Original Article
DOI: 10.1111/vco.12113
Immunohistochemical expression of heat shock proteins, p63 and androgen receptor in benign prostatic hyperplasia and prostatic carcinoma in the dog M. Romanucci1 , L. Frattone1 , A. Ciccarelli2 , L. Bongiovanni1 , D. Malatesta1 , C. Benazzi3 , C. Brachelente4 and L. Della Salda1 1
Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy Faculty of Political Sciences, University of Teramo, Teramo, Italy 3 Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell’Emilia (Bologna), Italy 4 Department of Biopathological Sciences and Hygiene of Animal and Food Production, Faculty of Veterinary Medicine, University of Perugia, Perugia, Italy 2
Abstract
Keywords androgen receptor, benign prostatic hyperplasia, dog, heat shock protein, p63, prostate cancer
This study compared heat shock proteins Hsp60, Hsp72 and Hsp73, along with p63 and androgen receptor (AR) immunoexpression between 16 cases of benign prostatic hyperplasia (BPH) and 11 prostatic carcinomas (PCa) in dogs. The proportion of Hsp60-positive cells was higher in PCa compared with BPH (P = 0.033), whereas the frequency and intensity of Hsp73 immunostaining did not differ significantly between the two groups. Hsp72-immunostained nuclei formed a discontinuous layer along the basement membrane in BPH, whereas cells in this layer in PCa were negative or weakly positive. Hsp72 nuclear score showed significant positive associations with both p63 (P = 0.016) and AR (P = 0.009) scores. Double immunofluorescence revealed Hsp72-p63 and Hsp72-AR co-expressions in basal cell nuclei. Aberrant cytoplasmic p63 immunolabelling was observed in 3 of 11 PCa cases. These results suggest a role of the combined expression of Hsp72, p63 and AR in basal epithelial cells in canine BPH and PCa.
Introduction
Correspondence address: Prof. L. Della Salda Faculty of Veterinary Medicine University of Teramo Piazza A. Moro 45, 64100 Teramo, Italy e-mail:
[email protected]
The canine prostate gland shares many morphological and functional similarities with its human counterpart. Apart from a few cases reported in domestic cats, human beings and dogs are the only species to spontaneously develop numerically important, clinically detectable prostate cancers, although the incidence of prostatic carcinoma (PCa) is much lower in dogs than in men1,2 and the precise cell of origin of canine PCa is unknown.3 Canine PCa is usually diagnosed at an advanced stage and is not responsive to castration. The common occurrence of bone metastases, as well as the androgen-independence of canine tumours, renders dogs with PCa relevant model for studying
© 2014 John Wiley & Sons Ltd
advanced, hormone-refractory PCa in men, so that pre-clinical studies in dogs may be useful for both human beings and dogs with PCa.2 Heat shock proteins (HSPs) are a group of highly conserved molecules which play a fundamental role in the maintenance of cellular homeostasis, under both physiological and stress conditions.4 The cytoprotective properties of most HSPs rely on their primary role as molecular chaperones in protein folding and stability, as well as on their anti-apoptotic functions.5,6 Hsp70 family members include the constitutively expressed Hsc70 (Hsp73 or HspA8) and the highly stress-inducible Hsp70 (Hsp72 or HspA1), which may be localized within the cytoplasm and/or the nucleus of all cells. Hsp70
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is often overexpressed in cancer cells and is essential for their survival, allowing stressed tumour cells to adapt to a hostile microenvironment.7 Hsp60 (HspD1) is a mitochondrial matrix protein, constitutively expressed in most mammalian cells. Unlike Hsp70 with established pro-survival functions, the role of Hsp60 is more controversial with both pro-survival and pro-apoptotic functions reported.8 Several studies have focused on the implications of Hsp60 and Hsp70 during human prostate tumourigenesis and cancer progression, as well as on their usefulness as prognostic markers of PCa.9 – 11 The information obtained from these studies may provide the scientific basis for the design of antitumour treatments targeting HSPs in combination with conventional therapies, which could improve the survival of PCa patients in the next future.12 However, no data are available concerning the expression of these HSPs in canine PCa. The first aim of this study was to compare the immunoexpression patterns of Hsp60, Hsp72 and Hsp73 between benign prostatic hyperplasia (BPH) and PCa in dogs, in order to investigate the possible involvement of these proteins in canine prostate carcinogenesis and provide useful information in comparative oncology for human beings and dogs. Given the reported transcriptional activities of the prostate basal cell marker p63 on the hsp70 gene,13,14 as well as the regulation of Hsp70 expression by androgen receptor (AR) signalling,15 this study secondly aimed to evaluate the possible relationship between the expression of these three molecules in benign and malignant lesions of the canine prostate gland, in order to provide further insights into the role of putative androgen-independent basal/stem cell-like cells in prostate carcinogenesis.
