This study aimed to evaluate imaging findings of cutaneous angiosarcoma (cAS) of the scalp compared with those of cutaneous squamous cell carcinoma (cSCC).This study included 15 patients with primary cAS and 10 with primary cSCC of the scalp. Seven patients with cAS and eight with cSCC underwent magnetic resonance imaging, and 11 patients with cAS and eight with cSCC underwent 18F-fluorodeoxyglucose-positron emission tomography/computed tomography imaging. Imaging findings for both pathologies were retrospectively reviewed and compared.All 15 cAS cases were elevated lesions with an obtuse angle, invading the subcutaneous fat tissue. Multiple lesions were observed in only five cAS cases (33%) and no cSCC cases. Maximum diameter-to-height ratio was significantly higher in cAS than in cSCC (3.3 ± 1.0 versus 2.3 ± 0.6; p < 0.01). On T2-weighted images, intratumoral hypointensity (86% versus 13%; p < 0.01) and mixed hyper- and hypointensity (71% versus 0%; p < 0.01) were observed more frequently in cAS than in cSCC. No significant differences were observed between cAS and cSCC regarding flow void (29% versus 25%; p = 0.656). Maximum standardized uptake values were marginally significantly lower in cAS than in cSCC (5.6 ± 3.1 versus 10.5 ± 6.6; p = 0.078).Cases of cAS of the scalp always exhibited flat elevated lesions with invasion of the subcutaneous fat tissue. Compared with cSCC, intratumoral hypointensity and mixed hyper- and hypointensity on T2-weighted images were more frequent in cAS. These findings will help with the differential diagnosis of cAS.
Purpose This study aimed to determine the MRI features of sporadic/simple lymphoepithelial cyst (SLEC) of the parotid gland. Methods Ten patients (seven men, three women; mean age, 60 years; age range, 38–77 years) with histopathologically and clinically proven SLEC of the parotid gland who underwent MRI before surgical resection were enrolled in this study. No enrolled patient had HIV infection or Sjögren syndrome. MRI findings of SLEC were retrospectively assessed. Results We confirmed 10 SLECs larger than 10 mm with a mean maximum diameter of 26.6 mm (range, 12–42 mm). Nine patients (90%) had a single cyst, and one (10%) had a large cyst accompanied by small cysts (<10 mm) within the ipsilateral parotid gland. Eight SLECs (80%) were unilocular, and two (20%) were bilocular, with complete septa. Among seven SLECs (70%) with internal septa, five unilocular SLECs (50%) had incomplete septa. Six SLECs (60%) had eccentric cyst wall thickening, and five (50%) were surrounded by small solid nodules that were isointense relative to lymph node. On T1-weighted images, all cyst contents were homogeneously hyperintense relative to cerebrospinal fluid. Conclusion SLECs of the parotid gland are usually single unilocular lesions. Internal septa, eccentric cyst wall thickening, and small solid nodules surrounding the lesion were often observed. Cyst contents are always homogeneously hyperintense on T1-weighted images.
This study aimed to assess the efficacy of MRI for differentiating between uterine submucosal polypoid adenomyomas (PAs) and endometrial polyps (EPs).MRI was used to examine 40 histopathologically confirmed benign polypoid endometrial tumors (8submucosal PAs and 32 EPs). Atypical PAs were excluded from this study. Quantitative measurements (maximum tumor diameter, maximum cyst diameter, number of cysts, and apparent diffusion coefficient values) and qualitative imaging findings (predominance of cystic or solid components as well as presence of cysts, hemorrhage, myometrial invasion, fluid-fluid level, and fibrous core) were correlated with the two pathologies.The predominance of cystic components (37% vs 6%; p < 0.05) was more frequently observed in PAs than in EPs. The frequency of cysts (88% vs 25%; p < 0.01), hemorrhage (50% vs 9%; p < 0.05), and myometrial invasion (25% vs 0%; p < 0.05) were significantly higher in PAs than in EPs. No significant differences were observed in terms of the maximum tumor diameter, maximum cyst diameter, number of cysts, apparent diffusion coefficient values, and presence of fluid-fluid level and fibrous core between PAs and EPs.The differences of MR findings with emphasis on cystic components and hemorrhage may be useful for differentiating between PAs and EPs.The predominance of cystic or solid components and the presence of cysts, hemorrhage, and myometrial invasion were useful MR findings for differentiating between PAs and EPs.
The cyclin-dependent kinase inhibitor 2A (CDKN2A)/alternate reading frame (ARF) locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19ARF in mice), encoding two unrelated proteins in alternative reading frames. Previous reports suggest that p16INK4a and p14ARF alterations independently exhibit differential roles, and p16INK4a is more closely associated with a poor prognosis in oral cancer. However, the role of p16INK4a-specific loss in oral squamous cell carcinogenesis remains unclear. The authors assessed chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced multistep oral squamous cell carcinogenesis in mice carrying p16INK4a-specific loss with retention of the p19ARF gene (p16INK4a−/−). 