In Silico Analysis Validates Proteomic Findings of Formalin-fixed Paraffin Embedded Cutaneous Squamous Cell Carcinoma Tissue

Abstract

Background: Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer but there are no comprehensive proteomic studies on this entity.

Materials and methods: We employed liquid chromatography coupled with tandem mass spectrometry (MS/MS) using formalin-fixed paraffin-embedded (FFPE) cSCC material to study the tumor and normal skin tissue proteomes. Ingenuity Pathway Analysis (IPA) was used to interpret the role of altered proteins in cSCC pathophysiology. Results were validated using the Human Protein Atlas and Oncomine database in silico.

Results: Of 1,310 unique proteins identified, expression of an average of 144 and 88 proteins were significantly (p<0.05) increased and decreased, respectively, in the tumor samples compared to their normal counterparts. IPA analysis revealed disruptions in proteins associated with cell proliferation, apoptosis, and migration. In silico analysis confirmed that proteins corresponding to 12 antibodies, and genes corresponding to 18 proteins were differentially expressed between the two categories, validating our proteomic measurements.

Conclusion: Label-free MS-based proteomics is useful for analyzing FFPE cSCC tissues.

Keywords: Cutaneous squamous cell carcinoma; formalin-fixed paraffin embedded; label-free mass spectrometry; laser capture microdissection; proteomics.

Figure 1. Laser capture microdissection (LCM) of formalin-fixed and paraffin-embedded (FFPE) cutaneous squamous cell carcinoma (cSCC) and morphologically normal tissues. Under direct microscopic visualization of the hematoxylin and eosin (H&E)-stained FFPE sections, cSCC masses (left panel) and adjacent morphologically normal tissues (right panel) were microdissected using the PALM Laser Microbeam instrument. The top panel shows the H&E-stained slides before LCM, middle panel after the LCM, and bottom panel the cells captured in the collecting tube; blue dots mark points where the laser pulses catapult the tissue into the collection tube.

Figure 2. Functional enrichment analysis and Ingenuity Pathway Analysis (IPA) of proteins changed in cutaneous squamous cell carcinoma (cSCC). A: Bar graph of top-10 subcellular locations overrepresented by proteins significantly changed in at least four patients with cSCC using enrichment analysis of proteins against the FunRich database (33). B: Heat map of the main biofunctions disrupted by proteins significantly changed in each patient as generated by IPA analysis: blue indicates inhibition and orange activation of functions in the corresponding patient; the higher the color intensity, the higher the disruption of biofunction; white boxes denote an absence of information. C: IPA upstream regulator analysis tool was used to assemble a network based on the top 16 upstream regulators with predicted activation or inhibition in cSCC. Molecules are represented as functional classes of the gene product as indicated in the legend. Individual patients are represented by different shapes, i.e. patient 1 by a circle, and the grey-scale is indicative of the level of significance.

Figure 3. In silico immunohistochemical expression of a subset of cutaneous squamous cell carcinoma (cSCC)-identified biomarkers. Surgical sections of cSCC and normal skin tissues were stained with S100 calcium binding protein A2 (S100A2), S100A11, mitochondrial superoxide dismutase [Mn] (SOD2), moesin (MSN), tenascin (TNC), fascin (FSCN1), nuclear factor erythroid 2-related 2 (NEF2L2), transcription factor AP-1 (JUN), pyruvate kinase PKM (PKM), transcription intermediary factor 1-alpha (TRIM24), peroxisomal acyl-coenzyme A oxidase 1 (ACOX1), galectin-7 (LGALS7) and apolipoprotein E (APOE) antibodies. Data are from The Human Protein Atlas online resource (www.proteinatlas.org).

Figure 4. Gene expression levels of S100 calcium binding protein A11 (S100A11) (A), fascin (FSCN1) (B), ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 (CD38) (C) and transcription factor AP-1 (JUN) (D) in independent cutaneous squamous cell carcinoma (cSCC), actinic keratosis (AK) and normal skin (control) samples from patient cohorts. mRNA levels were analyzed (Human Genome U133 Plus 2.0 Arrays) in silico using expression data from two cohorts (i) Nindl et al. (25) and (ii) Riker et al. (35). n Is the number of samples in each subgroup, dots in the box plots represent maximum and minimum values; error bars represent 1.5× interquartile range; significantly different at *p<0.05; **p<0.01; ***p<0.001.