Materials and methods Histological examination The study was carried out using formalin-fixed, paraffin wax-embedded samples from 28 canine prostate tissue samples, all from different dogs, including 16 BPH (7 cases of the benign diffuse glandular form and 9 cases of the complex form) and 11 primary PCa (pulmonary and lymph node
metastatic lesions were available for 1 case, although immunohistochemistry could not be performed for all the investigated molecules on the lymph node sample, due to insufficient tissue in the paraffin block). Mean ages of dogs in each category were as follows: BPH 9 years (range: 5–14.5 years; age unknown for one case); primary PCa 9.4 years (range: 2–12 years; age known for all cases). One necropsy sample of normal canine prostate gland (from an 1-year-old, sexually mature dog, dead of causes unrelated to the genital system), serving as a control, and one case of hyperplastic canine prostate gland showing multifocal lesions characterized by histological features of prostatic intraepithelial neoplasia (PIN), were also included. All tumour cases were supplied by the Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell’Emilia (Bologna), Italy and by the Department of Biopathological Sciences and Hygiene of Animal and Food Production, Faculty of Veterinary Medicine, University of Perugia, Perugia, Italy. Histological diagnosis was achieved using haematoxylin and eosin (HE)-stained slides, according to WHO guidelines.16
Immunohistochemistry Immunohistochemistry was performed using primary antibodies (Abs) specific for Hsp60 (1:100, clone LK-1, mouse anti-human monoclonal IgG1 , StressGen/Assay Designs, Ann Arbor, MI, USA), Hsp70/Hsp72 (1:100, clone C92F3A-5, mouse anti-human monoclonal IgG1 , StressGen/Assay Designs), Hsc70/Hsp73 (1:250, clone 1B5, rat anti-hamster monoclonal IgG2a , StressGen/Assay Designs), AR (1:500, rabbit anti-human polyclonal Ab, code sc-816, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p63 (1:400, clone 4A4, mouse anti-human monoclonal IgG2a , DAKO, Glostrup, Denmark), according to a previously described technique.17 Deparaffinized and rehydrated sections were incubated with 3% H2 O2 in absolute methanol for 45 min to inhibit endogenous peroxidase activity and then rinsed in 0.05 M Tris-buffered saline (TBS, pH 7.6) for 5 min. Antigen retrieval was performed by heat treatment in citrate buffer, pH 6.0 (for HSPs and p63) or Tris-EDTA buffer, pH 9.0 (for AR) in a microwave
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12113
HSPs, p63 and AR immunoexpression in benign and malignant canine prostatic lesions. 3
oven for 5 min (three and four cycles, respectively). After the last treatment, sections were left for 20 min in the buffer for cooling. To reduce non-specific binding, slides were then incubated with normal goat serum (code MR*HRP-650, Biospa, Milan, Italy) for 10 min at room temperature before overnight incubation with the primary antibody in a humidified chamber at 4 ∘ C. After rinsing with TBS, immune complexes were treated at room temperature for 10 min with secondary biotinylated goat anti-mouse/rabbit (ready-to-use, polyclonal, code MR*HRP-650, Biospa) or rabbit anti-rat (1:100, polyclonal, code E 0467, DAKO, Copenhagen, Denmark) Abs and subsequently detected using streptavidin–peroxidase (code MR*HRP-650, Biospa) for 10 min. Peroxidase activity was detected by a 5 min application of 0.1% H2 O2 in 3-3′ -diaminobenzidine solution (code D5905, Sigma–Aldrich, St. Louis, Mo, USA) followed by counterstaining with Mayer’’s haematoxylin (Merck, Darmstadt, Germany) for 1 min before rinsing, dehydrating and mounting. A negative control was performed in all instances by omitting the primary antibody and incubating tissue sections with TBS and/or replacing it by an antibody of irrelevant specificity (rabbit anti-human von Willebrand factor polyclonal Ab or mouse anti-human desmin monoclonal Ab, both from DAKO, Glostrup, Denmark). Sections of bovine urinary bladder known to display HSP expression18 (for HSPs) and sections of human BPH (for p63 and AR) were used as positive controls.