4NQO-treated p16−/− mice exhibited a higher incidence and multiplicity of oral squamous cell carcinoma (OSCC) development relative to 4NQO-treated wild-type mice. 4NQO-treated p16INK4a−/− OSCC cells exhibited higher proliferation and up-regulation of Arf, transcription factor E2f1, tumor protein p63 (tp63), and oncogenic ΔNp63, an isoform p63, compared with observations in 4NQO-treated wild-type OSCC cells. Furthermore, the overexpression of oncogenic ΔNp63 was associated with human OSCC. In conclusion, these results in mice indicate the biological significance of p16INK4a-specific loss with retention of p19ARF in oral squamous cell carcinogenesis, and ΔNp63 may be a potential target for OSCC. The cyclin-dependent kinase inhibitor 2A (CDKN2A)/alternate reading frame (ARF) locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19ARF in mice), encoding two unrelated proteins in alternative reading frames. Previous reports suggest that p16INK4a and p14ARF alterations independently exhibit differential roles, and p16INK4a is more closely associated with a poor prognosis in oral cancer. However, the role of p16INK4a-specific loss in oral squamous cell carcinogenesis remains unclear. The authors assessed chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced multistep oral squamous cell carcinogenesis in mice carrying p16INK4a-specific loss with retention of the p19ARF gene (p16INK4a−/−). 4NQO-treated p16−/− mice exhibited a higher incidence and multiplicity of oral squamous cell carcinoma (OSCC) development relative to 4NQO-treated wild-type mice. 4NQO-treated p16INK4a−/− OSCC cells exhibited higher proliferation and up-regulation of Arf, transcription factor E2f1, tumor protein p63 (tp63), and oncogenic ΔNp63, an isoform p63, compared with observations in 4NQO-treated wild-type OSCC cells. Furthermore, the overexpression of oncogenic ΔNp63 was associated with human OSCC. In conclusion, these results in mice indicate the biological significance of p16INK4a-specific loss with retention of p19ARF in oral squamous cell carcinogenesis, and ΔNp63 may be a potential target for OSCC. Head and neck cancer is the sixth most common type of cancer in humans,1Pai S.I. Westra W.H. Molecular pathology of head and neck cancer: implications for diagnosis, prognosis, and treatment.Annu Rev Pathol. 2009; 4: 49-70Crossref PubMed Scopus (307) Google Scholar and approximately 350,000 individuals are diagnosed with oral cancer annually worldwide.2Li J. Liang F. Yu D. Qing H. Yang Y. Development of a 4-nitroquinoline-1-oxide model of lymph node metastasis in oral squamous cell carcinoma.Oral Oncol. 2013; 49: 299-305Crossref PubMed Scopus (31) Google Scholar This disease occurs in the oral cavity in 48% of cases, 90% of which are oral squamous cell carcinoma (OSCC).3Jemal A. Siegel R. Ward E. Hao Y. Xu J. Murray T. Thun M.J. Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10196) Google Scholar Over the last few decades, the 5-year overall survival rate of patients with OSCC has remained at approximately 50% following initial therapy.4Leemans C.R. Braakhuis B.J.M. Brakenhoff R.H. The molecular biology of head and neck cancer.Nat Rev Cancer. 2011; 11: 9-22Crossref PubMed Scopus (1887) Google Scholar It is likely that this high mortality rate is attributed to its late diagnosis, recurrence, and resistance to therapy, whereas early detection followed by appropriate treatment can increase the cure rate to approximately 80%.2Li J. Liang F. Yu D. Qing H. Yang Y. Development of a 4-nitroquinoline-1-oxide model of lymph node metastasis in oral squamous cell carcinoma.Oral Oncol. 2013; 49: 299-305Crossref PubMed Scopus (31) Google Scholar,5Kao Y.-Y. Tu H.-F. Kao S.-Y. Chang K.-W. Lin S.-C. The increase of oncogenic miRNA expression in tongue carcinogenesis of a mouse model.Oral Oncol. 2015; 51: 1103-1112Crossref PubMed Scopus (31) Google Scholar The development of OSCC is a multistep process involving the progression from normal tissue to dysplasia, oral intraepithelial neoplasia (OIN), and invasive squamous cell carcinoma (SCC).6Tanaka T. Ishigamori R. Understanding carcinogenesis for fighting oral cancer.J Oncol. 2011; 2011: 603740Crossref PubMed Scopus (102) Google Scholar This progression of OSCC requires in the accumulation of multiple genetic, epigenetic, and chromosomal alterations, which are influenced by a patient's genetic/epigenetic predisposition, and by environmental influences, including tobacco, alcohol, and chronic inflammation.7Choi S. Myers J.N. Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy.J Dent Res. 2008; 87: 14-32Crossref PubMed Scopus (353) Google Scholar,8Gasche J.A. Goel A. Epigenetic mechanisms in oral carcinogenesis.Future Oncol. 2012; 8: 1407-1425Crossref PubMed Scopus (81) Google Scholar The p16INK4a and p14ARF (p19ARF in mice) (CDKN2A) genes are on the 9p21 region and have putative growth-suppressive activities. By investigating the functional relevance of these genes in the Rb and p53 pathways, the suppressive mechanism of malignant tumors can be elucidated.9Lopez F. Sampedro T. Llorente J.L. Hermsen M. Alvarez-Marcos C. Alterations of p14 (ARF), p15 (INK4b), and p16 (INK4a) genes in primary laryngeal squamous cell carcinoma.Pathol Oncol Res. 2017; 23: 63-71Crossref PubMed Scopus (8) Google Scholar The Ink4a/Arf locus encodes two proteins, p16INK4a and p14ARF, in overlapping reading frames.10Lowe S.W. Sherr C.J. Tumor suppression by Ink4a-Arf: progress and puzzles.Curr Opin Genet Dev. 2003; 13: 77-83Crossref PubMed Scopus (613) Google Scholar The p16 gene with loss of Arf products is a component of the tumor suppressor pathway that controls cell cycle, and loss or inactivation of the p16 gene with loss of the Arf gene has been reported in several malignancies, including those of the oral cavity.11Nemes J.A. Deli L. Nemes Z. Marton I.J. Expression of p16(INK4A), p53, and Rb proteins are independent from the presence of human papillomavirus genes in oral squamous cell carcinoma.