quantifiable proteomes of HepG2 (black points) and HC-04 (gray points) comparing protein levels before and after HGF treatment. These plots demonstrate the more drastic change in HC-04 following HGF treatment, suggesting that HC-04 is at a different point on the EMT spectrum that other common HCC. B, Observed protein regulation in naïve and hepatocyte growth factor (HGF) treated HepG2 cells in comparison to expected or unexpected changes of HC-04 cells following acute exposure to HGF (from Table I). With the exception of IF2B2, all HepG2 examples either changed in an expected manner based on the epithelial-mesenchymal transition, did not change more than 0.1 fold (NC), or were not detected. Data is based on three biological replicate experiments (shown in supplemental Table S7).

Fig. 6.

Plasmodium falciparum invasion efficiency is slightly decreased in HC-04 following HGF treatment. A–B, HC-04 cells were grown for 30 h in the absence (A) or presence (B) of HGF before the addition of P. falciparum NF54 sporozoites. After 24 h of incubation with the sporozoites in the continued presence or absence of HGF, inside-outside staining of the sporozoites revealed fewer sporozoites in HC-04 cells treated with HGF compared with those without HGF. A representative microscope field is shown for both treatments. Inset: A sporozoite inside a hepatocyte that is beginning to round-up to form an exoerythrocytic form. Red staining denotes a sporozoite inside an HC-04 cell; green denotes a sporozoite outside; yellow indicates an overlap. DAPI staining of the nuclei appears blue. C, Quantification of inside-outside staining of P. falciparum sporozoites that have invaded a HC-04 cell. Percent invasion efficiency is shown for experimental replicates that received HGF treatment, n = 5, and without HGF (naïve) treatment, n = 8. Error bars indicate the standard error of the mean.