Canine pulmonary adenocarcinoma tyrosine kinase receptor expression and phosphorylation

Abstract

Background: This study evaluated tyrosine kinase receptor (TKR) expression and activation in canine pulmonary adenocarcinoma (cpAC) biospecimens. As histological similarities exist between human and cpAC, we hypothesized that cpACs will have increased TKR mRNA and protein expression as well as TKR phosphorylation. The molecular profile of cpAC has not been well characterized making the selection of therapeutic targets that would potentially have relevant biological activity impossible. Therefore, the objectives of this study were to define TKR expression and their phosphorylation state in cpAC as well as to evaluate the tumors for the presence of potential epidermal growth factor receptor (EGFR) tyrosine kinase activating mutations in exons 18-21. Immunohistochemistry (IHC) for TKR expression was performed using a tissue microarray (TMA) constructed from twelve canine tumors and companion normal lung samples. Staining intensities of the IHC were quantified by a veterinary pathologist as well as by two different digitalized algorithm image analyses software programs. An antibody array was used to evaluate TKR phosphorylation of the tumor relative to the TKR phosphorylation of normal tissues with the resulting spot intensities quantified using array analysis software. Each EGFR exon PCR product from all of the tumors and non-affected lung tissues were sequenced using sequencing chemistry and the sequencing reactions were run on automated sequencer. Sequence alignments were made to the National Center for Biotechnology Information canine EGFR reference sequence.

Results: The pro-angiogenic growth factor receptor, PDGFRα, had increased cpAC tumor mRNA, protein expression and phosphorylation when compared to the normal lung tissue biospecimens. Similar to human pulmonary adenocarcinoma, significant increases in cpAC tumor mRNA expression and receptor phosphorylation of the anaplastic lymphoma kinase (ALK) tyrosine receptor were present when compared to the corresponding normal lung tissue. The EGFR mRNA, protein expression and phosphorylation were not increased compared to the normal lung and no activating mutations were identified in exons 18-21.

Conclusions: Canine pulmonary adenocarcinoma TKRs are detected at both the mRNA and protein levels and are activated. Further investigation into the contribution of TKR activation in cpAC tumorigenesis is warranted.

Figure 1

Relative TKR mRNA expression levels in canine normal lung tissue and companion lung cpAC biospecimens from three dogs. (A) Reverse transcriptase TKR cDNA transcripts in normal lung (N) and lung cpAC (T). GAPDH serves as a loading control. (B) Densitometry: the levels of tumor TKR cDNA normalized to total normal lung and GAPDH. Error bars, mean ± SEM. *EGFR and ALK cpAC/normal of three dogs: P < .05.

Figure 2

Representative micrographs of immunohistochemistry for selected TKRs and VEGF expression in TMA cores of normal lung and cpAC samples. The tissues were probed with the antibodies for EGFR, cKIT, PDGFRα, PDGFRβ, VEGFR2 and VEGF proteins. Left set of panels: Representative images of positive immunoreactivity for the TKRs and growth factor as shown by the brown staining in normal lung. Right set of panels: Representative images of positive immunoreactivity for the TKRs and growth factor as shown by the brown staining in neoplastic cells and/or stroma of the adenocarcinoma tissue. x 40; bar 50 μm.

Figure 3

Quantification of TKR immunopositivity in normal and tumor TMA cores using algorithm analysis software. (A) Positive pixel analysis is a digitalized pixel count in a user defined region (red square) of each core as shown in the visual representation of the TMA core for PDGFRα analysis (right). The bar graph represents the percent strong positive pixels for each TKR and VEGF (left). The fraction of strong stained pixels is the number of total pixels (blue and red) minus the negative pixels (blue) in the markup image of the TMA, divided by the number of total stained pixels. (B) Color deconvolution analyses of IHC digitalized images accounted for different staining densities of the entire core. The intensity range markup core of PDGFRα analysis (right) shows intensity ranges as weak positive staining (yellow), medium (orange), strong positive (red) and negative staining (blue). The biospecimen percent of positive pixels strongly stained for TKRs and VEGF are represented in the bar graph on the left. Each bar represents the mean positivity of 36 tumor (black) and 36 normal (grey) TMA cores from 12 dogs. Error bars, mean ± SEM. *cpAC vs. normal: P < .05.

Figure 4

Quantification of basal TKR and signaling node phosphorylation of cpAC and normal lung tissue. (A) Representative captured fluorescent image of a slide with four phosphorylation array profiles from paired normal and tumor protein lysates of two dogs. Differences between tumor and normal tissue fluorescence intensities for Akt/PKB/Rac at Thr308 are highlighted in the array set from the first dog, whereas differences between tumor and normal tissue fluorescence intensities for p44/42 MAPK are highlighted in the array set from the second dog (white arrows). The black arrow represents one row of the array spotted with three positive (black spots) and two negative (non-colored spots) controls. The entire array has 10 positive controls. (B-D) The protein array measured the phosphorylation status of the selected TKRs that were evaluated using IHC as well as 24 additional TKRs and 11 downstream signaling nodes. The phosphorylation measurement of PDGFR includes pan phosphorylation of both the alpha and beta forms of this receptor. Spot intensities were quantified using ImageQuant™ TL array analysis software. Each bar represents the mean fluorescence intensities obtained and therefore phosphorylation of 12 tumor (black) and 12 normal (grey) normal lung lysates from each dog. Error bars, mean ± SEM. *cpAC vs. normal: P < .05.