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 102: 344-352Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar,12Ribeiro D.A. Fracalossi A.C. Uatari S.A. Oshima C.T. Salvadori D.M. Imbalance of tumor suppression genes expression following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide.In Vivo. 2009; 23: 937-942PubMed Google Scholar Major inactivation of the p16INK4A/p14ARF genes results from promoter methylation, homozygous deletion, and intragenic mutation. Alterations affecting the INK4A/ARF locus can potentially disrupt the two main tumor suppression pathways, p16INK4A-Rb and p14ARF-p53. A previous study of OSCC in humans reported that 33% and 29% of cases examined (N = 384) exhibited p16INK4A deletion and DNA methylation, respectively, whereas p14ARF deletion and DNA methylation were observed in 12% and 18% of cases, respectively.13Sailasree R. Abhilash A. Sathyan K.M. Nalinakumari K.R. Thomas S. Kannan S. Differential roles of p16INK4A and p14ARF genes in prognosis of oral carcinoma.Cancer Epidemiol Biomarkers Prev. 2008; 17: 414-420Crossref PubMed Scopus (70) Google Scholar Although p16INK4A loss and silencing were associated with aggressiveness and poor prognosis, p14ARF silencing was associated with a lower rate of recurrence and good clinical outcome.13Sailasree R. Abhilash A. Sathyan K.M. Nalinakumari K.R. Thomas S. Kannan S. Differential roles of p16INK4A and p14ARF genes in prognosis of oral carcinoma.Cancer Epidemiol Biomarkers Prev. 2008; 17: 414-420Crossref PubMed Scopus (70) Google Scholar Furthermore, evidence supports the hypermethylation of p16INK4A and p14ARF to be p16INK4A as independent events, and each appears to contribute differentially during carcinogenesis.14Esteller M. Tortola S. Toyota M. Capella G. Peinado M.A. Baylin S.B. Herman J.G. Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status.Cancer Res. 2000; 60: 129-133PubMed Google Scholar The effect of loss of p16INK4a, but retention of p19ARF, is unclear in squamous cell carcinogenesis. Mice lacking p19ARF, but not p16INK4a, were observed to develop spontaneous tumors and to enhance tumor response to carcinogen. In addition, exposure to carcinogens, including DMBA, X-rays, and irradiation, rendered Arf-knockout (KO) mice more prone to tumors.15Ko A. Han S.Y. Song J. Dynamics of ARF regulation that control senescence and cancer.BMB Rep. 2016; 49: 598-606Crossref PubMed Scopus (23) Google Scholar However, Sharpless et al16Sharpless N.E. Ramsey M.R. Balasubramanian P. Castrillon D.H. DePinho R.A. The differential impact of p16(INK4a) or p19(ARF) deficiency on cell growth and tumorigenesis.Oncogene. 2004; 23: 379-385Crossref PubMed Scopus (167) Google Scholar reported that mice with p16INK4a−/− with retention of Arf had a significantly higher risk of developing tumors than wild-type (WT) mice following carcinogen treatment, but were less prone than p53−/− or p19ARF−/− animals. In addition, mice with p16INK4a−/− and retention of Arf did not spontaneously develop malignancy. Although the number of malignant lesions was increased in mice with p16INK4a−/− with retention of Arf, the tumor grade did not differ significantly from that in WT mice. However, the mechanisms underlying squamous cell carcinogenesis in mice lacking p16INK4a (p16INK4a−/−) but retaining p19ARF remain unclear. The function of p63 is also important for maintaining normal tissue and has been studied widely. p63 is essential for normal formation of the epidermis. In SCC of the head and neck, the expression of p63 has been reported to indicate a poor prognosis,17Rekhtman N. Ang D.C. Sima C.S. Travis W.D. Moreira A.L. Immunohistochemical algorithm for differentiation of lung adenocarcinoma and squamous cell carcinoma based on large series of whole-tissue sections with validation in small specimens.Mod Pathol. 2011; 24: 1348-1359Crossref PubMed Scopus (268) Google Scholar and the overexpression of p63 is associated with poor radiation response and shorter survival rates in OSCC.18Candi E. Rufini A. Terrinoni A. Dinsdale D. Ranalli M. Paradisi A. De Laurenzi V. Spagnoli L.G. Catani M.V. Ramadan S. Knight R.A. Melino G. Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.Cell Death Differ. 2006; 13: 1037-1047Crossref PubMed Scopus (221) Google Scholar,19Carvalho J.C. Thomas D.G. McHugh J.B. Shah R.B. Kunju L.P. p63, CK7, PAX8 and INI-1: an optimal immunohistochemical panel to distinguish poorly differentiated urothelial cell carcinoma from high-grade tumours of the renal collecting system.Histopathology. 2012; 60: 597-608Crossref PubMed Scopus (52) Google Scholar The protein expression of p63 originates from two promoters, giving rise to transactivating (TAp63) and nontransactivating (ΔNp63) isoforms.20Mukhopadhyay S. Katzenstein A.L. Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6.Am J Surg Pathol. 2011; 35: 15-25Crossref PubMed Scopus (245) Google Scholar The TAp63 isoforms act synergistically and/or subsequently to control differentiation of upper epidermal layers; TAp63 isoforms contain a p53-homologous N-terminal transactivation (TA) domain and have tumor suppressor properties similar to those of p53 in inducing growth arrest, apoptosis, and senescence. By contrast, ΔNp63 controls the expansion of progenitor cells in the basal layer; ΔNp63 isoforms lacking this TA domain are thought to possess oncogenic activities through the inhibition of TAp63, TAp73, and p53.18Candi E. Rufini A. Terrinoni A. Dinsdale D. Ranalli M. Paradisi A. De Laurenzi V. Spagnoli L.G. Catani M.V. Ramadan S. Knight R.A. Melino G. Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.Cell Death Differ. 2006; 13: 1037-1047Crossref PubMed Scopus (221) Google Scholar,21Nekulova M. Holcakova J. Nenutil R. Stratmann R. Bouchalova P. Muller P. Moukova L. Coates P.J. Vojtesek B. Characterization of specific p63 and p63-N-terminal isoform antibodies and their application for immunohistochemistry.Virchows Arch. 2013; 463: 415-425Crossref PubMed Scopus (24) Google Scholar Although it has been reported that the balance of TAp63 and ΔNp63 is disrupted in various types of cancer,9Lopez F. Sampedro T. Llorente J.L. Hermsen M. Alvarez-Marcos C. Alterations of p14 (ARF), p15 (INK4b), and p16 (INK4a) genes in primary laryngeal squamous cell carcinoma.Pathol Oncol Res. 2017; 23: 63-71Crossref PubMed Scopus (8) Google Scholar,15Ko A. Han S.Y. Song J. Dynamics of ARF regulation that control senescence and cancer.BMB Rep. 2016; 49: 598-606Crossref PubMed Scopus (23) Google Scholar,22Moore L. Venkatachalam S. Vogel H. Watt J.C. Wu C.L. Steinman H. Jones S.N. Donehower L.A. Cooperativity of p19ARF, Mdm2, and p53 in murine tumorigenesis.Oncogene. 2003; 22: 7831-7837Crossref PubMed Scopus (44) Google Scholar the status in OSCC remains to be elucidated. p16INK4a stimulates p14ARF by mediating pRb to activate E2F, and activation leads to increased tumor apoptosis.23Campisi J. d'Adda di Fagagna F. Cellular senescence: when bad things happen to good cells.Nat Rev Mol Cell Biol. 2007; 8: 729-740Crossref PubMed Scopus (2978) Google Scholar This phenomenon is caused by a decline in the function of tumor suppressor genes, including TAp63 and p53, by the activation of ΔNp63. In the present study, the authors investigated and identified the biological significance of p16INK4a−/− retaining p19ARF and Δp63 in mouse tongue carcinogenesis induced by 4NQO. p16INK4a−/− (FVB.129-Cdkn2atm2.1Rdp/Nci; strain no. 01XE4) mice were obtained from the Animal Production Area, National Cancer Institute-Frederick Cancer Research and Development Center (NCI-FCRDC, Frederick, MD). The mice were backcrossed to C57/BL6J mice for at least five generations. All mice were maintained under specific pathogen-free conditions with isolated ventilation cages in an air-conditioned room with a 12:12 light:dark cycle. The mice were bred and maintained on a basal diet, CE-2 (CLEA Japan, Tokyo, Japan) until termination of the study. Genotypes were identified by PCR analysis of tail DNA using allele-specific primers. All animal experiments and breeding were performed under the Regulations for Animal Experiments in Gifu University. The 4NQO carcinogen was dissolved in ethyl alcohol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) as a stock solution (20 mg/mL), stored at 4°C. The mice were administered drinking water containing 4NQO at a concentration of 0.1 mg/mL using light-shielded bottles. Human tissue sections were obtained from patients who underwent surgical resection or biopsy at Gifu University Hospital, Gifu, Japan. All study protocols were approved by the ethics committees of Gifu University, and written informed consent was obtained from all patients. The mice were sacrificed under anesthesia. From each animal, the tongue was removed and cut longitudinally. All tissues were fixed for 24 hours in neutral-buffered 10% formalin. The fixed samples were processed using standard methods, embedded in paraffin, sectioned at 3 μm, and stained with hematoxylin and eosin. The avidin–biotin–peroxidase complex technique was used for immunohistochemical analysis. The sections (3-μm thick) were cut, deparaffinized, rehydrated in phosphate-buffered saline, and placed in 10 mmol/L citrate buffer (pH 6.0), and the Decloaking Chamber NxGen (Biocare Medical, Pacheco, CA) was used (110°C, 2.5 minutes) for heat-induced antigen retrieval. The endogenous peroxidase activity was blocked by incubation for 10 minutes in 0.3% H2O2. Following washing three times with phosphate-buffered saline, nonspecific antibody binding was blocked with normal blocking serum for 40 minutes at room temperature. The sections were then incubated with the following primary antibodies overnight at 4°C: Ki-67 (1:200, rabbit-monoclonal, ab16667; Abcam, Cambridge, UK), cleaved caspase-3 (1:200, rabbit-polyclonal, #9661S; Cell Signaling Technology, Danvers, MA), PCNA (1:1000, mouse monoclonal, PC10; DakoCytomation, Glostrup, Denmark), total p63 (4A4) (1:400, mouse-monoclonal, ab735; Abcam), and TAp63 (1:200, rabbit-polyclonal, #618902; BioLegend, San Diego, CA). Subsequently, the sections were incubated with biotinylated secondary antibodies (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature, followed by incubation with avidin-coupled peroxidase (Vector Laboratories) for 30 minutes at room temperature. The sections were developed with 3,3′-diaminobenzidine (DAB) using the DAKO Liquid DAB Substrate-Chromogen system (DakoCytomation) and then counterstained with hematoxylin. No specific staining was observed in the negative control slides prepared without primary antibody. Human lung SCC and prostate tissues were used as positive controls for total p63 and TAP63 antibodies, respectively (Supplemental Figure S1). For fluorescent double-labeling, after deparaffinization, antigen retrieval, and blocking of the endogenous peroxidase activity and nonspecific antibody, the sections were incubated with the following primary cocktail antibodies overnight at 4°C: p63 (1:200, mouse-monoclonal, ab735; Abcam) and TAp63 (1:100, rabbit-polyclonal, #618902; BioLegend). The following day, the sections were rinsed in phosphate-buffered saline (3 × 5 minutes), followed by incubation with secondary antibody for 30 minutes at room temperature: goat anti-mouse IgG (1:200, Alexa Fluor 488, ab150117; Abcam, Cambridge, UK) and goat anti-rabbit IgG (1:200, Alexa Fluor 594, ab150084; Abcam) for double staining with the same species primary antibodies. The tissue sections were then incubated for 1 minute with DAPI (Molecular Probes, Eugene, OR) to reveal the nuclei. Finally, coverslips were mounted using Shandon Immu-Mount (Thermo Fisher Scientific, San Jose, CA). Tissues were removed and then immediately immersed into the liquid nitrogen for freezing and stocked at −80°C. The frozen tissues were homogenized using Bullet Blender (Next Advance, Averill Park, NY), and total RNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA). cDNA was synthesized from 0.4 μg of RNA, using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer's instructions. Real-time RT-PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems, USA) on a Step One Plus Real-Time PCR system (Applied Biosystems, Foster City, CA). The expression levels of mRNAs were normalized to the expression of β-actin using the 2−ΔΔCt method. All primers are listed in Table 1.Table 1List of Primers Used for Real Time RT-PCRMouse geneForward primerReverse primerp16INK4a5′-GTCGCAGGTTCTTGGTCACT-3′5′-GATCGCACGAACTTCACCAA-3′p19ARF5′-GAGCGGGGACATCAAGACAT-3′5′-AAGCTATGCCCGTCGGTCT-3′p16 suppressor pathway p215′-CGCTGTCTTGCACTCTGGT-3′5′-CGTTTTCGGCCCTGAGATGTT-3′ Cdk45′-AAGGTCACCCTAGTGTTTGAGC-3′5′-CCGCTTAGAAACTGACGCATTAG-3′ Cdk65′-GGCGTACCCACAGAAACCATA-3′5′-AGGTAAGGGCCATCTGAAAACT-3′ pRb5′-TGCATCTTTATCGCAGCAGTT-3′5′-GTTCACACGTCCGTTCTAATTTG-3′ Arf5′-CGCAGGTTCTTGGTCACTGT-3′5′-TGTTCACGAAAGCCAGAGCG-3′ p275′-TCAAACGTGAGAGTGTCTAACG-3′5′-CCGGGCCGAAGAGATTTCTG-3′ Ccnd15′-GCGTACCCTGACACCAATCTC-3′5′-CTCCTCTTCGCACTTCTGCTC-3′ E2F15′-GAGAAGTCACGCTATGAAACCTC-3′5′-CCCAGTTCAGGTCAACGACAC-3′ Trp535′-CTCTCCCCCGCAAAAGAAAAA-3′5′-CGGAACATCTCGAAGCGTTTA-3′p53-independent pathway Trp63(ΔNp63)5′-CTGGAAAACAATGCCCAGAC-3′5′-GAGGAGCCGTTCTGAATCTG-3′ Hif1α5′-GATGACGGCGACATGGTTTAC-3′5′-CTCACTGGGCCATTTCTGTGT-3′ Foxm15′-ATCACGGAGACGTTGGGAC-3′5′-CCACTGGATATTGGTTAAGCTGT-3′ Myc5′-GGAGGACCAAGCTCTGTATTCG-3′5′-GTAGCCAACACCCAGGGAT-3′ Mdm25′-GGATCTTGACGATGGCGTAAG-3′5′-AGGCTGTAATCTTCCGAGTCC-3′ Ctbp15′-ATCATTGGACTAGGTCGTGTGG-3′5′-CATAGAAGAGGACGTTGAAGCC-3′ Ctbp25′-TCGGTAGTGGCTACGACAAC-3′5′-CGCCGATACAGATTGAGAATGT-3′ Wrn5′-AAGGATTCTTGGTAGAGGTTCCC-3′5′-AGACTGCAAACTGGCTTCTCC-3′ Miz15′-TTCCCTTATTCCAGTTGATCCCC-3′5′-CCACTGCTGGTTATGACCCC-3′ Bckdha5′-CTCCTGTTGGGACGATCTGG-3′5′-CATTGGGCTGGATGAACTCAA-3′ Open table in a new tab The scoring of positive cells was performed independently by two pathologists (H.T. and A.Ha.). Ki-67, cleaved caspase-3, PCNA, 4A4, and TAp63 were counted in five randomly selected high-power fields at 40× magnification, and the counts were averaged. Continuous data are presented as means ± SD. Statistical differences between two groups were analyzed with the t-test or U-test according to the circumstances. P < 0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using MedCalc software version 12.4.0 (MedCalc Software, Ostend, Belgium) and StatMate V version 1.0 for Win&Mac Hybrid (ATMS Co., Ltd., Tokyo, Japan). p16INK4a/p14ARF (p16INK4a/p19ARF in mice) is among the most commonly mutated loci in human cancer, encoding two different tumor suppressors translated from alternatively spliced mRNAs (Figure 1A). p16INK4a is composed of exons 1a, 2, and 3, and is designated here as INK4A (inhibitor of cyclin-dependent kinase 4). p14ARF is encoded by exon 1b and ARFs of CDKN2A/ARF exons 2 and 3, herein referred to as ARF. KO mice with targeted deletion of specific Cdkn2A/Arf exons have disrupted p16INK4a or p19ARF genes, or both,24Serrano M. Lee H. Chin L. Cordon-Cardo C. Beach D. DePinho R.A. Role of the INK4a locus in tumor suppression and cell mortality.Cell. 1996; 85: 27-37Abstract Full Text Full Text PDF PubMed Scopus (1408) Google Scholar, 25Kamijo T. Zindy F. Roussel M.F. Quelle D.E. Downing J.R. Ashmun R.A. Grosveld G. Sherr C.J. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF.Cell. 1997; 91: 649-659Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar, 26Kamijo T. van de Kamp E. Chong M.J. Zindy F. Diehl J.A. Sherr C.J. McKinnon P.J. Loss of the ARF tumor suppressor reverses premature replicative arrest but not radiation hypersensitivity arising from disabled atm function.Cancer Res. 1999; 59: 2464-2469PubMed Google Scholar, 27Sharpless N.E. Bardeesy N. Lee K.H. Carrasco D. Castrillon D.H. Aguirre A.J. Wu E.A. Horner J.W. DePinho R.A. Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis.Nature. 