Double immunofluorescence Double immunofluorescence was also used to investigate Hsp72-p63 and Hsp72-AR nuclear co-expression in four selected cases (two BPH and two PCa). Tissue samples were treated as described for the immunohistochemical procedure. A sequential protocol was used for Hsp72-p63 double staining, whereas a mixture of primary Abs was used for Hsp72-AR. Primary Abs were applied overnight at 4 ∘ C. The first secondary Ab, biotinylated goat anti-mouse (for Hsp72) (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) was applied and incubated for 30 min at room temperature, and slides were then incubated with
Texas Red-conjugated avidin (Vector Laboratories) diluted 1:100 in a buffer composed of 0.1 M NaHCO3 and 0.15 M NaCl, pH 8.2–8.5, for 10 min at room temperature. An avidin/biotin blocking step was performed by incubating slides for 15 min with avidin and then biotin (Avidin/Biotin Blocking Kit, code SP-2001, Vector Laboratories) at room temperature. A further protein blocking step was performed by incubating slides with normal goat serum (Vector Laboratories) for 15 min at room temperature, before the overnight application of the second primary Ab. The second secondary Ab, biotinylated goat anti-rabbit (for AR) or anti-mouse (for p63) (1:200 dilution; Vector Laboratories) was applied and incubated for 30 min at room temperature, and slides were then incubated with fluorescein-conjugated avidin (1:100 dilution in 0.1 M NaHCO3 , 0.15 M NaCl buffer, pH 8.2–8.5; Vector Laboratories) for 10 min at room temperature. Nuclei were counterstained with 4′ ,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).
Quantification of immunolabelling and statistical analysis HSP immunohistochemical labelling of the cytoplasm and nucleus was assessed semiquantitatively as absent (0% labelled cells), low (>0–< 10% labelled cells), moderate (≥10–< 50% labelled cells), high (≥50%–< 75% labelled cells) or very high (≥75% labelled cells). The number of Hsp72-, p63- and AR-positive nuclei for each sample was also calculated in 10 randomly selected high-power (×400) fields, counting at least 1000 hyperplastic or neoplastic epithelial cells, and expressed as a percentage. Labelling intensity of all investigated molecules was graded as no (0), weak (1), moderate (2) or strong (3) labelling. Fisher’s exact test was used to compare BPH and PCa for semiquantitatively assessed proportion and intensity of HSP immunoreactivity, as well as for nuclear expression scores of Hsp72, p63 and AR. Cramer’s V was also calculated to measure the strength of association between the variables. Fisher’s exact test and Cramer’s V were also used to evaluate the associations among the nuclear expression scores of Hsp72, p63 and AR. For this purpose,
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12113
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the cases were grouped according to the nuclear score as follows:
