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. 2016 Aug 18;3(3):18.
doi: 10.3390/vetsci3030018.

p16, pRb, and p53 in Feline Oral Squamous Cell Carcinoma

Wachiraphan Supsavhad  1 , Wessel P Dirksen  2 , Blake E Hildreth  3 , Thomas J Rosol  4
Affiliations

p16, pRb, and p53 in Feline Oral Squamous Cell Carcinoma

Wachiraphan Supsavhad et al. Vet Sci. .

Abstract

Feline oral squamous cell carcinoma (FOSCC) is a highly aggressive head and neck cancer in cats, but the molecular pathogenesis of this cancer is still uncertain. In this study, p16, p53, and pRb proteins were detected and quantified by immunohistochemistry in forty-three FOSCC primary tumors and three FOSCC xenografts. p16 mRNA levels were also measured in three FOSCC cell lines (SCCF1, F2, and F3), which were consistent with their p16 immunoreactivity. Feline SCCF1 cells had very high levels of p16 protein and mRNA (55-fold greater) compared to SCCF2 and F3. A partial feline p16 cDNA sequence was amplified and sequenced. The average age of cats with FOSCC with high p16 immunoreactivity was significantly lower than the average age in the low p16 group. Eighteen of 43 (42%) FOSCCs had low p16 intensity, while 6/43 (14%) had high p16 immunoreactivity. Feline papillomavirus L1 (major capsid) DNA was not detected in the SCC cell lines or the FOSCCs with high p16 immunostaining. Five of 6 (83%) of the high p16 FOSCC had low p53, but only 1/6 (17%) had low pRb immunoreactivity. In summary, the staining pattern of p16, p53, and pRb in FOSCC was different from human head and neck squamous cell carcinoma and feline cutaneous squamous cell carcinoma. The majority of FOSCCs have low p16 immunostaining intensity, therefore, inactivation of CDKN2A is suspected to play a role in the pathogenesis of FOSCC. A subset of FOSCCs had increased p16 protein, which supports an alternate pathogenesis of cancer in these cats.

Keywords: feline; immunohistochemistry; oral; p16; p53; pRb; squamous cell carcinoma.

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Conflict of interest statement

All authors declared no potential conflicts of interest including financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence the research, authorship, and/or publication of this article. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1
Figure 1
Alignment between human, chimpanzee, rabbit, cat, cow and mouse p16 cDNA sequences. The 115-nucleotide RT-PCR amplicon spanning the junction between exons 1α and 2 of the cat p16 cDNA was aligned with the corresponding regions of p16 from the indicated species. The dot indicates the first nucleotide of exon 2. An asterisk (*) indicates a nucleotide that is identical between all the aligned sequences, a colon (:) indicates a nucleotide of strongly similar properties between aligned sequences and a period (.) indicates a nucleotide of weakly similar properties between aligned sequences. Alignment was performed using ClustalW2 software (European Bioinformatics Institute ftp server).
Figure 2
Figure 2
Relative p16 mRNA expression in 3 feline oral squamous cell carcinoma (FOSCC) cell lines compared to normal cat gingiva. All qRT-PCR was repeated in triplicate using 3 different passages of each cell line. The relative p16 mRNA expression in SCCF1 (n = 3) was significantly greater (55-fold) than in SCCF2 (n = 3), SCCF3 (n = 3), and normal gingiva (** p ≤ 0.0001). p16 mRNA was significantly reduced in the SCCF3 cells compared to normal gingiva (* p = 0.027).
Figure 3
Figure 3
FOSCC, oral tissue, cat, immunohistochemistry photomicrographs. Absent to low p16 immunoreactivity. Very low intensity of p16 immunostaining was present in less than 20% of the neoplastic cells (A); moderate p16 staining. Moderate levels of p16 immunoreactivity were present in the cytoplasm of some of the FOSCC cells. Small numbers of nuclei had a high intensity of p16 immunostaining (B); high intensity p16 staining. Both nuclear and cytoplasmic p16 immunostaining were prominent in the tumor cells (C); normal gingival epithelium, cat. Immunoreactivity to p16 protein in normal cat oral tissues. There was a moderate intensity of p16 staining in normal gingival epithelium (D); p16 IHC in a mouse xenograft tumor derived from the SCCF1 cell line. High p16 intensity (brown color) was observed in both the nuclei and cytoplasm of SCCF1 tumor cells (E); p16 IHC in a mouse xenograft tumor derived from the SCCF2 cell line. Low to absent p16 intensity was present in SCCF2 cells (F); p16 IHC in a mouse xenograft tumor derived from the SCCF3 cell line. Low to absent p16 staining intensity was observed (G). (DAB/hematoxylin).
Figure 4
Figure 4
FOSCC, oral tissue, cat. FOSCC with absent to low intensity of pRb staining. Less than 50% of the neoplastic cells had a low intensity of nuclear immunostaining to pRb (A); microscopic features of moderate pRb intensity in FOSCC. Moderate pRb staining intensity was present in more than 50% of neoplastic nuclei (B); high intensity of pRb in FOSCC. Almost all of the neoplastic nuclei have high intensity immunostaining for pRb protein (C); normal gingival epithelium, cat. pRb IHC of normal cat oral tissue. Moderate pRb staining intensity was present in epithelial cell nuclei (D); microscopic features of absent to low intensity of p53 staining in FOSCC. The immunoreactivity to p53 protein was absent from neoplastic nuclei (E); microscopic features of moderate p53 staining intensity in FOSCC. Moderately intense immunoreactivity to p53 was present in most of the neoplastic nuclei (F); microscopic features of high intensity p53 immunostaining in FOSCC. Prominent p53 immunostaining (dark brown color) was visible within neoplastic nuclei (G). Normal gingival epithelium, cat. Immunostaining of p53 protein in normal cat oral tissue. Low intensity p53 staining was present (H) (DAB/hematoxylin).
Figure 5
Figure 5
Immunohistochemistry of p16, p53, and pRb in spontaneous feline oral squamous cell carcinomas. The numbers in parentheses (percentages) indicate the 95% confidence intervals (CIs) of each group. No overlap between CIs indicates statistically significant differences between groups (p ˂ 0.05). Significantly different from absent to low group is indicated by (a) Significantly different from moderate group is indicated by (b) Significantly different from high group is indicated by (c).
Figure 6
Figure 6
The average age of cats with FOSCC with high intensity p16 staining group (n = 6, mean = 10.6 years, SD = 4.0) was significantly lower than the age of cats in the absent to low p16 immunostaining intensity group (n = 18, mean = 14.5 years, SD = 4.3) (* p < 0.05). There was no significant difference between the average ages of cats with absent to low and moderate p16 (n = 18) and between moderate and high p16 staining intensity. The plus sign represents the means, the middle line of the boxes indicates the medians, the boxes contain 50% of samples, and top and bottom error bars represent maximum and minimum age of cats in each group, respectively.
Figure 7
Figure 7
Feline p16 predicted cDNA sequence for exons 1α and 2. The feline p16 cDNA sequence shown was obtained from the updated cat genome sequence (Genbank accession NM 000077.4) after alignment with known cDNA sequences from other species. The sequenced feline p16 cDNA amplicon (Genbank accession: Banklt1884793 Seq1 KU508421) (115 nucleotides in bold) consists of the downstream end of exon 1α and the beginning of exon 2. The ATG start site of exon 1α is underlined.
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