2001; 413: 86-91Crossref PubMed Scopus (669) Google Scholar and develop a different spectrum of spontaneous tumors. To examine the functional role of p16INK4a-specific loss in oral squamous cell carcinogenesis in this study, the RNA expression level of tongue tissues was first confirmed in p16INK4a-specific homozygous KO (p16INK4a−/−) mice, which retain p19ARF, and wild-type (WT) mice by real-time RT-PCR. The results showed that the RNA expression level of p16INK4a was lower than WT mice and that of p19ARF was retained in the tongue tissues of p16INK4a−/− mice (Figure 1B). To elucidate whether deletion of the p16INK4a gene with retention of p19ARF is associated with squamous cell carcinogenesis in the oral cavity, the 4NQO model28Baba S. Yamada Y. Hatano Y. Miyazaki Y. Mori H. Shibata T. Hara A. Global DNA hypomethylation suppresses squamous carcinogenesis in the tongue and esophagus.Cancer Sci. 2009; 100: 1186-1191Crossref PubMed Scopus (37) Google Scholar was used for the development of invasive SCC in p16INK4a−/− and WT mice. A total of 25 p16INK4a−/− and 25 WT mice at 6 to 8 weeks of age were provided with drinking water containing 100 μg/mL 4NQO for 15 weeks (Figure 2A). Ten p16INK4a−/− and WT mice were provided with plain drinking water from birth until termination of the experiment, and there were no obvious abnormalities in any mice. There were no significant differences in the body weights of the 4NQO-treated p16INK4a−/− and WT mice at the end of experiment or any apparent differences in the liver, kidney, lung, or heart between the 4NQO-treated p16INK4a−/− and WT mice. Following 15 weeks of 4NQO administration, macroscopically, nodular, and polypoid tumors were observed in the dorsum and tip of the tongue in the 4NQO-treated p16INK4a−/− and WT mice (Figure 2B). Microscopically, those tongue tumors were identified as dysplasia, oral intraepithelial neoplasia (OIN), and OSCC (Figure 2C). However, there were no histopathologic differences between the 4NQO-treated p16INK4a−/− and WT mice. Histologic assessment of the squamous lesions in the tongue is summarized in Table 2. There were no significant differences in the incidence or multiplicity of early lesions, that is, dysplasia and OIN, between the p16INK4a−/− and WT mice. The incidence and multiplicity of OSCC in the p16INK4a−/− mice were significantly higher than in the WT mice (Table 1).Table 2Incidence and Multiplicities of Microscopic Tumors of the Tongue in 4NQO-Treated MiceGenotypeTongueDysplasiaOINOSCCIncidence, %WT1004820p16INK4a−/−10038.152.4Multiplicity, means ± SDWT3.32 ± 1.260.48 ± 0.500.20 ± 0.40p16INK4a−/−3.14 ± 1.490.52 ± 0.730.71 ± 0.76∗∗P < 0.05 versus OSCC of WT.4NQO, 4-nitroquinoline 1-oxide; OIN, oral intraepithelial neoplasia; OSCC, squamous cell carcinoma; WT, wild type. Open table in a new tab ∗P < 0.05 versus OSCC of WT. 4NQO, 4-nitroquinoline 1-oxide; OIN, oral intraepithelial neoplasia; OSCC, squamous cell carcinoma; WT, wild type. The above results indicate that p16INK4a-specific loss with retention of p19ARF enhanced the development of OSCC in mice. To investigate the mechanisms underlying the progression of OSCC carrying the depleted p16INK4a gene, the proliferation activity and apoptosis of tumor cells from 4NQO-treated mice were assessed. Immunohistochemical analyses for Ki-67, PCNA, and cleaved caspase-3 were conducted in the OSCC cells of 4NQO-treated p16INK4a−/− and WT mice (Figure 3). Ki-67 and PCNA are reliable immunohistochemical markers for evaluating cell proliferation in oral tumors.29Bologna-Molina R. Mosqueda-Taylor A. Molina-Frechero N. Mori-Estevez A.D. Sanchez-Acuna G. Comparison of the value of PCNA and Ki-67 as markers of cell proliferation in ameloblastic tumors.Med Oral Patol Oral Cir Bucal. 2013; 18: e174-e179Crossref PubMed Scopus (155) Google Scholar Cleaved caspase-3 is an immunohistochemical marker of the cell's entry point into the apoptotic signaling pathway.30Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3789) Google Scholar The Ki-67–positive cell ratio in OSCC cells of the 4NQO-treated p16INK4a−/− mice was significantly higher than that of the cells of the 4NQO-treated WT mice (Figure 3, A and B). Further, the PCNA-positive cell ratio in OSCC cells of the 4NQO-treated p16INK4a−/− mice was also significantly higher than 4NQO-treated WT mice (Figure 3, C and D). The cleaved caspase-3–positive cell ratio in OSCC cells of the 4NQO-treated p16INK4a−/− mice did not differ significantly from that of cells of the 4NQO-treated WT mice (Figure 3, E and F). Further, the p16INK4a RNA expression level in invasive SCC of the 4NQO-treated p16INK4a−/− mice was significantly lower than that in the WT mice (Supplemental Figure S2). These data indicate that the proliferation of cancer cells carrying depletion of the p16INK4a gene was associated with the progression of OSCC in mice. The mouse model initiated with 4-NQO is useful for investigating field cancerization in the oral cavity.31Miyamoto S. Yasui Y. Kim M. Sugie S. Murakami A. Ishigamori-Suzuki R. Tanaka T. A novel rasH2 mouse carcinogenesis model that is highly susceptible to 4-NQO-induced tongue and esophageal carcinogenesis is useful for preclinical chemoprevention studies.Carcinogenesis. 2008; 29: 418-426Crossref PubMed Scopus (25) Google Scholar The concept of field cancerization was conceived by Slaughter almost a decade prior to introducing the term in 1953.32Slaughter D.P. Southwick H.W. Smejkal W. Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin.Cancer. 1953; 6: 963-968Crossref PubMed Scopus (2827) Google Scholar Field cancerization has been established in oral cavity carcinogenesis. In carcinogenesis, the early genetic and/or epigenetic events might lead to clonal expansion of preneoplastic daughter cells in a particular tumor field. Subsequent genomic and/or epigenetic changes in some of these cells drive them toward the malignant phenotype.33Dakubo G.D. Jakupciak J.P. Birch-Machin M.A. Parr R.L. Clinical implications and utility of field cancerization.Cancer Cell Int. 2007; 7: 2Crossref PubMed Scopus (223) Google Scholar To evaluate whether the normal-looking squamous epithelial cells have high proliferative activity without any histologic changes after 4NQO exposure, cell proliferation in the normal-looking epithelia adjacent to SCC of the 4NQO-treated p16INK4a−/− and WT mice was assessed. There was no significant difference in the Ki-67–positive cell ratio in the normal-looking epithelia between the 4NQO-treated p16INK4a−/− and WT mice (Supplemental Figure S3). To analyze the impact of genes related to the p16-cyclinD/CDK4-pRb pathway in cell proliferation in the development of OSCC, the expression levels of genes, including p21, Cdk4, Cdk6, pRb, Arf, p17, Ccnd1, E2f1, and Trp53,23Campisi J. d'Adda di Fagagna F. Cellular senescence: when bad things happen to good cells.Nat Rev Mol Cell Biol. 2007; 8: 729-740Crossref PubMed Scopus (2978) Google Scholar were measured in tongue tissues from the 4NQO-treated mice by real-time RT-PCR analysis. RNA samples were obtained from noncancerous normal-looking epithelial tissues close to tongue tumors in 4NQO-treated p16INK4a−/− and WT mice. The RNA expression levels of Arf in p16INK4a−/− and WT mice without 4NQO exposure in tongue tissues were similar (Figure 1B). The RNA levels of Arf and E2f1 were significantly up-regulated in OSCC of the 4NQO-treated p16INK4a−/− mice compared with those of the 4NQO-treated WT mice (Figure 4A). However, the RNA levels of Trp53, p21WAF1/CIP1, Ccnd1, Cdk4, cdk6, pRb, and p27 did not differ significantly between the 4NQO-treated p16INK4a−/− and WT mice (Figure 4A). Subsequently, to clarify the role of the transcriptional up-regulation of Arf in OSCC in the 4NQO-treated p16INK4a−/− mice, the expression of genes, including p63, Hif1a, Foxm1, Myc, Mdm2, Ctbp1, Ctbp2, Wrn, Miz1, and Bckdha, which are located downstream of the p53-dependent and -independent pathways,15Ko A. Han S.Y. Song J. Dynamics of ARF regulation that control senescence and cancer.BMB Rep. 2016; 49: 598-606Crossref PubMed Scopus (23) Google Scholar was assessed by real-time RT-PCR analysis (Figure 4B). Among these genes, the RNA expression of p63 was significantly up-regulated in OSCC in the 4NQO-treated p16INK4a−/− mice compared with that in the 4NQO-treated WT mice (Figure 4B). To determine whether ΔNp63, the oncogenic isoform of p63, is up-regulated or not, the RNA expression levels of ΔNp63 in OSCC by real-time RT-PCR analysis was measured. The results showed that the RNA expression level of ΔNp63 was significantly up-regulated in OSCC of the 4NQO-treated p16INK4a−/− mice compared with that in the 4NQO-treated WT mice (Figure 4C). The above results suggest that deletion of p16INK4a with retention of Arf enhanced ΔNp63 expression. This might lead to the promotion of OSCC in the 4NQO OSCC model. To examine whether the up-regulation of oncogenic ΔNp63 also occurs in human oral squamous cell carcinogenesis, immunohistochemical staining was performed in normal-looking epithelium (n = 9), OIN (n = 9), and OSCC (n = 9) in human patients. However, there is no specific structural antibody for ΔNp63. Therefore, in the present study, two types of antibodies34Karni-Schmidt O. Castillo-Martin M. Shen T.H. Gladoun N. Domingo-Domenech J. Sanchez-Carbayo M. Li Y. Lowe S. Prives C. Cordon-Cardo C. Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression.Am J Pathol. 2011; 178: 1350-1360Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar were used for analyzing the expression of ΔNp63. The total p63 (clone: 4A4) antibody reacts specifically to ΔNp63 and TAp63, and TAp63 antibody reacts specifically to TAp63 (Figure 5A). Using these antibodies, it was possible to identify ΔNp63-positive cells (ΔNp63-positive cells = total p63-positive cells − TAp63-expressing cells). Total p63 (ΔNp63 + TAp63)-positive cells were present in the basal layer of the normal-looking epithelium and OIN, and were spread through the tumor nest in OSCC (Figure 5B). TAp63-positive cells were broadly present over the basal layer of the normal-looking epithelium and basal layer of the OIN, but were absent in OSCC (Figure 5B). Subsequently, double immunofluorescent staining for total p63 (ΔNp63 + TAp63) and TAp63 was performed. ΔNp63-positive cells [total p63-positive cells − TAp63-positive cells (Figure 5C)] were present in the basal layer of the normal-looking epithelium and OIN, whereas ΔNp63-positive cells were spread through the tumor nest in OSCC (Figure 5C). Finally, the total p63-positive, TAp63-positive, and ΔNp63-positive cell ratios were analyzed. The total p63-positive cell ratio in OSCC was significantly higher than that in the normal-looking epithelium and OIN (Figure 5D). The ΔNp63-positive cell ratio was significantly higher in OIN compared with that in the normal-looking epithelium and OSCC, The ΔNp63-positive cell ratio was significantly higher in OSCC compared with that in the normal-looking epithelium (Figure 5E). Taken together, the overexpression of oncogenic ΔNp63 was associated with the progression of OSCC in human oral squamous cell carcinogenesis. In this study, the depletion of p16INK4A with retention of p19ARF enhanced the progression of OSCC in oral squamous cell carcinogenesis. This was caused by the proliferation of cancer cells mediated by the up-regulation of oncogenic ΔNp63. Overexpression of ΔNp63 was observed in the progression of human oral squamous cell carcinogenesis (Figure 6). The relative roles of p16INK4A and p19ARF in the genesis of human oral tumors were examined. The chemical carcinogenesis experiments in the present study demonstrated that the p16INK4a tumor suppressor pathway is likely to be associated with increased malignant transformation to OSCC, although field cancerization on the proliferation of 4NQO-injured normal epithelial cells was not observed. This was similar to a study in humans in which p16INK4A deletion (low expression of p16INK4A and high p16INK4A promoter methylation) was associated with aggressive oral tumors.13Sailasree R. Abhilash A. Sathyan K.M. Nalinakumari K.R. Thomas S. Kannan S. Differential roles of p16INK4A and p14ARF genes in prognosis of oral carcinoma.Cancer Epidemiol Biomarkers Prev. 2008; 17: 414-420Crossref PubMed Scopus (70) Google Scholar However, the tumor suppressive function of p19ARF is p53-dependent, p19ARF/p53−/− mice have a tumor incidence similar to p53−/− mice.18Candi E. Rufini A. Terrinoni A. Dinsdale D. Ranalli M. Paradisi A. De Laurenzi V. Spagnoli L.G. Catani M.V. Ramadan S. Knight R.A. Melino G. Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.Cell Death Differ. 2006; 13: 1037-1047Crossref PubMed Scopus (221) Google Scholar p19ARF also inhibits the activities of various transcriptional factors, including E22f1, Hif1a, Foxm1, and Myc, through direct interaction, thus, preventing cell proliferation.35Wang Y. Dong Q.-Z. Fu L. Stoecker M. Wang E. Wang E.-H. Overexpression of CRKL correlates with poor prognosis and cell proliferation in non-small cell lung cancer.Mol Carcinog. 2013; 52: 890-899Crossref PubMed Scopus (35) Google Scholar In the current experiment, activating the E2F gene with the loss of p16INK4a may lead to activating the cell cycle. Usually, p53, which serves a major role in the suppressor pathway, induces atypical cell apoptosis, but there was no such change in this experiment. However, whether p16INK4A deletion with retention of p19ARF induced the progression of OSCC remains unclear. ΔNp63 and TAp63 belong to the p53/p63/p73 family and differ in the N-terminal transactivation domain. ΔNp63 is overexpressed and promotes cell survival in head and neck SCC, but the extent to which it regulates gene expression that contributes to the malignant phenotype of head and neck SCC is not well defined. In the current experiment, ΔNp63 was up-regulated in 4NQO-treated p16INK4a−/− mice and human OSCC, whereas the expression of p53 did not differ significantly between the 4NQO-treated p16INK4a−/− and WT mice. This was considered to be caused by a decline in the function of tumor suppressor genes, including TAp63 and p53, by the activation of ΔNp63. TAp63 acts synergistically and/or subsequently to control differentiation of the upper epidermal layers. TAp63 isoforms contain a p53-homologous N-terminal TA domain and have similar tumor suppressor properties to p53 in inducing growth arrest, apoptosis, and senescence. By contrast, ΔNp63 controls the expansion of progenitor cells in the basal layer, and ΔNp63 isoforms lacking this TA domain are reported to possess oncogenic activities through the inhibition of TAp63, TAp73, and p53.9Lopez F. Sampedro T. Llorente J.L. Hermsen M. Alvarez-Marcos C. Alterations of p14 (ARF), p15 (INK4b), and p16 (INK4a) genes in primary laryngeal squamous cell carcinoma.Pathol Oncol Res. 2017; 23: 63-71Crossref PubMed Scopus (8) Google Scholar,17Rekhtman N. Ang D.C. Sima C.S. Travis W.D. Moreira A.L. Immunohistochemical algorithm for differentiation of lung adenocarcinoma and squamous cell carcinoma based on large series of whole-tissue sections with validation in small specimens.Mod Pathol. 2011; 24: 1348-1359Crossref PubMed Scopus (268) Google Scholar Therefore, the altered expression of ΔNp63 may be responsible for the increased tumor susceptibility, suggesting that ΔNp63 may be a potential target of OSCC therapy. In summary, the loss of p16INK4a with retention of the expression of p19ARF in the oral cavity leads to the progression of OSCC from the proliferation of cancer cells via the up-regulation of Δp63 in mice. Although the status of p16 and its isoforms in human tissues is unknown in this study, similar events may be associated with human oral squamous cell carcinogenesis. Further analysis on the Δp63 signaling pathway will contribute to target therapy of OSCC. We thank Kyoko Takahashi, Ayako Suga, Reiko Kitazumi, and Masayoshi Shimizu for their technical assistance. Supplemental Figure S2p16INK4a RNA expression level of premalignant (hyperplasia〜dysplasia) and invasive squamous cell carcinoma (SCC) in 4-nitroquinoline 1-oxide (4NQO)-treated p16INK4a−/− and wild-type (WT) mice by real-time RT-PCR. Data are expressed as means ± SD. ∗P < 0.05 (U-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplemental Figure S3A: Hematoxylin and eosin (left) and immunohistochemistry (right) for marker of proliferation Ki-67 (Mki67) in normal-looking epithelial cells of 4-nitroquinoline 1-oxide (4NQO)-treated p16INK4a−/− and wild-type (WT) mice. B: Ki-67–positive cell ratios in normal-looking epithelial cells of 4NQO-treated p16INK4a−/− and WT mice at 15 weeks. Data are expressed as means ± SD. Scale bars: 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)
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